Optimization of livestock serum as an alternative to fetal bovine serum in cultured meat application

Optimization of livestock serum as an alternative to fetal bovine serum in cultured meat application | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Optimization of livestock serum as an alternative to fetal bovine serum in cultured meat application Da-Young Lee, Ermie Jr. Mariano, Ji Won Park, Seok NamKung, So Young Choi, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9212101/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Fetal bovine serum (FBS) is widely used for cultured meat production, but its fetal origin raises ethical concerns and contributes to high cost and batch variability, driving the demand for practical alternatives. This study evaluated livestock sera from bovine, porcine, and chicken sources through biochemical profiling, proliferation and differentiation assays, and long-term serial passaging of bovine satellite cells. All sera met essential biochemical and sterility requirements when properly processed. Bovine and porcine sera, unlike chicken serum, consistently supported robust proliferation, myogenic differentiation, and long-term expansion. Importantly, the final FBS substitutes were optimized through supplementation with lipid-enriched albumin or insulin–transferrin–selenium (25 µg/mL), which significantly enhanced cellular performance. Cost analysis revealed that replacing FBS with the optimized livestock-derived serum formulations reduced total medium cost by 62.6%. Collectively, optimized bovine- and porcine-based FBS substitutes represent effective, scalable, and ethically aligned alternatives for sustainable cultured meat bioprocessing. Fetal bovine serum substitutes Cultured meat Livestock serum Cell culture Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Key points • Bovine and porcine sera fully replaced FBS for bovine satellite cell • Optimized FBS substitutes supported stable proliferation during serial passaging • Replacing FBS reduced total culture medium cost by 62.6% Introduction In modern society, interest in animal welfare and environmental sustainability has been steadily increasing, leading many consumers to explore diverse protein options, including alternatives to conventional meat. In this context, cultured meat has gained attention as a promising supplementary protein source alongside traditional livestock products (Lee et al. 2025b ; Stout et al. 2022 ). Currently, fetal bovine serum (FBS) is an essential component of the cell culture process and accounts for the largest portion of production costs while facing supply instability, high prices, reduced experimental reproducibility due to batch-to-batch quality fluctuations, risk of contamination with animal-derived pathogens, and ethical controversies (Chelladurai et al. 2021 ; Lee et al. 2024 ; O'Neill et al. 2023 ; Skrivergaard et al. 2023 ; Stout et al. 2022 ). These poses significant obstacles to its commercialization as a food product and its social acceptance. FBS is a complex protein-rich mixture of metabolites, making it highly effective in maintaining cell growth and metabolism. It also contributes to enhancing the buffering capacity of the medium and reducing physical stress (Chelladurai et al. 2021 ; Lee et al. 2025a ; Weber et al. 2025 ). Previous studies indicate that FBS-level cell proliferation in serum-free media can only be achieved through supplementation with FBS or animal-derived proteins such as growth factors or fetuin (Lee et al. 2025c ; O'Neill et al. 2023 ; Skrivergaard et al. 2023 ; Stout et al. 2022 ). The combination of albumin, insulin, transferrin, and selenium is known to enhance cell culture performance and is widely used in serum-free media formulation (Dan-Jumbo et al. 2024 ; Francis 2010 ; Lee et al. 2025c ; Shiozuka and Kimura 2000 ; Skrivergaard et al. 2023 ). However, no study has systematically investigated (1) the livestock sera composition differences, (2) improvement of sera via supplementation, and (3) its effects in long-term application. Our research team has focused on blood by-products generated during the livestock slaughter process and has developed alternative serum utilizing these products (Lee et al. 2023 ; Lee et al. 2024 ; Lee et al. 2025a ). In this study, we developed a livestock serum–based culture medium supplemented with additives such as lipid-rich albumin (Albu) and insulin–transferrin–selenium (ITS) primarily for bovine satellite cell lines. Through this approach, we demonstrated that this formulation can be a practical and economical FBS alternative for cultured meat production. Materials and methods Manufacturing process of livestock serum for FBS substitutes FBS (Corning, 35-015-CV, Corning, NY, USA) and adult bovine serum (ABS, 16170078, Gibco, 15140122, Grand Island, NY, USA) were purchased from commercial sources. Livestock blood was collected from Hoengseong KC Co., Ltd. (Hoengseong, Korea), Woo-gyeung Livestock Co. (Hwaseong, Korea), and the Chung-Ang University Farm (Anseong, Korea). Bovine blood was obtained from seven Hanwoo cattle (females aged 31–50 months; males aged 30–33 months). Porcine blood was obtained from six LYD crossbred pigs (Landrace × Yorkshire dam × Duroc sire; males aged 180–190 days). Chicken blood was obtained from 300 Ross 308 broilers (males aged 42–43 days). All animal procedures were conducted in accordance with the guidelines of the Animal Experiment Ethics Committee of Chung-Ang University (approval number: 202401030030). Livestock sera were manufactured as FBS substitutes using a standardized, multi-step processing workflow developed to ensure reproducibility, biosafety, and suitability for cell culture applications (Fig. 1 ). The manufacturing procedure was established based on protocols described in our previous studies (Lee et al. 2023 ; Lee et al 2024 ), with additional modifications applied to ensure consistency. • Serum separation by coagulation and centrifugation Briefly, freshly collected blood was allowed to undergo natural coagulation and subsequently centrifuged at 1,977 × g for 10–15 min (Combi 514R, Hanil Scientific, Gimpo, Korea), thereby enabling efficient separation of the serum fraction. • Sterile clarification by membrane filtration The resulting supernatant was carefully vacuum-filtered through a 0.1 µm Polyethersulfone Membrane Filter (1214756, GVS, Lancaster, UK) to eliminate residual cellular debris and potential microbial contaminants. • Thermal inactivation for biosafety assurance To further enhance biological safety, serum samples were heat-inactivated at 55°C, either immediately post-filtration or immediately before experimental application, in strict parallel with the procedures used for commercial FBS (Lee et al. 2023 ). • Additional clarification and quality validation Additional centrifugation steps were incorporated to remove minute precipitates that could interfere with downstream cell culture applications. Following these multi-tiered purification and safety procedures, comprehensive quality assessments were conducted to verify suitability for cell culture. • Batch selection and storage of validated sera Only sera that passed all quality-control checkpoints were retained. The validated livestock serum preparations were aliquoted and stored at − 20°C under conditions identical to those used for heat-inactivated FBS and ABS until use. Quality profiling of commercial serum and FBS substitutes In this study, we conducted a comparative quality analysis to evaluate the suitability of FBS substitutes, including bovine serum (BoS), porcine serum (PoS), and chicken serum (ChS) as substitutes for FBS. Commercially available FBS and ABS were included as reference sera. Each production lot of commercially available culture sera is provided with a Certificate of Analysis, which confirms the electrophoretic profile, endotoxin concentration, sterility (e.g., absence of mycoplasma), and results of viral detection tests (Thermo Fisher Scientific New Zealand Ltd 2025). However, to directly compare commercial and livestock sera under the same analytical conditions, additional quality analyses were performed in this study. The heme concentration in each serum sample was measured using the Heme Assay Kit (ab272534, Abcam, Cambridge, UK) according to the manufacturer's recommendations, and absorbance was measured at 400 nm using a microplate reader (SpectraMax 190, Molecular Devices, Sunnyvale, CA, USA). The osmolality of the sera was measured using the OsmoTECH® XT. Mycoplasma contamination testing was performed using the Myco-Read™ Mycoplasma Detection Kit (SMD0172, Guri, Korea) according to the manufacturer's protocol. The kit includes a positive control, and samples were prepared by collecting culture fluid from bovine cell lines cultured for 72 h with each serum. Amplified products were run on a 2% agarose gel. Microbiological contamination in the sera was assessed before cell culture using four different Petrifilm™ plates (3M, Saint Paul, MN, USA): Petrifilm™ Aerobic Count (AC) plates, Escherichia coli (EC) plates, Staphylococcus aureus (STX) plates, and Yeast & Mold (YM). Samples were diluted 1:10 in sterile distilled water, applied to the Petrifilm™, and incubated at 35°C for 24–72 h. The pH of the sera was measured using a pH meter (SevenCompact pH/Ion Meter S220, Mettler Toledo, Columbus, OH, USA). The protein content of the sera was measured using the bicinchoninic acid (BCA) assay (Pierce™ BCA Protein Assay Kit, 23227, Thermo Scientific, Waltham, MA, USA). Samples were incubated with the BCA reagent at 37°C for 30 min and measured at 562 nm using a microplate reader (SpectraMax 190, Molecular Devices). Each serum sample was compared by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) at equal protein load. A 10% resolving gel and a 5% stacking gel were used, and samples were loaded alongside an ExcelBand™ Enhanced 3-color High Range Protein Marker (PM2610, SMOBIO Technology, Inc., Hsinchu City, Taiwan). Electrophoresis was performed at 80 V for 10 min, followed by 100 V for 90 min. The gels were subsequently stained with a Brilliant Blue R solution (B7920, Sigma-Aldrich, St. Louis, MO, USA). Bovine cell culture The immortalized bovine satellite cell line (iBSC1, Kerafast, Boston, USA) was used. The cell culture medium was prepared by adding 20% FBS (Corning, 35-015-CV) and 1% penicillin-streptomycin (15140122, Gibco, 15140122) to Dulbecco's modified Eagle's medium (DMEM). The medium was supplemented with basic fibroblast growth factor (bFGF, GR003, Welgene, Gyeongsan, Korea) at final concentrations of 2 ng/mL. Serum was added at 20% to all treatment groups for bovine cell line culture. For other treatment groups, FBS was replaced with an equivalent amount of livestock serum. AlbuMAX™ II Lipid-Rich BSA (11021029, Gibc) was diluted to a concentration of 10 mg/mL in 0.1% BSA solution. The ITS used in this study (100X, ITS-G, 41400045, Gibco) was diluted to 1X with 0.1% BSA solution. Each additive was added to the culture medium at three concentration levels (25, 50, and 75 µg/mL). For the combination groups, both additives were co-supplemented at 25 + 25 or 50 + 50 µg/mL. To ensure equal volume adjustment, the same amount of 0.1% BSA solution was added to the non-supplemented control groups. Differentiation of iBSC was induced in myogenic (differentiation) medium, which consists of DMEM supplemented with 1% P/S/A and 2% horse serum. All procedures involving animals were approved by the Animal Experiment Ethics Committee of Chung-Ang University (approval number: 202301020084). Cell growth in FBS substitute media The iBSC cultured on plates for 3 days were counted by staining with trypan blue (15250061, Gibco). To measure cell viability, 0.5 mg/mL of thiazolyl blue tetrazolium bromide (MTT, M2128, Sigma-Aldrich) was added to cells cultured for 3 days and reacted for 4 h. The MTT solution and medium were removed from the wells, and DMSO was added to dissolve the purple MTT−formazan crystals. The optical density of each well was measured at 540 nm using a microplate reader (SpectraMax 190, Molecular Devices). Immunostaining The differentiated cells were washed with 1X Dulbecco's phosphate-buffered saline (DPBS, 16777-644, Cytiva, Marlborough, MA, USA) and fixed in paraformaldehyde (4%) for 20 min. After washing twice more with 1X DPBS, the samples were treated with 0.2% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) for 20 min and blocked with 2% BSA in 1X DPBS for 2 h at room temperature. Cells were then incubated for 2 h at 4 ℃ with the following primary antibodies (MF20, 1:800, DSHB, Iowa City, IA, USA; desmin [DES], 1:1,500, ab32362, Abcam) dissolved in 1% BSA in 1X DPBS. After incubation, cells were treated with secondary antibodies (A90-116F and A120-101D4, BETHYL, Montgomery, TX, USA) for 1 h at room temperature, then stained with Hoechst 33342 (1:3,000, 561908, BD Biosciences, San Jose, CA, USA). The images were observed with a fluorescence microscope (KI-3000F, Korea Lab Tech, Namyangju, Korea). Western blotting Western blots were performed on cell lysates prepared in RIPA buffer containing phosphatase inhibitor (78420, Thermo Fisher Scientific) and protease inhibitor (87785, Thermo Fisher Scientific). The BCA Protein Assay Kit (Pierce™ BCA Protein Assay Kits, 23227, Thermo Fisher Scientific) was used to quantify the total protein in each sample. SDS-PAGE (8–10% polyacrylamide gels) was carried out. After transfer of the proteins to polyethersulfone (PES) membranes, the proteins were blocked with 5% BSA in PBS-T for 1 h and probed overnight with primary antibodies Pax7 (1:1,500, DSHB, Iowa City, IA, USA), MyoG (1:1,500, F5D, DSHB), Desmin (1:2,500, ab32362, abcam, Cambridge, UK), MF20 (1:1,500, DSHB), and β-actin (1:5,000, ab8227, Abcam). Then, the proteins were incubated with anti-mouse IgG and anti-rabbit IgG as appropriate. Specific signals were detected with an e-BLOT Touch Imager (e-BLOT Life Science, Shanghai, China). Statistical analysis Statistical analysis of the experimental data involved one-way analysis of variance (ANOVA) using SPSS 22.0 (IBM Corp., Armonk, NY, USA). Statistical significance was assessed using Tukey’s multi-range test, and the significance level of all data was evaluated based on P < 0.05. The results are presented as the average of triplicate experiments. Results Quality profiling of commercial and livestock sera The major challenge in utilizing livestock slaughter by-products is ensuring they meet the appropriate quality and safety standards for cell culture. Contamination by bacteria or mycoplasma can severely compromise cell growth, and because antibiotics are being phased out from cultivated meat production, ensuring sterility is critical. We therefore evaluated the biochemical quality and contamination status of livestock sera, as well as those of commercial sera, before their application in cell culture ( Fig. 2 ). Visual inspection ( Fig. 2 ) revealed clear differences in serum color: BoS and PoS displayed darker reddish tones compared with FBS, ABS, and ChS, suggesting hemolysis during blood collection. The serum protein concentration and SDS-PAGE electrophoresis results were similar to those of our previous studies ( Fig. 2 b, c ) ( Lee et al. 2023 ; Lee et al. 2025a ). ABS and BoS showed relatively similar protein band patterns compared to FBS, with more bands detected in the region below 60 kDa. Considering that ABS was obtained from sera of cattles aged 12−24 months, there was no significant difference in SDS-PAGE profiling compared to BoS obtained from older individuals (aged 31−59 months). Although SDS-PAGE enabled comparison of overall protein banding patterns, specific growth-promoting proteins (e.g., albumin, transferrin, insulin growth factor [IGF] family members) were not individually quantified. Future proteomic profiling will be necessary to identify the molecular determinants underlying differences in serum performance. Compared with FBS, the protein contents of ABS, BoS, and PoS were significantly higher. In contrast, ChS showed a distinct absence of bands below 45 kDa, and its protein concentration was also significantly lower. Adult blood contains higher protein levels than fetal or neonatal blood, and if blood is not promptly processed after collection, intracellular proteins may be released into the sample through cell lysis, which can further increase the measured protein concentration ( Bjelosevic et al. 2017 ; Lee et al. 2025a ; Halvey et al. 2021 ). Measurements of heme concentration showed that BoS contained the highest concentration (146.8 µM), followed by PoS, and then FBS, ABS, and ChS, which all showed much lower concentrations ( Fig. 2 d ). The high heme concentration observed in the serum is thought to be due to destruction of red blood cells during blood collection or damage during transport, which is consistent with the dark color of the serum ( Fig. 2 a ). Notably, the measured values far exceeded the recommended hemoglobin cut-off of < 4 mg/mL, indicating a considerable level of erythrocyte rupture in some livestock samples (Corning Inc 2025). Osmotic analysis ( Fig. 2 d ) revealed that most sera were within the physiological range (285–340 mOsm/kg), but BoS (358 mOsm/kg) and PoS (397.3 mOsm/kg) values were higher, presumably due to intracellular solute release from ruptured erythrocytes ( Fig. 2 e ). When evaluated against a broader quality criterion—acceptable osmolality of 280–365 mOsm/kg H₂O—BoS fell slightly above the upper threshold, while PoS substantially exceeded it (Corning Inc 2025). Previous work has shown that osmotic pressure in coagulated whole blood increases over time, with a faster rise when serum is not promptly separated ( Sureda-Vives et al. 2017 ). The pH of all treatment groups fell within the reference range (7.0–8.3) suggested by the commercial FBS Quality Assurance Certificate ( Fig. 2 f ) (Corning Inc 2025). However, the pH values were higher than the value (7.3) suggested by the FBS quality assurance certificate. Other treatment groups were also confirmed to have values higher than FBS ( Fig. 2 f ). Increased contact with air during blood (serum) collection and preprocessing and increased storage temperature (from frozen/refrigerated to room temperature) can decrease the solubility of carbon dioxide in blood, leading to an increase in pH ( Kirschbaum 2003 ). These mechanisms could explain the pH results observed in the present study. Mycoplasma testing ( Fig. 2 g ) yielded negative results for all serum types; an important finding given that mycoplasma is one of the most problematic contaminants in cell culture. Furthermore, microbial safety tests using AC, EC, STX, and YM Petrifilm™ plates ( Fig. 2 h ) showed no microbial growth after filtration, confirming that the applied processing system effectively eliminated viable microorganisms and preserved sterility. Media development and screening of Albu and ITS for iBSC proliferation To evaluate the potential of livestock serum as an alternative to FBS, iBSC were cultured for 3 days in media containing livestock serum obtained from bovine, porcine, or chicken blood by-products, either alone (raw) or supplemented with Albu and/or ITS. Proliferation was assessed by direct nuclear counting and MTT assay, and the results were compared with commercial serum groups (FBS and ABS) ( Fig. 3 ). In all analyses, the minimal supplementation principle was applied: when no significant differences were observed among treatment groups, the lowest effective concentration or single additive treatment was selected. Final concentrations (25 or 50 µg/mL) were selected because these levels maintained ≥ 70–80% of the proliferative activity supported by FBS, and higher concentrations provided no additional benefit while occasionally showing early signs of diminishing returns. All treatment groups completely replaced 20% FBS. In media containing BoS, even when certain supplementation conditions exceeded the cell proliferation rate of the FBS control medium, the treatment group that achieved ≥ 100% proliferation with the smallest additive dose was selected to maximize efficiency and reproducibility. Raw BoS itself supported iBSC proliferation at a level comparable to the CTL (control) condition, and supplementation with Albu or ITS alone produced a similar outcome, as neither additive significantly increased cell counts relative to CTL ( Fig. 3 b, c, left). This pattern was similarly observed in MTT absorbance–based metabolic activity ( Fig. 3 b, c, right). Although slight increases were visually noticeable, they were not statistically significant. Furthermore, co-addition of Albu and ITS did not produce synergistic effects and, in some cases, even resulted in minor reductions. Overall, BoS demonstrated a sufficiently strong baseline proliferative capacity in its raw form, and because additional supplementation did not produce meaningful improvements, 25 µg/mL was selected as an appropriate supplementation level for subsequent analyses based on the minimal supplementation principle. Raw PoS promoted iBSC proliferation at a level comparable to that of CTL. However, the proliferation level appeared numerically lower than in BoS-supplemented medium, a trend that did not reach statistical significance. Supplementation with Albu or ITS likewise did not result in meaningful increases in either cell counts or metabolic activity ( Fig. 3 b, c ). Moreover, the combined treatment of Albu and ITS did not produce synergistic effects and, occasionally, even resulted in a modest numerical decrease. Although slight visual increases or decreases were observed, they were not statistically supported. As with BoS, supplementation did not clearly enhance iBSC proliferation, and 25 µg/mL of each additive was considered adequate for downstream analyses according to the minimal supplementation principle. ChS showed a different response compared with BoS and PoS. In medium containing raw ChS, a weaker baseline proliferative capacity was observed, requiring relatively higher additive concentrations. The proliferation increase was dependent on the additive concentration, with a relatively notable effect observed when supplemented with ITS ( Fig. 3 d ). Nonetheless, the combination of Albu and ITS did not yield synergistic effects beyond single supplementation. In this screening assay, medium containing ChS with additive treatment produced reproducible proliferation levels of approximately 75–80% relative to the FBS control. A concentration of 50 µg/mL was considered sufficient to maintain cell growth and was selected for subsequent experiments. Thus, subsequent analyses used 25 µg/mL of Albu or ITS for BoS and PoS, and 50 µg/mL of Albu or ITS for ChS. Each Raw serum condition, along with FBS and ABS controls, was included, resulting in a total of 11 treatment groups. This strategy was adopted to minimize cost and formulation complexity while maintaining functional performance. Effects of optimized livestock serum–based culture medium on iBSC proliferation and differentiation Effect of selected additives on iBSC proliferation iBSCs were cultured for 3 days in media formulated with selected conditions for each livestock serum group. Cell counting and MTT-based metabolic activity analyses revealed no significant difference between the commercial controls, namely FBS and ABS ( Fig. 4 a, b ). In Day 3 cultures, the expression of the satellite cell-specific protein Pax7 was significantly higher in the ABS group ( Fig. 4 c, d ). In all BoS groups, BoS supported robust cell proliferation, similar to that observed in FBS control medium ( Fig. 4 ). All BoS treatment groups exhibited Pax7 protein expression levels approximately 150% of those of FBS, showing consistently elevated and comparable effects across BoS alone (BR), BoS+Albu (BA), and BoS + ITS (BI) ( Fig. 4 d ). These results suggest that BoS already provides near-optimal culture conditions, and supplementation may have limited visible impact due to a ceiling effect. Adult PoS supported iBSC proliferation at levels comparable to or greater than those observed with FBS ( Fig. 4 a, b ). Notably, on Day 3, the PoS+Albu (PA) and PoS + ITS (PI) treatments enhanced the effect of PoS alone (PR), resulting in more than a 200% increase in Pax7 protein expression compared with that observed in the FBS control group ( Fig. 4 ). This demonstrates that PoS not only supports stable proliferation but also retains responsiveness to exogenous Albu and ITS, thereby further enhancing proliferation and satellite cell characteristics. In contrast to BoS and PoS, the ChS group exhibited significantly lower proliferation across all treatments despite the use of higher additive concentrations ( Fig. 4 a ). Although ITS supplementation provided a statistically measurable improvement in proliferation ( Fig. 4 b ), the overall effect remained limited and did not reach the level of efficacy observed in the FBS. The Pax7 levels in ChS alone (CR) and ChS+Albu (CA) were comparable to those in FBS but markedly lower than in other serum groups. Moreover, unlike its effect on proliferation, ITS supplementation (ChS + ITS, CI) further reduced Pax7 expression ( Fig. 4 d ). Induction of myogenic differentiation after the proliferation phase After 3 days of proliferation, the medium was replaced with HS-containing differentiation medium, and cells were cultured for four additional days (total of 7 days). Proliferation was evaluated relative to the FBS, and differentiation was initiated simultaneously across all treatments, even when some groups (e.g., ChS) did not reach full confluence. MyoG was assessed at early differentiation (Day 5 and 7), and MF20 and Desmin at late differentiation (Day 7). The fusion index was calculated as the proportion of Desmin-positive multinucleated myotubes. In the FBS group, nearly all proliferated cells differentiated, resulting in the highest fusion index ( Fig. 5 a, b ). ABS and all livestock serum treatments showed significantly lower fusion indices than FBS, but differentiation occurred in all conditions ( Fig. 5 a, b ). The MF20-stained area was lower than the Desmin-stained area across groups; however, both showed similar trends, with the FBS group exhibiting the highest myotube area ( Fig. 5 c ). Western blot analysis further revealed that the BoS group exhibited MyoG expression similar to that of the ABS group at Day 5 across all treatments. However, a significant decrease in MyoG was observed in the BoS treatment groups, particularly in BA and BI, at Day 7 ( Fig. 5 e ). This trend was also observed in the PoS and ChS groups, with some treatments (PA, CA and CI) even exhibiting higher MyoG expression than the FBS group at Day 5. MyoG is known to increase as iBSCs enter differentiation and decrease toward the late stage. Therefore, the consistent decrease in MyoG observed at Day 7 compared with Day 5 suggests that iBSCs successfully transitioned from early to late differentiation in all serum groups ( Fig. 5 e ). In the BoS group, supplement supplementation-dependent differences in myotube area were not evident ( Fig. 5 c ). Muscle-specific protein expression analysis to assess late myotube formation showed that all BoS groups performed better than the ABS group, albeit BR showed similar effects to BA and BI despite the lack of supplementation ( Fig. 5 e ). The effects of all BoS groups were, higher than ABS, but only approximately 70% of those of FBS on average ( Fig. 5 ). Supplementation in the PoS groups increased muscle-specific protein formation compared with PR ( Fig. 5 c ). In the PA and PI groups, the myotube area markedly increased, reaching approximately 100% of the MF20 level and ~ 80% of the Desmin level, with both proteins showing no significant difference compared with FBS ( Fig. 5 c ). Muscle-specific protein expression in these groups was also higher than that observed in the ABS group ( Fig. 5 e ). Notably, MF20 expression in the PA group was equivalent to that of the FBS group, demonstrating its full replacement potential, whereas MF20 expression in the PI group remained at approximately 70% of the FBS-group level ( Fig. 5 e ). In the ChS group, Desmin was restored to levels similar to those in the FBS group through CA and CI supplementation, whereas MF20 expression remained markedly low ( Fig. 5 e ). Unlike Desmin, MF20 is strongly expressed only in sufficiently matured, thick myotubes ( Ciecierska et al. 2020 ; Costa et al. 2004 ). Thus, although structural integrity (DES) was recovered by supplementation, full maturation (MF20) did not reach FBS-group levels. These results are consistent with the proliferation differences observed (Section 3.3.1), suggesting that cell density before differentiation is a key determinant of myotube maturation efficiency. Long-term maintenance of proliferative potential during serial passaging of iBSCs With commercial serum (FBS and ABS), no significant decrease in cell proliferation was observed during four consecutive passages (Day 3–12). The overall proliferative capacity remained stable throughout the long-term culture period, maintaining levels comparable to the initial passage ( Fig. 6 , commercial serum). These results confirm that commercial serum supports long-term expansion of iBSCs without loss of proliferative potential. Similarly, BA and BI maintained a stable proliferative capacity during long-term culture. Although a slight fluctuation occurred after Day 6, this variation did not result in a statistically significant decrease compared with Day 3 ( Fig. 6 , ABS). Notably, previous research has suggested that albumin or ITS (or insulin) supplementation can enhance long-term proliferative stability in myogenic cells ( Lee et al. 2025c ; Mainzer et al. 2014 ; Stout et al. 2022 ). Consistent with this, both BA and BI in our study exhibited more sustained proliferation compared with BR, indicating that these supplements positively contribute to maintaining long-term expansion capacity. The PoS group also preserved a stable level of proliferative ability during repeated passaging. Although a slight change in proliferation was observed after Day 6, this variation did not result in a statistically significant decrease compared with Day 3 ( Fig. 6 , porcine adult serum). Notably, upon ITS supplementation, cells exhibited consistently stable proliferation throughout the entire culture period without significant variation. In contrast to BoS and PoS, ChS supplementation exhibited a marked decline in proliferative capacity over successive passages. After Day 9, the number of proliferated cells sharply decreased to less than half of the initial level ( Fig. 6 , adult ChS). ITS supplementation (CI) temporarily slowed this decline but did not prevent the overall reduction in proliferation. Interestingly, although ChS supplementation maintained approximately 70–80% of the proliferation level observed with the FBS control during short-term screening, this performance did not translate to long-term stability. The sharp decline highlights that short-term proliferation does not predict long-term maintenance, and serum quality cannot be reliably evaluated based on short-term or single-passage data alone. Given the strong species-specific compatibility observed in this study—ChS supporting avian satellite cells but not bovine satellite cells—the pronounced decline during long-term culture likely reflects this inherent biological mismatch. Cost evaluation of optimized livestock serum–based culture medium for hybrid cultured meat applications To determine the economic feasibility of the experimental sera, a comparative cost analysis was conducted based on the composition of each growth medium. Cost calculations were based on 2025 market prices from major suppliers and expressed as cost per 600 mL of complete medium. Although the preparation of livestock serum may entail specific processing costs, this study was based on the concept of using the large volume of blood typically discarded during slaughter. Filtration and labor costs were not included because they have a minimal impact on serum cost and are the same regardless of serum type. Therefore, the unit cost of livestock serum was set to zero in the cost analysis. Media prepared with FBS had the highest cost at 126.6 USD per 600 mL ( Fig. 7 ). Of this, the serum itself (79.3 USD) accounted for 62.7% of the total cost, while bFGF (26.7 USD) accounted for 21.1%. Basal medium and antibiotics contributed 14.5% and 1.5%, respectively ( Fig. 7 ). This again confirms that serum and growth factors are the major cost drivers in conventional cell culture media. By contrast, media containing ABS cost 64.2 USD —a 49.3% reduction compared with FBS-based media. In this case, the serum cost decreased sharply (16.9 USD), and the dominant cost components became bFGF (6.7 USD) and basal medium (18.7 USD) ( Fig. 7 ). When serum was replaced with livestock serum (serum cost = 0), the total medium cost decreased further to 47.4 USD —a 62.6% reduction relative to FBS-based media. Under these conditions, the cost structure shifted from serum-dependent to growth factor-dependent, with bFGF accounting for the largest proportion of total cost. Even when livestock serum was supplemented with low-cost additives such as AlbuMAX™ (.1 USD) and ITS (0.1 USD), the total cost increased only marginally— 47.4 USD, representing an increase of merely 0.46% ( Fig. 7 ). Despite this minimal cost increase, cell proliferation stability was improved, demonstrating the cost-effectiveness of combining livestock serum with low-cost supplements. Discussion In this study, we systematically evaluated livestock-derived sera with respect to their biochemical properties, proliferative capacity, differentiation potential, and long-term stability, in comparison with commercial FBS and ABS. Overall, bovine and porcine sera exhibited comparable or superior performance to FBS, whereas chicken serum displayed distinct limitations. Quality assessments of livestock serum revealed no microbial contamination, but most livestock serum still failed to meet the quality standards expected for FBS. Heme concentration, osmotic pressure, and pH outside the normal physiological range can both inhibit cell proliferation, metabolic activity, and growth factor secretion and cause damage such as endoplasmic reticulum stress or apoptosis ( Alhuthali et al. 2021 ; Ceccarini & Eagle 1971 ; Pethő et al. 2021 ; Tatsumi et al. 2002 ). The minor differences observed between livestock serum and commercial FBS appear to result largely from practical handling factors rather than intrinsic quality limitations. As evidence, most biochemical parameters in our serum samples were comparable to those of commercial products, and no mycoplasma or bacterial contamination was detected, confirming that livestock serum can already achieve a quality level suitable for cell culture. Variations in heme concentration and osmolality likely reflect routine factors such as transport conditions or processing time, which can be readily stabilized when industrial protocols and cold-chain systems are applied. With these procedures in place, consistent, high-quality livestock serum can be produced, further reinforcing its potential as a robust and reliable alternative to FBS. Albumin and ITS are the most commonly used cell additives in serum-free conditions. Albumin increases cell proliferation and sustains its effects ( Kobayashi & Takubo 2020 ; Skrivergaard et al. 2023 ; Stout et al. 2022 ). Albumin supports cellular energy metabolism by transporting fatty acids, suppresses oxidative stress by binding metal ions, and stabilizes growth factors as well as the osmotic and protein balance of the medium ( Francis 2010 ; Skrivergaard et al. 2023 ; Stout et al. 2022 ). Through these functions, albumin contributes to overall culture stability and promotes cell proliferation. Insulin stimulates cell proliferation and adhesion, and in combination with ITS, enhances cell culture stability ( Lee et al. 2025a ; Skrivergaard et al. 2023 ). Furthermore, because transferrin in ITS can supply iron to cells during cell proliferation, it may play a role in utilizing the abundant iron in BoS ( Lee et al. 2025a ; Mainzer et al. 2014 ; Shiozuka & Kimura 2000 ). Despite these well-established benefits, such effects were not apparent in the ChS group in our study. ChS required supplementation due to its low albumin content; however, the relatively low level of albumin added based on screening data likely did not fully compensate for the absolute albumin deficiency, resulting in a lower expected improvement in iBSC proliferation and Pax7 expression. In addition, ChS is commonly used without FBS in commercial avian satellite cell cultures, and our results suggest that these protocols are based on species-specific effects ( Hagiwara & Ozawa 1982 ; Kerafast 2023 ; Oh et al. 2022 ). These results are similar to our previous findings that ChS was the least effective for bovine satellite cells and C2C12 (mouse myoblasts) ( Lee et al. 2024 ; Lee et al. 2025a ). Even the addition of ITS, which supports satellite cell proliferation, can delay, but not completely prevent, this trend ( Skrivergaard et al. 2023 ; Wu et al. 2018 ). This indicates that responsiveness to ChS is species-dependent, with limited effects on iBSCs despite its effectiveness for avian satellite cells. Interestingly, the relationship between serum albumin content and the effects of additive supplementation was not straightforward. In our previous studies, the albumin content of collected BoS and PoS was approximately twice that of FBS, whereas that of ChS was approximately half. Despite this, supplementation with lipid-rich albumin and ITS in the porcine serum group produced a clear synergistic enhancement of satellite cell characteristics ( Lee et al. 2024 ; Lee et al. 2025a ). Albumin and ITS are generally known to promote proliferation in serum-free or low-serum systems, yet the magnitude of their effect differed substantially between serum types. In the BoS group, the strong proliferative capacity of BR and BI itself left little room for further enhancement, which explains the minimal additional response to supplementation. In contrast, the pronounced responsiveness observed in the PoS group suggests that, although PR also supported robust growth, it retained a metabolic or signaling margin that allowed synergistic effects upon supplementation. However, the precise biochemical factors, such as differences in the composition of unidentified effective proteins or growth factors in serum, remain unclear, and no existing literature provides a clear explanation for the specific response of PoS to albumin and ITS. These observations highlight an intriguing but still unresolved relationship between serum composition and supplement responsiveness, suggesting that targeted mechanistic studies will be necessary to fully elucidate this synergistic effect. Overall, ChS was ineffective in promoting iBSC proliferation, whereas BoS and PoS consistently supported robust cell growth and preserved satellite cell characteristics. In the BoS group, all treatments supported proliferation equivalent to FBS and induced approximately 150% of the Pax7 expression observed in FBS control medium, indicating that BoS provides inherently optimal culture conditions without requiring additional supplements. In the PoS group, responsiveness to supplementation was even more pronounced; the PA and PI treatments produced Pax7 levels exceeding 200% of those in FBS control medium, demonstrating a clear synergistic enhancement of satellite cell characteristics. Collectively, these findings highlight the strong functional potential of both BoS and PoS as practical alternatives to FBS, with PoS showing particular promise when combined with Albu and ITS. Insulin, a key component of ITS, is well known as a myogenic enhancer in differentiation media ( Dan-Jumbo et al. 2024 ; Sian et al. 2025 ). Previous studies have shown that albumin or insulin supplied only during proliferation can enhance both growth and differentiation potential after switching to differentiation medium ( Francis 2010 ; Lee et al. 2025c ; Stout et al. 2022 ). Thus, serum conditions that supported robust proliferation during the first 3 days likely primed cells to a metabolic state more favorable for differentiation, enabling more efficient myotube formation. Desmin expression was relatively uniform across groups, consistent with its early structural role before contractile protein accumulation ( Ciecierska et al. 2020 ; Costa et al. 2004 ). Previous reports show MHC levels can vary even when Desmin does not, indicating that Desmin alone cannot assess late myotube maturation ( Ciecierska et al. 2020 ; Costa et al. 2004 ). In our study, Desmin levels remained consistent across serum groups, whereas MF20 varied widely, suggesting distinct differences in maturation stage among treatments. MF20 expression was particularly low in the ChS group. Although Desmin-positive structures were present, MF20 expression differed markedly from other serum groups despite similar fusion index values ( Fig. 5 e ). Low cell density delays myotube formation and reduces MHC expression ( Murphy et al. 2016 ; Tanaka et al. 2011 ), and thinner myotubes show lower MHC expression ( Hsieh et al. 2020 ). Because the ChS group showed the lowest proliferation before differentiation among the groups, its reduced MF20 signal likely reflects delayed or incomplete maturation rather than failed differentiation, consistent with the discrepancy between Desmin and MF20 expression levels. Overall, although ChS treatment maintained early differentiation markers, it was less effective in driving full myotube maturation, likely due to insufficient pre-differentiation proliferation, and ITS supplementation was less effective than FBS. In contrast, BoS and PoS treatments supported successful progression from early to late differentiation. All BoS groups and PoS promoted myotube formation and muscle-specific protein expression similarly or better than ABS, and PR achieved effects comparable to FBS. These findings highlight the strong potential of BoS and PoS as functional alternatives to commercial serum and their potential for cost-effective cultured meat production through 100% replacement of FBS. This study has some limitations. A key limitation is the inability to distinguish whether lower MF20 results reflect reduced differentiation capacity or merely slower maturation. Additional time-resolved assays and functional measurements will be needed to determine whether these serum conditions limit or simply delay myotube maturation. Furthermore, supplementation requirements differed clearly by species—BR itself provided the most effective condition, Albu exerted the strongest enhancement in PoS, and ITS supplementation produced the most pronounced improvement in ChS—indicating underlying biochemical differences that warrant future investigation. Although ChS supplementation did not support long-term proliferation of bovine satellite cells, both BoS and PoS maintained stable proliferative capacity over four consecutive passages, comparable to that of commercial serum. Notably, supplementation further enhanced this stability: in the BoS group, BA and BI sustained higher proliferation than BR, and in the PoS group, all serum conditions—including PR, PA, and PI—maintained strong proliferative stability throughout subculture. Our observations suggest that species compatibility between cells and sera may contribute to long-term culture stability. Taken together, these findings demonstrate that BoS and PoS—particularly when fortified with Albu or ITS—provide strong, reliable, and cost-effective long-term support for iBSC expansion, highlighting their practical potential as high-performance alternatives to FBS for large-scale cultured meat production. Importantly, these functional outcomes were achieved alongside a substantial reduction in medium cost, indicating that performance improvements were not achieved at the expense of economic feasibility. Livestock blood is produced in large quantities during slaughter and is typically associated with disposal costs. Its use as a serum source, therefore, provides multiple advantages : • Reduced media costs (= zero purchase cost) • Reduced waste disposal cost • Reduced environmental burden and promotion of resource recycling Thus, livestock serum is not merely a low-cost alternative to FBS but a strategic resource capable of enabling a sustainable and circular cultured meat production system. Furthermore, using livestock serum enables a substantial reduction in media cost, and the resulting cultured biomass can be applied to the development of hybrid cultured meat products ( Fig. 7 ). As illustrated in the conceptual workflow ( Fig. 7 ), biomass generated using BoS and PoS can be combined with plant-based proteins, colorants, seasonings, and texturizing agents to achieve desirable sensory and structural properties (data not shown). The resulting mixture can then be molded and heat-set to form the final product, demonstrating how livestock serum-based cell cultivation can be integrated into practical downstream manufacturing. Furthermore, although livestock serum–based FBS substitutes already offer substantial cost advantages, future large-scale commercialization is expected to further reduce production costs and enable the stable supply of more uniform, high-quality products through improved resource utilization and economies of scale. We are currently advancing this process and conducting experiments to further optimize the quality of hybrid cultured meat. Overall, the approach of this study presents a sustainable and economically viable strategy that could support the industrial transition of hybrid cultured meat technology. Conclusion This study provided an integrated evaluation of livestock sera, including biochemical profiling, optimized supplementation strategies, functional assays, long-term passaging, and cost modeling, to assess their suitability as alternatives to FBS. Quality assessment revealed that livestock sera fully satisfied sterility requirements and exhibited biochemical characteristics largely comparable to commercial FBS. The remaining variations—such as elevated heme concentration, osmolality, or pH—were minor and primarily attributable to practical handling conditions rather than inherent limitations, and can be readily stabilized through standardized blood collection, processing, and cold-chain control. Among the three livestock sera, ChS showed the least applicability for bovine satellite cells, with low proliferative capacity, species-specific incompatibility, and a pronounced decline during long-term passaging. Its reduced ability to sustain growth and myotube maturation indicates that ChS is not a viable option for large-scale bovine cell expansion. In contrast to adult ChS, both BoS and PoS demonstrated strong, consistent, and durable performance across short-term proliferation, myogenic differentiation, and extended serial culture. ABS showed the highest intrinsic proliferative capacity without supplementation, and the BA and BI condition (each supplemented with 25 µg/mL of additives)) maintained the most stable long-term expansion, making it the most reliable BoS candidate for FBS replacement. Adult PoS also sustained robust growth across both short- and long-term culture, with the PR and PI condition (each supplemented with 25 µg/mL of additives) achieving proliferation and differentiation outcomes equivalent to or exceeding those of FBS. BA and BI: proliferation ≈ 150% of FBS; differentiation ≈ 70% of FBS PA: proliferation ≈ 200% of FBS; differentiation ≈ 100% of FBS PI: proliferation ≈ 230% of FBS; differentiation ≈ 80−100% of FBS Importantly, replacing FBS with livestock serum reduced total medium cost by 62.7%, substantially lowering production expenses while decreasing slaughterhouse waste-disposal burdens and improving resource circularity. With further optimization of collection and processing pipelines, consistent, high-quality livestock sera can be produced, establishing a scalable foundation for cost-efficient cultured meat bioprocessing. Collectively, these findings position BoS and PoS as scientifically validated, industrially scalable, and economically advantageous alternatives to FBS, offering strong potential to advance sustainable hybrid and fully cell-based meat production systems. Declarations CRediT authorship contribution statement Da - Young Lee: Investigation, Validation, Methodology, Writing - original draft, Writing - review & editing. Ermie Jr. Mariano: Investigation, Validation, Writing - review & editing. Ji Won Park: Investigation, Validation, Writing - review & editing. Seok Namkung: Investigation, Validation, Writing - review & editing. So Young Choi: Investigation, Validation, Writing - review & editing. Woo Jin Lee: Investigation, Validation, Writing - review & editing. Ye Won Shin: Investigation, Validation, Writing - review & editing. Chae Hyeon Bok: Investigation, Validation, Writing - review & editing. Sun Jin Hur: Supervision, Writing - review & editing. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Ethical Approval Not applicable Funding This work was conducted with the support of Chung-Ang University. This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry(IPET) through High Value-added Food Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs(MAFRA)(RS-2025-02215727). Data availability All data generated or analysed during this study are included in this published article. Additional raw datasets are available from the corresponding author upon reasonable request. References Alhuthali S, Kotidis P, Kontoravdi C (2021) Osmolality effects on CHO cell growth, cell volume, antibody productivity and glycosylation. Int J Mol Sci 22(7):3290. https://doi.org/10.3390/ijms22073290 Bjelosevic S, Pascovici D, Ping H, Karlaftis V, Zaw T, Song X, Molloy MP, Monagle P, Ignjatovic V (2017) Quantitative age-specific variability of plasma proteins in healthy neonates, children and adults. 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Front Toxicol 7:1612903. https://doi.org/10.3389/ftox.2025.1612903 Wu J, Matthias N, Lo J, Ortiz-Vitali JL, Shieh AW, Wang SH, Darabi R (2018) A myogenic double-reporter human pluripotent stem cell line allows prospective isolation of skeletal muscle progenitors. Cell Rep 25(7):1966–1981. https://doi.org/10.1016/j.celrep.2018.10.067 Additional Declarations No competing interests reported. Supplementary Files Supplementaryfile.xlsx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9212101","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":618838465,"identity":"85679b5b-c000-481f-985a-49f722ded23f","order_by":0,"name":"Da-Young Lee","email":"","orcid":"","institution":"Chung-Ang University","correspondingAuthor":false,"prefix":"","firstName":"Da-Young","middleName":"","lastName":"Lee","suffix":""},{"id":618838466,"identity":"e5e0ea09-c695-4a37-8e63-81f9dc0c4f48","order_by":1,"name":"Ermie Jr. Mariano","email":"","orcid":"","institution":"Chung-Ang University","correspondingAuthor":false,"prefix":"","firstName":"Ermie","middleName":"Jr.","lastName":"Mar","suffix":"Jr."},{"id":618838467,"identity":"3a3af9bb-23a5-43f6-be68-0abd65da89cf","order_by":2,"name":"Ji Won Park","email":"","orcid":"","institution":"Chung-Ang University","correspondingAuthor":false,"prefix":"","firstName":"Ji","middleName":"Won","lastName":"Park","suffix":""},{"id":618838469,"identity":"a682e17f-f006-42b4-ae0f-531f27b592dc","order_by":3,"name":"Seok NamKung","email":"","orcid":"","institution":"Chung-Ang University","correspondingAuthor":false,"prefix":"","firstName":"Seok","middleName":"","lastName":"NamKung","suffix":""},{"id":618838470,"identity":"4876fa96-4750-4388-a0b2-a271878411ba","order_by":4,"name":"So Young Choi","email":"","orcid":"","institution":"Chung-Ang University","correspondingAuthor":false,"prefix":"","firstName":"So","middleName":"Young","lastName":"Choi","suffix":""},{"id":618838471,"identity":"30592726-1ae5-4d20-bb6c-11b73863a856","order_by":5,"name":"Woo Jin Lee","email":"","orcid":"","institution":"Chung-Ang University","correspondingAuthor":false,"prefix":"","firstName":"Woo","middleName":"Jin","lastName":"Lee","suffix":""},{"id":618838472,"identity":"74dc02d4-466d-4e43-9acd-0e93c3b3ac80","order_by":6,"name":"Ye Won Shin","email":"","orcid":"","institution":"Chung-Ang University","correspondingAuthor":false,"prefix":"","firstName":"Ye","middleName":"Won","lastName":"Shin","suffix":""},{"id":618838473,"identity":"def61b80-1bcd-4254-848a-47f9ebf1966e","order_by":7,"name":"Chae Hyeon Bok","email":"","orcid":"","institution":"Chung-Ang University","correspondingAuthor":false,"prefix":"","firstName":"Chae","middleName":"Hyeon","lastName":"Bok","suffix":""},{"id":618838474,"identity":"e68a1e2a-04ab-4d3d-aa66-f80f0e58296c","order_by":8,"name":"Sun Jin Hur","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtUlEQVRIiWNgGAWjYBACAxAhUQHjHiBayxmStTC2kaLFXCL38QfLeXbyBgeYH35gOHOPsBbLGelmEpLbkg03HGAzlmC4UUyEw26ksTFIbjvAuOEAgxkDw4cEorQwf5Ccc8B+wwH2b0RrYZCQbDiQuOEAD9CWG8RoOfOMTULiWHLyzMM8xRIJZ4jRcjyN+bNEjZ1t3/H2jR8+HCNCCwgwS4BJICZSAzAmPxCrchSMglEwCkYmAADMyThvLhuPwAAAAABJRU5ErkJggg==","orcid":"","institution":"Chung-Ang University","correspondingAuthor":true,"prefix":"","firstName":"Sun","middleName":"Jin","lastName":"Hur","suffix":""}],"badges":[],"createdAt":"2026-03-24 12:53:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9212101/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9212101/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106960404,"identity":"0d0c47e6-99b5-457a-8232-8bbb967a9cf8","added_by":"auto","created_at":"2026-04-15 09:20:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":660368,"visible":true,"origin":"","legend":"\u003cp\u003eOverview of livestock serum for cell culture.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-9212101/v1/ce503e9cc9ab58cec9e52e5e.png"},{"id":106960391,"identity":"4aab8b99-d2d4-49a6-ae3f-a34f7f6bf24a","added_by":"auto","created_at":"2026-04-15 09:20:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":521844,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSerum quality analysis.\u003c/strong\u003e (a) Serum appearance (FBS, ABS, and livestock-derived sera). (b) SDS-PAGE protein profiling (7 μg). (c) Protein contents. (d) Heme concentration. (e) Osmolality. (f) pH. (g) Mycoplasma detection. (h) Microbial contamination assay. FBS, fetal bovine serum; ABS, adult bovine serum; BoS, bovine serum; PoS, porcine serum; ChS, chicken serum; AC, Aerobic Count (total aerobic bacteria); YM, Yeast and Mold; STX, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e; EC, \u003cem\u003eEscherichia coli\u003c/em\u003e/coliform Petrifilm. Statistical analysis was conducted using one-way ANOVA with post hoc multiple comparisons against FBS; \u003csup\u003ea–d\u003c/sup\u003e Means with different superscripts within the same row are significantly different (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-9212101/v1/dba84d277b59c63259ed83d7.png"},{"id":106807811,"identity":"a2d6db9c-3e7e-42d5-888c-13b2227fa2cf","added_by":"auto","created_at":"2026-04-13 15:49:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":949084,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScreening of additive effects on cell proliferation by cell counting and MTT assay in iBSC.\u003c/strong\u003e (a) Commercial serum (b) Bovine, (c) Porcine, (d) Chicken serum groups. Left: Cell counting, Right: MTT assay. CTL represents 20% FBS. Raw indicates cultures with 20% livestock-derived serum only, and additive-treated indicates cultures supplemented with the indicated additives FBS, Fetal bovine serum; ABS, Adult bovine serum; Albu, lipid-rich albumin (10 mg/ml); ITS, 1X insulin–transferrin–selenium. All data are expressed as percentage of the control group (CTL, set to 100%). Scale bars indicate 200 µm. Statistical analysis was conducted using one-way ANOVA with post hoc multiple comparisons against CTL (20% FBS); \u003csup\u003ea–c\u003c/sup\u003e Means with different superscripts within the same row are significantly different (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-9212101/v1/f2d600425b09c3379911bbfd.png"},{"id":106960021,"identity":"44f070c1-2791-45c7-b3ed-4512a91d99bd","added_by":"auto","created_at":"2026-04-15 09:17:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":625750,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExperimental workflow and comparative analysis of iBSC proliferation under different livestock serum conditions, including bovine, porcine, and chicken serum groups.\u003c/strong\u003e a) Cell counting, (b) MTT assay (absorbance), (c) Western blot, and (d) Quantification of protein expression determined by Western blot (Pax7). CTL is FBS, and all treatment groups had 20% added serum. FBS, Fetal bovine serum; ABS, Adult bovine serum; B: Bovine, P: Porcine, C: Chicken, R: RAW, A: lipid-rich albumin (B and P: 25 µg/mL, C: 50 µg/mL), I: 1X insulin–transferrin–selenium (B and P: 25 µg/mL, C: 50 µg/mL). Statistical significance conducted using one-way ANOVA with post hoc multiple comparisons ((b) was conducted only on day 3); \u003csup\u003ea–c\u003c/sup\u003e Means with different superscripts within the same row are significantly different (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-9212101/v1/6bfb65fd80dc2c6f4c837bb6.png"},{"id":106960284,"identity":"40530da6-e857-491b-ae7f-d7e8475c4055","added_by":"auto","created_at":"2026-04-15 09:19:45","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":464832,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExperimental workflow and comparative analysis of differentiation of iBSCs grown under various livestock serum conditions, including bovine, porcine, and chicken serum groups.\u003c/strong\u003e (a) Morphology of differentiated cells and immunofluorescence staining of Nuclei (blue), MF20 (red), and Desmin (green), (b) Fusion index (Desmin-based), (c) Quantification of myotubes area stained with immunofluorescence, (d) Western blot, and (e) Quantification of protein expression determined by Western blot (MyoG (Day 5 and 7), MF20 and Desmin (Day 7)). FBS, Fetal bovine serum; ABS, Adult bovine serum; B: Bovine, P: Porcine, C: Chicken, R: RAW, A: lipid-rich albumin (B and P: 25 µg/mL, C: 50 µg/mL), I: 1X insulin–transferrin–selenium (B and P: 25 µg/mL, C: 50 µg/mL). Statistical significance conducted using one-way ANOVA with post hoc multiple comparisons. Western blot data are expressed as percentage of the control group (FBS, set to 100%). Scale bars indicate 200 µm. Statistical analysis was conducted using one-way ANOVA with post hoc multiple comparisons against FBS; \u003csup\u003ea–c\u003c/sup\u003e Means with different superscripts within the same row are significantly different (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-9212101/v1/f272e0267591c8ca72306226.png"},{"id":106960547,"identity":"6d8130f9-095b-462f-8d1c-1ff9481283bd","added_by":"auto","created_at":"2026-04-15 09:21:43","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":645768,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLong-term proliferation of iBSCs during serial passaging (P1–P4) in different serum types.\u003c/strong\u003e (a) Commercial bovine serum group (b) Bovine, (c) Porcine, (d) Chicken adult serum. FBS, Fetal bovine serum; ABS, Adult bovine serum; B: Bovine, P: Porcine, C: Chicken, R: RAW, A: lipid-rich albumin (B and P: 25 µg/mL, C: 50 µg/mL), I: 1X insulin–transferrin–selenium (B and P: 25 µg/mL, C: 50 µg/mL). Raw indicates cells cultured with only livestock-derived serum, whereas additive-treated groups were cultured with 20% livestock-derived serum supplemented with the indicated additives. Statistical analysis was performed between successive subcultures of the same treatment group. Statistical analysis was conducted using one-way ANOVA with post hoc multiple comparisons Day 3; \u003csup\u003ea–c\u003c/sup\u003e Means with different superscripts within the same row are significantly different (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-9212101/v1/d1167ed045b933ff0d72b8d0.png"},{"id":106807814,"identity":"8b6e7d32-436b-4c60-baaf-5b4d5ebed166","added_by":"auto","created_at":"2026-04-13 15:49:21","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":546495,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCost comparison of culture media formulations and schematic workflow for hybrid cultivated-meat production.\u003c/strong\u003e 2025 market price, suppliers listed in the table or Methods (Section 2). Bottom image: Conceptual process for producing hybrid cultured meat products combining cultured meat grown with bovine and porcine serum with plant-based ingredients to achieve final texture and sensory qualities (unpublished data). bFGF: basic fibroblast growth factor; ITS, 1X Insulin–transferrin–selenium.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-9212101/v1/f3ab84f758c83ff657450745.png"},{"id":106962804,"identity":"4b791c4e-59ea-46eb-92af-534b14344bb0","added_by":"auto","created_at":"2026-04-15 09:39:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5809859,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9212101/v1/22ea0801-32a0-42a4-a0ed-a97dc9940613.pdf"},{"id":106807809,"identity":"419a4cbe-4f3d-42d4-ace2-8cdf1a4a853b","added_by":"auto","created_at":"2026-04-13 15:49:21","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1151919,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9212101/v1/17e4b6960e0705202633ba5c.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Optimization of livestock serum as an alternative to fetal bovine serum in cultured meat application","fulltext":[{"header":"Key points","content":"\u003cp\u003e\u0026bull; Bovine and porcine sera fully replaced FBS for bovine satellite cell\u003c/p\u003e\u003cp\u003e\u0026bull; Optimized FBS substitutes supported stable proliferation during serial passaging\u003c/p\u003e\u003cp\u003e\u0026bull; Replacing FBS reduced total culture medium cost by 62.6%\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eIn modern society, interest in animal welfare and environmental sustainability has been steadily increasing, leading many consumers to explore diverse protein options, including alternatives to conventional meat. In this context, cultured meat has gained attention as a promising supplementary protein source alongside traditional livestock products (Lee et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2025b\u003c/span\u003e; Stout et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Currently, fetal bovine serum (FBS) is an essential component of the cell culture process and accounts for the largest portion of production costs while facing supply instability, high prices, reduced experimental reproducibility due to batch-to-batch quality fluctuations, risk of contamination with animal-derived pathogens, and ethical controversies (Chelladurai et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Lee et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; O'Neill et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Skrivergaard et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Stout et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These poses significant obstacles to its commercialization as a food product and its social acceptance.\u003c/p\u003e \u003cp\u003eFBS is a complex protein-rich mixture of metabolites, making it highly effective in maintaining cell growth and metabolism. It also contributes to enhancing the buffering capacity of the medium and reducing physical stress (Chelladurai et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Lee et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2025a\u003c/span\u003e; Weber et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Previous studies indicate that FBS-level cell proliferation in serum-free media can only be achieved through supplementation with FBS or animal-derived proteins such as growth factors or fetuin (Lee et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2025c\u003c/span\u003e; O'Neill et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Skrivergaard et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Stout et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The combination of albumin, insulin, transferrin, and selenium is known to enhance cell culture performance and is widely used in serum-free media formulation (Dan-Jumbo et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Francis \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Lee et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2025c\u003c/span\u003e; Shiozuka and Kimura \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Skrivergaard et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, no study has systematically investigated (1) the livestock sera composition differences, (2) improvement of sera via supplementation, and (3) its effects in long-term application.\u003c/p\u003e \u003cp\u003eOur research team has focused on blood by-products generated during the livestock slaughter process and has developed alternative serum utilizing these products (Lee et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Lee et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Lee et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2025a\u003c/span\u003e). In this study, we developed a livestock serum\u0026ndash;based culture medium supplemented with additives such as lipid-rich albumin (Albu) and insulin\u0026ndash;transferrin\u0026ndash;selenium (ITS) primarily for bovine satellite cell lines. Through this approach, we demonstrated that this formulation can be a practical and economical FBS alternative for cultured meat production.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eManufacturing process of livestock serum for FBS substitutes\u003c/h2\u003e \u003cp\u003eFBS (Corning, 35-015-CV, Corning, NY, USA) and adult bovine serum (ABS, 16170078, Gibco, 15140122, Grand Island, NY, USA) were purchased from commercial sources. Livestock blood was collected from Hoengseong KC Co., Ltd. (Hoengseong, Korea), Woo-gyeung Livestock Co. (Hwaseong, Korea), and the Chung-Ang University Farm (Anseong, Korea). Bovine blood was obtained from seven Hanwoo cattle (females aged 31\u0026ndash;50 months; males aged 30\u0026ndash;33 months). Porcine blood was obtained from six LYD crossbred pigs (Landrace \u0026times; Yorkshire dam \u0026times; Duroc sire; males aged 180\u0026ndash;190 days). Chicken blood was obtained from 300 Ross 308 broilers (males aged 42\u0026ndash;43 days). All animal procedures were conducted in accordance with the guidelines of the Animal Experiment Ethics Committee of Chung-Ang University (approval number: 202401030030).\u003c/p\u003e \u003cp\u003eLivestock sera were manufactured as FBS substitutes using a standardized, multi-step processing workflow developed to ensure reproducibility, biosafety, and suitability for cell culture applications (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The manufacturing procedure was established based on protocols described in our previous studies (Lee et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Lee et al \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), with additional modifications applied to ensure consistency.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e• Serum separation by coagulation and centrifugation\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eBriefly, freshly collected blood was allowed to undergo natural coagulation and subsequently centrifuged at 1,977 \u0026times; \u003cem\u003eg\u003c/em\u003e for 10\u0026ndash;15 min (Combi 514R, Hanil Scientific, Gimpo, Korea), thereby enabling efficient separation of the serum fraction.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003e• Sterile clarification by membrane filtration\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe resulting supernatant was carefully vacuum-filtered through a 0.1 \u0026micro;m Polyethersulfone Membrane Filter (1214756, GVS, Lancaster, UK) to eliminate residual cellular debris and potential microbial contaminants.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003e• Thermal inactivation for biosafety assurance\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTo further enhance biological safety, serum samples were heat-inactivated at 55\u0026deg;C, either immediately post-filtration or immediately before experimental application, in strict parallel with the procedures used for commercial FBS (Lee et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003e• Additional clarification and quality validation\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAdditional centrifugation steps were incorporated to remove minute precipitates that could interfere with downstream cell culture applications. Following these multi-tiered purification and safety procedures, comprehensive quality assessments were conducted to verify suitability for cell culture.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e\u0026bull; Batch selection and storage of validated sera\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eOnly sera that passed all quality-control checkpoints were retained. The validated livestock serum preparations were aliquoted and stored at \u0026minus;\u0026thinsp;20\u0026deg;C under conditions identical to those used for heat-inactivated FBS and ABS until use.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eQuality profiling of commercial serum and FBS substitutes\u003c/h3\u003e\n\u003cp\u003eIn this study, we conducted a comparative quality analysis to evaluate the suitability of FBS substitutes, including bovine serum (BoS), porcine serum (PoS), and chicken serum (ChS) as substitutes for FBS. Commercially available FBS and ABS were included as reference sera. Each production lot of commercially available culture sera is provided with a Certificate of Analysis, which confirms the electrophoretic profile, endotoxin concentration, sterility (e.g., absence of mycoplasma), and results of viral detection tests (Thermo Fisher Scientific New Zealand Ltd 2025). However, to directly compare commercial and livestock sera under the same analytical conditions, additional quality analyses were performed in this study.\u003c/p\u003e \u003cp\u003eThe heme concentration in each serum sample was measured using the Heme Assay Kit (ab272534, Abcam, Cambridge, UK) according to the manufacturer's recommendations, and absorbance was measured at 400 nm using a microplate reader (SpectraMax 190, Molecular Devices, Sunnyvale, CA, USA). The osmolality of the sera was measured using the OsmoTECH\u0026reg; XT. Mycoplasma contamination testing was performed using the Myco-Read\u0026trade; Mycoplasma Detection Kit (SMD0172, Guri, Korea) according to the manufacturer's protocol. The kit includes a positive control, and samples were prepared by collecting culture fluid from bovine cell lines cultured for 72 h with each serum. Amplified products were run on a 2% agarose gel. Microbiological contamination in the sera was assessed before cell culture using four different Petrifilm\u0026trade; plates (3M, Saint Paul, MN, USA): Petrifilm\u0026trade; Aerobic Count (AC) plates, Escherichia coli (EC) plates, Staphylococcus aureus (STX) plates, and Yeast \u0026amp; Mold (YM). Samples were diluted 1:10 in sterile distilled water, applied to the Petrifilm\u0026trade;, and incubated at 35\u0026deg;C for 24\u0026ndash;72 h. The pH of the sera was measured using a pH meter (SevenCompact pH/Ion Meter S220, Mettler Toledo, Columbus, OH, USA). The protein content of the sera was measured using the bicinchoninic acid (BCA) assay (Pierce\u0026trade; BCA Protein Assay Kit, 23227, Thermo Scientific, Waltham, MA, USA). Samples were incubated with the BCA reagent at 37\u0026deg;C for 30 min and measured at 562 nm using a microplate reader (SpectraMax 190, Molecular Devices). Each serum sample was compared by sodium dodecyl sulfate\u0026ndash;polyacrylamide gel electrophoresis (SDS\u0026ndash;PAGE) at equal protein load. A 10% resolving gel and a 5% stacking gel were used, and samples were loaded alongside an ExcelBand\u0026trade; Enhanced 3-color High Range Protein Marker (PM2610, SMOBIO Technology, Inc., Hsinchu City, Taiwan). Electrophoresis was performed at 80 V for 10 min, followed by 100 V for 90 min. The gels were subsequently stained with a Brilliant Blue R solution (B7920, Sigma-Aldrich, St. Louis, MO, USA).\u003c/p\u003e\n\u003ch3\u003eBovine cell culture\u003c/h3\u003e\n\u003cp\u003eThe immortalized bovine satellite cell line (iBSC1, Kerafast, Boston, USA) was used. The cell culture medium was prepared by adding 20% FBS (Corning, 35-015-CV) and 1% penicillin-streptomycin (15140122, Gibco, 15140122) to Dulbecco's modified Eagle's medium (DMEM). The medium was supplemented with basic fibroblast growth factor (bFGF, GR003, Welgene, Gyeongsan, Korea) at final concentrations of 2 ng/mL. Serum was added at 20% to all treatment groups for bovine cell line culture. For other treatment groups, FBS was replaced with an equivalent amount of livestock serum.\u003c/p\u003e \u003cp\u003eAlbuMAX\u0026trade; II Lipid-Rich BSA (11021029, Gibc) was diluted to a concentration of 10 mg/mL in 0.1% BSA solution. The ITS used in this study (100X, ITS-G, 41400045, Gibco) was diluted to 1X with 0.1% BSA solution. Each additive was added to the culture medium at three concentration levels (25, 50, and 75 \u0026micro;g/mL). For the combination groups, both additives were co-supplemented at 25\u0026thinsp;+\u0026thinsp;25 or 50\u0026thinsp;+\u0026thinsp;50 \u0026micro;g/mL. To ensure equal volume adjustment, the same amount of 0.1% BSA solution was added to the non-supplemented control groups.\u003c/p\u003e \u003cp\u003eDifferentiation of iBSC was induced in myogenic (differentiation) medium, which consists of DMEM supplemented with 1% P/S/A and 2% horse serum. All procedures involving animals were approved by the Animal Experiment Ethics Committee of Chung-Ang University (approval number: 202301020084).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCell growth in FBS substitute media\u003c/h2\u003e \u003cp\u003eThe iBSC cultured on plates for 3 days were counted by staining with trypan blue (15250061, Gibco). To measure cell viability, 0.5 mg/mL of thiazolyl blue tetrazolium bromide (MTT, M2128, Sigma-Aldrich) was added to cells cultured for 3 days and reacted for 4 h. The MTT solution and medium were removed from the wells, and DMSO was added to dissolve the purple MTT\u0026minus;formazan crystals. The optical density of each well was measured at 540 nm using a microplate reader (SpectraMax 190, Molecular Devices).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eImmunostaining\u003c/h2\u003e \u003cp\u003eThe differentiated cells were washed with 1X Dulbecco's phosphate-buffered saline (DPBS, 16777-644, Cytiva, Marlborough, MA, USA) and fixed in paraformaldehyde (4%) for 20 min. After washing twice more with 1X DPBS, the samples were treated with 0.2% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) for 20 min and blocked with 2% BSA in 1X DPBS for 2 h at room temperature. Cells were then incubated for 2 h at 4 ℃ with the following primary antibodies (MF20, 1:800, DSHB, Iowa City, IA, USA; desmin [DES], 1:1,500, ab32362, Abcam) dissolved in 1% BSA in 1X DPBS. After incubation, cells were treated with secondary antibodies (A90-116F and A120-101D4, BETHYL, Montgomery, TX, USA) for 1 h at room temperature, then stained with Hoechst 33342 (1:3,000, 561908, BD Biosciences, San Jose, CA, USA). The images were observed with a fluorescence microscope (KI-3000F, Korea Lab Tech, Namyangju, Korea).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eWestern blots were performed on cell lysates prepared in RIPA buffer containing phosphatase inhibitor (78420, Thermo Fisher Scientific) and protease inhibitor (87785, Thermo Fisher Scientific). The BCA Protein Assay Kit (Pierce\u0026trade; BCA Protein Assay Kits, 23227, Thermo Fisher Scientific) was used to quantify the total protein in each sample. SDS-PAGE (8\u0026ndash;10% polyacrylamide gels) was carried out. After transfer of the proteins to polyethersulfone (PES) membranes, the proteins were blocked with 5% BSA in PBS-T for 1 h and probed overnight with primary antibodies Pax7 (1:1,500, DSHB, Iowa City, IA, USA), MyoG (1:1,500, F5D, DSHB), Desmin (1:2,500, ab32362, abcam, Cambridge, UK), MF20 (1:1,500, DSHB), and β-actin (1:5,000, ab8227, Abcam). Then, the proteins were incubated with anti-mouse IgG and anti-rabbit IgG as appropriate. Specific signals were detected with an e-BLOT Touch Imager (e-BLOT Life Science, Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis of the experimental data involved one-way analysis of variance (ANOVA) using SPSS 22.0 (IBM Corp., Armonk, NY, USA). Statistical significance was assessed using Tukey\u0026rsquo;s multi-range test, and the significance level of all data was evaluated based on \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The results are presented as the average of triplicate experiments.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eQuality profiling of commercial and livestock sera\u003c/h2\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eThe major challenge in utilizing livestock slaughter by-products is ensuring they meet the appropriate quality and safety standards for cell culture. Contamination by bacteria or mycoplasma can severely compromise cell growth, and because antibiotics are being phased out from cultivated meat production, ensuring sterility is critical. We therefore evaluated the biochemical quality and contamination status of livestock sera, as well as those of commercial sera, before their application in cell culture (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e).\u003c/span\u003e\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eVisual inspection (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e) revealed clear differences in serum color: BoS and PoS displayed darker reddish tones compared with FBS, ABS, and ChS, suggesting hemolysis during blood collection. The serum protein concentration and SDS-PAGE electrophoresis results were similar to those of our previous studies (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, c\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e) (\u003c/span\u003eLee et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Lee et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2025a\u003c/span\u003e). \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eABS and BoS showed relatively similar protein band patterns compared to FBS, with more bands detected in the region below 60 kDa. Considering that ABS was obtained from sera of cattles aged 12\u0026minus;24 months, there was no significant difference in SDS-PAGE profiling compared to BoS obtained from older individuals (aged 31\u0026minus;59 months). Although SDS-PAGE enabled comparison of overall protein banding patterns, specific growth-promoting proteins (e.g., albumin, transferrin, insulin growth factor [IGF] family members) were not individually quantified. Future proteomic profiling will be necessary to identify the molecular determinants underlying differences in serum performance. Compared with FBS, the protein contents of ABS, BoS, and PoS were significantly higher. In contrast, ChS showed a distinct absence of bands below 45 kDa, and its protein concentration was also significantly lower. Adult blood contains higher protein levels than fetal or neonatal blood, and if blood is not promptly processed after collection, intracellular proteins may be released into the sample through cell lysis, which can further increase the measured protein concentration (\u003c/span\u003eBjelosevic et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Lee et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2025a\u003c/span\u003e; Halvey et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eMeasurements of heme concentration showed that BoS contained the highest concentration (146.8 \u0026micro;M), followed by PoS, and then FBS, ABS, and ChS, which all showed much lower concentrations (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e). The high heme concentration observed in the serum is thought to be due to destruction of red blood cells during blood collection or damage during transport, which is consistent with the dark color of the serum (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e). Notably, the measured values far exceeded the recommended hemoglobin cut-off of \u0026lt;\u0026thinsp;4 mg/mL, indicating a considerable level of erythrocyte rupture in some livestock samples (Corning Inc 2025).\u003c/span\u003e\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eOsmotic analysis (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e) revealed that most sera were within the physiological range (285\u0026ndash;340 mOsm/kg), but BoS (358 mOsm/kg) and PoS (397.3 mOsm/kg) values were higher, presumably due to intracellular solute release from ruptured erythrocytes (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e). When evaluated against a broader quality criterion\u0026mdash;acceptable osmolality of 280\u0026ndash;365 mOsm/kg H₂O\u0026mdash;BoS fell slightly above the upper threshold, while PoS substantially exceeded it (Corning Inc 2025). Previous work has shown that osmotic pressure in coagulated whole blood increases over time, with a faster rise when serum is not promptly separated (\u003c/span\u003eSureda-Vives et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eThe pH of all treatment groups fell within the reference range (7.0\u0026ndash;8.3) suggested by the commercial FBS Quality Assurance Certificate (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e) (Corning Inc 2025). However, the pH values were higher than the value (7.3) suggested by the FBS quality assurance certificate. Other treatment groups were also confirmed to have values higher than FBS (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e). Increased contact with air during blood (serum) collection and preprocessing and increased storage temperature (from frozen/refrigerated to room temperature) can decrease the solubility of carbon dioxide in blood, leading to an increase in pH (\u003c/span\u003eKirschbaum \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2003\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e). These mechanisms could explain the pH results observed in the present study.\u003c/span\u003e\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eMycoplasma testing (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e) yielded negative results for all serum types; an important finding given that mycoplasma is one of the most problematic contaminants in cell culture. Furthermore, microbial safety tests using AC, EC, STX, and YM Petrifilm\u0026trade; plates (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e) showed no microbial growth after filtration, confirming that the applied processing system effectively eliminated viable microorganisms and preserved sterility.\u003c/span\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eMedia development and screening of Albu and ITS for iBSC proliferation\u003c/h2\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eTo evaluate the potential of livestock serum as an alternative to FBS, iBSC were cultured for 3 days in media containing livestock serum obtained from bovine, porcine, or chicken blood by-products, either alone (raw) or supplemented with Albu and/or ITS. Proliferation was assessed by direct nuclear counting and MTT assay, and the results were compared with commercial serum groups (FBS and ABS) (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e). In all analyses, the minimal supplementation principle was applied: when no significant differences were observed among treatment groups, the lowest effective concentration or single additive treatment was selected. Final concentrations (25 or 50 \u0026micro;g/mL) were selected because these levels maintained\u0026thinsp;\u0026ge;\u0026thinsp;70\u0026ndash;80% of the proliferative activity supported by FBS, and higher concentrations provided no additional benefit while occasionally showing early signs of diminishing returns. All treatment groups completely replaced 20% FBS.\u003c/span\u003e\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eIn media containing BoS, even when certain supplementation conditions exceeded the cell proliferation rate of the FBS control medium, the treatment group that achieved\u0026thinsp;\u0026ge;\u0026thinsp;100% proliferation with the smallest additive dose was selected to maximize efficiency and reproducibility. Raw BoS itself supported iBSC proliferation at a level comparable to the CTL (control) condition, and supplementation with Albu or ITS alone produced a similar outcome, as neither additive significantly increased cell counts relative to CTL (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, c, \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eleft). This pattern was similarly observed in MTT absorbance\u0026ndash;based metabolic activity (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, c, \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eright). Although slight increases were visually noticeable, they were not statistically significant. Furthermore, co-addition of Albu and ITS did not produce synergistic effects and, in some cases, even resulted in minor reductions. Overall, BoS demonstrated a sufficiently strong baseline proliferative capacity in its raw form, and because additional supplementation did not produce meaningful improvements, 25 \u0026micro;g/mL was selected as an appropriate supplementation level for subsequent analyses based on the minimal supplementation principle.\u003c/span\u003e\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eRaw PoS promoted iBSC proliferation at a level comparable to that of CTL. However, the proliferation level appeared numerically lower than in BoS-supplemented medium, a trend that did not reach statistical significance. Supplementation with Albu or ITS likewise did not result in meaningful increases in either cell counts or metabolic activity (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, c\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e). Moreover, the combined treatment of Albu and ITS did not produce synergistic effects and, occasionally, even resulted in a modest numerical decrease. Although slight visual increases or decreases were observed, they were not statistically supported. As with BoS, supplementation did not clearly enhance iBSC proliferation, and 25 \u0026micro;g/mL of each additive was considered adequate for downstream analyses according to the minimal supplementation principle.\u003c/span\u003e\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eChS showed a different response compared with BoS and PoS. In medium containing raw ChS, a weaker baseline proliferative capacity was observed, requiring relatively higher additive concentrations. The proliferation increase was dependent on the additive concentration, with a relatively notable effect observed when supplemented with ITS (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e). Nonetheless, the combination of Albu and ITS did not yield synergistic effects beyond single supplementation. In this screening assay, medium containing ChS with additive treatment produced reproducible proliferation levels of approximately 75\u0026ndash;80% relative to the FBS control. A concentration of 50 \u0026micro;g/mL was considered sufficient to maintain cell growth and was selected for subsequent experiments.\u003c/span\u003e\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eThus, subsequent analyses used 25 \u0026micro;g/mL of Albu or ITS for BoS and PoS, and 50 \u0026micro;g/mL of Albu or ITS for ChS. Each Raw serum condition, along with FBS and ABS controls, was included, resulting in a total of 11 treatment groups. This strategy was adopted to minimize cost and formulation complexity while maintaining functional performance.\u003c/span\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eEffects of optimized livestock serum\u0026ndash;based culture medium on iBSC proliferation and differentiation\u003c/h2\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003eEffect of selected additives on iBSC proliferation\u003c/h2\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eiBSCs were cultured for 3 days in media formulated with selected conditions for each livestock serum group. Cell counting and MTT-based metabolic activity analyses revealed no significant difference between the commercial controls, namely FBS and ABS (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e). In Day 3 cultures, the expression of the satellite cell-specific protein Pax7 was significantly higher in the ABS group (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, d\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e).\u003c/span\u003e\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eIn all BoS groups, BoS supported robust cell proliferation, similar to that observed in FBS control medium (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e). All BoS treatment groups exhibited Pax7 protein expression levels approximately 150% of those of FBS, showing consistently elevated and comparable effects across BoS alone (BR), BoS+Albu (BA), and BoS\u0026thinsp;+\u0026thinsp;ITS (BI) (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e). These results suggest that BoS already provides near-optimal culture conditions, and supplementation may have limited visible impact due to a ceiling effect.\u003c/span\u003e\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eAdult PoS supported iBSC proliferation at levels comparable to or greater than those observed with FBS (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e). Notably, on Day 3, the PoS+Albu (PA) and PoS\u0026thinsp;+\u0026thinsp;ITS (PI) treatments enhanced the effect of PoS alone (PR), resulting in more than a 200% increase in Pax7 protein expression compared with that observed in the FBS control group (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e). This demonstrates that PoS not only supports stable proliferation but also retains responsiveness to exogenous Albu and ITS, thereby further enhancing proliferation and satellite cell characteristics.\u003c/span\u003e\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eIn contrast to BoS and PoS, the ChS group exhibited significantly lower proliferation across all treatments despite the use of higher additive concentrations (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e). Although ITS supplementation provided a statistically measurable improvement in proliferation (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e), the overall effect remained limited and did not reach the level of efficacy observed in the FBS. The Pax7 levels in ChS alone (CR) and ChS+Albu (CA) were comparable to those in FBS but markedly lower than in other serum groups. Moreover, unlike its effect on proliferation, ITS supplementation (ChS\u0026thinsp;+\u0026thinsp;ITS, CI) further reduced Pax7 expression (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e).\u003c/span\u003e\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eInduction of myogenic differentiation after the proliferation phase\u003c/h2\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eAfter 3 days of proliferation, the medium was replaced with HS-containing differentiation medium, and cells were cultured for four additional days (total of 7 days). Proliferation was evaluated relative to the FBS, and differentiation was initiated simultaneously across all treatments, even when some groups (e.g., ChS) did not reach full confluence. MyoG was assessed at early differentiation (Day 5 and 7), and MF20 and Desmin at late differentiation (Day 7). The fusion index was calculated as the proportion of Desmin-positive multinucleated myotubes.\u003c/span\u003e \u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eIn the FBS group, nearly all proliferated cells differentiated, resulting in the highest fusion index (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, b\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e). ABS and all livestock serum treatments showed significantly lower fusion indices than FBS, but differentiation occurred in all conditions (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, b\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e). The MF20-stained area was lower than the Desmin-stained area across groups; however, both showed similar trends, with the FBS group exhibiting the highest myotube area (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e). Western blot analysis further revealed that the BoS group exhibited MyoG expression similar to that of the ABS group at Day 5 across all treatments. However, a significant decrease in MyoG was observed in the BoS treatment groups, particularly in BA and BI, at Day 7 (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e). This trend was also observed in the PoS and ChS groups, with some treatments (PA, CA and CI) even exhibiting higher MyoG expression than the FBS group at Day 5. MyoG is known to increase as iBSCs enter differentiation and decrease toward the late stage. Therefore, the consistent decrease in MyoG observed at Day 7 compared with Day 5 suggests that iBSCs successfully transitioned from early to late differentiation in all serum groups (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e).\u003c/span\u003e\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eIn the BoS group, supplement supplementation-dependent differences in myotube area were not evident (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e). Muscle-specific protein expression analysis to assess late myotube formation showed that all BoS groups performed better than the ABS group, albeit BR showed similar effects to BA and BI despite the lack of supplementation (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e). The effects of all BoS groups were, higher than ABS, but only approximately 70% of those of FBS on average (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e).\u003c/span\u003e\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eSupplementation in the PoS groups increased muscle-specific protein formation compared with PR (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e). In the PA and PI groups, the myotube area markedly increased, reaching approximately 100% of the MF20 level and ~\u0026thinsp;80% of the Desmin level, with both proteins showing no significant difference compared with FBS (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e). Muscle-specific protein expression in these groups was also higher than that observed in the ABS group (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e). Notably, MF20 expression in the PA group was equivalent to that of the FBS group, demonstrating its full replacement potential, whereas MF20 expression in the PI group remained at approximately 70% of the FBS-group level (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e).\u003c/span\u003e\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eIn the ChS group, Desmin was restored to levels similar to those in the FBS group through CA and CI supplementation, whereas MF20 expression remained markedly low (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e). Unlike Desmin, MF20 is strongly expressed only in sufficiently matured, thick myotubes (\u003c/span\u003eCiecierska et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Costa et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eThus, although structural integrity (DES) was recovered by supplementation, full maturation (MF20) did not reach FBS-group levels. These results are consistent with the proliferation differences observed (Section 3.3.1), suggesting that cell density before differentiation is a key determinant of myotube maturation efficiency.\u003c/span\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eLong-term maintenance of proliferative potential during serial passaging of iBSCs\u003c/h2\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eWith commercial serum (FBS and ABS), no significant decrease in cell proliferation was observed during four consecutive passages (Day 3\u0026ndash;12). The overall proliferative capacity remained stable throughout the long-term culture period, maintaining levels comparable to the initial passage (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ecommercial serum). These results confirm that commercial serum supports long-term expansion of iBSCs without loss of proliferative potential.\u003c/span\u003e\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eSimilarly, BA and BI maintained a stable proliferative capacity during long-term culture. Although a slight fluctuation occurred after Day 6, this variation did not result in a statistically significant decrease compared with Day 3 (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eABS). Notably, previous research has suggested that albumin or ITS (or insulin) supplementation can enhance long-term proliferative stability in myogenic cells (\u003c/span\u003eLee et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2025c\u003c/span\u003e; Mainzer et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Stout et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eConsistent with this, both BA and BI in our study exhibited more sustained proliferation compared with BR, indicating that these supplements positively contribute to maintaining long-term expansion capacity.\u003c/span\u003e\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eThe PoS group also preserved a stable level of proliferative ability during repeated passaging. Although a slight change in proliferation was observed after Day 6, this variation did not result in a statistically significant decrease compared with Day 3 (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eporcine adult serum). Notably, upon ITS supplementation, cells exhibited consistently stable proliferation throughout the entire culture period without significant variation.\u003c/span\u003e\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eIn contrast to BoS and PoS, ChS supplementation exhibited a marked decline in proliferative capacity over successive passages. After Day 9, the number of proliferated cells sharply decreased to less than half of the initial level (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eadult ChS). ITS supplementation (CI) temporarily slowed this decline but did not prevent the overall reduction in proliferation. Interestingly, although ChS supplementation maintained approximately 70\u0026ndash;80% of the proliferation level observed with the FBS control during short-term screening, this performance did not translate to long-term stability. The sharp decline highlights that short-term proliferation does not predict long-term maintenance, and serum quality cannot be reliably evaluated based on short-term or single-passage data alone. Given the strong species-specific compatibility observed in this study\u0026mdash;ChS supporting avian satellite cells but not bovine satellite cells\u0026mdash;the pronounced decline during long-term culture likely reflects this inherent biological mismatch.\u003c/span\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eCost evaluation of optimized livestock serum\u0026ndash;based culture medium for hybrid cultured meat applications\u003c/h2\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eTo determine the economic feasibility of the experimental sera, a comparative cost analysis was conducted based on the composition of each growth medium. Cost calculations were based on 2025 market prices from major suppliers and expressed as cost per 600 mL of complete medium. Although the preparation of livestock serum may entail specific processing costs, this study was based on the concept of using the large volume of blood typically discarded during slaughter. Filtration and labor costs were not included because they have a minimal impact on serum cost and are the same regardless of serum type. Therefore, the unit cost of livestock serum was set to zero in the cost analysis.\u003c/span\u003e \u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eMedia prepared with FBS had the highest cost at 126.6 USD per 600 mL (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e). Of this, the serum itself (79.3 USD) accounted for 62.7% of the total cost, while bFGF (26.7 USD) accounted for 21.1%. Basal medium and antibiotics contributed 14.5% and 1.5%, respectively (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e). This again confirms that serum and growth factors are the major cost drivers in conventional cell culture media. By contrast, media containing ABS cost 64.2 USD \u0026mdash;a 49.3% reduction compared with FBS-based media. In this case, the serum cost decreased sharply (16.9 USD), and the dominant cost components became bFGF (6.7 USD) and basal medium (18.7 USD) (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e). When serum was replaced with livestock serum (serum cost\u0026thinsp;=\u0026thinsp;0), the total medium cost decreased further to 47.4 USD \u0026mdash;a 62.6% reduction relative to FBS-based media. Under these conditions, the cost structure shifted from serum-dependent to growth factor-dependent, with bFGF accounting for the largest proportion of total cost. Even when livestock serum was supplemented with low-cost additives such as AlbuMAX\u0026trade; (.1 USD) and ITS (0.1 USD), the total cost increased only marginally\u0026mdash; 47.4 USD, representing an increase of merely 0.46% (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e). Despite this minimal cost increase, cell proliferation stability was improved, demonstrating the cost-effectiveness of combining livestock serum with low-cost supplements.\u003c/span\u003e\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eIn this study, we systematically evaluated livestock-derived sera with respect to their biochemical properties, proliferative capacity, differentiation potential, and long-term stability, in comparison with commercial FBS and ABS. Overall, bovine and porcine sera exhibited comparable or superior performance to FBS, whereas chicken serum displayed distinct limitations.\u003c/span\u003e \u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eQuality assessments of livestock serum revealed no microbial contamination, but most livestock serum still failed to meet the quality standards expected for FBS. Heme concentration, osmotic pressure, and pH outside the normal physiological range can both inhibit cell proliferation, metabolic activity, and growth factor secretion and cause damage such as endoplasmic reticulum stress or apoptosis (\u003c/span\u003eAlhuthali et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Ceccarini \u0026amp; Eagle \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1971\u003c/span\u003e; Pethő et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Tatsumi et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2002\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eThe minor differences observed between livestock serum and commercial FBS appear to result largely from practical handling factors rather than intrinsic quality limitations. As evidence, most biochemical parameters in our serum samples were comparable to those of commercial products, and no mycoplasma or bacterial contamination was detected, confirming that livestock serum can already achieve a quality level suitable for cell culture. Variations in heme concentration and osmolality likely reflect routine factors such as transport conditions or processing time, which can be readily stabilized when industrial protocols and cold-chain systems are applied. With these procedures in place, consistent, high-quality livestock serum can be produced, further reinforcing its potential as a robust and reliable alternative to FBS.\u003c/span\u003e \u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eAlbumin and ITS are the most commonly used cell additives in serum-free conditions. Albumin increases cell proliferation and sustains its effects (\u003c/span\u003eKobayashi \u0026amp; Takubo \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Skrivergaard et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Stout et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eAlbumin supports cellular energy metabolism by transporting fatty acids, suppresses oxidative stress by binding metal ions, and stabilizes growth factors as well as the osmotic and protein balance of the medium (\u003c/span\u003eFrancis \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Skrivergaard et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Stout et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eThrough these functions, albumin contributes to overall culture stability and promotes cell proliferation. Insulin stimulates cell proliferation and adhesion, and in combination with ITS, enhances cell culture stability (\u003c/span\u003eLee et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2025a\u003c/span\u003e; Skrivergaard et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eFurthermore, because transferrin in ITS can supply iron to cells during cell proliferation, it may play a role in utilizing the abundant iron in BoS (\u003c/span\u003eLee et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2025a\u003c/span\u003e; Mainzer et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Shiozuka \u0026amp; Kimura \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2000\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e).\u003c/span\u003e\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eDespite these well-established benefits, such effects were not apparent in the ChS group in our study. ChS required supplementation due to its low albumin content; however, the relatively low level of albumin added based on screening data likely did not fully compensate for the absolute albumin deficiency, resulting in a lower expected improvement in iBSC proliferation and Pax7 expression. In addition, ChS is commonly used without FBS in commercial avian satellite cell cultures, and our results suggest that these protocols are based on species-specific effects (\u003c/span\u003eHagiwara \u0026amp; Ozawa \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1982\u003c/span\u003e; Kerafast \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Oh et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eThese results are similar to our previous findings that ChS was the least effective for bovine satellite cells and C2C12 (mouse myoblasts) (\u003c/span\u003eLee et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Lee et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2025a\u003c/span\u003e). \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eEven the addition of ITS, which supports satellite cell proliferation, can delay, but not completely prevent, this trend (\u003c/span\u003eSkrivergaard et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Wu et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eThis indicates that responsiveness to ChS is species-dependent, with limited effects on iBSCs despite its effectiveness for avian satellite cells.\u003c/span\u003e\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eInterestingly, the relationship between serum albumin content and the effects of additive supplementation was not straightforward. In our previous studies, the albumin content of collected BoS and PoS was approximately twice that of FBS, whereas that of ChS was approximately half. Despite this, supplementation with lipid-rich albumin and ITS in the porcine serum group produced a clear synergistic enhancement of satellite cell characteristics (\u003c/span\u003eLee et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Lee et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2025a\u003c/span\u003e). \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eAlbumin and ITS are generally known to promote proliferation in serum-free or low-serum systems, yet the magnitude of their effect differed substantially between serum types.\u003c/span\u003e\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eIn the BoS group, the strong proliferative capacity of BR and BI itself left little room for further enhancement, which explains the minimal additional response to supplementation. In contrast, the pronounced responsiveness observed in the PoS group suggests that, although PR also supported robust growth, it retained a metabolic or signaling margin that allowed synergistic effects upon supplementation. However, the precise biochemical factors, such as differences in the composition of unidentified effective proteins or growth factors in serum, remain unclear, and no existing literature provides a clear explanation for the specific response of PoS to albumin and ITS. These observations highlight an intriguing but still unresolved relationship between serum composition and supplement responsiveness, suggesting that targeted mechanistic studies will be necessary to fully elucidate this synergistic effect.\u003c/span\u003e \u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eOverall, ChS was ineffective in promoting iBSC proliferation, whereas BoS and PoS consistently supported robust cell growth and preserved satellite cell characteristics. In the BoS group, all treatments supported proliferation equivalent to FBS and induced approximately 150% of the Pax7 expression observed in FBS control medium, indicating that BoS provides inherently optimal culture conditions without requiring additional supplements. In the PoS group, responsiveness to supplementation was even more pronounced; the PA and PI treatments produced Pax7 levels exceeding 200% of those in FBS control medium, demonstrating a clear synergistic enhancement of satellite cell characteristics. Collectively, these findings highlight the strong functional potential of both BoS and PoS as practical alternatives to FBS, with PoS showing particular promise when combined with Albu and ITS.\u003c/span\u003e \u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eInsulin, a key component of ITS, is well known as a myogenic enhancer in differentiation media (\u003c/span\u003eDan-Jumbo et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Sian et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ePrevious studies have shown that albumin or insulin supplied only during proliferation can enhance both growth and differentiation potential after switching to differentiation medium (\u003c/span\u003eFrancis \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Lee et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2025c\u003c/span\u003e; Stout et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eThus, serum conditions that supported robust proliferation during the first 3 days likely primed cells to a metabolic state more favorable for differentiation, enabling more efficient myotube formation.\u003c/span\u003e\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eDesmin expression was relatively uniform across groups, consistent with its early structural role before contractile protein accumulation (\u003c/span\u003eCiecierska et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Costa et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ePrevious reports show MHC levels can vary even when Desmin does not, indicating that Desmin alone cannot assess late myotube maturation (\u003c/span\u003eCiecierska et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Costa et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eIn our study, Desmin levels remained consistent across serum groups, whereas MF20 varied widely, suggesting distinct differences in maturation stage among treatments. MF20 expression was particularly low in the ChS group. Although Desmin-positive structures were present, MF20 expression differed markedly from other serum groups despite similar fusion index values (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e). Low cell density delays myotube formation and reduces MHC expression (\u003c/span\u003eMurphy et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Tanaka et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eand thinner myotubes show lower MHC expression (\u003c/span\u003eHsieh et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eBecause the ChS group showed the lowest proliferation before differentiation among the groups, its reduced MF20 signal likely reflects delayed or incomplete maturation rather than failed differentiation, consistent with the discrepancy between Desmin and MF20 expression levels.\u003c/span\u003e\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eOverall, although ChS treatment maintained early differentiation markers, it was less effective in driving full myotube maturation, likely due to insufficient pre-differentiation proliferation, and ITS supplementation was less effective than FBS. In contrast, BoS and PoS treatments supported successful progression from early to late differentiation. All BoS groups and PoS promoted myotube formation and muscle-specific protein expression similarly or better than ABS, and PR achieved effects comparable to FBS. These findings highlight the strong potential of BoS and PoS as functional alternatives to commercial serum and their potential for cost-effective cultured meat production through 100% replacement of FBS.\u003c/span\u003e \u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eThis study has some limitations. A key limitation is the inability to distinguish whether lower MF20 results reflect reduced differentiation capacity or merely slower maturation. Additional time-resolved assays and functional measurements will be needed to determine whether these serum conditions limit or simply delay myotube maturation. Furthermore, supplementation requirements differed clearly by species\u0026mdash;BR itself provided the most effective condition, Albu exerted the strongest enhancement in PoS, and ITS supplementation produced the most pronounced improvement in ChS\u0026mdash;indicating underlying biochemical differences that warrant future investigation.\u003c/span\u003e \u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eAlthough ChS supplementation did not support long-term proliferation of bovine satellite cells, both BoS and PoS maintained stable proliferative capacity over four consecutive passages, comparable to that of commercial serum. Notably, supplementation further enhanced this stability: in the BoS group, BA and BI sustained higher proliferation than BR, and in the PoS group, all serum conditions\u0026mdash;including PR, PA, and PI\u0026mdash;maintained strong proliferative stability throughout subculture. Our observations suggest that species compatibility between cells and sera may contribute to long-term culture stability.\u003c/span\u003e \u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eTaken together, these findings demonstrate that BoS and PoS\u0026mdash;particularly when fortified with Albu or ITS\u0026mdash;provide strong, reliable, and cost-effective long-term support for iBSC expansion, highlighting their practical potential as high-performance alternatives to FBS for large-scale cultured meat production.\u003c/span\u003e \u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eImportantly, these functional outcomes were achieved alongside a substantial reduction in medium cost, indicating that performance improvements were not achieved at the expense of economic feasibility. Livestock blood is produced in large quantities during slaughter and is typically associated with disposal costs. Its use as a serum source, therefore, provides multiple advantages\u003c/span\u003e:\u003c/p\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e\u0026bull; Reduced media costs (=\u0026thinsp;zero purchase cost)\u003c/h2\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003e\u0026bull; Reduced waste disposal cost\u003c/h2\u003e \u003cdiv id=\"Sec26\" class=\"Section4\"\u003e \u003ch2\u003e\u0026bull; Reduced environmental burden and promotion of resource recycling\u003c/h2\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eThus, livestock serum is not merely a low-cost alternative to FBS but a strategic resource capable of enabling a sustainable and circular cultured meat production system. Furthermore, using livestock serum enables a substantial reduction in media cost, and the resulting cultured biomass can be applied to the development of hybrid cultured meat products (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e). As illustrated in the conceptual workflow (\u003c/span\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e), biomass generated using BoS and PoS can be combined with plant-based proteins, colorants, seasonings, and texturizing agents to achieve desirable sensory and structural properties (data not shown). The resulting mixture can then be molded and heat-set to form the final product, demonstrating how livestock serum-based cell cultivation can be integrated into practical downstream manufacturing. Furthermore, although livestock serum\u0026ndash;based FBS substitutes already offer substantial cost advantages, future large-scale commercialization is expected to further reduce production costs and enable the stable supply of more uniform, high-quality products through improved resource utilization and economies of scale. We are currently advancing this process and conducting experiments to further optimize the quality of hybrid cultured meat. Overall, the approach of this study presents a sustainable and economically viable strategy that could support the industrial transition of hybrid cultured meat technology.\u003c/span\u003e\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study provided an integrated evaluation of livestock sera, including biochemical profiling, optimized supplementation strategies, functional assays, long-term passaging, and cost modeling, to assess their suitability as alternatives to FBS. Quality assessment revealed that livestock sera fully satisfied sterility requirements and exhibited biochemical characteristics largely comparable to commercial FBS. The remaining variations\u0026mdash;such as elevated heme concentration, osmolality, or pH\u0026mdash;were minor and primarily attributable to practical handling conditions rather than inherent limitations, and can be readily stabilized through standardized blood collection, processing, and cold-chain control.\u003c/p\u003e \u003cp\u003eAmong the three livestock sera, ChS showed the least applicability for bovine satellite cells, with low proliferative capacity, species-specific incompatibility, and a pronounced decline during long-term passaging. Its reduced ability to sustain growth and myotube maturation indicates that ChS is not a viable option for large-scale bovine cell expansion. In contrast to adult ChS, both BoS and PoS demonstrated strong, consistent, and durable performance across short-term proliferation, myogenic differentiation, and extended serial culture. ABS showed the highest intrinsic proliferative capacity without supplementation, and the BA and BI condition (each supplemented with 25 \u0026micro;g/mL of additives)) maintained the most stable long-term expansion, making it the most reliable BoS candidate for FBS replacement. Adult PoS also sustained robust growth across both short- and long-term culture, with the PR and PI condition (each supplemented with 25 \u0026micro;g/mL of additives) achieving proliferation and differentiation outcomes equivalent to or exceeding those of FBS.\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eBA and BI: proliferation\u0026thinsp;\u0026asymp;\u0026thinsp;150% of FBS; differentiation\u0026thinsp;\u0026asymp;\u0026thinsp;70% of FBS\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003ePA: proliferation\u0026thinsp;\u0026asymp;\u0026thinsp;200% of FBS; differentiation\u0026thinsp;\u0026asymp;\u0026thinsp;100% of FBS\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003ePI: proliferation\u0026thinsp;\u0026asymp;\u0026thinsp;230% of FBS; differentiation\u0026thinsp;\u0026asymp;\u0026thinsp;80\u0026minus;100% of FBS\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eImportantly, replacing FBS with livestock serum reduced total medium cost by 62.7%, substantially lowering production expenses while decreasing slaughterhouse waste-disposal burdens and improving resource circularity. With further optimization of collection and processing pipelines, consistent, high-quality livestock sera can be produced, establishing a scalable foundation for cost-efficient cultured meat bioprocessing. Collectively, these findings position BoS and PoS as scientifically validated, industrially scalable, and economically advantageous alternatives to FBS, offering strong potential to advance sustainable hybrid and fully cell-based meat production systems.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDa\u003c/strong\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003cstrong\u003eYoung Lee:\u003c/strong\u003e Investigation, Validation, Methodology, Writing - original draft, Writing - review \u0026amp; editing. \u003cstrong\u003eErmie Jr. Mariano:\u003c/strong\u003e Investigation, Validation, Writing - review \u0026amp; editing. \u003cstrong\u003eJi Won Park:\u003c/strong\u003e Investigation, Validation, Writing - review \u0026amp; editing. \u003cstrong\u003eSeok Namkung:\u003c/strong\u003e Investigation, Validation, Writing - review \u0026amp; editing. \u003cstrong\u003eSo Young Choi:\u003c/strong\u003e Investigation, Validation, Writing - review \u0026amp; editing. \u003cstrong\u003eWoo Jin Lee:\u003c/strong\u003e Investigation, Validation, Writing - review \u0026amp; editing. \u003cstrong\u003eYe Won Shin:\u003c/strong\u003e Investigation, Validation, Writing - review \u0026amp; editing. \u003cstrong\u003eChae Hyeon Bok:\u003c/strong\u003e Investigation, Validation, Writing - review \u0026amp; editing. \u003cstrong\u003eSun Jin Hur:\u003c/strong\u003e Supervision, Writing - review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was conducted with the support of Chung-Ang University. This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry(IPET) through\u0026nbsp;High Value-added Food Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs(MAFRA)(RS-2025-02215727).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article. Additional raw datasets are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlhuthali S, Kotidis P, Kontoravdi C (2021) Osmolality effects on CHO cell growth, cell volume, antibody productivity and glycosylation. 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Cell Rep 25(7):1966\u0026ndash;1981. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.celrep.2018.10.067\u003c/span\u003e\u003cspan address=\"10.1016/j.celrep.2018.10.067\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Fetal bovine serum substitutes, Cultured meat, Livestock serum, Cell culture","lastPublishedDoi":"10.21203/rs.3.rs-9212101/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9212101/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFetal bovine serum (FBS) is widely used for cultured meat production, but its fetal origin raises ethical concerns and contributes to high cost and batch variability, driving the demand for practical alternatives. This study evaluated livestock sera from bovine, porcine, and chicken sources through biochemical profiling, proliferation and differentiation assays, and long-term serial passaging of bovine satellite cells. All sera met essential biochemical and sterility requirements when properly processed. Bovine and porcine sera, unlike chicken serum, consistently supported robust proliferation, myogenic differentiation, and long-term expansion. Importantly, the final FBS substitutes were optimized through supplementation with lipid-enriched albumin or insulin\u0026ndash;transferrin\u0026ndash;selenium (25 \u0026micro;g/mL), which significantly enhanced cellular performance. Cost analysis revealed that replacing FBS with the optimized livestock-derived serum formulations reduced total medium cost by 62.6%. Collectively, optimized bovine- and porcine-based FBS substitutes represent effective, scalable, and ethically aligned alternatives for sustainable cultured meat bioprocessing.\u003c/p\u003e","manuscriptTitle":"Optimization of livestock serum as an alternative to fetal bovine serum in cultured meat application","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-13 15:49:16","doi":"10.21203/rs.3.rs-9212101/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b28f50fb-7ffd-46e7-9013-1173bcf9f51b","owner":[],"postedDate":"April 13th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-12T11:30:17+00:00","index":38,"fulltext":""},{"type":"reviewerAgreed","content":"224367368279826072215906213654585739727","date":"2026-05-04T19:26:39+00:00","index":37,"fulltext":""},{"type":"reviewerAgreed","content":"248582449260690432914445761489705634042","date":"2026-05-04T04:54:15+00:00","index":36,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-13T15:49:16+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-13 15:49:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9212101","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9212101","identity":"rs-9212101","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}