Development of a sustainable strategy for cultured fat production based on serum-free 3D culture of bovine adipose stem cells

Development of a sustainable strategy for cultured fat production based on serum-free 3D culture of bovine adipose stem cells | 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 Article Development of a sustainable strategy for cultured fat production based on serum-free 3D culture of bovine adipose stem cells Zongzhe Xuan, Qiuyue Peng, Mariia Borsuk, Rupali Prasad, Vladimir Zachar, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5611796/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Dec, 2025 Read the published version in Scientific Reports → Version 1 posted 6 You are reading this latest preprint version Abstract Cultivated meat provides a sustainable and ethical alternative to traditional livestock production, addressing global challenges in meat demand and environmental impact. Fat is recognized as an essential component of meat products, but strategies for culturing animal fat precursors still have major limitations, such as the need for a serum-containing medium and inefficient adipogenic cell maturation. In this study, a serum-free medium (SFM) formulation for the cultivation of bovine adipose-derived stem cells (bASC) was first validated by comparison with serum-containing media (FBS). After a 14-day induction period, the SFM increased bASC proliferation by 20% and triglyceride accumulation by 66%. When the SFM was applied to a three-dimensional (3D) spheroid culture system, it resulted in up to 34% higher triglyceride accumulation compared to 2D cultures. These results were accompanied by a significant increase in the expression of key adipogenic markers and lipid droplet formation, suggesting a synergistic potential of SFM and 3D spheroid strategies for bASC culture. Overall, the results of this study provide a scalable and sustainable platform for cultured fat production and are an important step towards overcoming some of the challenges in cultured meat production. Biological sciences/Biotechnology/Stem cell biotechnology Biological sciences/Biotechnology/Tissue engineering Biological sciences/Biochemistry/Lipids cultured fat serum-free medium 3D spheroid culture adipogenic differentiation bovine adipose stem cells sustainable meat production Figures Figure 1 Figure 2 Figure 3 Introduction Cultured or cultivated meat, produced from animal cells using tissue engineering methods, has gained significant attention in recent years due to its potential to offer a more ethical and sustainable alternative to conventional meat production 1 , 2 . Unlike plant-based meat substitutes, cultivated meat aims to replicate the sensory experience of traditional meat, including its texture, taste, and appearance 3 . While much of the research in cultivated meat focuses on muscle tissue as the primary protein source, the role of fat in meat cannot be overlooked. Fat contributes to the juiciness, flavor, and mouthfeel of meat, which are critical for consumer acceptance 4 . During cooking, for example, meat releases hundreds of volatile compounds, most of which originate from fats 5 . Additionally, fat is essential for creating the species-specific flavor that distinguishes the taste of different meats 6 . Therefore, producing and integrating tissue engineered fats into cultivated meat products is vital for achieving a product that meets consumer expectations 3 . In vivo, the lipids that contribute to the sensory properties of meat are primarily synthesized and stored within adipocytes, specialized cells that form adipose tissue. Adipocytes play a crucial role in lipid metabolism, through synthesis, accumulation, and release of fatty acids that are essential for the development and function of adipose tissue 7 . Adipose stromal/stem cells (ASCs) have been identified as a valuable source for generating adipocytes in vitro due to their inherent ability to differentiate into lipid-producing cells 8 , 9 . To support the growth and differentiation of ASCs in culture media are supplemented with fetal bovine serum (FBS), which provides the necessary growth factors and nutrients to promote efficient proliferation and adipogenic differentiation of the cells 10 , 11 . However, the cost of using culture and differentiation media containing FBS is a major limiting factor in scaling up production of cultivated meat 12 . Additionally, the reliance on animal-derived FBS raises ethical concerns and limits the sustainability of the process, conflicting with the overarching goals of cultivated meat production. With the aim of eliminating dependence on products of animal origin, while ensuring efficient cell growth and differentiation, the development of serum-free alternatives has been extensively studied in the context of expanding ASCs for clinical applications 13 – 15 . In the field of culture meat, serum-free media formulations are crucial for the development of a sustainable alternative to conventional meat. The majority of efforts have so far focused on optimizing media for muscle precursor cells, likely due to their central role in defining the texture and protein content of cultured meat products 16 – 18 . In contrast, serum-free strategies tailored specifically for bovine fat cells remain relatively underexplored, but recent studies have begun to address this gap. Mitić et al. introduced a simplified, cross-species serum-free formulation capable of supporting adipogenic differentiation 19 . Similarly, Messmer et al. applied single-cell transcriptomic analysis to identify bovine muscle-derived cell subtypes with adipogenic potential, providing insights for more targeted media development 20 . More recently, refinement of media composition has focused on replacing traditional components like glutamine and glucose with alternatives that minimize toxic byproducts such as ammonia, thereby enhancing both cell proliferation and adipogenic differentiation 21 . Traditionally, ASCs are cultured in two-dimensional (2D) monolayer systems, which do not accurately mimic the complex microenvironment of tissues 22 . Additionally, prolonged culture of ASCs in 2D conditions often results in a loss of replicative ability, colony-forming efficiency, and differentiation capacity 23 . A promising alternative is the cultivation of ASCs as three-dimensional (3D) tissue structures that better mimic the natural stem cell microenvironment 24 – 26 . Research with human ASCs has shown that 3D spheroids represent a more relevant system for the differentiation and maturation of the cells into the adipogenic lineage, producing cells that are more similar to mature adipocytes 27 – 29 . However, protocols developed for adipogenic differentiation of human and mouse cells showed low efficiency in inducing adipogenesis of bovine progenitor cells, emphasizing that there are significant differences between human and ruminant adipogenic processes 30 . For instance, human cells exhibit higher enzymatic activity for converting glucose to fatty acids, while bovine cells rely more on acetate due to their unique metabolic adaptations 31 . Furthermore, while more recent studies have begun to understand the effect of 3D culture in bovine adipogenic precursors 30 , 32 , these studies were conducted in serum-containing media. Studies have shown that human mesenchymal stromal cells form loose 3D aggregates but not solid spheroids after serum withdrawal 33 , which could be related to removal of the adhesion proteins contained in the medium, including fibronectin and vitronectin 34 . Therefore, it is not yet known whether the removal of serum influences the 3D cultivation of bovine ASCs. In this study, we address these challenges by comparing 2D and 3D culture methods for bovine ASCs, focusing on adipogenic differentiation in serum-free media. By characterizing these properties, we aim to provide a foundation for the efficient and sustainable production of cultivated fat, potentially paving the way for large-scale adoption of these methods in cultivated meat production. Results The growth and differentiation potential of bovine adipose-derived stem cells (bASCs) was investigated under serum-free conditions. Over passages 3 to 5, bASCs cultured in serum-free medium (SFM) showed a significantly higher proliferation capacity than those cultured in serum-containing medium (FBS). As shown in Fig. 1 , the doubling time of cells in SFM was significantly shorter, indicating a faster proliferation rate in the absence of serum. The percentage increase was approximately 20%. In addition, Ki-67 analysis revealed significantly higher expression of this proliferation marker in cells cultured in SFM after prolonged passage, further supporting the sustained proliferation potential of bASCs under these conditions (Figure S1 , Supplementary information file). Non-induced controls were maintained in growth medium (GM) for the duration of the experiment, while differentiation was induced using a two-step protocol consisting of induction medium (DM1) followed by maintenance medium (DM2). After initiation of adipogenic differentiation, bASCs in SFM exhibited a substantial increase in intracellular triglyceride accumulation, as shown in Fig. 1 . Compared to bASCs cultured in FBS, cells in SFM displayed significantly higher triglyceride content on days 8 and 14, indicating a more robust differentiation response. On day 14, when comparing the mean values of normalized triglyceride content (16.62 in SFM vs. 9.96 in FBS), it can be inferred that the percentage increase in lipid accumulation due to SFM was over 66%. This enhanced lipid accumulation was also confirmed by Oil Red O staining on day 14 (Fig. 1 ), in which cells cultured in SFM showed more intense staining of lipid droplets compared to their FBS counterparts. At the transcriptional level, adipogenic differentiation in SFM was further supported by the expression profiles of key adipogenic markers, PPAR-γ and FABP4 . As shown in Fig. 1 , while only minimal changes in gene expression were observed after 2 days of induction (DM1), cells cultured in SFM exhibited significant upregulation of both markers at later stages of differentiation in DM2 (day 8 and 14). On day 14, the expression of PPAR-γ and FABP4 was significantly higher in bASCs cultured in SFM than in FBS. The morphology and viability of spheroids formed from bASCs seeded at different initial densities were analyzed over a three-day culture period using SFM. Figure 2 illustrates the self-organization of cells into spheroids at different densities (5000, 10000, and 20000 cells per well) from day 1 to day 3. On day 1 after seeding, the spheroids exhibited a quasi-circular morphology characterized by well-defined edges, with almost all seeded cells seamlessly integrated into the spheroid structure. A clear seeding density-dependent morphological difference was observed, with higher initial cell densities leading to larger spheroids. Quantitative analysis of spheroid size, represented as Feret diameter, confirmed that spheroid size was directly correlated with initial cell density (Fig. 2 ). The doubling time of bASCs cultured in 3D spheroids was compared with different cell densities and with 2D monolayer cultures (Fig. 2 ). Cells cultured under 2D conditions showed a significantly shorter doubling time than cells in 3D spheroids, with the doubling time increasing significantly with increasing initial cell density in the spheroids. Cell viability was assessed using live/dead staining. Figure 2 shows representative images of both 3D spheroids and 2D monolayer cultures on days 1 and 3, with live cells shown in green and dead cells in red. In 3D cultures, the spheroids formed at higher densities (10k and 20k) exhibited more pronounced red staining, indicating a higher degree of cell death over time. In contrast, the 2D cultures maintained a higher overall viability over the same period. Quantitative analysis of cell viability confirmed the microscopy results, with cells in all 3D conditions consistently exhibiting lower viability compared to 2D cultures (Fig. 2 ). All three spheroid groups with different seeding densities appeared to have relatively high cell viability on day 1 (> 80%). Cell viability in 3D spheroids decreased significantly from day 1 to day 3, but there was no statistical difference in cell viability between the different seeding densities. Statistical analysis confirmed the significant differences in viability between 2D and 3D cultures and between day 1 and day 3 for the 3D conditions (### p < 0.001). Representative flow cytometry plots used for this quantification are provided in the supplementary information file (Figure S2). The adipogenic differentiation potential of bASCs was investigated under 3D spheroid culture conditions using SFM, with 2D monolayers as reference. As shown in Fig. 3 , adipogenic induction in 3D spheroids led to a significant increase in intracellular triglyceride content. After 8 days in DM2, the spheroids showed significantly higher triglyceride levels compared to the early induction phase (DM1) and the non-induced controls (GM). On day 14, the spheroids showed the highest triglyceride accumulation compared to the conventional 2D cultures. The percentage increase in triglyceride accumulation in 3D was over 34%. Further confirmation of adipogenic differentiation was provided by BODIPY 493/503 staining, which demonstrated the formation of lipid droplets. At 14 days post-induction, bASCs in 3D spheroids exhibited extensive lipid accumulation, with significantly greater lipid droplet coverage compared to 2D monolayers (Fig. 3 ). The 3D spheroid cultures exhibited a dense and organized distribution of lipid droplets, indicating a more mature adipogenic state. At the transcriptional level, after 8 days in DM2, the expression of PPAR-γ and FABP4 was significantly increased in the 3D culture compared to early differentiation stages and 2D monolayer culture (Fig. 3 ). At day 14, bASCs in 3D spheroids continued to exhibit much higher expression of PPAR-γ and FABP4 than their 2D counterparts, consistent with the increased lipid accumulation observed under 3D conditions. Discussion While most in vitro adipogenesis studies in the past have been based on serum-containing media, the focus in the cultured meat field has been on the development of serum-free media suitable for the expansion and differentiation of progenitor cells 2 . The use of FBS is associated with several challenges, including ethical concerns regarding animal welfare, risk of pathogen contamination, unpredictable supply and high costs 35 . Therefore, development and optimization of serum-free media is critical to achieve the scalability and sustainability required for large-scale cultured meat production 36 , 37 . The challenges associated with formulating serum-free media have been extensively addressed in previous research, with significant progress made in creating chemically defined media to support the differentiation of various progenitor cells, including bovine satellite cells 16 , 38 and adipogenic progenitor cells from several species 19 . In this study, the initial focus is on the validation of existing serum-free culture media for the expansion and differentiation of bASCs, with the main objective being to ensure that the media can support the culture of the cells under well-defined conditions. This validation step is critical to assess the viability of 3D culture conditions and provide a basis for future scalability and sustainability in the context of cultured meat production. In terms of cell proliferation, the SFM was more efficient than serum-containing media, as evidenced by a reduced doubling time and relatively higher expression of the proliferation marker Ki-67. This cell cycle-associated protein is expressed during the active phases of the cell cycle (G1-M), so an increased expression is an indication of active cell proliferation 39 . These findings highlight the validity of the SFM not only to replace FBS but also to enhance cell growth performance. Previous studies have investigated the role of serum in the proliferation and differentiation of bovine adipogenic progenitor cells, particularly muscle-derived fibroadipogenic progenitors (FAPs). While these cells differentiate efficiently under serum-free conditions, their expansion phase is normally carried out in serum-containing medium 40 . Notably, FAPs cultured without serum show comparable gene expression profiles to cells grown under serum-containing conditions 20 . Recent efforts have aimed to simplify adipogenesis protocols by reducing the number of inducers and differentiation phases to facilitate upscaling. 19 . For the differentiation phase, the approach in this study incorporated two distinct serum-free media: one specifically formulated for adipogenic induction and the other for maintenance. This dual-media system effectively supported both the early stages of adipocyte commitment and the later stages of lipid accumulation, as evidenced by higher expression levels of key adipogenic transcription factors, such as PPARγ and FABP4 . These transcription factors play a critical role in preadipocyte determination 41 , 42 . The separation of induction and maintenance phases allows for optimal regulation of the differentiation process, maximizing the efficiency of lipid accumulation in the absence of serum-derived growth factors, which have been shown to influence adipogenic outcomes unpredictably 19 . Studies on human ASCs demonstrated that using defined media in each phase (differentiation and long-term maintenance) could enhance cell adherence and lipid accumulation while maintaining cell functionality over extended culture periods 43 . Studies have shown that 3D culture conditions enhance the production of mature adipocytes from human and murine precursors 27 – 29 . However, bovine adipogenic models face unique challenges, including an often inconsistent adipogenic differentiation compared to other species, which makes the validation of 3D culture conditions particularly critical in the bovine context 30 . The results of this study show that bASCs rapidly formed stable spheroids within three days at all cell densities tested. The diameter of the spheroids decreased after their initial formation, which correlated with the density of cells seeded in each well and the incubation time. These observations are consistent with previous studies that have shown that the size of cell spheroids can be adjusted by controlling cell seeding density 44 . As in previous studies, the viability of bASCs decreased significantly under 3D spheroid culture conditions compared to 2D culture throughout the process of spheroid formation 45 . For spheroids in the diameter range between 200 and 500 µm, studies have shown that there is a gradual accumulation of metabolic waste and insufficient diffusion of oxygen/nutrients, which could be the main reason for the decrease in cellular viability 46 . Following the assessment of viability and growth, the results show that the 3D spheroid system provided an enhanced microenvironment for adipogenesis of bASCs, as evidenced by the increase in triglyceride content and larger lipid droplets within the spheroids after adipogenic induction. The expression of transcription factors PPARγ and FABP4 was consistently higher in spheroid cultures than in 2D monolayers, leading to more mature adipocytes with larger lipid droplets. The 3D environment may mimic aspects of the native extracellular matrix, offering a more physiologically relevant context that encourages adipogenic commitment and maturation 47 . Furthermore, low oxygen (hypoxic) conditions are known to modulate ASC behavior, particularly through enhanced proliferation and increased secretion of growth factors that can further promote differentiation 15 , 45 , 48 . Hypoxia and cell morphology can influence adipogenic differentiation, as shown by the improved adipogenic outcomes in ASCs when cytoskeletal tension and oxygen levels are reduced 49 . Thus, a combination of reduced cytoskeletal stress and limited oxygen diffusion within the dense spheroids may also have contributed to the increased adipogenic efficiency observed in 3D. The efficiency of adipogenic differentiation of bASCs is particularly important for cultured fat production 50 . Notably, the use of serum-free media in the 3D system proved critical for achieving this level of adipogenic maturity, further underscoring the advantages of 3D cultures over conventional 2D systems. While the presented 3D approach is effective for cell differentiation, large-scale production of cultured fat requires extensive cell expansion prior to the differentiation phase to generate substantial amounts of cellular material from limited donor tissue 51 . Although the 3D spheroid format supports adipogenic maturation, our data indicated that it is not optimal for cell expansion. This limitation suggests that a two-step strategy may be required for large-scale applications, e.g. initial expansion in bioreactors (such as suspension or hollow fiber bioreactors) followed by differentiation in 3D 52 . In addition, the reliability and reproducibility of stem cell sources are crucial 53 . In this study, the cells were isolated according to a standard protocol, which generally provides very consistent results in terms of yield of ASCs. The isolated bASCs were characterized using colony-forming unit–fibroblastic (CFU-F) and osteogenic differentiation assays to assess self-renewal and clonogenic capacity as well as multipotency (Supplementary information file). Clones were consistently formed at all seeding densities tested, demonstrating the self-renewal and proliferation capacity of the bASCs (Figure S3). Interestingly, even when the cells were seeded sparsely, the clonogenic efficiency was higher than previously reported 54 . In addition, a positive osteogenic differentiation confirmed the multipotency of the cells (Figure S4). One possible limitation is the fact that only cells from one donor were used. To ensure the robustness and reproducibility of the findings, future research should validate these results with cells from multiple animal donors and understand the behavior of the cells in long-term cultures, which may impact the efficiency of adipogenic differentiation and scalability for cultured fat production. In human ASCs, significant immunophenotypical and biological changes occur during in vitro expansion, underscoring the importance of monitoring cell behavior over extended culture periods 55 . In summary, this study presents a novel method for culturing bASCs into mature adipocytes in a 3D spheroid culture system under serum-free conditions. Compared to conventional 2D cultures in serum-containing media, the presented method enables efficient lipid accumulation. These results indicate the potential of this approach for scalable and sustainable cultured fat production. However, further validation with different cell donors is required to ensure the robustness and reproducibility of the protocol. Overall, this research contributes to the advancement of cultured meat technologies by addressing critical challenges in the cell maturation phase, potentially paving the way for large-scale adoption of these methods in cultured meat production. Materials and methods Isolation and culture of cells Primary bovine ASCs (bASCs) were isolated from the subcutaneous adipose tissue of 6-month-old Black Angus calf (collected at the abattoir) following previously described protocols 56,57 , which were adapted for processing excised tissues. As the material was obtained from a deceased animal as a by-product of a routine commercial practice, no ethical approval was required. This is in accordance with Directive 2010/63/EU on the protection of animals used for scientific purposes. In brief, tissue samples were transferred to a sterile hood and disinfected in 70% ethanol for 2 minutes. The samples were washed with PBS and subsequently excised from visible connective, muscle tissue and blood vessels in isolation medium comprising Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FBS), 12.5 µg/ml amphotericin B, and 500 U/ml penicillin/streptomycin (all from ThermoFisher Scientific). The part of the tissue containing only white fat cells was minced into small pieces (~2 mm 3 ) in isolation medium using sterile forceps and scissors. The tissue pieces were then digested with STEMxyme 1 solution (BioConcept Ltd, Switzerland) in a shaking incubator/water bath at 37°C for 2 hours. The resulting mixture was centrifuged at 80 g for 5 minutes at 4°C and the supernatant was filtered through cell strainers. The cell suspension was then incubated on ice in erythrocyte lysis buffer for 5 minutes. After careful washing and resuspension, the cells were plated and expanded in growth medium (GM) consisting of DMEM/F-12 nutrient mixture (DMEM/F-12, Gibco), supplemented with 10% FBS, and 100 U/ml penicillin/streptomycin. The cells were maintained in tissue culture flasks (Greiner Bio-one) in a standard humidified incubator at 37°C and 5% CO 2 . The culture medium was changed every second day. Cells were passaged using TrypLE select (Gibco, ThermoFisher Scientific) when they reached 90% confluence. Preparation of cultures for assessment of cell growth and differentiation To evaluate the performance of the serum-free media in terms of proliferation and differentiation, bASCs were seeded at a density of 5,000 cells/cm 2 on standard 6-well plates (Greiner, Bio-One) previously coated with a 0.2% gelatin solution. Once the bASCs were confluent, the cells were differentiated into mature adipocytes in two phases. In the first phase, bASCs were induced into preadipocytes by incubation with the first differentiation medium (DM1) for 48 hours. Subsequently, the preadipocytes were maintained in the second differentiation medium (DM2) for 12 days. The non-induced control groups were cultured in GM for the entire assessment period. Serum-containing DM1 was prepared according to the protocol of Kilroy et al 58 with minor modifications. The DMEM/F-12 nutrient mixture was supplemented with 15% FBS, 33 µM biotin, 17 µM pantothenate, 0.2 µM insulin, 1 µM dexamethasone, 0.5 mM isobutylmethylxanthine (IBMX), 5 µM rosiglitazone, and 100 U/ml penicillin/streptomycin. Serum-containing DM2 was prepared according to Mehta et al 11 using DMEM/F-12 supplemented with 10% FBS, a mixture of free fatty acids (5 μM pristanic acid, 1 μM phytanic acid, 50 μM myristoleic acid, 5 μM elaidic acid, 5 μM erucic acid, 5 μM oleic acid, and 150 μM palmitoleic acid) and 100 U/ml penicillin/streptomycin. The serum-free GM (FatSFM, Mirai International) consists of DMEM/F12 and supplements. Serum-free DM1 (FatDM1, Mirai International) consists of Glasgow’s modified Eagle medium (GMEM) and supplements. The serum-free DM2 (FatDM1, Mirai International) is based on GMEM, insulin and free fatty acids. The exact composition of the serum-free media is proprietary to Mirai International. To evaluate the influence of three-dimensional (3D) culture conditions on bASC behavior, experiments were performed exclusively in serum-free media formulations. After culturing the bASCs in serum-free GM in tissue culture flasks for at least 7 days, the cells were trypsinized and seeded on 96-well spheroid plates (BIOFLOAT, faCellitate) at three different densities (5000, 10000 and 20000 cells/well). These plates provide a highly defined cell-repellent surface on which spheroids can be formed without the need for a scaffold. To prevent spheroids from being lost during media changes, only half of the media volume was replaced with each media change. The control group comprised cells cultured in 6-well plates treated in the same way for consistency. The process of spheroid formation was monitored at different time points using a plate scanner with digital phase contrast (CytoSMART Omni, Axion). The device was set to scan the spheroids every six hours for 72 hours. The images were collected and then analyzed using Image J to quantify the process of spheroid change over time and compare the size of the different seeding densities. Proliferation assay To evaluate cell proliferation under different culture conditions, a Presto Blue assay (Invitrogen, Thermo Fisher Scientific) was performed with cells between the third and fifth passage. After 24, 48, 72 and 96 hours, Presto Blue reagent was added to the cell-containing wells at 10% of the total volume, followed by incubation at 37 °C for 3 hours. The negative controls consisted of cell-free wells. Subsequently, 100 µl of the solution was loaded (in duplicates) in a 96-well microplate. Fluorescence was measured using a multimode plate reader (EnSpire, PerkinElmer) at excitation and emission wavelengths of 560 nm and 610 nm, respectively. After fluorescence measurement, the cells were washed with PBS and fresh growth medium was added. Cell proliferation was expressed as doubling time based on the actual measurement of fluorescence intensity (n = 6), as previously reported 59 . Cell viability assessment To assess cell viability, live and dead cells were stained 1 and 3 days after seeding. Cells were first washed with PBS and incubated for 1 hour in a serum-free DMEM/F-12 nutrient mixture containing calcein-AM (1 μM) and propidium iodide (5 µM). The cells were then washed twice with serum-free DMEM/F-12. Samples were observed using a fluorescence microscope (Axio Observer Z1, Carl Zeiss) and images were captured using a digital camera (C11440 ORCA Hamamatsu) and ZEN 2012 software. Quantitative analysis of cell viability was performed using a flow cytometer (CytoFLEX, Beckman Coulter, Copenhagen, Denmark). The spheroids were harvested through a 40 µm BD Falcon cell strainer and subsequently dissociated with a cell dissociation reagent (StemPro Accutase, Gibco), followed by two washes with PBS. The dissociated cells were resuspended in PBS at a concentration of 1 × 10 6 cells/100 μl. To quantify the number of live and dead cells, the fixable viability dye 570 (FVS 570, BD Biosciences, Lyngby, Denmark) was added to the cell suspensions and incubated at room temperature in the dark for 15 minutes. Prior to analysis, the cells were washed and resuspended in flow staining buffer consisting of 0.1% bovine serum albumin (BSA) in PBS. The experiments were repeated three times, each time recording at least 2.5 × 10 4 individual cells. The data obtained were visualized and analyzed using the Kaluza 2.1 software package (Beckman Coulter, Indianapolis, IN, USA). Quantification of intracellular triglyceride and DNA content An adipogenesis test kit (MAK040-1KT, Sigma Aldrich) was used to determine the total cellular concentration of triglycerides. This assay is based on an enzymatic reaction that generates a product that is measured by spectrofluorometry. The assay was performed according to the manufacturer’s instructions. In brief, the lipid extraction reagent was added to the cells in 2D monolayers and 3D spheroids. The spheroids were disrupted by vigorous pipetting. The measurement was performed with a multimode plate reader (EnSpire, PerkinElmer) at excitation and emission wavelengths of 535 nm and 587 nm, respectively. The data were normalized to the DNA content of each sample. The DNA content of each well was measured using the AccuClear dsDNA quantification kit (Biotum) according to the manufacturer's instructions. In brief, 10 μL of each sample was added to 200 μL of working solution prepared by diluting the dye 100-fold with the DNA quantification buffer and mixed by pipetting. After a 5-minute incubation at room temperature in the dark, the plate was read on a multimode plate reader with excitation and emission settings of 468 and 507 nm, respectively. The results obtained were fitted to a DNA standard curve to determine the mass of DNA per sample (n = 3). RNA extraction and quantitative real-time RT-PCR analysis A quantitative real-time PCR was performed to investigate the transcriptional activity of genes involved in the differentiation of bASCs. The Aurum total RNA mini kit (Bio-Rad, Copenhagen, Denmark) was used to harvest total RNA from cultured cells. A nanoliter spectrophotometer (NanoDrop; Thermo Fisher Scientific, Wilmington, DE, USA) was used to determine RNA purity and concentration. The iScript cDNA synthesis kit (Bio-Rad, Copenhagen, Denmark) was used for the synthesis of complementary DNA (cDNA). Amplification reactions for each cDNA sample were performed in duplicate, in a final volume of 20 µl. Each reaction consisted of cDNA, IQ SYBR Green Supermix (Bio-Rad), and the target-specific primers (Table 1). The reaction was carried out on a QuantStudio TM 5 Real-Time PCR System (Thermo Fisher Scientific, Wilmington, DE, USA). The reaction was carried out for 40 amplification cycles: DNA denaturation was performed at 95 °C for 3 min, 95 °C for 10 s, and annealing and extension were performed at the corresponding annealing temperature for 30 s. One housekeeping gene (Ubiquitously Expressed Prefoldin Like Chaperone, UXT) was used to obtain the relative expression level of each gene in different samples (Table 1). Table 1: Genes, primer sequences, and annealing temperatures used in real time qRT-PCR analysis Gene symbol NCBI reference sequence Forward primer sequence Reverse primer sequence Annealing temperature PPAR-γ NM_181024.2 5′-ATG-GAT-GAC-CAC-TCC-CAT-GCC -3' 5′-ATC-TGC-AAC-CAT-CGG-GTC-AGC-3′ 61°C FABP4 NM_174314.2 5'-AGC-TGC-ACT-TCT-TTC-TCA-CCT-TGA-A-3' 5'-TTG-GCC-ATG-CCA-GCC-AGC-CAC-TTT-3' 64°C UXT NM_001037471.2 5'-GTT-CGG-AGA-GGA-GAG-GCA-AA-3' 5'-AGG-CCG-TCC-ATG-AAG-TTG-TT-3' 60°C Visualization of intracellular lipid accumulation Intracellular lipid droplets were visualized using both colorimetric and fluorometric assays. Cells were first fixed with 4% paraformaldehyde (AppliChem, Esbjerg, Denmark) in PBS for 10 minutes. Following fixation, for the colorimetric visualization, the cells were washed once with PBS and then stained with a 0.5% Oil Red O solution in ethanol for 30 minutes. Brightfield images were captured using an Axio-Observer Z1 microscope (Carl Zeiss) equipped with a digital camera (C11550 ORCA, Hamamatsu) and processed using ZEN 2012 software (Carl Zeiss). For fluorometric visualization, cells were incubated with 1 µg/ml BODIPY 493/503 in DMSO (Sigma-Aldrich) for 30 minutes at 37°C. Confocal images were acquired using an LSM 900 confocal laser scanning microscope (Carl Zeiss) with an Airyscan detector and subsequently processed with ZEN 3.4 software (Carl Zeiss) Statistical analysis Statistical analyses were performed with GraphPad Prism 9 (San Diego, USA). The normality of the data distribution was assessed using the Shapiro-Wilk test. For comparisons between two groups, an independent samples t-test was used. When more than two conditions were compared, one-way analysis of variance (ANOVA) was performed, followed by Tukey's post hoc test to assess differences between groups. Results are presented as mean ± standard error of the mean (SEM), with a p-value of < 0.05 considered statistically significant. Declarations Acknowledgments The authors would like to express their special thanks to Lisa Engen for her technical support with the qRT-PCR assay. This research was funded by grants from PROMEAT (E! 115596), an initiative supported by the EUROSTARS Program and Innovation Fund Denmark. The CytoSMART Omni device was purchased through a donation from Simon Fougner Hartmanns Familiefond and the Faculty of Medicine at Aalborg University. Author contributions ZX and CPP conceived and designed the analysis. ZX and QP collected the data. QP, MB, RP, and SKD contributed with data, materials and analysis tools. ZX, QP, and CPP performed the analysis. SKD and CPP acquired funding. ZX drafted the manuscript. VZ, SKD, and CPP critically revised the manuscript drafts. All authors have approved the final submitted version. 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Rep. 13 , 498 (2023). Stout, A. J. et al. Simple and effective serum-free medium for sustained expansion of bovine satellite cells for cell cultured meat. Commun. Biol. 5 , 466 (2022). Romar, G. A., Kupper, T. S. & Divito, S. J. Research Techniques Made Simple: Techniques to Assess Cell Proliferation. J. Invest. Dermatol. 136 , e1–e7 (2016). Dohmen, R. G. J. et al. Muscle-derived fibro-adipogenic progenitor cells for production of cultured bovine adipose tissue. npj Sci. Food 6 , 1–12 (2022). Rosen, E. D. The Molecular Control of Adipogenesis, with Special Reference to Lymphatic Pathology. Ann. N. Y. Acad. Sci. 979 , 143–158 (2002). Chawla, A., Schwarz, E. J., Dimaculangan, D. D. & Lazar, M. A. Peroxisome proliferator-activated receptor (PPAR) gamma: adipose-predominant expression and induction early in adipocyte differentiation. Endocrinology 135 , 798–800 (1994). Volz, A. C. & Kluger, P. J. Completely serum-free and chemically defined adipocyte development and maintenance. Cytotherapy 20 , 576–588 (2018). Yu, J., Hsu, Y. C., Lee, J. K. & Cheng, N. C. Enhanced angiogenic potential of adipose-derived stem cell sheets by integration with cell spheroids of the same source. Stem Cell Res. Ther. 13 , 1–14 (2022). Rybkowska, P. et al. The Metabolic Changes between Monolayer (2D) and Three-Dimensional (3D) Culture Conditions in Human Mesenchymal Stem/Stromal Cells Derived from Adipose Tissue. Cells 12 , 178 (2023). Nath, S. & Devi, G. R. Three-dimensional culture systems in cancer research: Focus on tumor spheroid model. Pharmacol. Ther. 163 , 94–108 (2016). Gupta, R. K. et al. Transcriptional control of preadipocyte determination by Zfp423. Nat. 2010 4647288 464 , 619–623 (2010). Prasad, M., Zachar, V., Fink, T. & Pennisi, C. P. Moderate hypoxia influences potassium outward currents in adipose-derived stem cells. PLoS One 9 , (2014). Schiller, Z. A., Schiele, N. R., Sims, J. K., Lee, K. & Kuo, C. K. Adipogenesis of adipose-derived stem cells may be regulated via the cytoskeleton at physiological oxygen levels in vitro. Stem Cell Res. Ther. 4 , 1–10 (2013). Yuen, J. S. K. et al. Perspectives on scaling production of adipose tissue for food applications. Biomaterials 280 , 121273 (2022). Zagury, Y., Ianovici, I., Landau, S., Lavon, N. & Levenberg, S. Engineered marble-like bovine fat tissue for cultured meat. Commun. Biol. 5 , 927 (2022). Yuen Jr, J. S. K. et al. Perspectives on scaling production of adipose tissue for food applications. Biomaterials 280 , 121273 (2022). Jara, T. C. et al. Stem cell-based strategies and challenges for production of cultivated meat. Nat. Food 4 , 841–853 (2023). Maldonado, V. V., Pokharel, S., Powell, J. G. & Samsonraj, R. M. Phenotypic and Functional Characterization of Bovine Adipose-Derived Mesenchymal Stromal Cells. Animals 14 , 1292 (2024). Peng, Q. et al. Multiplex Analysis of Adipose-Derived Stem Cell (ASC) Immunophenotype Adaption to In Vitro Expansion. Cells 10 , (2021). Alstrup, T., Eijken, M., Bohn, A. B., Møller, B. & Damsgaard, T. E. Isolation of Adipose Tissue–Derived Stem Cells: Enzymatic Digestion in Combination with Mechanical Distortion to Increase Adipose Tissue–Derived Stem Cell Yield from Human Aspirated Fat. Curr. Protoc. Stem Cell Biol. 48 , e68 (2019). Zachar, V., Rasmussen, J. G. & Fink, T. Isolation and growth of adipose tissue-derived stem cells. Methods Mol. Biol. 698 , 37–49 (2011). Kilroy, G. E. et al. Cytokine profile of human adipose-derived stem cells: Expression of angiogenic, hematopoietic, and pro-inflammatory factors. J. Cell. Physiol. 212 , 702–709 (2007). Pennisi, C. P., Zachar, V., Fink, T., Gurevich, L. & Fojan, P. Patterned polymeric surfaces to study the influence of nanotopography on the growth and differentiation of mesenchymal Additional Declarations No competing interests reported. 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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-5611796","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":477363876,"identity":"6127158b-11c4-4463-92f8-538a00cc2b84","order_by":0,"name":"Zongzhe Xuan","email":"","orcid":"","institution":"Aalborg University","correspondingAuthor":false,"prefix":"","firstName":"Zongzhe","middleName":"","lastName":"Xuan","suffix":""},{"id":477363877,"identity":"b7d4439c-15ac-4b88-8802-bf416d3c9b5a","order_by":1,"name":"Qiuyue Peng","email":"","orcid":"","institution":"Aalborg University","correspondingAuthor":false,"prefix":"","firstName":"Qiuyue","middleName":"","lastName":"Peng","suffix":""},{"id":477363879,"identity":"63235b19-1c38-46a5-868a-f9b7e7e0561a","order_by":2,"name":"Mariia Borsuk","email":"","orcid":"","institution":"Mirai International AG","correspondingAuthor":false,"prefix":"","firstName":"Mariia","middleName":"","lastName":"Borsuk","suffix":""},{"id":477363880,"identity":"c3f80639-20cb-43b2-97f3-d3ef448c4579","order_by":3,"name":"Rupali Prasad","email":"","orcid":"","institution":"Mirai International AG","correspondingAuthor":false,"prefix":"","firstName":"Rupali","middleName":"","lastName":"Prasad","suffix":""},{"id":477363883,"identity":"74264d3b-ea73-4e90-b47f-8ec11594ce5a","order_by":4,"name":"Vladimir Zachar","email":"","orcid":"","institution":"Aalborg University","correspondingAuthor":false,"prefix":"","firstName":"Vladimir","middleName":"","lastName":"Zachar","suffix":""},{"id":477363884,"identity":"f1ecd21a-c54b-4530-b499-f48a878d6531","order_by":5,"name":"Suman Kumar Das","email":"","orcid":"","institution":"Mirai International AG","correspondingAuthor":false,"prefix":"","firstName":"Suman","middleName":"Kumar","lastName":"Das","suffix":""},{"id":477363886,"identity":"702e95aa-b87b-4154-85c6-fed5872b7716","order_by":6,"name":"Cristian Pablo Pennisi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2ElEQVRIiWNgGAWjYNACAwYefgk4L41ILZIzGBgbSNAC0nWDWC38s5uPPS4oqJMxvt3+/AFDjV1iA3taAl4tEneOpRvPMDjMY3bnjGEDw7HkxAaeZwfwu0cix0yax+AAj9mNHKDD2JgTGyTSGwhoyf8G1FLHYzwj/WEDw796YrTksAG1MPMYSCQYNjC2HQZqScPvMKBfQA47zCNxI8dwRmLfceM2nmcJeLUAQ+yZNM+fOnv+GekPPnz4Vi3bz55mgFcLgwQyB2Q8G3716FpGwSgYBaNgFGADAIqqQTUFwzQdAAAAAElFTkSuQmCC","orcid":"","institution":"Aalborg University","correspondingAuthor":true,"prefix":"","firstName":"Cristian","middleName":"Pablo","lastName":"Pennisi","suffix":""}],"badges":[],"createdAt":"2024-12-09 21:53:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5611796/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5611796/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-28441-4","type":"published","date":"2025-12-29T15:58:31+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":85820583,"identity":"68c609fd-cedd-42e2-8932-131d1ebf9b36","added_by":"auto","created_at":"2025-07-02 06:29:06","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":348495,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth and adipogenic differentiation of bASCs in serum-free medium (SFM) and serum-containing medium (FBS). (a) Doubling time of cells at passages 3, 4, and 5. (b) Triglyceride accumulation normalized to cell number at different timepoints post-induction in growth medium (GM), differentiation medium for induction (DM1), and differentiation medium for maintenance (DM2). (c) Representative images of Oil Red O staining showing the formation of lipid droplets in bASCs at day 14 of adipogenic induction (scale bar = 100 μm). (d) Relative mRNA expression levels of adipogenic markers, peroxisome proliferator-activated receptor gamma (\u003cem\u003ePPAR-γ\u003c/em\u003e) and fatty acid-binding protein 4 (\u003cem\u003eFABP4\u003c/em\u003e), measured by RT-qPCR at different time points in GM, DM1, and DM2. (e) The diagram shows the assessment time points (d0, d2, d8 and d14) and the type of medium used in the induced and non-induced groups. Data are expressed as mean ± SEM (* \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5611796/v1/9bb57bc0d0db6a9d9023e164.png"},{"id":85819620,"identity":"a87a5a2e-cd38-41b3-923f-ea318c2b0d73","added_by":"auto","created_at":"2025-07-02 06:21:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":432646,"visible":true,"origin":"","legend":"\u003cp\u003eMorphology and viability of spheroids formed at different initial densities. (a) Representative phase-contrast microscopy images showing the morphological development of bASC spheroids. The spheroids were formed from three different initial seeding densities: 5000, 10000, or 20000 cells/well. The images were taken over three days of culture, labeled as d1, d2 and d3. Scale bar = 1 mm (b) Quantification of spheroid size for each seeding density. (c) Comparison of doubling times between 2D monolayer cultures and 3D spheroids. (d) Live/dead staining of bASCs cultured in 2D monolayers and 3D spheroids. Live cells are stained green, and dead cells are stained red (scale bar = 200 μm (3D) \u0026amp; 100 μm (2D)). (e) Quantitative analysis of cell viability across 2D and 3D cultures. Data are presented as mean ± SEM (* \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e\u003cstrong\u003e# # # \u0026nbsp;\u0026amp; \u003c/strong\u003e\u003c/sup\u003e*** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, \u003csup\u003e\u003cstrong\u003e# \u003c/strong\u003e\u003c/sup\u003eRepresents the comparative analysis between d3 and d1).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5611796/v1/399f173146cd8f6a0b67acfc.png"},{"id":85820584,"identity":"b0255aba-73ef-4e49-a93b-b7d4aab0f6ab","added_by":"auto","created_at":"2025-07-02 06:29:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":141468,"visible":true,"origin":"","legend":"\u003cp\u003eAdipogenic differentiation of bASCs in 3D spheroids and 2D monolayer cultures. (a) Normalized triglyceride content measured at different time points in serum-free growth medium (GM), differentiation medium for induction (DM1), and differentiation medium for maintenance (DM2). (b) Representative images of BODIPY 493/503 staining showing lipid accumulation (green) in bASCs cultured in 2D and 3D after 14 days of adipogenic induction (scale bar = 100 μm). (c) Relative mRNA expression levels of adipogenic markers, peroxisome proliferator-activated receptor gamma (\u003cem\u003ePPAR-γ\u003c/em\u003e) and fatty acid-binding protein 4 (\u003cem\u003eFABP4\u003c/em\u003e), measured by RT-qPCR at different time points in GM, DM1, and DM2. (e) The diagram shows the assessment time points (d0, d2, d8 and d14) and the type of medium used in the induced and non-induced groups. Data are expressed as mean ± SEM (* \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5611796/v1/101def5082771700cfc51226.png"},{"id":99545429,"identity":"6d1e318a-dfeb-464c-b03c-b50cb533fabd","added_by":"auto","created_at":"2026-01-05 16:07:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1684219,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5611796/v1/04df2f8e-9345-4d36-acfa-4429f531e873.pdf"},{"id":85820588,"identity":"ed47d994-899c-4d8c-840d-3f213e1be916","added_by":"auto","created_at":"2025-07-02 06:29:07","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":3065971,"visible":true,"origin":"","legend":"","description":"","filename":"Develpmentofasustainablestrategy...SupplinfoR2.docx","url":"https://assets-eu.researchsquare.com/files/rs-5611796/v1/0a592af64ba459f4cbb73687.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Development of a sustainable strategy for cultured fat production based on serum-free 3D culture of bovine adipose stem cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCultured or cultivated meat, produced from animal cells using tissue engineering methods, has gained significant attention in recent years due to its potential to offer a more ethical and sustainable alternative to conventional meat production \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Unlike plant-based meat substitutes, cultivated meat aims to replicate the sensory experience of traditional meat, including its texture, taste, and appearance \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. While much of the research in cultivated meat focuses on muscle tissue as the primary protein source, the role of fat in meat cannot be overlooked. Fat contributes to the juiciness, flavor, and mouthfeel of meat, which are critical for consumer acceptance \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. During cooking, for example, meat releases hundreds of volatile compounds, most of which originate from fats \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Additionally, fat is essential for creating the species-specific flavor that distinguishes the taste of different meats \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Therefore, producing and integrating tissue engineered fats into cultivated meat products is vital for achieving a product that meets consumer expectations \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn vivo, the lipids that contribute to the sensory properties of meat are primarily synthesized and stored within adipocytes, specialized cells that form adipose tissue. Adipocytes play a crucial role in lipid metabolism, through synthesis, accumulation, and release of fatty acids that are essential for the development and function of adipose tissue \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Adipose stromal/stem cells (ASCs) have been identified as a valuable source for generating adipocytes in vitro due to their inherent ability to differentiate into lipid-producing cells \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. To support the growth and differentiation of ASCs in culture media are supplemented with fetal bovine serum (FBS), which provides the necessary growth factors and nutrients to promote efficient proliferation and adipogenic differentiation of the cells \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. However, the cost of using culture and differentiation media containing FBS is a major limiting factor in scaling up production of cultivated meat \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Additionally, the reliance on animal-derived FBS raises ethical concerns and limits the sustainability of the process, conflicting with the overarching goals of cultivated meat production. With the aim of eliminating dependence on products of animal origin, while ensuring efficient cell growth and differentiation, the development of serum-free alternatives has been extensively studied in the context of expanding ASCs for clinical applications \u003csup\u003e\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. In the field of culture meat, serum-free media formulations are crucial for the development of a sustainable alternative to conventional meat. The majority of efforts have so far focused on optimizing media for muscle precursor cells, likely due to their central role in defining the texture and protein content of cultured meat products \u003csup\u003e\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. In contrast, serum-free strategies tailored specifically for bovine fat cells remain relatively underexplored, but recent studies have begun to address this gap. Mitić et al. introduced a simplified, cross-species serum-free formulation capable of supporting adipogenic differentiation\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Similarly, Messmer et al. applied single-cell transcriptomic analysis to identify bovine muscle-derived cell subtypes with adipogenic potential, providing insights for more targeted media development\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. More recently, refinement of media composition has focused on replacing traditional components like glutamine and glucose with alternatives that minimize toxic byproducts such as ammonia, thereby enhancing both cell proliferation and adipogenic differentiation\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTraditionally, ASCs are cultured in two-dimensional (2D) monolayer systems, which do not accurately mimic the complex microenvironment of tissues \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Additionally, prolonged culture of ASCs in 2D conditions often results in a loss of replicative ability, colony-forming efficiency, and differentiation capacity \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. A promising alternative is the cultivation of ASCs as three-dimensional (3D) tissue structures that better mimic the natural stem cell microenvironment \u003csup\u003e\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Research with human ASCs has shown that 3D spheroids represent a more relevant system for the differentiation and maturation of the cells into the adipogenic lineage, producing cells that are more similar to mature adipocytes \u003csup\u003e\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. However, protocols developed for adipogenic differentiation of human and mouse cells showed low efficiency in inducing adipogenesis of bovine progenitor cells, emphasizing that there are significant differences between human and ruminant adipogenic processes \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. For instance, human cells exhibit higher enzymatic activity for converting glucose to fatty acids, while bovine cells rely more on acetate due to their unique metabolic adaptations \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Furthermore, while more recent studies have begun to understand the effect of 3D culture in bovine adipogenic precursors\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, these studies were conducted in serum-containing media. Studies have shown that human mesenchymal stromal cells form loose 3D aggregates but not solid spheroids after serum withdrawal \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, which could be related to removal of the adhesion proteins contained in the medium, including fibronectin and vitronectin \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Therefore, it is not yet known whether the removal of serum influences the 3D cultivation of bovine ASCs. In this study, we address these challenges by comparing 2D and 3D culture methods for bovine ASCs, focusing on adipogenic differentiation in serum-free media. By characterizing these properties, we aim to provide a foundation for the efficient and sustainable production of cultivated fat, potentially paving the way for large-scale adoption of these methods in cultivated meat production.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThe growth and differentiation potential of bovine adipose-derived stem cells (bASCs) was investigated under serum-free conditions. Over passages 3 to 5, bASCs cultured in serum-free medium (SFM) showed a significantly higher proliferation capacity than those cultured in serum-containing medium (FBS). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the doubling time of cells in SFM was significantly shorter, indicating a faster proliferation rate in the absence of serum. The percentage increase was approximately 20%. In addition, Ki-67 analysis revealed significantly higher expression of this proliferation marker in cells cultured in SFM after prolonged passage, further supporting the sustained proliferation potential of bASCs under these conditions (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, Supplementary information file). Non-induced controls were maintained in growth medium (GM) for the duration of the experiment, while differentiation was induced using a two-step protocol consisting of induction medium (DM1) followed by maintenance medium (DM2). After initiation of adipogenic differentiation, bASCs in SFM exhibited a substantial increase in intracellular triglyceride accumulation, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Compared to bASCs cultured in FBS, cells in SFM displayed significantly higher triglyceride content on days 8 and 14, indicating a more robust differentiation response. On day 14, when comparing the mean values of normalized triglyceride content (16.62 in SFM vs. 9.96 in FBS), it can be inferred that the percentage increase in lipid accumulation due to SFM was over 66%. This enhanced lipid accumulation was also confirmed by Oil Red O staining on day 14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), in which cells cultured in SFM showed more intense staining of lipid droplets compared to their FBS counterparts. At the transcriptional level, adipogenic differentiation in SFM was further supported by the expression profiles of key adipogenic markers, \u003cem\u003ePPAR-γ\u003c/em\u003e and \u003cem\u003eFABP4\u003c/em\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, while only minimal changes in gene expression were observed after 2 days of induction (DM1), cells cultured in SFM exhibited significant upregulation of both markers at later stages of differentiation in DM2 (day 8 and 14). On day 14, the expression of \u003cem\u003ePPAR-γ\u003c/em\u003e and \u003cem\u003eFABP4\u003c/em\u003e was significantly higher in bASCs cultured in SFM than in FBS.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe morphology and viability of spheroids formed from bASCs seeded at different initial densities were analyzed over a three-day culture period using SFM. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e illustrates the self-organization of cells into spheroids at different densities (5000, 10000, and 20000 cells per well) from day 1 to day 3. On day 1 after seeding, the spheroids exhibited a quasi-circular morphology characterized by well-defined edges, with almost all seeded cells seamlessly integrated into the spheroid structure. A clear seeding density-dependent morphological difference was observed, with higher initial cell densities leading to larger spheroids. Quantitative analysis of spheroid size, represented as Feret diameter, confirmed that spheroid size was directly correlated with initial cell density (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The doubling time of bASCs cultured in 3D spheroids was compared with different cell densities and with 2D monolayer cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Cells cultured under 2D conditions showed a significantly shorter doubling time than cells in 3D spheroids, with the doubling time increasing significantly with increasing initial cell density in the spheroids. Cell viability was assessed using live/dead staining. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows representative images of both 3D spheroids and 2D monolayer cultures on days 1 and 3, with live cells shown in green and dead cells in red. In 3D cultures, the spheroids formed at higher densities (10k and 20k) exhibited more pronounced red staining, indicating a higher degree of cell death over time. In contrast, the 2D cultures maintained a higher overall viability over the same period. Quantitative analysis of cell viability confirmed the microscopy results, with cells in all 3D conditions consistently exhibiting lower viability compared to 2D cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). All three spheroid groups with different seeding densities appeared to have relatively high cell viability on day 1 (\u0026gt;\u0026thinsp;80%). Cell viability in 3D spheroids decreased significantly from day 1 to day 3, but there was no statistical difference in cell viability between the different seeding densities. Statistical analysis confirmed the significant differences in viability between 2D and 3D cultures and between day 1 and day 3 for the 3D conditions (### \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Representative flow cytometry plots used for this quantification are provided in the supplementary information file (Figure S2).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe adipogenic differentiation potential of bASCs was investigated under 3D spheroid culture conditions using SFM, with 2D monolayers as reference. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, adipogenic induction in 3D spheroids led to a significant increase in intracellular triglyceride content. After 8 days in DM2, the spheroids showed significantly higher triglyceride levels compared to the early induction phase (DM1) and the non-induced controls (GM). On day 14, the spheroids showed the highest triglyceride accumulation compared to the conventional 2D cultures. The percentage increase in triglyceride accumulation in 3D was over 34%. Further confirmation of adipogenic differentiation was provided by BODIPY 493/503 staining, which demonstrated the formation of lipid droplets. At 14 days post-induction, bASCs in 3D spheroids exhibited extensive lipid accumulation, with significantly greater lipid droplet coverage compared to 2D monolayers (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The 3D spheroid cultures exhibited a dense and organized distribution of lipid droplets, indicating a more mature adipogenic state. At the transcriptional level, after 8 days in DM2, the expression of \u003cem\u003ePPAR-γ\u003c/em\u003e and \u003cem\u003eFABP4\u003c/em\u003e was significantly increased in the 3D culture compared to early differentiation stages and 2D monolayer culture (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). At day 14, bASCs in 3D spheroids continued to exhibit much higher expression of \u003cem\u003ePPAR-γ\u003c/em\u003e and \u003cem\u003eFABP4\u003c/em\u003e than their 2D counterparts, consistent with the increased lipid accumulation observed under 3D conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWhile most in vitro adipogenesis studies in the past have been based on serum-containing media, the focus in the cultured meat field has been on the development of serum-free media suitable for the expansion and differentiation of progenitor cells \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The use of FBS is associated with several challenges, including ethical concerns regarding animal welfare, risk of pathogen contamination, unpredictable supply and high costs \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Therefore, development and optimization of serum-free media is critical to achieve the scalability and sustainability required for large-scale cultured meat production \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. The challenges associated with formulating serum-free media have been extensively addressed in previous research, with significant progress made in creating chemically defined media to support the differentiation of various progenitor cells, including bovine satellite cells \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e and adipogenic progenitor cells from several species \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. In this study, the initial focus is on the validation of existing serum-free culture media for the expansion and differentiation of bASCs, with the main objective being to ensure that the media can support the culture of the cells under well-defined conditions. This validation step is critical to assess the viability of 3D culture conditions and provide a basis for future scalability and sustainability in the context of cultured meat production.\u003c/p\u003e \u003cp\u003eIn terms of cell proliferation, the SFM was more efficient than serum-containing media, as evidenced by a reduced doubling time and relatively higher expression of the proliferation marker Ki-67. This cell cycle-associated protein is expressed during the active phases of the cell cycle (G1-M), so an increased expression is an indication of active cell proliferation\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. These findings highlight the validity of the SFM not only to replace FBS but also to enhance cell growth performance. Previous studies have investigated the role of serum in the proliferation and differentiation of bovine adipogenic progenitor cells, particularly muscle-derived fibroadipogenic progenitors (FAPs). While these cells differentiate efficiently under serum-free conditions, their expansion phase is normally carried out in serum-containing medium \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Notably, FAPs cultured without serum show comparable gene expression profiles to cells grown under serum-containing conditions \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Recent efforts have aimed to simplify adipogenesis protocols by reducing the number of inducers and differentiation phases to facilitate upscaling.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. For the differentiation phase, the approach in this study incorporated two distinct serum-free media: one specifically formulated for adipogenic induction and the other for maintenance. This dual-media system effectively supported both the early stages of adipocyte commitment and the later stages of lipid accumulation, as evidenced by higher expression levels of key adipogenic transcription factors, such as \u003cem\u003ePPARγ\u003c/em\u003e and \u003cem\u003eFABP4\u003c/em\u003e. These transcription factors play a critical role in preadipocyte determination \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. The separation of induction and maintenance phases allows for optimal regulation of the differentiation process, maximizing the efficiency of lipid accumulation in the absence of serum-derived growth factors, which have been shown to influence adipogenic outcomes unpredictably \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Studies on human ASCs demonstrated that using defined media in each phase (differentiation and long-term maintenance) could enhance cell adherence and lipid accumulation while maintaining cell functionality over extended culture periods \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eStudies have shown that 3D culture conditions enhance the production of mature adipocytes from human and murine precursors \u003csup\u003e\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. However, bovine adipogenic models face unique challenges, including an often inconsistent adipogenic differentiation compared to other species, which makes the validation of 3D culture conditions particularly critical in the bovine context \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. The results of this study show that bASCs rapidly formed stable spheroids within three days at all cell densities tested. The diameter of the spheroids decreased after their initial formation, which correlated with the density of cells seeded in each well and the incubation time. These observations are consistent with previous studies that have shown that the size of cell spheroids can be adjusted by controlling cell seeding density \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. As in previous studies, the viability of bASCs decreased significantly under 3D spheroid culture conditions compared to 2D culture throughout the process of spheroid formation \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. For spheroids in the diameter range between 200 and 500 \u0026micro;m, studies have shown that there is a gradual accumulation of metabolic waste and insufficient diffusion of oxygen/nutrients, which could be the main reason for the decrease in cellular viability \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFollowing the assessment of viability and growth, the results show that the 3D spheroid system provided an enhanced microenvironment for adipogenesis of bASCs, as evidenced by the increase in triglyceride content and larger lipid droplets within the spheroids after adipogenic induction. The expression of transcription factors \u003cem\u003ePPARγ\u003c/em\u003e and \u003cem\u003eFABP4\u003c/em\u003e was consistently higher in spheroid cultures than in 2D monolayers, leading to more mature adipocytes with larger lipid droplets. The 3D environment may mimic aspects of the native extracellular matrix, offering a more physiologically relevant context that encourages adipogenic commitment and maturation \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Furthermore, low oxygen (hypoxic) conditions are known to modulate ASC behavior, particularly through enhanced proliferation and increased secretion of growth factors that can further promote differentiation \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Hypoxia and cell morphology can influence adipogenic differentiation, as shown by the improved adipogenic outcomes in ASCs when cytoskeletal tension and oxygen levels are reduced \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Thus, a combination of reduced cytoskeletal stress and limited oxygen diffusion within the dense spheroids may also have contributed to the increased adipogenic efficiency observed in 3D. The efficiency of adipogenic differentiation of bASCs is particularly important for cultured fat production \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Notably, the use of serum-free media in the 3D system proved critical for achieving this level of adipogenic maturity, further underscoring the advantages of 3D cultures over conventional 2D systems.\u003c/p\u003e \u003cp\u003eWhile the presented 3D approach is effective for cell differentiation, large-scale production of cultured fat requires extensive cell expansion prior to the differentiation phase to generate substantial amounts of cellular material from limited donor tissue \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Although the 3D spheroid format supports adipogenic maturation, our data indicated that it is not optimal for cell expansion. This limitation suggests that a two-step strategy may be required for large-scale applications, e.g. initial expansion in bioreactors (such as suspension or hollow fiber bioreactors) followed by differentiation in 3D\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. In addition, the reliability and reproducibility of stem cell sources are crucial \u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. In this study, the cells were isolated according to a standard protocol, which generally provides very consistent results in terms of yield of ASCs. The isolated bASCs were characterized using colony-forming unit\u0026ndash;fibroblastic (CFU-F) and osteogenic differentiation assays to assess self-renewal and clonogenic capacity as well as multipotency (Supplementary information file). Clones were consistently formed at all seeding densities tested, demonstrating the self-renewal and proliferation capacity of the bASCs (Figure S3). Interestingly, even when the cells were seeded sparsely, the clonogenic efficiency was higher than previously reported \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. In addition, a positive osteogenic differentiation confirmed the multipotency of the cells (Figure S4). One possible limitation is the fact that only cells from one donor were used. To ensure the robustness and reproducibility of the findings, future research should validate these results with cells from multiple animal donors and understand the behavior of the cells in long-term cultures, which may impact the efficiency of adipogenic differentiation and scalability for cultured fat production. In human ASCs, significant immunophenotypical and biological changes occur during in vitro expansion, underscoring the importance of monitoring cell behavior over extended culture periods \u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn summary, this study presents a novel method for culturing bASCs into mature adipocytes in a 3D spheroid culture system under serum-free conditions. Compared to conventional 2D cultures in serum-containing media, the presented method enables efficient lipid accumulation. These results indicate the potential of this approach for scalable and sustainable cultured fat production. However, further validation with different cell donors is required to ensure the robustness and reproducibility of the protocol. Overall, this research contributes to the advancement of cultured meat technologies by addressing critical challenges in the cell maturation phase, potentially paving the way for large-scale adoption of these methods in cultured meat production.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eIsolation and culture of cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrimary bovine ASCs (bASCs) were isolated from the subcutaneous adipose tissue of 6-month-old Black Angus calf (collected at the abattoir) following previously described protocols\u003csup\u003e56,57\u003c/sup\u003e, which were adapted for processing excised tissues. As the material was obtained from a deceased animal as a by-product of a routine commercial practice, no ethical approval was required. This is in accordance with Directive 2010/63/EU on the protection of animals used for scientific purposes. In brief, tissue samples were transferred to a sterile hood and disinfected in 70% ethanol for 2 minutes. The samples were washed with PBS and subsequently excised from visible connective, muscle tissue and blood vessels in isolation medium comprising Dulbecco\u0026apos;s modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FBS), 12.5 \u0026micro;g/ml amphotericin B, and 500 U/ml penicillin/streptomycin (all from ThermoFisher Scientific). The part of the tissue containing only white fat cells was minced into small pieces (~2 mm\u003csup\u003e3\u003c/sup\u003e) in isolation medium using sterile forceps and scissors. The tissue pieces were then digested with STEMxyme 1 solution (BioConcept Ltd, Switzerland) in a shaking incubator/water bath at 37\u0026deg;C for 2 hours. The resulting mixture was centrifuged at 80 g for 5 minutes at 4\u0026deg;C and the supernatant was filtered through cell strainers. The cell suspension was then incubated on ice in erythrocyte lysis buffer for 5 minutes. After careful washing and resuspension, the cells were plated and expanded in growth medium (GM) consisting of DMEM/F-12 nutrient mixture (DMEM/F-12, Gibco), supplemented with 10% FBS, and\u0026nbsp;100 U/ml penicillin/streptomycin. The cells were maintained in tissue culture flasks (Greiner Bio-one) in a standard humidified incubator at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e. The culture medium was changed every second day. Cells were passaged using TrypLE select (Gibco, ThermoFisher Scientific) when they reached 90% confluence.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of cultures for assessment of cell growth and differentiation\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the performance of the serum-free media in terms of proliferation and differentiation, bASCs were seeded at a density of 5,000 cells/cm\u003csup\u003e2\u003c/sup\u003e on standard 6-well plates (Greiner, Bio-One) previously coated with a 0.2% gelatin solution. Once the bASCs were confluent, the cells were differentiated into mature adipocytes in two phases. In the first phase, bASCs were induced into preadipocytes by incubation with the first differentiation medium (DM1) for 48 hours. Subsequently, the preadipocytes were maintained in the second differentiation medium (DM2) for 12 days. The non-induced control groups were cultured in GM for the entire assessment period.\u003c/p\u003e\n\u003cp\u003eSerum-containing DM1 was prepared according to the protocol of Kilroy et al \u003csup\u003e58\u003c/sup\u003e with minor modifications. \u0026nbsp;The DMEM/F-12 nutrient mixture was supplemented with 15% FBS, 33 \u0026micro;M biotin, 17 \u0026micro;M pantothenate, 0.2 \u0026micro;M insulin, 1 \u0026micro;M dexamethasone, 0.5 mM isobutylmethylxanthine (IBMX), 5 \u0026micro;M rosiglitazone, and 100 U/ml penicillin/streptomycin. Serum-containing DM2 was prepared according to Mehta et al \u003csup\u003e11\u003c/sup\u003e using DMEM/F-12 supplemented with 10% FBS, a mixture of free fatty acids (5 \u0026mu;M pristanic acid, 1 \u0026mu;M phytanic acid, 50 \u0026mu;M myristoleic acid, 5 \u0026mu;M elaidic acid, 5 \u0026mu;M erucic acid, 5 \u0026mu;M oleic acid, and 150 \u0026mu;M palmitoleic acid) and 100 U/ml penicillin/streptomycin. The serum-free GM (FatSFM, Mirai International) consists of DMEM/F12 and supplements. Serum-free DM1 (FatDM1, Mirai International) consists of Glasgow\u0026rsquo;s modified Eagle medium (GMEM) and supplements. The serum-free DM2 (FatDM1, Mirai International) is based on GMEM, insulin and free fatty acids. The exact composition of the serum-free media is proprietary to Mirai International.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo evaluate the influence of three-dimensional (3D) culture conditions on bASC behavior, experiments were performed exclusively in serum-free media formulations. After culturing the bASCs in serum-free GM in tissue culture flasks for at least 7 days, the cells were trypsinized and seeded on 96-well spheroid plates (BIOFLOAT, faCellitate) at three different densities (5000, 10000 and 20000 cells/well). These plates provide a highly defined cell-repellent surface on which spheroids can be formed without the need for a scaffold. To prevent spheroids from being lost during media changes, only half of the media volume was replaced with each media change. The control group comprised cells cultured in 6-well plates treated in the same way for consistency. The process of spheroid formation was monitored at different time points using a plate scanner with digital phase contrast (CytoSMART Omni, Axion). The device was set to scan the spheroids every six hours for 72 hours. The images were collected and then analyzed using Image J to quantify the process of spheroid change over time and compare the size of the different seeding densities.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProliferation assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate cell proliferation under different culture conditions, a Presto Blue assay (Invitrogen, Thermo Fisher Scientific) was performed with cells between the third and fifth passage. After 24, 48, 72 and 96 hours, Presto Blue reagent was added to the cell-containing wells at 10% of the total volume, followed by incubation at 37 \u0026deg;C for 3 hours. The negative controls consisted of cell-free wells. Subsequently, 100 \u0026micro;l of the solution was loaded (in duplicates) in a 96-well microplate. Fluorescence was measured using a multimode plate reader (EnSpire, PerkinElmer) at excitation and emission wavelengths of 560 nm and 610 nm, respectively. After fluorescence measurement, the cells were washed with PBS and fresh growth medium was added. Cell proliferation was expressed as doubling time based on the actual measurement of fluorescence intensity (n = 6), as previously reported \u003csup\u003e59\u003c/sup\u003e. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell viability assessment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess cell viability, live and dead cells were stained 1 and 3 days after seeding. Cells were first washed with PBS and incubated for 1 hour in a serum-free DMEM/F-12 nutrient mixture containing calcein-AM (1 \u0026mu;M) and propidium iodide (5 \u0026micro;M). The cells were then washed twice with serum-free DMEM/F-12. Samples were observed using a fluorescence microscope (Axio Observer Z1, Carl Zeiss) and images were captured using a digital camera (C11440 ORCA Hamamatsu) and ZEN 2012 software.\u003c/p\u003e\n\u003cp\u003eQuantitative analysis of cell viability was performed using a flow cytometer (CytoFLEX, Beckman Coulter, Copenhagen, Denmark). The spheroids were harvested through a 40 \u0026micro;m BD Falcon cell strainer and subsequently dissociated with a cell dissociation reagent (StemPro Accutase, Gibco), followed by two washes with PBS. The dissociated cells were resuspended in PBS at a concentration of 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/100 \u0026mu;l. To quantify the number of live and dead cells, the fixable viability dye 570 (FVS 570, BD Biosciences, Lyngby, Denmark) was added to the cell suspensions and incubated at room temperature in the dark for 15 minutes. Prior to analysis, the cells were washed and resuspended in flow staining buffer consisting of 0.1% bovine serum albumin (BSA) in PBS. The experiments were repeated three times, each time recording at least 2.5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e individual cells. The data obtained were visualized and analyzed using the Kaluza 2.1 software package (Beckman Coulter, Indianapolis, IN, USA).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantification of intracellular triglyceride and DNA content\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAn adipogenesis test kit (MAK040-1KT, Sigma Aldrich) was used to determine the total cellular concentration of triglycerides. This assay is based on an enzymatic reaction that generates a product that is measured by spectrofluorometry. The assay was performed according to the manufacturer\u0026rsquo;s instructions. In brief, the lipid extraction reagent was added to the cells in 2D monolayers and 3D spheroids. The spheroids were disrupted by vigorous pipetting. The measurement was performed with a multimode plate reader (EnSpire, PerkinElmer) at excitation and emission wavelengths of 535 nm and 587 nm, respectively. The data were normalized to the DNA content of each sample. The DNA content of each well was measured using the AccuClear dsDNA quantification kit (Biotum) according to the manufacturer\u0026apos;s instructions. In brief, 10 \u0026mu;L of each sample was added to 200 \u0026mu;L of working solution prepared by diluting the dye 100-fold with the DNA quantification buffer and mixed by pipetting. After a 5-minute incubation at room temperature in the dark, the plate was read on a multimode plate reader with excitation and emission settings of 468 and 507 nm, respectively. The results obtained were fitted to a DNA standard curve to determine the mass of DNA per sample (n = 3).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA extraction and quantitative real-time RT-PCR analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA quantitative real-time PCR was performed to investigate the transcriptional activity of genes involved in the differentiation of bASCs. The Aurum total RNA mini kit (Bio-Rad, Copenhagen, Denmark) was used to harvest total RNA from cultured cells.\u0026nbsp;A nanoliter spectrophotometer (NanoDrop; Thermo Fisher Scientific, Wilmington, DE, USA) was used to determine RNA purity and concentration. The iScript cDNA synthesis kit (Bio-Rad, Copenhagen, Denmark) was used for the synthesis of complementary DNA (cDNA). Amplification reactions for each cDNA sample were performed in duplicate, in a final volume of 20 \u0026micro;l. Each reaction consisted of cDNA, IQ SYBR Green Supermix (Bio-Rad), and the target-specific primers (Table 1). The reaction was carried out on a\u0026nbsp;QuantStudio\u003csup\u003eTM\u003c/sup\u003e 5 Real-Time PCR System (Thermo Fisher Scientific, Wilmington, DE, USA). The reaction was carried out for 40 amplification cycles: DNA denaturation was performed at 95 \u0026deg;C for 3 min, 95 \u0026deg;C for 10 s, and annealing and extension were performed at the corresponding annealing temperature for 30 s. One housekeeping gene (Ubiquitously Expressed Prefoldin Like Chaperone, \u003cem\u003eUXT)\u0026nbsp;\u003c/em\u003ewas used to obtain the relative expression level of each gene in different samples (Table 1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1:\u0026nbsp;\u003c/strong\u003eGenes, primer sequences, and annealing temperatures used in real time qRT-PCR analysis\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"\" width=\"101%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 14.433%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGene symbol\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.5567%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNCBI reference sequence\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27.8351%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eForward primer sequence\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24.7423%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eReverse primer sequence\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.433%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAnnealing\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003etemperature\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 14.433%;\"\u003e\n \u003cp\u003e\u003cem\u003ePPAR-\u0026gamma;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.5567%;\"\u003e\n \u003cp\u003eNM_181024.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27.8351%;\"\u003e\n \u003cp\u003e5\u0026prime;-ATG-GAT-GAC-CAC-TCC-CAT-GCC -3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24.7423%;\"\u003e\n \u003cp\u003e5\u0026prime;-ATC-TGC-AAC-CAT-CGG-GTC-AGC-3\u0026prime;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.433%;\"\u003e\n \u003cp\u003e61\u0026deg;C\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 14.433%;\"\u003e\n \u003cp\u003e\u003cem\u003eFABP4\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.5567%;\"\u003e\n \u003cp\u003eNM_174314.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27.8351%;\"\u003e\n \u003cp\u003e5\u0026apos;-AGC-TGC-ACT-TCT-TTC-TCA-CCT-TGA-A-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24.7423%;\"\u003e\n \u003cp\u003e5\u0026apos;-TTG-GCC-ATG-CCA-GCC-AGC-CAC-TTT-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.433%;\"\u003e\n \u003cp\u003e64\u0026deg;C\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 14.433%;\"\u003e\n \u003cp\u003e\u003cem\u003eUXT\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.5567%;\"\u003e\n \u003cp\u003eNM_001037471.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27.8351%;\"\u003e\n \u003cp\u003e5\u0026apos;-GTT-CGG-AGA-GGA-GAG-GCA-AA-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24.7423%;\"\u003e\n \u003cp\u003e5\u0026apos;-AGG-CCG-TCC-ATG-AAG-TTG-TT-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.433%;\"\u003e\n \u003cp\u003e60\u0026deg;C\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eVisualization of intracellular lipid accumulation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIntracellular lipid droplets were visualized using both colorimetric and fluorometric assays. Cells were first fixed with 4% paraformaldehyde (AppliChem, Esbjerg, Denmark) in PBS for 10 minutes. Following fixation, for the colorimetric visualization, the cells were washed once with PBS and then stained with a 0.5% Oil Red O solution in ethanol for 30 minutes. Brightfield images were captured using an Axio-Observer Z1 microscope (Carl Zeiss) equipped with a digital camera (C11550 ORCA, Hamamatsu) and processed using ZEN 2012 software (Carl Zeiss). For fluorometric visualization, cells were incubated with 1 \u0026micro;g/ml BODIPY 493/503 in DMSO (Sigma-Aldrich) for 30 minutes at 37\u0026deg;C. Confocal images were acquired using an LSM 900 confocal laser scanning microscope (Carl Zeiss) with an Airyscan detector and subsequently processed with ZEN 3.4 software (Carl Zeiss)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analyses were performed with GraphPad Prism 9 (San Diego, USA). The normality of the data distribution was assessed using the Shapiro-Wilk test. For comparisons between two groups, an independent samples t-test was used. When more than two conditions were compared, one-way analysis of variance (ANOVA) was performed, followed by Tukey\u0026apos;s post hoc test to assess differences between groups. Results are presented as mean \u0026plusmn; standard error of the mean (SEM), with a p-value of \u0026lt; 0.05 considered statistically significant.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgments\u003c/h2\u003e\n\u003cp\u003eThe authors would like to express their special thanks to Lisa Engen for her technical support with the qRT-PCR assay. This research was funded by grants from PROMEAT (E! 115596), an initiative supported by the EUROSTARS Program and Innovation Fund Denmark. The CytoSMART Omni device was purchased through a donation from Simon Fougner Hartmanns Familiefond and the Faculty of Medicine at Aalborg University.\u003c/p\u003e\n\u003ch2\u003eAuthor contributions\u003c/h2\u003e\n\u003cp\u003eZX and CPP conceived and designed the analysis. ZX and QP collected the data. QP, MB, RP, and SKD contributed with data, materials and analysis tools. ZX, QP, and CPP performed the analysis. SKD and CPP acquired funding. ZX drafted the manuscript. VZ, SKD, and CPP critically revised the manuscript drafts. All authors have approved the final submitted version.\u003c/p\u003e\n\u003ch2\u003eData availability statement\u003c/h2\u003e\n\u003cp\u003eThe data generated during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003ch2\u003eAdditional information / Competing interests\u0026rsquo; statement\u003c/h2\u003e\n\u003cp\u003eAll authors declare no competing interests\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eYoung, J. F., Skrivergaard, S., Therkildsen, M. \u0026amp; Rasmussen, M. K. Cultured meat production\u0026mdash;Scale and quality. \u003cem\u003eCell Reports Sustain.\u003c/em\u003e \u003cstrong\u003e1\u003c/strong\u003e, 100012 (2024).\u003c/li\u003e\n\u003cli\u003eMartins, B. \u003cem\u003eet al.\u003c/em\u003e Advances and Challenges in Cell Biology for Cultured Meat. \u003cem\u003eAnnu. Rev. Anim. 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Patterned polymeric surfaces to study the influence of nanotopography on the growth and differentiation of mesenchymal \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":true,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"cultured fat, serum-free medium, 3D spheroid culture, adipogenic differentiation, bovine adipose stem cells, sustainable meat production","lastPublishedDoi":"10.21203/rs.3.rs-5611796/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5611796/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e Cultivated meat provides a sustainable and ethical alternative to traditional livestock production, addressing global challenges in meat demand and environmental impact. Fat is recognized as an essential component of meat products, but strategies for culturing animal fat precursors still have major limitations, such as the need for a serum-containing medium and inefficient adipogenic cell maturation. In this study, a serum-free medium (SFM) formulation for the cultivation of bovine adipose-derived stem cells (bASC) was first validated by comparison with serum-containing media (FBS). After a 14-day induction period, the SFM increased bASC proliferation by 20% and triglyceride accumulation by 66%. When the SFM was applied to a three-dimensional (3D) spheroid culture system, it resulted in up to 34% higher triglyceride accumulation compared to 2D cultures. These results were accompanied by a significant increase in the expression of key adipogenic markers and lipid droplet formation, suggesting a synergistic potential of SFM and 3D spheroid strategies for bASC culture. Overall, the results of this study provide a scalable and sustainable platform for cultured fat production and are an important step towards overcoming some of the challenges in cultured meat production.\u003c/p\u003e","manuscriptTitle":"Development of a sustainable strategy for cultured fat production based on serum-free 3D culture of bovine adipose stem cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-02 06:21:02","doi":"10.21203/rs.3.rs-5611796/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-27T11:33:14+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-24T01:55:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"139069517950709851276089559943035789228","date":"2025-06-16T02:46:14+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-13T14:46:08+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-12T05:18:41+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-06-11T10:32:30+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f300cf8f-378d-476c-a7ca-1adca61f3ce2","owner":[],"postedDate":"July 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":50693005,"name":"Biological sciences/Biotechnology/Stem cell biotechnology"},{"id":50693006,"name":"Biological sciences/Biotechnology/Tissue engineering"},{"id":50693007,"name":"Biological sciences/Biochemistry/Lipids"}],"tags":[],"updatedAt":"2026-01-05T16:04:20+00:00","versionOfRecord":{"articleIdentity":"rs-5611796","link":"https://doi.org/10.1038/s41598-025-28441-4","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-12-29 15:58:31","publishedOnDateReadable":"December 29th, 2025"},"versionCreatedAt":"2025-07-02 06:21:02","video":"","vorDoi":"10.1038/s41598-025-28441-4","vorDoiUrl":"https://doi.org/10.1038/s41598-025-28441-4","workflowStages":[]},"version":"v1","identity":"rs-5611796","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5611796","identity":"rs-5611796","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}