The Impact of Umbilical Cord Blood Platelet Lysate on Human corneal Endothelium During Organ Culture

The Impact of Umbilical Cord Blood Platelet Lysate on Human corneal Endothelium During Organ Culture | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The Impact of Umbilical Cord Blood Platelet Lysate on Human corneal Endothelium During Organ Culture Ivana Vidović, Marta Himelreich-Perić, Branka Golubić Ćepulić, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9427831/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Purpose To evaluate whether umbilical cord blood platelet lysate (UCB-PL) can preserve the morphology and function of human corneal endothelial cells during organ culture and serve as a xeno-free alternative to fetal bovine serum (FBS). Methods Paired human donor corneas were cultured for 28 days in α-MEM–based medium supplemented with either 2% FBS or 2% UCB-PL. Endothelial morphology, cell density, viability, and mosaic regularity were assessed at predefined time points. Metabolic activity was evaluated by measuring pH, glucose, and lactate concentrations in the culture media. Endothelial integrity, pump function, apoptosis, and proliferation were analyzed by whole-mount immunofluorescence staining for ZO-1, Na⁺/K⁺-ATPase, caspase-3, and Ki-67. Results Corneas stored in UCB-PL–supplemented medium demonstrated endothelial morphology, cell density decline, and viability comparable to those observed in FBS-supplemented medium throughout the culture period. No significant differences were detected between groups at any time point. Metabolic analysis showed sustained glucose availability and expected lactate accumulation in both media, without evidence of nutrient depletion. Immunofluorescence confirmed preserved endothelial junctional organization and pump protein expression, with no signs of endothelial apoptosis or proliferation. Conclusion UCB-PL preserves human corneal endothelial cell morphology and function during organ culture at levels equivalent to those achieved with FBS. These findings support the use of UCB-PL as a viable xeno-free alternative for corneal storage media, with potential implications for animal-free eye banking and corneal transplantation. cornea organ culture serum-free media umbilical cord blood platelet lysate Figures Figure 1 Figure 2 Figure 3 Figure 4 Key messages What is known: Fetal bovine serum (FBS) is a standard supplement in human corneal organ culture, but its animal origin presents regulatory and ethical limitations. What is new: • Umbilical cord blood platelet lysate (UCB-PL) maintains human corneal endothelial cell morphology and function at levels comparable to those achieved with FBS during in vitro organ culture. • Analysis of metabolic parameters and immunofluorescence markers (ZO-1, Na⁺/K⁺-ATPase, caspase-3, Ki67) demonstrates that UCB-PL preserves endothelial cell viability without evidence of nutrient depletion. • UCB-PL serves as a viable non-animal alternative for corneal storage media, supporting organ culture and potentially enabling transplantation without animal-derived components. 1. Introduction The success of corneal transplantation depends on the availability of high-quality donor tissue and effective storage methods that preserve the morphological and functional integrity of the corneal endothelium. In European eye banking practice, corneal organ culture using a basic culture medium supplemented with 2–10% fetal bovine serum (FBS) allows storage at + 31–37°C for up to seven weeks ( 1 , 2 ). FBS has long been used as a growth supplement in human cell culture due to its high content of biologically active factors that support cell survival and function ( 3 ). Despite its widespread use, FBS presents several limitations for clinical application. As a xenogeneic product, it carries potential risks such as alloimmunization to animal antigens and transmission of infectious agents. In addition, batch-to-batch variability related to season, geographic origin, and manufacturing processes may affect reproducibility. Ethical concerns regarding the use of bovine fetuses further contribute to the demand for alternative supplements ( 3 , 4 ). These limitations have driven ongoing efforts to develop serum-free or xeno-free organ culture media for human donor corneas intended for clinical use. Platelets play a central role in hemostasis, wound healing, and tissue regeneration ( 5 ). Their α-granules contain a broad range of growth factors and cytokines that can be released either physiologically during clot formation or artificially through platelet lysis induced by freeze–thaw cycles, sonication, or chemical treatment. Freeze–thaw cycling represents the most commonly used, efficient, and economical method for platelet lysis and growth factor release, resulting in platelet lysate. Consequently, human platelet lysate (hPL) has emerged as a promising animal-free growth supplement, rich in trophic factors and often produced from expired platelet concentrates, making it a potential substitute for recombinant growth factors or animal-derived products in vitro and in vivo ( 6 ). However, the routine use of peripheral blood-derived hPL is limited by the availability of platelet concentrates for laboratory and clinical applications ( 6 ). Umbilical cord blood (UCB), collected by venipuncture of umbilical cord vessels after delivery, is a well-established source of hematopoietic stem and progenitor cells and has been widely used in transplantation for hematological malignancies, immunodeficiencies, and metabolic disorders. However, approximately 80% of collected UCB units are deemed unsuitable for allogeneic transplantation due to insufficient cellular content. Despite this limitation, these units retain regenerative and anti-inflammatory properties, exhibit low immunogenicity, and carry a reduced risk of disease transmission ( 7 , 8 ). As such, UCB unsuitable for transplantation may represent a valuable alternative source for the production of umbilical cord blood platelet lysate (UCB-PL). UCB-PL has been successfully applied in regenerative medicine and in vitro cell culture, particularly for the expansion and proliferation of mesenchymal stromal cells ( 9 – 12 ). In ophthalmology, UCB-derived blood products have been used for the treatment of ocular surface disorders, including dry eye disease, chemical burns, and corneal ulcers, owing to their high concentration of growth factors involved in cell migration, proliferation, and tissue repair ( 9 , 13 ). These properties suggest that UCB-PL may be suitable for maintaining corneal integrity and supporting the biological characteristics of the human corneal endothelium during organ culture. The aim of this study was to evaluate and compare the effects of UCB-PL and standard FBS supplementation on the biological characteristics of human corneal endothelium during 28 days of organ culture, as well as to assess the feasibility of preserving human donor corneas in a xeno-free culture medium suitable for clinical eye banking and corneal transplantation. 2. Methods 2.1. Materials This prospective study included 14 human corneas obtained from seven donors whose tissues were deemed unsuitable for clinical transplantation. The study protocol was approved by the Institutional Review Board of the University Hospital Centre Zagreb (02/21 AG, 20 December 2021). Following procurement, corneas were stored in hypothermic preservation medium (Eusol-C, Alchimia, Italy) at + 4°C. Initial corneal quality assessment was performed in accordance with standard eye bank procedures and included slit-lamp biomicroscopy (BQ 900-LED, Haag-Streit International, Germany) and specular microscopy (EKA-10, Konan Medical, Japan) ( 14 ). Endothelial cell density (ECD) was determined using the center method, with at least 60 cells counted in a minimum of three different endothelial areas. ECD was calculated using the specular microscope software (KSS-EB10, Konan Medical, Japan). If the difference between counts exceeded 300 cells/mm², additional endothelial cells were counted. Macroscopic evaluation included assessment of medium turbidity and color, as well as the shape and size of the corneoscleral ring. Corneas with an ECD between 2000 and 2200 cells/mm², a coefficient of variation (CV) of cell size between 0.20 and 0.30, and more than 50% hexagonal endothelial cells were included in the study. 2.2. Study design The study was conducted in two phases. In the first phase, the feasibility of producing UCB-PL from individual UCB units that did not meet transplantation criteria was evaluated. In the second phase, a corneal organ culture medium supplemented with UCB-PL was developed and its effects on human corneal tissue were compared with those of the standard FBS-supplemented medium during a 28-day organ culture period at + 31°C. 2.2.1. Preparation of umbilical cord blood platelet lysate UCB-PL was prepared from individual donated UCB units (n = 10) that did not meet criteria for hematopoietic stem cell transplantation ( 15 ). Inclusion criteria for UCB units were a total nucleated cell count < 14 × 10⁸/L, volume ≥ 100 mL, and platelet count ≥ 150 × 10⁹/L. Written informed consent for UCB donation was obtained from all donors prior to collection, and serological screening was performed in accordance with established guidelines ( 16 ). Production of UCB-PL was performed in two steps. First, UCB platelet concentrates (UCB-PC) were prepared using a double-spin centrifugation protocol. Second, platelet lysate was generated by freeze–thaw cycling (− 80°C / +37°C), as previously described ( 10 ). A closed bag system (BioNest-CB, Meditalia, Italy) was used for UCB-PC production, yielding platelet concentrations ranging from 800 to 1200 × 10⁹/L ( 10 ). Initial quality control of UCB units included complete blood count analysis using a hematology analyzer (ADVIA 2120i, Siemens, Germany) and volume determination. The manufacturing process was monitored by weighing blood component bags (PGL 3002, Adam Equipment Co. Ltd., United Kingdom) and calculating weight and volume, followed by complete blood count analysis (ADVIA 2120i, Siemens, Germany). Component weights were calculated using specific gravities defined in the Standards for Blood Banks and Transfusion Services ( 17 ). The absolute number of total nucleated cells (TNCs) was calculated using the following formula: TNC × 10⁸ = white blood cell count (×10⁹/L) × collected blood volume (mL) ÷ 100 2.2.2. Organ culture media Two organ culture media were prepared: one supplemented with 2% FBS and the other with 2% UCB-PL. Both media were filtered through 0.2 µm filters (TPP Syringe Filters 99722, Sarstedt, Germany), aliquoted into 100 mL glass vials, and stored at − 20°C. The basic organ culture medium (1000 mL) consisted of α-minimum essential medium (α-MEM; Macopharma, France), supplemented with 2 mM L-glutamine and a penicillin–streptomycin–amphotericin B solution containing 100 IU/mL penicillin G, 0.1 mg/mL streptomycin, and 0.25 µg/mL amphotericin B (Sigma-Aldrich, Germany), following the protocol described by Armitage et al. ( 18 ). For the FBS-supplemented medium, 2% FBS (Sigma-Aldrich, Germany) was added to the basic medium. For the UCB-PL–supplemented medium, 2% UCB-PL derived from a single UCB donor and 2 IU/mL heparin (Heparin 25,000 IU/mL, Belupo, Croatia) were added. 2.2.3. Organ culture of human corneas After initial quality assessment, paired corneas from the same donor were assigned to different culture conditions. The right cornea was stored in medium supplemented with 2% FBS (n = 7), and the left cornea was stored in medium supplemented with 2% UCB-PL (n = 7). All corneas were cultured for 28 days at + 31°C in an incubator (Binder GmbH, Germany). 2.2.4. Assessment of corneal quality during organ culture Corneal quality during organ culture was evaluated on days 0, 7, 14, 21, and 28 using an inverted microscope (Axio Vert.A1, Zeiss, Germany). Endothelial assessment included evaluation of endothelial cell density, mosaic regularity, intercellular space swelling, and endothelial cell viability ( 14 ). Intercellular spaces were visualized using a 1.4% sucrose solution (SR-S, Alchimia, Italy). Endothelial cell viability was assessed by 0.25% trypan blue staining (TB-S, Alchimia, Italy), and trypan blue–positive cells were expressed as a percentage of total ECD. Corneal quality based on ECD was classified as follows: Excellent: > 3000 cells/mm² Very good: 2601–3000 cells/mm² Good: 2301–2600 cells/mm² Sufficient: 2001–2300 cells/mm² Poor: < 2000 cells/mm² Endothelial viability was calculated as the percentage of viable cells relative to total ECD. After organ culture, an endothelial cell mortality rate between 2% and 5% was considered acceptable ( 32 ). Intercellular space swelling was graded as regular, mild, moderate, or severe. Cell size and shape variation were assessed, and endothelial mosaic appearance was classified as regular, mildly pleomorphic, moderately pleomorphic, or severely pleomorphic. Metabolic parameters, including pH, glucose, and lactate concentrations, were measured in 1.8 mL medium samples using a GEM Premier 5000 analyzer (Instrumentation Laboratory, USA). On day 28, histological analysis of corneal endothelium was performed, and protein expression of ZO-1, Na⁺/K⁺-ATPase, caspase-3, and Ki-67 was evaluated by whole-mount immunofluorescence staining ( 19 , 20 ). Qualitative analysis of ZO-1 and Na⁺/K⁺-ATPase expression was performed on seven corneas stored in each medium, in technical duplicates, using a confocal microscope (Olympus FV100, 40× objective). Images were analyzed using Fiji/ImageJ software, and protein expression was assessed descriptively. 2.2.5. Statistical analysis Categorical variables are presented as absolute and relative frequencies. Normality of continuous variables was assessed using the Shapiro–Wilk test; however, due to the small sample size, non-parametric statistical methods were applied. Paired analyses were performed to account for the paired-cornea study design. Continuous variables are presented as medians with interquartile ranges. Differences between groups were analyzed using the Wilcoxon signed-rank test, with Hodges–Lehmann estimates of median differences and corresponding 95% confidence intervals. All tests were two-sided, with statistical significance set at α = 0.05. Statistical analysis was performed using MedCalc® Statistical Software version 23.3.4 (MedCalc Software Ltd., Ostend, Belgium; 2025). 3. Results 3.1. Corneal donor demographics The study included 14 corneas obtained from seven donors whose tissues were deemed unsuitable for clinical transplantation. The median donor age was 71 years (range: 69–73 years). The most frequent cause of death was neoplastic disease. The mean postmortem interval, defined as the time from circulatory arrest to tissue procurement, was 14 hours and 30 minutes. Donor characteristics are summarized in Table 1 . Table 1 Characteristics of cornea donors (n = 7) ID number of the donor Sex Age (yrs) Cause of death Type of donor Recoverd tissue Reasons for rejection for clinical use Postmortem interval (hh:mm) A M 64 Acute myocardial infarction DCD cornea results of communicable disease testing 12:08 B M 69 Aortic stenosis DCD cornea results of communicable disease testing 17:55 C Ž 68 Pancreatic cancer DCD cornea results of communicable disease testing 23:20 D Ž 71 Breast cancer DCD cornea results of communicable disease testing 19:15 E Ž 54 Subarachnoid hemorrhage DBD eye bulb medical history of the donor 14:30 F Ž 73 Breast cancer DCD cornea medical history of the donor 12:10 G M 80 Diseases of the respiratory system DCD cornea results of communicable disease testing 11:38 Median (range) n.a 71 (69–73) n.a n.a n.a n.a 14:30 (12:08–23:20) DBD – donor after brain death , DCD - donor after circulatoy death , postmortem interval - time from the death to tissue collection, n.a- not applicable 3.2. Umbilical cord blood platelet lysate characteristics Ten umbilical cord blood units were used for UCB-PL production. The median UCB volume was 105 mL (IQR 91.4–118.3), with a median absolute platelet count of 22.2 × 10⁹ (IQR 17.3–33.5). The median absolute leukocyte count was 1 × 10⁹ (IQR 0.8–1.3), and the median absolute erythrocyte count was 0.4 × 10¹² (IQR 0.3–0.4). Following double-spin centrifugation, the median volume of UCB platelet concentrate was 8.4 mL (IQR 7.12–11.91), with a median absolute platelet count of 7.3 × 10⁹ (IQR 4.66–13.51). Leukocyte contamination was negligible, with a median absolute leukocyte count of 0 × 10⁹ (IQR 0–0.01). UCB and UCB-PC characteristics are presented in Table 2 . Table 2 Characteristics of umbilical cord blood, platelet rich plasma and platelet concentrate (n = 10) Preparation Median (IQR) Volume (mL) Platelets (10 9 ) Leukocytes (10 9 ) Erythrocytes (10 12 ) Hemoglobin (g/L) UCB 105 (91,4–118,3) 22,2 ( 17 , 3 – 33 , 5 ) 1 (0,8 − 1,3) 0,4 (0,3 − 0,4) 106,5 (104–119,5) PRP 40,9 ( 36 , 7 – 43 , 6 ) 12 ( 7 , 8 – 17 , 5 ) 0 (0–0) 0 (0–0) 1 (0–1) UCB-PC 8,4 (7,12 − 11,91) 7,3 (4,66 − 13,51) 0 (0–0,01) 0,1 (0,07 − 0,14) 1 (0–2) UCB-PL n.a. 948 (615–1228) 0,58 (0,32–0,93) n.a. n.a. UCB - umbilical cord blood , PRP - platelet rich plasma , UCB-PC - umbilical cord blood platelate concentrat, n.a.-not applicable 3.3. Corneal quality assessment before organ culture Baseline quality assessment of all corneas included in the study demonstrated a median endothelial cell density of 2417 cells/mm² (IQR 2304–3067). The median coefficient of variation of cell size was 35 (IQR 32–39), and the median percentage of hexagonal endothelial cells was 59% (IQR 56.8–63.3). No statistically significant differences were observed between paired corneas prior to organ culture with respect to endothelial parameters, corneoscleral ring morphology, or slit-lamp findings. Baseline specular microscopy results are shown in Table 3 . Table 3 Cornea endothelial cell density during organ culture in media with fetal bovine serum and umbilical cord blood platelet lysate Days of organ culture Endothelial cell density Median (interquartile range) Estimate of the median difference† 95% CI of difference P* Media with FBS Media with UCB-PL 0 day 2400 (2312,5–2658,3) 2400 (2162–2850) -16,9 -133,3 to 133,3 0,60 7 day 2267 (2178,3–2541,7) 2309 (2125–2533) -33 -133 to 66,3 0,24 14 day 2133 (1988–2266) 2025 (1925–2138) -120,8 -273 to 25 0,08 21 day 1750 (1550–1967) 1775 (1575–1900) 6,3 -58,3 to 58,3 0,92 28 day 1467 (1225–1725) 1300 (1195–1463) -158 -367 to 8 0,11 * Wilcoxon test; †Hodges Lehmann median difference FBS- fetal bovine serum, UCB-PL umbilical cord blood platelet lysat, CI - Confidence Interval 3.4. Corneal quality during organ culture During organ culture, endothelial cell density gradually decreased in both media. No statistically significant differences in ECD were observed between corneas stored in FBS- and UCB-PL–supplemented media at any evaluated time point (days 0, 7, 14, 21, and 28) (Table 3 ; Figs. 1 and 2 ). Endothelial cell viability was high in both groups throughout the culture period. Median viability was 99.8% (IQR 99.6–99.9) at baseline and 100% (IQR 99.8–100) after 28 days, with no statistically significant differences between media at any time point. Endothelial mosaic architecture remained regular in the majority of corneas until day 7 in both media. From day 14 onward, mild to moderate pleomorphism was observed; however, severe pleomorphism was not detected in any cornea. Similarly, no statistically significant differences were identified between groups with respect to intercellular space swelling at any time point. 3.5. Metabolic changes during organ culture Glucose concentrations decreased progressively in both culture media over time. Statistically significant differences between media were observed at baseline (P = 0.02), day 7 (P = 0.02), day 14 (P = 0.04), and day 21 (P = 0.02), with higher glucose levels consistently measured in the UCB-PL–supplemented medium (Table 4 ). Table 4 Glucose concentration ​​(mmol/L) ​​during the organ culture Days of organ culture Glucose concentration (mmol/L) Median (interquartile range) Estimate of the median difference† 95% CI of difference P* Media with FBS Media with UCB-PL 0 day 5,3 (4,97–5,38) 5,9 (5,6–6,08) 0,48 0,3 to 1,2 0,02 7 day 3,6 ( 3 , 43 – 3 , 8 ) 4,1 (3,85–4,63) 0,55 0,2 to 0,9 0,02 14 day 2,6 (2,35–25,78) 2,8 (2,63–3,0) 0,30 0 to 0,55 0,04 21 day 1,4 (1,2–1,68) 1,7 (1,53–2,05) 0,30 0,1 to 0,5 0,02 28 day 0,8 (0,4–0,95) 0,8 (0,6–1,13) 0,13 -0,1 to 0,4 0,17 * Wilcoxon test; †Hodges Lehmann median difference, FBS-fetal bovine serum, UCB-PL umbilical cord blood platelet lysat, CI - Confidence Interval. Bold values denote statistical significance. Lactate concentrations increased in both media during organ culture. Statistically significant differences were detected on day 7 (P = 0.03) and day 21 (P = 0.04). Lactate levels were higher in the FBS-supplemented medium at baseline and day 7, whereas from day 14 onward, higher lactate concentrations were observed in the UCB-PL–supplemented medium (Table 5 ). Table 5 Lactate concentration ​​(mmol/L) during the organ culture Days of organ culture Lactate concentration (mmol/L) Median (interquartile range) Estimate of the median difference† 95% CI of difference P* Media with FBS Media with UCB-PL 0 day 0,4 (0,3–0,4) 0,3 (0,3–0,3) -0,1 -0,2 do 0 0,06 7 day 2,5 (2,2–3,03) 2,3 ( 2 , 1 – 2 , 6 ) -0,2 -0,5 do -0,05 0,03 14 day 4,4 ( 3 , 9 – 5 , 4 ) 4,5 (4,3–5,0) 0,08 -24,5 do 0,5 0,87 21 day 6,6 ( 5 , 8 – 7 , 3 ) 7,0 ( 6 , 1 – 7 , 6 ) 0,28 0 do 0,5 0,04 28 day 7,8 ( 7 , 1 – 8 , 2 ) 8,0 (7,0–8,9) 0,43 -0,35 do 0,7 0,24 CI - Confidence Interval. Bold values denote statistical significance; * Wilcoxon test; †Hodges Lehmann median difference, ​ FBS-fetal bovine serum, UCB-PL umbilical cord blood platelet lysate Medium pH values decreased over time in both groups; however, no statistically significant differences between media were observed at any time point. 3.6. Pathohistological analysis of corneal endothelium Whole-mount immunofluorescence analysis performed after 28 days of organ culture demonstrated preserved endothelial protein expression in both media. ZO-1 exhibited a characteristic continuous zigzag pattern along intercellular junctions, with interruptions at Y-shaped junctions between adjacent cells. Na⁺/K⁺-ATPase expression was localized to the lateral membranes of corneal endothelial and epithelial cells. Ki-67–positive nuclei were detected exclusively in the epithelial layer, indicating proliferative activity, whereas Ki-67 expression was absent in corneal endothelial cells. Cleaved caspase-3 expression was observed in a small number of epithelial cells but was not detected in the corneal endothelium. Representative images demonstrating Na⁺/K⁺-ATPase and ZO-1 expression and their co-localization with nuclear staining are shown in Figs. 3 and 4 . 4. Discussion The primary objective of corneal organ culture is to preserve the structural and functional integrity of the corneal endothelium under conditions that closely resemble the physiological environment ( 14 ). In European eye banking practice, donor corneas are commonly stored in MEM-based media supplemented with fetal bovine serum (FBS) at concentrations ranging from 2% to 10%, in accordance with recommendations of the European Eye Bank Association ( 21 ). Although FBS contains a broad spectrum of biologically active components that support cell survival, its xenogeneic origin raises concerns regarding batch variability, immunogenicity, and the potential transmission of infectious agents, including prions, viruses, and mycoplasmas ( 22 ). These limitations have prompted increasing interest in xeno-free alternatives such as human platelet lysate. Umbilical cord blood (UCB) represents a unique and underutilized source of biologically active factors. In contrast to peripheral blood-derived human platelet lysate (hPL), UCB-derived platelet lysate contains a distinct growth factor profile and exhibits low immunogenicity and a favorable safety profile ( 23 , 24 ). In the present study, we evaluated the impact of umbilical cord blood platelet lysate (UCB-PL) on human corneal endothelial cells during organ culture and assessed its suitability as a xeno-free substitute for FBS. Our results demonstrate that UCB-PL supports endothelial cell survival and function during 28 days of organ culture at a level comparable to standard FBS-supplemented medium. Endothelial cell density (ECD) decreased progressively in both media, consistent with previously reported endothelial cell loss during organ culture storage ( 28 , 29 ). Although a slightly greater reduction in ECD was observed in corneas stored in UCB-PL–supplemented medium, the difference was not statistically significant at any time point and did not adversely affect endothelial morphology or viability. These findings are clinically relevant, as corneal graft survival and postoperative transparency are strongly dependent on endothelial cell count and function ( 14 , 26 , 27 ). Endothelial cell viability remained above 99% throughout the culture period in both groups, well within the accepted threshold for clinical use ( 31 , 32 ). Morphological evaluation further confirmed preserved endothelial mosaic architecture, with regular cell patterns observed until day 7 and only mild to moderate pleomorphism developing thereafter. This pattern aligns with previous observations that endothelial cells compensate for cell loss through enlargement and migration rather than proliferation, given their post-mitotic nature ( 26 , 27 , 20 ). The absence of Ki-67 expression in endothelial cells in both media is consistent with this established biological behavior ( 47 , 40 ). Metabolic analysis revealed progressive glucose consumption and lactate accumulation in both culture media, reflecting sustained metabolic activity of the corneal endothelium during storage ( 36 , 37 ). Notably, glucose concentrations were consistently higher in the UCB-PL–supplemented medium at several time points, suggesting adequate nutrient availability throughout the culture period. Lactate accumulation and gradual pH reduction were observed in both media, in line with previous reports describing anaerobic glycolysis under in vitro storage conditions ( 36 – 38 ). Given the known tolerance of corneal endothelial cells to extracellular pH fluctuations within the range observed in this study, these metabolic changes are unlikely to compromise endothelial viability ( 14 ). Whole-mount immunofluorescence analysis provided further evidence of preserved endothelial function in both storage conditions. ZO-1 localization along intercellular junctions exhibited a characteristic zigzag pattern with interruptions at Y-shaped junctions, consistent with the known organization of tight junctions in the corneal endothelium ( 41 , 42 , 20 ). Proper localization of Na⁺/K⁺-ATPase along endothelial cell membranes was also maintained, supporting intact pump function essential for stromal deturgescence and corneal transparency ( 43 , 28 ). Importantly, no caspase-3 expression was detected in endothelial cells, indicating that apoptosis was not a major mechanism of endothelial cell loss during storage, in agreement with previous studies ( 28 , 44 , 45 ). An important practical aspect of this study is the use of UCB units that were unsuitable for hematopoietic stem cell transplantation. Repurposing these otherwise discarded units for UCB-PL production offers a sustainable and ethically favorable approach for generating growth factor–rich supplements for tissue banking applications ( 25 ). Nevertheless, several limitations should be acknowledged, including the small sample size, the use of a single UCB-PL concentration, and the restriction of the observation period to 28 days. Further studies are warranted to evaluate dose-dependent effects of UCB-PL, extend storage duration, and quantify growth factor composition in organ culture media. In conclusion, this study demonstrates that UCB-PL preserves the structural and functional integrity of the human corneal endothelium during organ culture at levels comparable to those achieved with FBS. These findings support the feasibility of implementing UCB-PL as a xeno-free supplement in corneal storage media, with potential benefits for standardized, animal-free eye banking and corneal transplantation. Conclusions This study demonstrates that supplementation of corneal organ culture medium with UCB-PL preserves the morphological and functional characteristics of the human corneal endothelium during in vitro storage. Endothelial cell viability, morphology, metabolic activity, and key functional protein expression were maintained at levels comparable to those observed with standard fetal bovine serum–supplemented medium. Given the critical role of endothelial cell integrity in determining corneal graft survival and postoperative transparency, these findings indicate that human donor corneas can be successfully preserved in a xeno-free storage medium without compromising endothelial quality. The use of UCB-PL offers a safe and effective alternative to animal-derived supplements and may contribute to reducing regulatory, ethical, and biological risks associated with FBS. Future studies should focus on standardizing UCB-PL production protocols, evaluating different supplement concentrations, extending storage durations, and characterizing growth factor composition in organ culture media. Such efforts may further support the implementation of UCB-PL–based, animal-free storage media in clinical eye banking and corneal transplantation. Declarations The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper. Conflict of interest All authors certify that they have no affiliations with or involvement in any organization or entity with any financial or non-financial interest in the subject matter or materials discussed in this manuscript. Competing interests The authors declare that they have no competing interests. Funding This research was funded by the Scientific Center of Excellence for Reproductive and Regenerative Medicine, Republic of Croatia, and by the European Union through the European Regional Development Fund, under grant agreement No. KK.01.1.1.01.0008, project „Reproductive and Regenerative Medicine - Exploring New Platforms and Potentials”. Ethical approval The Institutional Review Board of the University Hospital Centre Zagreb approved the protocol of this study, and it conformed to the tenets of the Declaration of Helsinki (02/21 AG from 20th December 2021) Informed consent Written informed consent was obtained from all the donor corneas that were included in this study Acknowledgement This research was funded by the Scientific Center of Excellence for Reproductive and Regenerative Medicine, Republic of Croatia, and by the European Union through the European Regional Development Fund, under grant agreement No. KK.01.1.1.01.0008, project „Reproductive and Regenerative Medicine - Exploring New Platforms and Potentials”. Author Contributions Conceptualization: Assoc. Prof. Branka Golubić Ćepulić MD, PhD, Mirna Golemović, MSc in Biology, PhD, Ivana Vidović, MD, PhD Methodology: Assoc. Prof. Branka Golubić Ćepulić MD, PhD, Mirna Golemović, MSc in Biology, PhD, Prof. Davor Ježek, MD, PhD; Assoc. Prof. Ines Bojanić, Ivana Vidović, MD, PhD Investigation: Ivana Vidović, MD, PhD; Marta Himelreich-Perić, MD, PhD Data curation: Ivana Vidović, MD, PhD; Marta Himelreich-Perić, MD, PhD Formal analysis: Assoc. Prof. Kristina Kralik, PhD Writing – original draft preparation: Ivana Vidović, MD, PhD; Marta Himelreich-Perić, MD, PhD Writing – review and editing: Ivana Vidović, MD, PhD; Marta Himelreich-Perić, MD, PhD Visualization: Ivana Vidović, MD, PhD; Marta Himelreich-Perić, MD, PhD Supervision: Assoc. Prof. Branka Golubić Ćepulić; Mirna Golemović, MSc in Biology, PhD, Prof. Davor Ježek, MD, PhD; Assoc. Prof. Ines Bojanić, MD, PhD All authors have read and agreed to the published version of the manuscript. References Møller-Pedersen T, Hartmann U, Møller H, Ehler N, Engelmann K. Evaluation of potential organ culture media for eye banking using human corneas. Br J Ophthalmol. 2001;85:1075–9. doi: 10.1136/bjo.85.9.1075 Ehlers H, Ehlers N, Hjortdal JO. 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Trends Biotechnol. 2020;38(1):13–23. doi: 10.1016/j.tibtech.2019.06.002 Bień A, Vermeulen J, Bączek B, Pięta M, Pięta B. Cord blood banking: balancing hype and hope in stem cell therapy. Eur J Midwifery. 2024;8:192930. doi: 10.18332/ejm/192930 Wang Q, Liu Y, Gao Y, Zhang M, Yang L, Hu J, Ren Y, Zhou Q, Zhu M. Comparison of the structural integrity and quality of corneal endothelium stored in organ culture storage medium versus Eusol-C. Cell Tissue Bank. 2024;25(3):839–50. doi: 10.1007/s10561-024-10138-z Christou I, Mallis P, Michalopoulos E, Chatzistamatiou T, Mermelekas G, et al. Evaluation of peripheral blood and cord blood platelet lysates in isolation and expansion of multipotent mesenchymal stromal cells. Bioengineering (Basel). 2018;5(1):19. doi: 10.3390/bioengineering5010019 Samarkanova D, Rodríguez L, Vives J, Coll R, Tahull E, Azqueta C, et al. Cord blood-derived platelet concentrates as starting material for new therapeutic blood components prepared in a public cord blood bank: from product development to clinical application. Blood Transfus. 2020;18(3):208–16. doi: 10.2450/2020.0305-19 Giannaccare G, Carnevali A, Senni C, Logozzo L, Scorcia V. Umbilical cord blood and serum for the treatment of ocular diseases: a review. Ophthalmol Ther. 2020;9:235–48. doi: 10.1007/s40123-020-00239-9 Valentini CG, Nuzzolo ER, Bianchi M, Orlando N, Iachininoto MG, Pinci P, et al. Cord blood platelet lysate: in vitro evaluation to support the use in regenerative medicine. Mediterr J Hematol Infect Dis. 2019;11(1):e2019021. doi: 10.4084/MJHID.2019.021 Tovar A, White I, Sabater A. Use of acellular umbilical cord-derived tissues in corneal and ocular surface diseases. Medicines (Basel). 2021;8(2):12. doi: 10.3390/medicines8020012 Jirsova K. Light and specular microscopy of the cornea. Cham: Springer; 2017. doi: 10.1007/978-3-319-48845-5 Golubić Ćepulić B, Bojanić I, Mazić S. Banke krvi iz pupkovine. Acta Med Croatica. 2009;63(3):245–50. European Directorate for the Quality of Medicines & HealthCare (EDQM). Guide to the preparation, use and quality assurance of blood components. 5th ed. Strasbourg: Council of Europe Publishing; 2020. Brecher ME, et al. Technical manual. 15th ed. Bethesda (MD): AABB; 2005. Armitage W, Jones M, Zambrano I, Carley F, Tole D. The suitability of corneas stored by organ culture for penetrating keratoplasty and influence of donor and recipient factors on 5-year graft survival. Invest Ophthalmol Vis Sci. 2014;55(2):784–91. doi: 10.1167/iovs.13-13386 Forest F, Thuret G, Gain P, Dumollard J, Peoc’h M, Perrache C, et al. Optimization of immunostaining on flat-mounted human corneas. Mol Vis. 2015;21:1345–56. He Z, Forest F, Gain P, Rageade D, Bernard A, Acquart S, et al. 3D map of the human corneal endothelial cell. Sci Rep. 2016;6:29047. doi: 10.1038/srep29047 European Eye Bank Association. Annual directory of the European Eye Bank Association. 28th ed. 2020. Immalaraju S, Goyal S, Jonnalagadda R. Towards the standardization of human platelet lysate production and its comparison to fetal bovine serum for human hematopoietic cell culture: a scoping review. Front Toxicol. 2025;7:1496231. doi: 10.3389/ftox.2025.1496231 Huang L, Critser PJ, Grimes BR, Yoder MC. Human umbilical cord blood plasma can replace fetal bovine serum for in vitro expansion of functional human endothelial colony forming cells. Cytotherapy. 2011;13(6):712–21. doi: 10.3109/14653249.2010.548380 Petsoglou C, Wen L, Hoque M, Zhu M, Valtink M, Sutton G, You J. Effects of human platelet lysate on the growth of cultured human corneal endothelial cells. Exp Eye Res. 2021;208:108613. doi: 10.1016/j.exer.2021.108613 Rebulla P, Pupella S, Santodirocco M, Greppi N, Villanova I, Buzzi M, et al. Multicentric standardisation of the clinical grade procedure for the preparation of allogeneic platelet concentrates from umbilical cord blood. Blood Transfus. 2016;14(1):73–9. doi: 10.2450/2015.0122-15 He Z, Okumura N, Sato M, Komori Y, Nakahara M, Gain P, Koizumi K, Thuret G. Corneal endothelial cell therapy: feasibility of cell culture from corneas stored in organ culture. Cell Tissue Bank. 2021;22(4):551–62. doi: 10.1007/s10561-021-09918-8 Nejepinska J, Juklova K, Jirsova K. Organ culture, but not hypothermic storage, facilitates the repair of the corneal endothelium following mechanical damage. Acta Ophthalmol. 2010;88(4):413–9. doi: 10.1111/j.1755-3768.2008.01490.x Crewe J, Armitage W. Integrity of epithelium and endothelium in organ cultured human corneas. Invest Ophthalmol Vis Sci. 2001;42(8):1757–61. Wang Q, Liu Y, Gao Y, Zhang M, Yang L, Hu J, Ren Y, Zhou Q, Zhu M. Comparison of the structural integrity and quality of corneal endothelium stored in organ culture storage medium versus Eusol-C. Cell Tissue Bank. 2024;25(3):839–50. doi: 10.1007/s10561-024-10138-z Parekh M, Peh G, Mehta JS, Ahmad S, Ponzin D, Ferrari S. Effects of corneal preservation conditions on human corneal endothelial cell culture. Exp Eye Res. 2019;179:93–101. doi: 10.1016/j.exer.2018.11.007 Gain P. Value of two mortality assessment techniques for organ cultured corneal endothelium: trypan blue versus TUNEL technique. Br J Ophthalmol. 2002;86(3):306–10. doi: 10.1136/bjo.86.3.306 Builles N, Kodjikian L, Burillon C, Damour O. Major endothelial loss from corneas in organ culture: importance of second endothelial count. Cornea. 2006;25(7):815–20. doi: 10.1097/01.ico.0000230253.62730.85 Filev F, Stein M, Schultheiss M, Fitzek ADE, Feuerstake J, Engel O, Hellwinkel OJC. Semiautomatic assessment of endothelial density and morphology in organ-cultured corneas: potential predictors for transplantation suitability and clinical outcome? Graefes Arch Clin Exp Ophthalmol. 2023;261:2593–602. doi: 10.1007/s00417-023-06079-0 Lužnik Z, Sun Z, Yin J, Benetz BA, Lass JH, Dana R. A standardized methodology for longitudinal assessment of corneal endothelial morphometry in eye-banked corneas. J Biol Methods. 2019;6(4):e120. doi: 10.14440/jbm.2019.304 Yazdani M. Tear film lipid layer and corneal oxygenation: a new function? Eye (Lond). 2023;37(17):3534–41. doi: 10.1038/s41433-023-02557-1 Redbrake C, Salla S, Frantz A, Reim M. Metabolic changes of the human donor cornea during organ culture. Acta Ophthalmol Scand. 1999;77(3):266–72. doi: 10.1034/j.1600-0420.1999.770304.x Li S, Shyam R, Ogando DG, Bonanno JA. Bicarbonate activates glycolysis and lactate production in corneal endothelial cells by increased pHi. Exp Eye Res. 2020;199:108193. doi: 10.1016/j.exer.2020.108193 Pang K, Lennikov A, Yang M. Hypoxia adaptation in the cornea: current animal models and underlying mechanisms. Anim Model Exp Med. 2021;4(4):300–10. doi: 10.1002/ame2.12192 Tsai MC, Kureshim A, Daniels JT. Establishment of an ex vivo human corneal endothelium wound model. Transl Vis Sci Technol. 2025;14(1):24. doi: 10.1167/tvst.14.1.24 He Z, Campolmi N, Ha Thi B, Dumollard J, Peoc’h M, Garraud O, et al. Immunolocalization of cell cycle proteins in corneal endothelial cells. Mol Vis. 2011;17:3494–511. Srinivas SP. Dynamic regulation of barrier integrity of the corneal endothelium. Optom Vis Sci. 2010;87(4):E239-54. Klyce SD. Endothelial pump and barrier function. Exp Eye Res. 2020;198:108068. doi: 10.1016/j.exer.2020.108068 Anney P, Charpentier P, Proulx P. Influence of intraocular pressure on the expression and activity of sodium-potassium pumps in the corneal endothelium. Int J Mol Sci. 2024;25:10227. doi: 10.3390/ijms251810227 Lin W, Chen M, Cisse Y, Chen X, Bai L. Roles and mechanisms of regulated necrosis in corneal diseases: progress and perspectives. J Ophthalmol. 2022;2022:2695212. doi: 10.1155/2022/2695212 Albon J, Tullo AB, Aktar S, Boulton ME. Apoptosis in the endothelium of human corneas for transplantation. Invest Ophthalmol Vis Sci. 2000;41(10):2887–93. Dare EV, Fung CK, McCanna DJ, Subbaraman LN, Jones LW. Establishment of optimal culture media in corneal epithelial wound healing models. J Cell Biotechnol. 2022;8(1):1–12. doi: 10.3233/JCB-210039 Smeringaiova I, Utheim TP, Jirsova K. Ex vivo expansion and characterization of human corneal endothelium for transplantation: a review. Stem Cell Res Ther. 2021;12:554. doi: 10.1186/s13287-021-02611-3 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 29 Apr, 2026 Reviews received at journal 28 Apr, 2026 Reviews received at journal 20 Apr, 2026 Reviewers agreed at journal 20 Apr, 2026 Reviewers agreed at journal 19 Apr, 2026 Reviewers agreed at journal 18 Apr, 2026 Reviewers agreed at journal 17 Apr, 2026 Reviewers invited by journal 17 Apr, 2026 Editor assigned by journal 16 Apr, 2026 Submission checks completed at journal 16 Apr, 2026 First submitted to journal 15 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9427831","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":626168547,"identity":"68d85ae0-7a84-4984-9056-52a183dad4fe","order_by":0,"name":"Ivana 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13:39:56","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9427831/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9427831/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107763532,"identity":"191ba895-059e-4cff-8da5-190969212496","added_by":"auto","created_at":"2026-04-25 00:09:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":183380,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure1..png","url":"https://assets-eu.researchsquare.com/files/rs-9427831/v1/9027d8422addf5dfe09eb7d1.png"},{"id":107763531,"identity":"013247c3-6281-4197-b734-06ff4217bfb1","added_by":"auto","created_at":"2026-04-25 00:09:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":941642,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure2..png","url":"https://assets-eu.researchsquare.com/files/rs-9427831/v1/63fde2e34ba0da9276613c66.png"},{"id":107869317,"identity":"2e456604-d80e-42f1-b11b-fe4ab69dbf3f","added_by":"auto","created_at":"2026-04-27 07:36:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1498666,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure3..png","url":"https://assets-eu.researchsquare.com/files/rs-9427831/v1/43549b1b83ad33f64d3dfe4d.png"},{"id":107763534,"identity":"78e45629-54c5-4a72-aeb6-1ba76732d97f","added_by":"auto","created_at":"2026-04-25 00:09:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1666056,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure4..png","url":"https://assets-eu.researchsquare.com/files/rs-9427831/v1/0764ceae56938dcc314ec97a.png"},{"id":107871761,"identity":"a788ad09-6cfe-49a9-9e20-00ba341c4a6a","added_by":"auto","created_at":"2026-04-27 07:54:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6858027,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9427831/v1/9156783c-b40c-4ef6-a312-a071703e78b9.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eThe Impact of Umbilical Cord Blood Platelet Lysate on Human corneal Endothelium During Organ Culture\u003c/p\u003e","fulltext":[{"header":"Key messages","content":"\u003cp\u003e\u003cstrong\u003eWhat is known:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFetal bovine serum (FBS) is a standard supplement in human corneal organ culture, but its animal origin presents regulatory and ethical limitations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWhat is new:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026bull; Umbilical cord blood platelet lysate (UCB-PL) maintains human corneal endothelial cell morphology and function at levels comparable to those achieved with FBS during \u003cem\u003ein vitro\u003c/em\u003e organ culture.\u003c/p\u003e\n\u003cp\u003e\u0026bull; Analysis of metabolic parameters and immunofluorescence markers (ZO-1, Na⁺/K⁺-ATPase, caspase-3, Ki67) demonstrates that UCB-PL preserves endothelial cell viability without evidence of nutrient depletion.\u003c/p\u003e\n\u003cp\u003e\u0026bull; UCB-PL serves as a viable non-animal alternative for corneal storage media, supporting organ culture and potentially enabling transplantation without animal-derived components.\u003c/p\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eThe success of corneal transplantation depends on the availability of high-quality donor tissue and effective storage methods that preserve the morphological and functional integrity of the corneal endothelium. In European eye banking practice, corneal organ culture using a basic culture medium supplemented with 2\u0026ndash;10% fetal bovine serum (FBS) allows storage at +\u0026thinsp;31\u0026ndash;37\u0026deg;C for up to seven weeks (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). FBS has long been used as a growth supplement in human cell culture due to its high content of biologically active factors that support cell survival and function (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDespite its widespread use, FBS presents several limitations for clinical application. As a xenogeneic product, it carries potential risks such as alloimmunization to animal antigens and transmission of infectious agents. In addition, batch-to-batch variability related to season, geographic origin, and manufacturing processes may affect reproducibility. Ethical concerns regarding the use of bovine fetuses further contribute to the demand for alternative supplements (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). These limitations have driven ongoing efforts to develop serum-free or xeno-free organ culture media for human donor corneas intended for clinical use.\u003c/p\u003e \u003cp\u003ePlatelets play a central role in hemostasis, wound healing, and tissue regeneration (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Their α-granules contain a broad range of growth factors and cytokines that can be released either physiologically during clot formation or artificially through platelet lysis induced by freeze\u0026ndash;thaw cycles, sonication, or chemical treatment. Freeze\u0026ndash;thaw cycling represents the most commonly used, efficient, and economical method for platelet lysis and growth factor release, resulting in platelet lysate. Consequently, human platelet lysate (hPL) has emerged as a promising animal-free growth supplement, rich in trophic factors and often produced from expired platelet concentrates, making it a potential substitute for recombinant growth factors or animal-derived products in vitro and in vivo (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). However, the routine use of peripheral blood-derived hPL is limited by the availability of platelet concentrates for laboratory and clinical applications (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eUmbilical cord blood (UCB), collected by venipuncture of umbilical cord vessels after delivery, is a well-established source of hematopoietic stem and progenitor cells and has been widely used in transplantation for hematological malignancies, immunodeficiencies, and metabolic disorders. However, approximately 80% of collected UCB units are deemed unsuitable for allogeneic transplantation due to insufficient cellular content. Despite this limitation, these units retain regenerative and anti-inflammatory properties, exhibit low immunogenicity, and carry a reduced risk of disease transmission (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). As such, UCB unsuitable for transplantation may represent a valuable alternative source for the production of umbilical cord blood platelet lysate (UCB-PL).\u003c/p\u003e \u003cp\u003eUCB-PL has been successfully applied in regenerative medicine and \u003cem\u003ein vitro\u003c/em\u003e cell culture, particularly for the expansion and proliferation of mesenchymal stromal cells (\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). In ophthalmology, UCB-derived blood products have been used for the treatment of ocular surface disorders, including dry eye disease, chemical burns, and corneal ulcers, owing to their high concentration of growth factors involved in cell migration, proliferation, and tissue repair (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). These properties suggest that UCB-PL may be suitable for maintaining corneal integrity and supporting the biological characteristics of the human corneal endothelium during organ culture.\u003c/p\u003e \u003cp\u003eThe aim of this study was to evaluate and compare the effects of UCB-PL and standard FBS supplementation on the biological characteristics of human corneal endothelium during 28 days of organ culture, as well as to assess the feasibility of preserving human donor corneas in a xeno-free culture medium suitable for clinical eye banking and corneal transplantation.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials\u003c/h2\u003e \u003cp\u003eThis prospective study included 14 human corneas obtained from seven donors whose tissues were deemed unsuitable for clinical transplantation. The study protocol was approved by the Institutional Review Board of the University Hospital Centre Zagreb (02/21 AG, 20 December 2021). Following procurement, corneas were stored in hypothermic preservation medium (Eusol-C, Alchimia, Italy) at +\u0026thinsp;4\u0026deg;C.\u003c/p\u003e \u003cp\u003eInitial corneal quality assessment was performed in accordance with standard eye bank procedures and included slit-lamp biomicroscopy (BQ 900-LED, Haag-Streit International, Germany) and specular microscopy (EKA-10, Konan Medical, Japan) (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Endothelial cell density (ECD) was determined using the center method, with at least 60 cells counted in a minimum of three different endothelial areas. ECD was calculated using the specular microscope software (KSS-EB10, Konan Medical, Japan). If the difference between counts exceeded 300 cells/mm\u0026sup2;, additional endothelial cells were counted.\u003c/p\u003e \u003cp\u003eMacroscopic evaluation included assessment of medium turbidity and color, as well as the shape and size of the corneoscleral ring. Corneas with an ECD between 2000 and 2200 cells/mm\u0026sup2;, a coefficient of variation (CV) of cell size between 0.20 and 0.30, and more than 50% hexagonal endothelial cells were included in the study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Study design\u003c/h2\u003e \u003cp\u003eThe study was conducted in two phases. In the first phase, the feasibility of producing UCB-PL from individual UCB units that did not meet transplantation criteria was evaluated. In the second phase, a corneal organ culture medium supplemented with UCB-PL was developed and its effects on human corneal tissue were compared with those of the standard FBS-supplemented medium during a 28-day organ culture period at +\u0026thinsp;31\u0026deg;C.\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1. Preparation of umbilical cord blood platelet lysate\u003c/h2\u003e \u003cp\u003eUCB-PL was prepared from individual donated UCB units (n\u0026thinsp;=\u0026thinsp;10) that did not meet criteria for hematopoietic stem cell transplantation (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Inclusion criteria for UCB units were a total nucleated cell count\u0026thinsp;\u0026lt;\u0026thinsp;14 \u0026times; 10⁸/L, volume\u0026thinsp;\u0026ge;\u0026thinsp;100 mL, and platelet count\u0026thinsp;\u0026ge;\u0026thinsp;150 \u0026times; 10⁹/L. Written informed consent for UCB donation was obtained from all donors prior to collection, and serological screening was performed in accordance with established guidelines (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eProduction of UCB-PL was performed in two steps. First, UCB platelet concentrates (UCB-PC) were prepared using a double-spin centrifugation protocol. Second, platelet lysate was generated by freeze\u0026ndash;thaw cycling (\u0026minus;\u0026thinsp;80\u0026deg;C / +37\u0026deg;C), as previously described (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). A closed bag system (BioNest-CB, Meditalia, Italy) was used for UCB-PC production, yielding platelet concentrations ranging from 800 to 1200 \u0026times; 10⁹/L (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eInitial quality control of UCB units included complete blood count analysis using a hematology analyzer (ADVIA 2120i, Siemens, Germany) and volume determination. The manufacturing process was monitored by weighing blood component bags (PGL 3002, Adam Equipment Co. Ltd., United Kingdom) and calculating weight and volume, followed by complete blood count analysis (ADVIA 2120i, Siemens, Germany). Component weights were calculated using specific gravities defined in the Standards for Blood Banks and Transfusion Services (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe absolute number of total nucleated cells (TNCs) was calculated using the following formula:\u003c/p\u003e \u003cp\u003eTNC \u0026times; 10⁸ = white blood cell count (\u0026times;10⁹/L) \u0026times; collected blood volume (mL)\u0026thinsp;\u0026divide;\u0026thinsp;100\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2. Organ culture media\u003c/h2\u003e \u003cp\u003eTwo organ culture media were prepared: one supplemented with 2% FBS and the other with 2% UCB-PL. Both media were filtered through 0.2 \u0026micro;m filters (TPP Syringe Filters 99722, Sarstedt, Germany), aliquoted into 100 mL glass vials, and stored at \u0026minus;\u0026thinsp;20\u0026deg;C.\u003c/p\u003e \u003cp\u003eThe basic organ culture medium (1000 mL) consisted of α-minimum essential medium (α-MEM; Macopharma, France), supplemented with 2 mM L-glutamine and a penicillin\u0026ndash;streptomycin\u0026ndash;amphotericin B solution containing 100 IU/mL penicillin G, 0.1 mg/mL streptomycin, and 0.25 \u0026micro;g/mL amphotericin B (Sigma-Aldrich, Germany), following the protocol described by Armitage et al. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFor the FBS-supplemented medium, 2% FBS (Sigma-Aldrich, Germany) was added to the basic medium. For the UCB-PL\u0026ndash;supplemented medium, 2% UCB-PL derived from a single UCB donor and 2 IU/mL heparin (Heparin 25,000 IU/mL, Belupo, Croatia) were added.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.2.3. Organ culture of human corneas\u003c/h2\u003e \u003cp\u003eAfter initial quality assessment, paired corneas from the same donor were assigned to different culture conditions. The right cornea was stored in medium supplemented with 2% FBS (n\u0026thinsp;=\u0026thinsp;7), and the left cornea was stored in medium supplemented with 2% UCB-PL (n\u0026thinsp;=\u0026thinsp;7). All corneas were cultured for 28 days at +\u0026thinsp;31\u0026deg;C in an incubator (Binder GmbH, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.2.4. Assessment of corneal quality during organ culture\u003c/h2\u003e \u003cp\u003eCorneal quality during organ culture was evaluated on days 0, 7, 14, 21, and 28 using an inverted microscope (Axio Vert.A1, Zeiss, Germany). Endothelial assessment included evaluation of endothelial cell density, mosaic regularity, intercellular space swelling, and endothelial cell viability (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Intercellular spaces were visualized using a 1.4% sucrose solution (SR-S, Alchimia, Italy). Endothelial cell viability was assessed by 0.25% trypan blue staining (TB-S, Alchimia, Italy), and trypan blue\u0026ndash;positive cells were expressed as a percentage of total ECD.\u003c/p\u003e \u003cp\u003eCorneal quality based on ECD was classified as follows:\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eExcellent: \u0026gt; 3000 cells/mm\u0026sup2;\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eVery good: 2601\u0026ndash;3000 cells/mm\u0026sup2;\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eGood: 2301\u0026ndash;2600 cells/mm\u0026sup2;\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eSufficient: 2001\u0026ndash;2300 cells/mm\u0026sup2;\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003ePoor: \u0026lt; 2000 cells/mm\u0026sup2;\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eEndothelial viability was calculated as the percentage of viable cells relative to total ECD. After organ culture, an endothelial cell mortality rate between 2% and 5% was considered acceptable (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Intercellular space swelling was graded as regular, mild, moderate, or severe. Cell size and shape variation were assessed, and endothelial mosaic appearance was classified as regular, mildly pleomorphic, moderately pleomorphic, or severely pleomorphic.\u003c/p\u003e \u003cp\u003eMetabolic parameters, including pH, glucose, and lactate concentrations, were measured in 1.8 mL medium samples using a GEM Premier 5000 analyzer (Instrumentation Laboratory, USA). On day 28, histological analysis of corneal endothelium was performed, and protein expression of ZO-1, Na⁺/K⁺-ATPase, caspase-3, and Ki-67 was evaluated by whole-mount immunofluorescence staining (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eQualitative analysis of ZO-1 and Na⁺/K⁺-ATPase expression was performed on seven corneas stored in each medium, in technical duplicates, using a confocal microscope (Olympus FV100, 40\u0026times; objective). Images were analyzed using Fiji/ImageJ software, and protein expression was assessed descriptively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.2.5. Statistical analysis\u003c/h2\u003e \u003cp\u003eCategorical variables are presented as absolute and relative frequencies. Normality of continuous variables was assessed using the Shapiro\u0026ndash;Wilk test; however, due to the small sample size, non-parametric statistical methods were applied. Paired analyses were performed to account for the paired-cornea study design.\u003c/p\u003e \u003cp\u003eContinuous variables are presented as medians with interquartile ranges. Differences between groups were analyzed using the Wilcoxon signed-rank test, with Hodges\u0026ndash;Lehmann estimates of median differences and corresponding 95% confidence intervals. All tests were two-sided, with statistical significance set at α\u0026thinsp;=\u0026thinsp;0.05.\u003c/p\u003e \u003cp\u003eStatistical analysis was performed using MedCalc\u0026reg; Statistical Software version 23.3.4 (MedCalc Software Ltd., Ostend, Belgium; 2025).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3. Results","content":" \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Corneal donor demographics\u003c/h2\u003e \u003cp\u003eThe study included 14 corneas obtained from seven donors whose tissues were deemed unsuitable for clinical transplantation. The median donor age was 71 years (range: 69\u0026ndash;73 years). The most frequent cause of death was neoplastic disease. The mean postmortem interval, defined as the time from circulatory arrest to tissue procurement, was 14 hours and 30 minutes. Donor characteristics are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCharacteristics of cornea donors (n\u0026thinsp;=\u0026thinsp;7)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eID number of the donor\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSex\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAge (yrs)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCause of death\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eType of donor\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRecoverd tissue\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eReasons for rejection for clinical use\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003ePostmortem interval (hh:mm)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAcute myocardial infarction\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDCD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ecornea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eresults of communicable disease testing\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e12:08\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAortic stenosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDCD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ecornea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eresults of communicable disease testing\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e17:55\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eŽ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePancreatic cancer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDCD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ecornea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eresults of communicable disease testing\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e23:20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eŽ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBreast cancer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDCD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ecornea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eresults of communicable disease testing\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e19:15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eŽ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSubarachnoid hemorrhage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDBD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eeye bulb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003emedical history of the donor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e14:30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eŽ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBreast cancer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDCD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ecornea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003emedical history of the donor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e12:10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDiseases of the respiratory system\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDCD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ecornea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eresults of communicable disease testing\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e11:38\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMedian\u003c/p\u003e \u003cp\u003e(range)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003en.a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e71\u003c/p\u003e \u003cp\u003e(69\u0026ndash;73)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003en.a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en.a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003en.a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003en.a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e14:30\u003c/p\u003e \u003cp\u003e(12:08\u0026ndash;23:20)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"8\"\u003eDBD \u0026ndash; \u003cem\u003edonor after brain death\u003c/em\u003e, DCD - \u003cem\u003edonor after circulatoy death\u003c/em\u003e, postmortem interval - time from the death to tissue collection, n.a- not applicable\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Umbilical cord blood platelet lysate characteristics\u003c/h2\u003e \u003cp\u003eTen umbilical cord blood units were used for UCB-PL production. The median UCB volume was 105 mL (IQR 91.4\u0026ndash;118.3), with a median absolute platelet count of 22.2 \u0026times; 10⁹ (IQR 17.3\u0026ndash;33.5). The median absolute leukocyte count was 1 \u0026times; 10⁹ (IQR 0.8\u0026ndash;1.3), and the median absolute erythrocyte count was 0.4 \u0026times; 10\u0026sup1;\u0026sup2; (IQR 0.3\u0026ndash;0.4). Following double-spin centrifugation, the median volume of UCB platelet concentrate was 8.4 mL (IQR 7.12\u0026ndash;11.91), with a median absolute platelet count of 7.3 \u0026times; 10⁹ (IQR 4.66\u0026ndash;13.51). Leukocyte contamination was negligible, with a median absolute leukocyte count of 0 \u0026times; 10⁹ (IQR 0\u0026ndash;0.01). UCB and UCB-PC characteristics are presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCharacteristics of umbilical cord blood, platelet rich plasma and platelet concentrate (n\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ePreparation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c6\" namest=\"c2\"\u003e \u003cp\u003eMedian (IQR)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVolume\u003c/p\u003e \u003cp\u003e(mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePlatelets\u003c/p\u003e \u003cp\u003e(10\u003csup\u003e9\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLeukocytes (10\u003csup\u003e9\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eErythrocytes (10\u003csup\u003e12\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eHemoglobin\u003c/p\u003e \u003cp\u003e(g/L)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eUCB\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e105 (91,4\u0026ndash;118,3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22,2 (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan additionalcitationids=\"CR4 CR5 CR6 CR7 CR8 CR9 CR10 CR11 CR12 CR13 CR14 CR15 CR16 CR17 CR18 CR19 CR20 CR21 CR22 CR23 CR24 CR25 CR26 CR27 CR28 CR29 CR30 CR31 CR32\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 (0,8\u0026thinsp;\u0026minus;\u0026thinsp;1,3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0,4 (0,3\u0026thinsp;\u0026minus;\u0026thinsp;0,4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e106,5 (104\u0026ndash;119,5)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePRP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40,9 (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan additionalcitationids=\"CR8 CR9 CR10 CR11 CR12 CR13 CR14 CR15 CR16 CR17 CR18 CR19 CR20 CR21 CR22 CR23 CR24 CR25 CR26 CR27 CR28 CR29 CR30 CR31 CR32 CR33 CR34 CR35 CR36 CR37 CR38 CR39 CR40 CR41 CR42\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12 (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan additionalcitationids=\"CR9 CR10 CR11 CR12 CR13 CR14 CR15 CR16\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0 (0\u0026ndash;0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0 (0\u0026ndash;0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1 (0\u0026ndash;1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eUCB-PC\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8,4 (7,12\u0026thinsp;\u0026minus;\u0026thinsp;11,91)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7,3 (4,66\u0026thinsp;\u0026minus;\u0026thinsp;13,51)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0 (0\u0026ndash;0,01)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0,1 (0,07\u0026thinsp;\u0026minus;\u0026thinsp;0,14)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1 (0\u0026ndash;2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eUCB-PL\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003en.a.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e948 (615\u0026ndash;1228)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0,58 (0,32\u0026ndash;0,93)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en.a.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003en.a.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eUCB - \u003cem\u003eumbilical cord blood\u003c/em\u003e, PRP - \u003cem\u003eplatelet rich plasma\u003c/em\u003e, UCB-PC - \u003cem\u003eumbilical cord blood platelate concentrat, n.a.-not applicable\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Corneal quality assessment before organ culture\u003c/h2\u003e \u003cp\u003eBaseline quality assessment of all corneas included in the study demonstrated a median endothelial cell density of 2417 cells/mm\u0026sup2; (IQR 2304\u0026ndash;3067). The median coefficient of variation of cell size was 35 (IQR 32\u0026ndash;39), and the median percentage of hexagonal endothelial cells was 59% (IQR 56.8\u0026ndash;63.3). No statistically significant differences were observed between paired corneas prior to organ culture with respect to endothelial parameters, corneoscleral ring morphology, or slit-lamp findings. Baseline specular microscopy results are shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCornea endothelial cell density during organ culture in media with fetal bovine serum and umbilical cord blood platelet lysate\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eDays of organ culture\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eEndothelial cell density\u003c/p\u003e \u003cp\u003eMedian (interquartile range)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eEstimate of the median difference\u0026dagger;\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e95% CI of difference\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eP*\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eMedia with FBS\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eMedia with UCB-PL\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0 day\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2400 (2312,5\u0026ndash;2658,3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2400 (2162\u0026ndash;2850)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-16,9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-133,3 to 133,3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0,60\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7 day\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2267 (2178,3\u0026ndash;2541,7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2309 (2125\u0026ndash;2533)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-133 to 66,3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0,24\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14 day\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2133 (1988\u0026ndash;2266)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2025 (1925\u0026ndash;2138)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-120,8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-273 to 25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0,08\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e21 day\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1750 (1550\u0026ndash;1967)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1775 (1575\u0026ndash;1900)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6,3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-58,3 to 58,3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0,92\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e28 day\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1467 (1225\u0026ndash;1725)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1300 (1195\u0026ndash;1463)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-158\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-367 to 8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0,11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e* Wilcoxon test; \u0026dagger;Hodges Lehmann median difference\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003cem\u003eFBS- fetal bovine serum, UCB-PL umbilical cord blood platelet lysat, CI - Confidence Interval\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Corneal quality during organ culture\u003c/h2\u003e \u003cp\u003eDuring organ culture, endothelial cell density gradually decreased in both media. No statistically significant differences in ECD were observed between corneas stored in FBS- and UCB-PL\u0026ndash;supplemented media at any evaluated time point (days 0, 7, 14, 21, and 28) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eEndothelial cell viability was high in both groups throughout the culture period. Median viability was 99.8% (IQR 99.6\u0026ndash;99.9) at baseline and 100% (IQR 99.8\u0026ndash;100) after 28 days, with no statistically significant differences between media at any time point.\u003c/p\u003e \u003cp\u003eEndothelial mosaic architecture remained regular in the majority of corneas until day 7 in both media. From day 14 onward, mild to moderate pleomorphism was observed; however, severe pleomorphism was not detected in any cornea. Similarly, no statistically significant differences were identified between groups with respect to intercellular space swelling at any time point.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Metabolic changes during organ culture\u003c/h2\u003e \u003cp\u003eGlucose concentrations decreased progressively in both culture media over time. Statistically significant differences between media were observed at baseline (P\u0026thinsp;=\u0026thinsp;0.02), day 7 (P\u0026thinsp;=\u0026thinsp;0.02), day 14 (P\u0026thinsp;=\u0026thinsp;0.04), and day 21 (P\u0026thinsp;=\u0026thinsp;0.02), with higher glucose levels consistently measured in the UCB-PL\u0026ndash;supplemented medium (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGlucose concentration ​​(mmol/L) ​​during the organ culture\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eDays of organ culture\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eGlucose concentration (mmol/L)\u003c/p\u003e \u003cp\u003eMedian (interquartile range)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eEstimate of the median difference\u0026dagger;\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e95% CI of difference\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eP*\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eMedia with FBS\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eMedia with UCB-PL\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0 day\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5,3 (4,97\u0026ndash;5,38)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5,9 (5,6\u0026ndash;6,08)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0,48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0,3 to 1,2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0,02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7 day\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3,6 (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4,1 (3,85\u0026ndash;4,63)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0,55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0,2 to 0,9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0,02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14 day\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2,6 (2,35\u0026ndash;25,78)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2,8 (2,63\u0026ndash;3,0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0,30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0 to 0,55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0,04\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e21 day\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1,4 (1,2\u0026ndash;1,68)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1,7 (1,53\u0026ndash;2,05)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0,30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0,1 to 0,5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0,02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e28 day\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0,8 (0,4\u0026ndash;0,95)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0,8 (0,6\u0026ndash;1,13)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0,13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-0,1 to 0,4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0,17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e* Wilcoxon test; \u0026dagger;Hodges Lehmann median difference, FBS-fetal bovine serum, UCB-PL umbilical cord blood platelet lysat,\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eCI - Confidence Interval. Bold values denote statistical significance.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eLactate concentrations increased in both media during organ culture. Statistically significant differences were detected on day 7 (P\u0026thinsp;=\u0026thinsp;0.03) and day 21 (P\u0026thinsp;=\u0026thinsp;0.04). Lactate levels were higher in the FBS-supplemented medium at baseline and day 7, whereas from day 14 onward, higher lactate concentrations were observed in the UCB-PL\u0026ndash;supplemented medium (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eLactate concentration ​​(mmol/L) during the organ culture\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eDays of organ culture\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eLactate concentration (mmol/L)\u003c/p\u003e \u003cp\u003eMedian (interquartile range)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eEstimate of the median difference\u0026dagger;\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e95% CI of difference\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eP*\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eMedia with FBS\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eMedia with UCB-PL\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0 day\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0,4 (0,3\u0026ndash;0,4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0,3 (0,3\u0026ndash;0,3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-0,1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-0,2 do 0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0,06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7 day\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2,5 (2,2\u0026ndash;3,03)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2,3 (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-0,2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-0,5 do -0,05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e0,03\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14 day\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4,4 (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4,5 (4,3\u0026ndash;5,0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0,08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-24,5 do 0,5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0,87\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e21 day\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6,6 (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7,0 (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5 CR6\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0,28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0 do 0,5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e0,04\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e28 day\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7,8 (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5 CR6 CR7\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8,0 (7,0\u0026ndash;8,9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0,43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-0,35 do 0,7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0,24\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eCI - Confidence Interval. Bold values denote statistical significance; \u003cem\u003e* Wilcoxon test; \u0026dagger;Hodges Lehmann median difference, ​\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003cem\u003eFBS-fetal bovine serum, UCB-PL umbilical cord blood platelet lysate\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eMedium pH values decreased over time in both groups; however, no statistically significant differences between media were observed at any time point.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Pathohistological analysis of corneal endothelium\u003c/h2\u003e \u003cp\u003eWhole-mount immunofluorescence analysis performed after 28 days of organ culture demonstrated preserved endothelial protein expression in both media. ZO-1 exhibited a characteristic continuous zigzag pattern along intercellular junctions, with interruptions at Y-shaped junctions between adjacent cells. Na⁺/K⁺-ATPase expression was localized to the lateral membranes of corneal endothelial and epithelial cells.\u003c/p\u003e \u003cp\u003eKi-67\u0026ndash;positive nuclei were detected exclusively in the epithelial layer, indicating proliferative activity, whereas Ki-67 expression was absent in corneal endothelial cells. Cleaved caspase-3 expression was observed in a small number of epithelial cells but was not detected in the corneal endothelium. Representative images demonstrating Na⁺/K⁺-ATPase and ZO-1 expression and their co-localization with nuclear staining are shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe primary objective of corneal organ culture is to preserve the structural and functional integrity of the corneal endothelium under conditions that closely resemble the physiological environment (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). In European eye banking practice, donor corneas are commonly stored in MEM-based media supplemented with fetal bovine serum (FBS) at concentrations ranging from 2% to 10%, in accordance with recommendations of the European Eye Bank Association (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Although FBS contains a broad spectrum of biologically active components that support cell survival, its xenogeneic origin raises concerns regarding batch variability, immunogenicity, and the potential transmission of infectious agents, including prions, viruses, and mycoplasmas (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). These limitations have prompted increasing interest in xeno-free alternatives such as human platelet lysate.\u003c/p\u003e \u003cp\u003eUmbilical cord blood (UCB) represents a unique and underutilized source of biologically active factors. In contrast to peripheral blood-derived human platelet lysate (hPL), UCB-derived platelet lysate contains a distinct growth factor profile and exhibits low immunogenicity and a favorable safety profile (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). In the present study, we evaluated the impact of umbilical cord blood platelet lysate (UCB-PL) on human corneal endothelial cells during organ culture and assessed its suitability as a xeno-free substitute for FBS.\u003c/p\u003e \u003cp\u003eOur results demonstrate that UCB-PL supports endothelial cell survival and function during 28 days of organ culture at a level comparable to standard FBS-supplemented medium. Endothelial cell density (ECD) decreased progressively in both media, consistent with previously reported endothelial cell loss during organ culture storage (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Although a slightly greater reduction in ECD was observed in corneas stored in UCB-PL\u0026ndash;supplemented medium, the difference was not statistically significant at any time point and did not adversely affect endothelial morphology or viability. These findings are clinically relevant, as corneal graft survival and postoperative transparency are strongly dependent on endothelial cell count and function (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEndothelial cell viability remained above 99% throughout the culture period in both groups, well within the accepted threshold for clinical use (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Morphological evaluation further confirmed preserved endothelial mosaic architecture, with regular cell patterns observed until day 7 and only mild to moderate pleomorphism developing thereafter. This pattern aligns with previous observations that endothelial cells compensate for cell loss through enlargement and migration rather than proliferation, given their post-mitotic nature (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). The absence of Ki-67 expression in endothelial cells in both media is consistent with this established biological behavior (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMetabolic analysis revealed progressive glucose consumption and lactate accumulation in both culture media, reflecting sustained metabolic activity of the corneal endothelium during storage (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Notably, glucose concentrations were consistently higher in the UCB-PL\u0026ndash;supplemented medium at several time points, suggesting adequate nutrient availability throughout the culture period. Lactate accumulation and gradual pH reduction were observed in both media, in line with previous reports describing anaerobic glycolysis under \u003cem\u003ein vitro\u003c/em\u003e storage conditions (\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Given the known tolerance of corneal endothelial cells to extracellular pH fluctuations within the range observed in this study, these metabolic changes are unlikely to compromise endothelial viability (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhole-mount immunofluorescence analysis provided further evidence of preserved endothelial function in both storage conditions. ZO-1 localization along intercellular junctions exhibited a characteristic zigzag pattern with interruptions at Y-shaped junctions, consistent with the known organization of tight junctions in the corneal endothelium (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Proper localization of Na⁺/K⁺-ATPase along endothelial cell membranes was also maintained, supporting intact pump function essential for stromal deturgescence and corneal transparency (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Importantly, no caspase-3 expression was detected in endothelial cells, indicating that apoptosis was not a major mechanism of endothelial cell loss during storage, in agreement with previous studies (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAn important practical aspect of this study is the use of UCB units that were unsuitable for hematopoietic stem cell transplantation. Repurposing these otherwise discarded units for UCB-PL production offers a sustainable and ethically favorable approach for generating growth factor\u0026ndash;rich supplements for tissue banking applications (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Nevertheless, several limitations should be acknowledged, including the small sample size, the use of a single UCB-PL concentration, and the restriction of the observation period to 28 days. Further studies are warranted to evaluate dose-dependent effects of UCB-PL, extend storage duration, and quantify growth factor composition in organ culture media.\u003c/p\u003e \u003cp\u003eIn conclusion, this study demonstrates that UCB-PL preserves the structural and functional integrity of the human corneal endothelium during organ culture at levels comparable to those achieved with FBS. These findings support the feasibility of implementing UCB-PL as a xeno-free supplement in corneal storage media, with potential benefits for standardized, animal-free eye banking and corneal transplantation.\u003c/p\u003e"},{"header":" Conclusions","content":"\u003cp\u003eThis study demonstrates that supplementation of corneal organ culture medium with UCB-PL preserves the morphological and functional characteristics of the human corneal endothelium during in vitro storage. Endothelial cell viability, morphology, metabolic activity, and key functional protein expression were maintained at levels comparable to those observed with standard fetal bovine serum\u0026ndash;supplemented medium.\u003c/p\u003e \u003cp\u003eGiven the critical role of endothelial cell integrity in determining corneal graft survival and postoperative transparency, these findings indicate that human donor corneas can be successfully preserved in a xeno-free storage medium without compromising endothelial quality. The use of UCB-PL offers a safe and effective alternative to animal-derived supplements and may contribute to reducing regulatory, ethical, and biological risks associated with FBS.\u003c/p\u003e \u003cp\u003eFuture studies should focus on standardizing UCB-PL production protocols, evaluating different supplement concentrations, extending storage durations, and characterizing growth factor composition in organ culture media. Such efforts may further support the implementation of UCB-PL\u0026ndash;based, animal-free storage media in clinical eye banking and corneal transplantation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e All authors certify that they have no affiliations with or involvement in any organization or entity with any financial or non-financial interest in the subject matter or materials discussed in this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e The authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e This research was funded by the Scientific Center of Excellence for Reproductive and Regenerative Medicine, Republic of Croatia, and by the European Union through the European Regional Development Fund, under grant agreement No. KK.01.1.1.01.0008, project \u0026bdquo;Reproductive and Regenerative Medicine - Exploring New Platforms and Potentials\u0026rdquo;.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e The Institutional Review Board of the University Hospital Centre Zagreb approved the protocol of this study, and it conformed to the tenets of the Declaration of Helsinki (02/21 AG from 20th December 2021)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed consent\u003c/strong\u003e Written informed consent was obtained from all the donor corneas that were included in this study\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the Scientific Center of Excellence for Reproductive and Regenerative Medicine, Republic of Croatia, and by the European Union through the European Regional Development Fund, under grant agreement No. KK.01.1.1.01.0008, project \u0026bdquo;Reproductive and Regenerative Medicine - Exploring New Platforms and Potentials\u0026rdquo;.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: Assoc. Prof. Branka Golubić Ćepulić MD, PhD, Mirna Golemović, MSc in Biology, PhD, Ivana Vidović, MD, PhD\u003cbr\u003e\u0026nbsp;Methodology: Assoc. Prof. Branka Golubić Ćepulić MD, PhD, Mirna Golemović, MSc in Biology, PhD, Prof. Davor Ježek, MD, PhD; Assoc. Prof. Ines Bojanić, Ivana Vidović, MD, PhD\u003cbr\u003e\u0026nbsp;Investigation: Ivana Vidović, MD, PhD; Marta Himelreich-Perić, MD, PhD\u003cbr\u003e\u0026nbsp;Data curation: Ivana Vidović, MD, PhD; Marta Himelreich-Perić, MD, PhD\u003cbr\u003e\u0026nbsp;Formal analysis: Assoc. Prof. Kristina Kralik, PhD\u003cbr\u003e\u0026nbsp;Writing \u0026ndash; original draft preparation: Ivana Vidović, MD, PhD; Marta Himelreich-Perić, MD, PhD\u003cbr\u003e\u0026nbsp;Writing \u0026ndash; review and editing: Ivana Vidović, MD, PhD; Marta Himelreich-Perić, MD, PhD\u003cbr\u003e\u0026nbsp;Visualization: Ivana Vidović, MD, PhD; Marta Himelreich-Perić, MD, PhD\u003cbr\u003eSupervision: Assoc. Prof. Branka Golubić Ćepulić; Mirna Golemović, MSc in Biology, PhD, Prof. Davor Ježek, MD, PhD; Assoc. Prof. Ines Bojanić, MD, PhD\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have read and agreed to the published version of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eM\u0026oslash;ller-Pedersen T, Hartmann U, M\u0026oslash;ller H, Ehler N, Engelmann K. Evaluation of potential organ culture media for eye banking using human corneas. Br J Ophthalmol. 2001;85:1075\u0026ndash;9. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1136/bjo.85.9.1075\u003c/span\u003e\u003cspan address=\"10.1136/bjo.85.9.1075\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEhlers H, Ehlers N, Hjortdal JO. 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Stem Cell Res Ther. 2021;12:554. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s13287-021-02611-3\u003c/span\u003e\u003cspan address=\"10.1186/s13287-021-02611-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"international-ophthalmology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"inte","sideBox":"Learn more about [International Ophthalmology](https://www.springer.com/journal/10792)","snPcode":"10792","submissionUrl":"https://submission.nature.com/new-submission/10792/3","title":"International Ophthalmology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"cornea, organ culture, serum-free media, umbilical cord blood platelet lysate","lastPublishedDoi":"10.21203/rs.3.rs-9427831/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9427831/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eTo evaluate whether umbilical cord blood platelet lysate (UCB-PL) can preserve the morphology and function of human corneal endothelial cells during organ culture and serve as a xeno-free alternative to fetal bovine serum (FBS).\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003ePaired human donor corneas were cultured for 28 days in α-MEM\u0026ndash;based medium supplemented with either 2% FBS or 2% UCB-PL. Endothelial morphology, cell density, viability, and mosaic regularity were assessed at predefined time points. Metabolic activity was evaluated by measuring pH, glucose, and lactate concentrations in the culture media. Endothelial integrity, pump function, apoptosis, and proliferation were analyzed by whole-mount immunofluorescence staining for ZO-1, Na⁺/K⁺-ATPase, caspase-3, and Ki-67.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eCorneas stored in UCB-PL\u0026ndash;supplemented medium demonstrated endothelial morphology, cell density decline, and viability comparable to those observed in FBS-supplemented medium throughout the culture period. No significant differences were detected between groups at any time point. Metabolic analysis showed sustained glucose availability and expected lactate accumulation in both media, without evidence of nutrient depletion. Immunofluorescence confirmed preserved endothelial junctional organization and pump protein expression, with no signs of endothelial apoptosis or proliferation.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eUCB-PL preserves human corneal endothelial cell morphology and function during organ culture at levels equivalent to those achieved with FBS. These findings support the use of UCB-PL as a viable xeno-free alternative for corneal storage media, with potential implications for animal-free eye banking and corneal transplantation.\u003c/p\u003e","manuscriptTitle":"The Impact of Umbilical Cord Blood Platelet Lysate on Human corneal Endothelium During Organ Culture","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-25 00:09:31","doi":"10.21203/rs.3.rs-9427831/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-04-29T15:00:28+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-29T03:57:54+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-20T09:27:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"304003193408325550112761598350924127573","date":"2026-04-20T09:17:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"137692761446241701638051741510101652921","date":"2026-04-19T12:45:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"286451422724750716642771518108915151818","date":"2026-04-18T05:43:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"99992253482082040161231341922683371595","date":"2026-04-17T13:41:59+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-17T11:31:15+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-16T09:09:59+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-16T09:09:30+00:00","index":"","fulltext":""},{"type":"submitted","content":"International Ophthalmology","date":"2026-04-15T13:28:25+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"international-ophthalmology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"inte","sideBox":"Learn more about [International Ophthalmology](https://www.springer.com/journal/10792)","snPcode":"10792","submissionUrl":"https://submission.nature.com/new-submission/10792/3","title":"International Ophthalmology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"55e06697-caa7-4610-8a5c-9150ccd0d1be","owner":[],"postedDate":"April 25th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-04-29T15:00:28+00:00","index":24,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-29T03:57:54+00:00","index":23,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-25T00:09:31+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-25 00:09:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9427831","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9427831","identity":"rs-9427831","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}