Alina Hagen Equine platelet lysate as synergistically active cell culture supplement in veterinary regenerative medicine Equine Clinic (Surgery, Orthopedics), Justus-Liebig-University Giessen Submitted in June 2023 132 pages, 6 figures, 2 tables, 2 publications, 397 references Keywords: mesenchymal stromal cells (MSC), platelet concentrate, platelet lysate, fetal bovine serum, equine, cell culture, cell fitness, functional properties, co-cultivation Introduction – Regenerative medicine and the clinical application of multipotent mesenchymal stromal cells (MSC) have gained tremendous importance in veterinary medicine. In order to obtain a sufficient number of MSC for a therapeutic application, their in vitro cultivation is required, as there is only a small amount of MSC in their tissue of origin. One critical aspect in the in vitro cultivation of MSC is the cell culture medium, which may strongly impact the quality and efficacy of the cell-based therapeutic. Currently, the critically discussed fetal bovine serum (FBS) is the gold standard as a basal medium supplement for the in vitro cultivation of MSC in animal species. However, the trend towards the use of xeno-free culture supplements is emerging, and replacement of FBS is recommended by the European Medicines Agency (EMA) and the International Society for Cell and Gene Therapy (ISCT). While several alternatives have been investigated, the use of human platelet lysate (hPL) in human medicine has shown promising results. Aims – The aim of the first study was to establish a scalable protocol for equine platelet lysate (ePL) production based on hPL production protocols and to compare the obtained ePL with FBS as cell culture supplement in equine MSC (eMSC) culture regarding the preservation of their basic characteristics. The potency and functional properties of MSC are strongly dependent on varying environmental factors. Therefore, the second study aimed to investigate the influence of ePL on cell fitness and pro-angiogenic potency of eMSC. Materials and methods – In the first in vitro study, whole blood from 20 healthy horses was harvested into blood collection bags. After screening the whole blood for pathogen contamination, the blood of 19 horses was included in the study. The platelet concentrates were prepared from whole blood using the buffy coat method and subsequently lysed by freeze/thaw cycles. During the manufacturing process, platelet, leukocyte, platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-1 (TGF-1) concentrations were determined. After preparation, the final pooled ePL was evaluated as a cell culture supplement for cultivation of adipose-derived eMSC of n = 4 donor horses, in comparison with FBS. The generation time, immunophenotype and trilineage differentiation potential of eMSC were investigated using the three different media supplements 10 % FBS, 10 % ePL and 2,5 % ePL. The second study analyzed if the cell fitness and pro-angiogenic potential of adipose-derived eMSC is comparably supported by ePL as by FBS medium. For this purpose, eMSC from n = 5 donor horses cultured in 10 % FBS, 10 % ePL or 2,5 % ePL medium were evaluated in terms of their apoptotic, necrotic and senescent state, genetic stability and pro-angiogenic potency. For analysis of pro-angiogenic potential in co-culture, equine endothelial cells were obtained from equine umbilical arteries using a digestion procedure. Additionally, an arterial ring assay was initiated based on arterial rings extracted from the umbilical arteries. An indirect transwell system was used for the co-cultivation of eMSC and equine endothelial cells. The pro-angiogenic potential of the eMSC was assessed by measuring the vascular endothelial growth factor-A (VEGF-A) concentration of the different cell culture supernatants after co-cultivation and by examining the gene expression of the angiogenesis-related factors using qRT-PCR. In addition, morphological analysis of the cells was performed after staining the eMSC with Phalloidin and the endothelial cells with lectin both in the arterial ring assay and after co-cultivation with eMSC. Statistical analysis was performed using IBM SPSS and GraphPad based on non-parametric tests. Results – The study 1 provides the first evaluation of a buffy-coat-based protocol for ePL production. In this process, a 4.2-fold increased platelet concentration (p < 0.05) and a 0.4-fold decreased leukocyte concentration (p < 0.05) was observed in the platelet concentrates in comparison to the whole blood. In addition, the concentrations of the growth factors PDGF-BB and TGF-1 were increased in the platelet concentrates (p < 0.05 for PDGF-BB and p < 0.01 for TGF-1) as well as in the platelet lysates (p < 0.01 for PDGF-BB and p < 0.05 for TGF-1). The age of the donors correlated negatively with the platelet counts in the platelet concentrates (p < 0.01 and r = –0.582) as well as with the PDGF-BB (p < 0.01 an r = –0.627) and TGF-1 (p < 0.05 and r = –0.483) concentrations in the concentrates. Evaluating the ePL as a cell culture supplement, it supported eMSC proliferation and basic characteristics similar to FBS when used in the same concentration (10 %). In contrast, during cultivation with 2.5 % ePL medium, the eMSC exhibited inconstant proliferation or even a complete loss of proliferation, as evidenced by a significantly prolonged generation time and altered confluency (p < 0.05). The study 2 demonstrated that eMSC cultured with 2,5 % ePL medium exhibited higher apoptosis levels than eMSC after cultivation in 10 % ePL medium (p < 0.05). The cell fitness of eMSC cultured with 10 % ePL medium was comparable to that of eMSC cultivated with FBS medium. Interestingly, the eMSC showed a higher genetic stability after cultivation with 10 % ePL medium. Roughly 8 % of the eMSC with FBS medium showed non-clonal chromosomal aberrations and only 4.8 % of the eMSC cultivated with 10 % ePL medium. Clonal aberrations of the eMSC were neither observed in 10 % FBS nor in 10 % ePL medium. In addition, it was demonstrated that ePL supported the pro-angiogenic properties of eMSC, because a significantly higher concentration of VEGF-A was released during cultivation with 10 % ePL medium (p < 0.05) and the VEGF-receptor 2 (VEGFR2) expression was increased as compared to FBS medium. In addition, the eMSC and ePL promoted the growth of vessel-like structures in two of the co-cultured samples. Furthermore, the migration and proliferation of equine endothelial cells in the arterial-ring assay was higher with 10 % ePL than with FBS medium. Conclusion – Based on the results in the present work, the ePL is a promising alternative to FBS as a cell culture supplement for in vitro cultivation of eMSC. In this regard, the buffy coat method has proven to be a useful procedure to produce a standardized ePL in large scales. However, preferably young donor horses should be selected to obtain the highest possible platelet and growth factor concentrations. The ePL supported the proliferation and basic characteristics of eMSC when used at the same concentration as FBS (10 %). In addition, ePL promoted the cell fitness of eMSC comparably to FBS, and the functional properties of eMSC were positively affected by ePL, as evidenced by the improved pro-angiogenic properties. Based on these results, the ePL can be recommended as a cell culture supplement for eMSC cultivation. However, further studies on the detailed composition of ePL are necessary and, due to the positive influence on the pro-angiogenic effect of eMSC, in vivo studies in the field of wound healing should also be conducted to establish ePL for therapeutic use alone or in combination with eMSC.