Scalable Production of Equine Platelet Lysate for Multipotent Mesenchymal Stromal Cell Culture

Abstract

Translation of multipotent mesenchymal stromal cell (MSC)-based therapies is advancing in human and veterinary medicine. One critical issue is the in vitro culture of MSC before clinical use. Using fetal bovine serum (FBS) as supplement to the basal medium is still the gold standard for cultivation of many cell types including equine MSC. Alternatives are being explored, with substantial success using platelet lysate-supplemented media for human MSC. However, progress lags behind in the veterinary field. The aim of this study was to establish a scalable protocol for equine platelet lysate (ePL) production and to test the ePL in equine MSC culture. Whole blood was harvested into blood collection bags from 20 healthy horses. After checking sample materials for pathogen contamination, samples from 19 animals were included. Platelet concentrates were prepared using a buffy coat method. Platelets, platelet-derived growth factor BB, and transforming growth factor β1 concentrations were increased in the concentrates compared with whole blood or serum (p < 0.05), while white blood cells were reduced (p < 0.05). The concentrates were lysed using freeze/thaw cycles, which eliminated the cells while growth factor concentrations were maintained. Donor age negatively correlated with platelet and growth factor concentrations after processing (p < 0.05). Finally, all lysates were pooled and the ePL was evaluated as culture medium supplement in comparison with FBS, using adipose-derived MSC from four unrelated donor horses. MSC proliferated well in 10% FBS as well as in 10% ePL. However, using 5 or 2.5% ePL entailed highly inconsistent proliferation or loss of proliferation, with significant differences in generation times and confluencies (p < 0.05). MSC expressed the surface antigens CD90, CD44, and CD29, but CD73 and CD105 detection was low in all culture media. Adipogenic and osteogenic differentiation led to similar results in MSC from different culture media. The buffy coat method is useful to produce equine platelet concentrate with increased platelet and reduced white blood cell content in large scales. The ePL obtained supports MSC expansion similar as FBS when used at the same concentration (10%). Further investigations into equine MSC functionality in culture with ePL should follow.

Rho/ROCK Inhibition Promotes TGF-β3-Induced Tenogenic Differentiation in Mesenchymal Stromal Cells

“…Therefore, we aimed to further investigate the role of ROCK in the context of TGF-β3-induced tenogenic differentiation, including experiments in tendon scaffold culture. The scaffold chosen for this study has been established, characterized, and used in several previous studies [19,22,26,28,36,37]. As it consists of native decellularized tendon tissue, it combines the two most important influencing factors of the ECM, topography and matrix components, and represents the closest possible approximation to the environment of native tendon tissue.…”

“…Chondrogenic differentiation was only evaluated in equine MSC due to limited human MSC cell numbers. Surface antigen staining and flow cytometry were performed as described previously for human [25] and equine [26] MSC. Additionally, the effect of the ROCK inhibitor Y-27632 on human and equine MSC was tested in preliminary experiments.…”

“…For image quantification, FIJI ImageJ was used. [26]. When testing the ROCK inhibitor Y-27632, a concentration of 10 μM was effective in disrupting the actin cytoskeleton and had no adverse effect on MSC viability, although a slight effect on proliferation was observed until day 3 (p > 0:05).…”

Mesenchymal Stromal Cells Adapt to Chronic Tendon Disease Environment with an Initial Reduction in Matrix Remodeling

“…The following are available online at . References [ 68 , 69 ] are cited in the supplementary materials.…”

Phospholipid Profiles for Phenotypic Characterization of Adipose-Derived Multipotent Mesenchymal Stromal Cells

“…The latter consisted of α Minimum Essential Medium (αMEM; Gibco ™ , ThermoFisher Scientific) supplemented with 2.5% human platelet lysate (hPL; PL Bioscience GmbH, Aachen, Germany; Lot: 18-002.08), 1 U/ml heparin (Ratiopharm, Ulm, Germany) and antibiotics. Correspondingly, eMSC from each donor were also cultured with 10% equine buffy-coat-derived platelet lysate (ePL; Hagen et al, 2021; same batch used for all experiments) and 1 U/ml heparin instead of FBS, but in the same DMEM as specified above. Furthermore, in order to estimate the influence of an extracellular matrix environment on the MSC phenotype, hMSC were additionally cultured in collagen-coated flasks (Corning ™ BioCoat ™ Collagen I; purchased from VWR International), again using FBS-supplemented DMEM.…”