Preparation of Fibrinogen-Depleted Human Platelet Lysate to Support Heparin-Free Expansion of Umbilical Cord-Derived Mesenchymal Stem Cells

SIMPLE SUMMARY: The use of fetal bovine serum (FBS) in the manufacturing and production of therapeutic products is discouraged by the regulatory authorities due to the risk of zoonoses and xenogeneic immune reactions. Human platelet lysate (hPL) is suggested as an alternative to FBS for in vitro cell expansion in accordance with good manufacturing practice (GMP) regulations. Since hPL contains a lot of fibrinogen and other coagulation factors, heparin is commonly added to prevent gel formation. However, heparin is typically derived from animals, and despite following the recommended maximum dose, gel and precipitate formation can still occur during storage and cell culture. Thus, an alternative preparation method is proposed in this study to prepare fibrinogen-depleted hPL (Fd-hPL) that supports heparin-free expansion of umbilical cord-derived mesenchymal stem cells (UC-MSCs). The findings revealed that Fd-hPL supported the in vitro expansion of UC-MSCs without compromising their growth and characteristics. Therefore, it has the potential to be used as a substitute for FBS for in the vitro culture of MSCs without introducing xenogeneic components. However, further research is necessary to fully understand the functionality and therapeutic potential of these expanded cells. ABSTRACT: Human platelet lysate (hPL) has high levels of fibrinogen and coagulation factors, which can lead to gel and precipitate formation during storage and cell culture. Heparin derived from animals is commonly added to minimize these risks, but cannot completely eliminate them. Thus, this study proposes an alternative method to prepare fibrinogen-depleted hPL (Fd-hPL) that supports heparin-free expansion of mesenchymal stem cells (MSCs). hPL was added to heparin to prepare heparin-hPL (H-hPL), whilst Fd-hPL was prepared by adding calcium salt to hPL to remove the fibrin clot. The concentrations of calcium, fibrinogen, and growth factors in H-hPL and Fd-hPL were compared. The effects of H-hPL and Fd-hPL on umbilical cord-derived MSCs (UC-MSCs) were assessed. The results showed that Fd-hPL possessed a significantly higher calcium concentration and a lower fibrinogen level than H-hPL. The concentrations of BDNF, TGF-β1, and PDGF-BB showed no significant difference between H-hPL and Fd-hPL, but Fd-hPL had a lower VEGF concentration. Fd-hPL retained the characteristics of UC-MSCs, as it did not affect the cell viability, proliferation, multilineage differentiation potential, or surface marker expression. In conclusion, Fd-hPL effectively supported the in vitro expansion of MSCs without compromising their characteristics, positioning it as a potential substitute for FBS in MSC culture.