Calcium chloride declotted human platelet lysate promotes the expansion of mesenchymal stromal cells and allows manufacturing of immunomodulatory active extracellular vesicle products.

Mesenchymal stromal cells (MSCs) exert immunomodulatory effects, primarily through released extracellular vesicles (EVs). For the clinical-grade manufacturing of MSC-EV products culture conditions need to support MSC expansion and allow the manufacturing of potent MSC-EV products. Traditionally, MSCs are expanded in fetal bovine serum-supplemented media. However, according to good manufacturing practice (GMP) guidelines the use of animal sera should be avoided. To this end, human platelet lysate (hPL) has been qualified as an animal serum replacement. Although hPL outcompetes animal sera in promoting MSC expansion, hPL typically contains components of the coagulation system that need to be inhibited or removed to avoid coagulation reactions in the cell culture. Commonly, heparin is utilized as an anticoagulant; however, higher concentrations of heparin can negatively impact MSC viability, and conventional concentrations alone do not sufficiently prevent clot formation in prepared media. To circumvent unwanted coagulation processes, this study compared various clotting prevention strategies, including different anticoagulants and calcium chloride (CaCl 2)-mediated declotting methods, which in combination with heparin addition was found effective. We evaluated the influence of the differently treated hPLs on the proliferation and phenotype of primary bone marrow-derived MSCs and identified the CaCl 2 -mediated declotting method as the most effective option. To determine whether CaCl 2 declotted hPL allows the manufacturing of immunomodulatory MSC-EV products, EVs were prepared from conditioned media of MSCs expanded with either conventional or CaCl 2 declotted hPL. In addition to metric analyses, the immunomodulatory potential of resulting MSC-EV products was assessed in a recently established multi-donor mixed lymphocyte reaction assay. Our findings conclusively show that CaCl 2 -declotted hPLs support the production of immunomodulatory-active MSC-EV products. [ABSTRACT FROM AUTHOR]