Human Platelet Releasate (hPR) - A xeno-free supplement for the expansion of stem cells

Abstract

Background & Aim Background Recent regulatory guidelines for cell therapy applications recommend the use of xeno-free materials to mitigate the risk of immune reaction to animal-derived additives i.e. fetal bovine serum (FBS). On this note, human platelet lysate (hPL) has been accepted as a xeno-free alternative for stem cell expansion. With several methods available to manufacture hPL, the lack of standardization amongst manufacturers has led to inconsistencies in expansion rate, phenotype, and immunomodulatory properties of stem cells; altering the intrinsic therapeutic potential of cells. Further, some platelet lysates induce “conditioning of cells”, where the cell expansion occurs with a bias towards a lineage of differentiation, as directed by the composition and concentration of growth factors, cytokines and chemokines present in the hPL. We have developed a cGMP process to manufacture human platelet releasate (hPR), a potential alternative to hPL and FBS. hPR is a xeno-free, non-conditioning media supplement derived from expired-human platelets. Methods, Results & Conclusion Methods We have developed a proprietary closed-system manufacturing process designed for optimal extraction of growth factors, cytokines and chemokines (Fig.1). Every lot is tested for sterility, endotoxin, mycoplasma, total protein content, and growth factors. The performance characteristics was evaluated by expanding bone marrow derived MSCs (BM-MSCs) in basal growth medium (DMEM) supplemented with hPR and benchmarked with equivalent strengths of three commercially available non-heparin-requiring human platelet lysates, AB serum, and FBS. Cell proliferation and population doubling time were measured using Luna II automated cell counter. Tri-lineage differentiation potential of these cells was assessed with adipogenesis, chondrogenesis and osteogenesis assay. The phenotypic, and biochemical markers of BM-MSCs were quantified using flow cytometry and multiplex ELISA quantibody assay (Fig.2). The potency of expanded cells was assessed by i) measuring human indoleamine 2, 3-dioxygenase (IDO) using kynurenine assay and luminex assay (IDO human ProcartaPlex), ii) mixed lymphocyte reaction assay (a lab-developed co-culture assay), and regulatory T-cell conversion assay using flow cytometry. Results We have developed a cGMP-compliant process to manufacture consistent lots of hPR. Our results indicate that the potency, and proliferation rate of MSCs is higher in basal medium supplemented with hPR compared to FBS or hPL (Fig.3).