Evaluation of human platelet lysate (HPL) as a substitute for foetal bovine serum (FBS) and recombinant proteins for the growth and maintenance of bone marrow derived mesenchymal stromal cells (BM-MSC)

Abstract

Aim To evaluate the potential of using HPL produced in Australia as a substitute for FBS during in vitro culture of BM-MSC. Background The replacement of FBS with an alternate serum source during the manufacture of cellular products for human therapeutics should minimise cost and exposure to the potential risk of animal products. Human AB sera serves as the current gold standard in clinical grade manufacture of cell therapies but suffers from high cost and limited supply. Here, we propose the use of HPL as an FBS replacement. Methods We compared BM-MSC cultured in FBS or HPL by assessing their growth, viability and phenotype by flow cytometry at P3 and P4. Three batches of HPL made from either apheresis or pooled platelet concentrates after expiry were tested. BM-MSC from a single donor were seeded at 1.8 × 103/cm2 in culture flasks pre-coated with 0.0001% human fibronectin, and passaged at 80% confluency. Cells were cultured in DMEM supplemented with 5% apheresis HPL, 10% pooled HPL or 10% Commercial HPL, compared to the Control media (10% FBS, 5 ng/mL PDGF, and 5 ng/mL EGF). As preliminary analysis revealed minimal variability between batches of HPL, we focused the remaining experiments on one batch of each type of HPL. Results The phenotype of all BM-MSC at P3 and P4 satisfied established MSC criteria, that is, >98% expression of CD73, CD90 and CD105, and lack of expression (i.e. 0.05 by one way ANOVA). In contrast, pooled HPL did not support BM-MSC growth beyond P3. This may be due to differences in the characteristics of apheresis and pooled HPL. Conclusion The growth and phenotype of BM-MSC cultured in either apheresis HPL or commercial HPL were comparable, suggesting apheresis HPL may prove a suitable alternative to FBS. A simpler preparation protocol of HPL for the end users will increase the likelihood of introducing HPL into current laboratory practice. The potential of pooled HPL warrants further investigation.