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Background & Aim To evaluate the suitability of human platelet lysate (HPL) as a replacement for foetal bovine serum (FBS) for the propagation of human bone marrow derived mesenchymal stromal cells (MSCs). Background FBS is used universally as a cell culture supplement. Currently the use of FBS is still tolerated, however its use in clinical trials is tightly regulated and the US Food and Drug Administration (FDA) is moving towards adopting European guidelines, requiring that xenogeneic materials be eliminated in the preparation of any human therapeutic product containing cultured cells. Therefore, the use of HPL as a replacement for FBS in MSC expansion was evaluated. Methods, Results & Conclusion Methods Two types of HPL were produced by freeze-thawing and pooling either 12 units of expired apheresis platelet concentrates or 10 units of expired pooled platelet concentrates (n=3 batches each). MSCs from two donors were seeded at 5000 cells/cm2 and cultured to approximately 75-80% confluency using DMEM supplemented with either apheresis HPL, pooled HPL or FBS. Similar to FBS, HPL was heat-inactivated for 60 min at 56°C prior to addition to DMEM. The optimum concentration of HPL was determined by titration (2.5%, 5%, 7.5%, or 10%) against MSC culture expansion. Cell proliferation rates and doubling time were determined. Post-thaw immunophenotype and viability (7AAD) of BM-MSCs were determined by flow cytometry. Tri-lineage differentiation capacity was confirmed by microscopy after in vitro stimulation for either 14 days (chondrogenic and adipogenic) or 28 days (osteogenic). Results All 3 batches of HPL demonstrated an optimal culture concentration of 5% for apheresis HPL and 7.5 % for pooled HPL. Both types of HPL induced faster MSC proliferation when compared with matched 10% FBS controls. Apheresis HPL consistently outperformed pooled HPL, presumably as it is richer in growth factors. MSCs expanded under optimum conditions showed >90% expression of CD73, CD90 and CD105, and all were negative for CD45, CD34, CD19, CD11b, and HLA-DR. Mean 7AAD viability was 90% for MSCs grown in apheresis HPL, 88% for pooled HPL, and 84% for FBS. One batch of each type of HPL was used for tri-lineage differentiation capacity analysis. All MSCs were able to differentiate into either chondrogenic, osteogenic, or adipogenic lineages. Conclusion Apheresis and pooled HPL induced higher MSC expansion than FBS. The use of HPL as a substitute for FBS in MSC propagation warrants further investigation. |
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