A novel method for maintaining the stability of freshly cultured Mesenchymal stem cells in clinical grade injection ready state without cryopreservation

Abstract

Mesenchymal Stem Cells (MSCs) are multipotent cells with low immuonogenecity, and dynamic tissue repair potential, which explains the overwhelming attention they have attracted in regenerative therapy. One notable challenge in MSCs therapy is the bench to bed timeline of freshly cultured MSCs; it does not exceed 24 h. For use after 24 h, MSC need to be cryopreserved - which can preserve the cells for years - but it is a costly and damaging process. Here we introduce a method to extend the bench to bed lifetime of MSCs up to 4 days without the high cost and cell damaging effects of cryopreservation. Our method is based on preserving the MSCs in human plasma. MSCs of 12 tissue samples - 4 adipose, 4 bone marrow and 4 Wharton’s jelly- were cultured and expanded in standard conditions. Cells harvested from passage 2 or 3 were washed, centrifuged, pelleted, and re-suspended in human plasma. Cell suspensions were refrigerated (5 ± 3 °C) or stored at room temperature (22 ± 3 °C) in a sterile, temperature controlled room. During the next 7 days, two tubes (one from each group) were examined every 24 h to assess MSCs viability and growth potential. On day 3, we assessed MSC cell surface markers and its differentiation potential to adipocyte and osteocyte tissues. Results were analyzed by computing the overall mean and applying the independent-samples t-test to those means. The sample means for both cell expansion and cell viability were compared between the two “refrigerator” and “room temperature” groups. Although there was a gradual decrease in cell growth potential between the cells stored for 1 day to those stored for 7 days, we show more than 80% of the cells remain alive for up to 4 days of storage in both groups. The cells reached 80% confluency in under 20 days for all samples stored up to 4 days. No significant differences were observed between the two groups (room temperature and refrigerator stored). The differentiation potential to adipocyte and osteocyte tested on day 3 were positive in all samples. The analysis of cell surface markers tested on day 3 were positive for CD90, CD105, CD73 and negative for CD34, CD45 and HLA-DR. We present a method of MSC culture medium using human plasma that can preserve their viability and growth potential for up to 4 days in both room and refrigerator temperatures without losing their stemness characteristics (we recommend use of 5 ± 3 °C). This novel method will allow rapid expansion and therapeutic use of MSCs. Since the cells can be maintained in clinical grade, injection ready state for several days, they can be transported across the globe.