Human mesenchymal ctem cells (hMSCs) were isolated from bone marrow aspirates obtained from a single unrelated donor who was not a human-leukocyte-antigen match to recipients. The hMSCs in Prochymal and Chondrogen products (Osiris Therapeutics, Inc., Baltimore, Maryland) were manufactured in a manner consistent with International Conference on Harmonization and Food and Drug Administration regulatory (FDA) guidelines. The donor was tested according to the FDA Donor Suitability Guidance before donation.
The hMSC preparation under evaluation was referred to as “Provacel,” which is the same investigational agent as Prochymal, a highly puriﬁed preparation of ex vivo-cultured adult hMSCs.
Bone marrow aspirate cell pellets are separated on a Percoll gradient (discontinuous, 1.073 g/ml) and cells at the interface (0.01-0.001% of the total cells) are cultured in medium containing FBS (10%). The adherent cells are grown to near confluence and re-plated until preparation for infusion. At this stage, the cultures are assessed for i) safety (absence of bacteria (especially mycoplasma), viral pathogens and endotoxin), ii) purity (lack of contaminating hematopoietic cells, and of residual animal proteins from processing), iii) function (a measure of MSC potency by expression of TNFR1 and ability to inhibit T cell IL2Ra expression during stimulation of T cells),iv) and phenotype.