Clinical-scale expansion of mesenchymal stromal cells: a large banking experience

Abstract

Background: Mesenchymal stromal cells (MSC) are largely investigated in clinical trials aiming to control inappropriate immune reactions (GVHD, Crohn's disease, solid organ transplantation). As the percentage of MSC precursors in bone marrow is very low, these must be expanded in vitro to obtain therapeutic cell doses. We describe here the constitution of an allogeneic human third-party MSC bank from screened healthy volunteer donors in compliance with quality specifications and ISCT-release criteria and report follow-up of different aspects of this activity since 2007.

Methods: 68 clinical-grade large-scale MSC cultures were completed and analyzed. The whole process was described, including volunteer donor screening, bone marrow collection, mononuclear cell isolation and expansion over 4 weeks, harvesting, cryopreservation, release, administration and quality controls of the cells (including microbiology, phenotype, and potency assays).

Results: From 59 validated donors, 68 cultures were completed (mean of final yields: 886 × 10(6) cells/culture) and a total of 464 MSC aliquots have been produced and stored in liquid nitrogen (mean of 132.8 × 10(6) cells/bag). Each MSC batch underwent extensive testing to verify its conformity with EBMT and ISCT release criteria and was individually validated. As of June 1 2015, 314 bags have been released and infused to patients included in 6 different clinical protocols. All thawed MSC units satisfied to release criteria and no infusion-related toxicity was reported.

Conclusion: In conclusion, despite low passage cultures, we have been able to create an allogeneic "off-the-shelf" MSC bank with a large number of frozen aliquots and report here an efficient clinical-grade MSC banking activity in place for more than 7 years. Our challenge now is to produce MSC in compliance with good manufacturing practices (GMP) as, in the meantime, MSC have become considered as advanced therapy medicinal products (ATMP). Another significant challenge remains the development of relevant potency assay.

Keywords: Banking; Clinical-grade; GMP; MSC; Mesenchymal stem cells.

Fig. 1

MSC clinical expansion. From ±50 mL of initial fresh BM, MNC cells were isolated by manual or automated ficoll isolation and seeded in T175 flasks. After a P0 expansion of 14 days, cells were harvested and re-loaded in new flasks for P1 expansion. One week later, cells were harvested and re-loaded according to the same scheme for P2 expansion before a final harvest around day 28. Quality controls were performed at different stages of the process

Fig. 2

MSC banking: production and release activity. a Production: From 59 validated donors, 68 cultures were completed allowing freezing of 464 MSC bags. From these, 430 are already validated, 18 non-compliant and 16 are still awaiting validation. b Release: Since 2007, 314 bags have been released. From those, 290 were MSC and 24 placebo; 187 were released on site and 103 were sent to other Belgian centers

Fig. 3

MSC immunosuppressive properties. Inhibition of PBMC proliferation by third party MSC: PBMC (100,000 or 50,000) were stimulated (S-PBMC) with anti-αCD3/CD28 microbeads during 4 days with or without irradiated (25 Gy) MSC (10/1 or 5/1 PBMC/MSC ratios) added at the beginning of the culture. Proliferation was assessed by analysis of the cell cycle by flow cytometry (N = 28). Result are expressed as the percentage of cells present in S + G2 M phases (a) and as the percentage of inhibition compared to the stimulated PBMC condition alone (b) for the 10/1 PBMC/MSC ratio

Fig. 4

MSC post-thaw viability and cell recovery. Pre- and post-thaw MSC viability (a), MSC number (b), as well as recovery of cell number (c) (N = 170)

Fig. 5

MSC post-thaw immunosuppressive properties. MSC bags were thawed (N = 5), washed and replated in flasks in complete media. Immunosuppressive properties (a) and cell recoveries (b) were evaluated at different time points after thawing