Background: Over the past decade, bone marrow-derived mesenchymal stromal cells (BM-MSCs) have been used therapeutically in a variety of clinical settings including graft versus host disease, ischemic/non-ischemic cardiovascular disease, ischemic stroke and as gene-delivery vehicles. Limitations with BM-MSCs include the declining number and differentiation potential of the cells with increasing donor age, the inconsistent quality of BM-MSC products and the invasiveness of the requisite BM aspiration procedure. Following a normal infant birth, the cord blood tissue (CBt) is typically discarded, thus collection of the starting material is non-invasive. CBt-MSCs can expand to higher numbers more rapidly than BM-MSCs, and have similar immunosuppressive properties. We therefore chose to develop a GMP-compliant procedure to generate large numbers of CBt-MSCs for clinical use.

Methods. CBt was obtained from consented healthy mothers of full term neonates following elective cesarean section. The CBt was transported in plasmalyte with penicillin/streptomycin. The CBt was cut into 7 equal fractions and incubated for 76 minutes in 370C in C-Tubes (Miltenyi) with various enzyme combinations including collagenase-NB4/6(Serva) and hyaluronidase (Sigma Aldrich) with or without DNase (Genentech) in the GentleMACS Octo Dissociator (Miltenyi). The cell suspension was filtered, washed and resuspended in alpha-MEM media containing 10% Platelet lysate, L-glutamine, heparin (complete media) with pen-strep and seeded into T175 flasks, then cultured until the MSCs were 80% confluent. The cells were harvested and expanded to P1 in T175 flasks to 80% confluence using complete media without antibiotics. After harvest of P1, the MSCs were analyzed for the expression of the typical MSC surface markers by flow cytometry and cryopreserved. Expansion was subsequently performed in a Quantum Bioreactor (Terumo) for 5-6 days. Immunosuppressive potential of CBt-MSCs was tested in vitro by CD4+ T cell proliferation assay (CFSE) and CD4+ T cell cytokine secretion assay. Half of the CBt-MSCs were preactivated with Interferon Gamma, then seeded into a 96 well plate and the others were seeded untreated. The following day, the MSCs were incubated with 10⁵ isolated CD4+ T cells at ratios of 1:1, 1:2, 1:10 and 1:20. CD3/28 beads (ThermoFisher Scientific) were added to all wells except the negative control. Isolated T cells were stained with CFSE for 10 minutes then incubated with 10% Fetal Bovine Serum (FBS) prior to co-culture with MSCs. After 72 hours, the wells were treated with BFA (10X), PMA (100X) and ionomycin (10X). Half of the wells were harvested, washed and stained with anti-CD4 (Biolegend) and Live-Dead dye (ThermoFisher Scientific). After Cytofix/Cytoperm Fixation and Permeabilization Solution (BD Biosciences), then 1X buffer were added, cells were stained for IL-2 (BD), TNF-alpha (BD) and Interferon gamma (BD Biosciences). On day 5, the remaining wells were harvested and stained with anti- CD4-APC (Biolegend) and Live-Dead (ThermoFisher Scientific). Flow cytometry was performed on all samples using the Fortessa X20 (BD Biosciences), then analyzed with FlowJo software.

Results

Following enzymatic digestion, the samples without DNase had poor P0 to P1 growth (less than 80% confluent by day 10) thus were eliminated. NB4, hyaluronidase and DNase became the standard enzyme combination. After seeding the Bioreactor with a median of 51 x 10E6 CBt-MSCs (range 45 to 62x10E6 cells), expansion in the Bioreactor for 5-6 days yielded a median of 1495 x 10E6 CBt-MSCs (range 1245 to 1935 x 10E6). The median doubling time (the time required for MSCS to proliferate and double in number) for CBt-MSCs was 28.2 hours (range 24.5 to 29.7) (n=3). The immunosuppression assay demonstrated that CBt MSCs inhibit the proliferation of CD4+ T cells in a dose-dependent manner. Moreover, CBt-MSCs reduce the cytokine expression on stimulated CD4+ T cells (interferon-gamma, tumor necrosis factor-a, IL-2) with each successive generation of CD4+ T-cells.

Conclusion: We standardized a GMP-compliant protocol for the isolation of MSCs from whole CBt. Large-scale expansion of CBt-MSCs with immunosuppressive properties can be generated quickly and efficiently in the Terumo bioreactor. Clinical trials with these cells are planned.

Disclosures

Kurtzberg: Gamida Cell: Research Funding.

Author notes

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Asterisk with author names denotes non-ASH members.

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