Abstract

Marrow stromal cells (MSC) are increasingly employed for human cellular therapy protocols. In some cases, it may be advantageous to use autologous MSC, but the feasibility of quickly generating sufficient MSC for autologous therapy has not been established. We investigated the feasibility of rapidly generating large numbers of autologous, early passage, MSC as a prerequisite for the clinical investigation of MSC in human ischemic stroke. Bone marrow (25–35 mL) was collected from 8 healthy volunteers under an IRB-approved protocol; a mononuclear cell (mnc) fraction prepared by ficol density gradient, a portion of the mnc fraction was suspended in low glucose DMEM with 20% pre-screened FBS and plated in T175 culture flasks at 0.2–0.4 × 106 mnc/cm2; maintained at 37 C in a 5% CO2 incubator; non-adherent cells were removed at 48 h and a portion of the cells were passaged when 80% confluent into additional flasks (12–14 days) at 5700 cells/cm2 and MSC were harvested with trypsin at 21 days. The MSC were washed x3 and resuspended in sterile saline for enumeration and suitability testing. MSC were identified according to ISCT criteria (

Dominici M,
Cytotherapy
2006
;
8
(4):
315
), including plastic adherence, morphology, growth characteristics, and phenotype by flow cytometry (CD73, CD90, CD105 positive; CD14, CD34, CD45, HLA-DR negative) and differentiation into adipocytes. The expected total cell yield was calculated based upon actual cell yields minus the volume of cells required for testing based upon a 70 kg patient.

Results: In 8 experiments using different donors, a median of 40 (range 20–225) × 106 mnc were available for plating from the original marrow. The median mnc recovery after ficol density gradient was 37% (range 23–130), but the median CFU-F recovery after ficol from the original marrow was 96% (94–96). The calculated MSC yield after primary culture was a median of 25 (10–53) × 106 cells. The calculated yield of MSC after 1st passage at 21 days was a median of 2.1 (0.9–4.4) × 106 MSC/kg (70 kg patient weight), and the median cell viability was 98.5% (92–97.9). At the end of 1st passage all samples yielded negative gram stains and cultures for mycoplasma, bacteria, fungal growth and the median endotoxin level was 0.027 EU/mL (0.02 – 0.10). The viability of the harvested MSC was well maintained in saline at 1 × 106 MSC/mL for 24 h at 4 C (85% +/− 9%) but not after 48 h (37% +/− 9%) or if stored at room temperature. The effects of supplementation with 5% human serum albumin on MSC storage for 24 h at 4 C were variable, ranging from 27% to 95% viability, depending on the albumin manufacturer (3 mfrs; n=4 each).

Conclusion: These studies suggest that sufficient MSC that are suitable for autologous human cellular therapy can be rapidly and reliably generated under GTP conditions, that the resultant MSC may be preserved for up to 24 h if stored at 4 C in either saline or pre-screened human serum albumin.

Author notes

Disclosure: No relevant conflicts of interest to declare.