The establishment of a bank of stored clinical bone marrow stromal cell products

Abstract

Background: Bone marrow stromal cells (BMSCs) are being used to treat a variety of conditions. For many applications a supply of cryopreserved products that can be used for acute therapy is needed. The establishment of a bank of BMSC products from healthy third party donors is described.

Methods: The recruitment of healthy subjects willing to donate marrow for BMSC production and the Good Manufacturing Practices (GMP) used for assessing potential donors, collecting marrow, culturing BMSCs and BMSC cryopreservation are described.

Results: Seventeen subjects were enrolled in our marrow collection protocol for BMSC production. Six of the 17 subjects were found to be ineligible during the donor screening process and one became ill and their donation was cancelled. Approximately 12 ml of marrow was aspirated from one posterior iliac crest of 10 donors; one donor donated twice. The BMSCs were initially cultured in T-75 flasks and then expanded for three passages in multilayer cell factories. The final BMSC product was packaged into units of 100 × 106 viable cells, cryopreserved and stored in a vapor phase liquid nitrogen tank under continuous monitoring. BMSC products meeting all lot release criteria were obtained from 8 of the 11 marrow collections. The rate of growth of the primary cultures was similar for all products except those generated from the two oldest donors. One lot did not meet the criteria for final release; its CD34 antigen expression was greater than the cut off set at 5%. The mean number of BMSC units obtained from each donor was 17 and ranged from 3 to 40.

Conclusions: The production of large numbers of BMSCs from bone marrow aspirates of healthy donors is feasible, but is limited by the high number of donors that did not meet eligibility criteria and products that did not meet lot release criteria.

Figure 1

Effects of donor age on the quantity of CD34+ cells and total nucleated cells (TNC) in the aspirated marrow. The relationship between the age of the donor and the quantity of TNCs (Panel A, open squares), percentage of leukocytes expressing CD34 (Panel A, filled circles) and the total number of CD34+ cells in the marrow aspirate (Panel B) are shown. R stands for correlation coefficient and P stands for P value both of which were calculated using a regression model.

Figure 2

Effect of donor age on the marrow aspirate CFE and the quantity of BMSCs harvested from the primary culture. The relationship between the age of each donor and the CFE of the aspirated marrow (solid circles) and the quantity of BMSCs harvested from the primary culture (open triangles) is shown in panel A. The effects of donor age and gender on the marrow aspirate CFE is shown in panel B and on the quantity of BMSCs from the primary harvest is shown in panel C. The relationship between marrow aspirate CFE and the quantity of BMSCs from the primary harvest is shown in panel D. R stands for correlation coefficient and P for P value. Both were calculated using a regression model. Differences between males and females were compared using t-tests. * indicates significant differences (p < 0.05)

Figure 3

Factors affecting the quantity of BMSCs from the final harvest. The relationship between the quantity of BMSCs from the final harvest and the quantity of BMSCs in the primary harvest are shown in Panel A and the relationship between BMSCs in final harvest and donor age are shown in Panel B. The effects of the quantity of TNC in the marrow aspirate (Panel C), marrow aspirate CFE (Panel D) and percentage of leukocytes expressing CD34 in the marrow aspirate (Panel E) on the quantity of BMSCs in the final harvest are also shown. R stands for correlation coefficient and P for P value. Both were calculated using a regression model. *indicates significant differences (p < 0.05)