Marrow Stromal Cell (MSC) Growth from Long Term Cryopreserved Bone Marrow.

Autologous MSC are under active investigation for a variety of potential clinical indications in regenerative medicine and gene therapy. The potential to derive autologous MSC from long-term cryopreserved bone marrow has not been fully elucidated. We report here the growth characteristics of MSC from bone marrow that was cryopreserved for more than 10 years.

Methods: We identified 3 deceased patients whose cryopreserved autologous bone marrow that was originally intended for autologous transplant, was to be discarded after fulfilling all accepted criteria for discard. The pts were age 46, 47, and 60 at the time of harvest, one had high risk and 2 had metastatic breast cancer (both to bone) and all had had multiple chemotherapy regimens prior to harvest. The marrow was cryopreserved between 1994–1995 in 10% DMSO using a controlled-rate freezer and stored in the vapor phase of liquid nitrogen until June, 2006 (11–12 years). The marrow was thawed in a 37 C waterbath, diluted with an equal volume of PBS, washed × 3 in PBS, resuspended in culture medium with 20% fetal bovine serum and plated at 5 × 10E6 mononuclear cells (mnc)/cm2 in 2 – 4 T175 flasks. Culture medium was changed in 48 hours and the plates were observed during subsequent culture medium changes every 3–4 days.

Results: At 2 – 4 days only scattered round cells were observed, but all samples grew scattered spindle-shaped cells were between days 4 – 12. Six of 8 primary cultures became > 70% confluent between days 11 and 21; 2 remained at < 40% confluence until replated (then achieving > 70% confluence at day 31). First passage cultures (n=12) became > 70% confluent in 4 – 10 days (median = 7) and 2^nd passage cultures were > 70% confluent in 7 – 10 days (median = 9). Second or 3^rd passage cultures yielded 2.1 – 4.2 × 10E6 MSC/T175 (n= 14; median 3.15) and were 89 – 96 % viable. Flow cytometry (n = 3) confirmed that 3^rd passage cells stained positive for CD105 and CD73 and negative for CD14 and CD45. Additional characterization of the differentiation potential of these presumptive MSC are in progress.

Conclusion: These studies indicate that cells typical for MSC can be readily grown from marrow that has been cryopreserved for > 10 years and suggest that even long-term stored bone marrow may serve as a source of cells for regenerative medicine.