Freezing Harvested hMSCs and Recovery of hMSCs from Frozen Vials for Subsequent Expansion, Analysis, and Experimentation

Human multipotential stromal cells (hMSCs) are easily isolated from bone marrow and can be expanded by up to 200-fold in culture. Cultures of hMSCs are heterogeneous mixtures of stem/progenitor cells and more mature cell types. The proportion of each cell type in a given culture depends on how the cells are maintained. To maintain their stem cell-like qualities, hMSCs should be plated at low seeding densities (60–150 cells/cm² ), lifted when between 60% and 80% confluent and should not be expanded beyond 4–5 passages. Thus, it is useful to establish a frozen bank of early passage cells. hMSCs store well in vapor phase liquid nitrogen (LN₂ ) and are easily recovered for further expansion. This chapter describes one method of establishing a bank of early passage hMSCs using a seed lot system and the subsequent recovery of hMSCs from frozen stocks. The recovered cells can then be harvested and used for analyses of identification, functionality, in vitro and/or in vivo experimentation, or further expanded.