Cryopreservation of human bone marrow cells by a modification of the two-step cooling technique

The first attempt to freeze human bone marrow cells with a two-step cooling method is reported. A simple and reliable way of obtaining stable first-step subzero freezing baths is described. One-milliliter samples each containing 20 X 10(6) bone marrow cells and 10% Me2SO were frozen in polypropylene cryotubes. Using these experimental conditions, the optimal freezing temperature was found to be in the range of -36 to 37.5 degrees C for BM progenitor cell (GM-CFC, CFUE, and BFUE) survival. Such temperatures were easily obtained in stable sludges of anisole or K2CO3 eutectic solution in water. The optimal holding time was 20 min before plunging tubes into liquid nitrogen. Similar or improved progenitor cell recoveries were observed compared with the conventional cooling technique. Adaptation of this two-step technique for the freezing of large volumes of BM cells for autografting is under investigation.