Unfractionated human marrow cell cryopreservation using dimethylsulfoxide and hydroxyethyl starch

Unfractionated bone marrow (BM) cells were cryopreserved in 1- to 2-ml aliquots using a mixture containing both 5% dimethylsulfoxide (DMSO) and 6% hydroxyethyl starch (HES) in an attempt to increase the viable cell yield and reduce the clumping after thawing, observed when 10% DMSO is used alone. Samples thawed after storage for 6 months in the vapor phase of liquid nitrogen, were assayed. Compared to prefreeze values, there was both a greater number of cells that excluded Trypan Blue (50 +/- 12 vs 28 +/- 12%, P less than .01) and a greater CFU-C Recovery (110 +/- 20 vs 89 +/- 35%, P less than .02) for cells in the DMSO/HES mixture, compared to those in 10% DMSO alone. No macroscopic clumping of the thawed cells was observed for those cryopreserved in the mixture in contrast to those in DMSO alone. Freezing was done without a rate-controlled freezing apparatus by simply placing the samples initially into a -80 degrees C freezer, and then later into a liquid nitrogen freezer. Additional samples stored in the DMSO/HES mixture were kept at only -80 degrees C, and when thawed 12 to 16 months later also gave an excellent CFU-C recovery (105 +/- 39% of prefreeze). The DMSO/HES mixture allows for a simplified BM cryopreservation technique that not only assures excellent recovery of CFU-Cs and eliminates clumping upon thawing, but also does not require either the use of a rate-controlled freezer or liquid nitrogen temperatures for storage up to a year.