Postcryopreservation growth of human CFU-GM: sequential examination of methodologic factors

Postcryopreservation growth of human CFU-GM might provide insight into the reconstitutive potential of human marrow preparations cryopreserved for subsequent autologous hematopoietic reconstitution. Ultimately, the utility of such measurements will depend on: whether comparison of pre- and postcryopreservation CFU-GM values is a guide to loss of reconstitutive potency during or following cryopreservation, and whether such measurements are consistent and reproducible. In our hands, direct, unmanipulated plating of CFU-GM after freezing and thawing resulted in extremely poor CFU-GM growth. Consequently, we undertook a set of sequential experiments designed to optimize technical conditions for growing human CFU-GM after cryopreservation. Utilizing stepwise examinations of washing, multiple plating factors, dilution rate, and diluent, we were able to increase consistently fractional recovery of postcryopreservation CFU-GM growth per 2 X 10(5) cells plated from 8% to 56%. Our results underscore the importance that technical factors (other than freezing conditions) play in the postcryopreservation growth of human CFU-GM.