Importance of a three-stage cooling regime and induced ice nucleation during cryopreservation on colony-forming potential and differentiation in mesenchymal stem progenitor cells from human fetal liver.

Colony-forming activity, as well as osteogenic and adipogenic capacities of primary human fetal liver (HFL) mesenchymal stem or progenitor cells (MSCs) were compared before and after cryopreservation using a standard three-step cooling protocol (Cryo3-S) or the same protocol with induced ice nucleation (Cryo3-IIN) and 5% and 10% w/v dimethyl sulphoxide (Me₂SO). Cell viability, using the Cryo3-S protocol with 5 % and 10 % Me₂SO, was about 60 to 70 % as assessed by the trypan blue staining method, but the ability to undergo growth in culture and form colonies was completely lost. Cryopreservation using Cryo3-IIN resulted in conservation of colony-forming MSCs. Colony-forming efficiency (CFE) of the cell samples cryopreserved with Cryo3-IIN and 5 % Me₂SO was on average 0.4 +/- 0.1 colonies per 10⁵ cells, whereas with 10% Me₂SO 1.6 +/- 0.7 colonies were obtained. HFL MSCs recovered after cryopreservation in the both groups demonstrated capacity to be expanded and induced into either osteogenic or adipogenic differentiation.