[Methods for freezing, thawing and viability estimation of hemopoietic stem cells]

Freezing and thawing of hemopoietic stem cells (CD34+) are indispensable stages between their collection from peripheral blood or bone marrow and transfusion to the patient who has previously undergone myeloablative chemotherapy. At present there are two methods of the cells freezing: non-controlled--placing CD34+ cells directly in the final temperature and rate-controlled--gradual cooling them at programmed speed. Non-controlled freezing leads to the considerable loss of cells viability (up to 50%), but it doesn't require the expensive equipment used for rate-controlled freezing (viability loss up to 10%). In order to reduce the loss resulting from intra-cellular crystallization of water the haemopoietic cells are mixed with one of the cryopreservative substances: dimethylsulphoxide-DMSO, hydro-xyethylstarch-HES, polyvinylpyrrolidone-PVP or glycerol. The most important moment of freezing procedure is the phase transition of water. The adequate shape of the cooling curve leads to a considerable reduction of the loss of the cells viability. Further cooling is the most effective when it takes place at max. speed of 5 degrees C/min. The storage of the frozen cells is the best in very low temperatures (-170-180 degrees C-vapour phase of liquid nitrogen), but mechanical freezers (-80 degrees C) are used, too. The thawing procedure should be very fast (up to 90 degrees C/min.) and the defrosted cells must be immediately transfused to the patient because of very high toxicity of cryopreservative agents to non-frozen cells. The cells viability estimation is carried out with trypan blue or cytofluorometrically after incubation with propidine iodide.