Author/Editor     Cukjati, Marko
Title     Programirano in nadzorovano zamrzovanje mononuklearnih celic z aparatom Nicool Plus PC
Translated title     Programmed and controlled rate freezing of mononuclear cells on Nicool Plus PC freezer
Type     monografija
Place     Ljubljana
Publisher     Univerza v Ljubljani, Medicinska fakulteta
Publication year     2006
Volume     str. 77
Language     slo
Abstract     Background Mononuclear cells (MNC) have a major role in cell therapy. Cryopreservation is required for long-term storage of MNC. Cell damage during freezing cannot be prevented but can be effectively reduced by using cryoprotectors and controlled rate freezing. The aim of the study was to establish and to validate a protocol for freezing MNC on Nicool Plus PC (Air Liquide) which would enable uniform freezing with predetermined freezing rate at different loads of apparatus. Materials and methods The thermal gradient has been set to -1 °C/min down to T2 (freezing point), -2°C/min down to -40°C and -10°C/min down to -130°C. The temperature of cell suspension and freezing chamber was measured in sec intervals. Temperature range of crystallisation (T2 to Tf) was determined according to analysis of freezing curves. Optimal Tx was determined at three different loads of the freezer (3, 7 and 14 bags) at which the freezing curve between T2 and Tf was closest to value of -2°C/min. 24 buffy coats with volume 100 ml each in 10% DMSO were frozen and subsequently measured for leukocytes recovery and MNC viability with trypan-blue dye exclusion test. Initial concentrations of different cell types in buffy coat and cell recovery and viability after freezing were checked for correlation. Our results were compared with results of some other studies of controlled and uncontrolled rate freezing. Results T2 and Tf were -5.57 and -10.58°C, respectively. Optimal Tx for 3, 7 and 14 bags was -65, -67 in -73°C, respectively. In the temperature range of T2 to -40°C the actual freezing rate was -1.46 to -2.85°C/min. The maximum crystallisation peak rise was 1.18°C above the freezing point of the suspension. Mean leucocyte recovery and mononuclear cell viability was 85.4% and 88.5%, respectively. Concentration of cells in the initial suspension had no influence on viability of MNC after freezing. (ABstact truncated at 2000 characters)
Descriptors     MONOCYTES