The development of the cell cryopreservation protocol with controlled rate thawing
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ORIGINAL PAPER
The development of the cell cryopreservation protocol with controlled rate thawing Tatyana M. Gurina . Alexandr V. Pakhomov . Anna L. Polyakova . Evgeniy I. Legach . Galyna A. Bozhok
Received: 12 February 2015 / Accepted: 9 September 2015 Ó Springer Science+Business Media Dordrecht 2015
Abstract Thawing in the water bath is often considered as a standard procedure. The thermal history of samples thawed in this way is poorly controlled, but cryopreservation and banking of cell-based products require standardization, automation and safety of all the technological stages including thawing. The programmable freezers allow implementation of the controlled cooling as well as the controlled thawing. As the cell damage occurs during the phase transformation that takes place in the cryoprotectant medium in the process of freezing–thawing, the choice of warming rates within the temperature intervals of transformations is very important. The goal of the study was to investigate the influence of warming rates within the intervals of the phase transformations in the DMSO-based cryoprotectant medium on the cell recovery and to develop a cryopreservation protocol with controlled cooling and warming rates. The temperature intervals of phase transformations such as melting of the eutectic mixture of the cryoprotectant solution (MEMCS), melting of the eutectic salt solution (MESS), melting of the main ice mass (MMIM), recrystallization before MEMCS, recrystallization before MESS and recrystallization before
T. M. Gurina A. V. Pakhomov A. L. Polyakova E. I. Legach G. A. Bozhok (&) The Institute for Problems of Cryobiology and Cryomedicine, The National Academy of Sciences of Ukraine, Kharkiv, Ukraine e-mail: [email protected]
MMIM were determined by thermo-mechanical analysis. The biological experiments were performed on the rat testicular interstitial cells (TIC). The highest levels of the cell recovery and metabolic activity after cryopreservation were obtained using the protocol with the high (20 °C/min) warming rate in the temperature intervals of crystallization of the eutectics as well as recrystallizations and the low (1 °C/min) warming rate in the temperature intervals of melting of the eutectics as well as MMIM. The total cell recovery was 65.3 ± 2.1 %, the recovery of the 3-beta-HSDpositive (Leydig) cells was 82.9 ± 1.8 %, the MTT staining was 32.5 ± 0.9 % versus 42.1 ± 1.7 %; 57.4 ± 2.1 % and 24.0 ± 1.1 % respectively, when compared to the thawing in the water bath. Keywords Cryopreservation DMSO Controlled thawing Temperature interval of phase transformation Cell recovery
Introduction The production of living cells for clinical therapy often involves cryopreservation and banking. One of the basic principles of GMP is the control of all aspects of the production process. Much attention in the cryopreservation practice is paid to the regulation of cooling regimens, the composition of the medium, the procedures of the cryoprotectant adding and washing, the
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Cell Tissue Bank
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