The development of the cell cryopreservation protocol with controlled rate thawing

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 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-HSD-positive (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.

What is the best protocol to cryopreserve immature mouse testicular cell suspensions?

“…In this study, samples were thawed in a 37°C water bath, which is considered a standard procedure. Controlled thawing is an unexplored, yet promising alternative that deserves to be studied (Gurina et al, 2015). Also, further study of vitrification is warranted as it is a fast and easy way to cryopreserve cell samples.…”

Cryopreservation of testicular tissue or testicular cell suspensions: a pivotal step in fertility preservation

“…Clinical grade cryopreservation and banking require standardization, automation and safety assessment of all the technological steps including thawing. In view of the implementation of a clinical procedure, controlled thawing is still an unexplored alternative (Gurina et al ., 2015). …”

Cryopreservation of Mesenchymal Stem Cells Using Medical Grade Ice Nucleation Inducer

“…Standard practice for cell recovery in cryovials is to encourage fast thawing utilizing a 37 • C water bath [23,24,35,40]. However, optimal heating rates within 96-well plates have yet to be defined.…”

“…Recently, in tandem with controlled rate freezers, controlled rate thawing has been applied to cryovials and cryobags when considering cells for use in therapies, although the accepted thawing rate is still high. While this has now been applied to testicular interstitial cells, pluripotent and mesenchymal stem cells [23,24] frozen in cryovials, there are no data on cryopreservation of MSC in a 96-well plate format. This could be used in developing cryosolutions or as a pre-prepared plate for toxicology testing or rapid distribution of cells.…”