This approach was motivated by evidence of electrolyte toxicity at high ZVADFMK concentrations that occurred during freezing [64]. In fact, increased electrolyte concentration, particularly the sodium ion concentration, is found to have adverse effects on cell viability and homeostasis in various aspects of biopreservation
by changing the ionization levels of the phospholipid head groups and their range of electrostatic interactions [44] and [99]. Nonetheless, even theoretically perfect protocols of cryopreservation did not result in 100% survival of cells all the time, which led Fahy to discuss the evidence of CPA toxicity as a major cause of inefficiencies in protocols of cryopreservation for cells and tissues in an article published in 1986 [27]. In liquidus-tracking, exposing cells to a low concentration of CPA at the lowest temperature possible, i.e. 0 °C, minimizes the concentration- and temperature-dependent
toxicity of the CPA. The time-dependent toxicity of the CPA would depend on the rate of the CPA diffusion into the cells. Then the suspension of cells in CPA can be cooled down to the freezing point of the solution to further suppress the temperature-dependent toxic effects. Sequentially, more CPA concentration is introduced to the suspension and time is allowed for equilibration. The steps of concentration increase selleck and temperature decrease continue until a vitrifiable concentration of the CPA is reached within the cells, at which point plunging the suspension of cells into liquid nitrogen vitrifies the system. This approach is feasible with cells in suspension considering that the size of the cells is much smaller compared to tissues (μm vs. mm). The CPA can cross the cell membrane and reach equilibrium within minutes [67] and [112]. In tissues, however, the time of diffusion and equilibration of CPA can take hours depending on the size and dimensions of the tissue [92] which is far too long for
protocols suggested by Farrant in terms CPA cytotoxicity. Elford and Walter were first SB-3CT to practice Farrant’s stepwise loading/cooling method using smooth muscle cells [24]. However, the interstitial concentration of the CPA at each step was unknown in that study. In the last of a series of three papers by Pegg [82], [83] and [84], he describes the first perfectly performed actual liquidus-tracking method. Discs of ovine cartilage with 0.7 mm thickness were vitrified through a 7-step vitrification protocol and the interstitial concentration of the Me2SO at each step during loading and washing steps was carefully measured. The reported recovery of the chondrocytes, about 57%, was assessed by 35S incorporation in a glycosaminoglycan (GAG) functional assay.
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