Novel Cryopreservation Method Reduces Procedural Toxicity

A new study describes how mathematical modeling could advance ice-free cryopreservation, i.e., vitrification, paving the way for optimized tissue and organ preservation.

Researchers at Oregon State University (OSU; Corvallis, USA) and Northern Illinois University (DeKalb, USA) designed a mathematical approach to identify and modify minimally toxic cryoprotective agent (CPA) equilibration procedures. To do so, they first developed a concentration- and temperature-dependent toxicity cost function by exposing adherent endothelial cells to a range of glycerol concentrations at 21 °C and 37 °C, and fitting the resulting viability data to a first order cell death model.

The resulting toxicity cost function was then numerically minimized to establish a state-constrained optimization routine intended to minimize cell damage during the cryopreservation procedure, a routine which involves initially exposing the adherent endothelial cells to a low concentration of cryoprotectant, and then allowing time for the cells to swell. Following the swelling stage, the tissue sample can then be rapidly vitrified by adding a high concentration of CPA, resulting in much less overall toxicity.

The researchers then conducted lab tests using the predicted optimal procedure routine, and succeeded in obtaining 81% cell recovery after exposure to the vitrification solutions, as well as successful vitrification with relatively slow cooling and warming rates of 50 °C/min and 130 °C/min. In comparison, similar cell tissues that underwent conventional multistep CPA equilibration procedures resulted in much lower yields, of only about 10%. The study was published on November 35, 2015, in PLOS One.

“The biggest single problem and limiting factor in vitrification is cryoprotectant toxicity, and this helps to address that,” said associate professor Adam Higgins, PhD, of the OSU School of Chemical, Biological, and Environmental Engineering. “The model should also help us identify less toxic cryoprotectants, and ultimately open the door to vitrification of more complex tissues, and perhaps complete organs.”

“Many more applications of vitrification could be possible, especially as future progress is made in the rapidly advancing field of tissue regeneration, in which stem cells can be used to grow new tissues or even organs,” concluded Dr. Higgins. “Tissues could be made in small amounts and then stored until needed for transplantation; organs being used for transplants could be routinely preserved until a precise immunological match was found for their use. Conceptually, a person could even grow a spare heart or liver from their stem cells and preserve it through vitrification.”

Cryopreservation is a process in which cells, whole tissues, or any other substances susceptible to damage caused by chemical reactivity or time are preserved by cooling to sub-zero temperatures so as to stop enzymatic activity. By default it should be considered that cryopreservation alters or compromises the structure and function of cells, unless it is proven otherwise for a particular cell population.

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Oregon State University
Northern Illinois University