Abstract
Cryopreservation is the most promising way for long-term storage of biological samples e.g., single cells and cellular structures. Among various cryopreservation methods, vitrification is advantageous by employing high cooling rate to avoid the formation of harmful ice crystals in cells. Most existing vitrification methods adopt direct contact of cells with liquid nitrogen to obtain high cooling rates, which however causes the potential contamination and difficult cell collection. To address these limitations, we developed a non-contact vitrification device based on an ultra-thin freezing film to achieve high cooling/warming rate and avoid direct contact between cells and liquid nitrogen. A high-throughput cell printer was employed to rapidly generate uniform cell-laden microdroplets into the device, where the microdroplets were hung on one side of the film and then vitrified by pouring the liquid nitrogen onto the other side via boiling heat transfer. Through theoretical and experimental studies on vitrification processes, we demonstrated that our device offers a high cooling/warming rate for vitrification of the NIH 3T3 cells and human adipose-derived stem cells (hASCs) with maintained cell viability and differentiation potential. This non-contact vitrification device provides a novel and effective way to cryopreserve cells at high throughput and avoid the contamination and collection problems.
Highlights
Cryopreservation has been widely used for long-term preservation of cells, aggregates, tissues and organs
To predict the effect of droplet size on the effectiveness of vitrification device, we carried out simulation to evaluate the crystallization using droplets with volumes in the range of 0.2 to 5 μ L, which were commonly applied for vitrification of embryos[35,47], oocytes[48] and mammalian cells[4,49]
We developed a novel non-contact vitrification device where the vitrification of cell-laden droplets was achieved through pool boiling of liquid nitrogen and heat transfer of an ultra-thin silver film
Summary
Cryopreservation has been widely used for long-term preservation of cells, aggregates, tissues and organs. Due to the small volume of the droplets, a significant high cooling rate was achieved at a relatively low CPA concentration (e.g., 4.5% ectoine17), which can potentially improve the efficiency of freezing and reduce the risk of toxicity from high CPA concentrations[5,18]. With this method, a variety of cells, such as red blood cells[17], hepatocytes cells[5], oocytes[19] and even whole blood[20] have been successfully preserved. Our findings suggest that this novel vitrification device can be used to vitrify cells at high throughput with low risk of contamination and cell loss
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