Emerging Technologies in Medical Applications of Minimum Volume Vitrification

Abstract

Cell/tissue biopreservation has broad public health and socio-economic impact affecting millions of lives. Cryopreservation technologies provide an efficient way to preserve cells and tissues targeting the clinic for applications including reproductive medicine and organ transplantation. Among these technologies, vitrification has displayed significant improvement in post-thaw cell viability and function by eliminating harmful effects of ice crystal formation compared to the traditional slow freezing methods. However, high cryoprotectant agent concentrations are required, which induces toxicity and osmotic stress to cells and tissues. It has been shown that vitrification using small sample volumes (i.e., <1 μl) significantly increases cooling rates and hence reduces the required cryoprotectant agent levels. Recently, emerging nano- and micro-scale technologies have shown potential to manipulate picoliter to nanoliter sample sizes. Therefore, the synergistic integration of nanoscale technologies with cryogenics has the potential to improve biopreservation methods.

Cryopreservation of Human Mesenchymal Stem Cells for Clinical Applications: Current Methods and Challenges

“…33 With a freezing rate of 1°C/min, a large number of MSCs can be easily frozen in one vial with the required CPA at low concentrations (<1.5 M). 79 Further, there is no direct contact between cells and nonsterile liquid nitrogen during the freezing process, significantly reducing the potential risk of contamination. Initially, MSCs loaded with CPAs were frozen using a nonprogrammable time freezing protocol in a -20°C freezer or freezing device called ''Mr.…”

“…Quartz microcapillaries, which provide a higher freezing rate of 250,000°C/min were developed, followed by cryoloops offering a freezing rate as high as 700,000°C/min. 79 The use of higher freezing/thawing rates permits vitrification using lower CPA concentrations, thereby reducing the potential for CPA toxicity and osmotic damage. To achieve a higher freezing rate, microscale (nanoliter) droplet vitrification systems (carrier-free systems) that involve generation of cell encapsulating CPA droplets, followed by direct injection into liquid nitrogen, have been developed.…”

“…To achieve a higher freezing rate, microscale (nanoliter) droplet vitrification systems (carrier-free systems) that involve generation of cell encapsulating CPA droplets, followed by direct injection into liquid nitrogen, have been developed. 79 To date, MSCs have not been preserved using nonequilibrium vitrification.…”

“…Reproduced with permission from Martin-Ibanez et al 84 which have the potential to cause chemical toxicity and osmotic shock to cells. 79 Moreover, vitrification might also lead to potential MSCs contamination with pathogenic agents, due to the direct exposure of the cell suspensions to nonsterile liquid nitrogen. Liquid nitrogen has been reported to have the issues of potential contamination with pathogens such as Aspergillus sp.…”

“…However, the recycling of the cells is labor-consuming as individual cell colonies need to be selected manually. 79,83 To date, the risk of contamination with pathogens should be reduced for the potential clinical use of vitrified MSCs. Given the prior concerns, the development of a sterile, closed, fully automated and high throughput system that allows MSCs to be vitrified without any direct exposure to liquid nitrogen is required.…”