Abstract
This study reports an alternative approach to achieve vitrification where cells are pre-desiccated prior to cooling to cryogenic temperatures for storage. Chinese Hamster Ovary (CHO) cells suspended in a trehalose solution were rapidly and uniformly desiccated to a low moisture content (<0.12 g of water per g of dry weight) using a spin-drying technique. Trehalose was also introduced into the cells using a high-capacity trehalose transporter (TRET1). Fourier Transform Infrared Spectroscopy (FTIR) was used to examine the uniformity of water concentration distribution in the spin-dried samples. 62% of the cells were shown to survive spin-drying in the presence of trehalose following immediate rehydration. The spin-dried samples were stored in liquid nitrogen (LN2) at a vitrified state. It was shown that following re-warming to room temperature and re-hydration with a fully complemented cell culture medium, 51% of the spin-dried and vitrified cells survived and demonstrated normal growth characteristics. Spin-drying is a novel strategy that can be used to improve cryopreservation outcome by promoting rapid vitrification.
Highlights
Vitrification is the direct transition from a liquid to an ice-free glassy state upon cooling
We developed an alternative approach to achieve vitrification without the need to incubate the cells in exceedingly high concentrations of CPA
In conclusion, the pre-desiccation of cells prior to vitrification at cryogenic temperature is an exciting approach in cryopreservation
Summary
Vitrification is the direct transition from a liquid to an ice-free glassy state upon cooling. A major bottleneck of the vitrification technique is that it requires high concentrations of cryoprotectants (CPAs) to avoid ice-nucleation during cooling. Such high concentrations (6–8M) of CPAs are toxic to the cells [1] and, as a result, multiple steps and elaborate protocols are required to load and unload CPAs into cells. This makes vitrification a complex and difficult process. The technique of spin-drying has previously been used by Chakraborty et al [2] to create ultra-thin films of trehalose
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