Cryopreservation of Spin-Dried Mammalian Cells

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.

Late embryogenesis abundant proteins protect human hepatoma cells during acute desiccation

“…The HepG2 cells provisioned with intracellular trehalose and LEA proteins are not stable at room temperature in the dried state and begin losing viability if not rehydrated within minutes; the loss of viability can be greatly retarded by immediate storage of the dried cells at liquid nitrogen temperatures (34). Our FTIR measurements for the spin-dried samples indicate a T g of approximately 4.5°C, which means that the cells do not exist in a vitrified state at room temperature.…”

“…To our knowledge, both the short-term membrane integrities and the long-term viabilities obtained after immediate rehydration in this study are the highest yet reported for mammalian cells dried uniformly to a water content below 0.12 g H 2 O/g dry weight. As we have discussed previously in more detail (34), traditional drying procedures result in substantial spatial nonuniformity, such that regions with higher residual water are prone to higher rates of chemical reactions that can foster unwanted metabolic processes, energy depletion, and cell degradation. Reduced molecular motion, such as that observed in glassy states, is an important requirement for storage of desiccated cells.…”

“…Spin-drying is another key step in the development of an improved dry-processing technique for mammalian cells (34), primarily because of the high degree of spatial uniformity in moisture content obtained in the samples and the rapid drying of cells. To our knowledge, both the short-term membrane integrities and the long-term viabilities obtained after immediate rehydration in this study are the highest yet reported for mammalian cells dried uniformly to a water content below 0.12 g H 2 O/g dry weight.…”

“…To avoid problems commonly encountered with evaporative drying of sessile droplets containing suspended cells, we have developed a spin-drying technique that yields rapid and uniform drying across the sample and low final water contents (33,34). Droplet drying is inherently slow, and water removal is spatially nonuniform, because a glassy skin forms at the sample interface when solutions of glass-forming solutes such as trehalose are dried (35,36).…”

A Raman Microspectroscopy Study of Water and Trehalose in Spin-Dried Cells

“…Nonetheless, the successful desiccation and long-term storage of mammalian cells at ambient temperature is yet to be achieved, with the most notable exception being the dried storage of anucleated platelets (1,(19)(20)(21)(22)(23)(24).…”