Sand-mediated ice seeding enables serum-free low-cryoprotectant cryopreservation of human induced pluripotent stem cells

Abstract

Human induced pluripotent stem cells (hiPSCs) possess tremendous potential for tissue regeneration and banking hiPSCs by cryopreservation for their ready availability is crucial to their widespread use. However, contemporary methods for hiPSC cryopreservation are associated with both limited cell survival and high concentration of toxic cryoprotectants and/or serum. The latter may cause spontaneous differentiation and/or introduce xenogeneic factors, which may compromise the quality of hiPSCs. Here, sand from nature is discovered to be capable of seeding ice above −10 °C, which enables cryopreservation of hiPSCs with no serum, much-reduced cryoprotectant, and high cell survival. Furthermore, the cryopreserved hiPSCs retain high pluripotency and functions judged by their pluripotency marker expression, cell cycle analysis, and capability of differentiation into the three germ layers. This unique sand-mediated cryopreservation method may greatly facilitate the convenient and ready availability of high-quality hiPSCs and probably many other types of cells/tissues for the emerging cell-based translational medicine.

Pollen derived macromolecules serve as a new class of ice-nucleating cryoprotectants

“…A small number of chemical nucleators, which are capable of nucleating ice near the melting point via heterogeneous nucleation, have also been deployed. These include Snomax™ 20 , 21 , which is derived from the bacterial plant pathogen Pseudomonas syringae , cholesterol crystals 22 , encapsulated silver iodide 23 , biologically inert minerals 24 and sand 25 . However, these existing chemical nucleators are usually insoluble and difficult to sterilise, or else are difficult to introduce into cryopreservation volumes in a reproducible fashion 11 .…”

The gelatin-based liquid marbles for cell cryopreservation

“…Therefore, cells would not be damaged by osmotic shock during a slow cooling process, even as extracellular ice precipitation gradually increases. Meanwhile, slow-freezing method not only allows sufficient time for intracellular water to gradually diffuse out of cells to reduce the formation of intracellular ice crystals, but also minimize the osmotic pressure damage [ 16 ]. In this work, the ice inhibition capacity of gelatin was investigated using a standard splat assay and DSC tests.…”

“…4 C). Thus, the stepwise freezing process was appropriate for the cell cryopreservation based on gelatin-based LM because the lower cooling rate not only could maintain the LM structure, but also allow water to sufficiently flow out of the cell to reduce the formation of cellular ice crystals [ 16 , 64 ]. Notably, the cell cryoprotection efficiency of in gelatin-based LMs was significantly higher than CS (p ​< ​0.001).…”

“…DMSO is intrinsically toxic, affects the epigenetic profile of cells and induces uncontrolled differentiation of stem cells, which results in phenotypic changes [ [13] , [14] , [15] ]. In addition, FBS from different donor species in the preservation solution would be an undesirable component for cell cryopreservation, which may introduce xenogeneic factors and cause spontaneous differentiation [ [15] , [16] , [17] ].…”

“…Recently, researchers have designed and synthesized CPAs for DMSO-free/FBS-free cryopreservation of cells [ 16 , [18] , [19] , [20] ]. Meanwhile, various innovative cryopreservation techniques, such as, cell printing, microfluidic platforms, and liquid marbles (LMs), have also been used to improve the viability and efficiency of cell cryopreservation [ [21] , [22] , [23] , [24] ].…”