Chemical approaches to cryopreservation

Abstract

Cryopreservation of cells and biologics underpins all biomedical research from routine sample storage to emerging cell-based therapies, as well as ensuring cell banks provide authenticated, stable and consistent cell products. This field began with the discovery and wide adoption of glycerol and dimethyl sulfoxide as cryoprotectants over 60 years ago, but these tools do not work for all cells and are not ideal for all workflows. In this Review, we highlight and critically review the approaches to discover, and apply, new chemical tools for cryopreservation. We summarize the key (and complex) damage pathways during cellular cryopreservation and how each can be addressed. Bio-inspired approaches, such as those based on extremophiles, are also discussed. We describe both small-molecule-based and macromolecular-based strategies, including ice binders, ice nucleators, ice nucleation inhibitors and emerging materials whose exact mechanism has yet to be understood. Finally, looking towards the future of the field, the application of bottom-up molecular modelling, library-based discovery approaches and materials science tools, which are set to transform cryopreservation strategies, are also included.

Recent Advances in Macroporous Hydrogels for Cell Behavior and Tissue Engineering

“…Recently, Muuray et al summarized the key factors of cell damage during freezing: (1) The formation of ice crystals in the ECM creates an osmotic gradient in the cell membrane, which leads to cell dehydration. (2) Concentration of cryoprotectants at low temperatures leads to osmotic shock or toxic damage to cells [ 133 ]. Common cell cryoprotectants include dimethyl sulfoxide and glycerol.…”

“…In addition, cryoprotectants have potential adverse effects on patients, including tonic-clonic seizure and cardiac arrest [ 135 ]. Many advanced cell cryopreservation agents have been developed, including vitrification agents, ice recrystallization inhibitors, macromolecular cryoprotectants, ice nucleators, cell encapsulation, intracellular CPA delivery, and the modulation of biochemical pathways [ 133 ]. Understanding and utilizing more advanced cell cryopreservation technology will help to expand the application effect and scope of cryogel in tissue engineering.…”

Assay-ready Cryopreserved Cell Monolayers Enabled by Macromolecular Cryoprotectants

“…To address the challenges of cryopreservation, natural ,− and synthetic − small molecules and macromolecules have been explored, which are inspired by extremophiles and capable of inhibiting ice recrystallization (IRI) and modulating ice growth and formation . IRI has been shown to benefit red blood cell cryopreservation; ,,, however, the magnitude of benefit during monolayer or suspension cryopreservation of nucleated cells is limited. − Protective osmolytes such as trehalose and l -proline can mitigate some damage during monolayer freezing, increasing the recovery of Neuro-2a cell monolayers from 13 to 53% and HepG2 cells from 13 to 42% .…”

Strategies in developing dimethyl sulfoxide (DMSO)-free cryopreservation protocols for biotherapeutics

“…The situation is considerably more critical during vitrification; a freezing method that has attracted heightened recognition as a faster and economic substitute to slow freezing as the unorganized liquid state of water is rapidly transformed to a glassy solid state without ice crystallization ( 10 ). In vitrification, high cooling rates and high concentrations (4–8 M) of cryoprotectants are usually required ( 11 ); enforcing on the exigency of using non-toxic cryoprotectants because increasing DMSO concentration is not advisable.…”