Cryopreservation refers to a process used to preserve biological constructs (like 2D and 3D cell assemblies) by storing them at low temperatures, states a study by a team of researchers from Inje University, Republic of Korea. Â
This study highlights the existence of cryoinjury: cell damage occurring due to phase changes of water in extracellular (outside the cell) as well as intracellular (inside the cell) environments. This can occur because of osmotic rupture (happens due to an imbalance between concentrations across the cell’s outer layer) and intracellular ice formation (IIF: ice crystal formation within cells during rapid freezing).
On cryopreservation of a biological sample, nucleation (a process where molecular aggregates form in a liquid, which eventually form crystals) starts to occur in the extracellular space first, and occurs later in the intracellular space (within the cell), explains an account on intracellular ice formation by ATP-Bio Engineering Research Center.
On rapid freezing cryopreservation, along with extracellular nucleation, intracellular nucleation takes place; leading to IIF, mentions the account. On slow freezing cryopreservation, the nucleation happens in the extracellular environment first; this draws out the liquid content present inside the cell and leads to the cell’s dehydration, this does not lead to IIF, in comparison.
A recent study by a team of researchers from University of Warwick, UK, Swedish University of Agricultural Sciences, Sweden, and Southern University of Science and Technology, China, published in Journal of the American Chemical Society (JACS) Au, highlights the fact that, though this removal of water from cells reduces the fatal effects—loss of cell viability, cell injury, and cell death—of IIF, it is difficult to implement and is unnecessary for volumes in the range of milliliters, in which ice can form at warm temperatures.
The article states that the nucleation temperature varies with cell type and that cryopreservation of small amounts of cells is often challenging. This could be addressed by inducing nucleation in the extracellular space to protect the intracellular environment. However, there are very few methods, which can aid in inducing nucleation at warmer temperatures, with challenges of their own: ice-nucleating protein from Pseudomonus syringae has insoluble components, feldspar and silver iodide are not easily soluble, and electrofreezing is impractical and stress-inducing to be utilized for cryopreservation of cells.
However, a polysaccharide, available on few pollen grains, can act as soluble nucleators, states the study; pollen-washing water (PWW) is sterilizable easily; this, when combined with dimethylsulfoxide (DMSO: a cryoprotective agent that can eliminate ice formation), has proved to significantly increase post-thaw recovery of cell monolayers (a layer of cells where cells grow side by side, and no cell grows above another).
In their study, the researchers used soluble polysaccharide-based nucleators with DMSO to chemically trigger extracellular nucleation for the cryopreservation of 2D and 3D cell assemblies of human cancer cells. Their results revealed that for the 2D (monolayer) and 3D cell assemblies (spheroids) the post-thaw recovery rate increased largely; with 1.5-2 fold increase in recovery rates for 3D cell assemblies (spheroids).
The authors believe that chemically triggered extracellular ice nucleation can mitigate intracellular damage by limiting ice propagation between cells, and this can improve cryopreservation of complex cell assemblies. 3D cell assemblies, like spheroids and organoids, can reduce the dependency on animal testing. They can give a more accurate response to medications being tested on them. Improving 3D cell assemblies’ storage and distribution will be a step forward towards reducing the necessity of preclinical trials.
