A highly active mineral-based ice nucleating agent supports in situ cell cryopreservation in a high throughput format

Abstract

Cryopreservation of biological matter in microlitre scale volumes of liquid would be useful for a range of applications. At present, it is challenging because small volumes of water tend to supercool, and deep supercooling is known to lead to poor post-thaw cell viability. Here, we show that a mineral ice nucleator can almost eliminate supercooling in 100 µl liquid volumes during cryopreservation. This strategy of eliminating supercooling greatly enhances cell viability relative to cryopreservation protocols with uncontrolled ice nucleation. Using infrared thermography, we demonstrate a direct relationship between the extent of supercooling and post-thaw cell viability. Using a mineral nucleator delivery system, we open the door to the routine cryopreservation of mammalian cells in multiwell plates for applications such as high throughput toxicology testing of pharmaceutical products and regenerative medicine.

Chemically Induced Extracellular Ice Nucleation Reduces Intracellular Ice Formation Enabling 2D and 3D Cellular Cryopreservation

“…It should be reiterated at this point that cryopreservation in 96-well plates is extremely challenging due to supercooling of the aqueous solution and there are few examples of it being achieved. , Cell recovery was measured both with 10% DMSO alone (−IN) and with induced ice nucleation (cryoprotectant containing 10% DMSO and PWW) (+IN). Previous work has suggested this nucleator increases the temperature of ice nucleation in wells which is significant in the cryobiological context. ,,, As would be expected, experiments show significant biological variability between individual measurements. To account for this variability and establish the statistical significance of our results we have analyzed our data using a mixed-effects model .…”

“…The solubility and sterile nature of this chemical nucleator will enable its widespread use in 2D and 3D cryopreservation protocols, and the simplicity of addition to established cryopreservation solutions makes this easy to deploy compared to insoluble nucleators. While this study clearly demonstrates the efficacy of PWW as a nucleator for cryopreservation of cell-based models, it has recently been shown that warmer nucleation temperatures than those achieved using PWW will likely yield further improvements to cryopreservation outcomes, motivating further research into biocompatible ice nucleators . Altogether, this work shows that the design, discovery, and understanding of chemical ice nucleators is essential to enable banking and distribution of increasingly complex cell-based models.…”

“…For instance, Lauterboeck et al found that a nucleation temperature of −10 °C was optimal for mesenchymal stromal cells, 19 while it has been shown very recently that nucleation at the melting point was most beneficial for cryopreservation of human hepatocyte carcinoma cells. 18 Nevertheless, it is clear that deep supercooling during cryopreservation impairs cells recovery. Cryopreservation of cells in smaller volumes of liquids often proves challenging, however.…”

“…The original studies on controlled rate cryopreservation employed seeding with ice crystals to ensure ice formed extracellularly . This method is difficult to implement at scale, and is often unnecessary for cryopreservation of milliliter scale volumes, in which ice tends to form at warm temperatures even in the absence of deliberately introduced ice nucelatiors. , The optimum temperature for ice nucleation is not well established and may vary by cell type. For instance, Lauterboeck et al found that a nucleation temperature of −10 °C was optimal for mesenchymal stromal cells, while it has been shown very recently that nucleation at the melting point was most beneficial for cryopreservation of human hepatocyte carcinoma cells .…”

Investigation of Rapid Rewarming Chips for Cryopreservation by Joule Heating

“…32,33 The customary practice for reducing the degree of supercooling during cryopreservation is "manual" nucleation, where touching the outside of the vessel with a very cold object locally cools the contents enough to trigger ice nucleation. 34,35 However, the degree of supercooling can be significantly reduced without manual nucleation during the cooling of the EHC. We speculate that this may be due to the EHC's high specific surface area, which greatly increases the heterogeneous ice nucleating sites, significantly reducing the degree of supercooling during cooling.…”