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

Abstract

Cryopreservation of biological material is vital for existing and emerging biomedical and biotechnological research and related applications, but there remain significant challenges. Cryopreservation of cells in sub-milliliter volumes is difficult because they tend to deeply supercool, favoring lethal intracellular ice formation. Some tree pollens are known to produce polysaccharides capable of nucleating ice at warm sub-zero temperatures. Here we demonstrated that aqueous extractions from European hornbeam pollen (pollen washing water, PWW) increased ice nucleation temperatures in 96-well plates from ≈ − 13 °C to ≈ − 7 °C. Application of PWW to the cryopreservation of immortalized T-cells in 96-well plates resulted in an increase of post-thaw metabolic activity from 63.9% (95% CI [58.5 to 69.2%]) to 97.4% (95% CI [86.5 to 108.2%]) of unfrozen control. When applied to cryopreservation of immortalized lung carcinoma monolayers, PWW dramatically increased post-thaw metabolic activity, from 1.6% (95% CI [− 6.6 to 9.79%]) to 55.0% (95% CI [41.6 to 68.4%]). In contrast to other ice nucleating agents, PWW is soluble, sterile and has low cytotoxicity meaning it can be readily incorporated into existing cryopreservation procedures. As such, it can be regarded as a unique class of cryoprotectant which acts by inducing ice nucleation at warm temperatures.

Cryopreservation of Liver-Cell Spheroids with Macromolecular Cryoprotectants

“…The as-prepared spheroids were cryopreserved in two formats, either directly within the agar molds or as a suspension in cryovials following their release from the molds at −80 °C. We have previously reported how suspension versus monolayer cell cryopreservation can lead to dramatically different outcomes, 33 and hence this comparison of freezing formats was important. Figure 2 A shows the post-thaw viability of spheroids following cryopreservation with 10% DMSO and a range of concentrations of the polyampholyte (structure shown in Figure 1 B).…”

“…However, in this experiment, nucleation onset was ∼−20 °C, whereas nucleation of larger volumes, such as those in vials, would occur at much warmer temperatures. 33 , 34 Thus, nucleation was mechanically induced at – 8 °C to attempt to mimic the conditions in vial freezing; however, no intracellular ice growth was observed, Figure S11 . Hence, further study is required to determine if the polymer modulates intracellular ice formation under the exact spheroid freezing conditions due to this mismatch of nucleation temperatures.…”

“… 17 , 24 , 25 Cryoprotectants inspired by ice-binding proteins, 26 , 27 which can modulate ice growth (recrystallization), have been discovered and applied to cryopreservation. 28 − 32 Controlled nucleation (inspired by ice nucleating proteins) has also been shown to benefit 2-D cell monolayer cryopreservation 33 , 34 and spheroid cryopreservation. 35 Vitrification, which uses larger volumes of cryoprotectants (including glycerol and DMSO), can be applied to larger cell models, but the large volume of cryoprotectants is technically challenging to administer uniformly and remove post-thaw.…”

“…For nucleated (mammalian) cells, the most common cryopreservation procedure is based on dimethyl sulfoxide (DMSO) (typically 10%), which protects cells by dehydration, replacing intracellular water, and reducing osmotic shock. − Cryopreserving cells in suspension using DMSO typically works well, with >80% post-thaw recoveries possible, but the cryopreservation of more complex models including cell monolayers and, even more so, spheroids remains a major technological challenge. During the freezing of 2- and 3-D cell models, there are challenges of nutrient/cryoprotectant transport to overcome, as well as ice nucleation and propagation across cell–cell contacts. , Fine-tuning traditional cryopreservation formulations can improve these outcomes, ,, but innovative cryoprotectants are now emerging that can address the damage mechanisms that DMSO does not. ,, Cryoprotectants inspired by ice-binding proteins, , which can modulate ice growth (recrystallization), have been discovered and applied to cryopreservation. − Controlled nucleation (inspired by ice nucleating proteins) has also been shown to benefit 2-D cell monolayer cryopreservation , and spheroid cryopreservation . Vitrification, which uses larger volumes of cryoprotectants (including glycerol and DMSO), can be applied to larger cell models, but the large volume of cryoprotectants is technically challenging to administer uniformly and remove post-thaw.…”

Assay-ready Cryopreserved Cell Monolayers Enabled by Macromolecular Cryoprotectants

“… 16 IIF is a particular problem for the cryopreservation of cells as monolayers (and spheroids), compared to suspension cells, as cell–cell contacts promote the propagation of intracellular ice, 17 − 19 which is usually fatal and contributes to the low cell recovery rates (although IIF is not always deleterious). 20 To increase post-thaw cell recovery, IIF can be reduced by directional freezing 21 or manually inducing nucleation at warmer temperatures, for example using N 2 ice mist, 22 or pollen extracts 23 however reproducibly inducing nucleation is difficult using manual methods and chemically defined nucleating agents are not available.…”