Cryopreservation and Rapid Recovery of Differentiated Intestinal Epithelial Barrier Cells at Complex Transwell Interfaces Is Enabled by Chemically Induced Ice Nucleation.

Cell-based models, such as organ-on-chips, can replace and inform in vivo (animal) studies for drug discovery, toxicology, and biomedical science, but most cannot be banked "ready to use" as they do not survive conventional cryopreservation with DMSO alone. Here, we demonstrate how macromolecular ice nucleators enable the successful cryopreservation of epithelial intestinal models supported upon the interface of transwells, allowing recovery of function in just 7 days post-thaw directly from the freezer, compared to 21 days from conventional suspension cryopreservation. Caco-2 cells and Caco-2/HT29-MTX cocultures are cryopreserved on transwell inserts, with chemically induced ice nucleation at warmer temperatures resulting in increased cell viability but crucially retaining the complex cellular adhesion on the transwell insert interfaces, which other cryoprotectants do not. Trans-epithelial electrical resistance measurements, confocal microscopy, histology, and whole-cell proteomics demonstrated the rapid recovery of differentiated cell function, including the formation of tight junctions. Lucifer yellow permeability assays confirmed that the barrier functions of the cells were intact. This work will help solve the long-standing problem of transwell tissue barrier model storage, facilitating access to advanced predictive cellular models. This is underpinned by precise control of the nucleation temperature, addressing a crucial biophysical mode of damage.

Understanding the impact of ammonium ion substitutions on heterogeneous ice nucleation.

Understanding the mechanisms underpinning heterogeneous ice nucleation in the presence of ionic inclusions is important for fields such as cryopreservation and for improved models of climate and weather prediction. Feldspar and ammonium are both present in significant quantities in the atmosphere, and experimental evidence has shown that feldspar can nucleate ice from ammonium-containing solutions at temperatures warmer than water alone. In recent work, Whale hypothesised that this increase in nucleation temperature is due to an increase in configurational entropy when an ammonium ion is included in the ice hydrogen bond network (T. F. Whale, J. Chem. Phys., 2022, 156, 144503). In this work, we investigate the impact of the inclusion of an ammonium ion on the hydrogen bond network by direct enumeration of the number of structures found using Rick's algorithm. We also determine the energy of these systems and thus compare the effects of enthalpy and entropy to test Whale's hypothesis. We find that the inclusion of an ammonium ion increases the total number of configurations under conditions consistent with a realistic surface charge. We also find that the enthalpic contribution is dominant in determining the location of the ammonium ion within the structure, although we note that this neglects other practicalities of ice nucleation.

The limit of macroscopic homogeneous ice nucleation at the nanoscale.

Nucleation in small volumes of water has garnered renewed interest due to the relevance of pore condensation and freezing under conditions of low partial pressures of water, such as in the upper troposphere. Molecular simulations can in principle provide insight on this process at the molecular scale that is challenging to achieve experimentally. However, there are discrepancies in the literature as to whether the rate in confined systems is enhanced or suppressed relative to bulk water at the same temperature and pressure. In this study, we investigate the extent to which the size of the critical nucleus and the rate at which it grows in thin films of water are affected by the thickness of the film. Our results suggest that nucleation remains bulk-like in films that are barely large enough accommodate a critical nucleus. This conclusion seems robust to the presence of physical confining boundaries. We also discuss the difficulties in unambiguously determining homogeneous nucleation rates in nanoscale systems, owing to the challenges in defining the volume. Our results suggest any impact on a film's thickness on the rate is largely inconsequential for present day experiments.

Proline-conditioning and chemically-programmed ice nucleation protects spheroids during cryopreservation.

Spheroids mimic 3-D tissue niches better than standard cell cultures. Cryopreserving spheroids, however, remains challenging as conventional cryoprotectants do not mitigate all damage mechanisms. Here chemically-programmed extracellular ice nucleation is used to prevent supercooling, alongside proline pre-conditioning, which are found to synergystically improve post-thaw recovery of spheroids. This validates the need to identify compounds and materials to address both biochemical and biophysical damage pathways beyond standard cryoprotectants.

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

3D cell assemblies such as spheroids reproduce the in vivo state more accurately than traditional 2D cell monolayers and are emerging as tools to reduce or replace animal testing. Current cryopreservation methods are not optimized for complex cell models, hence they are not easily banked and not as widely used as 2D models. Here we use soluble ice nucleating polysaccharides to nucleate extracellular ice and dramatically improve spheroid cryopreservation outcomes. This protects the cells beyond using DMSO alone, and with the major advantage that the nucleators function extracellularly and hence do not need to permeate the 3D cell models. Critical comparison of suspension, 2D and 3D cryopreservation outcomes demonstrated that warm-temperature ice nucleation reduces the formation of (fatal) intracellular ice, and in the case of 2/3D models this reduces propagation of ice between adjacent cells. This demonstrates that extracellular chemical nucleators could revolutionize the banking and deployment of advanced cell models.

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

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.

Poly(vinyl alcohol) Molecular Bottlebrushes Nucleate Ice.

Ice binding proteins (IBP) have evolved to limit the growth of ice but also to promote ice formation by ice-nucleating proteins (INPs). IBPs, which modulate these seemingly distinct processes, often have high sequence similarities, and molecular size/assembly is hypothesized to be a crucial determinant. There are only a few synthetic materials that reproduce INP function, and rational design of ice nucleators has not been achieved due to outstanding questions about the mechanisms of ice binding. Poly(vinyl alcohol) (PVA) is a water-soluble synthetic polymer well known to effectively block ice recrystallization, by binding to ice. Here, we report the synthesis of a polymeric ice nucleator, which mimics the dense assembly of IBPs, using confined ice-binding polymers in a high-molar-mass molecular bottlebrush. Poly(vinyl alcohol)-based molecular bottlebrushes with different side-chain densities were synthesized via a combination of ring-opening metathesis polymerization (ROMP) and reversible addition-fragmentation chain-transfer (RAFT) polymerization, using "grafting-to" and "grafting-through" approaches. The facile preparation of the PVA bottlebrushes was performed via selective hydrolysis of the acetate of the poly(vinyl acetate) (PVAc) side chains of the PVAc bottlebrush precursors. Ice-binding polymer side-chain density was shown to be crucial for nucleation activity, with less dense brushes resulting in colder nucleation than denser brushes. This bio-inspired approach provides a synthetic framework for probing heterogeneous ice nucleation and a route toward defined synthetic nucleators for biotechnological applications.

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

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.

The role of structural order in heterogeneous ice nucleation.

The freezing of water into ice is a key process that is still not fully understood. It generally requires an impurity of some description to initiate the heterogeneous nucleation of the ice crystals. The molecular structure, as well as the extent of structural order within the impurity in question, both play an essential role in determining its effectiveness. However, disentangling these two contributions is a challenge for both experiments and simulations. In this work, we have systematically investigated the ice-nucleating ability of the very same compound, cholesterol, from the crystalline (and thus ordered) form to disordered self-assembled monolayers. Leveraging a combination of experiments and simulations, we identify a "sweet spot" in terms of the surface coverage of the monolayers, whereby cholesterol maximises its ability to nucleate ice (which remains inferior to that of crystalline cholesterol) by enhancing the structural order of the interfacial water molecules. These findings have practical implications for the rational design of synthetic ice-nucleating agents.

Disordering effect of the ammonium cation accounts for anomalous enhancement of heterogeneous ice nucleation.

Heterogeneous nucleation of ice from supercooled water is the process responsible for triggering nearly all ice formation in the natural environment. Understanding of heterogeneous ice nucleation is particularly key for understanding the formation of ice in clouds, which impacts weather and climate. While many effective ice nucleators are known, the mechanisms of their actions remain poorly understood. Some inorganic nucleators have been found to nucleate ice at warmer temperatures in dilute ammonium solution than in pure water. This is surprising, analogous to salty water melting at a warmer temperature than pure water. Here, the magnitude of this effect is rationalized as being due to thermodynamically favorable ammonium-induced disordering of the hydrogen bond network of ice critical clusters formed on inorganic ice nucleators. Theoretical calculations are shown to be consistent with new experimental measurements aimed at finding the maximum magnitude of the effect. The implication of this study is that the ice-nucleating sites and surfaces of many inorganic ice nucleators are either polar or charged and, therefore, tend to induce formation of hydrogen-ordered ice clusters. This work corroborates various literature reports, indicating that some inorganic ice nucleators are most effective when nominally neutral, and implies a commonality in mechanism between a wide range of inorganic ice nucleators.

Polymer Self-Assembly Induced Enhancement of Ice Recrystallization Inhibition.

Ice binding proteins modulate ice nucleation/growth and have huge (bio)technological potential. There are few synthetic materials that reproduce their function, and rational design is challenging due to the outstanding questions about the mechanisms of ice binding, including whether ice binding is essential to reproduce all their macroscopic properties. Here we report that nanoparticles obtained by polymerization-induced self-assembly (PISA) inhibit ice recrystallization (IRI) despite their constituent polymers having no apparent activity. Poly(ethylene glycol), poly(dimethylacrylamide), and poly(vinylpyrrolidone) coronas were all IRI-active when assembled into nanoparticles. Different core-forming blocks were also screened, revealing the core chemistry had no effect. These observations show ice binding domains are not essential for macroscopic IRI activity and suggest that the size, and crowding, of polymers may increase the IRI activity of "non-active" polymers. It was also discovered that poly(vinylpyrrolidone) particles had ice crystal shaping activity, indicating this polymer can engage ice crystal surfaces, even though on its own it does not show any appreciable ice recrystallization inhibition. Larger (vesicle) nanoparticles are shown to have higher ice recrystallization inhibition activity compared to smaller (sphere) particles, whereas ice nucleation activity was not found for any material. This shows that assembly into larger structures can increase IRI activity and that increasing the "size" of an IRI does not always lead to ice nucleation. This nanoparticle approach offers a platform toward ice-controlling soft materials and insight into how IRI activity scales with molecular size of additives.

Cryopreservation of primary cultures of mammalian somatic cells in 96-well plates benefits from control of ice nucleation.

Cryopreservation of mammalian cells has to date typically been conducted in cryovials, but there are applications where cryopreservation of primary cells in multiwell plates would be advantageous. However excessive supercooling in the small volumes of liquid in each well of the multiwell plates is inevitable without intervention and tends to result in high and variable cell mortality. Here, we describe a technique for cryopreservation of adhered primary bovine granulosa cells in 96-well plates by controlled rate freezing using controlled ice nucleation. Inducing ice nucleation at warm supercooled temperatures (less than 5 °C below the melting point) during cryopreservation using a manual seeding technique significantly improved post-thaw recovery from 29.6% (SD = 8.3%) where nucleation was left uncontrolled to 57.7% (9.3%) when averaged over 8 replicate cultures (p < 0.001). Detachment of thawed cells was qualitatively observed to be more prevalent in wells which did not have ice nucleation control which suggests cryopreserved cell monolayer detachment may be a consequence of deep supercooling. Using an infra-red thermography technique we showed that many aliquots of cryoprotectant solution in 96-well plates can supercool to temperatures below -20 °C when nucleation is not controlled, and also that the freezing temperatures observed are highly variable despite stringent attempts to remove contaminants acting as nucleation sites. We conclude that successful cryopreservation of cells in 96-well plates, or any small volume format, requires control of ice nucleation.

High-speed imaging of ice nucleation in water proves the existence of active sites.

Understanding how surfaces direct nucleation is a complex problem that limits our ability to predict and control crystal formation. We here address this challenge using high-speed imaging to identify and quantify the sites at which ice nucleates in water droplets on the two natural cleavage faces of macroscopic feldspar substrates. Our data show that ice nucleation only occurs at a few locations, all of which are associated with micron-size surface pits. Similar behavior is observed on α-quartz substrates that lack cleavage planes. These results demonstrate that substrate heterogeneities are the salient factor in promoting nucleation and therefore prove the existence of active sites. We also provide strong evidence that the activity of these sites derives from a combination of surface chemistry and nanoscale topography. Our results have implications for the nucleation of many materials and suggest new strategies for promoting or inhibiting nucleation across a wide range of applications.

Unravelling the origins of ice nucleation on organic crystals.

Organic molecules such as steroids or amino acids form crystals that can facilitate the formation of ice - arguably the most important phase transition on earth. However, the origin of the ice nucleating ability of organic crystals is still largely unknown. Here, we combine experiments and simulations to unravel the microscopic details of ice formation on cholesterol, a prototypical organic crystal widely used in cryopreservation. We find that cholesterol - which is also a substantial component of cell membranes - is an ice nucleating agent more potent than many inorganic substrates, including the mineral feldspar (one of the most active ice nucleating materials in the atmosphere). Scanning electron microscopy measurements reveal a variety of morphological features on the surfaces of cholesterol crystals: this suggests that the topography of the surface is key to the broad range of ice nucleating activity observed (from -4 to -20 °C). In addition, we show via molecular simulations that cholesterol crystals aid the formation of ice nuclei in a unconventional fashion. Rather than providing a template for a flat ice-like contact layer (as found in the case of many inorganic substrates), the flexibility of the cholesterol surface and its low density of hydrophilic functional groups leads to the formation of molecular cages involving both water molecules and terminal hydroxyl groups of the cholesterol surface. These cages are made of 6- and, surprisingly, 5-membered hydrogen bonded rings of water and hydroxyl groups that favour the nucleation of hexagonal as well as cubic ice (a rare occurrence). We argue that the phenomenal ice nucleating activity of steroids such as cholesterol (and potentially of many other organic crystals) is due to (i) the ability of flexible hydrophilic surfaces to form unconventional ice-templating structures and (ii) the different nucleation sites offered by the diverse topography of the crystalline surfaces. These findings clarify how exactly organic crystals promote the formation of ice, thus paving the way toward deeper understanding of ice formation in soft and biological matter - with obvious reverberations on atmospheric science and cryobiology.

Correction: The enhancement and suppression of immersion mode heterogeneous ice-nucleation by solutes.

[This corrects the article DOI: 10.1039/C7SC05421A.].

The study of atmospheric ice-nucleating particles via microfluidically generated droplets.

Ice-nucleating particles (INPs) play a significant role in the climate and hydrological cycle by triggering ice formation in supercooled clouds, thereby causing precipitation and affecting cloud lifetimes and their radiative properties. However, despite their importance, INP often comprise only 1 in 103-106 ambient particles, making it difficult to ascertain and predict their type, source, and concentration. The typical techniques for quantifying INP concentrations tend to be highly labour-intensive, suffer from poor time resolution, or are limited in sensitivity to low concentrations. Here, we present the application of microfluidic devices to the study of atmospheric INPs via the simple and rapid production of monodisperse droplets and their subsequent freezing on a cold stage. This device offers the potential for the testing of INP concentrations in aqueous samples with high sensitivity and high counting statistics. Various INPs were tested for validation of the platform, including mineral dust and biological species, with results compared to literature values. We also describe a methodology for sampling atmospheric aerosol in a manner that minimises sampling biases and which is compatible with the microfluidic device. We present results for INP concentrations in air sampled during two field campaigns: (1) from a rural location in the UK and (2) during the UK's annual Bonfire Night festival. These initial results will provide a route for deployment of the microfluidic platform for the study and quantification of INPs in upcoming field campaigns around the globe, while providing a benchmark for future lab-on-a-chip-based INP studies.

The enhancement and suppression of immersion mode heterogeneous ice-nucleation by solutes.

Heterogeneous nucleation of ice from aqueous solutions is an important yet poorly understood process in multiple fields, not least the atmospheric sciences where it impacts the formation and properties of clouds. In the atmosphere ice-nucleating particles are usually, if not always, mixed with soluble material. However, the impact of this soluble material on ice nucleation is poorly understood. In the atmospheric community the current paradigm for freezing under mixed phase cloud conditions is that dilute solutions will not influence heterogeneous freezing. By testing combinations of nucleators and solute molecules we have demonstrated that 0.015 M solutions (predicted melting point depression <0.1 °C) of several ammonium salts can cause suspended particles of feldspars and quartz to nucleate ice up to around 3 °C warmer than they do in pure water. In contrast, dilute solutions of certain alkali metal halides can dramatically depress freezing points for the same nucleators. At 0.015 M, solutes can enhance or deactivate the ice-nucleating ability of a microcline feldspar across a range of more than 10 °C, which corresponds to a change in active site density of more than a factor of 105. This concentration was chosen for a survey across multiple solutes-nucleant combinations since it had a minimal colligative impact on freezing and is relevant for activating cloud droplets. Other nucleators, for instance a silica gel, are unaffected by these 'solute effects', to within experimental uncertainty. This split in response to the presence of solutes indicates that different mechanisms of ice nucleation occur on the different nucleators or that surface modification of relevance to ice nucleation proceeds in different ways for different nucleators. These solute effects on immersion mode ice nucleation may be of importance in the atmosphere as sea salt and ammonium sulphate are common cloud condensation nuclei (CCN) for cloud droplets and are internally mixed with ice-nucleating particles in mixed-phase clouds. In addition, we propose a pathway dependence where activation of CCN at low temperatures might lead to enhanced ice formation relative to pathways where CCN activation occurs at higher temperatures prior to cooling to nucleation temperature.

The role of phase separation and related topography in the exceptional ice-nucleating ability of alkali feldspars.

Our understanding of crystal nucleation is a limiting factor in many fields, not least in the atmospheric sciences. It was recently found that feldspar, a component of airborne desert dust, plays a dominant role in triggering ice formation in clouds, but the origin of this effect was unclear. By investigating the structure/property relationships of a wide range of feldspars, we demonstrate that alkali feldspars with certain microtextures, related to phase separation into Na and K-rich regions, show exceptional ice-nucleating abilities in supercooled water. We found no correlation between ice-nucleating efficiency and the crystal structures or the chemical compositions of these active feldspars, which suggests that specific topographical features associated with these microtextures are key in the activity of these feldspars. That topography likely acts to promote ice nucleation, improves our understanding of ice formation in clouds, and may also enable the design and manufacture of bespoke nucleating materials for uses such as cloud seeding and cryopreservation.

A marine biogenic source of atmospheric ice-nucleating particles.

The amount of ice present in clouds can affect cloud lifetime, precipitation and radiative properties. The formation of ice in clouds is facilitated by the presence of airborne ice-nucleating particles. Sea spray is one of the major global sources of atmospheric particles, but it is unclear to what extent these particles are capable of nucleating ice. Sea-spray aerosol contains large amounts of organic material that is ejected into the atmosphere during bubble bursting at the organically enriched sea-air interface or sea surface microlayer. Here we show that organic material in the sea surface microlayer nucleates ice under conditions relevant for mixed-phase cloud and high-altitude ice cloud formation. The ice-nucleating material is probably biogenic and less than approximately 0.2 micrometres in size. We find that exudates separated from cells of the marine diatom Thalassiosira pseudonana nucleate ice, and propose that organic material associated with phytoplankton cell exudates is a likely candidate for the observed ice-nucleating ability of the microlayer samples. Global model simulations of marine organic aerosol, in combination with our measurements, suggest that marine organic material may be an important source of ice-nucleating particles in remote marine environments such as the Southern Ocean, North Pacific Ocean and North Atlantic Ocean.