Academic literature on the topic 'Cryobiology'

ABSTRACT Cryo banking of spermatozoa is an essential aspect of fertility preservation. With the advancements in cryobiology and better understanding of cryoprotectants and assisted reproduction, indications for semen banking are expanding. The exponential developments that have occurred over the years in the field of cryopreservation have proved that frozen sperm is as good as fresh sperm in fertilizing oocytes. Semen banking has major role in fertility preservation in cancer patients.

Thesis (Ph. D.)--University of Missouri-Columbia, 2007.
"May 2007" The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. Includes bibliographical references.

Preservation of tissue structure, morphology and biomarkers is of utmost importance for pathological examination of biopsy specimens for diagnostic and therapeutic purposes. However current methods employed to evade tissue degradation and preserve biomarkers have several shortcomings that include irreproducibility, morphological artifacts and altered biomarker antigenicity. These artifacts may affect the analysis and subsequent diagnosis of the tissue pathology. This creates need for developing improved preservation methods that reproducibly maintain tissue morphology and biomarker antigenicity and are simple, rapid and inexpensive. Experiments conducted for testing the hypothesis that cryopreservation procedures yield high quality morphology and antigenicity showed that cryopreservation maintains tissue structure, morphology and antigenicity at equivalent or better levels compared to standard freezing techniques. In order to understand the mechanisms of osmotic transport in cellular systems upon exposure to multi-component solutions that are prevalent in virtification protocols, experimental studies were undertaken using microfluidics for single cell manipulation. The experimental data yielded permeability parameters in binary and ternary solutions for MC3T3-E1 murine osteoblasts for the first time. The hydraulic conductivity (L(p)) decreased with increasing concentrations but the solute permeability either increased or decreased with increasing solution concentration. The changes in hydraulic conductivity were consistent with previously published trends and conform to a functional relationship in the form of Arrhenius type relationship between L(p) and solution concentration. Further a theoretical model was developed from principles of linear irreversible thermodynamics to simulate multi--‐‑component mass transport across membrane. The model was successfully validated by comparison with experimental data for murine osteoblasts and showed good agreement between the numerical predictions and experimental observations. The modeling approach can be used to investigate the transport mechanisms, which show that in multicomponent osmotic transport response, the dynamics is dictated by slower moving solute.

Freeze-injury at the plasma membrane level has been identified as being crucial for the survival of living matter. Since plasma membranes consist of several micro domains that make the structure rather complex, this study attempted to use simple model membranes to investigate the changes of phospholipid bilayers at sub-zero temperatures. Egg yolk L-α-phosphatidylcholine (EPC) and 1, 2-dipalmitoyl-rac-glycero-3-phosphocholine (DPPC) that mimic plasma membranes in their unique ways were used to prepare large unilamellar vesicles (LUV), which were the model membranes of this study. At cooling rates of 0.5 and 10�C/min, LUV were freeze-concentrated in the unfrozen matrix as a result of the advancing extraliposomal ice front and the decreasing phase volume of the unfrozen matrix, both of which led to membrane lesion. At the slow cooling rate of 0.5�C/min, an additional freezing stress imposed by the osmotic gradient across the bilayers, due to the increase of solute concentration in the unfrozen matrix, promoted leakage of LUV. The gel-liquid crystal phase transition temperature of phospholipids played an important role in determining if the LUV could withstand freezing stress when the LUV were held at a defined sub-zero temperature for a given period of holding time. EPC LUV were more leaky than DPPC LUV when they were held at the high sub-zero temperatures and their leakage increased with increasing holding time. The leakiness of EPC LUV could be related to the fluid and deformable nature of the EPC above its phase transition temperature. In contrast, DPPC LUV with a higher gel-liquid crystal phase transition temperature compared to EPC may become increasingly fragile at lower sub-zero temperatures, which led to the increase of leakage when the DPPC LUV were held at the lower sub-zero temperatures. These results indicated that the determination of the fatty acid profile of the plasma membranes was essential to aid in developing the most suitable holding temperature and time during the cryopreservation of biological specimens. Adding to the integrity of LUV that depended on the gel-liquid crystal phase transition temperature of phospholipids, intraliposomal ice formation also depended on the phase transition temperature of phospholipids. Intraliposomal ice formation was only observed for DPPC LUV but not for EPC LUV. In addition to the extraliposomal ice formation, other physical changes such as the eutectic crystallization of sodium chloride (NaCl) and ice mixture on the stability of LUV were also investigated. The eutectic crystallization of NaCl/ice mixture was governed by the intra- and extraliposomal distribution of NaCl and was more likely to occur at the physiological NaCl concentrations compared to lower NaCl concentrations. The eutectic crystallization of NaCl/ice mixture further increased the leakage of LUV. The understanding of the freezing behaviour and the mechanisms of freeze-injury of LUV allowed the use of the current model membranes for further investigations of the cryoprotective actions of cryoprotective agents (CPA). Partial phase diagrams of sugar-salt-water, dimethyl sulfoxide (DMSO)-salt-water and ethylene glycol (EG)-salt-water systems that resembled extraliposomal solute compositions were constructed and the phase volume of ice and unfrozen matrix was estimated from the freezing curves. Ice reduction was the major mechanism by which the non-permeable and permeable CPA protected the LUV from freeze-injury. Other cryoprotective mechanisms of the non-permeable and permeable CPA through the dilution and spacing out of the LUV in the unfrozen matrix as well as the suppression of the eutectic crystallization of NaCl/ice mixture were not ruled out. Non-permeable CPA were more effective in preventing leakage of DPPC than EPC LUV. Unlike the non-permeable CPA, permeable CPA were more effective for EPC than DPPC LUV that had been subjected to freezing and thawing processes. At room temperature, however, DMSO and EG were detrimental to the stability of DPPC LUV. The choice of CPA is strictly dependent on the type of phospholipids that varied in their acyl chain length and phase transition temperature. In summary, this study provides insights of the freeze-injury of LUV and the cryoprotective mechanisms of the non-permeable and permeable CPA which are beneficial to the field of cryopreservation that often depends on empirical trial and error methods. By integrating a comprehensive molecular-based understanding, an optimal cryopreservation procedure could be designed.

With emerging trends of new cellular therapies, the need for quick access to cellular components is necessary. For most applications genetically compatible biological components are essential to prevent adverse immune responses and graft-versus host disease (GVHD). Since these biological components have a limited window to be used, techniques for long-term storage are needed. Cryopreservation is essential for this in the field of biobanking and regenerative medicine to allow for long-term storage of cell products. During this process, ice recrystallization is the major contributor to cell death and decreased cell viability post-thaw. Due to this, controlling ice growth and recrystallization is imperative to increasing cell survival and function. The Ben lab is focused on the synthesis of small molecule, carbohydrate-based cryoprotectants that function as ice recrystallization inhibitors (IRIs). Previously, many IRIs have been synthesized showing varying degrees of ice recrystallization inhibition (IRI). Through the structure-function work, a delicate balance between hydrophobic and hydrophilic portions on the same molecule must be met. These compounds are believed to disrupt hydrogen bonding networks present in the formation of ice, and control ice growth. While numerous types of functional groups on carbohydrate derivatives have been explored, many highly solvated functional groups (amines, sulfates, phosphates, etc.) have not been thoroughly investigated. Highly solvated functional groups should disrupt hydrogen bond networks due to their solvation and in theory, should illicit an IRI response. Sulfate groups have not previously been studied, but are present in several different biological processes, such as immune response and blood coagulation. This suggests that sulfated carbohydrates should be well tolerated biologically. Sulfate groups can also be easily installed on existing IRI active molecules through orthogonal protecting group chemistry. The first part of this thesis is focused on the synthesis and IRI activity of sulfated carbohydrates based upon previously synthesized, IRI active pyranose derivatives. When compared to their parent compounds, most of the sulfated derivatives were less active, but all compounds were incredibly soluble in aqueous media. These derivatives did not show much promise as new IRIs due to the length of their synthesis and reduced IRI activity compared to their parent compounds. The Ben lab has also developed a new class of IRI active carbohydrates: aldonamide derivatives. These compounds are open-chain carbohydrates with an amide bond, arising from the ring opening of a carbohydrate lactone with a substituted amine. While many of these compounds displayed high degrees of IRI activity, many were incredibly insoluble in aqueous systems (many with solubility limits under 50 mM). Since sulfate groups were able to greatly increase solubility with some derivatives retaining IRI activity, installing sulfate groups on existing aldonamide-based IRIs should increase their solubility. Additionally, since many of these derivatives display high degrees of IRI activity, a reduction in IRI activity can be tolerated. Similarly, to the sulfated pyranose derivatives, the presence of a sulfate group reduced the IRI activity compared to the parent compounds in most derivatives. Though some sulfated derivatives possessed a higher degree of IRI activity, all the derivatives experienced a drastic increase in solubility (over 200 mM in PBS). Some of the sulfated aldonamide derivatives were assessed for their ability to protect red blood cells (RBCs) during freezing with reduced glycerol concentrations (15% glycerol), although none of thew tested derivatives showed an improvement over existing IRIs explored by the Ben lab. Since the introduction of sulfate groups to existing IRIs drastically increased solubility in aqueous systems, but resulted in reduced IRI activity in most compounds, focus was switched to the addition of different hydrophilic functional groups. Amino functional groups were briefly explored with galactose-based pyranose IRIs, aldonamide derivatives had not been explored. Amino groups are present on many biological carbohydrates and should be well tolerated biologically. The addition of amino groups to aldonamide derivatives should increase solubility, with the amino derivatives ideally retaining some IRI activity. The amino aldonamide derivatives synthesized had high solubilities (>500 mM in PBS), but did possess lower degrees of IRI activity. Due to the high solubility these derivatives were initially assessed in the cryopreservation of RBCs with reduced glycerol concentrations. Initial experiments showed improvements over current IRIs, and the compounds were assessed in a number of other biological cryopreservation scenarios including articular cartilage, platelets, and hematopoietic stem/progenitor cells (HSPCs). While the compounds showed toxicity in these cell types, more studies need to be conducted for the cryopreservation of RBCs.

Le specie animali, sia domestiche che selvatiche, rappresentano un patrimonio di biodiversità genetica di inestimabile valore in tutto il mondo. Le loro caratteristiche intrinseche contribuiscono a sostenere la prosperità economica e il benessere umano, nonostante i profondi cambiamenti in atto a livello globale e locale. Conoscere, valorizzare e conservare queste preziose risorse genetiche è un dovere sociale, culturale, scientifico ed economico e rappresenta la chiave dell’adattamento e della sopravvivenza in un ambiente dominato dall’uomo. L’Italia, essendo caratterizzata da una grande varietà di paesaggi ambientali e climi estremamente diversi, è considerata uno dei paesi europei più ricchi di biodiversità animale con il maggior numero di endemismi. Tuttavia, l’effetto combinato di azioni antropiche dannose e l’introduzione dei moderni sistemi di produzione intensiva, associati all’aumento del reddito, della popolazione umana e all’urbanizzazione, hanno portato ad un aumento drammatico del numero di specie autoctone in via di estinzione, guidando il paese verso un irreversibile perdita di risorse genetiche. In questo scenario, il numero di razze avicole e cunicole italiane, così come le naturali popolazioni autoctone di Trota Mediterranea, sono attualmente in allarmante declino. Pertanto, negli ultimi due decenni, queste preziose risorse genetiche sono diventate il fulcro di importanti programmi di conservazione, finalizzati esclusivamente al mantenimento delle popolazioni in situ. Secondo l’Organizzazione delle Nazioni Unite per l’alimentazione e l’agricoltura (FAO), questa strategia di conservazione è considerata prioritaria per la salvaguardia delle popolazioni a rischio di estinzione. Tuttavia, a causa della complessità del mantenimento degli animali vivi, questo tipo di approccio risulta spesso troppo costoso ed il raggiungimento degli obiettivi genetici procede ad un ritmo relativamente lento. Di conseguenza, la strategia di conservazione ex situ in vitro sta ottenendo sempre più maggiore attenzione nell’ambito della gestione e conservazione delle risorse genetiche animali, come supporto integrato alla strategia in situ. In particolare, la possibilità di utilizzare il seme congelato nella pratica dell’inseminazione artificiale (AI), rappresenta un fattore chiave per assicurare la conservazione a lungo termine della diversità genetica, attraverso l’istituzione di una criobanca del seme. In questo contesto, l’urgente bisogno di istituire azioni nazionali volte a supportare l’attuazione di programmi di conservazione ex situ in vitro, ha recentemente promosso il finanziamento di tre progetti, di cui uno europeo 1) “Recovery of S. macrostigma: Application of innovative techniques and participatory governance tools in the rivers of Molise” (Life Nat.Sal.Mo); e due italiani 2) “Tutela della Biodiversità nelle razze Avicole Italiane” (TuBAvI); 3) “La Cunicoltura del Futuro: benessere e sostenibilità degli allevamenti cunicoli italiani” (Cun-Fu). I presenti progetti, sono caratterizzati da uno scopo comune, ossia quello di preservare e valorizzare le razze avicole e cunicole italiane (TuBAvI e Cun-Fu) e le popolazioni native di Trota Mediterranea (Life Nat.Sal.Mo), attraverso l’uso combinato di strategie di conservazione in situ ed ex situ. Pertanto, nell’ambito di questi progetti, la creazione delle prime criobanche del seme italiane finalizzate alla salvaguardia di questo prezioso patrimonio di biodiversità animale, ha rappresentato un importante pietra miliare. A tal riguardo, sono divenuti necessari studi sistematici dei principali fattori in grado di influenzare il successo delle procedure di crioconservazione del seme di tacchino, coniglio e Trota Mediterranea, al fine di ridurre il danno cellulare provocato dal processo di congelamento/scongelamento. Alla luce di questa premessa, le attività di ricerca incluse nella presente tesi di dottorato sono parte integrante dei tre progetti più estesi sopramenzionati, il cui scopo è stato quello di sviluppare efficaci procedure di riferimento per la crioconservazione del seme di queste preziose risorse genetiche italiane, al fine di garantirne la conservazione a lungo termine. Inoltre, in linea con il raggiungimento dei presenti obiettivi, è stato effettuato anche uno studio di proteomica comparativa che ha avuto come scopo quello di confrontare i pattern proteici del seme di coniglio fresco e crioconservato. La logica di questa ricerca è stata quella di svelare, per la prima volta, i principali meccanismi coinvolti nel danno criogenico del seme nella specie cunicola. I risultati di questi studi sono presentati sotto forma di sei manoscritti pubblicati e una bozza di articolo, opportunamente ripartiti in quattro sessioni come segue: • la sessione 1 include tre studi finalizzati ad ottimizzare e identificare una procedura di riferimento per la crioconservazione del seme di Trota Mediterranea, ed i risultati ottenuti dall’avvio della prima criobanca del seme per questa specie; • la sessione 2 include due studi il cui scopo è stato quello di identificare e standardizzare una procedura di riferimento per il congelamento del seme di tacchino, attraverso la valutazione della sensibilità al congelamento in vitro e della capacità fertilizzante in vivo. Inoltre, sono riportate nel dettaglio le attività svolte ed i risultati ottenuti dalla creazione della prima criobanca italiana del seme di razze avicole autoctone; • la sessione 3 include uno studio il cui obiettivo è stato quello di sviluppare una procedura di riferimento per la crioconservazione del seme di coniglio, attraverso la comparazione di due diluenti e tre differenti dosi di inseminazione. Inoltre, sono riportati i risultati ottenuti dall’avvio della prima criobanca italiana del seme di razze cunicole autoctone; • la sessione 4 include i risultati sperimentali ottenuti dalla prima analisi comparativa del profilo proteico del seme di coniglio fresco e congelato, finalizzato a valutare i principali meccanismi fisiologici e biochimici influenzati dalla procedura di criocoservazione del seme nella specie cunicola.
Both domestic and wild native animal species represent a genetic biodiversity heritage of inestimable value all over the world. Their valuable intrinsic properties contribute to support economic prosperity and human well-being, despite the deep changes that are “globally” and “locally” taking place. Knowing, valorizing and conserving these precious genetic resources is a social, cultural, scientific and economic imperative that is the key to adaptation and survival in a human dominated environment. Italy appears to be one of the richest countries in animal biodiversity within the European Union. Because of the presence of extremely different environments, it has the highest number and density of animal species characterized by a high rate of endemism. However, the combined effect of harmful anthropic actions and the introduction of modern intensive production methods, associated to increasing income, human population and urbanization, has led to a dramatic increase in the number of endangered native species, resulting in an irreversible loss of genetic resources. In this scenario, Italian biodiversity of local poultry and rabbit breeds, as well as native Mediterranean trout populations, is currently in an alarming decline. As result, over the last two decades, these valuable genetic resources becoming the focus of important national conservation programs, exclusively aimed at the maintenance of live populations. According to the Food and Agriculture Organization of the United Nations (FAO), the in situ conservation strategy is in fact considered a priority for the safeguard of endangered populations. However, this approach, is often too expensive, and the achievement of genetic goals is going on at a relatively slow pace. Therefore, the ex situ in vitro strategy, as integrated support to the in situ strategy, is getting more and more attention. In particular, the use of artificial insemination (AI) with cryopreserved semen has been recognized as the most widespread method for ensuring the long-term conservation of genetic diversity, through the establishment of semen cryobanks. In this context, the urgent need to establish national actions aimed at promoting ex situ conservation programs, has recently favored the financing of three important projects, one of which is an European project: 1) Recovery of S. macrostigma: Application of innovative techniques and participatory governance tools in the rivers of Molise - (Life Nat.Sal.Mo); and two national projects 2) Tutela della Biodiversità nelle razze Avicole Italiane – TuBAvI (MiPAAF); 3) La Cunicoltura del Futuro: benessere e sostenibilità degli allevamenti cunicoli italiani - Cun-Fu (MiPAAF). These projects have a common aim, which is to preserve and valorize the local poultry and rabbit breeds (TuBAvI and Cun-Fu projects), and native Mediterranean trout populations (Nat.Sal.Mo project), through the combined use of in situ and ex situ conservation strategies. Accordingly, milestones of the present projects are the creation of semen cryobanks for the safeguard of these precious Italian heritage. In this regard, systematic studies of the main factors affecting the cryopreservation procedures of turkey, rabbit and Mediterranean trout semen have become necessary, in order to reduce cell damages caused by freezing/thawing process. In the light of this, the present doctoral thesis is an integral part of the three more extended projects mentioned above. The research activities were focused on developing effective reference procedures in order to ensure the long-term conservation of this valuable Italian genetic diversity. In accordance with these goals, a comparative proteomic study on fresh and frozen rabbit semen was also performed in order to provide new knowledge concerning the main mechanisms involved in cryogenic sperm damage in this species. The results of these studies are presented in the form of six published manuscripts and one draft article divided into four sessions, as follows: • session 1 includes three studies designed to optimize and identify a reference procedure for semen freezing of native Mediterranean trout and the results obtained from the start-up of the semen cryobank for this species; • session 2 includes two studies aimed to identify and standardize a reference procedure for freezing turkey semen through the evaluation sensitivity to freezing in vitro and the fertilizing ability in vivo. Moreover, the activities performed for the implementation of the first Italian avian semen cryobank were also reported in detail; • session 3 includes one study designed to develop a reference procedure for rabbit semen cryopreservation by comparing two extenders and three different inseminating doses, followed by the results achieved from the creation of the first national semen cryobank for Italian rabbit breeds; • session 4 includes one draft article aimed to evaluate, for the first time, the changes in rabbit semen proteins induced by cryopreservation process, trought comparative analysis of the differential expression of proteins in fresh and frozen semen.

Intracellular ice formation (IIF), a major cause of cryoinjury in biological cells, is significantly more pronounced during freezing of tissue than during freezing of suspended cells. While extensive studies of IIF have been conducted for single cells in suspension, few have investigated IIF in tissue. Due to the increased complexity that arises from both cell-substrate and cell-cell interactions in tissue, knowledge of cryobiology of isolated cells cannot simply be extrapolated to tissue. Different theories have been hypothesized for the mechanisms of IIF in tissue, but none have been conclusively proven. Towards the goal of developing mathematical models to accurately predict the probability of IIF in tissues of one or more cell types, we have developed a novel high-speed video cryomicroscopy system capable of image acquisition at sampling rates up to 32,000 Hz. Specifically, the effects of cell adhesion to the substrate and cell-cell interactions were investigated with experimental (micropatterned endothelial cell constructs) and mathematical models (Monte Carlo simulations). We have reported the first direct observations of the IIF process recorded at unprecedented sub-millisecond and sub-micron resolution. For the majority of our experiments, IIF nucleation was determined to occur preferentially at the cell perimeter. This observation was not consistent with the commonly accepted hypotheses of ice nucleation in suspended cells and suggests that an alternative mechanism of IIF initiation is dominant in adherent cells. In addition, the kinetics of ice nucleation were shown to be influenced by time in culture, attached cell perimeter, fibronectin coating density, and degree of cell-cell contact. Moreover, an additional phenomenon, paracellular ice penetration was identified, and the frequency of formation was correlated with focal adhesion formation. The data and mathematical models presented in this thesis bring closer the goal of elucidating the primary mechanisms contributing to IIF in tissue; providing important contributions to both the fields of cryopreservation (minimizing IIF) and cryosurgery (maximizing IIF).

A large number of studies in cryobiology have focused on understanding the underlying biophysics at the cellular level to help predict survival outcome after cryopreservation or cryosurgery. While this behavior is increasingly well studied and understood in cells gaps remain in our understanding of how cells in tissues behave which can hamper freezing applications in tissues. This study compares freezing behavior in cells in suspension vs. attached (a model tissue) state to investigate any differences in cellular behavior in these two states.

In this study, a theoretical model is developed to simulate the biophysical events in the intracellular spaces considering the biphasic, i.e., poroelastic, behavior of the cytoplasm. Most previous studies in the cryobiology literature have modeled the biophysical response of cells to freezing assuming the spatial homogeneity of all physical properties within the intracellular space without considering fluid-structure interaction in both the intracellular and extracellular spaces. However, a few recent studies strongly indicate that spatial heterogeneity in the intracellular space occurs during freezing. We thus model the cytoplasm as a poroelastic material considering nanoscale fluid-structure interaction between the cytoskeleton and cytosol, and the effects of hierarchical fluid-structure interaction across the cell during freezing.

Intracellular ice formation (IIF) is of great importance in cryobiology but its mechanisms are still under debate. In this work a micro-layer chip with a sandwiched thin layer of medium was employed to facilitate the observation of IIF; and thermoelectric chips were used to induce a directional cooling along the chip. Remarkable phenomena of extracellular ice formation (EIF) triggering IIF were observed. The extracellular ice contacted and “penetrated through” and initiated the intracellular ice. In the aid of the fluorescent reagents, it was found that the cell membrane could keep intact even though the cell had undergone IIF, and the intact cell could have IIF again in the next freezing cycle, until the membrane was finally ruptured. The results shed light on the IIF mechanisms.

Intracellular ice formation (IIF) plays a critical role in cryobiology, though the underlying biophysical mechanisms are still not completely explicable. In this work, a directional freezing scheme integrated with microlayer cell culture was employed to allow directional freezing as well as high-power microscopic observation of IIF in a single optical plane. The initiation of IIF and its spreading within the cells were well observed. The fluorescent reagents were employed to label the cell membrane and nucleus. It was found that the cell membrane could keep intact even though the cell had undergone IIF, and the intact cells could have IIF again in the next freezing cycle until their membranes were finally disrupted. The results shed light on the relationship between IIF and the integrity of cell membrane.

Freezing is a common technique for preservation of isolated cells, and extending its applications to the preservation of tissues would have important implications for the storage and distribution of tissue engineered products. Unlike isolated cells in suspension, cells in tissue interact with each other, and this interaction is known to affect the outcome of tissue cryopreservation. As a consequence, our knowledge of the cryobiology of isolated cells cannot simply be extrapolated to tissues, and new models, which consider the interaction between cells, need to be developed. The model that we propose is based on previous quantitative analysis of intercellular ice propagation in a micropatterned two-cell system. We used Monte Carlo simulations to extrapolate the results from cell pairs to two-dimensional and three-dimensional tissues. Effects of tissue geometry, cellular connectivity, and degree of intercellular interaction were investigated.

Kinetic (very rapid) vitrification (KVF) is a very promising approach in cryopreservation (CP) of biological materials as it is simple, avoids lethal intracellular ice formation (IIF) and minimizes damaging dehydration effects of extracellular crystallization. Moreover, achieving the ultra-high rates, which would prevent IIF during cooling and devitrification during resuscitation, and achieve KVF for practically any type of cells with one protocol of cooling and re-warming would be the “Holy Grail” of cell cryobiology [3]. However such hyperrapid rates currently require very small sample size which, however, is insufficient for many applications such as stem cells, blood or sperm. As the result, even smallest droplets of 0.25 microliters cannot be vitrified sufficiently fast to avoid the use of potentially toxic external vitrification agents such as DMSO or EG due to the Leidenfrost effect (LFE). In this presentation, we describe an entirely new system for hyperfast cooling of one-two order of magnitude larger samples that we call “KrioBlastTM”, which completely eliminates LFE. We have successfully vitrified up to 4,000 microliters of 15% glycerol solutions, which theoretically corresponds to the critical cooling rate of hundreds of thousands °C/min. We believe that such a system can revolutionize the future cryobiological paradigm.

The ability to eliminate freezing damage using “vitrification” (or the formation of glass) has long been an area of intense interest in cryobiology. Typically vitrification is achieved when biological systems are cooled at rates ranging from ∼8,000 °C/min to ∼10,000 °C/min [1–5]. Using traditional cooling methods (immersion in liquid nitrogen), such high cooling rates are currently not achievable, in large tissue sections (∼cm’s). In the present study we investigate a novel method to achieve high cooling rates in large tissue sections by pulsed laser heating in conjunction with cryogenic temperatures, i.e. high cooling rates are achieved by the localized difference in temperature between the laser heated tissue (∼1000’s of °C) and the surrounding liquid nitrogen (∼−160 °C). Additionally, the use of pulsed lasers allows localized heating of the tissue coupled with small time scales of energy deposition (0.1 to 1 pico seconds) such that the heating/thermal damage in tissues is minimized. To amplify this idea further, we developed a numerical model to predict the temperature transients in tissues exposed to laser heating and cryogenic temperatures. Analysis of our numerical simulations suggest that a perturbation of ∼3500 °C in a 5mm thick tissue leads to cooling rates in excess of ∼8000 °C/min throughout the tissue slice. These results indicate the possibility of vitrifying large tissue sections of cryobiological relevance using a combination of laser heating and liquid nitrogen cooling.