Microplate-Based Cryopreservation of Adherent-Cultured Human Cell Lines Using Amino Acids and Proteins.

With the progression of regenerative medicine and cell therapy, the importance of cryopreservation techniques for cultured cells continues to rise. Traditional cryoprotectants, such as dimethyl sulfoxide and glycerol, are effective in cryopreserving suspended cells, but they do not demonstrate sufficient efficacy for two-dimensional (2D)-cultured cells. In the past decade, small molecules and polymers have been studied as cryoprotectants. Some L-amino acids have been reported to be natural and biocompatible cryoprotectants. However, the cryoprotective effects of D-amino acids have not been investigated for such organized cells. In the present study, the cryoprotective effects of D- and L-amino acids and previously reported cryoprotectants were assessed using HepG2 cells cultured on a microplate without suspending the cells. d-Proline had the highest cryoprotective effect on 2D-cultured cells. The composition of the cell-freezing solution and freezing conditions were then optimized. The d-proline-containing cell-freezing solution also effectively worked for other cell lines. To minimize the amount of animal-derived components, fetal bovine serum in the cell freezing solution was substituted with bovine serum albumin and StemFit (a commercial supplement for stem cell induction). Further investigations on the mechanism of cryopreservation suggested that d-proline protected enzymes essential for cell survival from freeze-induced damage. In conclusion, an effective and xeno-free cell-freezing solution was produced using d-proline combined with dimethyl sulfoxide and StemFit for 2D-cultured cells.

Adult Stem Cells Freezing Processes and Cryopreservation Protocols.

The development of simple but effective storage protocols for adult stem cells will greatly enhance their use and utility in tissue-engineering applications. Cryopreservation has shown the most promise but is a fairly complex process, necessitating the use of chemicals called cryoprotective agents (CPAs), freezing equipment, and obviously, storage in liquid nitrogen. The purpose of this chapter is to present a general overview of cryopreservation storage techniques and the optimal protocols/results obtained in our laboratory for long-term storage of adult stem cells using freezing storage.

Advances in Cryopreservatives: Exploring Safer Alternatives.

Cryopreservation of cells, tissues, and organs is widely used in the biomedical and research world. There are different cryopreservatives that are used for this process; however, many of them, such as DMSO, are used despite the problems they present, mainly due to the toxicity it presents to certain types of samples. The aim of this Review is to highlight the different types of substances used in the cryopreservation process. It has been shown that some of these substances are well-known, as in the case of the families of alcohols, sugars, sulfoxides, etc. However, in recent years, other compounds have appeared, such as ionic liquids, deep eutectic solvents, or certain polymers, which open the door to new cryopreservation methods and are also less toxic to frozen samples.

Engineered Test Tissues: A Model for Quantifying the Effects of Cryopreservation Parameters.

Engineered tissues are showing promise as implants to repair or replace damaged tissues in vivo or as in vitro tools to discover new therapies. A major challenge of the tissue engineering field is the sample preservation and storage until their transport and desired use. To successfully cryopreserve tissue, its viability, structure, and function must be retained post-thaw. The outcome of cryopreservation is impacted by several parameters, including the cryopreserving agent (CPA) utilized, the cooling rate, and the storage temperature. Although a number of CPAs are commercially available for cell cryopreservation, there are few CPAs designed specifically for tissue cryostorage and recovery. In this study, we present a flexible, relatively high-throughput method that utilizes engineered tissue rings as test tissues for screening the commercially available CPAs and cryopreservation parameters. Engineered test tissues can be fabricated with low batch-to-batch variability and characteristic morphology due to their endogenous extracellular matrix, and they have mechanical properties and a ring format suitable for testing with standard methods. The tissues were grown for 7 days in standard 48-well plates and cryopreserved in standard cryovials. The method allowed for the quantification of metabolic recovery, tissue apoptosis/necrosis, morphology, and mechanical properties. In addition to establishing the method, we tested different CPA formulations, freezing rates, and freezing points. Our proposed method enables timely preliminary screening of CPA formulations and cryopreservation parameters that may improve the storage of engineered tissues.

Chondrocyte Viability of Particulated Porcine Articular Cartilage Is Maintained in Tissue Storage After Cryoprotectant Exposure, Vitrification, and Tissue Warming.

OBJECTIVE: Vitrification of articular cartilage (AC) is a promising technique which may enable long-term tissue banking of AC allografts. We previously developed a 2-step, dual-temperature, multi-cryoprotectant agent (CPA) loading protocol to cryopreserve particulated AC (1 mm3 cubes). Furthermore, we also determined that the inclusion of ascorbic acid (AA) effectively mitigates CPA toxicity in cryopreserved AC. Prior to clinical translation, chondrocytes must remain viable after tissue re-warming and before transplantation. However, the effects of short-term hypothermic storage of particulated AC after vitrification and re-warming are not documented. This study evaluated the chondrocyte viability of post-vitrified particulated AC during a 7-day tissue storage period at 4 °C. We hypothesized that porcine particulated AC could be stored for up to 7 days after successful vitrification without significant loss of cell viability, and these results would be enhanced when cartilage is incubated in storage medium supplemented with clinical grade AA. DESIGN: Three experimental groups were examined at 5 time points: a fresh control (only incubated in medium), a vitrified - AA group, and a vitrified + AA group (N = 7). RESULTS: There was a mild decline in cell viability but both treatment groups maintained a viability of greater than 80% viable cells which is acceptable for clinical translation. CONCLUSION: We determined that particulated AC can be stored for up to 7 days after successful vitrification without a clinically significant decline in chondrocyte viability. This information can be used to guide tissue banks regarding the implementation of AC vitrification to increase cartilage allograft availability.

Trehalose in Biomedical Cryopreservation-Properties, Mechanisms, Delivery Methods, Applications, Benefits, and Problems.

Cells and tissues are the foundation of translational medicine. At present, one of the main technological obstacles is their preservation for long-term usage while maintaining adequate viability and function. Optimized storage techniques must be developed to make them safer to use in the clinic. Cryopreservation is the most common long-term preservation method to maintain the vitality and function of cells and tissues. But, the formation of ice crystals in cells and tissues is considered to be the main mechanism that could harm cells and tissues during freezing and thawing. To reduce the formation of ice crystals, cryoprotective agents (CPAs) must be added to the cells and tissues to achieve the cryoprotective effect. However, conventional cryopreservation of cells and tissues often needs to use toxic organic solvents as CPAs. As a result, cryopreserved cells and tissues may need to go through a time-consuming washing process to remove CPAs for further applications in translational medicine, and multiple valuable cells are potentially lost or killed. Currently, trehalose has been researched as a nontoxic CPA due to its cryoprotective ability and stability during cryopreservation. Nevertheless, trehalose is a nonpermeable CPA, and the lack of an effective intracellular trehalose delivery method has become the main obstacle to its use in cryopreservation. This article illustrated the properties, mechanisms, delivery methods, and applications of trehalose, summarized the benefits and limits of trehalose, and summed up the findings and research direction of trehalose in biomedical cryopreservation.

Reconsideration of the safety and effectiveness of human oocyte cryopreservation.

Mature oocyte cryopreservation (OC) has become increasingly common since the American Society for Reproductive Medicine declared OC to no longer be experimental. Utilization of the open vitrification protocol has led to a marked improvement in the efficacy of oocyte cryopreservation. However, the safety and effectiveness of this cryopreservation method remain controversial. A previous report stated that among all initiated recipient cycles, the live-birth rate among recipients of all ages was significantly higher when using fresh donor oocytes (FDOs) rather than cryopreserved donor oocytes (CDOs). Confounding patient characteristics were noted as possible causes. OC stands as an acceptable elective medical intervention for preserving fertility in women. To further understand the effects of OC on the live birth rate resulting from fresh versus cryopreserved donor oocytes, reported data from the Society for Assisted Reproductive Technology from 2013 to 2020 were analyzed. The mean of the mean live-birth rate in all ages resulting from FDOs was 49.0% (44.6-53.3%) versus 41.0% (39.1-43.2%) for CDOs (difference, 8.0% [95% confidence interval, 5.35-10.57%], p value < 0.001). The lower live-birth rate observed for CDOs versus FDOs has been consistent throughout past decades. While there has been no reported increase in the aneuploidy rate for CDOs compared to FDOs, differences in the nondisjunction separation rate among different chromosomes were described in a recent report. Open vitrification culture medium usually contains high concentrations of cryoprotectants, such as 15% dimethyl sulfoxide (DMSO) and 15% ethylene glycol (EG). Recent studies showed that tissue culture with 0.1% DMSO or 10% EG resulted in deregulation of gene expression, disruption of epigenetic imprints, and accumulation of reactive oxygen species. The addition of melatonin, which can remove reactive oxygen species from vitrification medium, was shown to improve CDOs qualities and functions to conditions similar to those of FDOs; however, there were insufficient data to conclude that melatonin could improve the lower live-birth rate. These factors that affect live birth rates, birth defects, birth weights and developmental health cannot be ignored and perhaps need to be studied again and followed when evaluating the true effectiveness of human oocyte cryopreservation.

Lactulose in combination with soybean lecithin has a cryoprotective effect on probiotic taxa of bifidobacteria and Lactobacillaceae.

Lactulose is commonly used in pharmacy for constipation and hepatic encephalopathy treatment. The prebiotic effect of lactulose is also often mentioned. However, its cryoprotective effect in combination with lecithin on the main representatives of probiotics has not been tested yet. The 12 taxa of bifidobacteria and Lactobacillaceae members were used for the purpose. These were mixed in a ratio of 1:1 with lactulose + lecithin (finally 5.0% and 1.25%, respectively; LL). The 25% glycerol (G+) solution and cultures themselves were applied as positive and negative controls, respectively. Bacterial suspensions were stored at a mild freezing temperature (-20°C) until the end of the experiment (210th day). The LL solution had a comparable (insignificant difference at the P-value = 0.05) cryoprotective effect as the positive control in five of six bifidobacteria and in three of six representatives of Lactobacillaceae. The better cryoprotective effect was revealed in other Lactobacillaceae. At the end of the experiment, the generally accepted therapeutic minimum (>107 Colony Forming Units/mL) was determined in LL solution in five bifidobacteria and four Lactobacillaceae strains. The presented results improve knowledge about long-term mild cryopreservation of the most commonly used probiotics and could contribute to developing new forms of (nutri)synbiotics.

Infrared spectroscopic analysis of hydrogen-bonding interactions in cryopreservation solutions.

BACKGROUND: In this study we investigated hydrogen bonding interactions in hydrated and frozen solutions of different cryoprotective agents (CPAs) including dimethyl sulfoxide, glycerol, ethylene glycol, propylene glycol, and trehalose. We also investigated the effect of CPAs on ice crystal growth during storage and correlated this with storage stability of liposomes. METHODS: FTIR spectroscopy was used to study hydrogen bonding interactions in CPA solutions in H2O and D2O, and their thermal response was analyzed using van 't Hoff analysis. The effect of CPAs on ice crystal growth during storage was investigated by microscopy and correlated with storage stability of liposomes encapsulated with a fluorescent dye. RESULTS: Principal component analyses demonstrated that different CPAs can be recognized based on the shape of the OD band region only. Chemically similar molecules such as glycerol and ethylene glycol closely group together in a principal component score plot, whereas trehalose and DMSO appear as condensed separated clusters. The OH/OD band of CPA solutions exhibits an overall shift to higher wavenumbers with increasing temperature and changed fractions of weak and strong hydrogen interactions. CPAs diminish ice crystal formation in frozen samples during storage and minimize liposome leakage during freezing but cannot prevent leakage during frozen storage. CONCLUSIONS: CPAs can be distinguished from one another based on the hydrogen bonding network that is formed in solution. DMSO-water mixtures behave anomalous compared to other CPAs that have OH groups. CPAs modulate ice crystal formation during frozen storage but cannot prevent liposome leakage during frozen storage.

Comparison of dilute and nondilute osmotic equilibrium models for erythrocytes.

Successful cryopreservation requires the addition of cryoprotective agents (CPAs). The addition of permeating CPAs, such as glycerol, is associated with some risk to the cells and tissues. These risks are both related to the CPA themselves (CPA toxicity) and to the volume response of the cell (osmotic damage). To minimize the potential for damage during cryopreservation, mathematical models are often employed to understand the interactions between protocols and cell volume responses. In the literature, this volume response is usually captured using ideal and dilute approximations of chemical potential and osmolality, an approach that has been called into question for cells in high concentrations of CPAs. To address this, the relevance of non-ideal and non-dilute models has been explored in a number of cell types in the presence of permeating CPAs. However, it has not been explored in erythrocytes, which have a cytosolic hemoglobin content of more than 20% by volume and are cryopreserved in 40% glycerol. Because hemoglobin has been suggested to be a highly non-ideal solute, if the non-ideal and non-dilute transport model is relevant to any cells, it should be relevant to erythrocytes. Here we investigate the use, and accuracy, of both the dilute and non-dilute models in predicting cell volume changes during CPA equilibration in erythrocytes, and demonstrate that using published values for the non-ideal and non-dilute model, applied to erythrocytes, leads to model predictions inconsistent with experimental data, whereas dilute approximations align well with experimental data.

The Role of Aquaporin 7 in the Movement of Water and Cryoprotectants in Bovine In Vitro Matured Oocytes.

Aquaglyceroporins are known as channel proteins, and are able to transport water and small neutral solutes. In this study, we evaluate the effect of exposure of in vitro matured bovine oocytes to hyperosmotic solutions containing ethylene glycol (EG), dimethyl sulfoxide (Me2SO) or sucrose on the expression levels of AQP3, AQP7 and AQP9. Moreover, we studied whether artificial protein expression of AQP7 in bovine oocytes increases their permeability to water and cryoprotectants. Exposure to hyperosmotic solutions stimulated AQP3 and AQP7 but not AQP9 expression. Oocytes exposed to hyperosmotic Me2SO solution exhibited upregulated AQP3 expression, while AQP7 expression was upregulated by EG hyperosmotic exposure. Microinjection of oocytes at the germinal vesicle stage with enhanced green fluorescent protein (EGFP) or EGFP+AQP7 cRNAs resulted in the expression of the corresponding proteins in ≈86% of the metaphase-II stage oocytes. AQP7 facilitated water diffusion when bovine MII oocytes were in presence of Me2SO solution but not EG or sucrose solution. However, the overexpression of this aquaporin did not increase membrane permeability to Me2SO or EG. In summary, cryoprotectant-induced increase of AQP3 and AQP7 expression could be one of the mechanisms underlying oocyte tolerance to hyperosmotic stress. Water diffusion appears to be improved when AQP7 overexpressed oocytes are exposed to Me2SO, shortening the time required for oocytes to achieve osmotic balance with cryoprotectant solutions.

Factors affecting rabbit sperm cryopreservation: a mini-review.

Rabbits are an important animal species for meeting the nutritional requirements of the world's growing population due to the high conversion rate of feed. In most countries, the rabbit industry currently relies on artificial insemination with fresh or chilled and frozen-thawed spermatozoa. Various factors during the freezing process, including diluents, sperm preparation and freezing techniques, antioxidants, sudden temperature changes, ice formation and osmotic stress, have been proposed as reasons for the poor sperm quality post thaw. Despite the extensive progress reached in the field of rabbit sperm cryopreservation, new methodological approaches that could overcome problems in sperm cryopreservation are necessary. The aim of this review was to describe the factors that affect the cryopreservation of rabbit sperm.

Effectiveness of Clinical-Grade Chondroitin Sulfate and Ascorbic Acid in Mitigating Cryoprotectant Toxicity in Porcine Articular Cartilage.

High concentrations of cryoprotective agents (CPAs) are required to achieve successful vitrification of articular cartilage; however, CPA cytotoxicity causes chondrocyte death. To reduce CPA toxicity, supplementation with research-grade additives, in particular chondroitin sulfate (CS) and ascorbic acid (AA), have previously been shown to improve chondrocyte recovery and metabolic function after exposure to CPAs at hypothermic conditions. However, it is necessary to evaluate the pharmaceutical equivalent clinical grade of these additives to facilitate the supplementation of additives into future vitrification protocols, which will be designed for vitrifying human articular cartilage in tissue banks. We sought to investigate the effectiveness of clinical-grade CS, AA, and N-acetylcysteine (NAC) in mitigating toxicity to chondrocytes during CPA exposure and removal, and determine whether a combination of two additives would further improve chondrocyte viability. We hypothesized that clinical-grade additives would exert chondroprotective effects comparable to those of research-grade additives, and that this protective effect would be enhanced if two additives were combined when compared with a single additive. The results indicated that both clinical-grade and research-grade additives significantly improved cell viability (p < 0.10) compared with the negative control (CPA with no additives). CS, AA, and NAC+AA increased cell viability significantly (p < 0.10) compared with the negative control. However, NAC, NAC+CS, and CS+AA did not improve cell viability when compared with the negative control (p > 0.10). We demonstrated that supplementation with clinical-grade CS or AA significantly improved chondrocyte viability in porcine cartilage subjected to high CPA concentrations, whereas supplementation with clinical-grade NAC did not benefit chondrocyte viability. Supplementation with clinical-grade additives in CPA solutions can mitigate CPA toxicity, which will be important in translating previously developed effective protocols for the vitrification of articular cartilage to human tissue banks.

Avaliação da aplicabilidade de polímeros sintéticos na vitrificação de tecido ovariano bovino / Evaluation of the applicability of synthetic polymers in the vitrification of bovine ovarian tissue

A preservação da fertilidade em pacientes com câncer objetiva assegurar a saúde reprodutiva. A criopreservação de tecido ovariano é a única técnica disponível para meninas pré-púberes e para casos em que o tratamento não pode ser adiado. A técnica de vitrificação está associada a uma melhor preservação de fragmentos do córtex ovariano quando comparada ao congelamento lento. Estudos preliminares demonstraram que a combinação de polímeros sintéticos na vitrificação preservou melhor o tecido e os folículos secundários no córtex de ovários de macacos, por serem miméticos às proteínas naturais responsáveis pela proteção conferida a alguns organismos durante o inverno. A técnica de vitrificação associada a polímeros sintéticos é uma alternativa promissora, mas ainda não está disponível um protocolo padrão que demonstre resultados consistentes. Assim, este trabalho teve como objetivo avaliar a aplicabilidade de polímeros sintéticos na criopreservação por vitrificação de tecido ovariano bovino. Ovários bovinos foram obtidos a partir de animais abatidos para consumo em um abatedouro local. O córtex foi extraído e cortado em fragmentos. Os fragmentos foram divididos em três grupos (controle fresco, vitrificação com (CP) e sem (SP) adição de polímeros sintéticos). Os fragmentos de tecido de todos os grupos antes e após aquecimento foram fixados em paraformaldeído a 4%, corados com hematoxilina e eosina para avaliação de morfologia, contagem e observação do estágio folicular. Parte dos fragmentos tiveram seus folículos secundários isolados mecanicamente e cultivados em matriz de alginato até atingirem o estágio antral. Durante o cultivo, para análise de viabilidade, foram avaliados a sobrevida, crescimento e formação de antro folicular. Para avaliação da funcionalidade folicular, o meio de cultivo foi coletado para posterior dosagem de esteróides ovarianos. Então, os três grupos foram comparados estatisticamente. Os tecidos ovarianos vitrificados apresentaram uma morfologia com sinais de injúria, com espaços vazios e menos densos, além de exibirem uma menor porcentagem de folículos normais quando comparados ao tecido fresco (Fresco x CP p<0,0001; Fresco x SP p=0,0004). Contudo, não foi observada diferença entre os grupos vitrificados com e sem polímeros (CP x SP p = 0,7173). Os folículos que passaram pela vitrificação apresentaram uma sobrevida similar entre si e menor que o controle fresco (χ²(2) = 19,87; p< 0,0001). Todos os grupos avaliados foram semelhantes na taxa de formação de antro (χ²(1) = 0,6569; p< 0,4176). Em todos os grupos houve crescimento folicular durante o cultivo. No entanto, os folículos frescos e com adição de polímeros aumentaram de diâmetro durante todo o cultivo, ao passo que os folículos sem adição de polímeros cresceram apenas na primeira semana. No fim do cultivo, os folículos que passaram pelo processo de vitrificação produzem menos hormônios que os frescos (p < 0,05), mas sem diferença entre SP e CP. A partir desses resultados, é possível concluir que a combinação do uso de polímeros sintéticos na vitrificação de tecido ovariano é uma técnica promissora, que poderá proteger o desenvolvimento folicular, mas são necessários mais estudos que possam aperfeiçoar esse protocolo.

The preservation of fertility in cancer patients aims to ensure reproductive health. Ovarian tissue cryopreservation is the only technique available for prepubescent girls and for cases where treatment cannot be delayed. The vitrification technique is associated with better preservation of ovarian cortex fragments when compared to slow freezing. Preliminary studies in the cortex of monkeys' ovaries have shown that the combination of synthetic polymers in vitrification is better to preserve the tissue and secondary follicles, as they are mimetic to the natural proteins responsible for the protection during the winter in some organisms. The vitrification technique associated with synthetic polymers is a promising alternative, but a standard protocol that demonstrates consistent results is not yet available. Thus, this work aimed to evaluate the applicability of synthetic polymers in cryopreservation by vitrification of bovine ovarian tissue. Bovine ovaries were obtained at a local abattoir. The cortex was extracted and cut into fragments. The fragments were divided into three groups (fresh control, vitrification with (CP) and without (SP) addition of synthetic polymers). Tissue fragments from all groups before and after heating were fixed in 4% paraformaldehyde, stained with hematoxylin and eosin for morphology assessment, counting and observation of the follicular stage. Part of the fragments had their secondary follicles mechanically isolated and cultivated in alginate matrix until they reached the antral stage. During cultivation, for viability analysis, survival, growth and follicular antrum formation were evaluated. To evaluate the follicular functionality, the culture medium was collected for later measurement of ovarian steroids. The vitrified ovarian tissues presented a morphology with signs of injury, with empty and less dense spaces, in addition to showing a lower percentage of normal follicles when compared to fresh tissue (Fresh control x CP p< 0.0001). All groups evaluated were similar in the rate of antrum formation (χ²(1) = 0.6569; p< 0.4176). In all groups there was follicular growth during cultivation. However, fresh and polymer-added follicles increased in diameter throughout the cultivation, whereas follicles without polymer additions grew only in the first week. At the end of cultivation, the follicles that underwent the vitrification process produced less hormones than the fresh ones (p < 0.05), but there was no difference between SP and CP. From these results, it is possible to conclude that the combination of the use of synthetic polymers in the vitrification of ovarian tissue is a promising technique, which may protect follicular development, but further studies are needed to improve this protocol.

Differentiation of Human iPS Cells Into Sensory Neurons Exhibits Developmental Stage-Specific Cryopreservation Challenges.

Differentiation of human induced pluripotent stem cells (hiPSCs) generates cell phenotypes valuable for cell therapy and personalized medicine. Successful translation of these hiPSC-derived therapeutic products will rely upon effective cryopreservation at multiple stages of the manufacturing cycle. From the perspective of cryobiology, we attempted to understand how the challenge of cryopreservation evolves between cell phenotypes along an hiPSC-to-sensory neuron differentiation trajectory. Cells were cultivated at three different stages to represent intermediate, differentiated, and matured cell products. All cell stages remained ≥90% viable in a dimethyl sulfoxide (DMSO)-free formulation but suffered ≥50% loss in DMSO before freezing. Raman spectroscopy revealed higher sensitivity to undercooling in hiPSC-derived neuronal cells with lower membrane fluidity and higher sensitivity to suboptimal cooling rates in stem cell developmental stages with larger cell bodies. Highly viable and functional sensory neurons were obtained following DMSO-free cryopreservation. Our study also demonstrated that dissociating adherent cultures plays an important role in the ability of cells to survive and function after cryopreservation.

New Alternative Mixtures of Cryoprotectants for Equine Immature Oocyte Vitrification.

Equine oocyte vitrification would benefit the growing in vitro embryo production programs, but further optimization of the protocol is necessary to reach clinical efficiency. Therefore, we aimed to perform a direct comparison of non-permeating and permeating cryoprotective agents (CPAs) during the vitrification and warming of equine immature oocytes. In the first experiment, cumulus oocytes complexes (COCs) were vitrified comparing sucrose, trehalose, and galactose in combination with ethylene glycol (EG) and dimethyl sulfoxide (DMSO). In the second experiment, the COCs were vitrified using three mixtures of permeating CPAs in a 50:50 volume ratio (ethylene glycol-dimethyl sulfoxide (ED), propylene glycol-ethylene glycol (PE), and propylene glycol-dimethyl sulfoxide (PD)) with galactose and warmed in different galactose concentrations (0.3 or 0.5 mol/L). Overall, all the treatments supported blastocyst formation, but the developmental rates were lower for all the vitrified groups in the first (4.3 to 7.6%) and the second (3.5 to 9.4%) experiment compared to the control (26.5 and 34.2%, respectively; p < 0.01). In the first experiment, the maturation was not affected by vitrification. The sucrose exhibited lower cleavage than the control (p = 0.02). Although the galactose tended to have lower maturation than trehalose (p = 0.060) and control (p = 0.069), the highest numerical cleavage and blastocyst rates were obtained with this CPA. In the second experiment, the maturation, cleavage, and blastocyst rates were similar between the treatments. Compared to the control, only the ED reached similar maturation (p = 0.02) and PE similar cleavage (p = 0.1). The galactose concentration during warming did not affect the maturation, cleavage, or blastocyst rates (p > 0.1), but the PE-0.3 exhibited the highest blastocyst rate (15.1%) among the treatments, being the only one comparable to the control (34.2%). As such, PE-galactose provides a valuable option for equine immature oocyte vitrification and should be considered for the future optimization of the protocol.

Effects of storage media, supplements and cryopreservation methods on quality of stem cells.

Despite a vast amount of different methods, protocols and cryoprotective agents (CPA), stem cells are often frozen using standard protocols that have been optimized for use with cell lines, rather than with stem cells. Relatively few comparative studies have been performed to assess the effects of cryopreservation methods on these stem cells. Dimethyl sulfoxide (DMSO) has been a key agent for the development of cryobiology and has been used universally for cryopreservation. However, the use of DMSO has been associated with in vitro and in vivo toxicity and has been shown to affect many cellular processes due to changes in DNA methylation and dysregulation of gene expression. Despite studies showing that DMSO may affect cell characteristics, DMSO remains the CPA of choice, both in a research setting and in the clinics. However, numerous alternatives to DMSO have been shown to hold promise for use as a CPA and include albumin, trehalose, sucrose, ethylene glycol, polyethylene glycol and many more. Here, we will discuss the use, advantages and disadvantages of these CPAs for cryopreservation of different types of stem cells, including hematopoietic stem cells, mesenchymal stromal/stem cells and induced pluripotent stem cells.

Cryoprotectant agents for ovarian tissue vitrification: Systematic review.

Studies on the cryopreservation of ovarian tissue usually compare slow freezing versus vitrification and aim to optimize protocols, evaluate combinations or concentrations of cryoprotectant agents (CPAs), exposure time, and the addition of synthetic polymers. This systematic review aimed to identify the different CPAs used for the vitrification of human or primate ovarian tissue and to compare their results in terms of follicular survival and functional preservation. We searched Pubmed and EMBASE for randomized clinical trials or cohort studies comparing CPAs for human and/or primate ovarian vitrification. The highest rate of morphologically normal follicles after cryopreservation was 98% and was obtained with a combination of 27% ethylene glycol (EG) plus 27% glycerol, in addition to non-permeable synthetic polymers. The use of dimethyl sulfoxide (DMSO) in relatively low concentrations combined with EG and other CPAs yielded more than 90% of intact follicles after vitrification. The methods and outcomes varied largely among studies, making it difficult to combine their results. While there is no definite answer to what is the best combination of CPAs for vitrification of human ovarian tissue, the data reviewed here suggest that current vitrification techniques are able to preserve the integrity of most follicles.

Carboxylated Poly-l-Lysine as a Macromolecular Cryoprotective Agent Enables the Development of Defined and Xeno-Free Human Sperm Cryopreservation Reagents.

In human sperm cryopreservation, test yolk buffer and human serum albumin have been used as permeating macromolecular-weight cryoprotectants. In clinical reproductive medicine, human serum albumin is frequently used because of low risks of zoonoses and allergic reactions. However, the risk of allogeneic infectious diseases exists, and the supply may be unstable because human serum albumin is derived from human blood. Therefore, the development of xeno-free human sperm cryopreservative reagents that could overcome the aforementioned problems is warranted. We succeeded in developing a new xeno-free and defined sperm cryopreservation reagent containing glycerol, carboxylated poly-l-lysine, and raffinose. The cryopreservation reagent was not significantly different in terms of sperm motility, viability, and DNA fragmentation and was comparable in performance to a commercial cryopreservation reagent containing human serum albumin. Moreover, the addition of saccharides was essential for its long-term storage. These results may help elucidate the unknown function of macromolecular-weight permeating cryoprotective agents.

Cryopreservation of porcine urethral tissue: Storage at -20°C preserves the mechanical, failure and geometrical properties.

Cryopreservation is required to preserve the native properties of tissue for prolonged periods of time. In this study, we evaluate the impact that 4 different cryopreservation protocols have on porcine urethral tissue, to identify a protocol that best preserves the native properties of the tissue. The cryopreservation protocols include storage in cryoprotective agents at -20 °C and -80 °C with a slow, gradual, and fast reduction in temperature. To evaluate the effects of cryopreservation, the tissue is mechanically characterised in uniaxial tension and the mechanical properties, failure mechanics, and tissue dimensions are compared fresh and following cryopreservation. The mechanical response of the tissue is altered following cryopreservation, yet the elastic modulus from the high stress, linear region of the Cauchy stress - stretch curves is unaffected by the freezing process. To further investigate the change in mechanical response following cryopreservation, the stretch at different tensile stress values was evaluated, which revealed that storage at -20 °C is the only protocol that does not significantly alter the mechanical properties of the tissue compared to the fresh samples. Conversely, the ultimate tensile strength and the stretch at failure were relatively unaffected by the freezing process, regardless of the cryopreservation protocol. However, there were alterations to the tissue dimensions following cryopreservation that were significantly different from the fresh samples for the tissue stored at -80 °C. Therefore, any study intent on preserving the mechanical, failure, and geometric properties of urethral tissue during cryopreservation should do so by freezing samples at -20 °C, as storage at -80 °C is shown here to significantly alter the tissue properties.