Optimal Cryopreservation Protocol Research Articles

An Update on Cryopreservation of Adipose Tissue.

Currently, fat transplantation occurs immediately after harvesting procedures. Because low rates of fat graft take are well reported in the literature, many patients require multiple surgical procedures for fat graft harvest. These subsequent procedures lead to increased cost, donor-site morbidity, and patient discomfort in the long term. The ability to preserve our patients' own adipose aspirate would allow us to counteract these shortcomings and ultimately improve the clinical outcome after fat grafting. Unfortunately, there is no optimal and practical adipose tissue cryopreservation protocol for use by the plastic surgeon at the present time. Because of this dilemma, the senior author (L.L.Q.P.) has investigated this concept in an effort to create a protocol that is both technically sound and clinically achievable to allow for the long-term preservation of adipose tissue. In this article, the authors aim to outline this effort, review current clinical applications that have been reported in the literature, and detail exciting future perspectives in the use of preserved lipoaspirates for repeated fat grafting procedures or in the form of cell-based therapy engineered for reconstructive endeavors for their patients.

A model protocol for the cryopreservation and recovery of motile lizard sperm using the phosphodiesterase inhibitor caffeine.

Reproductive technologies such as genome storage and assisted reproduction have a significant role to play in ending or reversing species extinctions. However, such technologies for non-model organisms (i.e. non-mammalian species) are poorly developed. This is particularly true for the reptiles, in which there is a dearth of successful protocols for cryopreserving reptile spermatozoa, despite limited attempts. We investigated sperm cryopreservation in the Australian lizard Varanus panoptes with the objective of addressing the unmet need for an optimized cryopreservation protocol for the spermatozoa of squamate reptiles. We tested the efficacy of two cryoprotectants [dimethyl sulfoxide (DMSO) and glycerol] as well supplementation with a phosphodiesterase inhibitor (caffeine) to promote post-thaw motility. For cryopreservation, sperm were cooled in straws suspended in liquid nitrogen vapour for 5 minutes (approximately −135°C), before being plunged into liquid nitrogen (approximately −196°C), and later thawed in a water bath at 35°C. Samples were incubated post-thaw for 10 minutes in the presence or absence of 10 mM of caffeine. Both cryoprotectant type and concentration significantly affected percent sperm motility pre-freezing, with DMSO being less cytotoxic than glycerol and motility decreasing at higher concentrations of both cryoprotectant types. While cold shock did not significantly affect sperm motility, both cryoprotectant type and concentration did significantly impact the motility of post-thawed spermatozoa. Thus, mid-range concentrations (10% v/v) of DMSO and glycerol yielded a greater post-thaw motility compared with 5 and 20% v/v, while DMSO proved superior to glycerol. The addition of caffeine resulted in a significant recovery of post-thaw motility for both cryoprotectants, with higher rates of motility being associated with higher cryoprotectant concentrations. These protocols provide a significant step forward for in situ and ex situ management of threatened reptiles and add to recent evidence that reptilian sperm may have the full range of phosphorylation-mediated cellular mechanisms associated with capacitation, motility and metabolic regulation found in mammalian sperm.

Turning Cells into Reagents: The Preparation of Assay Ready Cells for Routine Use in Bioassays.

Assay ready cells are cryopreserved at a highly functional state and can be used in cell-based assay without prior cultivation or cell passaging. Basically, like any other reagent, the cells are applied to the assay instantly after thawing. Introduced initially in the drug discovery process where assay ready Frozen Instant Cells help to streamline cell-based high-throughput screening campaigns, the methodology now has been accepted for a much broader scope of applications and industries. The preparation of assay ready Frozen Instant Cells is not so much a piece of magic but a combination of good cell culture practice, careful handling, and individually optimized cryopreservation protocols. Here, a standard protocol is presented, how HepG2 cells are frozen in an assay ready quality to be used as a reagent in routine cell-based assays.

Cryopreservation of banana shrimp (Fenneropenaeus merguiensis) spermatophores with supplementation of medicinal plant extracts: Development of a programmable controlled-rate method and a practical method

The objectives of this study were to generate the optimal cryopreservation protocols (programmable controlled-rate and practical methods) for banana shrimp (Fenneropenaeus merguiensis) spermatophores and investigate the efficacy of plant extracts (moringa, Moringa oleifera Lam and ginger, Zingiber officinale Roscoe) on sperm survival and bacterial abundance in cryostored spermatophores. Spermatophores were exposed to various cryoprotectants at different concentrations and equilibrium times prior to examining sperm viability. A minimal toxicity to banana shrimp sperm was observed in spermatophores suspended in 5% DMSO. The optimal freezing protocol using a programmable controlled-rate freezer included exposure of spermatophores in calcium-free saline (Ca–F saline) containing 5% DMSO with 20 min equilibration time, and then utilization of a one-step freezing at the rate of −2 °C min−1 from 25 °C to −80 °C and holding for 30 s before storage in a liquid nitrogen tank. Highest sperm viability of post-thawed spermatophores was obtained after thawing at 30 °C for 2 min in a water bath. Cryopreservation of F. merguiensis spermatophores using a practical method was achieved by suspending spermatophores in Ca–F saline containing 5% DMSO with equilibration time of 20 min at 25 °C and placing at 4 cm over liquid nitrogen surface (a freezing rate of about −2.5 °C min−1) prior to plunging into liquid nitrogen. In order to evaluate the efficacy of plant extracts on sperm survival and bacterial abundance of cryostored spermatophores, two freezing protocols (programmable and practical methods) with ethanolic moringa and ginger extracts (0.1 mg mL−1 each) were developed for long-term cryostorage. Moringa and ginger extracts had no detrimental effect on F. merguiensis sperm due to retaining high viable sperm without significant differences (P > 0.05) compared to the control after a 12-month cryostorage. Supplementation of moringa or ginger extract into sperm extender resulted in a reduction of bacterial abundance in cryostored spermatophores. This report, for the first time, indicates the promising potential of plant extracts as an alternative method for controlling bacterial contaminants in cryopreserved spermatophores of penaeid shrimp.

Cryopreservation of human umbilical vein and porcine corneal endothelial cell monolayers

Cryopreservation of endothelium is one of the major challenges in the cryopreservation of complex tissues. Human umbilical vein endothelial cells (HUVECs) in suspension are available commercially and recently their post-thaw cell membrane integrity was significantly improved by cryopreservation in 5% dimethyl sulfoxide (Me2SO) and 6% hydroxyethyl starch (HES). However, cryopreservation of cells in monolayers has been elusive. The exact mechanisms of damage during cell monolayer cryopreservation are still under investigation. Here, we show that a combination of different factors contribute to significant progress in cryopreservation of endothelial monolayers. The addition of 2% chondroitin sulfate to 5% Me2SO and 6% HES and cooling at 0.2 or 1 °C/min led to high membrane integrity (97.3 ± 3.2%) immediately after thaw when HUVECs were cultured on a substrate with a coefficient of thermal expansion similar to that of ice. The optimized cryopreservation protocol was applied to monolayers of primary porcine corneal endothelial cells, and resulted in high post-thaw viability (95.9 ± 3.7% membrane integrity) with metabolic activity 12 h post-thaw comparable to unfrozen control.

Comparative study on cellular and molecular responses in oyster sperm revealed different susceptibilities to cryopreservation

The Portuguese (Crassostrea angulata) and Pacific oyster (Crassostrea gigas), both from the genus Crassostrea, are two important species for production and conservation. Although they have common characteristics, different susceptibilities to rearing conditions have been described in these species. Overall, in the case of C. angulata, only a few remaining populations are present in the south of Portugal and Spain. The preservation of genetic material from improved stocks or from the original population is crucial in oysters to prevent the potential impacts of epidemic diseases and natural disasters. Sperm cryopreservation in oysters has progressed in recent years. However many issues, such as protocol standardization, are still unsolved for the application of research results. In the present study a sperm cryopreservation protocol, previously published in C. angulata by our group, were analyzed in terms of cellular and molecular damage, in an effort to determine the most sensible parameters to standardize the cryopreservation protocols for both species. Different approaches in the analysis of sperm quality were performed for the first time in this genus to detect different susceptibilities between the two valuable species. Our results revealed that our previously published protocol containing 10% DMSO as cryoprotectant is more suitable comparing to 10% EG in both species. In addition, an integrative analysis was performed in both oyster species comparing all cellular parameters and C.gigas showed a higher susceptibility to cryopreservation using this optimized protocol. Moreover, higher susceptibility to transcript degradation was detected in C. gigas using this optimized cryopreservation protocol. This study highlights the importance of using different techniques and exhaustive analyses for selecting the most suitable cryopreservation protocol and its standardization, thus ensuring the total safety of the technique.

Effects of different diluents, cryoprotective agents, and freezing rates on sperm cryopreservation in Epinephelus akaara

We investigated various factors, including cryoprotective agents (CPAs), diluents, and freezing rates, to develop an optimal cryopreservation protocol for Epinephelus akaara sperm. In experiments using 10% dimethyl sulfoxide (DMSO), glycerol, and methanol with various diluents, 10% DMSO and 300 mM glucose yielded the highest post-thaw sperm motility. The combination of 10% glycerol and 300 mM sucrose yielded significantly higher post-thaw sperm motility than did combinations using other diluents. Glycerol and DMSO at a concentration of 10% as CPAs with 300 mM glucose as the diluent resulted in the highest MSR and sperm activity index (SAI). An investigation to determine the effects of glycerol and DMSO concentrations on post-thaw sperm survival rate revealed no significant differences among 5, 10, 15, and 20% concentrations of either CPA. In assessing the effects of CPA concentration on the fertilization rate, the 10% concentration yielded the highest fertilization rate (81.4 ± 4.3%) in DMSO, whereas 15% was the optimal concentration for glycerol (fertilization rate = 66.7 ± 6.1%). The hatching rate was also highest in 10% DMSO (40.1 ± 0.4%) and in 15% glycerol (27.8 ± 2.3%). In conclusion, the optimal rates of post-thaw sperm motility, fertilization, and hatching were achieved when E. akaara sperm were cryopreserved in a diluent of 300 mM glucose with 10% DMSO as the CPA at a freezing rate of −5 °C/min. We therefore recommend this protocol for the cryopreservation of E. akaara sperm.

Cryobiology of ovarian tissues: known knowns and known unknowns.

To model the cryobiological responses of cells and tissues, the cellular membrane permeability characteristics are often measured at suprazero temperatures as well as at subzero temperatures with and without the presence of extra-cellular ice. These measured membrane permeability characteristics are then used to predict the responses of cells and tissues for a given thermal insult with the ultimate aim of mitigating the damage caused during the freeze-thaw process. This brief review articles summarizes efforts from my research group over the past 15 years as related to cryobiology of mammalian ovarian tissue sections, i.e., the known knowns as well as the critical cryobiological knowledge that is still lacking to rationally design optimal cryopreservation protocols for ovarian tissues, i.e., the known unknowns.

A fluorescence ratio-based method to determine microalgal viability and its application to rapid optimization of cryopreservation

The utility of microalgal biomass and bioproducts depends on long-term maintenance of certain physiological or biochemical features of the species. While unique characteristics may not be durably maintained with general subculture, cryopreservation methods better prevent alterations from desired characteristics. Post-thaw viability is critical to establishing microalgal cultures, and there is a critical need to effectively and rapidly evaluate microalgal viability after the post-thawing process. In the present study, we developed a rapid assay based on the change of fluorescence ratio to determine microalgal viability post-thaw. It was shown that the assessment of microalgal viability by the fluorescence ratio method correlated well with that of the FDA-staining (R2 = 0.978) and regrowth method (R2 = 0.976), demonstrating that the present method could be applied in the high-throughput detection of viability of microalgal strains. Subsequent to establishing this method, we aimed to find out optimal cryopreservation protocol for each strain from a group of 125 microalgal strains. The viability of these strains under different treatments was quickly evaluated by the fluorescence ratio method. Of these strains, 95 attained post-thaw viability over 60%. DMSO was a suitable cryoprotectant for most strains at a concentration ≤10%. Based on the dataset, the relative contribution of 3 variables-genus, cryoprotectants and concentration to post-viability was analyzed with the Random Forest (RF) classification method. All variables together could explain 97.8% of the viability, and type and concentration of cryoprotectant could explain 59.1% in Chlorophyta. This study provided a new approach for viability assay and demonstrated that this method can facilitate to find out the optimal protocols for cryopreservation of microalgal strains.

Directional freezing for the cryopreservation of adherent mammalian cells on a substrate.

Successfully cryopreserving cells adhered to a substrate would facilitate the growth of a vital confluent cell culture after thawing while dramatically shortening the post-thaw culturing time. Herein we propose a controlled slow cooling method combining initial directional freezing followed by gradual cooling down to -80°C for robust preservation of cell monolayers adherent to a substrate. Using computer controlled cryostages we examined the effect of cooling rates and dimethylsulfoxide (DMSO) concentration on cell survival and established an optimal cryopreservation protocol. Experimental results show the highest post-thawing viability for directional ice growth at a speed of 30 μm/sec (equivalent to freezing rate of 3.8°C/min), followed by gradual cooling of the sample with decreasing rate of 0.5°C/min. Efficient cryopreservation of three widely used epithelial cell lines: IEC-18, HeLa, and Caco-2, provides proof-of-concept support for this new freezing protocol applied to adherent cells. This method is highly reproducible, significantly increases the post-thaw cell viability and can be readily applied for cryopreservation of cellular cultures in microfluidic devices.

Cryopreservation of sperm from the coral Acropora humilis

Corals are sensitive to minute changes in their environments, and their continued existence is substantially threatened by the increasing number of destructive anthropogenic activities and unprecedented rates of climate change. Although cryopreservation has been successfully to preserve mammalian gametes for decades, coral cryopreservation was attempted for the first time less than 15 years ago, and freezing protocols exist for only a handful of coral species. The present study developed a cryopreservation protocol for the sperm of the common Indo-Pacific reef-builder Acropora humilis. Colonies of reefs of Sattahip Bay, Chonburi Province, Thailand were collected from 3 m depth with a mesh net during a spawning event. Immediately after collection, the sperm were isolated and subjected to a two-step freezing method featuring DMSO, polyethylene glycol, or methanol as the cryoprotectant. Viability and motility were assessed via a bioluminescence technique and a “computer-assisted semen analysis, and it was found that a 15-min equilibration with 2 M DMSO followed by cooling at 41.7 °C was the optimum cryopreservation protocol for A. humilis sperm. The post-thaw sperm achieved 45% fertilization success, and 35% of the fertilized eggs developed into blastopore larvae. The present optimized protocol can therefore facilitate the preservation of sperm for future propagation efforts of this species and provide an experimental platform for optimizing cryopreservation protocols for gametes of other scleractinian coral species.

Solea senegalensis sperm cryopreservation: New insights on sperm quality.

Cryopreservation of Senegalese sole sperm can represent an alternative to overcome some reproductive problems of this species. However, it is important to guarantee the safe use of cryopreserved sperm by selecting an appropriate protocol according to a high demand quality need to be ensured. It has been demonstrated that traditional assays such as motility and viability do not provide enough information to identify specific damage caused by cryopreservation process (freezing and thawing). Specific tests, including lipid peroxidation and DNA damage, should be performed. In the present study, motility and lipid peroxidation were performed as specific tests allowing us to discard cryopreservation conditions such as methanol as internal cryoprotectant and bovine serum albumin as external cryoprotectant. In addition, a caspase 3/7 detection by flow cytometry was performed to analyze apoptosis activity in the best selected conditions. Moreover, new highly sensitive tests based on transcript number detection have recently been described in fish sperm cryopreservation. For this reason, a transcript level detection assay was performed on certain oxidative and chaperone genes related to fertilization ability and embryo development (hsp70, hsp90BB, hsp90AA, gpx) to select the best cryopreservation conditions. DMSO+ egg yolk proved to be the best cryoprotectant combination in terms of transcript level. This study describes an optimized cryopreservation protocol for Solea senegalensis sperm demonstrating for the first time that transcript degradation is the most sensitive predictor of cell status in this species after cryopreservation.

Cryopreservation of Hepatocyte Microbeads for Clinical Transplantation

Intraperitoneal transplantation of hepatocyte microbeads is an attractive option for the management of acute liver failure. Encapsulation of hepatocytes in alginate microbeads supports their function and prevents immune attack of the cells. Establishment of banked cryopreserved hepatocyte microbeads is important for emergency use. The aim of this study was to develop an optimized protocol for cryopreservation of hepatocyte microbeads for clinical transplantation using modified freezing solutions. Four freezing solutions with potential for clinical application were investigated. Human and rat hepatocytes cryopreserved with University of Wisconsin (UW)/10% dimethyl sulfoxide (DMSO)/5% (300 mM) glucose and CryoStor CS10 showed better postthawing cell viability, attachment, and hepatocyte functions than with histidine–tryptophan–ketoglutarate/10% DMSO/5% glucose and Bambanker. The 2 freezing solutions that gave better results were studied with human and rat hepatocytes microbeads. Similar effects on cryopreserved microbead morphology (external and ultrastructural), viability, and hepatocyte-functions post thawing were observed over 7 d in culture. UW/DMSO/glucose, as a basal freezing medium, was used to investigate the additional effects of cytoprotectants: a pan-caspase inhibitor (benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone [ZVAD]), an antioxidant (desferoxamine [DFO]), and a buffering and mechanical protectant (human serum albumin [HSA]) on RMBs. ZVAD (60 µM) had a beneficial effect on cell viability that was greater than with DFO (1 mM), HSA (2%), and basal freezing medium alone. Improvements in the ultrastructure of encapsulated hepatocytes and a lower degree of cell apoptosis were observed with all 3 cytoprotectants, with ZVAD tending to provide the greatest effect. Cytochrome P450 activity was significantly higher in the 3 cytoprotectant groups than with fresh microbeads. In conclusion, developing an optimized cryopreservation protocol by adding cytoprotectants such as ZVAD could improve the outcome of cryopreserved hepatocyte microbeads for future clinical use.

Dimethyleacetamide improves the cryosurvivability of Indian red jungle fowl (Gallus gallus murghi) sperm

It was hypothesized that dimethyleacetamide (DMA) can be used as an alternate to glycerol for cryopreservation of Indian red jungle fowl semen. Four concentrations of DMA (4%, 6%, 8% and 10%) in extender were compared with previously optimized cryopreservation protocol based on 20% glycerol (control) for Indian red jungle fowl. Sperm motility, plasma membrane integrity, viability, and acrosome integrity were assessed at the stage of post-dilution, cooling, equilibration, and freeze-thawing. The whole experiment was repeated/replicated for five times independently. Sperm motility, plasma membrane integrity, viability and acrosome integrity were recorded highest (P < 0.05) at post-dilution, cooling, equilibration, and freeze-thawing in extender having 6% DMA compared to control and other experimental extenders. The highest (P < 0.05) recovery rates of all aforementioned parameters were also recorded in extender having 6% DMA; thus, 6% DMA was further compared with control (20% glycerol) for fertility after artificial insemination. Eggs were collected for five days after artificial insemination with semen cryopreserved in extender containing 6% DMA and control. The higher no. of fertilized eggs, fertility, no. of hatched eggs, hatch (%) and hatchability were recorded with semen cryopreserved in extender having 6% DMA compared to control. It is concluded that 6% DMA maintained higher post-thaw quality and fertility of Indian red jungle fowl semen and is a better replacement of glycerol.

Complete protection against cryodamage of cryopreserved whole bovine and human ovaries using DMSO as a cryoprotectant

PurposeThis study aims to determine the optimal cryopreservation protocol for whole ovaries intended for preservation of fertility in women.MethodsWe investigated the optimal cryopreservation procedure for whole ovaries in a bovine model. The following parameters were investigated to determine their effect on ovarian tissue viability: type of cryoprotectant, administration route of the cryoprotectant (perfusion and/or submersion), and the maximum tolerable interval between death of the animal and start of the cryopreservation process. The resulting optimal cryopreservation procedure for bovine ovaries was subsequently tested on human ovaries. In vitro glucose uptake, histology, and immunohistochemistry were used to assess the integrity of the ovarian tissue.ResultsStarting the cryopreservation procedure (including perfusion with and submersion in DMSO) within 10–15 min after death of the animal proved critical, resulting in a 90–100% protection level against cryodamage. When cryopreserving human ovaries using the same protocol, over 95% protection against cryodamage was observed on all tissue levels. In addition, no apparent morphological damage to either the follicles or the vascular endothelium was observed.ConclusionOur findings suggest that using the optimized protocol presented in this paper allows good cryopreservation of whole human ovaries and represents an important step in considering whole ovary autotransplantation for clinically applied fertility preservation.

Cryobanking Mesenchymal Stem Cells.

Cryopreservation and storage of culture-expanded mesenchymal stem cells (MSCs) is essential for a biobank to maintain a collection of cell lines for research and clinical use. Optimization of cryopreservation protocols and methods to minimize damage to cells during freezing and thawing is critical to ensure reliable availability of viable cells. Controlling the freezing rate and the use of appropriate cryoprotectant, as well as stable storage temperature, can minimize the negative effects on cell viability. In this chapter, protocols for cryopreserving MSCs are described.

Cryopreservation: Vitrification and Controlled Rate Cooling.

Cryopreservation is the application of low temperatures to preserve the structural and functional integrity of cells and tissues. Conventional cooling protocols allow ice to form and solute concentrations to rise during the cryopreservation process. The damage caused by the rise in solute concentration can be mitigated by the use of compounds known as cryoprotectants. Such compounds protect cells from the consequences of slow cooling injury, allowing them to be cooled at cooling rates which avoid the lethal effects of intracellular ice. An alternative to conventional cooling is vitrification. Vitrification methods incorporate cryoprotectants at sufficiently high concentrations to prevent ice crystallization so that the system forms an amorphous glass thus avoiding the damaging effects caused by conventional slow cooling. However, vitrification too can impose damaging consequences on cells as the cryoprotectant concentrations required to vitrify cells at lower cooling rates are potentially, and often, harmful. While these concentrations can be lowered to nontoxic levels, if the cells are ultra-rapidly cooled, the resulting metastable system can lead to damage through devitrification and growth of ice during subsequent storage and rewarming if not appropriately handled.The commercial and clinical application of stem cells requires robust and reproducible cryopreservation protocols and appropriate long-term, low-temperature storage conditions to provide reliable master and working cell banks. Though current Good Manufacturing Practice (cGMP) compliant methods for the derivation and banking of clinical grade pluripotent stem cells exist and stem cell lines suitable for clinical applications are available, current cryopreservation protocols, whether for vitrification or conventional slow freezing, remain suboptimal. Apart from the resultant loss of valuable product that suboptimal cryopreservation engenders, there is a danger that such processes will impose a selective pressure on the cells selecting out a nonrepresentative, freeze-resistant subpopulation. Optimizing this process requires knowledge of the fundamental processes that occur during the freezing of cellular systems, the mechanisms of damage and methods for avoiding them. This chapter draws together the knowledge of cryopreservation gained in other systems with the current state-of-the-art for embryonic and induced pluripotent stem cell preservation in an attempt to provide the background for future attempts to optimize cryopreservation protocols.

Chapter 19 Cryopreservation Effect on Genetic Function: Neonatal Outcomes.

Cryopreservation is a well-established technique commonly used in clinical practice. It is used widely for the conservation of gametes and embryos that will be used later for insemination or in vitro fertilization. However, several studies have shown that this technique can produce changes in messenger RNA levels, in the epigenome and induce DNA damage. Although the embryo has potent mechanisms for DNA repair, and molecular changes in spermatozoa are not necessarily reflected in the embryo, it is important to explore new molecular tests and diagnostic tools to design optimal cryopreservation protocols and avoid undesirable molecular alterations. This chapter describes a protocol to quantify the lesions produced by cryopreservation using a protocol previously published by Rothfuss.

Improvement of the cryopreservation protocols for the dusky grouper, Epinephelus marginatus

The dusky grouper, Epinephelus marginatus, is a potential species for aquaculture production although the limited number of males kept in captivity has been the cause of some constraints in its production. Sperm cryopreservation emerged as a solution for this problem. However, spermatozoa can be severely affected by freezing/thawing processes and poor sperm quality is a limiting factor in reproduction success. The present study aimed at evaluating two main aspects in the design of a cryopreservation protocol-extender additives (taurine, glucose, cholesterol, BSA) and sperm containers (0.5mL straws, 2mL cryovials and 5mL macrotubes). Sperm quality was assessed through the evaluation of the percentage of motile cells, viable cells, DNA fragmentation, lipid peroxidation and apoptosis. Some specific techniques, such as Caspase 3/7 detection, which provides information on membrane integrity and cell death, detecting early and late apoptotic and necrotic events, were required to establish an optimized cryopreservation protocol for this species. Taurine was the most suitable cryopreservation additive in terms of viable cells and cholesterol presented the highest percentage of necrotic cells in this study. Caspase 3/7 assay enabled us to detect necrotic damage induced by cryopreservation. Statement of relevanceThe development of reproductive tools in dusky grouper, a potential species for aquaculture production, emerges as important tool to decrease the number of wild males maintained in captivity. A cryopreservation protocol was previously described for this species although several constraints in terms of sperm quality were detected. Our work provided new evidences that cryopreservation protocols can be improved through the addition of certain additives and use of appropriate sperm containers. Specific sperm analysis was crucial to identify and establish the most suitable conditions for breeders management and species conservation purposes.

Small-Molecule Ice Recrystallization Inhibitors Improve the Post-Thaw Function of Hematopoietic Stem and Progenitor Cells.

The success of hematopoietic stem cell transplantation depends in part on the number and the quality of cells transplanted. Cryoinjuries during freezing and thawing reduce the ability of hematopoietic stem and progenitor cells (HSPCs) to proliferate and differentiate after thawing. Up to 20% of the patients undergoing umbilical cord blood (UCB) transplant experience delayed or failed engraftment, likely because of the inadequate hematopoietic potency of the unit. Therefore, the optimization of cryopreservation protocols, with an emphasis on the preservation of HSPCs, is an important issue. Current protocols typically utilize a 10% dimethyl sulfoxide cryoprotectant solution. This solution ensures 70–80% post-thaw cell viability by diluting intracellular solutes and maintaining the cell volume during cryopreservation. However, this solution fails to fully protect HSPCs, resulting in the loss of potency. Therefore, a new class of cryoprotectants (N-aryl-d-aldonamides) was designed and assessed for the ability to inhibit ice recrystallization and to protect HSPCs against cryoinjury. Several highly active ice recrystallization inhibitors were discovered. When used as additives to the conventional cryoprotectant solution, these nontoxic small molecules improved the preservation of functionally divergent hematopoietic progenitors in the colony-forming unit and long-term culture-initiating cell assays. By contrast, structurally similar compounds that did not inhibit ice recrystallization failed to improve the post-thaw recovery of myeloid progenitors. Together, these results demonstrate that the supplementation of cryopreservation solution with compounds capable of controlling ice recrystallization increases the post-thaw function and potency of HSPCs in UCB. This increase may translate into reduced risk of engraftment failure and allow for greater use of cryopreserved cord blood units.