Non-permeating Cryoprotectants Research Articles

Investigating the cryoprotective efficacy of fructans in mammalian cell systems via a structure-functional perspective

Fructans have long been known with their role in protecting organisms against various stress factors due to their ability to induce controlled dehydration and support membrane stability. Considering the vital importance of such features in cryo-technologies, this study aimed to explore the cryoprotective efficacy of fructans in mammalian cell systems where structurally different fructan polymers were examined on in vitro cell models derived from organs such as the liver, frequently used in transplantation, osteoblast, and cord cells, commonly employed in cell banking, as well as human seminal fluids that are of vital importance in assisted reproductive technology. To gain insights into the fructan/membrane interplay, structural differences were linked to rheological properties as well as to lipid membrane interactions where both fluorescein leakage from unilamellar liposomes and membrane integrity of osteoblast cells were monitored. High survival rates obtained with human endothelial, osteoblast and liver cells for up to two months clearly showed that fructans could be considered as effective non-permeating cryoprotectants, especially for extended periods of cryopreservation. In trials with human seminal fluid, short chained levan in combination with human serum albumin and glycerol proved very effective in preserving semen samples across multiple patients without any morphological abnormalities.

Effect of non-permeable cryoprotectant sucrose on the development of spotted knifejaw (Oplegnathus punctatus) embryos

In this study, the toxicity of sucrose to Oplegnathus punctatus embryos was evaluated. Embryos at the 4–6 somite, tail-bud, heart formation, and heart-beating stages were exposed to 0, 0.5, 1,1.5, 2, 2.5, or 3 M sucrose for 1 h. Survival rates of embryos at the tail-bud, heart formation, and heart-beating stages after rehydration for 1 h were not affected by treatment with 2 M sucrose (the maximum concentration). Embryos at the tail-bud, heart formation, and heart-beating stages were exposed to 2 M sucrose for 0, 30, 60, 90, 120, 150, or 180 min. Long-term developmental indicators, including rates of survival, hatching, swimming, and malformation, were evaluated for 4 days after rehydration. Based on the survival rates 10 min after rehydration, the longest tolerance time for embryos at the three stages was 120 min. Based on long-term developmental indicators, the longest tolerance times were 60 min at the tail-bud, 60 min at the heart formation stage and 30 min at the heart beating stage. The malformation rates increased as the treatment time increased. The malformation rates were 100% when embryos were exposed to sucrose for ≥120 min. Malformation was divided into larval and embryonic abnormality. As the exposure time increased for tail-bud stage embryos, the rate of larval malformation increased. Treatment at heart formation and heart-beating stages resulted in higher rates of failure to hatch at exposure time. Based on these results, toxicity tests of non-permeable cryoprotectant in embryos requires the observation of development for at least 2 days after rehydration. Based on long-term observation, it was concluded that dehydration before freezing was not the direct cause of larvae deformity that hatched from frozen-thawing embryo. These results provide a reference for the singly use of representative non-permeable cryoprotectant sucrose.

Oxidative assessment of thawed donkey semen with seminal plasma, permeable and non-permeable cryoprotectants

During cryopreservation, donkey semen is exposed to oxidative and osmotic damage that alters the plasma membrane and causes alteration of sperm metabolism. In equids, the elimination of seminal plasma (SP) is a necessary process for the conservation of sperm in artificial conditions such as freezing, however, it is known that SP has an important role in physiological processes for sperm functions such as protection, transport, antioxidant defense, regulation of the inflammatory response in the uterus and interaction with the oocyte. The objective of this study was to evaluate total antioxidant capacity (TAC), reactive oxygen species (ROS) production and mitochondrial membrane potential of sperm, with different alternatives for donkey semen cryopreservation with permeable, non-permeable cryoprotectants and SP. Samples of semen were collected from ten Colombian Creole donkeys (Equus asinus), three ejaculates were collected from each donkey, for a total of 30 ejaculates. Eight treatments (T) were used with different combinations of 5% of dimethylformamide (DMF), 200 mM of sucrose (SUC), 1% of bovine serum albumin (BSA) and 10% of homologous seminal plasma (SP), as following: T1: DMF, T2: DMF/SP, T3: SAC/BSA, T4: SAC/BSA/SP, T5: DMF/SAC/BSA, T6: DMF/SAC/BSA/SP, T7: DMF/BSA, T8: DMF/BSA/SP. Besides the treatment, sperm was extended in EquiPlus® and 5% of clarified egg yolk, until a sperm concentration of 100 × 106 spermatozoa/mL. Posthawing, TAC was evaluated by the cationic radicalmethod (ABTS) and by the method of oxygen trapping capacity (ORAC) methodologies, total reactive oxygen species (ROS) production, superoxide anion (O2-) and hydroxyl radical (OH-) content were evaluated by spectrofluorimetry; sperm viability and mitochondrial membrane potential (ΔΨM) were measured by flow cytometry. For statistical analysis, a general linear model was fitted for each dependent variable. Normal distribution of variables was validated with the Kolmogorov–Smirnov test. Means were compared by the Tukey's test. Treatments T3 and T4 presented higher TAC (p<0.05) (ABTS and ORAC) compared to T1. For ΔψM, the lowest mean value was 9.1% for T4 (p<0.05), corresponding to one of the treatments without permeable cryoprotectant and SP. For sperm viability, the lowest values were found in both treatments with non-permeable cryoprotectant. Moreover, the treatments that were supplemented with SP showed higher production of ROS (p<0.05). In conclusion, supplementation of donkey semen with seminal plasma for cryopreservation does not alter the total antioxidant capacity; additionally, freezing semen with non-permeable cryoprotectants generates low sperm viability, low mitochondrial membrane potential and a high amount of reactive oxygen species and the composition of the freezing extender for donkey semen with seminal plasma influences the generation of total reactive oxygen species.

Cryopreservation of testicular cell in striped catfish (Pangasianodon hypophthalmus) and its effects on apoptosis, germ-cell specific gene expression and germ cell transplantability

Cryopreservation of testicular tissue would provide a useful tool to maintain spermatogonia, a precursor of gametes, for fish regeneration by germ cell transplantation. This study aimed to develop a cryopreservation method for spermatogonia by freezing the testicular tissue of striped catfish (Pangasianodon hypophthalmus) and testing whether cryopreserved spermatogonia exhibit transplantability. Freezing cryomedium containing L-15 medium and 1.3 M DMSO yielded the highest spermatogonia recovery rate after thawing. Supplementation with glucose and non-permeating cryoprotectants was not necessary to improve the recovery rate. We determined that an optimised equilibration time for cryopreserved testes in cryomedium included 60 min of equilibrium on ice before slow freezing (−1 °C/min), and subsequent thawing at 10 °C for 4–8 min. An Annexin V affinity assay and flow cytometry analysis were performed to identify apoptosis in cryopreserved testicular cells. Our results showed that cryoinjury of spermatogonia during cryopreservation could lead to apoptosis. Additionally, to determine the characteristics of cryopreserved spermatogonia at the molecular level, RT-qPCR was performed to quantify the expression of cxcr4b, dazl, dnd1, dnd2, nanos1, nanos2, and vasa. The results showed that compared to fresh testicular cells, frozen cells exhibited lower expression levels of cxcr4b and dazl. However, there were no significant differences in dnd1, dnd2, nanos1, and nanos2 expression levels. Although the expression level of vasa was lower in cryopreserved testes, both fresh and cryopreserved testicular cells showed positive vasa expression using in situ hybridisation. Furthermore, cryopreserved spermatogonia were transplanted into allogeniec recipient larvae and compared with fresh spermatogonia. Although the incorporation rate was lower than that of fresh spermatogonia, cryopreserved spermatogonia could be incorporated into the genital ridge of recipient larvae, demonstrating the transplantability of cryopreserved spermatogonia. Taken together, a method for cryopreservation of testicular tissue of striped catfish was optimised, and the cryopreserved testicular cells retained several spermatogenic stem cell characteristics, as well as transplantability.

Cryopreservation: A Comprehensive Overview, Challenges, and Future Perspectives.

Cryopreservation is the most prevalent method of long-term cell preservation. Effective cell cryopreservation depends on freezing, adequate storage, and correct thawing techniques. Recent advances in cryopreservation techniques minimize the cellular damage which occurs while processing samples. This article focuses on the fundamentals of cryopreservation techniques and how they can be implemented in a variety of clinical settings. The article presents a brief description of each of the standard cryopreservation procedures, such as slow freezing and vitrification. Alongside that, the membrane permeating and nonpermeating cryoprotectants are briefly discussed, along with current advancements in the field of cryopreservation and variables influencing the cryopreservation process. The diminution of cryoinjury incurred by the cell via the resuscitation process will also be highlighted. Inthe end applicationof cryopreservation techniques in many fields, with a special emphasis on stem cell preservation techniques and current advancements presented. Furthermore, the challenges while implementing cryopreservation and the futuristic scope of the fields are illustrated herein. The content of this review sheds light on various ways to enhance the output of the cell preservation process and minimize cryoinjury while improving cell revival.

Donkey semen cryopreservation: Alternatives with permeable, non-permeable cryoprotectants and seminal plasma.

Cryopreservation of semen is an important technique to preserve genetic material. Yet, pregnancy rates in jennies after artificial insemination with frozen-thawed donkey semen are poor. This condition has been attributed to the impact of permeable cryoprotectants, that could cause high post-breeding endometritis. Removal of seminal plasma (SP) prior to semen freezing process is another contributing factor. SP is involved in a multitude of sperm functions and events preceding fertilization and has a mediating effect of sperm capacitation and postcoital uterine inflammatory response. The aim of this study was to evaluate different alternatives in donkey semen cryopreservation with permeable, non-permeable cryoprotectants, BSA and SP. Thirty ejaculates from 10 donkeys were cryopreserved with different combinations of dimethylformamide (DMF, 5%), sucrose (SUC, 200 mM) and homologous SP (10%): DMF (T1), DMF/SP (T2), SUC/BSA (T3), SUC/BSA/SP (T4), DMF/SUC/BSA (T5), DMF/SUC/BSA/SP (T6), DMF/BSA (T7) and DMF/BSA/SP (T8). After thawing, sperm motility and kinetics were assessed by computerized semen analysis. Sperm vitality (SV) was evaluated by fluorescence microscopy, functional membrane integrity (FMI) by the HOST test, abnormal morphology by eosin-nigrosin staining and sperm membrane stability by flow cytometry. For statistical analysis, sperm quality indexes (SQi) were obtained, general linear models were carried out and mean comparisons were made by the Tukey test. T1, T2, T5, T6, and T7 had higher and equivalent results for motility, most kinetic parameters and function membrane integrity. Cryopreservation of donkey semen without permeable cryoprotectant (T3 and T4) showed a reduction in motility, kinetics, SV, FMI and SQi. T5 showed a reduction in progressive motility, sperm velocities, IMF and SQi compared to other DMF treatments. T6 and T8 achieved higher SQi values compared to T1, but they were not different compared to T2 and T7. T1 had a smaller sperm population with low-M540 compared to T3. It is concluded that the use of permeable cryoprotectant is essential to achieve higher post-thaw quality of donkey semen. In addition, the combined use of BSA, SUC and/or PS may provide additional sperm protection compared to the individual use of DMF.

Canine sperm vitrification with nonpermeable cryoprotectants and coconut water extender.

This study was aimed to assess the efficiency of coconut water extender with addition of soy lecithin and sucrose as nonpermeable cryoprotectants for canine semen vitrification, using a simple method that yields a high survival rate of spermatozoa for clinical use. Twelve ejaculates from 12 adult normozoospermic dogs were collected separately by digital manipulation and only the second semen fraction was used in this study. After evaluation of volume, concentration, viability, total and progressive motility, velocity parameters and morphology, semen was diluted with a coconut water extender (50% (v/v(volume per volume)) coconut water, 25% (v/v) distilled water and 25% (v/v) 5% anhydrous monosodium citrate solution) with addition of soy lecithin and fructose at 1% and 0.25M sucrose until final concentration of 100x106 spermatozoa/ml. After equilibration at 5ºC for 60 minutes, semen was vitrified by "direct dropping method" into liquid nitrogen in spheres with a volume of 30 μl. After a week of storage the spheres were devitrified as three of them were dropped into 0.5 mL of CaniPlus AI medium (Minitüb, Germany), which was previously warmed in a water bath at 42ºC for 2 minutes and evaluated about the above mentioned parameters. It was found that vitrification resulted in a lower percentage of viable sperms, normal morphology, total and progressive motilities (p<0.05), but most of velocity parameters (VCL, VSL, VAP, LIN, ALH and BCF) did not differ (p>0.05) compared to fresh semen samples. In conclusion, our results demonstrate that vitrification with coconut water extender with addition of 1% soy lecithin and 0.25M sucrose as cryoprotectants, has an excellent potential for routine canine sperm cryopreservation.

Development of cryopreservation media for the slow-freezing of cultured primordial germ cells in chicken.

Conservation of chicken germplasm is crucial in supporting commercial breeds for sustainable egg and meat production and preserving the genetic diversity of indigenous breeds for future breeding. Cryopreservation of chicken fertilized eggs or embryos is not feasible, owing to the large yolk-laden structure of the eggs. Primordial germ cells (PGCs), the embryonic precursors of gametes, are the best candidates for the cryobanking of chicken germplasm. Effective cryobanking of chicken PGCs requires an optimal cryopreservation protocol. Cryomedia containing dimethyl sulfoxide (DMSO) or DMSO combined with serum have been widely used for the cryopreservation of chicken PGCs. However, as cryoprotectants are yet to be optimized for chicken PGCs, the efficacy of cryomedia can be further improved. Here, we investigated the cryoprotective effects of propylene glycol (PG), an alternative to DMSO, on chicken PGCs. We found that the addition of non-permeable cryoprotectants, such as trehalose or chicken serum, to DMSO or PG cryomedia improved the recovery and survival rates of post-thawed PGCs. We further investigated the cryoprotective effects of trehalose and chicken serum and found that these additives have different cryoprotective actions. Based on these findings, we designed two different cryomedia: DTS, including 5% DMSO, 0.3 M trehalose, and 1% chicken serum, and PTS, including 7.5% PG, 0.1 M trehalose, and 5% chicken serum. Among the different PGC lines and freshly isolated PGCs, the cryomedia showed similar post-thaw recovery rates. Following transplantation, post-thawed male PGCs can colonize gonads and differentiate into functional sperm. We successfully revived the offspring of Kurokashiwa, a rare chicken breed in Japan, with cryopreserved PGCs. In conclusion, we developed two different cryomedia that achieved > 50% recovery of viable PGCs after thawing while maintaining germline competency.

Ultra-Rapid Freezing Using Droplets Immersed into Liquid Nitrogen in Bull Sperm: Evaluation of Two Cryoprotective Disaccharides and Two Warming Temperatures.

The present study analyzes the effects of different disaccharide concentrations and two thawing temperatures on the characteristics of ultrarapid frozen (URF) bovine sperm, compared with conventional slow-frozen (CF) sperm. For URF sperm, samples were diluted in media comprising 2% bovine serum albumin (BSA) and various nonpermeable cryoprotectants. Five groups were compared: control (without cryoprotectant), sucrose 0.15 M, sucrose 0.3 M, trehalose 0.15 M, and trehalose 0.3 M. In addition, the influence of warming temperatures, 37°C and 65°C, was analyzed. The aspect of different diluents (by drops) immersed in liquid nitrogen was also evaluated. Sperm quality was assessed by measuring motility, viability, acrosome status, and membrane lipid peroxidation (LPO). Moreover, the cryoresistance rate (CR) was determined. The drops immersed in liquid nitrogen showed that crystallization occurred, but not vitrification. CF sperm exhibited significantly higher scores for total motility (TM) and progressive motility (PM), viability, and acrosome integrity, in contrast with URF samples. Cryoprotectants for URF sperm showed a significant (p ≤ 0.05) influence on the TM and PM, viability, acrosome integrity, and CR, but not on LPO. Sperm viability was reduced after ultrarapid freezing, and the control samples were observed to have significantly lower values than those treated with disaccharides. Samples supplemented with 0.3 M sucrose exhibited higher LPO when they were thawed at 37°C. In short, a limited number of spermatozoa were able to maintain their motility and other functional attributes after ultrarapid freezing, but disaccharides showed a moderate protective effect. Samples with trehalose and sucrose at 0.15 and 0.3 M, respectively, showed higher sperm quality than samples containing only BSA. In sum, the function of spermatozoa was moderately maintained when disaccharides were used for ultrarapid freezing, although motility was significantly reduced. In addition, thawing temperatures did not modify the sperm values, suggesting that the easier procedure, that is, 37°C for 30 seconds, can be used.

Efecto del proceso de equilibrio en espermatozoides caninos después de la vitrificación con dilutor a base de agua de coco con adición de lecitina de soya y sacarosa como crioprotectores no permeables

The aim of the present study was to evaluate the effect of equilibration process on canine sperms after vitrification using coconut water extender with addition of soy lecithin and sucrose as nonpermeable cryoprotectants. Twelve ejaculates were collected separately by digital manipulation from 12 adult dogs. Only the second fraction of the ejaculate was used in this study, which was evaluated about volume, concetration, viability, total and progressive motility, kinetic parameters and morphology. After evaluation, semen was diluted with a coconut water extender (50% coconut water (v/v), 25% (v/v) distilled water and 25% (v/v) 5% anhydrous monosodium citrate solution) with addition of soy lecithin and fructose at 1% (v/v) and 0.25 M sucrose until final concentration of 100x106 spermatozoa/ml. Samples were divided into three aliquots and each of them was processed at different regimens: without equilibration, 5ºC for 30 minutes and 5ºC for 60 minutes and then vitrified by dropping 30 μl of sperm suspension directly into liquid nitrogen. Sperm pellets were devitrified at least one week later as three of them were dropped into 0.3 mL of CaniPlus AI (Minitüb, Germany), which was previously warmed in a water bath at 37ºC for 2 minutes. Sperm concentration and motility parameters were assayed using a computer-aided sperm analysis (CASA) system, viability-by supravital staining technique and morphology parameters were evaluated in Haemacolor® stained semen samples. In conclusion, our results demonstrate that when vitrification and coconut water extender with addition of 1% soy lecithin and 0.25 M sucrose as cryoprotectants were used, presence of equilibration time of 60 minutes returned the best canine sperm quality results.

Sperm cryopreservation of Prochilodus brevis using different concentrations of non-permeable cryoprotectants.

The action of substances with non-permeable cryoprotectant potential, besides glucose, has not yet been studied for the species Prochilodus brevis. The objective of this work was to evaluate the action of four non-permeable cryoprotectants on this species sperm cryopreservation. Five pools were cryopreserved in a solution of 5% glucose and 10% dimethyl sulfoxide (Me2SO) associated or not (control) with cryoprotectants egg yolk (5, 10 or 12%), soy lecithin (2.5, 7.5 or 10%), sucrose (5, 10 or 20%) and lactose (5, 8 or 15%). After thawing, samples were evaluated for sperm kinetics (total motility, motility duration, velocities, and wobble - WOB), morphology and membrane and DNA integrity. The treatments containing egg yolk improved significantly (P<0.05) results when compared the control for the membrane integrity parameter. When compared to other treatments, egg yolk, at any concentration, presented higher results (P<0.05) for membrane integrity, total motility, curvilinear velocity (VCL) and average path velocity (VAP) parameters. Egg yolk also showed the best results for WOB, but it did not differ from 5% and 8% lactose and 5% and 20% sucrose. Soy lecithin had the lowest percentages of morphologically normal sperm (P<0.05), while the other treatments did not differ from each other. There was no difference regarding DNA integrity data. Thus, 5% egg yolk is indicated as a non-permeable cryoprotectant for P. brevis, in association with 5% glucose and 10% Me2SO.

Trehalose glycopolymers for cryopreservation of tissue-engineered constructs

The development of an effective cryopreservation method to achieve off-the-shelf and bioactive tissue-engineered constructs (TECs) is important to meet the requirements for clinical applications. The trehalose, a non-permeable cryoprotectant (CPA), has difficulty in penetrating the plasma membranes of mammalian cells and has only been used in combination with other cell penetrating CPA (such as DMSO) to cryopreserve mammalian cells. However, the inherent cytotoxicity of DMSO results in increasing risks with respect to cryopreserved cells. Therefore, in this study, permeable trehalose glycopolymers were synthesised for cryopreservation of TECs. The trehalose glycopolymers exhibited good ice inhibiting activities and biocompatibilities. Furthermore, the viability and function of TECs after cryopreservation with 5.0 wt% S2 were similar to those of the non-cryopreserved TECs. We developed an effective preservation strategy for the off-the-shelf availability of TECs.

Cryopreservation of dog epididymal spermatozoa by conventional freezing or ultra-rapid freezing with nonpermeable cryoprotectant

This study was aimed to assess the effectiveness of two methods for cryopreservation of dog epididymal spermatozoa, one by conventional freezing (CF) with shortening both equilibration and cooling times, and the other by ultra-rapid freezing (URF) with nonpermeable cryoprotectant. Sixty epididymides were recovered from thirty orchiectomized adult dogs and the sperm samples were retrieved by retrograde flushing using TCG-EY (tris, citric acid, glucose + 20% egg yolk) extender and then 20 pools were conformed. Each pool was divided into 2 aliquots and then cryopreserved by CF and URF methods respectively. The CF method maintained the cooled-pool samples for 2h (1h without and 1h with 5% glycerol) and then were frozen by liquid nitrogen (LN2) vapors for 2 min. The URF method cryopreserved the cooled-pool samples using TCG-EY+250 mM sucrose, equilibrating during 30 min (5 °C) and submerging 30-μL drops directly in LN2. The results showed that the URF method produced a lower percentage of total and progressive motilities and acrosome integrity (P < 0.05) than the CF method. However, the kinetic variables (curvilinear and straight-line velocities, straightness, linearity, wobble, amplitude of lateral head displacement, and beat-cross frequency) and plasma membrane integrity did not differ (P > 0.05) between both cryopreservation methods. Unlike the URF method, the width, area and perimeter of sperm head were reduced after the CF method (P < 0.05). In conclusion, despite the low motility achieved after the ultra-rapid freezing method, the similar values of kinetic, viability and head morphometric dimensions to those obtained after conventional freezing, suggest that ultra-rapid freezing with sucrose may be a useful alternative for the cryopreservation of canine epididymal sperm.

Synthetic zwitterions as efficient non-permeable cryoprotectants

Cryopreservation of cells is necessary for long periods of storage. However, some cell lines cannot be efficiently cryopreserved, even when optimized commercial cryoprotectants are employed. Previously, we found that a low-toxic synthetic zwitterion aqueous solution enabled good cryopreservation. However, this zwitterion solution could not cryopreserve some cells, such as human kidney BOSC cells, with good efficiency. Therefore, details of the cryoprotective effect of the zwitterions and optimization based on its mechanisms are required. Herein, we synthesized 18 zwitterion species and assessed the effects of the physical properties of water/zwitterion mixtures. Non-cell-permeable zwitterions can inhibit ice crystal formation extracellularly via direct interaction with water and intracellularly via dehydration of cells. However, cells that could not be cryopreserved by zwitterions were insufficiently dehydrated in the zwitterion solution. Dimethyl sulfoxide (DMSO) was combined as a cell-permeable cryoprotectant to compensate for the shortcomings of non-cell-permeable zwitterions. The water/zwitterion/DMSO (90/10/15, v/w/w) could cryopreserve different cells, for example freezing-vulnerable K562 and OVMANA cells; yielding ~1.8-fold cell viability compared to the case using a commercial cryoprotectant. Furthermore, molecular dynamics simulation indicated that the zwitterions protected the cell membrane from the collapse induced by DMSO.

Bovine Fetal Mesenchymal Stem Cells Obtained From Omental Adipose Tissue and Placenta Are More Resistant to Cryoprotectant Exposure Than Those From Bone Marrow.

Recent studies have shown promise for the development of cellular therapies with mesenchymal stem cells (MSCs) in livestock species, specifically bovines, and cryopreservation is highly relevant for the advancement of these applications. The use of permeable and/or non-permeable cryoprotectant solutions is necessary to reduce cell damage during freezing and thawing, but these same compounds can also cause negative effects on MSCs and their therapeutic properties. Another important factor to consider is the tissue source of MSCs, since it is now known that MSCs from different tissues of the same individual do not behave the same way, so optimizing the type and concentration of cryoprotectants for each cell type is essential to achieve a large and healthy population of MSCs after cryopreservation. Furthermore, sources of MSCs that could provide great quantities, non-invasively and without ethical concerns, such as placental tissue, have great potential for the development of regenerative medicine in livestock species, and have not been thoroughly evaluated. The objective of this study was to compare the viability of bovine fetal MSCs extracted from bone marrow (BM), adipose tissue (AT), and placenta (PT), following their exposure (15 and 30 min) to several solutions of permeable (dimethyl sulfoxide and ethylene glycol) and non-permeable (trehalose) cryoprotectants. Viability assays were performed with Trypan Blue to assess post-exposure plasma membrane integrity. The apoptotic potential was estimated analyzing the mRNA abundance of BAX and BCL-2 genes using quantitative rt-PCR. Based on the results of the study, BM-MSC exhibited significantly lower viability compared to AT-MSC and PT-MSC, at both 15 and 30 min of exposure to cryoprotectant solutions. Nevertheless, viability did not differ among treatments for any of the cell types or timepoints studied. BCL-2 expression was higher in BM-MSC compared to AT-MSC, however, BAX/BCL-2 ratio did not differ. In conclusion, AT-MSC and PT-MSC were more resistant that BM-MSC, which showed higher sensitivity to experimental conditions, regardless of the exposure times, and cryoprotectant solutions used in the study.

Cryopreservation of embryos of humpback grouper (Cromileptes altivelis) using combinations of non-permeating cryoprotectants

In this study, Cromileptes altivelis embryos were successfully preserved using non-permeable cryoprotectants for the first time. We compared the toxicity of single non-permeable cryoprotectants and combination of non-permeable cryoprotectants on C. altivelis embryos. Five non-permeable cryoprotectants at 1 M (i.e., trehalose, sucrose, lactose, fructose, and glucose) were used to treat embryos in the somite, tail-bud, or heart-beating stages for 1 h. Survival rates and rates of normal embryonic development after treatment with trehalose, sucrose, or lactose were all greater than 86.97% and 86.01%, respectively. Levels of toxicity of trehalose, sucrose, and lactose were lower than those of fructose and glucose in C. altivelis embryos. Sucrose and trehalose were combined at different ratios (total concentration 1 M) and used to treat embryos for 1 h. Rates of survival and normal development for the somite, tail-bud, or heart-beating stages did not significantly differ among groups treated with sucrose + trehalose at ratios of 1:1 (S1T1), 1:3 (S1T3), and 3:1 (S3T1). Embryos treated with S1T1 had a little higher survival rate than those of the other treatment ratios(P > 0.05). Survival rates of embryos treated with T1S1, sucrose, and trehalose were 93.86 ± 4.17%, 91.58 ± 3.15%, and 92.79 ± 2.45%, respectively. S1T1 was less toxic than sucrose or trehalose alone under the same conditions. Various cryopreservation-related factors (e.g., the cryoprotectant solution, embryonic stage, equilibrium time, thawing temperature, and time) were screened. The optimal protocol was the treatment of embryos at the tail-bud and heart-beating stages with 1 M S1T1 for 2–10 min and thawing at 26 °C for 10 s after freezing. In total, 21 viable embryos were recovered after freezing and thawing, with a survival rate of 0.21–7.55% and maximum survival time of 5 days. Our results demonstrate that it is feasible to cryopreserve C. altivelis embryos using non-permeable cryoprotectants and provide a reference for the use of combinations of cryoprotectants for the species.

Addition of disaccharides and dimethyl sulfoxide to sodium alginate gel improves viability of immobilized Saccharomyces boulardii after cryopreservation

Gel-immobilized microorganisms are increasingly used in microbiological industries. Currently, the problem of developing technologies for long-term storage of microorganisms immobilized in gel carriers remains urgent. Low-temperature storage is the most effective method of preserving microorganisms. The viability of immobilized cells is affected by cryoprotective properties of gel matrix as well as cooling regimes. Therefore, the effects of incorporation of cryoprotective agents in alginate gel and cooling regimes on the viability of immobilized Saccharomyces boulardii cells after cryopreservation were studied. Incorporation of non-permeable cryoprotectants (sucrose, lactose, and trehalose) and permeable one dimethyl sulfoxide (DMSO) in alginate gel beads promoted an increase in the viability of immobilized cells after freeze-thawing. The highest viability rates of the gel-immobilized cells were observed in the alginate gel beads incorporating combinations of DMSO (5 – 10 % v/v) and one of the disaccharides (10 – 20 % w/v). In all experiments, slow cooling provided significantly higher viability if compared to the rapid immersion of the samples into liquid nitrogen.

Development of an Effective Cryopreservation Protocol for Blue Catfish Oogonia

AbstractLong‐term storage of oogonia and germ‐line stem cells provides an alternative to the limitations associated with cryopreserving eggs of important fish species. These cell types are less vulnerable to the stresses of freezing. Cryopreservation has enormous potential for aquaculture advancement, but protocols must be developed for each species and cell type since its success hinges on various input factors. Blue Catfish Ictalurus furcatus were selected as the test species in this study because of the need to improve fry production of Blue Catfish ♂ × Channel Catfish I. punctatus ♀ hybrids, which can be facilitated by storing oogonia in gene banks. Our objective was to develop a freezing protocol for oogonia of this species. We tested different permeating and nonpermeating cryoprotectants, concentrations of these agents, and freezing rates. We proved that all three factors influenced postthaw recovery of oogonia. Of the permeating cryoprotectants, 1.0 M dimethyl sulfoxide resulted in the most live cells with the highest viability percentages, and adding 0.2 M lactose with 10% egg yolk further improved the results. There were also specific interactions in which the effects of concentration and freezing rate varied among the cryoprotectant treatments. The most effective freezing rate was −1.0°C/min, and cell viability was reduced at −2.5°C/min and −5.0°C/min. From these results, we propose adding 1.0 M dimethyl sulfoxide with 0.2 M lactose and 10% egg yolk to cryomedia and freezing it at a rate of −1.0°C/min. By developing a cryopreservation protocol for a commonly cultured catfish, this work may guide the development of protocols for other species of interest.

Development of Sperm Cryopreservation Protocols for Sharks and Rays: New Tools for Elasmobranch Conservation

Elasmobranchs are one of the most endangered vertebrate groups on the planet, but despite this situation the use of reproductive techniques in elasmobranch conservation strategies has been scarce. Among these techniques, sperm preservation is a potential tool for ex situ conservation and aquaria sustainability. However, there are no widespread preservation protocols for elasmobranch sperm, and shark sperm cryopreservation has never been achieved before. Here we present the establishment of successful cryopreservation protocols for elasmobranch sperm, tested in several species. We have formulated a sperm extender that can be used for different elasmobranch species, capable of maintaining sperm motility for several weeks. Additionally, we achieved the cryopreservation of sperm by previously diluting it in our extender and supplementing it with different combinations of cryoprotectants. The effects of methanol and dimethyl sulfoxide as permeating cryoprotectants were evaluated, as well egg yolk as a non-permeating cryoprotectant. Sperm quality was assessed by studying the motility and membrane integrity post-thawing, demonstrating its effectiveness in the 10 species tested, including two which are considered Critically Endangered. This is the first time that shark sperm cryopreservation has been reported, broadening our knowledge of the reproductive techniques that can be applied to elasmobranchs and laying the foundations for the first cryobanks for shark and ray sperm. Outcomes from this study will be useful for ex situ conservation efforts developed by public aquaria. A regular supply of frozen sperm will reduce the problems that result from the transport of specimens, inbreeding or lack of synchronized reproductive cycles in captivity.

Toward embryo cryopreservation-on-a-chip: A standalone microfluidic platform for gradual loading of cryoprotectants to minimize cryoinjuries.

Embryo vitrification is a fundamental practice in assisted reproduction and fertility preservation. A key step of this process is replacing the internal water with cryoprotectants (CPAs) by transferring embryos from an isotonic to a hypertonic solution of CPAs. However, this applies an abrupt osmotic shock to embryos, resulting in molecular damages that have long been a source of concern. In this study, we introduce a standalone microfluidic system to automate the manual process and minimize the osmotic shock applied to embryos. This device provides the same final CPA concentrations as the manual method but with a gradual increase over time instead of sudden increases. Our system allows the introduction of the dehydrating non-permeating CPA, sucrose, from the onset of CPA-water exchange, which in turn reduced the required time of CPA loading for successful vitrification without compromising its outcomes. We compared the efficacy of our device and the conventional manual procedure by studying vitrified-warmed mouse blastocysts based on their re-expansion and hatching rates and transcription pattern of selected genes involved in endoplasmic reticulum stress, oxidative stress, heat shock, and apoptosis. While both groups of embryos showed comparable re-expansion and hatching rates, on-chip loading reduced the detrimental gene expression of cryopreservation. The device developed here allowed us to automate the CPA loading process and push the boundaries of cryopreservation by minimizing its osmotic stress, shortening the overall process, and reducing its molecular footprint.