In situ cryopreservation of human embryonic stem cells in gas-permeable membrane culture cassettes for high post-thaw yield and good manufacturing practice

Current strategies for marine pollution monitoring are based on the integration of chemical and biological techniques. The sea urchin embryo-larval bioassays are among the biological methods most widely used worldwide. Cryopreservation of early embryos of sea urchins could provide a useful tool to overcome one of the main limitations of such bioassays, the availability of high quality biological material all year round. The present study aimed to determine the suitability of several permeant (dimethyl sulfoxide, Me₂SO; propylene glycol, PG; and ethylene glycol, EG) and non-permeant (trehalose, TRE; polyvinylpyrrolidone, PVP) cryoprotectant agents (CPAs) and their combination, for the cryopreservation of eggs and embryos of the sea urchin Paracentrotus lividus. On the basis of the CPAs toxicity, PG and EG, in combination with PVP, seem to be most suitable for the cryopreservation of P. lividus eggs and embryos. Several freezing procedures were also assayed. The most successful freezing regime consisted on cooling from 4 to −12 °C at 1 °C/min, holding for 2 min for seeding, cooling to −20 °C at 0.5 °C/min, and then cooling to −35 °C at 1 °C/min. Maximum normal larvae percentages of 41.5% and 68.5%, and maximum larval growth values of 42.9% and 60.5%, were obtained for frozen fertilized eggs and frozen blastulae, respectively.     

Cryopreservation of Endothelial Cells in Various Cryoprotective Agents and Media – Vitrification versus Slow Freezing Methods

Vitrification of endothelial cells (MHECT-5) has not previously been compared with controlled slow freezing methods under standardized conditions. To identify the best cryopreservation technique, we evaluated vitrification and standardized controlled-rate -1°C/minute cell freezing in a -80°C freezer and tested four cryoprotective agents (CPA), namely dimethyl sulfoxide (DMSO), ethylene glycol (EG), propylene glycol (PG), and glycerol (GLY), and two media, namely Dulbecco''s modified Eagle medium Ham’s F-12 (DMEM)and K⁺-modified TiProtec (K⁺TiP), which is a high-potassium-containing medium. Numbers of viable cells in proliferation were evaluated by the CellTiter 96® A_Queous One Solution Cell Proliferation Assay (Promega Corporation, Mannheim, Germany). To detect the exact frozen cell number per cryo vial, DNA content was measured by using Hoechst 33258 dye prior to analysis. Thus, results could be evaluated unconstrained by absolute cell number. Thawed cells were cultured in 25 cm² cell culture flasks to confluence and examined daily by phase contrast imaging. With regard to cell recovery immediately after thawing, DMSO was the most suitable CPA combined with K⁺TiP in vitrification (99 ±0.5%) and with DMEM in slow freezing (92 ±1.6%). The most viable cells in proliferation after three days of culture were obtained in cells vitrificated by using GLY with K⁺TiP (308 ±34%) and PG with DMEM in slow freezing (280 ±27%).     

Permeability of the equine embryonic capsule to ethylene glycol and glycerol in vitro

Poor survival of cryopreservation by equine expanded blastocysts may involve low penetration of the embryonic capsule by cryoprotective agents (CPAs). This study characterized the permeation and accumulation rates of the CPAs ethylene glycol (EG) and glycerol (GLY) across isolated capsule in vitro, using a dual-chambered Valia-Chien permeation apparatus. Pieces of Days 14 to 18 ±1 capsules separated media in the “donor” chamber containing either 1.5 M EG (n = 6), 0.74 M EG (n = 5), 0.87 M GLY (n = 7), or 0.15 M NaCl (saline, SAL) (n = 6), from the “recipient” chamber. Concentrations of CPA, determined by gas chromatography, allowed calculation of the capsule's apparent permeability (P_app) to those CPAs. Permeation of capsule by 1.5 M EG was significantly more rapid than by 0.87 M GLY, or 0.74 M EG; permeation by both CPAs was significantly slower than by SAL. Accumulation of CPA in the recipient chamber depended more on initial donor chamber concentration, rather than type, of CPA. Accumulation rates for CPAs and SAL were linear only when capsule was present, demonstrating that their permeation through capsule was more complex than simple diffusion. Successful cryopreservation of equine expanded blastocysts has been previously linked to lengths of step-wise exposures to CPAs. Based on the present results, we inferred that alternative CPAs, more capable of permeating the capsule, or alternative methods of ensuring CPA entry into the cells, may also be required.     

Cryopreservation of kangaroo spermatozoa using alternative approaches that reduce cytotoxic exposure to glycerol

Alternative techniques for the cryopreservation of kangaroo spermatozoa that reduced or eliminated the need for glycerol were investigated including; (1) freezing spermatozoa with 20% glycerol in pre-packaged 0.25 mL Cassou straws to enable rapid dilution of the glycerol post-thaw, (2) investigating the efficacy of 20% (v/v) dimethyl sulphoxide (DMSO) and dimethylacetamide (DMA—10%, 15% and 20% v/v) as cryoprotectants and (3) vitrification of spermatozoa with or without cryoprotectant (20% v/v glycerol, 20% v/v DMSO and 20% v/v DMA). Immediate in-straw post-thaw dilution of 20% glycerol and cryopreservation of spermatozoa in 20% DMSO produced no significant improvement in post-thaw viability of kangaroo spermatozoa. Spermatozoa frozen in 20% DMA showed post-thaw motility and plasma membrane integrity of 12.7 ± 1.9% and 22.7 ± 5.4%, respectively, while kangaroo spermatozoa frozen by ultra-rapid freezing techniques showed no evidence of post-thaw viability. The use of 10–20% DMA represents a modest but significant improvement in the development of a sperm cryopreservation procedure for kangaroos.     

Toxicity tests of cryoprotecting agents for Mytilus galloprovincialis (Lamark, 1819) early developmental stages

Global aquaculture production of blue mussel has increased over last years. This work reaffirms the great potential of cryopreservation technique on mussel industry and overcome economic barriers a cause of a traditional and rudimentary management and continue growing. The aim of this work is to set some preliminary basis attending to toxicity of cryoprotecting agents (CPAs) on different development stages of Mytilus galloprovincialis as a start point to develop a stable cryopreservation protocol. Toxicity tests were carried out by using common CPAs (dimethyl-sulfoxide (Me₂SO), glycerol, (GLY), propylene glycol (PG) and ethylene glycol (EG)) in a range from 0.5 to 3 M on fertilized egg, trochophore larva, and D-larva of Mytilus galloprovincialis. Results evidenced more resistance of older development stages to toxicity. Of all CPAs tested, toxicity testing highlights PG or EG as suitable CPAs for cryopreservation of early development stages; whereas D-larva was unaffected by any of the CPAs tested. Preliminary cryopreservation trials were developed to obtain information into cell cryoprotection. Further research should be focused on membrane permeability and other parameters, such as the balance between toxicity and cryoprotective effect of CPAs.     

Quantitative investigations on the effects of exposure durations to the combined cryoprotective agents on mouse oocyte vitrification procedures

Vitrification by using two-step exposures to combined cryoprotective agents (CPAs) has become one of the most common methods for oocyte cryopreservation. By quantitatively examining the status of oocytes during CPA additions and dilutions, we can analyze the degree of the associated osmotic damages. The osmotic responses of mouse MII oocyte in the presence of the combined CPAs (ethylene glycol, EG, and dimethyl sulfoxide, DMSO) were recorded and analyzed. A two-parameter model was used in the curve-fitting calculation to determine the values of hydraulic conductivity (L(p)) and permeability (P(s)) to the combined CPAs at 25°C and 37°C. The effects of exposure durations and the exposure temperatures on the cryopreservation in terms of frozen-thawed cell survival rates and subsequent development were examined in a series of cryopreservation experiments. Mouse MII oocytes were exposed to pretreatment solution (PTS) and vitrification solution (VS) at specific temperatures. The PTS used in our experiment was 10% EG and 10% DMSO dissolved in modified PBS (mPBS), and the VS was EDFS30 (15% EG, 15% DMSO, 3 × 10(-3) M Ficoll, and 0.35 M sucrose in mPBS).The accumulative osmotic damage (AOD) and intracellular CPA concentrations were calculated under the different cryopreservation conditions, and for the first time, the quantitative interactions between survival rates, subsequent development rates, and values of AOD were investigated.     

Cryobiology of rat embryos I: determination of zygote membrane permeability coefficients for water and cryoprotectants, their activation energies, and the development of improved cryopreservation methods

New rat models are being developed at an exponential rate, making improved methods to cryopreserve rat embryos extremely important. However, cryopreservation of rat embryos has proven to be difficult and expensive. In this study, a series of experiments was performed to characterize the fundamental cryobiology of rat fertilized 1-cell embryos (zygotes) and to investigate the effects of different cryoprotective agents (CPAs) and two different plunging temperatures (T(p)) on post-thaw survival of embryos from three genetic backgrounds. In the initial experiments, information on the fundamental cryobiology of rat zygotes was determined, including 1) the hydraulic conductivity in the presence of CPAs (L(p)), 2) the cryoprotectant permeability (P(CPA)), 3) the reflection coefficient (sigma), and 4) the activation energies for these parameters. P(CPA) values were determined for the CPAs, ethylene glycol (EG), dimethyl sulfoxide (DMSO), and propylene glycol (PG). Using this information, a cryopreservation method was developed and the cryosurvival and fetal development of Sprague-Dawley zygotes cryopreserved in either EG, DMSO, or PG and plunged at either -30 or -80 degrees C, were assessed. The highest fetal developmental rates were obtained using a T(p) of -30 degrees C and EG (61.2% +/- 2.4%), which was not different (P > 0.05) from nonfrozen control zygotes (54.6% +/- 3.0%).     

Molecular Mechanism of the Synergistic Effects of Vitrification Solutions on the Stability of Phospholipid Bilayers

The vitrification solutions used in the cryopreservation of biological samples aim to minimize the deleterious formation of ice by dehydrating cells and promoting the formation of the glassy state of water. They contain a mixture of different cryoprotective agents (CPAs) in water, typically polyhydroxylated alcohols and/or dimethyl sulfoxide (DMSO), which can damage cell membranes. Molecular dynamics simulations have been used to investigate the behavior of pure DPPC, pure DOPC, and mixed DOPC-β-sitosterol bilayers solvated in a vitrification solution containing glycerol, ethylene glycol, and DMSO at concentrations that approximate the widely used plant vitrification solution 2. As in the case of solutions containing a single CPA, the vitrification solution causes the bilayer to thin and become disordered, and pores form in the case of some bilayers. Importantly, the degree of thinning is, however, substantially reduced compared to solutions of DMSO containing the same total CPA concentration. The reduction in the damage done to the bilayers is a result of the ability of the polyhydroxylated species (especially glycerol) to form hydrogen bonds to the lipid and sterol molecules of the bilayer. A decrease in the amount of DMSO in the vitrification solution with a corresponding increase in the amount of glycerol or ethylene glycol diminishes further its damaging effect due to increased hydrogen bonding of the polyol species to the bilayer headgroups. These findings rationalize, to our knowledge for the first time, the synergistic effects of combining different CPAs, and form the basis for the optimization of vitrification solutions.     

Molecular Mechanism of the Synergistic Effects of Vitrification Solutions on the Stability of Phospholipid Bilayers

The vitrification solutions used in the cryopreservation of biological samples aim to minimize the deleterious formation of ice by dehydrating cells and promoting the formation of the glassy state of water. They contain a mixture of different cryoprotective agents (CPAs) in water, typically polyhydroxylated alcohols and/or dimethyl sulfoxide (DMSO), which can damage cell membranes. Molecular dynamics simulations have been used to investigate the behavior of pure DPPC, pure DOPC, and mixed DOPC-β-sitosterol bilayers solvated in a vitrification solution containing glycerol, ethylene glycol, and DMSO at concentrations that approximate the widely used plant vitrification solution 2. As in the case of solutions containing a single CPA, the vitrification solution causes the bilayer to thin and become disordered, and pores form in the case of some bilayers. Importantly, the degree of thinning is, however, substantially reduced compared to solutions of DMSO containing the same total CPA concentration. The reduction in the damage done to the bilayers is a result of the ability of the polyhydroxylated species (especially glycerol) to form hydrogen bonds to the lipid and sterol molecules of the bilayer. A decrease in the amount of DMSO in the vitrification solution with a corresponding increase in the amount of glycerol or ethylene glycol diminishes further its damaging effect due to increased hydrogen bonding of the polyol species to the bilayer headgroups. These findings rationalize, to our knowledge for the first time, the synergistic effects of combining different CPAs, and form the basis for the optimization of vitrification solutions.     

Dose–injury relationships for cryoprotective agent injury to human chondrocytes

Vitrification of articular cartilage (AC) could enhance tissue availability but requires high concentrations of cyroprotective agents (CPAs). This study investigated relative injuries caused by commonly used CPAs. We hypothesized that the in situ chondrocyte dose–injury relationships of five commonly used CPAs are nonlinear and that relative injuries could be determined by comparing cell death after exposure at increasing concentrations. Human AC samples were used from four patients undergoing total knee arthroplasty surgery. Seventy μm slices were exposed in a stepwise protocol to increasing concentrations of 5 CPAs (max = 8 M); dimethyl sulfoxide (Me₂SO), glycerol (Gly), propylene glycol (PG), ethylene glycol (EG), and formamide (FM). Chondrocyte viability was determined by membrane integrity stains. Statistical analysis included t-tests and nonlinear least squares estimation methods. The dose–injury to chondrocytes relationships for all CPAs were found to be nonlinear (sigmoidal best fit). For the particular loading protocol in this study, the data identified the following CPA concentrations at which chondrocyte recoveries statistically deviated significantly from the control recovery; 1 M for Gly, 4 M for FM and PG, 6 M for Me₂SO, and 7 M for EG. Comparison of individual means demonstrated that Gly exposure resulted in the lowest recovery, followed by PG, and then Me₂SO, FM and EG in no specific order. The information from this study provides an order of damage to human chondrocytes in situ of commonly used CPAs for vitrification of AC and identifies threshold CPA concentrations for a stepwise loading protocol at which chondrocyte recovery is significantly decreased. In general, Gly and PG were the most damaging while DMSO and EG were among the least damaging.     

Survival of tissue balls from the coral Pocillopora damicornis L. exposed to cryoprotectant solutions

In this study, the tolerance of tissue balls (TBs, 100–300 μm in diameter) from the coral Pocillopora damicornis produced using mechanical excision to exposure to cryoprotectant (CPA) solutions was tested. TBs were treated for 20 min at room temperature with solutions of ethylene glycol (EG), methanol (Met), glycerol (Gly) or dimethyl sulfoxide (Me₂SO) at concentrations between 1.0 and 4.5 M. Two parameters were used to evaluate the survival of TBs following CPA treatment. The Undamaged Duration of Tissue Balls (expressed in h) corresponded to the time period during which the membrane surface of TBs remained smooth and their motility was preserved. Tissue Ball Regression (expressed in μm/h) corresponded to the size reduction of TBs over time. TBs tolerated exposure to all CPAs tested at the three lower concentrations employed (1.0 M, 1.5 M and 2.0 M). No survival was achieved following exposure to a 4.5 M CPA solution. At concentrations of 3.0 and 4.0 M, higher Undamaged Duration of Tissue Balls and lower Tissue Ball Regression were obtained following treatment with EG compared to the other three CPAs. Our experiments show that TBs constitute a good experimental material to evaluate CPA toxicity on corals using large numbers of samples. Performing preliminary experiments with TBs may allow reducing the number of tests carried out with less easily available coral forms such as planulae, thereby preserving larval stocks.     

Cryoprotectant delivery and removal from murine insulinomas at vitrification-relevant concentrations

Development of optimal cryopreservation protocols requires delivery and removal of cryoprotective agents (CPAs) in such a way that negative osmotic and cytotoxic effects on cells are minimized. This is especially true for vitrification, where high CPA concentrations are employed. In this study, we report on the determination of cell membrane permeability parameters for water (L(p)) and solute (P(s)), and on the design and experimental verification of CPA addition and removal protocols at vitrification-relevant concentrations for a murine insulinoma cell line, betaTC-tet cells. Using membrane permeability values and osmotic tolerance limits, mathematical modeling and computer simulations were used to design CPA addition and removal protocols at high concentrations. The cytotoxic effects of CPAs were also evaluated. Cells were able to tolerate the addition and removal of 2.5M dimethyl sulfoxide (DMSO) and 2.5M 1,2 propanediol (PD) in single steps, but required multi-step addition and removal with 3.0M DMSO, 3.0M PD, and a vitrification-relevant concentration of 3.0M DMSO+3.0M PD. Cytotoxicity studies revealed that betaTC-tet cells were able to tolerate the presence of single component 6.0M DMSO and 6.0M PD and to a lesser extent 3.0M DMSO+3.0M PD. These results determine the time and concentration domain of CPA exposure that cells can tolerate and are essential for designing cryopreservation protocols for free cells as well as cells in engineered tissues.     

Comparison of actual vs. synthesized ternary phase diagrams for solutes of cryobiological interest

Phase diagrams are of great utility in cryobiology, especially, those consisting of a cryoprotective agent (CPA) dissolved in a physiological salt solution. These ternary phase diagrams consist of plots of the freezing points of increasing concentrations of solutions of cryoprotective agents (CPA) plus NaCl. Because they are time-consuming to generate, ternary diagrams are only available for a small number of CPAs. We wanted to determine whether accurate ternary phase diagrams could be synthesized by adding together the freezing point depressions of binary solutions of CPA/water and NaCl/water which match the corresponding solute molality concentrations in the ternary solution. We begin with a low concentration of a solution of CPA+salt of given R (CPA/salt) weight ratio. Ice formation in that solution is mimicked by withdrawing water from it which increases the concentrations of both the CPA and the NaCl. We compute the individual solute concentrations, determine their freezing points from published binary phase diagrams, and sum the freezing points. These yield the synthesized ternary phase diagram for a solution of given R. They were compared with published experimental ternary phase diagrams for glycerol, dimethyl sulfoxide (DMSO), sucrose, and ethylene glycol (EG) plus NaCl in water. For the first three, the synthesized and experimental phase diagrams agreed closely, with some divergence occurring as wt% concentrations exceeded 30% for DMSO and 55% for glycerol, and sucrose. However, in the case of EG there were substantial differences over nearly the entire range of concentrations which we attribute to systematic errors in the experimental EG data. New experimental EG work will be required to resolve this issue.     

Potentiation of proline accumulation in oilseed rape leaf discs exogenously supplied with combinations of PEG and cryoprotective agents is associated with overproduction of ABA

Physiological processes involved in the control of the magnitude of the stress-induced proline (Pro) response of higher plants are not fully understood. Here we are dealing with Pro accumulation by rape leaf discs (RLDs) treated in vitro, under light conditions, with dual unusual systems containing low concentrations of the non-permeant polymer PEG 6000 (PEG) added with readily permeant cryoprotective agents (CPAs). At osmotically active concentrations, dimethylsulfoxide (DMSO), glycerol, 1,3-propanediol, ethylene glycol and dimethylformamide (DMF) behaved as very poor inducers of the Pro response when provided alone. On the contrary when all those substances (apart DMF) were supplied at onset of treatments, in combination with PEG, Pro levels greatly exceeded what could be predicted through simple additivity of the effects of those substances provided individually. This suggested potentiation effects at the level of some component(s) of Pro metabolism involved in stress-induced Pro accumulation. We have also demonstrated that exogenous ABA could substitute for DMSO (but not PEG), this other binary system also inducing (through potentiation) high rate of Pro accumulation. The strinking similarity between the responses induced with PEG + DMSO and PEG + ABA suggested that DMSO induced increases in the endogenous amount of ABA which might be, at its turn, acting as exogenously supplied ABA. Additional amounts of ABA actually accumulated in leaf discs treated with the dual system PEG + DMSO. Other CPAs tested in this study, except dimethylformamide (DMF), might also be acting on the same way. Potentiating effects, associated with enhanced amount of ABA, were also found to result from combinations of PEG with a range of organic and inorganic substances which mimick to some extent the cytosolute composition of plant cells. Together, our results gain some insight into the physiological mechanisms involved in the control of stress-induced Pro accumulation and strongly suggest their relationship with stress-induced changes in ABA content.     

Measurement of the biophysical properties of porcine adipose-derived stem cells by a microperfusion system

Adipose-derived stem cells (ADSCs), which are an accessible source of adult stem cells with capacities for self-renewal and differentiation into various cell types, have a promising potential in tissue engineering and regenerative medicine strategies. To meet the clinical demand for ADSCs, cryopreservation has been applied for long-term ADSC preservation. To optimize the addition, removal, freezing, and thawing of cryoprotective agents (CPAs) applied to ADSCs, we measured the transport properties of porcine ADSCs (pADSCs). The cell responses of pADSCs to hypertonic phosphate-buffered saline and common CPAs, dimethyl sulfoxide, ethylene glycol, and glycerol were measured by a microperfusion system at temperatures of 28, 18, 8, and −2 °C. We determined the osmotically inactive cell volume (V_b), hydraulic conductivity (L_p), and CPA permeability (P_s) at various temperatures in a two-parameter model. Then, we quantitatively analyzed the effect of temperature on the transport properties of the pADSC membrane. Biophysical parameters were used to optimize CPA addition, removal, and freezing processes to minimize excessive shrinkage of pADSCs during cryopreservation. The biophysical properties of pADSCs have a great potential for effective optimization of cryopreservation procedures.     

Successful pregnancy following transfer of feline embryos derived from vitrified immature cat oocytes using 'stepwise' cryoprotectant exposure technique

Oocyte cryopreservation is the desired tool for the ‘long-term’ storage of female genetic potential especially for endangered/valuable species. This study aims at examining the ability of different cryoprotectant (CPA) and CPA exposure techniques to protect immature feline oocytes against cryoinjury during vitrification. Immature oocytes were submitted to different CPA exposure techniques: 1) 2-step DMSO, 2) 4-step DMSO, 3) 2-step EG, 4) 4-step EG, 5) 2-step EG plus DMSO and 6) 4-step EG plus DMSO. Non-CPA treated, non-vitrified oocytes served as controls. The oocytes were then submitted either to in vitro maturation (Experiment 1, n = 334) or to vitrification/warming (Experiment 2, n = 440). The stage of nuclear maturation was subsequently determined. In Experiment 3, the vitrified immature oocytes (n = 254) were matured and fertilized in vitro, and their developmental competence was assessed. A total of 424 embryos derived from vitrified immature oocytes were transferred into the oviduct of 6 recipient queens (Experiment 4).Vitrification reduced significantly the meiotic and developmental competence of immature cat oocytes compared with the non-vitrified controls. The EG alone or a combination of EG and DMSO yielded higher maturation rates than DMSO, irrespective of the CPA equilibration techniques used. The 4-step EG vitrification resulted in the highest maturation rate (37.6%) but cleavage and blastocyst rates were significantly lower than the non-vitrified controls (24.8% and 30.2% vs 62.5% and 49.3%, respectively). Pregnancy was established in recipients receiving embryos derived from non-vitrified and vitrified/warmed immature oocytes. It is concluded that the stepwise CPA exposure technique can be successfully applied for vitrification of immature cat oocytes, in terms of in vitro development but it is likely to affect in utero development.     

Immature bovine oocyte cryopreservation: comparison of different associations with ethylene glycol, glycerol and dimethylsulfoxide

Research on different cryoprotectants and their associations is important for successful vitrification, since greater cryoprotectant concentration of vitrification solution may be toxic to oocytes. The aim of the present research was to compare the efficiency of immature bovine oocyte vitrification in different associations of ethylene glycol (EG), glycerol and dimethylsulfoxide (Me(2)SO). In the first experiment, oocytes were exposed to the cryoprotectant for either 30 or 60s in final solutions of EG+DMSO1 (20% EG+20% Me(2)SO) or EG+DMSO2 (25% EG+25% Me(2)SO) or EG+GLY (25% EG+25% glycerol). In the second experiment, the oocytes were vitrified in open pulled straws (OPS) using 30s exposure of final solutions of EG+DMSO1 or EG+DMSO2 or EG+GLY. Maturation rates of 30s exposure groups were not different from the control, but 60s cryoprotectant exposure was toxic, decreasing maturation rates. The vitrification with EG+DMSO2 resulted in enhanced maturation rate (29.2%) as compared with EG+DMSO1 (11.7%) and EG+GLY (4.3%) treatments. These data demonstrate that concentration and type of cryoprotectant have important effects on the developmental competence of vitrified oocytes.     

Vitrification of collared peccary ovarian tissue using open or closed systems and different intracellular cryoprotectants

This study aimed to evaluate different vitrification methods using distinct cryoprotectants (CPAs) for the preservation of collared peccary ovarian preantral follicles (PFs). Ovarian pairs from six females were fragmented and three fragments (fresh control group) were immediately evaluated for morphology, viability, cell proliferation capacity (assessed by quantifying the number of argyrophilic nucleolus organizer regions – NORs), and apoptosis (by the identification of activated caspase-3 expression). The remaining 18 fragments were vitrified using the solid surface vitrification (SSV) method or the ovarian tissue cryosystem (OTC) with 3 M ethylene glycol (EG), 3 M dimethylsulfoxide (DMSO), or a combination of the two (1.5 M EG/1.5 M DMSO). After two weeks, samples were rewarmed and evaluated as described previously. The OTC with any of the CPAs provided a similar conservation of morphologically normal PFs as the fresh control group (75.6 ± 8.6%); however, the SSV was only efficient with DMSO alone (63.9 ± 7.6%). Regarding the viability or cell proliferation, all tested groups provided post rewarming values similar to those observed for the fresh control group, 84.0 ± 2.9% viable cells with 2.0 ± 0.2 NORs. Related to apoptosis analysis, only the OTC with EG (46.7%) and the SSV method with EG (43.4%) or the combination of EG and DMSO (33.4%) provided similar values to those found for the fresh control group (36.7%). Our findings indicate the utilization of a closed system, the OTC, with 3 M EG as the CPA for the vitrification of collared peccary ovarian tissue.     

Cryoprotectants for the vitrification of corneal endothelial cells

The objective of this work was to select and test systematically possible cryoprotective agents (CPAs) and to obtain a suitable formula for vitrification of corneal endothelial cells (CECs). Fresh bovine CECs were isolated and tested with an optimized vitrification protocol with multi-step CPA loading and removal. Three types of CPAs components, i.e. the penetrating CPAs, sugars and macromolecular compounds, were experimentally evaluated using the viability assayed by trypan blue. Dimethyl sulfoxide, ethylene glycol (EG), 1,2-propanediol, 2,3-butanediol, acetamide and ethylene glycol monomethyl ether were chosen as the penetrating CPA components. Sugars including xylose, fructose, mannose, glucose, maltose, sucrose and trehalose were tested. Ficoll (MW 7 kDa), dextran (MW 7 kDa), chondroitin sulfate (CS, MW 18-30 kDa), bovine serum albumin (MW 68 kDa) and polyethylene glycol (MW 6 kDa, 10 kDa and 20 kDa) were chosen as the macromolecular compounds. CECs were also preserved by slow freezing as a control. The results showed that EG was the most suitable penetrating CPA component and glucose the most suitable sugar, and CS the most suitable macromolecule. The optimized concentrations for each component in the vitrification solution were 52% (w/w) EG, 8% (w/w) glucose and 3% (w/w) CS. The CEC survival rate of 89.4 ± 2.1% (mean ± SD) was obtained using this formula and established vitrification protocol which was comparable to that by slow freezing.     

Use of luminometry and flow cytometry for evaluating the effects of cryoprotectants in the gorgonian coral endosymbiont Symbiodinium

Cryopreservation has consistently proven to be a viable method for the storage of a wide variety of biological material, and there has been a recent focus on the cryopreservation of Symbiodinium spp. given the role of these dinoflagellates in the biology of the coral hosts with which they regularly associate. The present study aimed to identify the effect of various cryoprotectants (CPAs) on clade G Symbiodinium isolated from the whip coral Junceella fragilis by analyzing (i) dinoflagellate cell membrane integrity (via SYTOX® staining) and (ii) metabolic function (via an ATP assay) after cryopreservation. At low concentrations (1 M), none of the CPAs tested were harmful to the dinoflagellates at up to 60 min of incubation, and methanol and DMSO were the least harmful; neither caused a significant effect on cell ATP content even after 30 min incubations at 4 M concentrations. Detrimental effects of the CPAs increased in the following order: MeOH (lowest) = DMSO < EG < PG < Gly. With respect to the different assays, the ATP bioassay was more sensitive to CPA exposure than SYTOX staining. Given these findings, MeOH and DMSO should see more widespread use in cryopreservation protocols for clade G Symbiodinium, as well as other dinoflagellates.