Studies on the cryopreservation of Dictyocaulus viviparus (Nematoda) third-stage larvae

Various cooling (0.1-5,100 degrees C min-1) and warming (20-6,800 degrees C min-1) rates, stepped cooling schedules and four cryoprotective additives (dimethyl sulphoxide, methanol, ethanediol and glycerol) were investigated in cryopreservation studies with Dictyocaulus viviparus third-stage larvae. Exsheathment with sodium hypochlorite was essential to achieve significant survival. With uninterrupted cooling, highest survival (30% normally motile) was achieved with rates of 10-70 degrees C min-1. Survival was higher (50-75%) using 1 degree C min-1 to -10 degrees C followed by plunging into liquid nitrogen. The optimum warming rate was 6,800 degrees C min-1. The use of cryoprotectants led to marginally lower survival while varying the suspending media had no significant effect on survival. X-irradiated, exsheathed third-stage larvae cryopreserved by the optimum protocol yielded 38.3 +/- 4.2% survival. Two calves each infected with 45,000 (15,000 viable) exsheathed, unirradiated, cryopreserved third-stage larvae harboured 494 worms (1.1% infectivity) and 355 worms (0.8%) at necropsy. Numbers of first-stage larvae in the faeces reached 420/g and 105/g respectively 27 days after infection.     

Water content in an engineered dermal replacement during permeation of Me2SO solutions using rapid MR imaging

The successful cryopreservation of cell and tissues typically requires the use of specialized solutions containing cryoprotective agents. At room temperature, the introduction of a cryopreservation solution can result in cell damage/death resulting from osmotic stresses and/or biochemical toxicity of the solution. For tissues, the permeation and equilibration of a cryoprotective solution throughout the tissue is important in enhancing the uniformity and consistency of the postthaw viability of the tissue. Magnetic resonance (MR) is a common nondestructive technique that can be used to quantitate the temporal and spatial composition of water and cryoprotective agents in a three-dimensional system. We have applied a recently developed rapid NMR imaging technique to quantify the transport of water in an artificial dermal replacement upon permeation of dimethyl sulfoxide (Me2SO) solutions. Results indicate that the rate of water transport is slower in the presence of Me2SO molecules. Furthermore, the transport is concentration-dependent, suggesting that Me2SO tends to retain bound water molecules in the tissue. Moreover, water transport decreases with decreasing temperature, and the presence of cells tends to increase water transport.     

Factors influencing renal cryopreservation. I. Effects of three vehicle solutions and the permeation kinetics of three cryoprotectants assessed with rabbit cortical slices

A renal cortical slice model was used to assess the effects on viability of three vehicle solutions-Krebs-Henseleit (K-H), solution A, and RPS-2--at 25 degrees C. After 120 min incubation no differences in [K+]/[Na+] ratios were found. Tracer techniques were used to study the osmotic effects and permeation kinetics at 25 degrees C of three cryoprotectants (dimethyl sulfoxide (Me2SO), ethylene glycol, and glycerol) and the effect of the vehicle solution (K-H or RPS-2) on Me2SO kinetics. It was found that Me2SO was most permeable and ethylene glycol least, and that ethylene glycol had unusual effects which suggest that it may not act as a simple solute. Differences were found when Me2SO was introduced in K-H and RPS-2 that are believed to be related to the binding properties of Me2SO to cell constituents.     

Viability of cryopreserved human skin allografts: effects of transport media and cryoprotectant

Human skin allografts can be preserved by different methods. In our clinical practice, human skin allografts are harvested on multi-organ and tissue donors, transferred at +4°C in Ringer Lactate, cryopreserved with 15% Glycerol and held in the vapor phase of a liquid nitrogen freezer until delivery to the burn center. The aim of this experimental study was to evaluate the impact of transport medium and cryoprotectant on the viability of human skin allografts. For this purpose, we compared skin samples harvested from 19 multi-organ and tissue donors with two different transport media and two different cryoprotectants. Viability was assessed by the MTT assay after harvesting at laboratory reception, during storage (at +4°C) at day 2 and day 7, and after cryopreservation and thawing. Histopathological analysis was performed for each MTT assay. Results indicate that, when stored at +4°C, skin retains more viability with RPMI, whereas Glycerol and DMSO are equivalent cryoprotectants regardless of the transport medium. In conclusion, our protocol could be improved by the utilization of RPMI as transport medium.     

Effect of amino acids on goat cauda epididymal sperm cryopreservation using a chemically defined model system

Studies were carried out to analyze the cryoprotecting efficacy of several amino acids by use of a chemically defined synthetic medium (modified Ringer's solution) and goat cauda epididymal sperm as the model system. Motile goat cauda sperm dispersed in the synthetic medium were subjected to a freezing protocol in a computer-controlled bio-freezer, cooling 0.25 degrees C x min(-1) to 5 degrees C, 5 degrees C x min(-1) to -20 degrees C, and 20 degrees C x min(-1) to -100 degrees C, prior to being plunged into liquid nitrogen. In the absence of amino acids, sperm cells completely lost their flagellar motility. Of all the amino acids tested, l-alanine showed maximal cryoprotection potential. l-Alanine at 135 mM offered optimum cryoprotection potential: recovery of sperm forward motility and total motility were 14 +/- 2% and 19 +/- 2%, respectively. l-Glutamine, l-proline, and glycine at optimum concentration (100-150 mM) cryopreserved approx. 11-17% total motility of the sperm cells, whereas amino acids such as l-arginine, l-lysine, and l-histidine offered little cryoprotection (0-5%) to the cells. Increasing the amino acid concentration beyond the optimum level sharply decreased the recovery of the sperm motility, which therefore showed a biphasic cryoprotection profile. Addition of amino acids enhanced (approx. 7-10%) the cryoprotection efficacy of the well-known cryoprotectants glycerol and a combination of glycerol and dimethyl sulfoxide. The presence of glycerol caused a marked reduction (from 100-150 mM to 20-70 mM levels) in the optimal cryoprotective concentration of the amino acids. The combined cryoprotecting action of glycerol, dimethyl sulfoxide, and amino acids provided motility recovery as high as 52%. The observation that amino acids and dimethyl sulfoxide had an additive effect in augmenting the cryoprotecting potential of glycerol suggests that the mechanism of their action is different from that of glycerol. This cocktail of cryoprotectants may be useful for cryopreservation of semen of various species.     

Metabolic changes in rat skin during preservation and storage in glycerol buffer at -196 degrees C.

The penetrating cryoprotective glycerol dissolved in different concentrations in buffer medium effectively protects skin tissue against freeze-thaw injury when progressively cooled to ?196 °C followed by a fast warming-up rate. Upon incubation of the tissue after storage, the incorporation of [2-¹⁴C]glycine into the proteins, of [6-³H]thymidine into DNA, and α-[1-¹⁴C]aminoisobutyric acid transport through the cell membrane are reduced compared to freshly incubated skin. An essential loss in metabolic activity occurs during exposure of the skin to the preserving buffer. Although the length of storage does not seem to affect the final viability, the actual freezing and thawing procedures are particularly damaging to tissue already injured by previous exposure to buffer containing cryoprotective agents.     

Cryopreservation of Giardia lamblia with dimethyl sulfoxide using a Dewar flask

This study examined the effect of varying freezing conditions on the human intestinal parasite Giardia lamblia (Portland-1 strain) using a constant vacuum in a Dewar flask and an ethanol bath to regulate the cooling rate. The cryopreservation of the trophozoite stage was investigated. Dimethyl sulfoxide (Me2SO), the cryoprotective agent of choice, was added directly to Giardia growth medium. Me2SO toxicity assays were conducted on those concentrations used in the freezing protocol. The results of this study indicated a 6.5% (v/v) Me2SO concentration yields a 90% survival based upon organism motility. A 30.9% cell viability was obtained by freezing in medium without a cryoprotective agent. Recommendations are offered concerning alternate viability criteria.     

A comparative study of the morphology and viability of hyphae of Penicillium expansum and Phytophthora nicotianae during freezing and thawing

The changes in morphology of Penicillium expansum Link and Phytophthora nicotianae Van Breda de Haan during freezing and thawing in a growth medium with and without the cryoprotective additive glycerol were examined with a light microscope fitted with a temperature-controlled stage. Viability of 0.5-1.0 mm diameter colonies of both fungi was determined after equivalent rates of cooling to -196 degrees C in the presence or absence of glycerol. In P. expansum shrinkage occurred in all hyphae at rates of cooling of less than 15 degrees C min-1; at faster rates intracellular ice nucleation occurred. The addition of glycerol increased the rate of cooling at which 50% of the hyphae formed intracellular ice from 18 degrees C min-1 to 55 degrees C min-1. This species was particularly resistant to freezing injury and recovery was greater than 60% at all rates of cooling examined. At rapid rates of cooling recovery occurred in hyphae in which intracellular ice had nucleated. In contrast, during the cooling of Ph. nicotianae in the growth medium, shrinkage occurred and no samples survived on thawing from -196 degrees C. However, on the addition of glycerol, shrinkage during freezing decreased and viable hyphae were recovered upon thawing; at rates of cooling over 10 degrees C min-1 the loss of viability was related to glycerol-induced osmotic shrinkage during cooling rather than to the nucleation of intracellular ice.     

Effect of chemical modifications on freeze denaturation of lactate dehydrogenase

Lactate dehydrogenase (LDH) was chemically ethyl-acetimidated (EA-), dimethyladipimidated (DMA-), carbamylated, acetylated, acetoacetylated, or succinylated in order to alter the ionic charges on the epsilon-amino group of lysine residues. Acetylation, acetoacetylation, and succinylation, which change the positive charge at the lysine side chains to a negative one, inactivated the enzymic activity, but the rest of the modifications exerted no such inactivating effects. The active modified enzymes were subjected to freeze denaturation study, using the enzymic activity as an indication of the degree of the denaturation. The active enzymes were diluted with deionized water and stored in a freezer (-23 degrees C) for 1-3 days. Enzymic activity was assayed immediately after thawing. All the modified enzymes retained their activity even after the 3-day frozen storage, while the control or native enzyme lost its activity within 1 day of storage. Furthermore, the modified LDHs freeze-stored in 0.2 M monosodium glutamate (MSG) or 0.2 M lysine-hydrochloride (Lys-HCl) retained their activity. The cryoprotective effects exerted by the modifications and by 0.2 M MSG seemed to be synergistic, whereas those exerted by the modifications and by 0.2 M Lys-HCl did not. The mechanisms of cryoprotection and freeze denaturation are discussed in relationship with the cryoprotective effect exerted by already known cryoprotectants, such as sucrose or dimethyl sulfoxide.     

Studies on the cryopreservation of eggs of Angiostrongylus cantonensis

The possibility of cryopreserving the eggs of Angiostrongylus cantonensis collected from the uterus of female worms was investigated. Eggs were cultured in NCTC 109 medium containing 50% rat serum, and various growth stages, from one-cell eggs to embryonated eggs, were used in this study. As a cryoprotective agent, dimethylsulphoxide (Me2SO) was added to the medium at a final concentration of 1 M. Eggs suspended in 0.2 ml of the medium at 37 degrees C were cooled to 0 degrees C at a rate of 1 degree C min-1, then an equal volume of 2M-Me2SO solution was added. After equilibration for 15 min, the freezing procedures were started. In the freezing procedures, the effectiveness of (i) a seeding process, (ii) different cooling and warming rates and (iii) the relationship between the growth stages of the eggs and their tolerance to freezing at -20 degrees C were investigated. It was found the highest level of survival could be obtained with 32-cell eggs cooled at a rate of 0.3 degrees C min-1 or more slowly with seeding at -4 degrees C and warming at a rate of 5 degrees C min-1. Survival was influenced more by cooling rate than by warming rate. Using these optimum conditions, the survival of eggs was then investigated following cooling to various temperatures. While more than 50% of eggs were found to survive cooling to -30 degrees C, extremely low survival was noted from lower temperatures.     

Toxicity and protective efficiency of cryoprotectants to flounder (Paralichthys olivaceus) embryos

With the purpose of finding an ideal cryoprotectant or combination of cryoprotectants in a suitable concentration for flounder (Paralichthys olivaceus) embryo cryopreservation, we tested the toxicities, at culture temperature (16 degrees C), of five most commonly used cryoprotectants-dimethyl sulfoxide (Me2SO), glycerol, methanol (MeOH), 1,2-propylene glycol (PG) and ethylene glycol (EG). In addition, cryoprotective efficiency to flounder embryos of individual and combined cryoprotectants were tested at -15 degrees C for 60 min. Five different concentrations of each of the five cryoprotectants and 20 different combinations of these cryoprotectants were tested for their protective efficiency. The results showed that the toxicity to flounder embryos of the five cryoprotectants are in the following sequence: PG < MeOH < Me2SO < glycerol < EG (P < 0.05); whereas the protective efficiency of each cryoprotectant, at -15 degrees C for a period of 60 min, are in the following sequence: PG > Me2SO approximately MeOH approximately glycerol > EG (greater symbols mean P < 0.05, and approximate symbols mean P > 0.05). Methanol combined with any one of the other cryoprotectants gave the best protection, while ethylene glycol combined with any one of the other cryoprotectants gave the poorest protection at -15 degrees C. Toxicity effect was concentration dependent with the lowest concentration being the least toxic for all five cryoprotectants at 16 degrees C. For PG, MeOH and glycerol, 20% solutions gave the best protection at -15 degrees C; whereas a 15% solution of Me2SO, and a 10% solution of EG, gave the best protection at -15 degrees C.     

Cell viability improves following inhibition of cryopreservation-induced apoptosis

A new concept in cryopreservation solution design was developed that focuses on the use of an intracellular-type, hypothermic maintenance medium coupled with additives that inhibit cryopreservation-induced apoptosis. HypoThermosol' (HTS), a hypothermic (4 degrees C) maintenance medium utilized in the long-term storage of cell, tissue, and organ systems, was tested for cryoprotective capability on a renal cell line (Madin-Darby Canine Kidney cells). HTS and HTS derivatives were tested against conventional cell culture medium (Dulbecco's Minimal Essential medium, DME) as the cryoprotectant carrier solution because (1) cells are exposed to an extended state of hypothermia during the freeze-thaw process, and (2) HTS is designed to protect cells exposed to a hypothermic state. Cells separately cryopreserved in either HTS or DME + 5% dimethyl sulfoxide (DMSO) yielded equivalent 24-h postthaw survival (approximately 30%) and 5-d recovery (approximately 90%). Cells cryopreserved in CryoStor CS 5, a HTS derivative containing 5% DMSO, yielded approximately 75% 24-h postthaw survival and recovery to 100% within 3 d. DNA gel electrophoresis was performed to determine the mechanisms of cell death contributing to cryopreservation failure. Cells preserved in DME (DMSO-free) died primarily through necrosis, whereas cells preserved in either DME + 5% DMSO, HTS, or CryoStor CS 5 died through a combination of apoptosis and necrosis. This observation led to the inclusion of an apoptotic inhibitor designed to improve cryopreservation outcome. MDCK cells cryopreserved in CryoStor CS 5 supplemented with an apoptotic inhibitor (Caspase I Inhibitor V), hereafter termed CryoStor CS 5N, resulted in a 24-h postthaw survival and recovery rate exceeding that of any other cryoprotective solution tested (85%). We conclude that: (1) the use of HTS (a dextran-based, intracellular-type solution) without DMSO can yield postthaw viability equivalent to that of standard DMSO-based cryopreservation methods, (2) postthaw viability can be significantly increased through the use of an intracellular-type solution in conjunction with DMSO, (3) the use of HTS allows for cryopreservation to be accomplished with reduced levels of cryoprotectants, and (4) the regulation of apoptosis is essential for the improvement of cryopreservation outcome.     

The effect of freezer type, cryoprotectant, and processing methods on viability of frozen embryos

Two hundred forty excellent-quality blastocysts flushed from 53 superovulated Holstein heifers were frozen by 1 of 16 procedures in a 2 x 2 x 2 x 2 factorial design. The main effects included a simple, inexpensive, portable mechanical freezing unit instead of a programmable Liquid Nitrogen (LN) freezer for freezing bovine embryos, cryoprotective agents dimethyl sulfoxide (DMSO) and glycerol, addition rates of the cryoprotectants and freezing rates. Embryo viability was assessed morphologically and by fluorescein diacetate (FDA) evaluation. Neither the type of freezer, the cryoprotectant nor the rate of cryoprotectant addition affected embryo viability. Embryo survival after 12 h of incubation was higher (P < 0.05) using a conventional freezing rate than a two-step method (37.2 vs 16.5%). The correlation coefficient between viability evaluation methods (morphology vs FDA) was influenced by cryoprotectant and embryo processing methods and ranged from -0.13 to +0.70. This study indicates that more simplified embryo freezing equipment and handling procedures may provide protection equal to that of more complicated, expensive equipment and more time-consuming methods. Economical on-farm embryo freezing is feasible.     

Postfreeze viability of human renal epithelial carcinoma cells related to phase transformations in ternary solutions

The postfreeze viability of human renal epithelial carcinoma cells frozen in solutions based on a complex physiologic support medium to which additions of NaCl and a cryoprotective agent, either glycerol or dimethyl sulfoxide (DMSO) were made, have been determined by a dye exclusion technique. The support medium consisted of either Eagle's Minimum Essential Medium with Hanks' salts added (MEM) or this same medium supplemented with 20 vol% heat-inactivated fetal calf serum (MEM + FCS). Glycerol was found to be an ineffective cryoprotective agent for these cells, while DMSO was highly effective. Addition of NaCl along with the DMSO further improved the viability of cells frozen at −196 °C. Freezing and thawing rates were found to be important with a slow freezing rate, 2.5 °C/min, and a rapid thawing rate, 240°C/min, yielding the best results.Maximum viability occurred in solutions containing 80 to 95 wt.% (MEM + FCS) with the balance being DMSO and NaCl in the weight ratio of 9:1. In addition to primary ice formation, two nonequilibrium glassy phases were observed during DTA studies of these solutions (10). The exintence of these vitreous states reduces the chances thet cells will be exposed to hypertonic concentrations of salt in the extracellur fluids during freezing-out of primary ice.     

Cryopreservation of sperm from the marine shrimp Sicyonia ingentis

Sperm from a marine shrimp, Sicyonia ingentis, were frozen to -196 degrees C using a variety of cooling rates and cryoprotectants. A cooling rate of 1 degree C/min resulted in minimal cell breakage. Sperm samples were frozen in solutions of known membrane stabilizers--trehalose, sucrose, proline, and glycerol. These compounds were somewhat effective but a dramatic increase in sperm viability was seen when DMSO was present in the freezing medium. Sperm viability was assessed using the in vitro acrosome reaction technique of Griffin et al. (1987). The highest sperm survival (56%) was obtained with samples frozen at 1 degrees C/min in a 5% (v/v) DMSO solution. No decrease in viability was seen in sperm samples stored in liquid nitrogen (-196 degrees C) for 1 month.     

Cryopreservation of seabream (Sparus aurata) spermatozoa

The aim of this research was to optimize protocols for freezing spermatozoa of seabream (Sparus aurata). All the phases of the cryopreservation procedure (sampling, choosing the cryoprotective extender, cooling, freezing, and thawing) were studied in relation to the species of spermatozoa under examination, so as to be able to restore on thawing the morphological and physiological characteristics of fresh semen. Seabream spermatozoa were collected by stripping and transported to the laboratory chilled (0-2 degrees C). Five cryoprotectants, dimethyl sulfoxide (Me(2)SO), ethylene glycol (EG), 1,2-propylene glycol (PG), glycerol, and methanol, were tested at concentrations between 5 and 15% by volume to evaluate their effect on the motility of semen exposed for up to 30 min at 26 degrees C. The less toxic cryoprotectants, 10% EG, 10% PG, and 5% Me(2)SO, respectively, were added to 1% NaCl to formulate the extenders for freezing. The semen was diluted 1:6 with the extender, inserted into 0.25-ml plastic straws by Pasteur pipette, and frozen using a cooling rate of either 10 or 15 degrees C/min to -150 degrees C followed by transfer and storage in liquid nitrogen (-196 degrees C). The straws were thawed at 15 degrees C/s. On thawing, the best motility was obtained with 5% Me(2)SO, although both 10% PG and EG showed good results; no differences were found between the two freezing gradients, although semen frozen with the 10 degrees C/min gradient showed a slightly higher and more prolonged motility.     

Permeability of cultured megakaryocytopoietic cells of the rat to dimethyl sulfoxide

The permeability of the membrane of the rat megakaryocytopoietic cell to dimethyl sulfoxide was measured to assess its availability to the intracellular compartment. The method used was osmotic, and measured the initial loss of cell water followed by a reswelling to isotonic volume when cells were placed in culture media containing 0.6 M DMSO. Values for the hydraulic coefficient, Lp, the permeability of the membrane to DMSO, wRT , and the reflection coefficient were calculated from the equations of Kedem and Katchalsky . The average value at 25 degrees C for Lp was 0.46 micron min-1 atm1 ; wRT was 9.3 micron min-1, and the reflection coefficient was 0.65. At these cell volumes, 50% equilibration occurred in 5 sec. Cells equilibrated in 0.6 M DMSO increased their volume of osmotically inactive water. Coupled with this phenomenon of stabilization of water was a reduction in the hydraulic coefficient by 50%. These findings are discussed in the context of current hypotheses about cellular viability during freezing and thawing in the presence and absence of cryoprotectants.     

Cryopreservation of heart cells from the eastern oyster

Summary Conditions were developed to cryopreserve cells from pronase-dissociated atria and ventricles of eastern oysters (Crassostrea virginica). The effect of three concentrations (5, 10, 15%) of the cryoprotectants (dimethyl sulfoxide, glycerol, and propylene glycol), three thawing temperatures (25, 45, 75°C), and three cooling rates (slow, medium, fast) were compared. Cells were frozen at −80°C and plunged in liquid nitrogen. Thawed cells were seeded in 96-well plates and primary cultures were evaluated after 3 d by measuring the metabolic activity using a tetrazolium compound, 3-(4,5-dimethylthiazol-2-yl)-5-( 3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, and by comparing the relative spreading of cells between treatments. The best conditions for freezing and thawing of cells for each cryoprotectant were selected and a final study was performed to compare cryoprotectants. For this final study, we measured the number of cells and their viability 3 d after thawing, in addition to determining cell metabolic activity and cell spreading. Primary cultures of cells frozen without cryoprotectant and of nonfrozen cells were used as controls in all studies. Atrial cells were best cryopreserved with glycerol at a concentration of 10%, a medium cooling rate, and thawing at 45°C. After thawing, atrial cells showed 53±5% of the metabolic activity, 84±5% of the number, and 92±2% of the viability of nonfrozen cells. For ventricular cells, 10% glycerol, a medium cooling rate, and thawing at 25°C yielded the best results. The thawed ventricular cells showed 83±5% of the metabolic activity, 91±5% of the number, and 96±2% of the viability of nonfrozen cells.     

Long-term (24h) cooling of ovarian fragments in the presence of permeable cryoprotectants prior to freezing: Two unsuccesful IVF-cycles and spontaneous pregnancy with baby born after re-transplantation

Cancer is the second major cause of death in the world. The problem of post-cancer infertility plays a significant role, because chemotherapy can be gonadotoxic. Cryopreservation of ovarian tissue before cancer therapy with re-implantation after convalescence is the potential key solution to this problem. The aim of this study was to test the viability of cryopreserved human ovarian cortex after long-term cooling in culture medium composed of permeable cryoprotectants. Ovarian fragments from sixteen patients were randomly divided into two groups. After the operation, tissue pieces assigned to both groups were cooled to 5 °C for 22–24 h, frozen and thawed. Group 1 pieces (n = 32) were cooled before cryopreservation in the standard culture medium, and Group 2 pieces (n = 32) were cooled in the freezing medium (culture medium+6% ethylene glycol+6% dimethyl sulfoxide+0.15 M sucrose). Freezing was performed in standard 5 ml cryo-vials with ice formation at −9 °C, cooling from −9 to −34 °C at a rate of −0.3 °C/min and plunging at −34 °C into liquid nitrogen. After thawing in a 100 °C (boiling) water bath, the removal of cryoprotectants was performed in 0.5 M sucrose with 20 min exposure in sucrose and 30 min stepping rehydration. The effectiveness of the pre-freezing cooling of tissue was evaluated by the development of follicles (histology). Six months after the autotransplantation, oocytes from the twenty-seven-year old, hormonally stimulated patient were retrieved and fertilized with her partner sperm through the intracytoplasmic spermatozoa injection (ICSI). For groups 1 and 2, 93.5 ± 1.9% and 96.4 ± 2.0% of the preantral follicles, respectively, were morphologically normal (P > 0.1) (with a tendency toward increasing in quality in Group 2). Six months after the auto-transplantation, two ICSI cycles resulted in the gathering and transplantation of high quality embryos, but no pregnancy had been established. Thirteen months after the auto-transplantation, the patient became spontaneously pregnant and delivered a healthy baby girl at term. Long-term (24 h) cooling of ovarian tissue to 5 °C before cryopreservation in the presence of permeable cryoprotectants simplifies the protocol of cryopreservation and has a tendency of increasing of the cells viability after thawing.     

Insights into the cryoprotective mechanism of dimethyl sulfoxide for phospholipid bilayers.

Dimethyl sulfoxide (Me2SO) is a widely used cryoprotectant for biological structures such as membranes. Despite hundreds of studies on the effects of this molecule, surprisingly little is known about its cryoprotective mechanism. This study investigates the ability of various Me2SO analogs to serve as cryoprotectants for liposomes. The data show that an increase in hydrophobicity progressively reduces the cryoprotective effect of sulfoxides. Additional experiments using phospholipid vesicles of varying composition demonstrate the Me2SO is markedly less effective on liposomes carrying a net negative charge. In fact, cryoprotection by Me2SO was virtually eliminated in vesicles composed of 30% phosphatidylserine (a negatively charged lipid). Based on these results, we suggest that the polar sulfoxide moiety of Me2SO interacts electrostatically with phospholipid membranes and that this interaction is critical for Me2SO's cryoprotective effect for membranes.