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PubMed1998

Comparison of glycerol, other polyols, trehalose, and raffinose to provide a defined cryoprotectant medium for mouse sperm cryopreservation.

Most procedures for mouse sperm cryopreservation have utilized raffinose to provide hypertonicity for cell desiccation prior to freezing and glycerol to block intracellular ice formation. Trehalose has been shown in other cell systems to provide positive protection to the plasma membrane and so was examined as a replacement for raffinose. Comparison of 3 and 6% glycerol and 7.5 and 20% sugar showed that 6% glycerol and 7.5% sugar gave maximal protection consistently and so were adopted as standard. Comparison of raffinose and trehalose at this concentration showed trehalose to give significantly better recovery of intact cells: 48 +/- 6% for trehalose, 36 +/- 9% for raffinose (+/- SE, n = 5; arc sine transformed data; P < 0.01). Less hydrophilic polyols should prove more permeant to the membrane than glycerol, enter the cell rapidly, and so possibly inhibit lethal intracellular ice formation effectively. We hypothesized that one of these polyols plus glycerol would be a more effective cryoprotectant than glycerol alone. The polyols tested as supplements to 6% glycerol were propane-1,2-diol, propane-1,3-diol, 1,1,1-tris-(hydroxymethyl)ethane (THME), and 2-ethyl-2-(hydroxymethyl)-propane-1,3-diol (EHMP). With 6% glycerol and 7.5% raffinose or trehalose, the two diols and THME gave less cryoprotection than with glycerol alone, and EHMP reduced postthaw membrane integrity to nil, thus invalidating the hypothesis. Comparison of bicarbonate-containing medium MJB to bicarbonate-free medium NTP, both with 6% glycerol/7.5% trehalose, showed no difference in recovery of membrane-intact cells. For ease of pH maintenance, NTP was chosen for studies of addition prefreeze and removal postthaw of 6% glycerol/7.5% trehalose cryoprotectant with in vitro fertilization as endpoint. Three protocols for cryoprotectant handling were tested: serial addition/dilution; dialysis addition and removal; and dialysis addition and direct insemination without cryoprotectant removal. The last proved significantly superior (P < 0.01), giving 62% fertilized eggs, normalized to controls, compared to 21% for dialysis addition and removal and 32% for serial addition and dilution. The glycerol/trehalose combination thus provides a defined cryoprotectant which, when used with addition by dialysis prefreeze and direct insemination postthaw, yields a satisfactory yield of fertilized eggs in an in vitro fertilization system.

Animals↗

PubMed2003

Long-term preservation of mouse spermatozoa after freeze-drying and freezing without cryoprotection.

The widespread production of mice with transgenes, disrupted genes and mutant genes, has strained the resources available for maintaining these mouse lines as live populations, and dependable methods for gamete and embryo preservation in these lines are needed. Here we report the results of intracytoplasmic sperm injection (ICSI) with spermatozoa freeze-dried or frozen without a cryoprotectant after storage for periods up to 1.5 years. Freeze-dried samples were stored at 4 degrees C. Samples frozen without cryoprotection were maintained at -196 degrees C. After storage, spermatozoa were injected into the oocytes by ICSI. Zygotic chromosomes and fetal development at Day 15 of gestation were examined after 0, 1, 3, 6, 9, and 12 mo of sperm storage. When fresh spermatozoa were used for ICSI, 96% of resultant zygotes contained normal chromosomes, and 58% of two-cell embryos transferred developed to normal viable fetuses. Similar results were obtained when spermatozoa were frozen without cryoprotection and then used for ICSI (87% and 45%, respectively; P > 0.05) and after 12 mo of sperm storage (mean of six endpoints examined: 87% and 52%, respectively; P > 0.05). Freeze-drying decreased the proportion of zygotes with normal karyoplates (75% vs. 96%; P < 0.001) and the proportion of embryos that developed into fetuses (35% vs. 58%; P < 0.001), but similar to freezing, there was no further deterioration during 12 mo of storage (mean of six endpoints examined: 68% and 34%, respectively; P > 0.05). Live offspring were obtained from both freeze-dried and frozen spermatozoa after storage for 1.5 yr. The results indicate that 1) the freeze-drying procedure itself causes some abnormalities in spermatozoa but freezing without cryoprotection does not and 2) long-term storage of both frozen and freeze-dried spermatozoa is not deleterious to their genetic integrity. Freezing without cryoprotection is highly successful, simple, and efficient but, like all routine sperm storage methods, requires liquid nitrogen. Liquid nitrogen is also required for freeze-drying, but sperm can then be stored at 4 degrees C and shipped at ambient temperatures. Both preservation methods are successful, but rapid freezing without cryoprotection is the preferred method for preservation of spermatozoa from mouse strains carrying unique genes and mutations.

Animals↗

PubMed1998

Thermal analysis of cryoprotective solutions for red blood cells.

A differential scanning calorimeter was used to study the thermal behavior of glycerol-water solutions (binary system) and the more complex glycerol-based cryoprotective solutions that are used clinically in order to examine the cryoprotective role of glycerol in preserving frozen red blood cells. The melting and glass transition temperatures for the clinically used cryoprotective solutions were as expected, based on the nonequilibriumphase diagram for cryoprotective solutions incorporating isotonic phosphate-buffered saline. Two zones were identified in which solidification occurred without the formation of ice crystals: a glassy state that is crystallographically amorphous was found for glycerol concentrations between 40 and 55% in the binary system and between 45 and 60% in the complex system; a glassy state in the complete absence of ice was found at glycerol concentrations greater than 55% for the binary system or 60% for the complex system. In clinical practice, cryoprotectants are used at initial concentrations lower than those at which these two glassy states occur but there is an increase in the effective glycerol concentration inside and outside the cells as ice forms during the freezing process.

Blood Preservation↗

PubMed2005

Study on fish embryo responses to the treatment of cryoprotective chemicals using impedance spectroscopy.

Investigations using electrical impedance spectroscopy to measure the responses of fish embryos to the cryoprotective chemicals, methanol and dimethyl sulphoxide (DMSO), were carried out. Zebrafish (Danio rerio) embryos were used as a model to study the newly proposed technique. The normalised permittivity and conductivity changes of the embryos were measured continuously over a 20-min period in a customised embryo-holding chamber. The normalised permittivity and conductivity spectra were obtained during embryo exposure to different concentrations of methanol (1.0, 2.0 and 3.0 M) and DMSO (0.5, 1.0 and 2.0 M) solutions. The results showed significant permittivity and conductivity changes after embryo exposure to methanol and DMSO at the optimum embryo loading level (six embryos). Embryos in different concentrations of methanol and DMSO also resulted in quantitative responses shown in the normalised permittivity and conductivity spectra. The results demonstrated that fish embryo membrane permeability to cryoprotective chemicals could be monitored in real-time. The measurement of permittivity at a lower frequency range (10-10(3) Hz) and conductivity at a higher frequency range (10(4)-10(6) Hz) during fish embryo exposure to cryoprotective chemicals using impedance spectroscopy can be used as a new tool for the fast screening of most effective cryoprotective chemicals. The results from the present study also demonstrated the possibility of quantifying the level of cryoprotective chemicals penetrating the fish embryos.

Animals↗

PubMed1985

Effect of cryoprotectants on the viability and function of unfrozen human polymorphonuclear cells.

High concentrations of membrane permeable cryoprotectants are necessary to protect human polymorphonuclear leukocytes from osmotic stress injury during freezing, but there are reports that some cryoprotectants are chemically toxic. Cells were exposed to various concentrations of glycerol, dimethyl sulfoxide, or ethylene glycol for 5 min to 2 hr at 37, 22 or 0 degree C, adding or removing the cryoprotectant either slowly or rapidly. Assays included cell number recovery, membrane integrity, phagocytosis, microbicidal ability, and chemotaxis. We conclude that (1) 1 and 2 M concentrations generally are not toxic if they are added and removed slowly at 22 degrees C; (2) addition and removal of glycerol at 0 degree C was injurious even at 1 M; (3) slow addition and removal allowed better recovery than rapid addition or removal; (4) salt concentration in cryoprotectant solutions should be adjusted to isotonic on the basis of moles per liter of solution, rather than moles per kilogram of water; (5) the toxicity reported by other investigators can be largely explained by osmotic stress or dilution shock rather than chemical toxicity; and (6) ethylene glycol is the easiest cryoprotectant to add to and remove from these cells.

Blood Preservation↗

PubMed2006

Plant vitrification solution 2 lowers water content and alters freezing behavior in shoot tips during cryoprotection.

Plant shoot tips do not survive exposure to liquid nitrogen temperatures without cryoprotective treatments. Some cryoprotectant solutions, such as plant vitrification solution 2 (PVS2), dehydrate cells and decrease lethal ice formation, but the extent of dehydration and the effect on water freezing properties are not known. We examined the effect of a PVS2 cryoprotection protocol on the water content and phase behavior of mint and garlic shoot tips using differential scanning calorimetry. The temperature and enthalpy of water melting transitions in unprotected and recovering shoot tips were comparable to dilute aqueous solutions. Exposure to PVS2 changed the behavior of water in shoot tips: enthalpy of melting transitions decreased to about 40 J g H2O(-1) (compared to 333 J g H2O(-1) for pure H2O), amount of unfrozen water increased to approximately 0.7 g H2O g dry mass(-1) (compared to approximately 0.4 g H2Og dry mass(-1) for unprotected shoot tips), and a glass transition (T(g)) at -115 degrees C was apparent. Evaporative drying at room temperature was slower in PVS2-treated shoot tips compared to shoot tips receiving no cryoprotection treatments. We quantified the extent that ethylene glycol and dimethyl sulfoxide components permeate into shoot tips and replace some of the water. Since T(g) in PVS2-treated shoot tips occurs at -115 degrees C, mechanisms other than glass formation prevent freezing at temperatures between 0 and -115 degrees C. Protection is likely a result of controlled dehydration or altered thermal properties of intracellular water. A comparison of thermodynamic measurements for cryoprotection solutions in diverse plant systems will identify efficacy among cryopreservation protocols.

Cryopreservation↗

PubMed2005

Survival of mint shoot tips after exposure to cryoprotectant solution components.

Many plant species can be cryopreserved by treating shoot tips with complex cryoprotectant solutions before rapidly cooling them to liquid nitrogen temperatures. Plant vitrification solution 2 (PVS2), a commonly selected cryoprotectant, can be lethal with extended exposure times. To determine potentially toxic combinations, we have exposed mint shoot tips to one-, two-, three-, and four-component solutions of PVS2 chemicals (30% glycerol, 15% ethylene glycol, 15% dimethyl sulfoxide, and 0.4 M sucrose) at 0 and 22 degrees C. Overall, solution exposures at 22 degrees C were more damaging than exposures at 0 degree C. Solutions with glycerol, particularly in combination with ethylene glycol and dimethyl sulfoxide, were also damaging. Cryoprotectant solutions PGluD (10% PEG8000, 10% glucose, and 10% dimethyl sulfoxide) and PVS3 (50% glycerol, 50% sucrose) were less damaging than PVS2 at 22 degrees C. When plant cryoprotectants are characterized on a toxicological and biophysical basis, less damaging cryoprotectant solutions could be developed.

Cryopreservation↗

PubMed2006

Cryoprotectant permeability of aquaporin-3 expressed in Xenopus oocytes.

It has been shown that aquaporin-3, a water channel, is expressed in mouse embryos. This type of aquaporin transports not only water but also neutral solutes, including cell-permeating cryoprotectants. Therefore, the expression of this channel may have significant influence on the survival of cryopreserved embryos. However, permeability coefficients of aquaporin-3 to cryoprotectants have not been determined except for glycerol. In addition, permeability coefficients under concentration gradients are important for developing and improving cryopreservation protocols. In this study, we examined the permeability of aquaporin-3 to various cryoprotectants using Xenopus oocytes. The permeability of aquaporin-3 to cryoprotectants was measured by the volume change of aquaporin-3 cRNA-injected oocytes in modified Barth's solution containing either 10% glycerol, 8% ethylene glycol, 10% propylene glycol, 1.5 M acetamide, or 9.5% DMSO (1.51-1.83 Osm/kg) at 25 degrees C. Permeability coefficients of aquaporin-3 for ethylene glycol and propylene glycol were 33.50 and 31.45 x 10(-3) cm/min, respectively, which were as high as the value for glycerol (36.13 x 10(-3) cm/min). These values were much higher than those for water-injected control oocytes (0.04-0.11 x 10(-3) cm/min). On the other hand, the coefficients for acetamide and DMSO were not well determined because the volume data were poorly fitted by the two parameter model, possibly because of membrane damage. To avoid this, the permeability for these cryoprotectants was measured under a low concentration gradient by suspending oocytes in aqueous solutions containing low concentrations of acetamide or DMSO dissolved in water (0.20 Osm/kg). The coefficient for acetamide (24.60 x 10(-3) cm/min) was as high as the coefficients for glycerol, ethylene glycol, and propylene glycol, and was significantly higher than the value for control (6.50 x 10(-3) cm/min). The value for DMSO (6.33 x 10(-3) cm/min) was relatively low, although higher than the value for control (0.79 x 10(-3) cm/min). This is the first reported observation of DMSO transport by aquaporin-3.

Animals↗

PubMed2002

Osmotic properties of spermatozoa from felids producing different proportions of pleiomorphisms: influence of adding and removing cryoprotectant.

The spermatozoon of felids (cats) survives cryopreservation inconsistently. Using ejaculates from three species (domestic cat [normospermic versus teratospermic], the normospermic serval and the teratospermic clouded leopard), this study (1) determined the influence of adding and removing two permeating cryoprotectants (glycerol and dimethylsulfoxide) and (2) assessed the impact of one-step versus multi-step cryoprotectant removal on sperm motility and membrane integrity. Spermatozoa were exposed in a single step to various anisotonic solutions or to 1M solutions of glycerol or dimethylsulfoxide. In both cases, sperm then were returned to near isotonic conditions in a single or multi-step with de-ionized water, Ham's F10 medium or saline. Percentage of sperm motility was measured subjectively, and plasma membrane integrity was assessed using a dual fluorescent stain and flow cytometry. Sperm motility was more sensitive to anisotonic conditions than membrane integrity. Rapid dilution into various test solutions and removal of cryoprotectant with de-ionized water reduced (P<0.01) sperm motility compared to control spermatozoa maintained in Ham's F10. Exposing sperm from all species to a 1M solution of either cryoprotectant resulted in >85% spermatozoa retaining intact membranes. However, return to isotonicity with de-ionized water in a single step or multiple steps always caused severe plasma membrane disruption. In contrast, sperm motility and membrane integrity in all species and populations remained unaffected (P>0.05) when spermatozoa were returned to isotonicity in multiple steps with Ham's F10 medium or 0.9% sodium chloride. Results demonstrate that: (1) felid spermatozoa are resistant to hypertonic stress; (2) sperm motility is more sensitive to changes in osmolality than membrane integrity; and (3) removal of cryoprotectant in multiple steps with an isotonic solution minimizes loss of sperm motility and membrane disruption in both normospermic and teratospermic males.

Animals↗

PubMed2001

Membrane permeability of human oocytes in the presence of the cryoprotectant propane-1,2-diol.

OBJECTIVE: To determine the permeability of unfertilized human oocytes to water and the cryoprotectant propane-1,2-diol over a range of temperatures and to use these data to predict osmotic responses under given conditions. DESIGN: Laboratory-based study. SETTING: Teaching hospital. PATIENT(S): Infertility patients donating unfertilized oocytes in excess of those required for treatment. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Water and cryoprotectant permeability were determined from measurements of oocyte volume excursions on exposure to 1.5 M propane-1,2-diol at 30 degrees C, 24 degrees C, and 10 degrees C. RESULT(S): Permeability of human oocytes to water and cryoprotectant increased as temperature increased. The predicted response of oocytes, based on these data, closely matched the measured response of an oocyte on exposure to a widely used method for addition of cryoprotectant before freezing. CONCLUSION(S): Commonly used cryopreservation protocols involving slow cooling in the presence of propane-1,2-diol cause potentially damaging excursions in cell volume on exposure to cryoprotectant. Modifications that can be expected to reduce cell volume excursions, based on oocyte permeability data, are suggested.

Cell Membrane Permeability↗

PubMed1993

Platelet activating factor and pentoxifylline as human sperm cryoprotectants.

OBJECTIVE: To explore whether the addition of platelet-activating factor (PAF) or pentoxifylline before cryopreservation improves the recovery of motile viable sperm and what role cyclic adenosine monophosphate (cAMP) plays in this recovery. DESIGN: Washed sperm was cryopreserved in the absence of and in the presence of PAF and pentoxifylline. After 2 weeks these samples were quick-thawed and evaluated before and after washing for sperm motility and other motion characteristics. Sperm viability and cAMP concentration were determined to compare the effects of these cryoprotectants. RESULTS: When sperm samples were cryopreserved in the presence of PAF or pentoxifylline, an improvement in the recovery of motile sperm in unwashed and washed post-thaw samples was observed. There were 38% more motile sperm recovered with PAF and 15% more with pentoxifylline when compared with untreated samples. In comparison with the unwashed samples, sperm motility in post-thaw samples was lowered by the washing procedure. When PAF was used as a cryoprotectant, a significant improvement in the linearity and straight line velocity of the post-thaw sperm was observed. When pentoxifylline was used as a cryoprotectant, lateral head displacement was significantly improved in the post-thaw samples than in the control group. Both PAF-and pentoxifylline-treated samples contained a greater number of viable sperm than the control. The cAMP concentrations in post-thaw samples were 12-fold higher in pentoxifylline-treated samples and 4-fold higher in PAF-treated samples when compared with the untreated control. A 4-fold decrease in cAMP concentration was observed in post-thaw control samples compared with fresh-washed sperm. CONCLUSIONS: The results of this study suggest that both PAF and pentoxifylline are useful cryoprotectants for the increased recovery of motile, viable sperm. Although increased recovery of motile sperm in pentoxifylline-treated samples is related to higher cAMP levels, the cryoprotective effect of PAF does not appear to be due to increased cAMP.

Cell Survival↗

PubMed1999

Cellular alteration after dilution of cryoprotective solutions used for the vitrification of in vitro-produced bovine embryos.

Embryo quality of in vitro-produced bovine blastocysts was assessed at several steps of a vitrification procedure in which glycerol and ethylene glycol were used as cryoprotectants (3-step equilibration with cryoprotectants followed by vitrification, dilution of the cryoprotectants in 0.85 M galactose then in embryo transfer freezing medium [ETF], and finally co-culture for periods). To visualize cell membrane alterations, double staining was performed using a cell permeant fluorochrome (bisbenzimide--BIS) and a nonpermeant one (propidium iodide--PI). In Experiment 1, the effect of the vitrification procedure on the hatching rate and total cell number was assessed 72 h after treatment. Hatching rate and the number of stained nuclei were decreased in comparison with untreated embryos when blastocysts were exposed to the whole procedure with or without vitrification (respectively 42 and 53% vs 76% for hatching and 128 +/- 17 and 141 +/- 17 vs 226 +/- 13 for stained nuclei). In Experiment 2, the effect of cryoprotectants and their dilution was evaluated on membrane permeability and total cell numbers at various steps of the vitrification procedure. Blastocysts exposed only to cryoprotectant solutions and stained immediately after dilution in galactose showed no modification. After dilution in ETF, the total number of stained nuclei decreased, and the number of blastomeres showing membrane permeabilization (PI-stained) increased (respectively, 74 +/- 5 vs 110 +/- 5 and 32 +/- 2% vs 0.1 +/- 1.8%). In Experiment 3, we demonstrated that the total number of stained nuclei after ethanol fixation (membrane permeabilization) was higher when embryos treated up to dilution in ETF were stained with PI than when the same embryos were stained with BIS. This suggests that, for unknown reasons, some nuclei of the treated embryos were not stained with BIS. Membrane permeabilization and inability of BIS to stain some nuclei were the most obvious alterations probably induced by osmotic shock at dilution. This hypothesis is supported by the fact that the introduction of a further dilution step in 0.42 M galactose (Experiment 4) before dilution in ETF decreased the proportion of cells permeant to PI and increased the hatching rate after 72 h of co-culture. In conclusion, double staining with BIS and PI allowed for discrimination between different types of cellular injuries after the various steps of our vitrification protocol. It represents a useful tool for adjusting equilibration and dilution conditions during a cryopreservation procedure.

Animals↗

PubMed1991

New cryoprotectant for cryorefractive surgery.

Cryorefractive surgeries, keratomileusis, keratophakia, and epikeratophakia cause destruction of keratocytes, which may result in postoperative corneal haze. We examined the effects of two cryoprotectants on keratocyte survival following freeze injury. We compared the ability of CPTES and the standard cryoprotectant KM-26 to prevent keratocyte death by altering the length of time corneal tissue was exposed to the cryoprotectant. When corneal stroma was immersed in CPTES for five minutes prior to freezing, 66.5% of the keratocytes survived; when tissue was immersed in KM-26 for the same length of time, 27.5% survived (P less than .01). Immersion for one to 30 minutes in CPTES prior to freezing produced keratocyte viabilities that were 40% to 80% of those of fresh, unfrozen tissue; immersion in KM-26 produced keratocyte viabilities of 20% to 60%. We compared the ability of these cryoprotectants to reduce corneal haze following freeze injury using our rabbit model of lamellar keratoplasty. The postoperative data were comparable to those in the cell culture experiments. Based on our findings in rabbit corneas, a cryoprotective medium such as CPTES may promote cell survival and thereby speed recovery from cryorefractive procedures in humans.

Animals↗

PubMed2003

Cryopreservation of follicles in human ovarian cortical tissue. Comparison of serum and human serum albumin in the cryoprotectant solutions.

BACKGROUND: Cryopreservation of follicles in ovarian cortical tissue has been suggested as a method for preserving fertility for young women who need to undergo cytotoxic therapy. Varying compositions of cryoprotectant solutions have been used to prevent tissue damage during cryopreservation and thawing. We compared human serum [20% (v/v)] and human serum albumin (HSA) (25 mg/ml) in cryoprotectant solutions containing propanediol and sucrose to evaluate whether serum-free medium could be used for this purpose. METHODS: Biopsies of ovarian cortical tissue were obtained from 23 subjects after informed consent. Fourteen underwent Caesarean section and nine underwent sterilization by laparoscopy. The cortical tissue was cut into pieces of 1-1.5 mm(3 )and cryopreserved in cryoprotectant solutions containing serum or HSA. After thawing, a total of 1318 follicles were analysed using light microscopy, transmission electron microscopy (TEM) or live/dead assay. RESULTS: Viability of the follicles was 99.3% in freshly dissected tissue. After thawing, 65% of the follicles and 75% of the oocytes were viable with serum, and 69 and 74%, respectively, with HSA. No significant differences were observed between results in solutions containing serum versus HSA. TEM showed similar results; however, poor survival of stromal tissue was evident in this analysis. The live/dead assay showed 82% viability after thawing for both groups. No benefits were seen from post-thawing culture for 4 h before histological preparation. CONCLUSIONS: A cryoprotectant solution containing HSA was equally effective as one containing serum. Good viability of follicles was confirmed when using propanediol and sucrose as cryoprotectants, with a large number of follicles.

Adult↗

PubMed2004

DNA integrity and motility of human spermatozoa after standard slow freezing versus cryoprotectant-free vitrification.

BACKGROUND: In contrast to the technique of conventional freezing, the vitrification of spermatozoa requires high cooling rates (720 000 degrees K/min), which could be damaging for spermatozoa. The aim of our study was to compare slowly frozen and vitrified spermatozoa in terms of their post-thaw DNA integrity and motility. METHODS: Semen samples were prepared according to the routine swim-up technique and divided into aliquots for comparison of fresh, conventionally frozen and vitrified spermatozoa from the same ejaculate in the presence or absence of cryoprotectants. Spermatozoa motility and DNA integrity were determined. RESULTS: The motility of spermatozoa conventionally (slowly) frozen with a cryoprotectant was similar to that recorded for spermatozoa vitrified in the absence of cryoprotectant (47 versus 52%). The DNA integrity was unaffected by the cryopreservation method or presence of cryoprotectants. CONCLUSION: The vitrification of human spermatozoa in the absence of conventional cryoprotectants is indeed feasible. The DNA integrity of vitrified sperm is comparable with that shown by standard slow-frozen/thawed spermatozoa, yet the method is quick and simple and does not require special cryobiological equipment.

Cryopreservation↗

PubMed1995

Effect of cryoprotectants and their concentration on post-thaw survival and development of rapid frozen-thawed pronuclear stage mouse embryos.

Experiments have been conducted to develop a simple rapid-freezing protocol for pronuclear stage mouse embryos. The effect of type of cryoprotectant (dimethyl-sulphoxide (DMSO) or 1,2-propanediol (PROH)) and concentration (ranging from 3.5 to 8.0 mol/l with 0.5 mol/l sucrose) on the post-thaw morphological survival rate, on cleavage rate on development to the blastocyst stage were studied. Further, in-vivo viability of embryos frozen using the most effective cryoprotectant concentration (PROH at 7.0 mol/l) was compared with viability of non-frozen embryos. The type of cryoprotectant and its concentration influenced the survival and development of embryos to the blastocyst stage in vitro. The best development with PROH was achieved at 7.0 mol/l (66%, 128/193), whereas with DMSO the best development was achieved at 6.0 mol/l (42%, 71/171). The rates of survival and cleavage did not differ between the two best cryoprotectant concentrations (P > 0.01) but the proportion of embryos which developed to blastocyst was higher (P < 0.001) with PROH at 7.0 mol/l compared with DMSO at 6.0 mol/l. The rates of survival and development were higher (P < 0.001) with DMSO at 3.5 and 6.0 mol/l compared with similar concentrations of PROH. The cleavage and development, however, was higher (P < 0.001) at 7.0 mol/l PROH compared with the same concentration of DMSO. At 8.0 mol/l the survival and development was not different (P > 0.01) between DMSO and PROH. The rate of implantation and the percentage of live fetuses at autopsy of the recipients receiving non-frozen embryos was higher (63 and 41% respectively) than in those receiving frozen-thawed embryos (53 and 37% respectively), but not significantly different. It may be concluded that the concentration range of cryoprotectants which allows acceptable embryo viability after freezing and thawing is very narrow. The rapid protocol using dehydration in 0.25 mol/l and 0.5 mol/l sucrose followed by exposure to 7.0 mol/l PROH and 0.5 mol/l sucrose for 45 s was the most effective for cryopreservation of pronuclear stage mouse embryos.

Animals↗

PubMed2005

Influence of fermentation time, cryoprotectant and neutralization of cell concentrate on freeze-drying survival, storage stability, and acid and bile exposure of Bifidobacterium animalis ssp. lactis cells produced without milk-based ingredients.

AIMS: To investigate the stability of Bifidobacterium animalis ssp. lactis VTT E-012010 (=Bb-12) during freeze-drying, storage and acid and bile exposure. The effect of harvesting time and composition and pH of the cryoprotectant on the survival was evaluated. The procedure was performed by using a milk-free culture medium and cryoprotectants to produce cells for nonmilk-based applications. METHODS AND RESULTS: Bifidobacterial cells were grown in fermenters in general edible medium for 15 or 22 h. The cell mass was freeze-dried either as non-neutralized or neutralized using sucrose, betaine or reconstituted skim milk (control) as cryoprotectants. For stability studies freeze-dried powders were stored at 37, 5 and -20 degrees C for 2-6 months. In addition, acid and bile tolerance of the powders was tested. Sucrose-formulated B. animalis ssp. lactis preparations had an excellent stability during storage at refrigerated and frozen temperatures for 5-6 months. They also had a good survival during storage at 37 degrees C for 2 months as well as during exposure to pH 3 and 1% bile acids. No difference was observed between 15 and 22 h grown cells or between non-neutralized and neutralized cells. Betaine proved to be a poor cryoprotectant compared with sucrose. CONCLUSIONS: Fermentation time and neutralization of cell concentrate before freeze-drying had no impact on the storage stability and bile and acid tolerance of freeze-dried bifidobacterial cells. The nonmilk-based production protocol using sucrose as a cryoprotectant yielded powdery preparations with excellent stability in adverse conditions (storage at elevated temperatures and during acid and bile exposure). SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate that it is feasible to develop nonmilk-based production technologies for probiotic cultures. This provides new possibilities for the development of nondairy-based probiotic products.

Bifidobacterium↗

PubMed1992

Influence of the cryoprotective agents glycerol and hydroxyethyl starch on red blood cell ATP and 2,3-diphosphoglyceric acid levels.

Because hydroxyethyl starch (HAES) is used for volume replacement therapy and as a cryoprotectant for frozen red blood cells (RBCs), this compound, in contrast to glycerol, does not require labor-intensive removal from thawed cells prior to transfusion. We here report the effect of both glycerol and HAES on the RBC organic phosphates ATP and 2,3-diphosphoglyceric acid (2,3-DPG). The CPD-A1-stabilized RBCs of 20 healthy donors (3 females, 17 males) were separately frozen in either 40% glycerol or 6% HAES, of molecular weight 200,000. ATP and 2,3-DPG concentrations were determined in CPD-A1 RBCs before addition of cryoprotectant and in cryopreserved thawed RBCs after 24 h storage at -80 degrees C (glycerol) and -196 degrees C (HAES). It appears that HAES, but not glycerol, significantly reduces ATP concentrations whereas both lead to a reduction of 2,3-DPG concentrations; this reduction was more pronounced with glycerol than with HAES. Experiments with the blood of 6 donors demonstrated that HAES affects autohemolysis by 16%, in contrast to glycerol, after which cryoprotectant autohemolysis was affected by 3.1% only. RBC recoveries were comparable using glycerol or HAES as cryoprotectants. A distinct pattern of reduction of 2,3-DPG levels by glycerol and less by HAES, and of ATP levels by HAES but not by glycerol, emerges. Our findings may be of importance if HAES is to be introduced as a convenient cryoprotectant.

2,3-Diphosphoglycerate↗