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PubMed1999

Freezing of testicular tissue as a minced suspension preserves sperm quality better than whole-biopsy freezing when glycerol is used as cryoprotectant.

Frozen-thawed testicular spermatozoa have been used successfully for ICSI, especially in cases of obstructive azoospermia with normal spermatogenesis. Fewer attempts, however, have been made to check whether these rather immature spermatozoa, in a different environment with several other cell types present, have cryobiological requirements other than those of ejaculated spermatozoa. This is the reason why the freezing protocols and cryoprotectants (glycerol) used for freezing testicular tissue are based on experience with semen freezing. This study aimed to assess whether cryosurvival and/or motility was influenced by freezing of testicular tissue either as an intact biopsy or as a shredded tissue suspension, when glycerol was used as cryoprotectant. Freezing of testicular tissue as a suspension preserved motility (type B + C) significantly better than freezing of whole biopsies (9.2% vs. 4.0%). Similar observations have been made for vitality (39.3% vs. 25.4%). Centrifugation on 50% Percoll in order to remove the cryoprotectant resulted in a huge loss of spermatozoa (or late spermatids) and should therefore be especially avoided in cases of testicular failure. On the basis of these observations, mincing of the testicular biopsies before freezing may be advocated. Testicular spermatozoa seem to be better preserved when frozen in suspension, at least when slowly permeating glycerol is used as a cryoprotectant.

Biopsy↗
PubMed1998

Permeation of human ovarian tissue with cryoprotective agents in preparation for cryopreservation.

The recent improvements in the treatment of cancer by chemo- and radiotherapy have led to a significant increase in the survival rates of patients with malignant disease, but at the expense of distressing side effects. One major problem, especially for younger patients, is that aggressive therapy destroys a significant proportion of the follicular population, which can result in either temporary or permanent infertility. Freeze-banking pieces of ovarian cortex prior to treatment is one strategy for preserving fecundity. When the patient is in remission, fertility could, theoretically, be restored by autografting the thawed tissue at the orthotopic site or by growing isolated follicles to maturity in vitro. Recent studies have found good follicular survival in frozen-thawed human ovarian tissue but to optimize the process an effective cryopreservation method needs to be developed. An essential part of such a technique is to permeate the tissue with a cryoprotectant to minimize ice formation and the extent of this equilibration is an important determinant of post-thaw cellular survival. In the current study, we have investigated the diffusion of four cryoprotective agents into human tissue at both 4 degrees C and 37 degrees C. We have also studied the effect of adding different concentrations of the non penetrating cryoprotective agent, sucrose, to the freezing media using the release of lactate dehydrogenase as a measure of its protective effect. At 4 degrees C propylene glycol and glycerol penetrated the tissue significantly slower than either ethylene glycol or dimethyl sulphoxide. At the higher temperature of 37 degrees C all four cryoprotectants penetrated at a faster rate, however concern about enhanced toxicity prevents the use of these conditions in practice. Thus, the results suggest that the best method of preparing tissue for freezing is exposure for 30 min to 1.5 M solutions of ethylene glycol or dimethyl sulphoxide at 4 degrees C; this achieved a mean tissue concentration that was almost 80% that of the bathing solution. We also report that the addition of low concentrations of sucrose to the freezing medium does not have a significant protective effect against freezing injury.

Adult↗
PubMed1989

Comparative results on survival of human and animal eggs using different cryoprotectants and freeze-thawing regimens. I. Mouse and hamster.

The achievement of successful pregnancies and births after in-vitro fertilization and embryo transfer of frozen-thawed human oocytes has stimulated further work on improving the established methodology. The present investigation was conducted on 1837 mouse oocytes, 1785 mouse pronuclear stage embryos, 1400 hamster oocytes and 1024 hamster pronuclear-stage embryos. In an effort to study the advantages and disadvantages of a newly introduced, 1,2-propanediol (1,2-PROH)-based system over the conventionally used dimethylsulphoxide (DMSO)-based methodology, a direct, prospective comparison between the two cryoprotectants was undertaken in a randomized trial. The combination of 1,2-PROH and DMSO potentiates their cryoprotective effect on mouse and hamster eggs. 1,2-PROH seems to improve cryopreservation in the animal system, though not significantly. Four different protocols were used to evaluate the effects of two changing experimental parameters. These were, firstly, the intermediary temperature attained before placing the cells into liquid nitrogen and, secondly, the modification of the method of adding and removing the cryoprotectant. The morphological survival rate, fertilization rate and developmental rate were significantly better in the low intermediary temperature compared with the high intermediary temperature system of cryopreservation. In addition, the rate of zona pellucida breakdown diminishes considerably in the former compared with the latter system. The 'rapid' sucrose method of cryoprotectant equilibrium and removal showed, in most groups, results which were lower than, or equal to, the traditionally used 'multiple-step', 'slow' method.

Animals↗
PubMed1999

Effects of commonly used cryoprotectants on glycogen phosphorylase activity and structure.

The effects of a number of cryoprotectants on the kinetic and structural properties of glycogen phosphorylase b have been investigated. Kinetic studies showed that glycerol, one of the most commonly used cryoprotectants in X-ray crystallographic studies, is a competitive inhibitor with respect to substrate glucose-1-P with an apparent Ki value of 3.8% (v/v). Cryogenic experiments, with the enzyme, have shown that glycerol binds at the catalytic site and competes with glucose analogues that bind at the catalytic site, thus preventing the formation of complexes. This necessitated a change in the conditions for cryoprotection in crystallographic binding experiments with glycogen phosphorylase. It was found that 2-methyl-2,4-pentanediol (MPD), polyethylene glycols (PEGs) of various molecular weights, and dimethyl sulfoxide (DMSO) activated glycogen phosphorylase b to different extents, by stabilizing its most active conformation, while sucrose acted as a noncompetitive inhibitor and ethylene glycol as an uncompetitive inhibitor with respect to glucose-1-P. A parallel experimental investigation by X-ray crystallography showed that, at 100 K, both MPD and DMSO do not bind at the catalytic site, do not induce any significant conformational change on the enzyme molecule, and hence, are more suitable cryoprotectants than glycerol for binding studies with glycogen phosphorylase.

Adenosine Monophosphate↗
PubMed1989

Extraction from prawn shells of substances cryoprotective for Vibrio cholerae.

Substances cryoprotective for Vibrio cholerae were detected from prawn shells immersed in phosphate-buffered saline. This cryoprotective activity was heat resistant and sensitive to treatment with trypsin. For the exhibition of its full activity, the presence of Mg ion was indispensable. The cryoprotective activity of this substance was more active than that of other known cryoprotectants, like glycerol or serum.

Animals↗
PubMed2004

Cryoprotection mechanisms of polyethylene glycols on lactate dehydrogenase during freeze-thawing.

The purpose of this study was to explore the cryoprotection mechanisms of high molecular weight polyethylene glycols (PEGs) (eg, PEG 4000 and PEG 8000) on lactate dehydrogenase (LDH). Ultraviolet activity assays, circular dichroism (CD) spectroscopy, gel filtration, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), (14)C-PEG 4000 labeling and binding, and cryostage microscopic study were conducted. Different molecular weights and concentrations of PEGs in LDH formulations were treated by freeze-thawing. Higher molecular weights and concentrations of PEGs in LDH-PEG formulations obtained better activity and secondary structure recoveries of LDH after freeze-thawing. Insoluble aggregation of LDH was not observed in gel filtration studies. SDS-PAGE results suggested surface characteristic modifications of LDH by the larger molecular weight PEGs. The 14C-PEG 4000 labeling and binding study showed extensive nonspecific interactions between the PEG 4000 and LDH molecules in a concentration-dependent manner. The bound LDH-PEG 4000/free PEG 4000 ratio increased when LDH or PEG 4000 concentrations increased. Cryostage microscopic study showed that PEG 8000 delayed the ice crystallization and eutectic transition of LDH formulation. It appeared that multiple mechanisms were at work during PEGs' cryoprotection of LDH. It was unclear whether the delayed eutectic characteristics of PEGs contributed to LDH cryoprotection. The favorable interaction, rather than preferential exclusion, between LDH and PEGs (eg, 4000) cryoprotected LDH.

Animals↗
PubMed2005

The effect of freeze-drying with different cryoprotectants and gamma-irradiation sterilization on the characteristics of ciprofloxacin HCl-loaded poly(D,L-lactide-glycolide) nanoparticles.

In the present study, the influence of freeze-drying with several cryoprotective agents and gamma (gamma)-irradiation sterilization on the physicochemical characteristics of ciprofloxacin HCl-loaded poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles was evaluated. Nanoparticles were prepared by W/O/W emulsification solvent evaporation followed by high-pressure homogenization. They were freeze-dried in the presence of 5.0% (w/v) mannitol, trehalose or glucose, with 5.0% (w/v) or 15.0% (w/v) dextran as cryoprotectants. The nanoparticles were irradiated at a dose of 25 kGy using a 60Co source. The following physicochemical properties of the formulations were investigated: the ratio of particle size before (initial) and after freeze-drying, the ease of reconstitution of the nanoparticle suspensions and the drug-release profiles of irradiated and non-irradiated nanoparticles. The antibacterial activity against Pseudomonas aeruginosa was measured. The freeze-drying process induced a significant increase in particle size when no cryoprotectant was employed. Similar results were observed when cryoprotectants were added to the formulation. Only when mannitol was used was no significant size increase measured. Moreover, for formulations with dextran, reconstitution after freeze-drying was difficult by manual agitation and particle size could not be determined because of aggregation. After gamma-sterilization no significant difference in mean particle size was observed, but reconstitution was more difficult and drug release was influenced negatively. Ciprofloxacin HCl incorporated in the nanoparticles was still effective against the micro-organism selected after freeze-drying and gamma-sterilization.

Anti-Bacterial Agents↗
PubMed2000

Cryoprotective activities of group 3 late embryogenesis abundant proteins from Chlorella vulgaris C-27.

The nucleotide sequence of hiC12, isolated as a cDNA clone of hardening-induced Chlorella (hiC) genes, was identified. The clone encodes a late embryogenesis abundant (LEA) protein having six repeats of a 11-mer amino acid motif, although in a slightly imperfect form. To overexpress the hiC61) and hiC12 genes, their coding regions were PCR amplified and subcloned into a pGEX-1lambdaT vector. The HIC6 and HIC12 proteins were expressed as GST fusion proteins in E. coli, then purified. The two HIC proteins were found to be effective in protecting a freeze-labile enzyme, LDH, against freeze-inactivation. On a molar concentration basis, they were about 3.1 x 10(6) times more effective in protecting LDH than sucrose and as effective as BSA. Cryoprotection tests with five kinds of chain-shortened polypeptides, synthesized based on the 11-mer amino acid motif of the HIC6 protein showed that the cryoprotective activity decreased with a decrease in the repeating units of the 11-mer motif. In fact, cryoprotective activities of three kinds of single 11-mer amino acids were very low even at high concentrations. All the results suggested that the sufficiently repeated 11-mer motif is required for the cryoprotective activities of Chlorella LEA proteins.

Amino Acid Sequence↗
PubMed1985

A new instant cryoprotectant for human sperm.

A new instant cryoprotectant, mainly containing culture medium, human serum albumin, and kallikrein, and its laboratory handling in the cryoprotection of semen are described. A comparative study distributed to three cryobiological centers using either egg yolk citrate medium or instant cryoprotectant showed that neither had a significantly better or worse mean percentage recovery rate. The advantage of the new instant cryoprotectant presented consists clinically in the absence of egg yolk and technically in its easy handling. Therefore, ICP is judged as an alternative to ECM in the cryopreservation of human sperm.

Citrates↗
PubMed2001

Development of a cryopreservation procedure employing a freezer bag for pancreatic islets using a newly developed cryoprotectant.

One of the most important requirements for success in clinical islet transplantation is the use of a large number of viable donor islets. To achieve this, the ability to cryopreserve islets and to establish an islet bank are critical. Previously, we developed a two-step cryopreservation procedure with freezing tubes utilizing low and high concentrations of dimethyl sulfoxide (DMSO) and using a fully automated cryomachine for human pancreatic islets and porcine islet-like cell clusters (ICCs). Based on these experiments, we developed a simple and efficient cryopreservation procedure of a freezer bag for isolated islets using a fully automated computer-controlled cryomachine with a newly developed cryoprotectant consisting of ethylene glycol (EG) instead of DMSO for decreasing injury of the islets by freezing. A 250 ml Cryocyte blood freezer bag and our newly developed cryoprotectant containing ethylene glycol (EG) were used in the freezing procedure. The islets were frozen by a fully automated computer-controlled cryomachine (GE 9,000) with our original program of slow cooling. Nucleation occurred at -8 degrees C, and the frozen islets were stored at -196 degrees C in a liquid nitrogen tank. The frozen-stored islets were subsequently rapidly thawed in a 37 degrees C water bath and cultured before viability testing. In vitro function, the stimulation index of insulin release during the static incubation test for rat islets cryopreserved in a freezer bag vs. nonfrozen islets as control, was 2.13 +/- 0.42 and 2.02 +/- 0.38 (94.8% compared with control), respectively (n = 5, p = NS). The islet recovery compared with the nonfrozen control group was 85% (n = 5) in insulin content. When 1000 rat islets cryopreserved in a freezer bag were transplanted into the renal capsule of diabetic athymic mice, all the mice became normoglycemic within 7 days from transplantation. Before nephrectomy, the intravenous glucose torelance test (IVGTT) was performed. The fractional decay constant of the glucose level (K value) of the frozen-thawed group was 0.42 +/- 0.06%/min. A histological study of renal subcapsular grafts demonstrated the morphological integrity of the islets. These results demonstrate the utility of our cryopreservation procedure of a freezer bag for isolated islets using a fully automated computer-controlled cryomachine with a newly developed cryoprotectant for the maintenance of viability and function of frozen-stored islets both in culture and after transplantation. Cryopreservation using freezer bags with the new cryoprotectant is an effective and simple method for making an islet bank for clinical trials of islet transplantation.

Animals↗
PubMed2000

[Effects of different cryoprotectants on biological properties of hematopoietic cells derived from cord blood].

OBJECTIVE: To search for effective cryoprotectants that can be used in cord blood bank to cryopreserve hematopoietic cells. METHODS: Four kinds of combined reagents were used to cryopreserve cord blood cells. The effects of these cryoprotectants on biological properties of hematopoietic cells were monitored and compared after the cells were refrigerated for 1 to 4 months. The detected biological properties included the number of total nucleated cells (TNC), CD(34)(+) cells and CFCs. RESULTS: The cryoprotectants of Dextran-40 + 10%DMSO resulted in the highest recovery rates, TNC, CD(34)(+) cells and CFCs were 77.8%, 78.5% and 87.2%, respectively. The combination of 10%DMSO + 6% AB serum + 5%HES led to the lowest recovery rates. CONCLUSION: Dextran-40 + 10%DMSO is an ideal cryoprotectant combination for cryopreserving hematopoietic cells in the cord blood bank.

Antigens, CD34↗
PubMed2005

[Influence of cryoprotectant with glycerol and freezing-thawing procedure on the motility of human sperm].

OBJECTIVE: To investigate the effects of cryoprotectant with glycerol and the freezing-thawing procedure on the motility of METHODS: The motion characteristics of human sperm from 18 selected specimens were assessed by computer-assisted human sperm. sperm analysis before and after adding hyper-osmolarity cryoprotectant with glycerol and the freeze-thaw procedure, and the data were evaluated in pairs. RESULTS: The adding of cryoprotectant caused an increase in the proportion of rapid linear motile sperms (P < 0.05) and sperm velocities, including VCL, VSL and VAP (P < 0.005). But no changes were observed in the proportion of progressively motile sperms and sperm motility. Compared with the data from pre-frozen samples, velocities of post-thawed sperms and the percentage of motile sperms in each grade significantly declined (P < or = 0.01), so did ALH, while WOB, LIN and STR remained unchanged (P > 0.05). Significant differences in ALH, WOB, LIN and STR were observed only in comparison between the post-thawed and pre-treated samples. CONCLUSION: The number of Grade a sperms and sperm velocity increased after adding hyper-osmolarity cryoprotectant with glycerol. The sperm motile potential was impaired and even entirely destroyed in some cases by cryodamage. The influences on the sperm motion were differently induced in freezing-thawing procedures.

Cryopreservation↗
PubMed1991

The ultrastructure of cryo-sections and intact vitrified cells--the effects of cryoprotectants and acceleration voltage on beam induced bubbling.

Chemically fixed pancreas was infiltrated with various cryoprotectants to obtain homogeneously vitrified samples upon cooling. The suitability of these samples for cryoultramicrotomy was tested. Contrast was hardly detectable initially in thin cryo-sections but increased upon irradiation, irrespective of the cryoprotectant (glycerol, propylene glycol, methanol) used. Contrast and beam damage were analyzed in vitrified thin films from collagen, phospholipid vesicles and various concentrations of glycerol. Glycerol increased the beam sensitivity of both collagen and phospholipid vesicles, but diminished the contrast between matrix and lipid vesicles or collagen fibers. The effects of glycerol as observed in thin films explain some of the effects of cryoprotectants in thin cryo-sections. To reduce beam damage in vitrified specimens two approaches are proposed. Firstly, when vitrified films are prepared, dilute suspensions should be used without cryoprotectant. In some cases, such as (thin) intact cells, the composition of the suspended material can only be marginally influenced. Then a second approach can be used involving the application of higher accelerating voltages (e.g. 300 kV). This has two advantages; the increase in mean free path-length of the electrons causes less beam damage on one hand and allows better resolution of thick specimens on the other hand. Micrographs from E. coli bacteria vitrified from suspension illustrate some of the potentials of "intermediate voltage" cryo-electron microscopy.

Animals↗
PubMed1981

Cryoprotected Lactobacillus casei: an approach to standardization of microbiological assay of folic acid in serum.

Folate-depleted, cryoprotected preparations of Lactobacillus casei are stable for at least eight months at -18 degrees C, capable of reproducible growth and suitable as a ready source of inoculum for measurement of folates in physiological fluids. Cryoprotected microorganisms can be a commercially available laboratory reagent, thus simplifying and further standardizing the microbiological assay of various nutriments. Standard folate growth curves of cryoprotected L. casei, prepared at intervals over eight months, are superimposable, have a low blank, and should eliminate the variations encountered with the continuous passages of microorganisms required for the classic microbiological assays. Serum folate values obtained by use of the cryoprotected L. casei fall into the same diagnostic groups as determined by the classic microbiological assay.

Biological Assay↗
PubMedDate not supplied

Effect of cryoprotectants on freezing, lyophilization, and storage of lyophilized recombinant alpha 1-antitrypsin formulations.

Stabilizing effect of several commonly used cryoprotectants, namely lactose, sucrose, and polyvinyl-pyrrolidone (PVP), on recombinant alpha 1-antitrypsin (rAAT) were studied. A solution of rAAT in a phosphate/citrate buffer (pH 7.0) was made with and without a given cryoprotectant and filled into small vials. The vials were frozen on shelf, at -40 degrees C in a lyophilizer. At the end of the freezing cycle, half of the batch was thawed at ambient temperature and the other half was continued to the completion of lyophilization. Sample vials taken before freezing, after thawing, and after lyophilization were analyzed for total rAAT protein, monomeric content, and elastase-inhibitory activity. Results indicated that neither freeze-and-thaw nor lyophilization caused any damage to rAAT, contrary to what was generally believed. The control formulation (i.e., without a cryoprotectant) performed as good as those containing cryoprotectants. Freezing rates and protein concentration in formulation did not influence the stability of rAAT either. Lyophilized rAAT samples retained the activity and purity during the 12 month stability period, at room temperature and 2-8 degrees C.

Buffers↗
PubMedDate not supplied

Freeze-thaw studies of a model protein, lactate dehydrogenase, in the presence of cryoprotectants.

The objective of this study was to investigate the behavior of lactate dehydrogenase (LDH) upon freezing and thawing, alone or in the presence of several selected cryoprotectants. Also, the influence of the freezing rate on retainment of LDH activity was investigated. It was observed that fast freezing caused less loss of LDH activity than slow freezing. The probable mechanisms of loss of activity after freeze-thaw cycles were discussed. Selected cryoprotectants were evaluated for their ability to protect LDH during freeze-thaw cycles. Surface tension and pH change measurements upon freezing of the cryoprotectant solutions were carried out. Based on the results of these experiments, a potential mechanism of cryoprotection has been developed.

Cryopreservation↗
PubMed1998

Current status of vitrification of embryos and oocytes in domestic animals: ethylene glycol as an emerging cryoprotectant of choice.

The cryopreservation of mammalian embryos has become an integral part of methods to control animal reproduction. Numerous vitrification solutions have been formulated with ethylene glycol in combination with macromolecules, sugars and other cryoprotective agents. These indicate that a study of ethylene glycol as a cryoprotectant of choice in vitrification studies would be promising. To understand the cryobiology of ethylene glycol, several factors have to be studied. These are: cryoprotectant toxicity, osmotic stress and temperature at exposure. Understanding these factors could lead to the formulation of vitrification protocols that would lead to higher viability rates after cooling. First, ethylene glycol must be used as the sole cryoprotectant in a solution without macromolecules and sugars. Second, partial dehydration and permeation prior to cooling to subzero temperatures must be studied to achieve accurate exposure and a one-step dilution method. Third, the toxic effects of ethylene glycol must be overcome without sacrificing its vitrification properties by combining step-wise exposure at appropriate temperatures, low concentration and decreased volume. Fourth, the long-term effects of ethylene glycol on exposed or vitrified embryos must be determined. Lastly, the influence of culture on the viability of vitrified embryos must be studied to improve viability rates after warming.

Animals↗
PubMed1992

Freezing preservation of the mammalian cardiac explant. V. Cryoprotection by ethanol.

We studied the colligative cryoprotective effect of ethanol (EtOH) in preserving the isolated rat heart frozen at -3.4 degrees C or unfrozen at -1.4 degrees C. Addition of 4.7% (v/v) EtOH to a cardioplegic solution, CP-14, raised the osmolality from 280 to 1100 mOsm/kg H2O and lowered the melting point from -0.52 to -2.1 degrees C. Freezing of the cardiac explant at -3.4 degrees C for 6 h resulted in 34.3 +/- 1.9% of the tissue water as ice; recovery of cardiac output (CO) was 50%. Polyethylene glycol, which at 5% (w/v) has been shown to cryoprotect the hearts during freezing at -1.4 degrees C, did not improve the protective effect of 4.7% EtOH. CP-14 + 4.7% EtOH did not freeze at -1.4 degrees C. After 6 h storage, CO in hearts flushed with CP-14 + 4.7% EtOH oxygenated with 95% O2/5%CO2 returned to almost control level and was much higher than that in hearts flushed with 100% O2 saturated-CP-14 + 4.7% EtOH. Storage of 8 and 12 h reduced CO to 87 +/- 9 and 60 +/- 5% of control. By employing EtOH as a colligative cryoprotectant, we preserved the adult mammalian heart frozen at -3.4 degrees C or unfrozen at -1.4 degrees C, suggesting that this small molecular weight, penetrating substance may be a suitable cryoprotectant for long-term storage of the cardiac explant at high subzero temperatures.

Animals↗