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PubMed2000

Highly purified CD34+ cells isolated using magnetically activated cell selection provide rapid engraftment following high-dose chemotherapy in breast cancer patients.

The primary objective of this study was to evaluate the safety of infusion of CD34+ cells, selected using a clinical scale magnetically activated cell sorting device, assessed by time to hematological engraftment and incidence of adverse events. Secondary objectives included evaluation of device performance in terms of purity and recovery of the CD34+ cell product. Breast cancer patients suitable for transplantation received cyclophosphamide and filgrastim for mobilisation, followed by three leukaphereses. The products of the first two leukaphereses underwent CD34+ cell selection. The product of the third leukapheresis was cryopreserved unmanipulated. Following high-dose cyclophosphamide, thiotepa and carboplatin, selected CD34+ cells were infused. In 54 patients who received selected cells only, the median time to platelet recovery and neutrophil recovery was 11 days (range 5-51) and 9 days (range 5-51), respectively. There were no adverse events associated with infusion of selected cells. A total of 126 leukapheresis samples was available before and after selection for central CD34+ analysis. The median purity was 96.1% (27.4-99.4) and the median recovery was 52. 3% (15.2-146.3). These data show that cells selected using magnetically activated cell selection provide safe and rapid engraftment after high-dose therapy. Bone Marrow Transplantation (2000) 25, 243-249.

Adolescent↗
PubMedDate not supplied

Characteristics of fresh gander semen and its susceptibility to cryopreservation in six generations derived from geese inseminated with frozen-thawed semen.

The paper summarises seven years experiments designed to determine the effect of continuous insemination with frozen-thawed semen on fresh semen quality and sperm susceptibility to freezing stress in succeeding generations. During course of experiments, semen was collected from 10-12 White Koluda ganders at the age of 8-9 months, then subjected to freezing and used after thawing for insemination of 10 geese in order to obtain the subsequent generation of males. Semen was diluted 1 to 0.5 (v/v) with EK diluent, equilibrated for 15 min at +4 degrees C, mixed with 6 percent (v/v) of dimethyl-formamide (DMF), frozen to temp. -140 degrees C at a rate 60 degree C per min and then transferred into liquid nitrogen container. Semen samples were thawed prior to insemination in a 60 degree C water-bath. It is difficult to conclude whether freezing stress affected the fresh semen quality, since average volume of SQF (index comprising ejaculate volume, sperm concentration and percentage of live normal cells) varied between generations from 19.3 to 56.2. Continuous goose reproduction by insemination with frozen-thawed semen resulted in significant increase (P less than 0.01) in spermatozoa resistance to cryoinjury in every subsequent generation. In the relation to adequate fresh semen the percentage of live morphologically intact spermatozoa which withstood freezing procedure increased from 27.2 in first generation to 74.4 in sixth generation.

Animals↗
PubMed2000

Functional capacity and fertilizing longevity of frozen-thawed scimitar-horned oryx (Oryx dammah) spermatozoa in a heterologous in vitro fertilization system.

This study was conducted to determine if cryopreservation and thawing reduces the quality of scimitar-horned oryx spermatozoa and thus might be responsible for sub-optimal artificial insemination (Al) efficiency. Functional capacity of frozen thawed oryx spermatozoa was compared in a heterologous bovine in vitro fertilization (IVF) system after being prepared by four methods. Fertilizing longevity was also assessed after thawing and pre-incubating spermatozoa for 12 or 24 h before IVF. Sperm characteristics (viability, morphology, acrosomal and capacitation status) were superior for samples prepared by Percoll centrifugation and standard swim-up compared with microdrop swim-up and wash methods. Regardless of variation in sperm characteristics over time, fertilization success and embryo development were high and did not differ among treatments. Fertilization and cleavage success for spermatozoa pre-incubated for 12 h before IVF were comparable with that achieved with non-incubated spermatozoa. Even 24 h after thawing, spermatozoa were capable of fertilizing oocytes, but percentage fertilization and embryo cleavage were significantly lower than for spermatozoa pre-incubated for 12 h. Overall, functional capacity of oryx spermatozoa after thawing appears comparable with that of domestic bull spermatozoa. When used for Al, frozen-thawed oryx spermatozoa should be capable of fertilizing oocytes in females ovulating 12 or even 24 h after insemination, providing sperm transport mechanisms are adequate. The functional capacity and fertilizing longevity of oryx sperm after thawing is high, and therefore unlikely to be responsible for decreased Al efficiency in the scimitar-horned oryx.

Animals↗
PubMed2003

Sperm cryopreservation in three species of endangered gazelles (Gazella cuvieri, G. dama mhorr, and G. dorcas neglecta).

Long-term storage of semen by cryopreservation, with high recovery rates on thawing, is essential for the establishment of genetic resource banks of endangered species. The purpose of the present study was to evaluate various diluents for the cryopreservation of spermatozoa from three species of gazelles (genus Gazella) in a captive breeding program. The diluents compared were Tes (N-tris(hydroxymethyl)methyl-2 aminoethane sulfonic acid)-Tris with 5% egg yolk and 6% glycerol (TEST) and Triladyl, yolk-citrate, Tris-trehalose, and Tris-lactose-all of them with 20% egg yolk and 6% (Triladyl) or 8% glycerol. Semen was obtained by electroejaculation from 12 G. cuvieri, 12 G. dama, and 13 G. dorcas males. Samples with less than 50% motile sperm, positive endosmosis, or acrosome integrity were not used. Diluted samples were loaded into 0.25-ml straws, cooled slowly to 5 degrees C over 1.5 h (-0.16 degrees C/min), equilibrated at that temperature for 2 h, frozen in nitrogen vapors for 10 min, and plunged into liquid nitrogen. Subsamples were assessed fresh, after refrigeration-equilibration, after freezing and thawing, and 2 h after thawing. Differences were seen between diluents, with best overall recovery rates after freezing and thawing found with Triladyl, TEST, and Tris-trehalose in G. cuvieri, TEST in G. dama, and Triladyl and TEST in G. dorcas. Differences were observed between species in the ability to withstand freezing and thawing, with best results seen in G. dorcas, intermediate results in G. dama, and worst results in G. cuvieri. These differences were inversely related to the average values of inbreeding of these populations. The underlying mechanism responsible for these differences may be a differential resistance to osmotic shock.

Acrosome↗
PubMed2006

Effects of frozen storage on the structure of sarcocysts in pig muscle and implications in taxonomic studies.

UNLABELLED: The morphology of the cyst wall of Sarcocystis has unique characteristics that can be used in species identification. To find a suitable way to preserve Sarcocystis cyst samples for species identification, by light microscopy and electron microscopy, we recorded the morphological changes in the cysts of Sarcocystis suihominis and Sarcocystis miescheriana from pig muscle, induced by storage at -20 degrees C. Comparisons were made between fresh cysts and those subjected to frozen storage for periods of 3 days, 20 days and 30 days. RESULTS: cyst wall of the two Sarcocystis species appeared unaffected by storage. There was no obvious change in the length, nor in the width of the protrusions after storage (P>0.05), but the structure of the bradyzoite in the sarcocyst was in many cases disintegrated at -20 degrees C in 20 days for S. miescheriana and 30 days for S. suihominis. To our knowledge this is the first report that Sarcocystis cyst in muscle can be stored at -20 degrees C before and remain suitable for ultrastructural morphological study. Consequently, this paper proposes freezing as a convenient storage method for samples used in taxonomic studies of Sarcocystis.

Animals↗
PubMed1998

Evaluation by high-performance liquid chromatography of the hydrolysis of human milk triacylglycerides during storage at low temperatures.

The effect of various storage methods on the stability of the triacylglyceride fraction of human milk was evaluated. Samples were treated as follows: Group I--stored at -20 degrees C for 4 months, group II--heated for 1.5 min at 80 degrees C and stored at -20 degrees C for 4 months, group III--stored at -80 degrees C for 4 months and thawed rapidly at room temperature (25 degrees C) just before analysis and group IV--stored at -80 degrees C for 2 months, thawed rapidly at room temperature (25 degrees C), then stored at -80 degrees C for a further 2 months and finally thawed rapidly at 25 degrees C just before analysis. The absence of hydrolysis products in group II and group III indicated that these storage procedures were satisfactory even when samples were rapidly thawed for a short time (group IV). Only storage at -20 degrees C without previous heat treatment led to the hydrolysis of triacylglycerides and the appearance of free fatty acids (group I). On the other hand, the effect that freezing and thawing had over the lipolysis grade was studied. Samples were treated as follows: group V--stored at -20 degrees C for 2 months, thawed slowly at refrigerator temperature (5 degrees C), held at this temperature for one week and stored for a further month at -20 degrees C. Freezing and thawing activated lipolysis and increased the production of free fatty acids, monoacylglycerides and diacylglycerides. Milk samples were analyzed by reversed-phase HPLC with a ternary gradient of acetonitrile-dichloromethane-acetone and an evaporative light-scattering detector.

Chromatography, High Pressure Liquid↗
PubMed1991

Haptoglobin phenotyping from older bloodstains by enzyme immunoassay and haptoglobin phenotypes within a Nebraska Caucasian population.

Enzyme immunoassay and Western blotting (electrophoretic) techniques were used to determine haptoglobin (HP) phenotypes from older bloodstains. Serum was collected from liquid blood and the HP phenotypes were determined. Bloodstains were prepared from these specimens and stored at various temperatures for several months. The stains were extracted and applied to gradient polyacrylamide gels. The Western blotting technique was used to achieve the transfer of HP bands from the gels to the nitrocellulose membranes. Enzyme immunoassay with goat anti-HP antiserum and rabbit anti-goat immunoglobulin peroxidase were used to identify the HP bands from the extracted samples. Enzyme immunoassay was found to be clearly more sensitive than o-dianisidine or o-tolidine in detecting HP bands from diluted serum samples. The haptoglobin frequency in a Caucasian population in Nebraska was calculated. The frequencies of Phenotypes 1, 2-1, and 2 were found to be 15.8, 48.4, and 35.8%, respectively.

Blood Preservation↗
PubMed1990

6-Thioguanine-resistant T-lymphocyte autoradiographic assay. Determination of variation frequencies in individuals suspected of radiation exposure.

We used the autoradiographic assay to assess human in vivo somatic cell gene mutation at the hypoxanthine guanine phosphoribosyl transferase (HGPRT) locus in T-lymphocytes. Cells able to incorporate tritiated thymidine in vitro in the presence of 6-thioguanine were enumerated in order to determine 6-thioguanine-resistance (TGr) variant frequencies in cryopreserved lymphocytes from 17 normal control individuals, from 3 persons suspected to have been exposed to 60Co in an accident in Cd. Juárez (Mexico), studied 24 months after the accident, and from 4 individuals who were in Kiev during the radiation accident in Chernobyl (U.S.S.R.); 2 of them were studied 1 month after the accident, and again 1 year after the first sampling, the other 2 were studied 13 months after the accident. The data obtained indicate that this assay may be useful in any laboratory of cytogenetics for human population monitoring and that its use following accidental exposure to ionizing radiation should be further evaluated.

Accidents↗
PubMed2002

Effect of freezing temperature, at which straws were plunged into liquid nitrogen, on the post-thaw motility and acrosomal status of ram spermatozoa.

The present study was conducted to observe the effect of initial freezing temperature on subsequent survival and acrosomal integrity of Malpura and Bharat Merino ram spermatozoa during post-thawing incubation. Semen samples were diluted in TEST-yolk-glycerol extender, loaded in 0.25 ml straws and cooled down to -25, -75 or -125 degrees C freezing temperature using a programmable cell freezer. Computer assisted sperm analysis and acrosomal integrity of thawed samples were assessed after thawing and at hourly intervals during incubation at 37 degrees C for 4 h. The percentage of motile cells in samples frozen at -125 degrees C were 80.3 and 63.7 after post-thawing and -thawing incubation, compared to 75.9 and 39.7 at -25 degrees C or 73.9 and 51.8 at -75 degrees C temperatures, respectively. The spermatozoa with normal acrosome were also significantly, respectively, higher in samples frozen at -125 degrees C, compared to -25 and -75 degrees C temperatures. There were no significant breed variations on percentage of motile, percentage of rapidly motile cells, percentage of normal acrosomes, curvilinear velocity and lateral head displacement except straight line velocity and average path velocity of spermatozoa. The results indicated that -125 degrees C initial freezing temperature conferred the best cryopreserving ability to ram spermatozoa for post-thawing thermoresistance test compared to -25 or -75 degrees C freezing temperature.

Acrosome↗
PubMed1996

Home ovulation testing in a donor insemination service.

The use of home ovulation testing kits in donor insemination (DI) has been proposed to increase patient and clinic convenience while not compromising fecundity rates. Such a system was introduced into our DI service in December 1994, and we here report an audit of experience over 6 months. Patients were offered home or laboratory luteinizing hormone (LH) testing, and those requesting home testing were asked to store an aliquot of tested urine for subsequent assay in the laboratory allowing retrospective analysis of the accuracy of cycle timing. Insemination using cryopreserved semen was performed on the day home testing predicted ovulation, or on the day an LH surge was detected in the laboratory, and on the following day. Pregnancy rates were significantly reduced in home testers: 3.4% per cycle (174 cycles, 64 women) versus 12.7% (110 cycles, 53 women) over the same time period (P < 0.005, 95% confidence interval 6.5-18.9). Urine samples from 140 cycles from 51 women using home testing were analysed. There were insufficient data in nine to allocate the cycle. Of home tested cycles, 37 (28%) were inseminated on a day other than the first day of the LH surge. In 13 of these insemination was performed after the first day of the LH surge. Incorrect treatment was associated with high baseline LH, but those with 'late' treatment had low basal LH concentrations, similar to those correctly treated. Analysis of individual urine samples showed that the positive predictive value of home testing was 72%. These results suggest that home ovulation testing results in reduced chance of pregnancy, with increased frustration for both patients and clinic staff. This may be particularly so in women with high baseline LH concentrations.

Adult↗
PubMed1993

Increased frequency of alloantigen-reactive helper T lymphocytes is associated with human cardiac allograft rejection.

Endomyocardial biopsy (EMB) is the standard method of monitoring heart transplant recipients for the development of allograft rejection. To date, noninvasive methods to detect cardiac allograft rejection have lacked adequate sensitivity and specificity for wide clinical application. In this study, limiting dilution analysis (LDA) was used to quantitate the number of donor alloantigen-reactive helper T lymphocytes (HTLs) in the peripheral blood of cardiac transplant recipients. Cadaveric donor splenocytes were cryopreserved, providing a source of donor alloantigenic stimulation for these assays. Peripheral blood mononuclear cells were harvested from cardiac transplant recipients before transplantation and at the time of EMB. LDA of donor-reactive HTLs was conducted simultaneously on all time points to minimize experimental variation, and these data were related to EMB scores. Frequencies of donor-reactive HTLs in pretransplant samples were highly variable, ranging from 1/1381 to < 1/200,000, and correlated poorly with the degree of HLA disparity. During episodes of moderate rejection, donor-specific HTL frequencies increased an average of 6 times their post-transplant baseline frequency. Additionally, 10-fold increases in HTL frequencies were seen preceding EMB-diagnosed rejection in several individuals. These data indicate that episodes of allograft rejection are associated with increases in the number of circulating donor-reactive HTL which are frequently detected before the development of histologically defined rejection. Thus, monitoring HTL frequencies may serve as a non-invasive method for detecting and predicting cardiac allograft rejection. Furthermore, this assay may provide a valuable means of assessing the in vivo efficacy of various immunosuppressive therapies.

Graft Rejection↗
PubMed2002

A broad and strong humoral immune response to donor HLA after implantation of cryopreserved human heart valve allografts.

Cryopreserved human heart valves are used for valve replacement in patients with congenital or acquired heart disease. Although no blood group or human leukocyte antigens (HLA) matching is performed and no immunosuppression is administered, the clinical results are relatively good. After valve replacement, the majority of the patients develop HLA antibodies, whereas a smaller group of patients shows valve-related events at the long term after right ventricular outflow tract reconstruction. Therefore, we hypothesized that not the mere presence, but rather the titers of antidonor HLA antibodies may be related to valve allograft failure. The presence and specificity of HLA class I antibodies were determined by complement-dependent microlymphocytotoxicity (CDC) test in longitudinally taken peripheral blood samples of 35 valve allograft recipients. In eight patients with an antibody response specific against donor-HLA class I, the titers were measured by this CDC method after stepwise dilution of the plasma. Panel reactive antibodies of more than 10% were found in 31 of 35 (89%) valve allograft recipients. From these 31 patients, 24 (77%) developed donor-specific HLA class I antibodies. All eight selected patients had detectable donor-specific antibody titers, ranging from 1:2 to 1:8,000. Two donor valve recipients before retransplantation had (donor-specific) HLA antibodies and showed high titers of 1:256 and 1:8,000 shortly after the second allograft valve replacement, which was associated with an early graft failure in the latter patient. We conclude that transplantation of cryopreserved human heart valve allografts leads to a broad and strong humoral response, which is probably the result of a lack of immunosuppressive therapy after valve transplantation. Patients receiving a second or following valve allograft appeared to be sensitized and developed early and high allo-antibody titers after second valve allograft implantation. Valve failure was diagnosed in a patient with extremely high titers. These findings suggest that preoperative cross-matching may identify patients with high donor-specific HLA antibody titers and may reduce the risk for early recurrent graft failure.

Adolescent↗
PubMed2000

Use of frozen-thawed testicular sperm for intracytoplasmic sperm injection.

OBJECTIVE: To determine the feasibility of using frozen-thawed testicular spermatozoa for intracytoplasmic sperm injection. DESIGN: Prospective clinical study. SETTING: A university hospital. PATIENT(S): One hundred seventy-five azoospermic men participating in a routine intracytoplasmic sperm injection program. INTERVENTION(S): The men underwent testicular biopsy for cryopreservation of tissue to be used in consecutive intracytoplasmic sperm injection treatment cycles. Their female partners underwent controlled ovarian hyperstimulation for conventional IVF treatment. MAIN OUTCOME MEASURE(S): Fertilization and pregnancy rates. RESULT(S): In 77% of the patients, spermatozoa could be harvested from the testis by an open testicular biopsy technique and used for intracytoplasmic sperm injection after freezing and thawing of testicular tissue. Histopathologic evaluation revealed a Sertoli cell-only pattern in 21%, maturation arrest in 60%, and hypospermatogenesis in 19% of the patients. In 2. 9% of the patients, carcinoma in situ or a germ cell tumor was detected. In all patients, viable spermatozoa could be visualized after the tissue samples were thawed. One hundred thirty-five intracytoplasmic sperm injection treatment cycles were performed, with a fertilization rate of 45% and a clinical pregnancy rate of 30% per oocyte retrieved. CONCLUSION(S): The use of frozen-thawed testicular tissue allows ovarian stimulation of the female partner to be timed and avoids cancellation of ovum pick-up when spermatozoa cannot be retrieved.

Chromosome Aberrations↗
PubMed1998

Sublethal damage during cryopreservation of rainbow trout sperm

Although cellular damage during cryopreservation of freshwater fish spermatozoa has been reported in several studies, there is a lack of correlation between this damage and the fertility rates of eggs using postthawed milt. The apparent lack of such correlation may be due to other undetected sublethal cryodamage, which could affect the cell functionality and viability. This may be extremely important for freshwater fish spermatozoa whose ability to fertilize the egg requires dilution in water or hypoosmotic solutions, an hazardous environment for the cells. This study tested the change in cell permeability during cryopreservation, using Hoechst 33258 to assess cell permeability. The permeability of spermatozoa at different times after dilution in several hypoosmotic media were investigated. In the first trial, fresh semen, sperm diluted in freezing media (CPT), and freeze/thawed semen were studied. Three CPT were tested (Me2SO, DMA, and methanol). In the second trial, the addition of egg yolk as a membrane stabilizer was investigated. Samples were frozen at -20 degreesC/min in a programmable cooler and thawed in a 25 degreesC water bath. Dilution in the CPTs slightly increased the susceptibility of cells to damage but freezing/thawing caused a dramatic increase in the fragility of cells, which were killed in a few seconds after their contact with the hypoosmotic solutions. Egg yolk provided a significant protection to the membrane, allowing the cells a greater and more prolonged survival in the fertilization media. Samples frozen with Me2SO displayed the best results. These results are consistent with the achieved fertility rates that demonstrated sublethal cryodamage in the function of the sperm membrane that was not detected by standard procedures. Copyright 1998 Academic Press.

Journal Article↗
PubMed2001

Determination of optimal transport conditions for biopsies of human articular cartilage as well as for suspensions of cultured chondrocytes used in autologous transplantation.

Autologous transplantation of chondrocytes is currently being promoted as a novel approach for the treatment of deep cartilage lesions. Briefly, the method involves enzyme-mediated release of chondrocytes from cartilage biopsies, the expansion of cells by in vitro cultivation and their re-implantation into the defect. The success of this technique depends on many factors including transport conditions for both, cartilage biopsies from the operating hall to the laboratory and the return transport of final suspension of cultured chondrocytes. To determine the extent of cellular damage in biopsies, chondrocytes were enzymatically isolated following a few days of tissue preservation in different tissue culture media. The proportion of dead cells was assessed by Trypan blue staining and counting. The viability was not dependant of the type of the medium used and remained approximately 50% in all samples, even after 72 h. To develop optimal conditions for transport of final chondrocyte suspension, isolated cells were firstly grown in monolayer cultures. Cell suspensions in media with different additives were injected into special glass containers used for the transport and left at 4 degrees C or 25 degrees C for up to 3 days. During this period every 24 h the samples were taken and viability as well as apoptosis levels were assessed. Viability of cells in suspensions at 25 degrees C decreased significantly and became inadequate already after 48 h. In contrast to that, the proportion of viable cells at 4 degrees C remained above 80% even after 48 h. In the majority of the samples, culture medium containing serum and vitamin C provided the best conditions for long-term preservation of chondrocytes.

Apoptosis↗
PubMed1993

Interrelationships among fluorometric analyses of spermatozoal function, classical semen quality parameters and the fertility of frozen-thawed bovine spermatozoa.

Cryopreserved spermatozoa from 8 bulls were used to examine the interrelationships among flow cytometric spermatozoal quality assessments and classical semen quality parameters and nonreturn rate estimates of fertility. The integrity of the sperm cell membrane and the functional capacity of the mitochondria were quantified by flow cytometry after concurrent staining with carboxydimethylfluorescein diacetate (CDMFDA), propidium iodide (PI), and rhodamine 123 (R123). For each sample a total of 10,000 stained spermatozoa were simultaneously quantified for the intensity of their green and red fluorescence. Three straws from each bull were each examined initially and following incubation at 37 degrees C for 3 hours to assess the rate of senescence. The proportion of spermatozoa retaining membrane integrity and having functional mitochondria, as determined by CDMFDA and R123 staining, were compared with classical semen quality assessments (sperm motility, acrosomal status, cellular and head morphology, presence of vacuoles/craters and cytoplasmic droplets) and with fertility (nonreturn to estrus rates). For individual ejaculates nonreturn rates, the range was from 61.8 to 78.8%, whereas the cumulative rates of several ejaculates for each bull ranged from 71.3 to 83.5%. The proportion of spermatozoa with functional membranes and mitochondria were positively correlated with the percentage of spermatozoa with normal morphology (r=0.82; P=0.01) and motility after 4 hours of incubation (r=0.78; P=0.02), but not with the estimates of fertility. The actual number of spermatozoa per straw staining with CDMFDA and R123 after 4 hours of incubation at 37 degrees C was correlated with the percentage of spermatozoa with normal morphology (r=0.73; P=0.04). Multiple regression equations indicated that combinations of semen quality measurements could be useful in estimating fertilizing potential.

Journal Article↗
PubMed1992

Cryo-electron microscopy of vitrified muscle samples.

A great deal of information on the 3-dimensional structure of the protein assemblies involved in muscle contraction has been obtained using conventional transmission electron microscopy. In recent years, developments in cryo-electron microscopy have facilitated work with fully hydrated, non-chemically fixed specimens. It is shown how this technique can be used to visualize muscle sarcomere filaments in quasi-native conditions, to access hitherto inaccessible states of the crossbridge cycle, and to obtain new high resolution structural information on their 3-dimensional protein structure. A short introduction to the crossbridge cycle and its biochemically accessible states illustrates the problems amenable to studies using the electron microscope, as well as the possibilities offered by cryo-microscopy on vitrified samples. Work on vitrified cryo-sections and myosin filament suspensions demonstrates the accessibility of crossbridge states and gives implications on the gross structural features of myosin filaments. Recent studies on actin filaments and myosin (S1) decorated actin filaments provide the first high resolution data on vitrified samples. The use of photolabile nucleotide precursors allows the trapping of short lived states in the millisecond time range, thereby visualizing intermediate states of the crossbridge cycle.

Animals↗
PubMed2006

Fracture formation in vitrified thin films of cryoprotectants.

As a part of an ongoing effort to study the continuum mechanics effects associated with cryopreservation, the current report focuses on fracture formation in vitrified thin films of cryoprotective agents. The current study combines experimental observations with continuum mechanics analysis. Experimental results have been developed using a new imaging device, termed a "cryomacroscope", which has been recently presented by the current research team. A newly developed liquid nitrogen-based cooling stage is presented in this paper. The samples under investigation are 0.5 ml droplets of cryoprotective agents, having a characteristic diameter of 20 mm and a characteristic thickness of 1.5 mm. Tested samples included dimethyl sulfoxide (DMSO) in a concentration range from 6 to 8.4M, and the cryoprotectant cocktails VS55 and DP6. Some samples contained small bovine muscle segments, having a characteristic dimension of 1mm, in order to study stress concentration effects. Experimental results show that the onset of fracturing in vitrified films of cryoprotectants is very consistent, occurring over a small temperature range. Fracture pattern, however, was affected by the cooling rate. The presence of tissue segments did not affect the onset temperature of fracture, but affected the fracture pattern. The continuum mechanics analysis solidified the hypothesis that fracture is driven by thermal stress, not by temperature per se, and allowed fracture strain to be inferred from observed fracture temperature. In conjunction with the current report, additional photos of fracture formation in thin films are available at .

Cryoprotective Agents↗