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PubMed2001

Successful xenografting of cryopreserved primary pancreatic cancers.

In order to assess the suitability of cryopreserved neoplastic tissues for xenografting into nude (nu/nu) mice, we compared the take rate in 28 samples of pancreatic ductal carcinoma. Eleven fresh samples were implanted in nu/nu mice, and 17 were frozen in cryopreserving solution and implanted at a later time. All samples were examined for the presence of neoplastic tissue in cryostat sections. A total of 15 tumors grew in the animals; five from the freshly implanted samples and ten from those cryopreserved. Ten xenografted tumors were characterized for alterations in p53, K-ras, and p16 genes, which were found in six, eight, and nine cases, respectively. Our results demonstrate that the take rate for xenografting is comparable between cryopreserved and fresh tissue samples. The procedure allows for the exchange of tumor material between institutions and permits the establishment of centralized facilities for the storage of an array of different primary tumor samples suitable for the production of in vivo models of cancers.

Adult↗
PubMed1996

Long-term cryopreservation of bone marrow for autologous transplantation.

Little is known about the effect of long-term cryopreservation on the viability of hematopoietic stem cells (HSC) or on the success of autologous bone marrow transplantation. Although progenitor cell assays such as culture of CFU-GM after thawing can be predictive of engraftment, the most rigorous assay for the cryosurvival of HSC is engraftment after reinfusion of stem cells. We retrospectively evaluated the engraftment data for 36 patients with hematologic malignancies or solid tumors treated at the Fred Hutchinson Cancer Research Center between 1981 and 1993 who received bone marrows stored for 2 years or more. The median duration of cryopreservation for this study group was 2.7 years (range 2.0-7.8). Ninety-seven percent of patients in the study group achieved a granulocyte count of > or = 0.5 x 1.0(9)/1 at a median of 19 days (range 10-115) vs 86% of control group (selected by diagnosis and date of storage) at a median of 20 days (P = 0.14). Seventy percent of patients in the study group achieved a platelet count > or = 20 x 10(9)/1 at a median of 27 days (range 9-69) vs 74% of control group at a median of 23 days (P = 0.47). Also, samples of 28 marrows cryopreserved for a median of 4.4 years (range 2.0-7.8) were cultured to determine if a loss of hematopoietic progenitors relative to duration of storage could be detected. The storage length was not predictive for the quantity of colonies formed (P = 0.57 for BFU-E-derived colonies; P = 0.65 for CFU-GM-derived colonies). We found no consistent detrimental effect of long-term cryopreservation on the success rate of autologous bone marrow transplantation. This report confirms previous reports that marrow cells cryopreserved for several years are capable of engrafting. Therefore, bone marrow cells may be stored at an early appropriate time before the side-effects of multiple cycles of chemotherapy and radiotherapy on hematopoietic tissues are incurred.

Adolescent↗
PubMed2002

Effect of pentoxifylline on motility and membrane integrity of cryopreserved human spermatozoa.

The purpose of this study was to examine the effects of pentoxifylline used before and after semen cryopreservation-thawing on sperm motility and membrane integrity. Twenty-four semen samples were split into four equal aliquots. Aliquots were incubated at 37 degrees C for 30 min, followed by cryopreservation with TEST-yolk freezing medium using slow programmable freezing protocol. After 2 weeks the sperm samples were thawed, washed twice in Quinn's Sperm Washing Medium (modified HTF with 5.0 mg/mL Human Albumin) and incubated at 37 degrees C for 30 min. Aliquots were treated by adding 3 mmol/L pentoxifylline to: (1) fresh sperm samples during incubation period prior to cryopreservation, (2) sperm samples as a supplement to the cryoprotectant prior to cryopreservation, and (3) thawed sperm samples during incubation period. One aliquot received no treatment (control group). The addition of 3 mmol/L pentoxifylline to fresh semen during incubation period prior to cryopreservation significantly decreased progressive and total motility compared with controls. However, the addition of 3 mmol/L pentoxifylline to cryopreserved semen after thawing significantly increased progressive and total motility compared with controls. After post-thaw, no differences in motion characteristics between sperm samples treated by adding 3 mmol/L pentoxifylline as a supplement to the cryoprotectant and control groups were observed. Post-thaw hypoosmotic swelling (HOS) test scores did not improve with the addition of pentoxifylline compared with the control group. It is concluded that pentoxifylline enhanced post-thaw motility of cryopreserved human spermatozoa when added after thawing. No improvement was found by freezing sperm with pentoxifylline.

Cell Membrane↗
PubMed2004

Cryoprotectant permeation through human articular cartilage.

OBJECTIVE: The cryopreservation of intact articular cartilage is constrained by minimal chondrocyte survival. It was the aim of the present study to gain an insight into the permeation kinetics of cryoprotectants through cartilage. This knowledge is essential for achieving adequate tissue permeation prior to cooling. DESIGN: The diffusion coefficients and penetration rates through human articular cartilage of dimethyl sulfoxide (Me(2)SO) and glycerol at different temperatures (4 degrees C, 17 degrees C, 27 degrees C and 37 degrees C) and at two concentrations [10% (v/v) and absolute state] were measured using diffusion nuclear magnetic imaging. Deuterated water (D(2)O) was used as a control substance. RESULTS: Glycerol penetrated faster than Me(2)SO at all temperatures and at rates that were comparable to those for D(2)O. The penetration rate of each agent increased with increasing temperature. The diffusion coefficients for glycerol and Me(2)SO increased with increasing temperature and decreased at the higher concentration, but the differences between each agent were not significant. CONCLUSIONS: The classical cryopreservation protocols expose cartilage samples to Me(2)SO at a too low temperature and/or for an insufficient time period for optimal cell survival. When considering the penetration rate, glycerol appears to be a more efficient cryoprotective agent than Me(2)SO. The present study demonstrates the power of nuclear magnetic resonance technology to elucidate key physiological factors in cryobiology.

Adult↗
PubMed2000

The Israel National Skin Bank: Quality Assurance and Graft Performance of Stored Tissues.

The Israel National Skin Bank (INSB) was founded jointly by the Israel Defense Forces (IDF) Medical Corps and the Ministry of Health in 1986. The prime purpose of the Skin Bank is to treat burn victims incurred at war or during mass casualty incidences. The INSB Protocol is comprised of international skin bank protocols and our previous and present research results. They provide the framework for selecting optimal guidelines for procurement, processing, preservation, storage and evaluation of transplantation performance of viable skin grafts. For evaluation and direct comparison of graft performance of glycerolized or cryopreserved skin stored for long periods, we have applied a mouse recipient model developed by us. This model assesses graft performance before the rejection process takes place. The in vivo design has inherent clinical relevance, which is especially appealing. Cryopreserved skin performed better than glycerolized skin (p > 0.027), but fresh skin performed significantly better than cryopreserved skin (p > 0.003), as analyzed by the Mann-Whitney non-parametric test. Then graft performance of skin specimens were cryopreserved by programmed or stepwise freezing and stored at -80 degrees C or in liquid nitrogen for 1 and 6-10 months was evaluated. The average score of skin preserved by programmed freezing and stored in liquid nitrogen is the highest for both storage periods. This method has a highly significant advantage (p < 0.007) over the others for 6-10 months storage, evaluated by graft adherence. Several interaction factors determine the quality of cryopreserved skin. Highly significant is the interaction factor/'combined effect' of sample variability with the method of cryopreservation or with the storage period. Finally, the results of paired comparison of selected histology criteria of cryopreserved to fresh skin indicated that storage of skin for up to 5 years did not impair significantly its performance compared to fresh skin, whereas, after six years of storage, there was a highly significant (p < 0.001) impairment in skin quality. We offer a simplified in vivo model and analysis for cryopreserved skin graft performance, suggesting that the evaluation procedures, which are issues of great interest in skin banking, may help future skin banks to make informed choices and decisions regarding quality issues.

Journal Article↗
PubMed1996

Interleukin 10 production by human melanoma.

Interleukin 10 (IL-10) has the physiological role of down-regulating cell-mediated immunity. We have recently reported that mRNA for IL-10 was present in most metastatic melanoma tissues. The purpose of this investigation was to determine whether melanoma metastases produce IL-10 protein. Single-cell suspensions were prepared by enzymatic dissociation of 28 lymph node metastases and 7 s.c. metastases and cryopreserved. Of these 35 samples, 30 produced IL-10 after a 24-h incubation (median, 125.1 pg/ml). IL-10 production was slightly diminished after 25 Gy irradiation but almost completely abrogated after modification with the hapten dinitrophenyl. After 7 or 14 days in tissue culture, melanoma cells continued to produce IL-10 but only at about 10% of the levels of freshly dissociated tissues. Moreover, of eight melanoma cell lines established from these cultures, only one produced IL-10 protein. To determine whether IL-10 was produced by melanoma cells or tumor-associated leukocytes, single-cell suspensions were fractionated with anti-CD45 antibody-conjugated magnetic beads. In four of five samples, IL-10 production was increased by depletion of leukocytes, suggesting that the primary source was the melanoma cells themselves. This was confirmed by immunohistochemical staining of cytospin preparations and frozen tissue sections. Finally, 10 of 55 patients with clinically evident metastases showed elevations of circulating IL-10; three patients who had been melanoma-free developed high serum IL-10 levels, concurrent with the appearance of distant metastases. These data indicate that production of IL-10 is characteristic of metastatic melanomas and raise the possibility that this cytokine allows tumors to avoid or to modulate immunological attack.

Cryopreservation↗
PubMedDate not supplied

Sperm morphology and preparation method affect bovine embryonic development.

This study was conducted to evaluate the effect of sperm separation methods of semen samples collected from bulls subjected to scrotal insulation on embryonic development after in vitro fertilization (IVF) and to determine whether IVF results would be affected by various heparin concentrations. Morphologically abnormal semen samples were obtained and cryopreserved from Holstein bulls following scrotal insulation for 48 hours. Standard protocols using the Percoll gradient (90%/45%) method and the swim-up method were used to separate spermatozoa fractions in experiment I. The pellet (A(p)) and the 45% layer (B(p)) were isolated from the Percoll separation, while for the swim-up separation, the supernatant (A(s)) and the interphase (B(s)) were isolated. The overall blastocyst rate for our laboratory control semen was 23.1 +/- 2.1% for Percoll separations (A(p) and B(p)) and 18.2 +/- 2.0% for swim-up (A(s) and B(s)) separations. This rate was higher (P <.01) than the rate observed for the semen from the bull that had the greatest response to scrotal insult 5 days prior to the insult, when it was 9.2 +/- 2.1% for the Percoll separation and 20.7 +/- 2.3% for the swim-up separation, while semen from 27 days after scrotal insulation (D +27) resulted in no blastocyst formation for the Percoll separation and a 4.2 +/- 2.1% rate for the swim-up separation. In experiment II, semen was sampled from the bulls that responded in the greatest and least degrees to scrotal insult 5 days before scrotal insulation (D -5) and on days 23 (D +23) and 34 (D +34) after scrotal insulation. These samples were exposed to IVF mediums with 3 different heparin concentrations (0.1, 1.0, and 10 microg/mL). There was a significant difference (P <.05) in developmental scores between the D -5 (1.08 +/- 0.08), D +23 (0.9 +/- 0.08), and D +34 (0.8 +/- 0.08) samples, but no differences were observed in blastocyst formation based on the number of cleaved embryos. Increasing the heparin concentration resulted in higher (P <.01) embryonic developmental scores. In conclusion, when semen samples with high percentages of abnormal spermatozoa are used for IVF, semen separation preparation methods affect results. Our results show that the separation methods used under these conditions were inadequate in their ability to provide potentially competent sperm for IVF. However, selecting appropriate sperm separation procedures could improve in the IVF embryonic development of semen from bulls used in artificial insemination. Also, an increase in the heparin concentration was able to partially overcome deficiencies, which suggests that morphologically abnormal spermatozoa undergo capacitation despite possible structural changes to the plasma membrane.

Animals↗
PubMed2005

Apoptosis in cryopreserved human ovarian tissue obtained from cancer patients: a tool for evaluating cryopreservation utility.

Fertility preservation is of major importance for women with cancer in whom ovarian function may be disturbed by the use of potentially sterilizing chemotherapeutic drugs and/or pelvic irradiation. Cryopreservation of ovarian cortical tissue is one of the potential options for preserving fertility among these women. Cryopreserved thawed human ovarian tissue can be autografted either orthotopically or heterotopically, but may also be transplanted first into an animal host with subsequent maturation and collection of oocytes. The objective of this study was to investigate the prevalence of ovarian follicular apoptosis in fresh and frozen/ thawed human ovarian tissue as a measure of follicular viability. The study group included 6 women with cancer who underwent ovarian tissue cryopreservation (OTCP). Ovarian tissue samples (n = 2) were obtained from each woman with one sample undergoing evaluation for apoptosis immediately following removal (control, group A) and the other evaluated for apoptosis following freezing/thawing (group B). Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) and 4'6' diamido-2-phenylindole hydrochloride (DAPI) staining methods were used to investigate follicular apoptosis. Morphological changes in the same samples were evaluated in hematoxylin and eosin (H&E)-stained sections. In each slide, only primordial and primary follicles were evaluated for abnormal morphology and apoptosis. Abnormal morphology was demonstrated in 23.8+/-8.7% of group A follicles compared to 48.3+/-11.2% of group B follicles (p < 0.05). Apoptosis was demonstrated in 25.4+/-8.4% of group A follicles compared to 60.9+/-6.0% of group B follicles (p < 0.05). We have shown that the ovarian follicles in group B demonstrated a higher incidence of apoptosis compared to those of group A. Therefore, the data suggest that follicular apoptosis might be a consequence of the freezing and thawing procedure. This may be used as a method for evaluating and comparing the outcome of different freezing/thawing protocols.

Adult↗
PubMed1995

The predictive value of computer-assisted semen analysis in the context of a donor insemination programme.

In this paper we examine the value of both conventional and computer-assisted semen analysis (CASA) using the Hamilton-Thorn HTM-S 2030 in predicting the in-vivo fertility of cryopreserved donor semen. Semen samples were examined prospectively and data on the conventional criteria of semen quality, sperm morphometry and movement were collected. Of 61 ejaculates identified, 33 achieved pregnancies ('successful') and 28 failed to do so ('unsuccessful'), despite insemination into at least four different normal female recipients. When the post-thaw semen profiles were compared, no differences were observed between the two groups in respect of the conventional criteria of semen quality determined by conventional laboratory techniques; however, there were differences in respect of both morphometry and movement characteristics determined by the HTM-S. When multiple logistic regression was used to examine the ability of the variables measured to predict the achievement of pregnancy, the conventional criteria of semen quality were of no value (chi 2 = 6.67, P = 0.353). However, the CASA assessment successfully predicted outcome in 86.9% of cases (chi 2 = 44.3, P = 0.0021). It was concluded that CASA assessment is of significant value in predicting the ability of an ejaculate to achieve pregnancy.

Autoanalysis↗
PubMed2003

Ultra-rapid freezing of very low numbers of sperm using cryoloops.

BACKGROUND: With the availability of ICSI, men with severe oligozoospermia (<5x10(6)/ml) are able to reproduce. Current methods for cryopreservation of severe oligozoospermic samples are labour intensive and costly. The objective of this study was to evaluate whether freezing small numbers of motile sperm (approximately 100) was feasible using cryoloops. METHODS: Initial tests assessed the effect of various dilutions of cryoprotectants on pre-freezing sperm motility. Several solutions were further evaluated for their ability to cryoprotect sperm during ultra-rapid freezing. Sperm were placed on cryoloops and held in liquid nitrogen vapour for 5 min prior to freezing (ultra-rapid freezing) or directly submerged into liquid nitrogen. Using the optimal cryoprotectant and technique from these experiments, ultra-rapid and standard slow-rate freezing protocols were compared. RESULTS: Optimal sperm survival was seen when sperm in cryoloops were placed in liquid nitrogen vapour in test yolk buffer with 12% v/v glycerol versus other cryoprotectants. Using this cryoprotectant, post-thaw sperm motility is comparable between ultra-rapid and slow-rate freezing methods. CONCLUSION: Ultra-rapid freezing of very low numbers of sperm is feasible using cryoloops suspended in liquid nitrogen vapour for 5 min.

Cell Survival↗
PubMed2003

Semen collection, cryopreservation and artificial insemination in the dromedary camel.

Collection of semen with a bovine artificial vagina (AV) was attempted with each of 14 camels over a period of 2 years. Semen samples were evaluated, extended and cryopreserved. Frozen thawed semen, diluted cooled semen or whole semen was used to inseminate some female camels which were induced to ovulate with hCG. Males ejaculated semen into the AV in 74.6% collection attempts. The male copulated for at least 200s in 62.9% attempts. The remaining copulations were of shorter duration. Similarly, 49.3% ejaculates were at least 3ml of semen. Libido and donation of semen improved from December onwards and reached a peak after mid January with peak performance persisting until April. It declined during May. The majority of camels had lost libido and refuse to donate semen by the end of May. Camel semen is in gel form. While 35.9% of 203 semen samples exhibited no individual sperm motility, 28.5% exhibited low to fair grade individual sperm motility and only 35.4% exhibited >50% sperm motility. Differences existed between animals (P<0.01) and months (P<0.05) of collection, while effect of copulation time was not significant. Mass motility was not observed in camel semen. Individual sperm motility develops after liquefaction of semen. Addition of caffeine but not chymotrypsin improved the individual motility. The mean live percent sperm count and normal acrosome were 73.3+/-1.0 and 92.0+/-0.5, respectively. Only 51.1% of 45 semen samples with pre-freeze motility of >50% and 25% of 16 semen samples from low pre-freeze motility group with an overall success of 44.2% of 61 semen samples were successfully preserved. Wide variation was observed in the freezability of semen from different males. Attempts to impregnate female camels with liquid semen, frozen thawed semen and whole semen after hCG induced ovulation resulted in 0/10, 1/13 and 4/10 pregnancies.

Acrosome↗
PubMed1986

In vivo isolation and maintenance of some wild strains of European hard tick spirochetes in mammalian and arthropod hosts. A parasitologist's view.

Methods and results of isolation and experimental maintenance of Borrelia strains from indigenous Ixodes ricinus ticks are described. Out of 12 different proved rodent species and breeding variants the mongolian jird or gerbils (Meriones unguiculatus) offers most perfectly all the necessary qualities to produce a transitory not progressive microscopically apparent spirochetemia when splenectomized previously. Using a laboratory breed of Ixodes ricinus the occurrence of transstadial and transovarial transmission could be confirmed in the vector tick. Each developmental stage of ticks can be infected via blood ingestion on spirochetemic mammalian hosts but each stage can infect vice versa its blood donors equally well. Needle borne transmissions from gerbil to gerbil by i.p. administration of infected blood are particularly successful after previous deep-freezing and cryopreservation of the blood samples or tissue suspensions. Attempts are described to infect other tick species than I. ricinus, fleas and mites, respectively, via blood meal. The problem of the spirochetes perfect adaptation to the host will be discussed.

Animals↗
PubMed2002

Design and validation of an enzyme-linked immunospot assay for use in clinical trials of candidate HIV vaccines.

The enzyme-linked immunosorbent (ELISPOT) assay, which enumerates peripheral blood mononuclear cells (PBMCs) releasing interferon gamma (IFN-gamma) on specific antigen stimulation, is becoming the assay of choice for evaluation of vaccine-induced cell-mediated immune responses in many clinical trials. A properly conducted trial requires the assays to be validated, especially should the trial lead to vaccine licensure. Here, the design and validation of an ELISPOT assay are described for use in clinical trials of candidate human immunodeficiency virus (HIV) vaccines, using a particular immunogen termed HIVA. This assay employs eight pools of 20 to 23 peptides each: seven pools are derived from the immunogen and one pool is derived from cytotoxic T cell epitopes of common human viruses serving as an internal positive control. The validation determined that first, the overall variation of a positive response of approximately 500 spot-forming units (SFU)/10(6) cells was 21%, while second, the average of 5 SFU/10(6) cells was detected for the seven HIVA-derived pools in HIV-uninfected individuals; third, a positive response to a peptide added to the assay pools was not occluded by the other pool peptides; fourth, the frequencies detected in fresh PBMCs were 2- to 3-fold higher compared with the same samples that had been cryopreserved; and finally, all seven HIV-derived pools induced IFN-gamma responses in PBMCs isolated from HIV-infected individuals. The limits of the validation of assays involving biological responses of living cells are discussed.

AIDS Vaccines↗
PubMed2002

Oncologists' attitudes and practices regarding banking sperm before cancer treatment.

PURPOSE: The goal of this study was to survey oncologists in three different practice settings to determine their knowledge, attitudes, and practices regarding referring patients to bank sperm before cancer treatment. METHODS: A postal survey about knowledge, attitudes, and practices regarding banking sperm before cancer treatment was sent to 718 oncology staff physicians and fellows at two cancer centers and at sites in a Community Clinical Oncology Program. RESULTS: The return rate was 24% and did not differ by institution, oncologic specialty, or sex. Fellows were significantly more likely to participate (37%) than staff physicians (20%). Ninety-one percent of respondents agreed that sperm banking should be offered to all men at risk of infertility as a result of cancer treatment, but 48% either never bring up the topic or mention it to less than a quarter of eligible men. Neither greater knowledge about sperm banking nor seeing large numbers of eligible men yearly increased the likelihood of discussing the option. Barriers cited included lack of time for the discussion, perceived high cost, and lack of convenient facilities. Oncologists reported they would be less likely to offer sperm banking to men who were homosexual, HIV-positive, had a poor prognosis, or had aggressive tumors. Oncologists overestimated the costs of sperm banking and the number of samples needed to make cryopreservation worthwhile. CONCLUSION: Sperm banking should be offered as an option to all men at risk of infertility because of their cancer treatment. Clearer practice standards could help oncologists increase their knowledge about sperm banking and avoid dependence on biased patient selection criteria.

Attitude of Health Personnel↗
PubMed1998

p53 gene mutation in the bone-marrow of a patient with diffuse mixed cell type lymphoma at diagnosis predicting eventual progression to large cell lymphoma.

Mutations of the p53 tumor suppressor gene have been used as molecular genetic markers of disease and serve as a prognostic indicator in various malignancies including non-Hodgkin's lymphoma (NHL). Alterations in the p53 gene were investigated in a bone marrow sample from a NHL patient admitted for autologous bone marrow transplantation. Diffuse mixed small and large cell NHL, was initially diagnosed which eventually progressed to large cell lymphoma at relapse following poly-chemotherapy. A sequential technique of polymerase chain reaction-mediated single-strand conformational polymorphism (PCR-SSCP) of the p53 gene revealed a shift in one band of exon 6 in the bone marrow, collected at the time of initial diagnosis. No mutations were detected in exons 5, 7, 8 and 9. Direct sequencing of exon 6 detected a single base change from G to C resulting in an amino acid substitution from glycine to histidine. Results of this study and data reviewed from other publications suggest that the missense p53 mutation seen in this patient at the time of diagnosis may perhaps have been used to predict the eventual outcome of the disease. This could, therefore, serve as an important genetic disease marker particularly in bone marrow or peripheral blood samples initially collected and cryopreserved for future possible autologous transplantation.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2002

Separation of motile populations of spermatozoa prior to freezing is beneficial for subsequent fertilization in vitro: a study with various mouse strains.

Success with in vitro fertilization (IVF) using inbred strains of mice varies considerably and appears to be related to the proportion of motile spermatozoa present in epididymal sperm samples of different strains. In this study, motile spermatozoa were separated from the original samples using a column of Sephadex G25. IVF rates were compared between separated and nonseparated samples of epididymal spermatozoa before and after cryopreservation. Oocytes and spermatozoa were obtained from FVB, DBA/2, C57BL/6J, and BALB/c inbred mice; and from F1 (C57BL/6J;ts DBA/2) hybrid mice, and isogenic gametes were used for IVF. These strains of mice were chosen because of their common use in transgenesis and mutagenesis studies. Dulbecco PBS was used for sperm separation on Sephadex, 18% raffinose, and 3% skim milk for cryopreservation; T6 medium for IVF; and mKSOM(AA) for embryo culture. There was a marked improvement in the rate of fertilization using fresh spermatozoa after motile spermatozoa were separated in C57BL/6J and BALB/c strains (92% vs. 58%, 79% vs. 44%) but no differences were found in fertilization rates between separated and nonseparated spermatozoa in F1, FVB, and DBA/2 strains (99% vs. 83%, 95% vs. 93%, 86% vs. 87%, respectively). After cryopreservation, higher rates of fertilization were obtained with separated motile samples in all strains; the greatest improvements were obtained with spermatozoa from C57BL/6J and BALB/c strains (40% vs. 16% and 51% vs. 14% for separated and nonseparated spermatozoa, respectively). No differences were found between the proportions of 14.5-day fetuses developing from embryos derived from separated and nonseparated spermatozoa with or without cryopreservation (33% to 46%). In conclusion, the fertility of frozen-thawed mouse epididymal spermatozoa improves significantly when highly motile populations of spermatozoa are separated for freezing.

Animals↗
PubMed2004

Effect of cryopreservation on the immunogenicity of umbilical cord blood cells.

Cryopreservation is the accepted method for long-term storage of cord blood (CB) cells. We evaluated the effects of using different cooling rates (1, 5, 7.5 and 10 degrees C/min) on CB cell allostimulatory and alloproliferative function, antigen expression and clonogenic potential. Significant decreases (P<0.001-0.003) in viable cell recovery observed between fresh CB cells and CB cells cryopreserved at each cooling rate tested. Reductions in clonogenic potential of CB cells cryopreserved at cooling rates of 1, 5, 7.5 and 10 degrees C/min were 44%, 76%, 88% and 93% respectively, compared to fresh controls. FACS analysis indicated no changes in percentages CD34+ cells or lymphocytes. Two sets of mixed lymphocyte reactions were carried out for each CB sample. It was observed that allostimulatory and alloproliferative function varied following cryopreservation at different cooling rates (1 and 5 degrees C/min). Interestingly, there was a significant decrease (P<0.001-0.04) in the alloproliferative function of six of the seven CB samples following cryopreservation using a cooling rate of 5 degrees C/min. Cooling rates between 1 and 5 degrees C/min may provide immunomodulation of CB with maintenance of haematopoietic progenitor cells function.

Antigens, CD34↗
PubMedDate not supplied

[Micromycetes and actinobacteria under conditions of many years of natural cryopreservation].

Almost all of the investigated samples of the Arctic and Antarctic permafrost sediments of different genesis with ages from 5-10 thousand to 2-3 million years were found to contain viable micromycete and bacterial cells. The maximum amounts of viable cells of fungi (up to 10(4) CFU/g air-dried sample) and bacteria (up to 10(7)-10(9) CFU/g air-dried sample) were present in fine peaty sediment samples taken from different depths. The identified micromycetes belonged to more than 20 genera of the divisions Basidiomycota, Ascomycota, and Zygomycota, and some represented mitosporic fungi. Thawing the samples at 35 and 52 degrees C allowed the number of detected fungal genera to be increased by more than 30%. Aerobic heterotrophic prokaryotes were dominated by coryneform, nocardioform, and spore-forming microorganisms of the order Actinomycetales. Analysis of the isolated fungi and actinomycetes showed that most of them originated from the microbial communities of ancient terrestrial biocenoses.

Antarctic Regions↗