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PubMed2002

Reproductive potential of fresh and cryopreserved epididymal and testicular spermatozoa in consecutive intracytoplasmic sperm injection cycles in the same patients.

OBJECTIVE: To determine if the cryopreservation of epididymal and testicular spermatozoa alters their reproductive potential by examination of patients who underwent consecutive cycles of ICSI using fresh and then cryopreserved spermatozoa. DESIGN: Retrospective review. SETTING: Tertiary care university hospital. PATIENT(S): One hundred sixty-two consecutive cycles of ICSI were analyzed. Thirteen patients were identified as having undergone treatment with freshly retrieved epididymal spermatozoa; these patients subsequently underwent treatment with spermatozoa cryopreserved from that cycle. Eighteen patients underwent ICSI with freshly retrieved testicular spermatozoa; these patients subsequently underwent treatment with spermatozoa cryopreserved from that cycle. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Fertilization rates and pregnancy rates. RESULT(S): The fertilizing capacity of epididymal spermatozoa remained unchanged after cryopreservation and subsequent thawing, with fertilization rates of 58% and 57% for fresh and cryopreserved spermatozoa, respectively. Testicular spermatozoa, however, showed a significant decrease in fertilizing capacity after cryopreservation when compared with freshly retrieved spermatozoa (52% and 71%, respectively). Pregnancy rates appeared unaffected by the cryopreservation of epididymal spermatozoa (fresh, 3/13; frozen, 2/13) or testicular spermatozoa (fresh, 2/18; frozen, 5/18). CONCLUSION(S): This study offers further evidence that motile epididymal spermatozoa retain their fertilizing capacity after cryopreservation. The data presented on testicular spermatozoa suggest that although cryopreservation may reduce the fertilizing capacity of testicular spermatozoa, there is no decrease in pregnancy rates.

Adult↗
PubMed2003

Legal considerations for cryopreservation of sperm and embryos.

OBJECTIVE: To summarize the case law for cryopreservation of sperm and embryos and make recommendations for desirable characteristics that should be included in a cryopreservation clinic's disposition agreements. DESIGN: A literature review of case law and legal review articles was performed. SETTING: Academic research center. PATIENT(S): None. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): None. RESULT(S): Two court cases involving cryopreserved semen and five cases involving cryopreserved embryos are reviewed. CONCLUSION(S): The state of the law surrounding cryopreservation is recent and unsettled. Disposition agreements can provide cryopreservation clinics some degree of certainty in this unsettled area of law. Those drafting cryopreservation disposition agreements are assisted by lessons learned from cryopreservation cases and insight gained from law review articles. It is evident that cryopreservation agreements will be most successful and most likely to be enforced if they are unambiguous, consistent with public policy, and include: a duration provision, each individual's contact information, the individual's provisions for use of their gametes in case of death, and responsibilities of cryopreservation clinic and individuals. Individuals must enter into agreements with a true understanding of the contained provisions.

Contracts↗
PubMed1995

Effects of cryopreservation on the proliferation and anticoagulant activity of human saphenous vein endothelial cells.

Human saphenous veins were cryopreserved in 4% human albumin and 10% dimethyl sulfoxide. The effect of cryopreservation on endothelial cells was studied in terms of the anticoagulant activity of thrombomodulin and in terms of cell proliferation. After storage for 2 weeks at -150 degrees C, 0.45 +/- 0.07 x 10(5) endothelial cells/cm2 were detected in cryopreserved veins and 1.03 +/- 0.04 x 10(5) endothelial cells/cm2 in fresh veins (p < 0.01). The thrombin-catalyzed activation of protein C decreased after cryopreservation, indicating altered thrombomodulin activity in the endothelial cells. On a cell number basis, the release of soluble thrombomodulin was three times higher from the cryopreserved endothelium than from the fresh endothelium (p < 0.05). The amount of spontaneous release of von Willebrand factor from the endothelial surface was not significantly different between fresh and cryopreserved veins. Endothelial cells were cultured from fresh veins and from their cryopreserved counterparts. On plating of endothelial cells in primary culture, the number of adhered cells was 0.9 +/- 0.09 x 10(3) cells/cm2 from fresh veins and 0.25 +/- 0.03 x 10(3) cells/cm2 from cryopreserved veins (p < 0.01). The positive immunohistochemical stain for von Willebrand factor indicated that the endothelial cell character was maintained after cryopreservation. The endothelial desquamation with loss of anticoagulant function and the slow proliferation of surviving cells in vitro suggest an impaired endothelial healing in vivo. The loss of anticoagulant activity complicates the problems of the exposure of thrombogenic subendothelial matrix to blood in implanted cryopreserved veins.

Blood Coagulation↗
PubMed2005

Eligibility of cryopreserved human embryos for stem cell research in Canada.

OBJECTIVE: To determine whether cryopreserved embryos in Canadian in vitro fertilization (IVF) clinics available (in theory) for research use are actually eligible (in practice) for stem cell research in accordance with the Assisted Human Reproduction Act, the Canadian Institutes of Health Research's Human Pluripotent Stem Cell Research: Guidelines for CIHR-Funded Research, and the Tri-Council Policy Statement: Ethical Conduct for Research Involving Humans. METHODS: Copies of current and past patient information sheets and consent forms for embryo cryopreservation, the future disposition of cryopreserved embryos, and the donation of cryopreserved embryos for research were requested from Canadian IVF clinics. Fourteen of 24 clinics (response rate 58%) provided one or more of the requested documents. RESULTS: A review of the documents currently in use showed that, as of May 2005, all 14 responding clinics require an advance directive for the eventual disposition of cryopreserved embryos. Ten of these 14 clinics allow the donation of cryopreserved embryos for research. Only three of these 10 clinics, however, satisfy in writing the disclosure requirements for embryo research at the time of initial consent for the cryopreservation and future disposition of embryos. Only one clinic specifically identifies the option of embryonic stem cell research. CONCLUSION: Only three of the responding Canadian IVF clinics provide written details about future donation of cryopreserved embryos for research use and about the need for re-contact in accordance with relevant legislation and guidelines. Two of these three clinics, however, include disposition options that may not be legally available. In the event of death or loss of contact, it will not be possible to get project-specific research consent from the legitimate embryo providers as required by law. These same two clinics also fail to mention embryonic stem cell research as one of the research categories. It follows that we have identified only one Canadian IVF clinic with cryopreserved embryos unequivocally eligible for stem cell research, provided the forms we reviewed were used to obtain consent for the cryopreservation of those embryos.

Canada↗
PubMed2000

Cartilaginous injury limits cryopreservation of tracheal isograft.

OBJECTIVE: Histological recovery of tracheal grafts after cryopreservation was investigated using a rat heterotopic tracheal transplant model in order to evaluate the clinical applicability of tracheal cryopreservation. METHODOLOGY: Heterotopic syngeneic tracheal transplantation was performed between F344 rats. Recipient animals received either a non-cryopreserved or a cryopreserved tracheal graft for direct comparison with regard to the effect of cryo-injury. In the non-cryopreserved group (CP(-)), tracheal segments were transplanted syngeneically between F344 rats immediately after harvest. Grafts were implanted into the abdominal space and wrapped with the greater omentum. In the cryopreserved group (CP(+)), grafts were implanted after cryopreservation for 7 days at -85 degrees C. Recipient rats were killed on days 7, 14, 21, 28, and at 2 months after surgery. Epithelial regeneration and cartilage changes were evaluated using a semiquantitative four grade scoring system. RESULTS: Squamous epithelium without ciliated structure was observed on day 7 in both groups. Bronchial epithelium was then regenerated gradually and normally ciliated epithelium was observed on day 28 in both groups. The condition of the epithelium was still well maintained in the CP(-) group at 2 months post-transplantation; however, a severe epithelial defect was observed in the CP(+) group. Bronchial cartilage showed a normal shape and mostly viable chondrocytes with proliferative cell nuclear antigen (PCNA) positive staining at all time points in the CP(-) group until 2 months after surgery. However, in the CP(+) group, a massive loss of viable chondrocytes was observed at 2 months post-transplantation. Macroscopically, CP(+) grafts showed a diminished structure without satisfactory airway lumen at 2 months. CONCLUSION: The epithelium of a tracheal graft can be temporarily recovered after implantation followed by 7 days cryopreservation. However, bronchial cartilage may be severely damaged by freezing, which results in late destruction with loss of viable chondrocytes. It is suggested here that establishing a method of safe cryopreservation for tracheal cartilage will be imperative to making tracheal cryopreservation possible.

Abdomen↗
PubMed1993

Differential effect of cryopreservation on natural killer cell and lymphokine-activated killer cell activities.

The use of lymphokine-activated killer (LAK) cell therapy in delayed treatment requires the use of cryopreserved effector cells. The purpose of this study was to determine the optimal cryopreservation protocol for the maintenance of cytotoxic activity in mononuclear cells (MNCs). MNCs were cryopreserved with dimethyl sulfoxide or 1,2-propanediol before and after 3 days of culture with recombinant interleukin 2. The effects of cryopreservation on cell recovery, LAK cell and natural killer (NK) cell cytotoxic activities, and surface antigen markers were studied. Recovery of nonactivated MNCs was higher with 1,2-propanediol than with dimethyl sulfoxide (p < 0.05). Cytotoxic activities, measured with a 51Cr release assay, significantly decreased after thawing, on both activated cells (76.3%; range, 35.8-92.2%) and fresh cells (54.6%; range, 17.5-75.4%). A 6-day kinetic test was used to compare the cytotoxic activity of cryopreserved and fresh cells. The results showed different patterns for NK cells (cryopreserved cells had lower levels of activity than fresh cells) and LAK cells (cryopreserved cells had higher levels of activity than fresh cells). Phenotype changes of effector cells in culture, with and without cryopreservation, were monitored by flow cytometry using monoclonal antibodies. These results were compared with changes in the cytotoxicity of cells with and without cryopreservation. After thawing, there was a decrease in MNCs expressing CD14 and CD56. Recovery of the CD56 marker correlates with increased cytotoxic activity. Despite some loss of NK cell activity, it is concluded that MNCs may be successfully cryopreserved before their use in immunotherapeutic treatment.

CD4-CD8 Ratio↗
PubMed2001

Collagen synthesis and collagenase activity of cryopreserved heart valves.

OBJECTIVE: Durability of the valve seems to be dependent on the remodeling ability of the valve itself, which is controlled by both collagen synthesis and collagenolytic activity of valvular fibroblasts and endothelial cells. However, the balance of collagen synthesis and collagenolysis of the cryopreserved valve has not yet been clearly revealed. Thus, we assessed the collagen synthesis and collagenolysis ability of the cryopreserved valve. METHODS: Twelve valves were divided into 2 groups: freshly harvested valves (n = 6) and cryopreserved valves (n = 6). We measured the collagen content using Sirius red, a dye selective to the collagen. Collagen synthesis was evaluated by means of the tritiated proline incorporation method. Noncollagenase-digestible counts, which represent protein synthesis, and collagenase-digestible counts, which represent collagen synthesis, were estimated. Collagenase activity of the valves was assessed by gelatin zymography. RESULTS: The collagen content of the cryopreserved group was maintained. The noncollagenase-digestible counts of the cryopreserved group decreased from 3862 +/- 1180 counts/mg to 1174 +/- 1362 counts/mg, and the collagenase-digestible counts of the cryopreserved group were 831 +/- 762 counts/mg compared with the value of 1062 +/- 136 counts/mg for the freshly harvested group. The collagenase activity of the cryopreserved group was observed at the same level as that of the freshly harvested group, despite the serious endothelial damage of the cryopreserved valves. CONCLUSIONS: Although the collagen synthesis of cryopreserved valves was relatively maintained, the protein synthesis was highly diminished, and the collagenolysis ability was activated immediately after the thawing process. These results imply that the cryopreservation procedure itself may cause the collagen metabolism to be on the degradable side, which will lead to valve failure.

Animals↗
PubMed2002

Storage of cane toad (Bufo marinus) sperm for 6 days at 0 degrees C with subsequent cryopreservation.

We investigated the recovery of motility of cane toad (Bufo marinus) sperm after storage for 6 days at 0 degree C (on ice) and after subsequent cryopreservation. Sperm suspensions were prepared from testes macerated in either simplified amphibian Ringer (SAR) or 10% (w/v) sucrose diluents, with 15% or 20% (v/v) glycerol or Me2SO as cryoprotectants, and were stored for 6 days. Alternatively, suspensions were prepared in either SAR or 10% (w/v) sucrose diluent and stored for 6 days, after which some of these suspensions were kept in diluents alone or, alternatively, had 15% or 20% (v/v) glycerol or Me2SO added. All treatments (suspensions) were then cryopreserved. Sperm motility was measured at Day 1 and Day 6 (before and after cryopreservation). A substantial and variable proportion (range 0%-40%) of sperm was immotile in suspensions immediately after preparation from testes macerates. Sperm stored in either SAR or 10% (w/v) sucrose diluent maintained approximately 75% motility for 6 days, but few sperm survived cryopreservation. After storage and cryopreservation, recovery of motility was substantially higher with Me2SO than in glycerol. However, both cryoprotectants exhibited toxicity at high concentrations. Glycerol was more toxic than Me2SO in 10% (w/v) sucrose than in SAR diluent, both before and after cryopreservation. The addition of some cryoprotectants to suspensions before storage gave greater recovery both before and after cryopreservation. After cryopreservation, the highest rate of sperm recovery was in suspensions with 10% (w/v) sucrose and 15% (v/v) Me2SO added prior to storage (mean (+/-SEM) 46 +/- 7% relative to initial; 39 +/- 6% absolute). Sperm were also stored for 6 days at 0 degrees C in suspensions or testes (with suspensions then prepared from testes) and cryopreserved. Sperm maintained higher recovery and membrane integrity both before and after cryopreservation when stored in suspensions rather than in testes.

Animals↗
PubMed1994

The contribution of embryo cryopreservation to in-vitro fertilization/gamete intra-fallopian transfer: 8 years experience.

In this paper, the authors summarized their experience with embryo cryopreservation over an 8-year period. The results, therefore, reflect the long-term benefit of embryo cryopreservation to the overall in-vitro fertilization/gamete intra-Fallopian transfer (IVF/GIFT) programme and to the women who had embryos cryopreserved. The stable survival rate of thawed embryos and pregnancy rate, especially over the past 4 years, suggests that the results can reliably be used to evaluate the efficacy of the embryo cryopreservation programme. The ongoing pregnancy rate of frozen/thawed embryo transfer is 10.9%, comparable with the ongoing pregnancy rate of fresh IVF/embryo transfer in our unit over the same period. In addition to those factors known to affect the pregnancy rate in fresh IVF/GIFT cycles, such as age of the recipients and number of embryos transferred, the major factor affecting the efficacy of the cryopreservation programme is the number of oocytes retrieved in the initial stimulation cycle, and the number of embryos available for cryopreservation. The storage time of cryopreserved embryos will also have a significant effect on the realization of the total potential of embryo cryopreservation. Overall the contribution of cryopreservation to our IVF/GIFT programme is substantial, increasing pregnancy rate by 4%, while the greater net benefit, of course, is for the women who had embryos cryopreserved (pregnancy rate increased by 7%), especially for those who returned for frozen/thawed embryo transfer cycles (pregnancy rate increased by 11%).

Adult↗
PubMed2000

Live rhesus offspring by artificial insemination using fresh sperm and cryopreserved sperm.

Artificial insemination (AI) and the cryopreservation of sperm with full reproductive capabilities are vital in the armamentarium of infertility clinics and reproductive laboratories. Notwithstanding the fantastic successes with AI and sperm cryopreservation in numerous species, including humans and cattle, these assisted reproductive technologies are less well developed in other species of importance for biomedical research, such as genetically modified mice and nonhuman primates. To that end, AI at high efficiency in the rhesus macaque (Macaca mullata) and the successful cryopreservation of rhesus sperm is presented here, as are the complexities of this primate model due to differences in reproductive tract anatomy and gamete physiology. Cryopreservation had no effect on the ability of sperm to fertilize oocytes in vitro or in vivo. Post-thaw progressive motility was not affected by cryopreservation; however, acrosome integrity was lower for cryopreserved (74.1%) than for fresh sperm (92.7%). Fertilization rates did not differ when fresh (58.1%; n = 32/55) or cryopreserved sperm (63.8%; n = 23/36) were used for in vitro fertilization. Similarly, pregnancy rates did not differ significantly after AI with fresh (57.1%; n = 8/14) or cryopreserved sperm (62.5%; n = 5/8). Seven live rhesus macaques were born following AI with fresh sperm, and three live offspring and two ongoing pregnancies were obtained when cryopreserved sperm were used. Cryopreservation of rhesus sperm as presented here would allow for the cost-effective storage of lineages of nonhuman primates with known genotypes. These results suggest that either national or international centers could be established as repositories to fill the global needs of sperm for nonhuman primate research and to provide the experimental foundation on which to explore and perfect the preservation of sperm from endangered nonhuman primates.

Acrosome Reaction↗
PubMed2001

Use of cryopreserved pronuclear embryos for the production of transgenic mice.

A series of experiments was conducted to test the hypothesis that an improved cryopreservation protocol for pronuclear stage mouse embryos will produce transgenic (Tg) mice by pronuclear gene injection at a rate not significantly different from noncryopreserved embryos. In the first experiment, three cryoprotective agents (CPAs) (dimethyl sulfoxide [DMSO], propylene glycol [PG], ethylene glycol [EG]) and two cryopreservation protocols, currently used for pronuclear embryos, were compared in regard to their ability to maintain post-thaw morphological integrity and in vitro developmental competence. In the second and third experiments, the optimal cryopreservation protocol determined from the first experiment was used to evaluate in vitro developmental competence of pronuclear embryos following green fluorescence protein gene injection and in vivo developmental competence as well as the gene integration rates. Survival (morphological integrity and development to two cells) of embryos cryopreserved in the presence of DMSO was higher (P < 0.05) than those cryopreserved with either PG or EG. Postinjection developmental competence (development to two cells) of cryopreserved CBA, C57B6/JxCBA-F1 and noncryopreserved (control) embryos was not different (P > 0.05). Postinjection blastocyst formation rate of cryopreserved and noncryopreserved C57B6/JxCBA-F1 embryos was similar (P > 0.05); however, noncryopreserved CBA embryos resulted in a higher blastocyst formation than controls (P < 0.05). While there was no difference in the percentage of transgenic fetuses between cryopreserved and control CBA embryos (P > 0.05), cryopreserved C57B6/JxCBA-F1 embryos resulted in lower transgenic fetuses than control (P < 0.05). These results indicate that the use of cryopreserved mouse pronuclear embryos can be a useful and efficient approach to the production of Tg mice.

Animals↗
PubMedDate not supplied

Antigenicity of cryopreserved arterial allografts: comparison with fresh and glutaraldehyde treated grafts.

The use of cryopreserved aortic allografts in cardiovascular surgery is widespread and has resulted in excellent outcomes. However, it is controversial whether cryopreservation suppresses the antigenicity of tissue. We designed experimental models to study whether the cryopreservation process alters antigenicity in comparison with that found in fresh and glutaraldehyde treated tissues. Fresh, cryopreserved, and glutaraldehyde treated thoracic aorta from Brown Norway rats were subcutaneously implanted into Lewis rats. Inflammatory cells infiltrating around the grafts were measured on days 7, 14, 28, and 56 after implantation. The glutaraldehyde treated grafts showed significantly less infiltration than the fresh or cryopreserved grafts (p < 0.005). No significant difference was detected between the fresh and cryopreserved grafts. Another study examined the effect of modifications of the aortic allograft on subsequent allogeneic skin graft antigenicity. Subcutaneous implantation of fresh, cryopreserved, and glutaraldehyde treated aortic grafts from Brown Norway into Lewis rats resulted in subsequent skin graft rejection at 4.4+/-0.7, 5.1+/-0.8, and 6.6+/-2.1 days, respectively. There was no significant difference between the fresh and cryopreserved groups; whereas skin grafts in the glutaraldehyde group survived longer than those in the cryopreserved group. These results indicate that cryopreservation had no significant influence on antigenic suppression of arterial allografts.

Animals↗
PubMed2000

Cell viability improves following inhibition of cryopreservation-induced apoptosis.

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

Animals↗
PubMedDate not supplied

Analysis of outcomes of cryopreserved surgically retrieved sperm for IVF/ICSI.

We evaluated our experience to date with in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) after either cryopreserved sperm or sperm produced on the date of IVF/ICSI was used. We performed a retrospective statistical analysis of data derived from 188 women undergoing IVF/ICSI cycles using surgically retrieved sperm. A total of 318 IVF/ICSI treatment cycles with 3280 ova were performed using testicular sperm extraction (TESE, 304 cycles) or microsurgical epididymal sperm aspiration (MESA, 14 cycles). Sperm obtained at time of IVF/ICSI (fresh) or thawed cryopreserved sperm samples were used in 38 and 280 of the ICSI cycles, respectively. For IVF/ICSI cycles using both TESE and MESA sperm, the fertilization rate was 59.9% for cryopreserved sperm, and 53.6% when fresh sperm was used (chi2 P-alpha < .02, Cramer's 0.04). The fertilization rate for the TESE group alone was 60.0% for cryopreserved sperm and 55.1% for fresh sperm (chi2P-alpha = .075). Cohen effect size was computed at 0.03; yielding for P-beta = .8, 6597 ova would be required to demonstrate similarity between fresh and cryopreserved sperm in the TESE group. To demonstrate superiority of cryopreserved sperm in this group at a P-alpha significance level of .05, 7524 ova would be necessary. The pregnancy rate for the TESE group was 27.3% for cryopreserved sperm and 27% for fresh sperm. Further analysis of the pregnancy data in this group, using the methods described, yielded a chi2 P-alpha and power of 0.971 (effect size calculated at 0.002). While our fertilization rates for cryopreserved sperm are greater in analyses of surgically derived sperm, based on the 7 years required to obtain data on 3280 ova, full numerical resolution of the issue of whether cryopreserved sperm is superior or similar will not be available until approximately 2010. However, we believe these results, along with the similarity shown in pregnancy rates achieved with both types of sperm, clearly indicate that cryopreserved sperm is not inferior to fresh sperm.

Cryopreservation↗
PubMed2004

Long-term function of cryopreserved rat hepatocytes in a coculture system.

The goal of this study was to investigate postpreservation long-term function of cryopreserved primary rat hepatocytes using the hepatocyte/3T3-J2 fibroblast coculture system. The long-term function of thawed hepatocytes cocultured with fibroblasts was evaluated and compared with hepatocytes cultured without fibroblasts. Fresh isolated primary rat hepatocytes were frozen at a controlled rate (-1 degrees C/min) up to -80 degrees C, and then stored in liquid nitrogen for up to 90 days. Thawed hepatocytes were thereafter cocultured with 3T3-J2 murine fibroblasts and cocultivation was monitored for 14 days. The viability of fresh isolated hepatocytes was 91.4%, and that of cryopreserved hepatocytes was 82.1%. Cellular morphology and polarity, which were determined by the localization of actin filaments and connexin-32, were successfully maintained in cryopreserved hepatocytes following cryopreservation. Albumin and urea synthesis reached the maximum level and became stable after day 7 in coculture in both fresh and cryopreserved hepatocytes. Urea synthesis of cryopreserved hepatocytes was maintained 89.0% of nonfrozen fresh control, and albumin production of cryopreserved hepatocytes was 63.7% of control in coculture. Cytochrome P450 activity, which was measured by deethylation of ethoxyresorufin, was also maintained in cryopreserved hepatocytes at 88.6% of nonfrozen fresh control in coculture. The retention of synthetic and detoxification activities was verified to be well preserved during extended low-temperature storage (90 days). Both fresh control and cryopreserved hepatocytes cultured without fibroblast did not retain their synthetic and detoxification functions in long-term culture. These data illustrate that, through the utilization of our cryopreservation procedure, primary hepatocyte function was successfully maintained when placed into coculture configuration following thawing.

Actin Cytoskeleton↗
PubMed2000

Clinical implications of intracytoplasmic sperm injection using cryopreserved testicular spermatozoa from men with azoospermia.

OBJECTIVE: To investigate whether sperm obtained by testicular sperm extraction (TESE) and cryopreserved well before intracytoplasmic sperm injection (ICSI) can serve as an effective sperm source. STUDY DESIGN: The role of cryopreserved testicular spermatozoa was evaluated in a retrospective analysis of consecutive ICSI cycles using fresh or cryopreserved sperm; they were followed by prospective, planned treatment using cryopreserved sperm with a modified ICSI procedure. Sixteen men (22 cycles) with obstructive or nonobstructive azoospermia were included in the retrospective analysis. Another 25 men (29 cycles) were in the planned treatment group. Following these series, the pregnancy outcomes were compared between ICSI cycles with fresh or cryopreserved testicular sperm. RESULTS: In the retrospective phase, 14 ICSI cycles were performed using fresh sperm, with 8 using cryopreserved sperm. There were no statistically significant differences between the two groups in any outcome measure. Planned treatment with cryopreserved sperm resulted in a fertilization rate of 84% and an embryo transfer rate of 89%. Thirteen couples (44%) achieved pregnancy (five ongoing, six delivered). These rates were similar to those in the retrospective phase of the study. All couples in the planned cryopreservation group had multiple aliquots (6.5 +/- 2.1) of sperm remaining after the first cycle. CONCLUSION: Cryopreserved sperm obtained by TESE can be used as an effective sperm source in ICSI cycles. Planned cryopreservation allows multiple aliquots to be stored for use in subsequent cycles and thus avoids the need for repeat biopsies.

Adult↗
PubMed2002

Cryopreservation of osteochondral allografts: dimethyl sulfoxide promotes angiogenesis and immune tolerance in mice.

BACKGROUND: Although transplantation of cryopreserved bone allografts has become a routine procedure in orthopaedic surgery, biological and immunological impairment remains an unsolved problem that causes clinical failures. Experimental and clinical evidence has indicated that bone grafts that are revascularized early remain viable and contribute to union at the recipient site. Unprotected cryopreservation, used in most bone banks to reduce graft antigenicity, is associated with complete loss of graft viability, potentially contributing to graft failure. The differences in the survival of various cell types during cryopreservation with use of dimethyl sulfoxide, particularly the increased sensitivity of leukocytes to fast freezing, has resulted in a new approach to modulate immunogenicity. On the basis of this concept, it was proposed that a reduction in the immune response and enhanced revascularization of osteochondral allografts could be achieved by rapid cryopreservation with dimethyl sulfoxide. To test this hypothesis, angiogenesis and immune tolerance were quantified in a murine model with use of intravital microscopy. METHODS: Fresh osteochondral tissue and osteochondral tissue that had been cryopreserved with and without dimethyl sulfoxide was transplanted into dorsal skinfold chambers as isografts and as allografts in presensitized and nonsensitized recipient mice. To quantify angiogenesis, the onset of hemorrhages in the vicinity of the grafts and the revascularization of the grafts were determined by means of intravital fluorescence microscopy. To determine the recipient's intravascular immune response to the grafts, the leukocyte-endothelium interaction was assessed on the twelfth day after transplantation. RESULTS: Nine of nine fresh isografts were revascularized at a mean (and standard deviation) of 57 +/- 33 hours, eight of nine isografts that had been cryopreserved with dimethyl sulfoxide were revascularized at 98 +/- 50 hours, and zero of nine isografts that had been cryopreserved without dimethyl sulfoxide were revascularized. Seven of seven fresh allografts were revascularized at 53 +/- 6 hours, and ten of ten allografts that had been cryopreserved with dimethyl sulfoxide were revascularized at 82 +/- 29 hours. However, signs of revascularization faded in four of the seven fresh allografts whereas reperfusion was maintained in the majority (seven) of the ten grafts frozen in the presence of dimethyl sulfoxide. Similar to the findings associated with unprotected frozen isografts, zero of ten unprotected frozen allografts were revascularized. None of the allografts that had been transplanted into presensitized recipients were revascularized, regardless of whether they had been implanted fresh (nine grafts) or had been implanted after protected (eight grafts) or unprotected (nine grafts) freezing. Quantification of the leukocyte-endothelium interaction revealed a reduction in the intravascular immune response to frozen allografts (both protected and unprotected) compared with fresh allografts. CONCLUSION: Osteochondral allografts that had been pretreated by cryopreservation with dimethyl sulfoxide demonstrated improved angiogenesis induction and enhanced immune tolerance compared with unprotected frozen grafts. A selective reduction in donor passenger leukocytes is the proposed mechanism underlying this phenomenon. CLINICAL RELEVANCE: In the absence of presensitization, cryopreservation with dimethyl sulfoxide appears to reduce the immune response to allografts and to enhance their revascularization; in the presence of presensitization, alternatives to allograft transplantation should be considered since the allografts will be exposed to a deleterious immune response.

Animals↗
PubMedDate not supplied

Effect of benzyladenine on recovery of cryopreserved shoot tips of grapevine and citrus cultured in vitro.

The effect of N6-benzyladenine (BA) on the recovery of cryopreserved shoot tips of the LN33 hybrid (Vitis L.) and Troyer citrange [Poncirus trifoliata (L.) Raf. x Citrus sinensis [L.] Osbeck.] cultured in vitro was examined. For the LN33 hybrid, the presence of BA in the recovery medium was essential for survival of control and cryopreserved shoot tips, although the BA concentration did not influence the survival percentage. BA at 5, 2, and 5 microM or higher induced callus formation in control, and shoot tips cryopreserved by vitrification, and by encapsulation-dehydration, respectively. While a BA concentration of 4 microM was found optimal for recovery of control shoot tips, 1 and 2-4 microM produced the best recovery of shoot tips cryopreserved by vitrification and encapsulation-dehydration, respectively. A similar pattern of effect of BA on recovery was found for 'Troyer' citrange. Low survival of control and cryopreserved shoot tips was observed with a BA-free recovery medium. The addition of BA to the recovery medium significantly increased survival. The BA concentration that induced callus formation in shoot tips cryopreserved by encapsulation-vitrification was higher than that which induced it in those cryopreserved by encapsulation-dehydration. Recovery of control shoot tips was best with an addition of 6-10 microM BA to the medium. Optimal recovery of shoot tips cryopreserved by encapsulation-vitrification and encapsulation-dehydration was achieved with 3-4 and 2 microM BA, respectively. Results from the present study suggest that an optimal BA concentration for recovery of control shoot tips may be different from that for cryopreserved shoot tips; furthermore, the optimal BA concentration for recovery of cryopreserved shoot tips may also differ among different cryogenic procedures.

Adenine↗