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At least 19 recordsLinked to original sources
PubMed2005

Normal birth after transfer of cryopreserved human embryos generated by microinjection of cryopreserved testicular spermatozoa into cryopreserved human oocytes.

OBJECTIVE: To report the first birth after transfer of cryopreserved embryos generated by intracytoplasmic sperm injection of cryopreserved testicular spermatozoa into cryopreserved human oocytes. DESIGN: Case report. SETTING: Tertiary center for reproductive technology. PATIENT(S): A 36-year-old woman with primary infertility of 3 years' duration and a 37-year-old man with congenital bilateral absence of the vas deferens. INTERVENTION(S): Cryopreservation of human embryos after oocytes and sperm thawing. MAIN OUTCOME MEASURE(S): Live birth. RESULT(S): A healthy, normal female infant with a birth weight of 2,950 g was born by cesarean section at 38 weeks' gestation, with normal 6-month follow-up. CONCLUSION(S): Embryo cryopreservation can lead to successful results, even with the use of cryopreserved gametes.

Adult↗
PubMed2007

Live birth following transfer of a cryopreserved embryo generated from a cryopreserved oocyte and a cryopreserved sperm: case report.

As is the case with non-frozen oocytes, the efficient and successful use of cryopreserved oocytes in human assisted reproduction is, in part, dependent on the ability to apply selection criteria when choosing the 'best' embryos for transfer from a cohort. In many cases this, in turn, will necessitate the cryopreservation of non-transferred embryos to minimise the risk of multiple pregnancy. It is therefore important to establish that an embryo, generated by fertilization of a frozen-thawed oocyte, can be capable of surviving subsequent cryopreservation while retaining the potential for normal development. In this case report, we document the delivery of a normal male infant following transfer of a frozen-thawed embryo, generated by the fertilization of a frozen-thawed oocyte by a frozen-thawed sperm.

Adult↗
PubMed2006

[Cryopreserved human heart cell transplantation--effect of cryopreservation on cell proliferation and immunogenicity].

UNLABELLED: We studied the possibility that cryopreserved human heart cells could be transplanted with some advantage. METHOD: Cells derived human atrial tissues (control group (n = 13)), cryopreserved cells (cell-cryopreservation group (n = 23), and cells derived cryopreserved tissue (tissue-cryopreservation group (n = 29)) were cultured for 15 days. The cell proliferation was compared between control and cryopreservation group by growth curves. BasicFGF, TGFbeta-1, IL-6, IL-8, and cell cycle were measured at pre and post-cryopreservation. Mixed Lymphocyte-heart cell culture established by PBL and heart cells was evaluated by PBL proliferation. Transplanted cells were evaluated by visually and histology. RESULTS: Cryopreserved cells were proliferated much more than control cells (p < 0.0001). The growth factors were increased, and cytokines were decreased by cryopreservation (p < 0.05). The cryopreserved cell cycles were shifted to G2 + M from G1 + G0 period. Cryopreserved cells stimulated PBL less than non-cryopreserved cells (p < 0.0001). Transplanted cryopreserved cell were survived. CONCLUSION: Cryopreserved human heart cells can be transplanted, and proliferated much more than non-cryopreserved cells. Cryopreservation enables the human cells to be more prolific, and reduced the immunogenicity of them. The transplanted cryopreserved cells survived and formed cardiac-like tissue.

Animals↗
PubMed1999

Cryopreservation of specific pathogen-free (SPF) pig islet cells: effect of culture time before cryopreservation and after thawing.

UNLABELLED: The aim of this study was to determine the optimal conditions (effect of culture time before and after cryopreservation) for cryopreservation of specific pathogen-free pig islet cells. METHODS: (1) Glucose-induced insulin secretion by fresh islet cells cultured for 10 days was compared to that by islet cells cryopreserved 7 days after isolation and cultured 3 days after thawing. (2) Islet cells were cryopreserved 1, 7, or 14 days after isolation and cultured 3, 7, 14, or 21 days after thawing. Islet cell number, insulin content, and insulin response under perifusion tests were investigated. RESULTS: (1) Insulin response by cryopreserved islet cells was identical to that by fresh islet cells (basal/stimulation index: 2. 13 +/- 0.19 vs 2.17 +/- 0.16, n = 4, NS), although the amount of secreted insulin was reduced by 40% (area under the curve: 2136 +/- 198 pM/10(4) cells/180 min vs 3564 +/- 636 pM/10(4) cells/180 min, P = 0.104). (2) Cell number 6 days after thawing was reduced by 54, 40, and 63% when cryopreservations were carried out at D1, D7, and D14. (3) Insulin content in cultured or cryopreserved islet cells increased between 7 and 14 days of culture. (4) Whatever the culture time before and after cryopreservation, insulin secretion in response to glucose was maintained. The insulin release was the highest for islet cells cryopreserved 14 days after isolation and cultured 14 days after thawing (stimulation index: 6.19 +/- 2.68). CONCLUSIONS: SPF pig islet cells remained functional after cryopreservation in polyethylene glycol and it may be important to culture islet cells over 14 days before and after cryopreservation.

Animals↗
PubMed2004

The viability and function of cryopreserved hepatocyte spheroids with different cryopreservation solutions.

We sought to evaluate the viability and function of cryopreserved hepatocyte spheroids using different cryopreservation solutions in order to elucidate the efficiency of cryopreservation. Hepatocytes isolated from a Sprague-Dawley rat were formed into spheroids by 24 hours of rotational culture. The spheroids were cryopreserved using a programmed linear freezer in a liquid nitrogen tank for 24 hours in four different cryopreservation solutions: UW solution (UW), William E media (WE), fetal bovine serum (FBS), and a mixture (MIX). After thawing, they were cultured for 4 days. With each hepatocyte spheroid, the viability using the MTT assay and hepatocyte-specific functions, such as ammonia clearance, urea nitrogen synthesis, and albumin secretion, were analyzed. The viabilities of cryopreserved hepatocyte spheroids were 64.8% +/- 10.2% (UW), 33.2% +/- 9.7% (WE), 69.3% +/- 8.7% (FBS), and 48.4% +/- 15.5% (MIX). Ammonia clearance of spheroids cyropreserved in UW solution was 0.93 +/- 0.13 mmol/L per well per day, which was not significantly different from freshly cultured spheroids. From the aspect of urea nitrogen synthesis, spheroids cryopreserved with UW, FBS, and MIX solution were not significantly different from freshly cultured spheroids. The amount of albumin secretion by the UW cryopreserved spheroids was significantly greater than that of other cryopreserved spheroids. Cryopreserved hepatocyte spheroids in UW solution were not significantly different from freshly cultured spheroids with respect to viability and function. UW solution was superior to other cryopreservation solutions for viability and functions.

Animals↗
PubMed2004

Cryopreservation of viable hepatocyte monolayers in cryoprotectant media with high serum content: metabolism of testosterone and kaempherol post-cryopreservation.

Little work in the literature focuses on the cryopreservation of primary hepatocytes as monolayer cultures, yet this technique offers many distinct advantages over other cryopreservation systems, including high recovery, high post-thaw nutrient penetration, and low numbers of trapped dead cells. This article investigates the cryopreservation of primary rat hepatocytes at -78 degrees C attached as monolayers to collagen coated culture dishes, and describes efforts to increase post-thaw viability and function through manipulation of the freeze/thaw protocol. Different concentrations of foetal calf serum (FCS) with 10% (v/v) dimethyl sulphoxide (ME2SO) were tested as cryopreservation media, and high cryoprotectant serum levels were found to be important in maintaining membrane integrity and function in the cryopreserved rat hepatocyte monolayer cultures. Cultures cryopreserved with 90% (v/v) FCS plus 10% (v/v) ME2SO maintain 79.7+/-6.5% of the monolayer area as viable cells with normal morphology (by image analysis), 112.7+/-14.2% protein concentration, 55.4+/-4.2% carboxyfluorescein diacetate de-acetylation, 27.2+/-7.5% kaempherol glucuronidation (a measure of UDP-glucuronosyl transferase activity), and 39.3+/-7.3% testosterone hydroxylation (a measure of cytochrome P-450 activity) compared with non-cryopreserved controls. This method of cryopreservation may provide a simple, convenient means of long-term storage of hepatocytes for in vitro metabolism studies.

Animals↗
PubMed2000

Allogenicity of cryopreserved human fibroblasts: cryopreservation does not downregulate the allogenicity of fibroblasts making up the matrices of allografts.

BACKGROUND: Although cryopreserved tissue allografts are being widely used, long-term degeneration of implanted cryopreserved allografts has become a problem. Although immunologic rejection has been suggested to play a part in this degeneration, cryopreserved allografts are considered to be less immunogenic than fresh allografts. OBJECTIVE: We investigated the effect of cryopreservation on the allogenicity of the fibroblasts that make up the matrices of allografts. METHODS: Fibroblast cell strains obtained from surgically resected lung specimens were used. Allogenicity-related antigens expressed on the cell surface (human leukocyte antigen 1, human leukocyte antigen 2, and intercellular adhesion molecule 1), stimulation indices during 1-way mixed lymphocyte-fibroblast cell culture, and proliferation indices of freshly passaged fibroblasts and cryopreserved fibroblasts stored for 1, 4, and 24 weeks were examined. Flow cytometric analysis with monoclonal antibodies was used to test for cell surface antigens, and a colorimetric methyl-thiazol-diphenyl-tetrazolium assay was used to assess stimulation indices and fibroblast proliferation indices. The effect of exogenous interferon-gamma on the degree of expression of human leukocyte antigen 1, human leukocyte antigen 2, and intercellular adhesion molecule 1 was examined simultaneously. RESULTS: The proliferation indices of fibroblasts were well maintained by cryopreservation. Expression of human leukocyte antigen 1, human leukocyte antigen 2, and intercellular adhesion molecule 1 by fibroblasts was significantly upregulated by interferon-gamma, and cryopreservation did not downregulate this expression. CONCLUSION: Our study suggests that although the fibroblast cell component may be beneficial in restoring allograft function properties initially, it may render the implanted allograft more immunogenic, ultimately resulting in greater rejection and inflammatory responses by the host and, in turn, degeneration of the graft.

Analysis of Variance↗
PubMed2001

Successful transplantation and engraftment of peripheral blood stem cells after cryopreservation, positive and negative purging procedures, and a second cryopreservation cycle.

Transplantation of peripheral blood stem cells (PBSC), positively and/or negatively selected immediately after harvest, has become a widely applied therapeutic option in hematological or oncological patients. The following case of peripheral blood stem cell transplantation represents the first case of successful transplantation of PBSC, cryopreserved twice and purged after cryopreservation. PBSC were harvested in a 44-year-old female patient with a low-grade non-Hodgkin's lymphoma stage IV after mobilization with chemotherapy and G-CSF. A total number of 15.2 x 10(6) CD34+ cells/kg bodyweight was harvested with a 36.9% contamination of tumor cells coexpressing CD5 and CD20. After subsequent chemotherapy cycles and cyclophosphamide mobilization, only 0.77 x 10(6) CD34+ cells/kg bodyweight, not sufficient for transplantation, were achieved after positive selection. Therefore, 10.8 x 10(6) cryopreserved CD34+ cells/kg bodyweight were thawed and a positive selection was carried out with the BAXTER Isolex 300i machine. Before additional negative selection, the 0.77 x 10(6) positively selected CD34+ cells/kg bodyweight from the second mobilization were added. A total quantity of 4.4 x 10(6) CD34+ cells/kg bodyweight with a purity of 93.1% representing a recovery of 38% was obtained. Cells were again cryopreserved, stored and retransfused after conditioning the patient with TBI and high-dose cyclophosphamide. The patient engrafted with a WBC count > 1000/microliter on day eight and a platelet count > 20,000/microliter without transfusion support on day 12 post-transplantation. This case indicates that purging procedures can successfully be carried out with cryopreserved cell material and that purified CD34+ cells can be cryopreserved a second time before transplantation, without affecting their hematopoietic capacity.

Adult↗
PubMed2004

Human oocyte cryopreservation: a valid alternative to embryo cryopreservation?

Embryo cryopreservation has become an ethical necessity due to the way human in vitro fertilization (IVF) infertility therapy has developed. Limited embryonic implantation has by necessity driven IVF therapy to adopt ways to maximize the harvest of oocytes following ovarian hyperstimulation with its attendant risks. Collection of more oocytes has allowed more embryos to be generated to compensate for poor embryonic viability, often leading to transfer of multiple embryos to increase per transfer pregnancy rates. In an era of improving embryonic viability and prevailing trend toward single embryo transfers, production of excessive numbers of surplus embryos appears increasingly inappropriate. At which stage embryo cryopreservation can be undertaken most effectively remains controversial. Embryo cryopreservation nevertheless represents the current solution to the problem of excessive embryo production, but inherently raises ethical concerns for certain couples uncomfortable with what they might perceive to be "experimental" cryostorage, who in extreme circumstances may even choose to limit the number of oocytes inseminated to obviate the production of spare embryos. On a more practical level, cryostored embryos are co-owned by two people who may separate, and as such the embryos then face an uncertain fate, commonly decided in courts of law. Oocyte cryopreservation, if consistent and successful, offers a way to avoid the above complications of routine IVF therapy. Oocytes may need to be cryostored in the event of unforeseen non-production of sperm during IVF therapy, allowing a more measured consideration of donor sperm use or other means of sperm retrieval. Beyond IVF for infertility therapy using a couple's own gametes, oocyte cryopreservation provides a wonderful opportunity to optimize donor oocyte cryo-banking, reducing costs and improving convenience. Meanwhile, frozen embryo donation is an approach that many couples are uncomfortable with, and allows only for retrospective donor screening, and less gametic choice. Advances in ovarian tissue cryopreservation will probably provide the best approach for long term storage of female gametes for women wishing to elect to prolong their reproductive potential, or prior to cancer therapy. However, improved consistency with vitrification technology through standardization of protocols and cell-carrying systems is bringing routine single oocyte cryostorage, at all stages of egg maturity, closer to reality. This, coupled with in vitro maturation, will aid development of oocyte collection protocols using minimal amounts of gonadotropins. All of which will help drive IVF programs to consider cryostorage of excess oocytes and not embryos, inseminating post-thaw/warming only a limited number of oocytes at any one time, in anticipation of the need for only one or two embryos at transfer. The question then is how close are we to being able to provide routine clinical application of human oocyte cryostorage, and when will it be appropriate?

Cryopreservation↗
PubMed2002

The added value of embryo cryopreservation to cumulative ongoing pregnancy rates per IVF treatment: is cryopreservation worth the effort?

PURPOSE: To calculate the added benefit of a cryopreservation program to the cumulative ongoing pregnancy rate over a maximum of three cycles of IVF. METHODS: A total of 1251 couples beginning their first IVF treatment between January 1995 and December 1999 were evaluated. Ongoing pregnancies from fresh and subsequent cryopreserved embryo transfer cycles were analyzed. Pregnancies arising from the cryopreservation cycle were considered to augment the cumulative pregnancy rate when no ongoing pregnancy arose from the fresh embryo transfer cycle. RESULTS: The ongoing pregnancy rate per cryopreserved embryo transfer was 11.7%. The cumulative ongoing pregnancy rate following three successive started fresh IVF cycles was 42.5%. When pregnancies arising from the transfer of thawed cryopreserved embryos were included, the cumulative ongoing pregnancy rate increased to 43.8%, rising to 44.8% when extrapolated data from as yet unthawed embryos was included. CONCLUSIONS: When analyzed in these terms, the supplementary benefit of cryopreserving supranumerical embryos appears limited.

Adult↗
PubMed1998

Long-term cryopreservation: successful trilineage engraftment after autologous bone marrow transplantation with bone marrow cryopreserved for seven years.

Successful autologous bone marrow transplantation (ABMT) and peripheral blood stem cell transplantation depend on safe hematopoietic stem cell (HSC) cryopreservation and storage. Several successful methods for cryopreservation and storage have been established and are commonly used all over the world. However, little is known about the effects of long-term cryopreservation on the capacity to sustain a complete immunohematopoietic engraftment. Several authors have investigated stem cell viability after cryopreservation and storage for more than 5 years and reported preclinical good viabilities in terms of dye-exclusion or colony-forming capability in vitro. Only two studies using BM cryopreserved for more than 5 years for transplantation are reported, but they did not provide proof of trilineage engraftment. In February 1997 at our institution, a patient with relapsed non-Hodgkin's lymphoma underwent ABMT with BM harvested in February 1990. He achieved a granulocyte count > 500 x 10(6)/L on day 21 and a self-supporting platelet count > 20 x 10(9)/L on day 30. After day 29, his hemoglobin level was satisfactory without need of transfusion support. This successful trilineage engraftment with cryopreserved BM that had been stored for 7 years suggests that HSC are able to maintain their capability to reconstitute hematopoiesis for a long time.

Bone Marrow↗
PubMed1999

Development of optimal techniques for cryopreservation of human platelets. I. Platelet activation during cold storage (at 22 and 8 degrees C) and cryopreservation.

Using the current blood bank storage conditions at 22 degrees C, the viability and function of human platelets can be maintained for only 5 days. This does not allow for the necessary and extensive banking of platelets needed to treat patients afflicted with thrombocytopenia, a side effect of many invasive surgeries such as cardiopulmonary bypass or bone marrow transplantation. The development of optimal techniques for long-term cryopreservation and banking of human platelets would provide the ability to greatly extend the viable life of the platelet and would fulfill an increasing and urgent need in many clinical applications. To determine the optimal techniques for platelet preservation, the expression of an activation marker, phosphatidylserine, on the platelet membrane during storage at 22 and 8 degrees C as well as during the different freezing preservation processes was examined using flow cytometry and annexin V binding assay. Human platelets were identified by both CD41 and light scatter in flow cytometry. In cryopreservation experiments, effects of the following factors on platelet activation were evaluated: (a) cryoprotective agents (CPAs) type: dimethyl sulfoxide (Me2SO), ethylene glycol (EG), and propylene glycol (PG), (b) CPA concentration ranging from 0 to 3 M, and (c) ending temperatures of a slow cooling process at -1 degrees C/min. Our results demonstrated that (a) approximately 50% of platelets were activated on days 7 and 16 at 22 and 8 degrees C, respectively; (b) platelets were not significantly activated after 30-min exposure to 1 M Me2SO, EG, and PG at 22 degrees C, respectively, and (c) there was a significant difference in cryoprotective efficacy among these three CPAs in preventing platelets from cryoinjury. After being cooled to -10 degrees C, 74% of the cryopreserved platelets survived (nonactivated) in 1 M Me2SO solution, while in 1 M EG and 1 M PG solutions, 62 and 42% of the platelets survived, respectively. Using the information that Me2SO consistently yields higher percentages of nonactivated platelets and does not seem to be cytotoxic to platelets for 30-min exposure time, this was found to be the optimal cryoprotective agent for platelets. In addition, significant Me2SO toxicity to platelets was not noted until Me2SO concentrations exceeded 2 M. Finally, a concentration of 1 M Me2SO proved to be the most effective at all cryopreservation ending temperatures tested (-10, -30, -60, and -196 degrees C). In conclusion, under the present experimental conditions, a storage temperature of 8 degrees C appeared to be much better than 22 degrees C. Although the potential chemical toxicity of 1 M Me2SO, EG, or PG is negligible, 1 M Me2SO was found to be optimum for cryopreservation of human platelets. PG has the least cryoprotective function for low-temperature platelet survival.

Blood Platelets↗
PubMed1999

Platelet cryopreservation using a reduced dimethyl sulfoxide concentration and second-messenger effectors as cryopreserving solution.

Cryopreservation of platelets is of great interest since it could extend to years the shelf life of therapeutic platelet concentrates (PCs) and facilitate stockpiling and inventory control in blood banking. We have compared the cryopreservation of PCs by the standard method using 6% Me(2)SO as cryoprotectant with the method of freezing employing low concentrations of Me(2)SO (2%) plus ThromboSol, a mixture of second-messenger effectors that protect platelets from cold damage. PC pools were treated either with 6% Me(2)SO or with ThromboSol and 2% Me(2)SO and then placed directly in a -80 degrees C freezer or in the vapor phase of a liquid nitrogen freezer (-120 degrees C). After storage for 1 week or for 3 months, samples were removed, thawed, and analyzed. Measurements included cell recovery, biochemical parameters, membrane glycoproteins (GPs), platelet aggregation, and binding of radiolabeled von Willebrand factor (vWF) and fibrinogen. PCs cryopreserved with ThromboSol and 2% Me(2)SO displayed a platelet recovery (90%) equivalent to those frozen with 6% Me(2)SO. Following either cryopreservation procedure, platelets showed increased surface expression of P-selectin and moderate loss of GP Ibalpha in comparison to fresh platelets. The aggregatory response to ristocetin and the binding of vWF were similar in platelets frozen by either procedure. Finally, both methods promoted comparable impairment of the reactivity of platelets to thrombin, aggregation and binding of fibrinogen and vWF, compared to that of fresh platelets. In summary, cryopreservation of PCs using reduced Me(2)SO concentration and ThromboSol yields platelets with in vitro functional characteristics equivalent to those of cells frozen with the conventional method using 6% Me(2)SO.

Blood Platelets↗
PubMed2004

Cryopreservation of human spermatozoa: comparison of two cryopreservation methods and three cryoprotectants.

OBJECTIVE: To evaluate the ability of two cryopreservation methods and three cryoprotectants to preserve sperm quality. DESIGN: A prospective clinical study. SETTING: Male infertility clinic at a tertiary healthcare center. PATIENT(S): Twenty infertile men and 10 healthy donors. INTERVENTION(S): In the first experiment, semen was cryopreserved by either the Irvine Scientific method (IS) or the Cleveland Clinic Foundation (CCF) method. In the second experiment, semen was cryopreserved by the IS method and one of three cryoprotectants: TES and Tris yolk buffer, Sperm Freezing Medium, or Enhance Sperm Freeze. MAIN OUTCOME MEASURE(S): Postthaw sperm motility, cryosurvival, and kinematics. RESULT(S): Percentages of postthaw sperm motility and cryosurvival were higher in the IS cryopreservation method compared with in the CCF method (15.94 +/- 9.19 vs. 12.07 +/- 7.31 and 47.42 +/- 17.44 vs. 35.76 +/- 17.56). However, the CCF method resulted in significantly better sperm kinematics. Postthaw motility in the donors and patients was highest in the samples frozen in TES and Tris yolk buffer medium. CONCLUSION(S): The IS method was associated with more flash freezing compared with the CCF method and resulted in better preservation of sperm motility and a higher cryosurvival rate. TES and Tris yolk buffer was most effective at protecting sperm from the negative effects of the cryopreservation process. This may be due to the presence of egg yolk along with glycerol.

Cryopreservation↗
PubMed2003

Immunogenicity of decellularized cryopreserved allografts in pediatric cardiac surgery: comparison with standard cryopreserved allografts.

BACKGROUND: Recognition of the immunogenicity of standard cryopreserved allografts has led to the development of new decellularized allografts (CryoValve SG; CryoLife, Inc, Kennesaw, Ga). This preliminary study examined the HLA antibody response to these decellularized allografts and compared it with the response to standard allograft material. METHODS: We prospectively measured the frequency of panel-reactive HLA class I (HLA-A, HLA-B, and HLA-C) and class II (HLA-DR/DQ) alloantibodies in 14 children (age 8.5 +/- 7.9 years) receiving decellularized, cryopreserved allografts, including 6 undergoing allograft patch insertion and 8 with a valved pulmonary allograft. We compared them with 20 historical control subjects (age 1.7 +/- 2.4 years) undergoing implantation of standard cryopreserved allografts, 8 with valves and 12 with allograft patch. All patients had panel-reactive antibody levels measured before and at 1, 3, and 12 months after the operation. HLA class I and class II panel-reactive antibody levels were determined with a sensitive flow cytometry technique. RESULTS: We found panel-reactive antibody levels in decellularized allografts to be elevated slightly from preoperative levels for both class I and class II antibodies at 1, 3, and 12 months (P >.05). The panel-reactive antibody level for both class I and class II antibodies were significantly lower for decellularized allografts as compared to standard allografts. Functionally, the allografts were similar with decellularized valved grafts showing a peak echo-determined systolic gradient of 13 +/- 15 mm Hg at 8 +/- 2.6 months postoperatively as compared to a gradient of 24 +/- 18 mm Hg measured 12 +/- 6 months postoperatively in standard allografts (P =.11). CONCLUSIONS: Decellularized grafts elicited significantly lower levels of class I and class II HLA antibody formation at 1, 3, and 12 months after implantation than did standard cryopreserved allografts. Early hemodynamic function of decellularized grafts was similar to that of standard cryopreserved allograft valves. Further experience is necessary to determine whether the reduced immunogenicity of decellularized allografts will truly allow tissue ingrowth and improved long-term durability in patients.

Adolescent↗
PubMed1998

Live mice from cryopreserved embryos derived in vitro with cryopreserved ejaculated spermatozoa.

This study was undertaken to try to reduce the number of animals required to maintain mouse strains by banking of embryos or spermatozoa. The principal objective was to cryopreserve ejaculated mouse spermatozoa, using a method recently developed for epididymal spermatozoa. Within 30 min after mating, ejaculated spermatozoa were flushed from the uterus of mated females; shortly afterwards, epididymal spermatozoa were also collected from the same males that had mated with the females. The average values for spermatozoal motility and viability of ejaculated specimens of nine males were 43 and 46%, respectively, and for epididymal specimens, the corresponding values were 60 and 52%. In experiment 1, ejaculated or epididymal spermatozoa were incubated with oocytes for 0.5 to 4 h. As evidenced by development into two-cell embryos within 24 h, kinetics of fertilization of the two spermatozoa types were similar. In experiment 2, ejaculated and epididymal spermatozoa of three males were separately cryopreserved in medium containing raffinose, glycerol, and egg yolk. Samples were cooled and seeded at -4 degrees C, cooled to -70 degrees C at 20 degrees C/min, and then were placed into liquid nitrogen for storage. When cryopreserved epididymal or ejaculated spermatozoa were thawed at > 1,000 degrees C/min and used for in vitro fertilization, > 60% of oocytes cleaved, and approximately 95% of cleaved embryos developed into morulae or blastocysts. When embryos produced with cryopreserved spermatozoa were transferred into recipients, 18 and 22 live pups were obtained from 62 and 54 embryos resulting from ejaculated or epididymal spermatozoa, respectively. This study documented the feasibility of cryopreserving ejaculated spermatozoa as an effective alternative to preserving germ plasm from genetically valuable mice.

Animals↗
PubMed1999

[The improvement on related condition of corneal cryopreservation and roles of cryopreserved corneas in emergent keratoplasty].

PURPOSE: To study the cryopreserved corneal endothelium viability on the condition that a series of related factors on long-term corneal cryopreservation were modified. The frozen corneas were used for emergent penetrating keratoplasty on poor condition patients. To evaluate the role of cryopreserved corneas on clinical application, especially on emergent keratoplasty. METHODS: Residual peripheral corneas were stained with Trypan blue and Alizarin red in order to assess endothelium survival rate after central grafts were punched. Specular microscope was used to measure corneal thickness and count endothelium density. The transparent rate of grafts, infective control rate, retinal restorative rate and the recovery degree of visual acurity were emphasized respectively. RESULTS: Endothelium survival rate of grafts was 82.1% averagely. Postoperative endothelium density was 1,642 cells/mm2 and graft thickness was 0.59 mm averagely. The epithelial defects healed after 2-5 days of the operation and edematous grafts were reclear after 2-4 weeks of the operation in all grafts, except for chemical burn and thermal burn. The transparent rate was 87.5%. Infective controll rate was 90.63%. Retinal restorative rate was 55.56%. Some useful visual acurity can be obtained when the infection was under controlled and the retinal was reattached. CONCLUSION: Modified long-term corneal cryopreservation is effective in maintaining endothelium viability and integrity. Furthermore, it could provide donors anytime because of it's unlimited preservative duration. Our study emphasizes especially the role of cryopreserved corneas in emergent keratoplasty.

Adolescent↗
PubMed1997

Cryopreservation of mouse spermatozoa. II. Relationship between survival after cryopreservation and osmotic tolerance of spermatozoa from three strains of mice.

A procedure to cryopreserve mouse spermatozoa has been derived to bank the genetics of valuable strains of mice in a practical way. The primary objective of this study was to apply the cryopreservation method developed for spermatozoa of strain B6D2F1 to those of strains 129/J and C57BL6/J. Using the capability of spermatozoa to fertilize oocytes in vitro as the criterion of survival, we found differences in survival after cryopreservation among the three strains. Blastocysts were obtained after in vitro fertilization of oocytes with frozen spermatozoa from B6D2F1 (51%) and 129/J (12%); none was obtained from C57BL6/J. Transfer of embryos into recipients resulted in the birth of 69 live pups from 164 embryos produced with frozen B6D2F1 spermatozoa and 11 pups from 35 embryos produced with 129/J spermatozoa. To seek an explanation of these differences among the three strains, spermatozoa were exposed to anisotonic solutions ranging from 5 to 3200 mOsm; viability of spermatozoa was assessed by a double stain using flow cytometry. Mouse spermatozoa tolerated exposure to solutions of osmolalities between 200 and 400 mOsm, but were damaged when exposed to solutions exceeding this range. Spermatozoa from C57BL6/J were the most sensitive: 20, 35, and 40% of C57BL6/J, 129/J, and B6D2F1 spermatozoa survived exposure to an 800 mOsm solution, respectively. This study suggests that there is a genetic basis for sensitivity of mouse spermatozoa to osmotic shock and freezing injury. Nevertheless, the birth of live pups produced with frozen spermatozoa from 129/J as well as with spermatozoa from B6D2F1 mice indicates that cryopreservation of spermatozoa can be used to preserve the genetics of valuable strains of mice.

Animals↗