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PubMed2002

Histological and immunohistological study of cryopreserved aortic valve grafts: the possibility of a clinical application for cryopreserved aortic valve xenograft.

The influence of preservation techniques on xenogeneic immune responses was examined to investigate the possibility of their clinical use. Mongrel dogs and pigs were used as allogeneic and discordant xenogeneic donors of aortic valve grafts, respectively. The allografts and xenografts were implanted in the abdominal aorta of mongrel dogs after cryopreservation or after fresh preservation. Allografts and xenografts were explanted at Days 7 and 42 for histological and immunohistochemical examination. In the fresh preserved allografts and the fresh preserved xenografts, the media of aortic wall grafts were significantly acellular at 7 days. Significant aortic medial destruction and neointimal proliferation with significant destruction of the valve leaflets was observed at 42 days. However, no differences were found among the preservation techniques in the xenogeneic grafts. To alleviate the lack of homografts, approaches such as the tissue-engineered graft may become an alternative in patients who are acceptable candidates.

Animals↗
PubMedDate not supplied

[Indications for the cryopreservation of ovarian tissue. Study Group for the Cryopreservation of Ovarian Tissue].

A number of questions should be envisioned before proposing the indications for clinical use of ovarian tissue cryopreservation (OTC). With what goal is ovarian tissue being preserved? To protect ovarian function which includes the ovarian endocrine secretions and the monthly release of a mature oocyte assuring the fertility of the patient; preservation of fertility is in fact the key goal. To whom should OTC be offered? In theory, to all women whose ovarian function is threatened in a physiological, pathological or iatrogenic manner. In practice, the threat to the follicular reservoir occurring with cancer chemotherapy is the best established indication. Other indications may in the future also be deemed appropriate-when human clinical use of OTC will have proven itself effective. For the present, it is preferable to reserve OTC use to patients under 35 years old who, in whatever manner, would have undergone a very severe cutback of their follicular reserve. What quantity of ovarian tissue should be extracted? It seems to us preferable to only remove one ovary, thereby leaving the patients with the possibility of undergoing spontaneous fertility, so that the frozen tissue need only be used if the ovary left in place should lose its function. How should use of the frozen tissue be directed? Among the three theoretic possibilities: of tissue autograft, follicular maturation in vitro, and heterograft, only the autograft appears realizable in the near future. However, presently, pregnancies using this technology have only been obtained in three animal species. For the near future, it appears to us that resort to OTC must remain infrequent and only performed in the setting of clinical research protocols. Priority should be given to continued basic research as many key points of OTC practice still need to be clarified.

Cryopreservation↗
PubMed2005

Rapid cryopreservation of five mammalian and one mosquito cell line at -80 degrees C while attached to flasks in a serum free cryopreservative.

Cell culturing, and the requisite storage of cell lines at ultra-low temperatures, is used in most laboratories studying or using eukaryotic proteomics, genomics, microarray, and RNA technologies. In this study we have observed that A72(dog), CRFK(cat), NB324K(human), MCF7(human), WI38(human), and C636(mosquito) cells were effectively cryopreserved at -80 degrees C while attached to the substratum of 25 cm2 tissue culture flasks. This was accomplished using a serum free crypreservative recently developed by Corsini and co-workers. The technique allows for significant savings of time and money in laboratories that rapidly process numerous cell lines.

Journal Article↗
PubMed1994

[The influence of different cryopreservation buffers on the fertilizing ability of human sperm "in vitro". II. Evaluation of the fertilizing potential of human sperm after cryopreservation].

We tried to define a potential ability of human spermatozoa for fertilization after 3 month preservation of samples in a liquid nitrogen. The functional test were applied: penetration of sperm to cervical mucus ("Penetrak") and sperm penetration to hamster zona-free oocytes. We would like to emphasize that presented way of cryo-preservation in 3 selected media with fast cooling of samples allow to maintain fertilizing ability of spermatozoa and subsequent insemination.

Adult↗
PubMed1986

Cryopreservation of dog platelets with dimethyl sulfoxide: therapeutic effectiveness of cryopreserved platelets in the treatment of thrombocytopenic dogs, and the effect of platelet storage at -80 degrees C.

Dog platelets were frozen with 6% dimethyl sulfoxide at 2-3 degrees C per minute in a -80 degrees C mechanical freezer. The frozen platelets were stored at -80 degrees C for as long as 39 months. After storage at -80 degrees C for less than 1 year, platelet in vitro freeze-thaw-wash recovery values were 70%, and in vivo survival values 1 to 2 hr after transfusion were 40% those of fresh platelets. After 2 years or longer storage, in vitro freeze-thaw-wash recovery values were 60%, and in vivo survival values 1 to 2 hr after transfusion were 20% those of fresh platelets. These results indicate that significant deterioration of the dog platelets occurred between the first and second year of storage at -80 degrees C. Platelets that were stored frozen at -80 degrees C for less than 1 year and washed before transfusion into lethally irradiated thrombocytopenic dogs were hemostatically effective.

Animals↗
PubMed1999

Successful tracheal transplantation using cryopreserved allografts in a rat model.

OBJECTIVES: The purpose of this study was to determine the appropriate cryopreservation period of tracheal allografts based on morphological and immunological findings and to test the possibility of tracheal transplantation in rats using cryopreserved allografts without immunosuppression. METHODS: Morphological and immunological studies were performed to compare the differences between non-cryopreserved grafts and cryopreserved grafts. Orthotopic tracheal transplantation using cryopreserved allografts, non-cryopreserved allografts, and non-cryopreserved autografts was performed and the rejection score of each group was evaluated. RESULTS: Epithelial cells were lost when the grafts were cryopreserved for more than 20 days. Immunohistochemical staining of the trachea revealed that the MHC classII antigen was expressed on normal epithelium. These findings suggest that cryopreservation for more than 20 days decreased the antigeneicity of allografts because of epithelial desquamation. All rats that received allografts cryopreserved for more than 20 days survived until the scheduled sacrifice day. Microscopically, cryopreserved allografts that had been preserved for more than 20 days had a significantly lower rejection score than that of non-cryopreserved allografts (P < 0.05). CONCLUSIONS: We conclude that the appropriate period for cryopreservation of allografts would be 20 days or more, because cryopreservation for more than 20 days depleted epithelium, which possessed the MHC classII antigen. Therefore, a longer period of cryopreservation decreases the antigeneicity of allografts. Rat tracheal transplantations using cryopreserved allografts is possible without immunosuppression when the grafts have been cryopreserved for more than 20 days.

Animals↗
PubMed2003

Cryopreservation of sperm: indications, methods and results.

PURPOSE: We review the history of the cryopreservation of human sperm. The current methods of cryopreservation and indications are highlighted, and the success rates of the various uses of cryopreserved sperm are reviewed. Potential adverse sequelae of human cryopreservation are also discussed. MATERIALS AND METHODS: Indications for cryopreservation include several applications, namely homologous and donor insemination, cryopreservation prior to surgical infertility treatment, intraoperative cryopreservation, postoperative cryopreservation, cryopreservation prior to treatment for malignancies and nonmalignant diseases, as well as premortem and postmortem cryopreservation. RESULTS: With the use of intracytoplasmic sperm injection, open microsurgical epididymal sperm aspiration and testicular sperm extraction with cryopreserved sperm in men with obstructive azoospermia yields pregnancy rates similar to those using fresh sperm. Intraoperative cryopreservation is recommended for complex reconstructive cases with lower patency rates, including vasoepididymostomy and vasoepididymostomy to the efferent ductule. In addition, sperm banking is strongly recommended for all patients with malignant disease who may wish to have children, since a small but significant percent of these men later use their banked semen. Although there is concern about the transfer of genetically damaged sperm after cryopreservation, no proven increase in genetic or phenotypic abnormalities in offspring has been identified. CONCLUSIONS: Sperm cryopreservation has revolutionized the field of assisted reproduction. Cryopreserving sperm avoids the need for additional surgery in couples undergoing repeated in vitro fertilization/intracytoplasmic sperm injection cycles. Moreover, it provides hope for men undergoing chemotherapy, radiation or radical surgery who once had no chance for future fertility.

Cryopreservation↗
PubMed2005

A novel method of cryopreservation of rat and human hepatocytes by using encapsulation technique and possible use for cell transplantation.

Encapsulated hepatocyte transplantation is a promising approach to cell transplantation without immunosuppression as an alternative to whole organ liver transplantation. However, the shortage of donor cells for hepatocyte transplantation has not been resolved, and at this critical point, it seems necessary to establish a method of hepatocyte cryopreservation to allow clinical application of hepatocyte transplantation and the development of a bioartificial liver system in the near future. In this study we demonstrated that cryopreserved microencapsulated rat and human hepatocytes can retain their hepatic function and that cryopreserved microencapsulated human hepatocytes transplanted into rat spleen remain viable without immunosuppression. Rat and human hepatocytes were isolated by a collagenase digestion method, and they were microencapsulated with poly-L-lysine. The microencapsulated rat hepatocytes were transferred to culture medium (DMEM containing 10% FBS and 10% DMSO) and immediately frozen in liquid nitrogen. A warm water bath (37 degrees C) was used to thaw the microencapsulated hepatocytes. Hepatic function, drug metabolism, and cell morphology were assessed after 90 days of cryopreservation. After 1 week of cryopreservation, microencapsulated hepatocytes were cultured for up to 2 weeks to assess their hepatic function and morphology. The morphology of human hepatocytes was assessed after 30 days of cryopreservation. Cryopreserved human hepatocytes were transplanted into rat spleen to assess their morphology. Cryopreserved microencapsulated hepatocytes retained their viability and were strongly positive for expression of albumin, OAT2, CYP3A2, and CYP3A9. Two weeks after cultivation, the cryopreserved microencapsulated rat hepatocytes had retained their hepatic function (urea synthesis). Cryopreserved microencapsulated human hepatocytes also mainly survived and retained their hepatic function for at least 30 days after cryopreservation. Moreover, entrapped cryopreserved human hepatocytes also survived and expressed albumin in rat spleen after transplantation. We demonstrated a novel method of long-term cryopreservation of rat and human hepatocytes by using an encapsulation technique, with retention of biological activity and excellent survival of the cryopreserved microencapsulated human hepatocytes transplanted into rat spleen. We believe that this novel approach to hepatocytes cryopreservation provides a new direction in encapsulated cell therapy with the goal of clinical application in the near future.

Alginates↗
PubMed2005

Phase I and II metabolism and carbohydrate metabolism in cultured cryopreserved porcine hepatocytes.

Primary porcine hepatocytes were cryopreserved using freezing boxes or a programmable freezer (PF). Upon thawing and culturing in 12-well plates cryopreserved hepatocytes were compared with their fresh controls on days 1 and 2 after plating. Cryopreserved hepatocytes attached approximately as well as fresh hepatocytes and useful cultures were obtained. In cryopreserved hepatocytes, coumarin 7-hydroxylation, 6beta-testosterone hydroxylation and p-nitrophenol glucuronidation were reduced to about 10-40, 35 and 40%, respectively, compared to their fresh counterparts. Glycogen synthesis in cryopreserved hepatocytes was reduced to about 30% on day 1 of culture and about 47% on day 2 of culture compared to the synthesis in fresh hepatocytes. Both fresh and cryopreserved hepatocytes increased the synthesis by twofold in response to stimulation with insulin. Reduced basal levels of glycogen and of glycogen synthesis could be explained by an increased energy demand in cryopreserved hepatocytes needing to repair damages caused by cryopreservation. Glycogenolysis was reduced to about 50% in cryopreserved hepatocytes and gluconeogenesis to about 40% of the glucose production in fresh hepatocytes. In both fresh and cryopreserved hepatocytes the glucose production from glycogenolysis and gluconeogenesis, respectively, was increased fourfold in response to stimulation with glucagon. Overall, the hepatocytes cryopreserved in boxes had a tendency to perform better than hepatocytes cryopreserved in a programmable freezer. In conclusion, the cryopreserved hepatocytes were metabolic active; however, to a lower extent than the fresh hepatocytes, although, the cryopreserved hepatocytes responded as well as the fresh hepatocytes to insulin and glucagon.

Animals↗
PubMed2004

Parasite cryopreservation by vitrification.

Parasitic protozoa and helminths and parasitic/vector insects each have distinct requirements for cryopreservation. Most parasitic protozoa respond to cryopreservation stresses similarly to other single cell suspensions, but few species are currently routinely cryopreserved by protocols specifically designed for vitrification. With slow equilibrium cooling, some protozoa osmotically dehydrated by solutes concentrated in the residual unfrozen fraction will survive by vitrifying. Several species of helminths, together with insect embryos cannot be cryopreserved by slow cooling protocols and have an absolute requirement for vitrification. Studies incorporating slow cooling and stepped cooling of both protozoa and helminths, particularly the intraerythrocytic stages of malaria and the schistosomula larvae of Schistosoma mansoni have aided in the design of vitrification protocols for parasites. For helminths, the most widely used cryopreservation protocol, originally successful for cryopreserving S. mansoni schistosomula, consists of the addition of ethanediol in two steps, followed by rapid cooling (approximately 5100 degrees C min(-1)) to -196 degrees C. This technique exploits the temperature-dependent differential in permeability of the cryoprotectant additive (CPA) to first permeate into the organism at 37 degrees C followed by a dehydration-mediated internal CPA increase in concentration resulting from incubation in a second higher CPA concentration at 0 degree C. Samples are rapidly warmed/diluted (approximately 14,000 degrees C min(-1)) to recover the organisms from liquid nitrogen storage. Variations on this technique have also been successful in cryopreserving the larvae and adult worms of filariae, muscle stage larvae of Trichinella spp., the infective stages of gastro-intestinal nematode parasites and insect embryos. Other protocols where the dehydration step precedes CPA addition have been used to cryopreserve entomogenous nematode larvae by vitrification. Techniques that utilize high concentrations of CPA cocktails and slower cooling, developed for the vitrification of mammalian embryos, have been applied to the cryopreservation of parasitic protozoa, but with limited success to date. Where cryopreservation by classical slow cooling methods is possible, vitrification has enhanced the levels of survival obtained. And vitrification has enabled the successful cryopreservation of those parasitic species not able to be cryopreserved by traditional methods. Since a limited number of parasitic organisms has been cryopreserved using vitrification protocols, there is considerable scope for further improvement in the cryopreservation techniques used for many parasitic species.

Animals↗