Refrigeration of donor cells in preparation for bovine somatic nuclear transfer.
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In mammals, preparation of donor cells for somatic nuclear transfer is very important because the character of the donor cell directly affects the efficiency and outcome of transfer. The protocols used most commonly for donor preparation are (i) disaggregating cells from fresh tissue 1-2 h before micromanipulation or (ii) trypsinizing cultured cells temporarily, after special treatments for 3-8 days (for example, serum starvation). In this study, a new simple protocol was designed, whereby the donor cells (cumulus cells) used in bovine somatic nuclear transfer were refrigerated. In brief, cultured cells at 80-100% confluency were detached using trypsin, washed by centrifugation, aliquoted into different vials and refrigerated at 4 degrees C. The density of viable cells was decreased after day 1 of refrigeration; however, the rate of decrease tended to slow down with increasing duration of refrigeration. Cells refrigerated for 15 days were seeded at a density of 5 x 10(4) ml(-1) and reached 70% confluency after day 2 of culture. Most cells had the normal number of chromosomes (2n = 60). Cells chilled at 4 degrees C for different durations were removed from refrigeration and immediately subjected to micromanipulation. The in vitro development of reconstructed embryos (fusion rates, cleavage rates, morula and blastocyst rates) indicated that there were no significant differences among treatment groups regardless of the duration of refrigeration (0-2 weeks) of the donor cells. Reconstructed embryos were transferred into the uteri of recipient cows. No significant differences were observed in established early pregnancies between embryos derived from the non-refrigerated donor cells and those derived from refrigerated donor cells. This study indicates that refrigeration of donor cells for 1-2 weeks is a feasible protocol for preparing donor cells for bovine somatic nuclear transfer, and does not compromise development in vitro and early development in vivo. (+info)
Effect of three different media on serum free culture of donor corneas and isolated human corneal endothelial cells.
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BACKGROUND: Removal of bovine serum from organ culture medium is necessary because of the variability in serum composition and the potential risk of infection. Two specific endothelial cell media (F99 and Endothelial-SFM) were compared with the routinely used medium MEM for their use in serum free cultivation of human corneal endothelial cells (HCEC) and donor corneas. METHODS: HCEC were incubated in three test media with or without increasing serum content and a growth assay was performed. Seven pairs of donor corneas were cultured in each of three media for 3 weeks, one cornea with serum supplementation and one without. Endothelial cell density was determined once each week. Trypan blue staining of the endothelium and vital staining of keratocytes was performed after 3 weeks. RESULTS: All three media promoted proliferation of cultured HCEC when supplemented with serum. Endothelial cell density of donor corneas was comparable after 3 weeks of cultivation in the different media. Only corneas cultured in medium MEM without serum exhibited a higher endothelial cell loss. Trypan blue staining of the endothelium after cultivation revealed the lowest number of damaged cells on corneas cultured in the medium Endothelial-SFM. The highest densities of keratocytes were found in corneas cultured in Endothelial-SFM and the lowest densities occurred after culture in MEM. CONCLUSION: After incubation in Endothelial-SFM even under serum free conditions corneas were found to be of higher quality with respect to endothelial cell survival, cell membrane integrity, and keratocyte density. This medium may replace MEM, which is routinely used in European eye banks but requires supplementation with serum. (+info)
Viral studies on human brain culture retrieved from cold storage.
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Tissue cultures established from brain tissues of six patients with various chronic, degenerative diseases did not show evidence of viral agents. Sucrose gradient analysis of radioactively labeled culture fluid from two patients failed to reveal the presence of any incomplete or defective virus. Extracellular fluids from three brain cultures contained a factor able to stimulate deoxyribonucleic acid synthesis of Vero cells. (+info)
Short-term preservation of mouse oocytes at 5 degrees C.
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The temporary preservation of oocytes without freezing would be useful for some experiments. ICR mouse oocytes were kept in a preservation medium under mineral oil for 1, 2, 3, 4 or 7 days at 5 degrees C, and 1 or 2 days at 37 degrees C. In vitro fertilization was attempted on oocytes rinsed with TYH medium after preservation. More than 70% of morphologically normal oocytes were recovered from each preservation group. Fertilization rates of oocytes preserved for 1, 2, 3, 4 or 7 days at 5 degrees C were 69.9, 66.5, 45.3, 26.7 and 8.8% respectively. Fertilization rates of oocytes preserved for 1 or 2 days at 37 degrees C were 9.6 and 1.6%, respectively. Preservation of oocytes at 5 degrees C has some capability as a method of short-term storage without freezing. (+info)
Quantitative gene expression analysis in renal biopsies: a novel protocol for a high-throughput multicenter application.
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BACKGROUND: Recent advances in gene expression analysis may add the quantification of mRNA species in renal biopsies to routine diagnostic procedures in nephrology. METHODS: A systematic evaluation was performed on the relevant steps required to efficiently obtain cDNA from renal biopsies for high-throughput reverse transcription-polymerase chain reaction (RT-PCR) based mRNA quantification. RESULTS: The protocol preserves mRNA integrity by a novel RNase inhibitor and allows meticulous microdissection followed by maximal RNA recovery from tissue samples. Reverse transcription was optimized to give the best yield from minimal starting material. RNA quantity and quality were systematically investigated by real-time RT-PCR and electrophoresis on a microfluidic system, respectively. The reported procedure offers high RNA preservation and increases the yield of cDNA significantly compared to former protocols. CONCLUSION: The simplicity of biopsy material acquisition combined with the centrally performed processing makes this protocol suitable for a wide spectrum of expression analysis in diverse clinical settings. (+info)
Spurious dyserythropoiesis.
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We documented the occurrence and severity of dyserythropoiesis as an artifact of storage in bone marrow aspirates collected in EDTA. Bone marrow samples were obtained from 7 patients without myelodysplasia. Specimens were stored at room (20 degrees C-24 degrees C) or refrigerated (1 degrees C-6 degrees C) temperature and examined for dyserythropoiesis at 0, 1, 2, and 3 days. Initial specimens showed few dyserythropoietic abnormalities; nuclear aberrations occurred in 1.07%+/-0.06% (mean +/- SEM) of the erythroid population. At room temperature, dyserythropoietic changes increased significantly with each day of storage. Nuclear and cytoplasmic alterations occurred; the former are diagnostically more important in the diagnosis of myelodysplastic syndromes. Cytoplasmic changes were more extensive than nuclear abnormalities. The mean +/- SEM percentage of erythroblasts with cytoplasmic vacuoles increased with each day of storage: day 0, 1.1%+/-0.2%; day 1, 22.1%+/-1.8%; day 2, 29.4%+/-2.0%; day 3, 35.6%+/-1.9%. Nuclear shape changes increased to 6.21%+/-1.12%, 11.36%+/-1.12%, and 12.85%+/-1.20% on days 1, 2, and 3, respectively. After 1 day of storage, sufficient dysplastic changes occur to cause difficulty in the diagnosis of a myelodysplastic syndrome. Changes are inhibited significantly by refrigerated storage. (+info)
Stimulatory effect of the lysosomal stabilizer, chloroquine, on the respiration and motility of fresh and aged bovine spermatozoa.
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The lysosomal stabilizer and anti-malarial agent, chloroquine, stimulated the respiration and motility of fresh and aged bovine spermatozoa stored in vitro. Duplication of these effects by the phosphodiesterase inhibitors, theophylline and caffeine, suggested that enhancement of sperm activity by chloroquine may be mediated by cyclic AMP. (+info)
Preservation of fertility in nature and ART.
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Individuals may regard reproduction as optional but sufficient number of them must be productive to perpetuate the species. The reproductive system is surprisingly vulnerable and depends, among other things, on a limited endowment of oocytes, controlled proliferation of spermatogonial stem cells and the genetic integrity of both. The developmental competence of oocytes and spermatogonial stem cells is maintained by evolved mechanisms for cellular detoxification and genomic stability, and excess or damaged cells are eliminated by apoptosis. Gonadal failure as a result of germ cell depletion can occur at any age, and from the effects of chemical cytotoxicity, disease and infection as well as genetic predisposition. Among extrinsic factors, alkylating agents and ionizing radiation are important causes of iatrogenic gonadal failure in young women and men. In animal models, there is evidence that hormonal manipulation, deletion of genes involved in apoptotic pathways and dietary manipulation can protect against natural and induced germ cell loss, but evidence in humans is absent or unclear. Assisted reproductive technologies (ARTs) provide an ensemble of strategies for preserving fertility in patients and commercially valuable or endangered species. Semen cryopreservation was the first technology for preserving male fertility, but this cannot serve prepubertal boys, for whom banking of testicular biopsies may provide a future option. In sterilized rodents, cryopreserved spermatogonial stem cells can recolonize seminiferous tubules and reinitiate spermatogenesis, and subcutaneous implantation of intact tubules can generate spermatozoa for fertilization in vitro by intracytoplasmic sperm injection. Transplantation of frozen-banked ovarian tissue is well-established for restoring cyclicity and fertility and is currently undergoing clinical evaluation for cancer patients. When restoration of natural fertility is unnecessary or reimplantation is unsafe, it is desirable to culture the germ cells from thawed tissue in vitro until they reach the stage at which they can be fertilized. Low temperature banking of immature germ cells is potentially very versatile, but storage of embryos and, to a lesser extent, mature oocytes is already practised in a number of species, including humans, and is likely to remain a mainstay for fertility preservation. (+info)