Cryopreservation of brain tissue for primary culture.

Factors affecting recovery of brain cells from cryopreserved cerebral tissues of fetal rats were examined based on yields of viable cells on cell culture. Favorable preservation was obtained with freezing small pieces (less than 1 mm cube) of brain tissues rather than whole tissues or dissociated single cells, and use of 10% dimethylsulfoxide as a cryoprotectant in liquid nitrogen. As for cell preparation procedures, cell survival was improved when tissues were heated at 32 degrees C during papain digestion and centrifugation. Under favorable conditions, the number of brain cells recovered from cryopreserved tissues corresponded to 20-30% of those from fresh control tissues. Immunocytochemical characteristics of cultured neurons, astrocytes, and oligodendrocytes from cryopreserved and fresh tissues were indistinguishable. Semi-quantitive analyses of microtubule-associated protein-2 (MAP-2) and synaptophysin revealed that there was no difference in the amounts of these markers between cultures from both fresh and cryopreserved tissues. These results suggest that most of all cell types including neurons were equally susceptible to the cryopreservation procedures. We concluded that cryopreservation in liquid nitrogen is an effective method for preservation of embryonic brain tissues for later use in cell culture studies.     

A Novel Approach for Ovine Primary Alveolar Epithelial Type II Cell Isolation and Culture from Fresh and Cryopreserved Tissue Obtained from Premature and Juvenile Animals

The in vivo ovine model provides a clinically relevant platform to study cardiopulmonary mechanisms and treatments of disease; however, a robust ovine primary alveolar epithelial type II (ATII) cell culture model is lacking. The objective of this study was to develop and optimize ovine lung tissue cryopreservation and primary ATII cell culture methodologies for the purposes of dissecting mechanisms at the cellular level to elucidate responses observed in vivo. To address this, we established in vitro submerged and air-liquid interface cultures of primary ovine ATII cells isolated from fresh or cryopreserved lung tissues obtained from mechanically ventilated sheep (128 days gestation—6 months of age). Presence, abundance, and mRNA expression of surfactant proteins was assessed by immunocytochemistry, Western Blot, and quantitative PCR respectively on the day of isolation, and throughout the 7 day cell culture study period. All biomarkers were significantly greater from cells isolated from fresh than cryopreserved tissue, and those cultured in air-liquid interface as compared to submerged culture conditions at all time points. Surfactant protein expression remained in the air-liquid interface culture system while that of cells cultured in the submerged system dissipated over time. Despite differences in biomarker magnitude between cells isolated from fresh and cryopreserved tissue, cells isolated from cryopreserved tissue remained metabolically active and demonstrated a similar response as cells from fresh tissue through 72 hr period of hyperoxia. These data demonstrate a cell culture methodology using fresh or cryopreserved tissue to support study of ovine primary ATII cell function and responses, to support expanded use of biobanked tissues, and to further understanding of mechanisms that contribute to in vivo function of the lung.     

Cell culture and in vitro studies of fresh and cryopreserved human insulinoma

Summary Dispersed cells from both fresh and cryopreserved human insulinoma have been maintained in cell culture. Initial yield of viable cells was 50% for fresh and 25% for cryopreserved tissue. Viability of cells in culture was documented by increasing numbers of cells (doubling time approximately 5 d initially and 2 d at the sixth subculture for both fresh and cryopreserved tissue) and continued release of insulin over time (approximately 100 ng/ml per 10⁵ cells at 10 d and 175 ng/ml per 10⁵ cells at 30 d of culture for both fresh and cryopreserved tissue). Evidence that cells growing in culture were beta cells was provided by: (a) recovery of intracellular and extracellular immunoreactive insulin (IRI), (b) electron microscopic morphology, and (c) immunohistochemical staining. Cells from fresh insulinoma incubated with increasing concentrations of extracellular glucose released increasing amounts of IRI up to approximately 15 mM glucose, which paralleled changes in plasma insulin obtained during a preoperative glucose tolerance test. Under an Intergovernmental Personnel Act Exchange from the Department of Surgery, University of California, Davis, Sacramento Medical Center.     

Viability of pulp stromal cells in cryopreserved deciduous teeth

The cryopreservation of exfoliated deciduous teeth and harvesting of stem cells from them as required would reduce the costs and efforts associated with banking stem cells from primary teeth. The aim of this study was determine whether the viability of pulp stromal cells from deciduous teeth was influenced by the cryopreservation process itself or the period of cryopreservation. In total, 126 deciduous teeth were divided into three groups: (1) fresh, (2) cryopreserved for <3 months (cryo<3), and (3) cryopreserved for 3–9 months (cryo3–9). The viability of the pulp tissues was compared among the three groups by evaluating the outgrowth from pulp tissues and cell activity within those pulp tissues. In addition, the terminal deoxynucleotidyl transferase-mediated dUTP–biotin nick end labeling (TUNEL) assay was performed to compare cell apoptosis within fresh pulp tissue and pulp tissue that had been cryopreserved for 4 months. The outgrowth from and cell activity within the pulp tissues did not differ significantly between the fresh and cryo<3 pulp tissues. However, these parameters were significantly reduced in the cryo3–9 pulp tissue. In TUNEL assay, 4-month cryopreserved pulp tissues has more apoptotic cells than fresh group. In conclusion, it is possible to acquire pulp stromal cells from cryopreserved deciduous teeth. However, as the period of cryopreservation becomes longer, it is difficult to get pulp cells due to reduced cell viability.     

Dimethyl sulfoxide maintains structure and function of cryopreserved equine endometrial explants

Availability of viable frozen-thawed endometrial tissues could facilitate detailed studies into physiologic and disease processes influencing the endometrium. This study was designed to investigate the cryosurvival of equine endometrial tissue. Previous studies in the human and horse have focused on cryopreservation of dissociated endometrial cells. To our knowledge, there are no studies on cryopreservation of endometrial explants. Our objectives were to 1) determine the influence of differing concentrations of the permeating cryoprotectant dimethyl sulfoxide (Me₂SO) on viability, structural integrity, and gene expression of cryopreserved equine endometrial tissues prior to and following a 5-day explant culture in vitro and 2) examine the influence of low (1000 mg/L dextrose) vs high (4500 mg/L dextrose) glucose medium during in vitro culture. Both 10% and 20% (v/v) concentrations of Me₂SO maintained viability following cryopreservation and in vitro culture. In addition, gene expression remained unaltered following cryopreservation with either 10% or 20% Me₂SO. However, tissue structural integrity was slightly reduced compared to the fresh control. Furthermore, there was no difference in structural integrity, cell viability, or gene expression between low and high glucose medium during in vitro culture. Although E-cadherin and Ki67 gene expression was not different among fresh, 10% Me₂SO, and 20% Me₂SO treatments prior to or following tissue culture, estrogen receptor-α and progesterone receptor gene expression were reduced in all groups after explant culture. This is the first report of successful cryopreservation of equine endometrial explants.     

Cells isolated from cryopreserved dental follicle display similar characteristics to cryopreserved dental follicle cells

Dental follicle tissue is a promising resource of mesenchymal stem cells for cytotherapeutic approaches and tissue engineering applications. There are two procedures for banking of human dental follicle stem cells have been reported. Conventional method requires cell isolation, expansion and immediate cryopreservation. Whereas dental follicle stem cells can be isolated from cryopreserved dental follicle fragments. The aim of this study was to compare the characteristics of dental follicle cells isolated from cryopreserved fragments (DFCs-CF) with dental follicle cells recovered from cryopreserved cells (DFCs-CC). Dental follicle fragments obtained after mechanical disaggregation were divided into two parts, with one part maintained in culture, while another part underwent cryopreservation. Dental follicle fragments and dental follicle cells from fresh tissue were stored in liquid nitrogen for 3 months. After thawing, the isolation, morphology, proliferation, cell cycle, colony-forming-unit ability, stemness-related marker expression, apoptosis, and multi-lineage differentiation potential of DFCs-CF were tested compared with DFCs-CC. DFCs-CF expressed mesenchymal stem cells marker, proliferated well, showed similar levels of mRNA for stemness- and apoptosis-related genes and exhibited the capacity of multi-lineage differentiation similar to those of DFCs-CC. These results imply that cryopreservation of dental follicle fragments is an effective banking method for isolation of dental follicle cells.     

Human platelet lysate enhances the proliferative activity of cultured human fibroblast-like cells from different tissues

Several studies have shown the presence of fibroblast-like cells in the stromal fraction of different tissues with a high proliferative and differentiation potential. Platelet alpha granules contain growth factors released into the environment during activation. The effects of different supplements for culture medium (human serum, bovine serum and platelet lysate) on cultured human fibroblast-like cells from bone marrow, adipose tissue, trabecular bone and dental pulp have been compared. Expression of typical stromal and hematopoietic markers was analyzed and proliferative rates were determined. Flow cytofluorometry showed a homogenous pattern in serial-passaged cells, with a high level of stromal cell-associated markers (CD13, CD90, CD105). The presence of platelet lysate in culture media increased the number of cell generations obtained regardless of cell source. This effect was serum-dependent. Cell-based therapies can benefit by the use of products from human origin for “ex vivo” expansion of multipotent cells.     

Alginate beads as a tool to handle,cryopreserve and culture isolated human primordial/primary follicles

Background

One major concern of grafting cryopreserved ovarian tissue to restore fertility in cancer patients is the possibility of reintroducing tumor cells. Cryopreservation of isolated primordial/primary follicles (PFs) may circumvent this problem. The aim of our work was to compare dimethyl sulfoxide (ME₂SO) and ethylene glycol (EG) as cryoprotectants (CPAs) for slow-freezing of isolated human PFs in alginate.

Methods

Ovarian biopsies from four women were processed for follicle isolation. PFs were embedded in alginate (5–15 per group). Follicles were frozen-thawed using 1.4 M ME₂SO or 1.5 M EG as CPAs. Fresh and cryopreserved isolated follicles were in vitro cultured (IVC) for 7 days. At different time periods (after isolation, cryopreservation and IVC), follicles were evaluated with live/dead assay (using fluorescent probes) and diameter measurement. Follicle viability was calculated according to the percentage of dead follicular cells and the presence of a live/dead oocyte.

Results

A total of 841 PFs were isolated, embedded in alginate and cryopreserved with ME₂SO (n = 424) or EG (n = 259), or used as controls (n = 158). After 7 days of IVC, a significant increase in follicle size was observed in the fresh and ME₂SO groups, but not in the EG group. The percentage of totally viable PFs was not significantly different before or after seven days of culture in fresh (100% and 82%) or ME₂SO (93.2% and 85.1%) tissue. The EG group showed significantly lower viability before (63.9%) and after IVC (66.2%) than the fresh and ME₂SO groups.

Conclusions

Our results show that 1.4 M ME₂SO yields better preservation of isolated PF viability after thawing and 7 days of IVC than 1.5 M EG. Alginate constitutes an easy, safe hydrogel matrix to handle and cryopreserve isolated human follicles using ME₂SO as a CPA.     

Survival of human bone marrow progenitor cells after freezing: Improved detection in the colony-formation assay

If cryopreserved suspensions of human bone marrow were stimulated by human placental conditioned medium in the same way as fresh unseparated marrows, less than 40% of granulopoietic progenitor cells (CFU_c) was identified. By adding α-thioglycerol (0.6 mM) to the culture medium, the concentration of detectable CFU_c in cryopreserved bone marrow was increased by a factor of 3.4, and the recovery of CFU_c after cryopreservation rose to 90%. The low recovery of CFU_c after freezing in the absence of α-thioglycerol is due to the destruction of accompanying cells. Noncolony-forming cells normally present in the fresh human marrow promote colony formation in cultures stimulated by placental conditioned medium. Their effect can be replaced by α-thioglycerol. It is concluded that, in order to detect all CFU_c independent of the cellular composition of the marrow suspension, this supplement is essential for CFU_c cultures stimulated by conditioned medium.     

Optimizing methods for human testicular tissue cryopreservation and spermatogonial stem cell isolation

Cryopreservation of testicular tissue before cancer therapy for fertility preservation in prepubertal boys with cancer is of great interest in reproductive medicine. Isolation of spermatogonial stem cells (SSCs) from cryopreserved tissues would be a suitable cell source to re-establish spermatogenesis after cancer therapy. We herein establish optimized protocols for cryopreservation of human testicular tissue and isolation of SSCs from cryopreserved tissue. We developed a freezing protocol that provided high testicular cell viability and supported structural integrity and tubular epithelium coherence similar to fresh tissue. Then, we established a protocol that allowed efficient isolation of functional SSCs from cryopreserved tissues. Isolated cells were found on the testicular basement membrane after xenotransplantation. Our results demonstrated the preservation of testicular tissue structure and high cell viability with efficient isolation of SSCs after testicular cryopreservation, which is promising for future therapeutic applications in fertility preservation.     

一种简单的人脐带间充质干细胞分离培养方法

目的 建立一种简单的人脐带间充质干细胞分离培养方法.方法 取新鲜脐带,剪成5 cm长的小段,直接剪碎为糊状,加入含10％胎牛血清的DMEM/F12在培养瓶中培养,光学显微镜下观察细胞的生长特征,运用流式细胞仪检测分析细胞的抗原标志表达,并检测其体外多向分化潜能.结果 运用不剥离血管组织、不用酶消化的组织贴块培养法可以从...     

Cryopreservation of adipose-derived stromal/stem cells using 1–2% Me2SO (DMSO) in combination with pentaisomaltose: An effective and less toxic alternative to comparable freezing media

Mesenchymal stromal/stem cells (MSCs) derived from bone marrow, umbilical cord and especially adipose tissue are increasingly being explored for their therapeutic potential to treat a wide variety of diseases. A prerequisite for most allogeneic off-the-shelf and some autologous MSC therapies is the ability to safely and efficiently cryopreserve cells during production or for storage prior to treatment. Dimethyl sulfoxide (Me₂SO) is still the commonly used gold standard cryoprotectant (CPA). However, undesirable cellular impacts and side effects of Me₂SO have led to an increasing demand for the development of safe and effective alternatives.This study investigated the effect of pentaisomaltose as a CPA for cryopreservation of adipose-derived stromal/stem cells (ASCs). We compared pentaisomaltose-based freezing media containing 1% Me₂SO (PIM1) or 2% Me₂SO (PIM2) to our in-house freezing media formulation containing 10% Me₂SO (STD10) and to CryoStor freezing media containing 2% or 10% Me₂SO (CS2 and CS10). We assessed the recovery of viable ASCs, their phenotype, differentiation potential, proliferation potential, and migratory potential. Further, their immunomodulatory potential was assessed by measuring their ability to suppress T cell proliferation and express immunomodulatory markers.The results showed that the post-thaw viability of ASCs cryopreserved with STD10, CS10 and PIM2 was improved compared to that of CS2. The recovery of ASCs with PIM1 and PIM2 was also improved compared to that of CS2. Proliferation and migration were comparable among the tested freezing media. The results showed no difference in the induction of PDL1, PDL2 or IDO1 expression. Nevertheless, the potential of cryopreserved ASCs to suppress T cell proliferation was reduced when the Me₂SO concentration was reduced (CS10>STD10>CS2 and PIM2>PIM1).Altogether, the migratory and immunomodulatory potential combined with improved recovery indicate that the addition of pentaisomaltose in the freezing media may allow for the reduction of the Me₂SO concentration to 2% while retaining a more potent cell product that what is recovered using comparable freezing media. With the desire to reduce the amount of Me₂SO, these results suggest that 2% and potentially even 1% Me₂SO in combination with 10% pentaisomaltose could be an effective and less toxic alternative to comparable freezing media.     

Cryopreservation of calf testicular tissues with knockout serum replacement

To enrich bovine gonocytes from cryopreserved testicular tissues, the cryoprotection effects of the freezing media containing knockout serum replacement (KSR) were examined. Using Minimum essential medium (MEM) + 10% dimethyl sulfoxide (Me₂SO) as the basic medium, calf testicular tissues were cryopreserved in media containing 0, 5, 10, 20, 40, 90% KSR and 5% fetal bovine serum (FBS) respectively. Morphologically, the seminiferous cords and interstitium were well preserved in all groups. The gonocytes were all glial cell line-derived neurotrophic factor (GDNF) family receptor α-1 (GFRα-1) positive. The recovery rates in all KSR groups were higher than that of the 10% Me₂SO group, while comparable to the 5% FBS group. The enriched gonocytes expressed gonocyte marker GFRα-1 typically. Collectively, supplementation of 5–10% KSR can achieve comparable cryoprotective effects with using 5% FBS, which is useful in future study due to its defined formulation that is more consistent in quality and stable in supply.     

Markers of stemness in equine mesenchymal stem cells: a plea for uniformity

Mesenchymal stromal cells (MSC) are a very promising subpopulation of adult stem cells for cell-based regenerative therapies in veterinary medicine. Despite major progress in the knowledge on adult stem cells during recent years, a proper identification of MSC remains a challenge. In human medicine, the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy (ISCT) recently proposed three criteria to define MSC. Firstly, cells must be plastic-adherent when maintained under standard culture conditions. Secondly, MSC must express CD73, CD90 and CD105, and lack expression of CD34, CD45, CD14 or CD11b, CD79α or CD19 and MHC class II antigens. Thirdly, MSC must be able to differentiate into osteoblasts, adipocytes and chondroblasts in vitro. Successful isolation and differentiation of equine MSC from different sources such as bone marrow, fat tissue, umbilical cord blood, Wharton's Jelly or peripheral blood has been widely reported. However, their unequivocal immunophenotyping is hampered by the lack of a single specific marker and the limited availability of monoclonal anti-horse antibodies, which are two major factors complicating successful research on equine MSC. Detection of gene expression on mRNA level is hereby a valuable alternative, although the need still exists to test several antibody clones in search for cross-reactivity. To date, commercial antibodies recognizing equine epitopes are only available for CD13, CD44 and MHC-II. Moreover, as the expression of certain adult stem cell markers may differ between species, it is mandatory to define a set of CD markers which can be uniformly applied for the identification of equine MSC.     

Effects of cell storage and passage on basal and oxytocin-regulated prostaglandin secretion by equine endometrial epithelial and stromal cells

Cell cultures are useful for determining the responses of specific cell types to various factors under controlled conditions and for obtaining a better understanding of in vivo physiologic processes. The aims of the present study were (i) to establish methodologies for isolation, culture and cryopreservation of equine endometrial epithelial and stromal cells; and (ii) to determine the effect of passage and cryopreservation on endometrial cell physiology, based on their basal and oxytocin (OT)-stimulated prostaglandin (PG) release. Epithelial and stromal cells were obtained by enzymatic digestion of equine endometrium collected from Days 2-5 of the estrous cycle (n = 16). Primary epithelial and stromal cells, as well as cryopreserved cells were stimulated with OT (10⁻⁷m) for 24 h. The concentrations of PGE₂ and PGF_2α in the culture medium were measured by enzyme-linked immunosorbent assay (EIA). Oxytocin increased PGE₂ and PGF_2α release by primary cultures of unfrozen epithelial cells until passage I (P < 0.01) and by the primary culture of unfrozen and cryopreserved/thawed stromal cells until passage IV (P < 0.01). Cryopreserved/thawed stromal cells cultured up to passage IV and unfrozen epithelial cells derived from passage I have physiological properties similar to those observed in primary culture and may be successfully used for in vitro studies of PG secretion.     

Human immunodeficiency virus type 1 infection of human placenta: potential route for fetal infection.

To determine the potential role of the placenta in transmission of human immunodeficiency virus (HIV) from mother to fetus, the ability of human placental tissue to support HIV type 1 (HIV-1) infection was examined. HIV-1-seronegative first-trimester placentas were maintained in culture and infected with HIV-1. Virus production, measured by HIV-1 antigen release into the supernatant, and HIV-1 DNA, identified by polymerase chain reaction, were detected for at least 12 days postinfection. Western immunoblot analysis showed Gag proteins, precursor p55, and cleavage products p24 and p17 in HIV-1-infected tissues. Double labeling of placental villi with antibodies to CD4 and placental trophoblast-specific alkaline phosphatase indicated that trophoblasts express CD4 antigen. Additionally, immunostaining of HIV-1-infected tissues with anti-p24 antibodies demonstrated HIV-1 protein expression in placental trophoblasts. Evaluation of human chorionic gonadotropin and progesterone production by the placental cultures indicated that there was a 90% decrease in human chorionic gonadotropin and a 70% decrease in progesterone production in HIV-1-infected cultures in comparison with controls. These data demonstrate that trophoblastic cells of human placenta tissue express CD4 and are susceptible to HIV-1 infection; also, placental endocrine function is decreased by HIV-1 infection. Thus, the placenta may serve as a reservoir of HIV-1 infection during pregnancy contributing to infection of the fetus, and decreased placental hormone production may result in impaired fetal development.     

Cryopreservation of amniotic fluid-derived stem cells using natural cryoprotectants and low concentrations of dimethylsulfoxide

Amniotic fluid-derived stem cells (AFSCs) are a potential cell source for therapeutic applications. They can be easily mass produced, cryopreserved and shipped to clinics for immediate use. However, one major obstacle to the manufacturing of clinical grade stem cells is the need for current good manufacturing practices for cryopreservation, storage, and distribution of these cells. Most current cryopreservation methods used for stem cells include the potentially toxic cryoprotectant (CPA) dimethylsulfoxide (Me₂SO) in the presence of animal serum proteins that prevent direct use of these cells in human therapeutic applications. To avoid any potential cryoprotectant related complications, it will be essential to develop non-toxic CPAs or reduce CPA concentration in the freezing media used. In this study, we assessed the use of disaccharides, antioxidants and caspase inhibitors for cryopreservation of AFSCs in combination with a reduced concentration of Me₂SO. The thawed cells were tested for viability with MTT assays and a growth curve was created to measure population doubling time. In addition, we performed flow cytometry analysis for cell surface antigens, RT-PCR for mRNA expression of stem cell markers, and assays to determine the myogenic differentiation potential of the cells. A statistically significant (p < 0.05) increase in post-thawed cell viability in solutions containing trehalose, catalase and _ZVAD-fmk with 5% Me₂SO was observed. The solutions containing trehalose and catalase with 5% or 2.5% (v/v) Me₂SO produced results similar to those for the control (10% (v/v) Me₂SO and 30% FBS) in terms of culture growth, expression of cell surface antigens and mRNA expression of stem cell markers in AFSCs cryopreserved for a minimum of 3 weeks. Thus, AFSCs can be cryopreserved with 1/4 the standard Me₂SO concentration with the addition of disaccharides, antioxidants and caspase inhibitors. The use of Me₂SO at low concentrations in cell freezing solutions may support the development of clinical trials of AFSCs.     

Cryopreservation of fetal skin is improved by extracellular trehalose

In this study, we tested a non-permeating cryoprotectant, trehalose, in combination with dimethyl sulfoxide (Me(2)SO) in the cryopreservation of human fetal skin and compared it to Me(2)SO and glycerol, protocols that are routinely used by skin banks. The viability of fetal skin from four groups (fresh, and cryopreserved with glycerol, Me(2)SO, or trehalose/Me(2)SO) were evaluated using an in vitro membrane integrity assay and by transplantation to immunodeficient mice. The membrane integrity assay showed a 90% integrity in fresh, unfrozen fetal skin. The number of intact cells dropped to 23 and 44% in fetal skin cryopreserved with glycerol and Me(2)SO, respectively. When trehalose was added to the cryopreservation medium containing Me(2)SO, the membrane integrity rose to 65%. When transplanted to immunodeficient mice, fetal skin cryopreserved with trehalose/Me(2)SO showed a graft performance indistinguishable from fresh unfrozen fetal skin and strikingly better graft take than that of fetal skin cryopreserved with Me(2)SO or glycerol only. These results suggest that cryopreservation protocols routinely used the skin banks can be improved by combining sugars such as trehalose with a permeating cryoprotectant.     

Evaluation of cell viability and apoptosis in human amniotic fluid-derived stem cells with natural cryoprotectants

A previous study demonstrated that disaccharides, antioxidants, and caspase inhibitors can be used in freezing solutions to reduce the concentration of Me₂SO from the current standard of 10% (v/v) to 5% (v/v) or 2.5% and to eliminate fetal bovine serum (FBS) for the cryopreservation of human amniotic fluid-derived stem cells (AFSCs). Hence, this study investigated whether an irreversible inhibitor of caspase enzymes, benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone (zVAD-fmk), could be used in post-thaw culture media to increase the survival rate of AFSCs. Our results showed that AFSCs cryopreserved in freezing solution containing trehalose, catalase, and 5% (v/v) Me₂SO and then supplemented with zVAD-fmk in the post-thaw culture media showed similar post-thawing viability, proliferation, and apoptosis than cells cryopreserved in the control solution (10% (v/v) Me₂SO and 20% FBS). The caspase-3 activity in all the cryopreservation solutions tested was similar to that of the control. Caspase-3, caspase-8, caspase-9, and PARP expression was not found in the cryopreserved cells. In addition, no difference was found in the survival rate and apoptosis between short-term (3 weeks) and long-term (1 year) storage of AFSCs cryopreserved in the solutions used in this study. The results of the present study demonstrate that recovery of cryopreserved cells was enhanced by using a caspase inhibitor in the post-thaw culture media.     

In vitro growth of oocyte-granulosa cell complexes isolated from cryopreserved ovine tissue.

A culture system has been designed in which enzymatically isolated oocyte-granulosa cell complexes from fresh and frozen-thawed ovine ovarian tissue can be grown to antral size in vitro. Oocyte-granulosa complexes ranging from 100 to 240 microns in diameter were dissected from stromal tissue and grown individually in serum-free medium for 30 days. Complexes < 190 microns generally excluded their oocytes or lost three-dimensional structure early in the culture period. In contrast, complexes isolated from fresh or frozen-thawed tissue and measuring 190-240 microns on the day of isolation formed antral cavities in 25 +/- 9% and 18 +/- 6% (mean +/- SEM) of cases, respectively. The effect of gonadotrophin supplementation to the culture medium was tested on frozen-thawed oocyte-granulosa cell complexes only. In cultures supplemented with both FSH and LH or FSH alone, there was no significant difference in the number of oocyte-granulosa cell complexes that formed antral cavities (18 +/- 7%). However, antrum formation was significantly less frequent in cultures lacking gonadotrophin stimulation (7 +/- 4%). All oocyte-granulosa cell complexes maintained a three-dimensional structure throughout culture and developed a functional P450 aromatase enzyme complex, as revealed by the induction of oestradiol production during 8 days of culture after antrum formation in serum-free medium containing testosterone. Oocytes recovered after 30 days of culture were viable and had increased in diameter from 78 +/- 2 microns on the day of isolation, to 131 +/- 3 microns at the end of culture. These results show that oocyte-granulosa cell complexes isolated from cryopreserved ovarian tissue can be grown to antral size in vitro with similar efficiency to those isolated from fresh tissue.