RESUMO
During cryopreservation of ovarian tissue, the conditions of freezing and thawing are big factors controlling the survival rate of oocytes obtained. However, the conditions and procedures as they pertain to ovarian follicles and oocytes have not been established. Thus, we tried to determine the appropriate freeze-thaw times using the vitrification method with ethylene glycol and DMSO as cryoprotective agents and dd Y female mouse ovaries. The maturity rate from GV to the metaphase-II (MII) stage was 62.8% with ethylene glycol and 69.3% using DMSO, while the controls (GV oocytes obtained from a fresh ovary) showed a maturation rate of 83.6% (46/55). MII oocytes obtained by culturing GV oocytes in vitro showed a 64.3% (18/28) fertility rate via in vitro fertilization and a developmental rate into a 2 cell stage embryo of 35.7% (10/28) and into a 4-cell stage, 7.1% (2/28). However, development beyond the 8 cell stage embryo was not observed. A significant difference was not recognized between control (fresh) and ovarian tissues that had been frozen/thawed with respect to their ability to produce hormones. It is concluded that the vitrification method was effective for both freezing ovarian tissues and preserving its functional ability (maturation and capacitation).
Assuntos
Criopreservação/métodos , Oócitos , Ovário , Animais , Células Cultivadas , Crioprotetores , Dimetil Sulfóxido , Desenvolvimento Embrionário e Fetal , Estradiol/metabolismo , Etilenoglicol , Feminino , Fertilização , Camundongos , Microscopia Eletrônica , Oócitos/fisiologia , Oócitos/ultraestrutura , Ovário/fisiologia , Ovário/ultraestrutura , Progesterona/metabolismoRESUMO
The cell line designated HHUS was established from human uterine cervical keratinizing squamous cell carcinoma. The HHUS cell line was subcultivated more than 70 times within 3 years. The cultured cells, polygonal or spindle, with neoplastic and pleomorphic features, appeared epithelial in shape, with a pavement-like arrangement and grew in multi-layers without contact inhibition. The chromosome number was varied from 40 to 88, and the modal number was stable in diploid range. The cultured cells produced keratinizing squamous cell carcinomas by heterotransplantation into the subcutis of nude mice. The HHUS cells were characterized as producing large amounts of SCC, in vitro and possessing HPV-59 DNA genomes.
Assuntos
Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/virologia , Técnicas de Cultura de Células/métodos , DNA Viral/isolamento & purificação , Papillomaviridae/isolamento & purificação , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/virologia , Animais , Antígenos de Neoplasias/análise , Carcinoma de Células Escamosas/genética , Linhagem Celular Tumoral , Mapeamento Cromossômico , Meios de Cultura/química , Citocinas/análise , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Transplante de Neoplasias , Papillomaviridae/classificação , Papillomaviridae/genética , Reação em Cadeia da Polimerase , Sorotipagem , Neoplasias do Colo do Útero/genéticaRESUMO
Regenerative medical treatment with embryonic stem cells (an ES cell) is a goal for organ transplantation. Structures that are tubular in nature (i.e. blood capillaries) were induced from early embryonic stem (EES) cells in vitro using embryotrophic factor (ETFs). In addition, cardiac muscle cells could be identified as well. However, differentiation of EES cells into a complete cardiovascular system was difficult because 3 germ layer primordial organs are directed embryologically in various ways and it is not possible to guide only cardiovascular organs. Thus, we introduced ETFs after the formation of an embryoid body and were successful in cloning cell clusters that beat, thus deriving only cardiovascular organs. The application of this to the treatment of various cardiovascular diseases is promising.
Assuntos
Sistema Cardiovascular/embriologia , Embrião de Mamíferos/citologia , Organogênese , Células-Tronco/citologia , Animais , Diferenciação Celular , Células Cultivadas , Células Clonais , Feminino , Camundongos , Camundongos EndogâmicosRESUMO
Cryopreservation of ovarian tissues containing many immature oocytes occurs in both gamete/embryo research and clinical medicine. Using vitrification, we studied factors related to meiosis after cryopreservation using the COCs (cumulus oocyte complexes) and preantral follicles obtained from cryopreserved ovarian tissues. COCs were isolated and cultured for 17 approximately 19 hr. Thereafter, Metaphase II stage (MII stage) oocytes and fertilized oocytes after IVF were observed at a rate of 76.5% and 60.0%, respectively. Preantral follicles (100 approximately 130 microm in diameter) were isolated and cultured in alpha MEM containing hFSH, ITS, and FBS. HCG and EGF were added to the media to stimulate ovulation on the 12th day of culture. The survival rates of the follicles obtained from the frozen/thawed ovaries were 66.4%. After 12 days of culture, the diameter of the follicles isolated from fresh (620.2 +/- 11.3 microm) and frozen/thawed ovaries (518.7 +/- 15.1 microm) differed as did the estradiol concentrations (3474.2 +/- 159 pg/ml vs. 1508.2 +/- 134 pg/ml). After in vitro ovulation, MII stage oocytes were observed in 84.5% of the fresh group and 60.5% of the frozen/thawed group while the fertilization rate was 74.2% and 53.5%, respectively. These studies demonstrate that cryopreservation of mouse ovarian tissues by vitrification did not affect the oocyte's ability to undergo meiosis. Thus, this technique may become a powerful tool for the preservation of the female gamete.
Assuntos
Criopreservação/métodos , Meiose , Metáfase , Oócitos/citologia , Folículo Ovariano/citologia , Ovário/citologia , Animais , Separação Celular , Sobrevivência Celular , Células Cultivadas , Meios de Cultura , Feminino , Fertilização in vitro , Camundongos , Camundongos Endogâmicos , Ovário/fisiologia , OvulaçãoRESUMO
A cell line designated HUUCLEC was established from a human uterine cervical lymphoepithelial carcinoma obtained from a 61-year-old Japanese woman. The cell line has grown slowly without interruption and serial passages were successively carried out 60 times within 3 years. The cultured cells were spindle or round in shape, showing anaplastic and pleomorphic features, a pavement cell arrangement and multilayering without contact inhibition. The population doubling time of the HUUCLEC line was 72 hours while the chromosomal number varied widely and showed aneuploidy. The modal chromosomal number was stable at the triploid range and marker chromosomes were present; the Ebstein-Barr virus was absent in the cultured cells.
Assuntos
Carcinoma de Células Escamosas/patologia , Neoplasias do Colo do Útero/patologia , Animais , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/virologia , Cricetinae , Feminino , Humanos , Cariotipagem , Mesocricetus , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Transplante de Neoplasias , Papillomaviridae/isolamento & purificação , Células Tumorais Cultivadas , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/virologiaRESUMO
A cell line with the characteristics of hepatocytes was established from rat early embryonic stem cells (REES). This cell line was established using a new novel method of Ishiwata et al. from two cell embryos taken from the spontaneous dwarf rat (SDR). The hepatocyte cell line (REES-hep) was instituted from dark red colored tissue in embryos during embryogenesis using REES cell line cultured in the presence of embryotrophic factors. These cell lines were cultured with DMEM/F12 medium supplemented 10% FBS and 1 ng/ml of LIF. They were found to maintain their diploid state, were characterized with 42 normal chromosomes and proliferated to confluence; contact inhibition was also present. These cells produced albumin when cultured using a collagen sponge gel system and reconstructed in a funicular form resembling the cell cords of liver. The cells also produced albumin and bilirubin when transplanted into the spleen of SDR Reconstruction of a REES-hep cell line from early embryonic stem cells should help in treating hepatic insufficient patients. It will be valuable for further research, as an introduction to cell transplantation and application for use in a bio-hybrid typed liver apparatus.