骨髓间充质干细胞无血清培养

为建立一种化学成分明确的、能用于体外扩增骨髓间充质干细胞的无血清培养基, 且骨髓间充质干细胞经无血清培养扩增后仍能保持其多向分化的潜能。采用密度梯度离心结合贴壁法从1月龄新西兰大白兔股骨中分离骨髓间充质干细胞, 比较在含10%胎牛血清的培养基(SCM)和自制的化学成分明确的无血清培养基(CDSFM)中骨髓间充质干细胞的形态、增殖能力, 以及扩增后的骨髓间充质干细胞的细胞周期、集落形成能力和成骨、成脂肪分化能力。经过10 d的培养, 骨髓间充质干细胞在自制的无血清培养基中扩增了50倍, 在含10%胎牛血清的培养基中扩增了40倍。在无血清和有血清培养基中扩增后的细胞中G0/G1期比例分别为(80.31%±0.6%)和(75.24%±4.0%), 两者无显著差异(P>0.05)。无血清培养扩增后的骨髓间充质干细胞集落形成率(12.7%±4.0%)低于有血清培养组(28.7%±4.2%), 两者比较差异显著(P<0.01)。经过无血清培养扩增的骨髓间充质干细胞在成骨、成脂肪诱导分化培养基中能够分化成成骨和脂肪细胞。自制的化学成分明确的无血清培养基能够在体外培养扩增骨髓间充质干细胞, 并且维持其干细胞特性, 可以用于细胞治疗以及生物医学研究。     

In vitro proliferation and terminal differentiation of quail neural crest cells in a defined culture medium

We report the formulation of a culture medium, medium MCDB202-21, that supports the in vitro proliferation of quail neural crest cells and their differentiation into melanocytes and adrenergic neuroblasts in the complete absence of serum and chick embryo extract. McKeehan & Ham's medium MCDB 202 was supplemented with hormones, stimulators of metabolism, vitamins, trophic factors, transport molecules, and small molecular nutrients.     

In vitro culture of shoots of Pinus caribaea on a liquid medium

Shoot apices of a clone of Pinus caribaea Morelet were cultured and multiplied in vitro by supporting them with their basal cut ends immersed in a liquid nutrient medium.The initial heights of explants and their initial numbers of leaves were positively correlated with the numbers of buds and shoots produced by the explants after a bud induction phase and after a shoot elongation phase. The final numbers of buds and shoots were positively correlated with reductions in the quantities of phosphorus detected in the media and negatively correlated with the numbers of brown leaves produced on the explants.In a comparison between the growth of shoot explants on liquid and solid media, shoots incubated on the liquid medium showed significantly greater increases in length in a four-week period than those cultured on solid medium.This technique, using liquid media, provides a system in which both the nutrient utilization and the growth rates of isolated pine tissues can be readily assessed. Furthermore, the multiplication rate of the tissue can be predicted following the observation of correlated characters early in the micropropagation cycle.     

In vitro development of theDrosophila chorion in a chemically defined organ culture medium

Summary TheDrosophila chorion is produced normally in isolated follicles in Robb's chemically defined culture medium. The complex architecture of the shell developed in vitro from follicles as young as early stage 10 is completely normal morphologically. In addition, the time required for in vitro development closely approximates that observed for in vivo development. Comparisons of insect culture media developed by Robb, Grace, Schneider, and Echalier show large variations in their ability to supportDrosophila chorion development.     

Mouse oocyte maturation in vitro at a low concentration of pyruvate in the culture medium

Mouse oocytes isolated from antrum--containing follicles were cultivated for 17-19 hours in the media with different concentrations of pyruvate. Decrease of pyruvate concentration down to 0.03 mM significantly increased the number of oocytes with intact germinal vesicles. However, in 50% of oocytes desintegration of nuclear membrane and condensation of chromosomes occurred even at a concentration of 0.02 mM.     

In vitro cultivation of Dipetalonema viteae third-stage larvae: evaluation of culture media, serum, and other supplements

Third-stage larvae of Dipetalonema viteae obtained from the tick vector developed to the fourth stage in several cell-free culture systems. Survival and development of larvae in a number of commercially available cell culture media, supplemented with serum and other defined and undefined components, were compared. All cultures were gassed with 5% carbon dioxide in nitrogen. Best survival, growth and development were obtained in stationary cultures containing 1:1 (v/v) mixtures of NCTC 135, either RPMI 1640 or Iscove's Modified Dulbecco's Medium, and a supplement of 20% non-heat-inactivated fetal bovine serum. The importance of the medium composition and physical environment of the culture system, for the survival, growth and development of D. viteae was demonstrated.     

In vitro culture of tetrathyridia of Mesocestoides corti using a gel based diphasic medium

Tetrathyridia of Mesocestoides corti were cultured in vitro in a diphasic medium consisting of a liquid medium (CMRL Sigma) and a thixotropic nutrient gel (Oxoid). Tests demonstrated that a 50% medium/gel mixture produced optimum conditions for the survival and development of tetrathyridia. Established anthelminthic drugs were inoculated into the gel which demonstrated that this system can be used for preliminary anthelminthic drug screening. The development and survival of the tetrathyridia were influenced by the addition of pepsin, trypsin and liver peptone to the culture media. The development and maturation of proglottids were observed in addition to asexual reproduction by the process of budding. Tetrathyridia maintained in vitro and reinfected into both mouse and rat hosts retained their viability.     

In vitro culture of mouse primordial germ cells

Germ cells were isolated from mouse fetal gonads 11 1/2-16 1/2 days post coitum (dpc), and exposed to various methods of in vitro culture. From 13 1/2 dpc onwards, both male and female germ cells survived well at 37 degrees C for several days. During the culture period the proportion of female germ cells in meiosis increased and later stages of meiotic prophase were seen. The gonadal environment is therefore not essential for the progress of meiosis. Male germ cells in vitro did not enter meiosis. Germ cells isolated from gonads 11 1/2 or 12 1/2 dpc did not survive at 37 degrees C in any of the three culture systems used (Petri dishes, microtest plate wells, drops under oil); cell density, substrate and culture medium were varied, and several additives tested, but no improvement in viability was detected. Below 30 degrees C, on the other hand, 11 1/2 and 12 1/2 day germ cells survived in vitro for at least a week. They did not enter meiosis in culture, but continued to undergo mitotic proliferation.     

漏斗多孔菌液体菌种培养基及其栽培条件

为实现漏斗多孔菌资源化利用,设计单因素试验,以菌丝生物量、菌球密度和菌球直径为指标,获得漏斗多孔菌Polyporus arcularius液体菌种培养基配方为马铃薯(去皮)200g、玉米粉20.0g、蛋白胨5.0g、KH2PO43.0g、 K2HPO41.0g,MgSO4·7H2O 1.5g,初始pH 5.0,并优化培...     

In vitro growth characteristics of rat mesothelioma cells in culture

The study reports morphological growth characteristics and chromosome analysis of neoplastic rat pleural mesothelial cells (RPMC) isolated from a mesothelioma-bearing rat. The pleural mesothelioma was induced by intrapleural injection of chrysotile fibers. Neoplastic RPMC were cultured by the standard methods used for normal RPMC. Neoplastic RPMC cultures had a population doubling time of 19 hr versus 30 hr for the normal cells. Plating efficiency in liquid medium was almost 100%. Cultures of neoplastic RPMC were anchorage-independent since 70% of the seeded cells formed colonies after one week; after the second week, colony size was enhanced but colony recovery was not. The serum dependence of neoplastic cells was less than that of the normal cells. 79 out of 100 metaphase cells analyzed had 41 to 43 chromosomes, and the modal number was 42 (38%). A large metacentric chromosome was observed in 77 of the 100 neoplastic metaphase cells analyzed, but not in any normal metaphase cells. In nude mice, the neoplastic RPMC were tumorigenic.     

In vitro cultivation of an insect Microsporidian Tubulinosema ratisbonensis in mammalian cells

Tubulinosema ratisbonensis is a microsporidian pathogen of Drosophila melanogaster belonging to the family Tubulinosematidae. The microsporidia in this family mainly cause infections in invertebrate hosts, but two members of this family, Brachiola vesicularum and Brachiola algerae, have been found to cause infections in humans as well. Moreover, B. algerae can be transmitted to immunodeficient mice and grows in mammalian cell cultures. Thus, the examination of the opportunistic properties of other members of the family Tubulinosematidae is important. Spores of T. ratisbonensis, isolated from infected fruit flies, were used to inoculate mammalian and insect cell cultures. Parasite growth was only seen in human lung fibroblasts. No growth was seen in Vero cells or insect cell cultures. Comparison of growth kinetics at 31 degrees C and 37 degrees C showed that there were fewer and smaller parasitic foci in cultures incubated at 37 degrees C. Transmission electron microscopy revealed the typical ultrastructure of T. ratisbonensis, and scanning electron microscopy showed oval or slightly pyriform spores, with some spores having extruded their polar tubes. The PCR-amplified sequences of rDNA fragments from infected cell cultures were 100% identical to the original T. ratisbonensis rRNA sequence. As T. ratisbonensis is able to proliferate in mammalian cell cultures, it may have the opportunistic properties of other members of the family Tubulinosematidae.     

In vitro cultivation and differentiation of fetal liver stem cells from mice

During embryonic development, pluripotent endoderm tissue in the developing foregut may adopt pancreatic fate or hepatic fate depending on the activation of key developmental regulators. Transdifferentiation occurs between hepatocytes and pancreatic cells under specific conditions. Hepatocytes and pancreatic cells have the common endodermal progenitor cells. In this study we isolated hepatic stem/progenitor cells from embryonic day (ED) 12-14 Kun-Ming mice with fluorescence-activated cell sorting (FACS). The cells were cultured under specific conditions. The cultured cells deploy dithizone staining and immunocytochemical staining at the 15th, 30th and 40th day after isolation. The results indicated the presence of insulin-producing cells. When the insulin-producing cells were transplanted into alloxaninduced diabetic mice, the nonfasting blood glucose level was reduced. These results suggested that fetal liver stem/progenitor cells could be converted into insulin-producing cells under specific culture conditions. Fetal liver stem/progenitor cells could become the potential source of insulin-producing cells for successful cell transplantation therapy strategies of diabetes.     

Isolation,growth requirements,cloning, prostacyclin production and life-span of human adult endothelial cells in low serum culture medium

Summary Endothelial cells from autopsy and biopsy specimens from a variety of adult human vascular tissue were harvested by collagenase treatment and gentle swabbing of the lumenal surface. Nutrient medium MCDB 107 containing a partially purified brain-derived growth factor (5 μg/ml), epidermal growth factor (10 ng/ml) and only 2% (v/v) fetal bovine serum supported clonal and long-term serial culture (17.6 to 26.1 cumulative population doublings) of endothelial cells from vena cava, thoracic aorta and tibial arteries at a 70% rate of success. Cumulative doublings of the cell population from eight cultures were inversely proportional to age of donor of the vascular tissue from which cells were isolated. Heparin had an enhancing effect on cell growth that varied with cell strain. Prostacyclin production of human adult endothelial cell cultures was stimulated by aracidonate and thrombin by 17 to 20 and 2 to 3-fold respectively. Endogenous and stimulated rates of prostacyclin production by human adult endothelial cells were 2 to 3 times that of human adult smooth muscle cells and 20 to 30 times that of human fibroblasts. The work was supported by Public Health Service Grant AGO3275 and Grant No. 1718 from the Council for Tobacco Research. Editor's statement This paper provides an opportunity for relatively rapid, easy growth and cloning of endothelial cells from various human specimens which are more difficult to deal with than those obtained from an intact artery or intact umbilical vein. Russel Ross