小鼠胚胎干细胞在六种培养体系的培养观察

目的　观察小鼠胚胎干细胞在六种培养体系中的生长情况。方法　小鼠胚胎干细胞 (ESD3细胞株 )在以下六种培养体系中培养 :1 .原代小鼠胚胎成纤维细胞 (MEF)有血清培养 ,2 .MEF无血清培养 ,3.SNL细胞有血清培养 ,4.LIF(白血病抑制因子 )有血清无饲养层培养 ,5.LIF无血清无饲养层培养 ,6.大鼠肝细胞 (BRL)条件培养基培养。经体外培养 1 0代后 ,观察其克隆形态 ,同时进行碱性磷酸酶检测并将ES细胞接种于裸小鼠皮下 ,观察ESD3的未分化状态和多潜能性。结果　六种培养体系培养的ESD3具有典型的ES细胞克隆形态 :巢状 (集落状 )隆起生长 ,边缘清楚 ,表面平滑 ,结构致密 ;AKP强阳性 ;裸小鼠体内形成了由多种组织构成的畸胎瘤。结论　六种培养体系均能支持ESD3生长 ,并能保持其未分化性和多潜能性 ,为ES细胞的应用研究奠定了良好的基础。     

用BRL-3A条件培养基培养鸡胚胎干细胞的初步研究

以CEF细胞、MEF细胞和STO细胞为饲养层,用:BRL-3A条件培养基培养寿光鸡X期胚盘细胞,并对获得的细胞克隆进行了鉴定,证明其具有ES细胞的克隆形态、PAS染色阳性、AKP染色阳性、能分化为多种类型的细胞、能参与受体胚胎的发育并形成羽色嵌合体.实验结果表明:BRL-3A条件培养基能促进鸡ES细胞克隆的形成,用饲养层比不用饲养层更有利于克隆的形成,且STO细胞和MEF细胞饲养层的培养效果要好于CEF细胞饲养层(P<0.05);挑克隆传代法比全消化法更有利于克隆的形成和未分化状态的维持(JP<0.05).     

大鼠心脏细胞条件培养基对小鼠ES细胞特性的维持

以C19-2和MESPU-13为供试细胞,用克隆测试、传代培养等方法对17种细胞的条件培养基进行了筛选,结果表明,大鼠心脏细胞的条件培养基（RH-CM）具有显著抑制小鼠ES细胞分化、维持其二倍体核型、促进ES细胞贴壁生长的作用。经RH-CM培养10代和20代的小鼠ES细胞在体内外分化能力上仍保留了原ES细胞的多方向分化潜能和特征;RH-CM也可作为小鼠ES细胞培养基的添加物,用含70%RH-CM的ES细胞培养基和小鼠胚胎原代成纤维细胞饲养层（PMEF）培养ES细胞,可长期有效地维持其未分化状态和二倍体核型。RT-PCR检测到大鼠心脏细胞有LIF mRNA表达。     

bFGF诱导MEF生成的条件培养基对人胚胎干细胞生长的影响

为了探讨低浓度bFGF诱导小鼠胚胎成纤维细胞（MEF）生成的条件培养基对人胚胎干细胞（hESC）生长分化的影响,以系列浓度的bFGF作用于MEF上,收集条件培养基（bFGF—MCM）,用于hES2细胞的无滋养层培养。以不添加bFGF而收集的MEF条件培养基（MCM）为阴性对照．同样浓度的bFGF添加于SR培养基（bFGF—SR）为空白对照。通过形态学特征和碱性磷酸酶染色法对hES2的生长分化状态进行评估。结果发现．培养一周内,未分化hES2克隆的比率,阴性对照为23％：空白对照组为13％-31％。当bFGF浓度为0．1,0．3,1,4ng／ml时,bFGF—MCM组未分化克隆的比率分别为44％,74％,77％和78％,与阴性和空白对照组相比,未分化克隆的比率均有不同程度提高．差异有统计学意义（P〈0．01）。该结果揭示了经bFGF诱导的MEF细胞所产生的条件培养基具备了维持hES细胞正常生长而不分化的能力。对bFGF—MCM的深入分析．有望更好地了解hESC的生长与分化机制。     

分离和克隆小鼠ES细胞集落的主要影响因素

目的　研究不同培养条件分离和克隆小鼠ES细胞集落的效率。方法　以PMEF饲养层、NIH3T3细胞饲养层或培养液中加入LIF为培养条件 ,分离和克隆昆明小鼠ES细胞集落 ,比较其效率。结果　饲养层的培养条件明显优于培养液中加入LIF的培养条件 ;有饲养层的培养条件下 ,桑椹胚的ES细胞集落出现率显著低于囊胚 ;两种饲养层培养囊胚 ,其ES细胞集落的出现率差异无显著性。结论　以PMEF或NIH3T3细胞作饲养层 ,培养昆明小鼠的囊胚 ,适时离散ICM ,是比较理想的分离ES细胞集落的方法。     

BALB/c小鼠胚胎干细胞系建立的方法学探讨

以小鼠胚胎成纤维细胞（ME）为饲养层,以大鼠心脏细胞条件下培养基（RH－CM）为ES细胞培养基,全面、详尽地对BALB／c小鼠ES细胞的纱和培养方法进行了探讨,成功地建立了一套建立和培养BALB/c小鼠ES细胞系的新方法。这一培养条件不但有效地维持了ES细胞的未分化状态和正常二倍体核型,而且维持了其作为能性胚胎干细胞的一系列特征。实验设计了两种离散方法和两种浓度的消化兴,用来离散增殖的ICM和ICM离散后出现的ES集落。两种离散方法即“一次离散法”,和“多次离散法”,两种浓度的消化液即0．25％。Trypsin－0．04％。EDTA和．005％。Trypsin-0.888％EDTA;同时对ICM离散时机、RH－CM在BALB／c小鼠ES细胞建系和培养中作用进行了探讨。结果表明：在低浓度消化液作用下,采用“多次离散法”离散增殖4天的ICM和ES集落的方法建立BALC／c小鼠的ES细胞系是理想的;从细胞形态、集落形态、增殖生长能力、核型检测、碱性磷酸酶测定以及体内外分化能力表明,所建立的9个BALB/c小鼠ES细胞系符合小鼠胚胎干细胞的一系列特征。     

615小鼠ES细胞集落的分离及初步鉴定

目的　分离和培养 6 15小鼠的ES细胞集落 ,为建系打下基础。方法　以PMEF为饲养层分离 6 15小鼠的ES细胞集落 ,进行无饲养层培养 ,并对其进行初步鉴定。结果　ES细胞集落的出现率和传代成功率为 2 2 6 %和 0 94 % ,其ALP染色阳性 ,具有稳定的二倍体核型 ,可自发分化为多种类型的细胞。结论　成功分离和培养了6 15小鼠的ES细胞集落     

小鼠胚胎干细胞的培养

ＥＳ细胞的培养应满足促进细胞增殖和抑制细胞分化而保持其高度未分化潜能。我们用胚胎成纤维细胞作为饲养层,培养基ＤＭＥＭ中加白血病抑制因子的方法来培养ＥＳ细胞。培养的ＥＳ细胞呈集落状生长,细胞排列紧密,呈未分化状态。胚胎成纤维细胞作为饲养层是分离培养ＥＳ细胞最普遍使用而且有效的方法。本文还围绕ＥＳ细胞培养中的促进增殖和抑制分化这两个方面的影响因素进行了讨论。     

饲养层细胞对绵羊胚胎干细胞体外培养的影响

家畜胚胎干细胞(embryonic stem cell,ES细胞)的研究进展缓慢,绵羊ES细胞的研究虽早有报道,但仍未建立可稳定传代的细胞系。在已建立的绵羊体外受精发育体系的基础上,摸索了饲养层(Feeder)细胞对绵羊ES细胞生长的影响,包括在一定的丝裂霉素浓度下处理Feeder的时间、细胞种类、代数、接种密度及新鲜制备和冷冻复苏后的Feeder细胞,通过试验比较研究,目的在于筛选合适的饲养层细胞,为建立绵羊ES细胞体外培养体系奠定基础。结果表明,10μg/ml丝裂霉素C处理2~2.5h获得的1~5代的SEF和1~3代的MEF及两者的1∶1混合细胞都能较好地支持绵羊ES细胞的生长。     

用饲养层分离胚胎干细胞集落的实验初探

目的 用饲养层分离胚胎干细胞集落。方法 用胚龄为13~14 d的小鼠胚胎分离原代成纤维细胞,制成饲养层,用于囊胚的培养。结果 小鼠原代胚胎成纤维细胞（PMEF）贴壁能力较好,增殖快,易铺层。囊胚和内细胞团（ICM）在饲养层上贴壁生长良好,当培养4~5 d时,其增殖率为16/28（57%）。在ICM离散48 h后,各种胚胎干细胞（ES）集落开始出现。此种集落经碱性磷酸酶染色成阳性。结论 用饲养层分离胚胎干细胞获得初步成功。     

Effects of feeder layer and BRL conditioned medium on mouse embryonic stem cells

In vitro growth and maintenance of embryonic stem (ES) cell lines derived from ICM cells of various blastocysts of 129 strain mice,the sustenance of their pluripotency and normal karyotype depend on the feeder layer of mouse embryonic fibroblasts (MEF).Compared with the feeder layer of MEF cells,medium conditioned by Buffalo rat liver cells (BRL-CM) is able to maintain pluripotency and karyotypic normality of ES cells only in short term cell propagation.Besides,ES cells grown in BRL-CM are also capable of aggregation with 8-cell embryos of Swiss strain and develop into germ line chimaeras.Modification to the method of aggregating ES cells with early embryos by making a hole in agar layer on the top of MEF feeder cells was shown to be more converient and efficient than the conventional microdrop method.     

Influence of feeder layer type on the efficiency of isolation of porcine embryo-derived cell lines

Experiments were conducted to determine the effects of feeder layers composed of different cell types on the efficiency of isolation and the behavior of porcine embryo-derived cell lines. Inner cell masses (ICM) isolated from 7- to 8-d-old embryos were plated on feeder layers composed of Buffalo rat liver cells (BRL), a continuous cell line of murine embryonic fibroblasts (STO), STO combined with BRL at a 9:1 and 1:1 ratio, STO with BRL-conditioned medium (STO + CM), porcine embryonic fibroblasts (PEF), PEF combined with BRL at a 9:1 and 1:1 ratio, porcine uterine epithelial cells (PUE), murine embryonic fibroblasts (MEF), or an epithelial-like porcine embryo-derived cell line (PH3A). It was found that embryo-derived cell lines could be isolated only from the STO and the STO with BRL-conditioned medium treatments. The isolated cell lines were of epithelial-like and embryonic stem cell-like (ES-like) morphology. The feeders tested had an effect on the behavior of plated ICM. Some feeders, represented by PUE, BRL, STO:BRL (1:1), PEF:BRL (1:1), and PH3A, did not promote attachment of the ICM to the feeder layer; others, represented by STO and MEF, allowed attachment, differentiation and proliferation. On PEF feeders the ICM spread onto the feeder layer after attachment without apparent signs of proliferation or differentiation. None of the feeders tested increased the efficiency of isolation or the growth characteristics of embryo-derived (both ES-like and epithelial-like) cell lines over that of STO feeders.     

Use of BRL-conditioned medium in combination with feeder layers to isolate a diploid embryonal stem cell line

Summary The derivation of a karyotypically normal embryonal stem (ES) cell line, E14, from inner cell masses (ICMs) isolated by immunosurgery from 129/Ola late mouse blastocysts is described. Disaggregated ICMs were cultured on mitotically-arrested fibroblast feeder layers in droplets of medium conditioned with Buffalo rat liver (BRL) cells under oil. BRL-conditioned medium inhibits the differentiation of established embryonal carcinoma (EC) and ES cell lines which can be maintained indefinitely in the complete absence of feeder cells (Smith and Hooper 1987). At clonal densities, however, a combination of BRL-conditioned medium and a feeder layer was most effective in preventing the differentiation of E14 cells. This effect was less pronounced at higher passage suggesting it may be particularly important to use a combination in the early stages of isolation. Once established, E14 has been maintained in BRL-conditioned medium alone. In non-conditioned medium on agarose, E14 cells formed embryoid bodies which when allowed to reattach differentiated into a wide variety of tissues. An HPRT-deficient sub line of E14, E14TG2a, has been demonstrated to form germline chimaeras with high efficiency after injection into blastocysts (Hooper et al. 1987). The modifications to the ES cell isolation procedure described here may improve the efficiency with which karyotypically normal lines can be derived.     

潮霉素抗性的小鼠胚胎干细胞滋养层的制备

目的：获得潮霉素抗性的小鼠胚胎干细胞滋养层。方法：利用基因组整合了潮霉素B磷酸转移酶基因的小鼠品系Smad4hygro＋/-,从受精14d的小鼠胚胎中制备原代胚胎成纤维细胞;鉴定基因型后,挑选一株潮霉素B磷酸转移酶基因杂合型胚胎成纤维细胞,用丝裂霉素C处理,以获得潮霉素抗性的滋养层细胞。结果：经潮霉素B加压处理后,杂合型滋养层细胞可存活2周左右,并可维持胚胎干细胞的生长。结论：制备的滋养层细胞可用于潮霉素抗性胚胎干细胞的筛选。     

Culturing in vitro produced blastocysts in sequential media promotes ES cell derivation

Embryonic stem (ES) cell lines are routinely derived from in vivo produced blastocysts. We investigated the efficiency of ES cells derivation from in vitro produced blastocysts either in monoculture or sequential culture. Zygotes from hybrid F1 B6D2 mice were cultured in vitro to the blastocyst stage in Potassium (K(+)) simplex optimised medium (KSOM) throughout or in KSOM and switched to COOK blastocyst medium on day 3 (KSOM-CBM). Blastocysts were explanted on a feeder layer of mitomycin C-inactivated murine embryonic fibroblasts (MEF) in TX-WES medium for ES cell derivation. Sequential KSOM-CBM resulted in improved blastocyst formation compared to KSOM monoculture. ES cells were obtained from 32.1% of explanted blastocsyts cultured in KSOM-CBM versus 18.4% in KSOM alone. ES cell lines were characterized by morphology, expression of SSEA-1, Oct-4 and alkaline phosphatase activity, and normal karyotype. These results indicate that in vitro culture systems to produce blastocysts can influence the efficiency of ES cell line derivation.     

Feeder cell density—A key parameter in human embryonic stem cell culture

Summary A key issue in human embryonic stem (ES) cell culture that has largely been ignored is the high degree of variability in the murine embryonic fibroblast (MEF) feeder cell density, which has been reported by different studies and protocols. Presumably, too low a feeder cell density would result in insufficient levels of secreted factors, extracellular matrix, and cellular contacts provided by the feeder cells for the maintenance of human ES cells in the undifferentiated state. Too high a feeder cell density, on the other hand, may result in a more rapid depletion of nutrients and oxygen within the in vitro culture milieu, as well as physically hinder the attachment and growth of ES colonies during serial passaging. Preliminary investigations by our group revealed that an elevated MEF cell density of 32,000 cells/cm², above the recommended value of 20,000 cells/cm², appeared to be highly detrimental to the attachment and growth of serially passaged ES colonies of the H9 line (WiCell Research Institute Inc., Wilmington, MA, USA). At the edge of ES colonies that have attached to the higher density feeder layer (32,000 cells/cm²), the ES cells appear to stack up to form a “bulge.” This was not observed under the recommended feeder cell density of 20,000 cells/cm². By contrast, other established ES cell lines are routinely propagated at much higher feeder densities of 60,000 to 70,000 cells/cm². This report briefly discusses the issue of MEF feeder cell density in relation to our preliminary observations, and the results of other studies.     

Isolation and culture of embryonic stem cells from porcine blastocysts

This study was conducted to establish embryonic stem (ES) cell lines from porcine blastocysts. Blastocysts were collected from China miniature pigs at day 7-9 of pregnancy. Embryos were either directly (intact embryos) cultured on mitomysin C-inactivated murine embryonic fibroblasts (MEF) as feeder layers, or were used to isolate the inner cell masses (ICM) by enzyme digestive method and then cultured. It was found that enzyme digestive method could isolate ICMs without any damages of cells in all blastocysts (28). All ICMs attached to the feeder layers. Primary cell colonies were formed in 68% of ICM culture and 28% of intact blastocyst culture. Two ES cell lines derived from ICM passed six subcultures (passages). These cells morphologically resembled mouse ES cells and consistently expressed alkaline phosphatase activity. When the ES cells were cultured in a medium without feeder layer and leukemin inhibitory factor, they differentiated into several types of cells including neuron-like, smooth muscle-like, and epithelium-like cells. Some cells formed embryoid bodies in a suspension culture. These results indicate that porcine ES cell line can be established under the present experimental conditions and these ES cells are pluripotent.     

In vitro development of bovine embryos in Buffalo rat liver- or bovine oviduct-conditioned medium

A culture system for bovine embryos was developed using Buffalo rat liver cell (BRL) line-conditioned medium without serum. Zygotes, obtained by in vitro maturation and fertilization of oocytes, were cultured either in unconditioned medium (TCM 199 or DMEM/F12) or in the same medium conditioned by bovine oviduct or BRL cells. No serum was added during conditioning or during embryo culture. The DMEM/F12 medium was superior to TCM 199 for development of bovine embryos to the 5 to 8-cell stage: on average between 50 and 57% of the embryos reached this stage after 2 d of culture in DMEM/F12 or in conditioned medium, while 36% reached this stage in TCM 199. Further development to the blastocyst stage was enhanced by conditioning. The highest percentage of blastocysts was achieved in DMEM/F12 medium conditioned with BRL cells (30%). The yield of blastocysts was similar in TCM 199 and in DMEM/F12 media conditioned with bovine oviduct cells (22 versus 20%), but after conditioning with BRL cells, DMEM/F12 medium yielded a higher percentage of blastocysts than TCM 199 (30 versus 18%). This might be explained by the fact that viability of BRL cells was better in DMEM/F12 medium than in TCM 199 when serum was omitted. Blastocysts produced in BRL-conditioned medium had a higher number of cells than blastocysts obtained in bovine oviduct-conditioned medium, and their transfer to recipients led to pregnancies and birth of calves. In conclusion, culture of bovine embryos in DMEM/F12 medium conditioned with BRL cells without serum led to the development of good-quality blastocysts and is thus a promising method for producing embryos for the study of potential embryotrophic factors. The use of rat liver cell lines guarantees against bovine viruses and allows for better production of embryos.