共查询到18条相似文献，搜索用时 216 毫秒
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研究不同培养体系对胎牛成纤维细胞体外培养的影响及用牛血清白蛋白代替血清培养胎牛成纤维细胞的可行性。利用M199、DMEM、α-MEM、DMEM/F124种培养体系通过组织块贴壁培养对成纤维细胞体外培养液进行筛选,以α-MEM组细胞生长状况较好。分别用含2、4、6、8、10mg/mL BSA的α-MEM培养液对胎牛成纤维细胞进行原代及传代培养,5种浓度的BSA对原代培养时细胞开始游离出组织块的时间影响不明显,均在培养后的48h有成纤维细胞和上皮细胞混合游离出,但在传代培养时,胎牛成纤维细胞在8mg/mL BSA浓度的α-MEM中贴壁率较高。结果表明:培养胎牛成纤维细胞时,可用BSA代替血清,较适宜的培养体系为含8mg/mL BSA的α-MEM培养液。 相似文献
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荷斯坦奶牛耳组织成纤维细胞分离培养及冷冻保存方法研究 总被引：5，自引：0，他引：5
试验对荷斯坦奶牛耳组织成纤维细胞的分离、体外培养及5种冷冻保存方法进行了研究。结果表明:用DMEM/F12 20%胎牛血清 100IU/mL双抗的完全培养液进行组织块培养,能获得良好的荷斯坦奶牛耳组织成纤维细胞原代培养物;成纤维细胞混合培养物经TrypsinEDTA(0.25%Trypsin,1mmol/LEDTA.4Na)消化所收集到的细胞主要为成纤维细胞,经2~3代传代,可得到纯化的荷斯坦奶牛耳组织成纤维细胞。纯化培养的成纤维细胞在冷冻保护剂和血清成分相同的条件下,选用5种不同的冷冻保存方法进行冷冻保存,其中使用70%细胞悬液 20%胎牛血清 10%DMSO的冷冻保存悬液,先在4℃预冷平衡0.5h,接着在液氮罐口的气态氮中悬挂4h,然后沉入液氮的方法冻存的细胞,解冻后,经台盼蓝染色后进行细胞活力分析,活细胞率为86.68%;培养48h后细胞贴壁率为86.40%,明显高于其他几种方法。 相似文献
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从荷斯坦奶牛乳房无菌采取乳腺组织,通过不同的方法分离、培养、纯化牛乳腺上皮细胞,研究乳腺上皮细胞的体外培养效果.结果表明:组织块接种、0.25%的胰酶和100 u/mL透明质酸酶的混合液37℃消化组织块3 h.可以得到大量细胞用于原代培养.在以DMEM/F12为基础培养液,在培养液中添加10%的胎牛血清、100μg/mL的双抗、表皮生长因子、胰岛素、转铁蛋白、硒酸钠等,组成的完全培养液中奶牛乳腺上皮细胞生长良好.上皮细胞显微结构显示:奶牛乳腺上皮细胞在体外培养过程中舍有不同的细胞类型,大多数上皮细胞呈多角形,细胞之间连接成片,细胞界限明显,部分细胞界限不明显. 相似文献
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马皮肤成纤维细胞的体外培养与冷冻保存 总被引：1，自引：0，他引：1
本研究利用小组织块直接培养法得到了成年马皮肤成纤维细胞的原代培养物,再用酶消化法处理,能够纯化马皮肤成纤维细胞。成纤维细胞的冷冻保存,首先采用手工冷冻法,选用含有不同浓度保护剂如：二甲基亚砜（DMSO）、乙二醇（EG）、甘油（GC））和新生牛血清（NCS）的12种冷冻液对马皮肤成纤维细胞进行冷冻保存;其次用2种冷冻方法对马皮肤成纤维细胞进行冷冻保存,以24 h贴壁率评价冻存效果。结果表明,10% DMSO和20% NCS的DMEM冻存液,对马皮肤成纤维细胞的冻存效果好(24 h贴壁率84.98%)。从冷冻方法来看,程序冷冻法(86.32%）优于手工冷冻法（79.98%)(P<0.05)。 相似文献
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牛胎儿成纤维细胞的分离培养 总被引：1，自引：0，他引：1
研究了牛胎儿组织成纤维细胞的分离、培养、纯化方法和生长特征,并对培养细胞的冷冻保存和复苏进行了观察.采取组织块培养法进行原代培养,在接种第5天到第6天成纤维细胞生长旺盛;用0.25%胰蛋白酶消化,传代培养细胞生长状态良好;用液氮冷冻法保存传代细胞,解冻后持续传代至第12代仍生长良好,第8代细胞冻存前和复苏后的活率分别为97.0% 和94.3%,无显著差异(P>0.05);分离纯化的胎牛成纤维细胞的生长曲线都正常;成功地建立了牛胎儿成纤维细胞系.该细胞可经多次传代培养和冷冻保存. 相似文献
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从胚胎发育阶段、饲养层和培养体系等方面对影响绵羊类ES细胞分离、克隆效率的因素进行探讨。结果显示：致密桑葚胚和囊胚的ICM增殖率高于囊胚和孵化囊胚。绵羊类ES细胞在同源绵羊胎儿成纤维细胞（SEF）上生长比较缓慢,最终传代次数也低于小鼠胎儿成纤维细胞（MEF）组。培养液中同时添加胎牛血清（FBS）和Knock-out血清替代品（KSR）,绵羊类ES传至7代,添加了碱性成纤维细胞生长因子（bFGF）后,最高可传至8代,而单纯添加KSR或FBS,分别传至4代和5代。对类ES细胞进行AKP染色、核型分析、体外分化试验,证实分离的类ES细胞符合ES细胞的主要特征,而且表达多潜能性细胞因子Nanog。由此认为,致密桑葚胚和囊胚更适合绵羊类ES细胞的体外分离和培养,而且MEF更适合于绵羊类ES细胞的分离传代,培养液中添加5%FBS和15%KSR,比较适合类ES细胞的分离传代,bFGF对绵羊类ES细胞的增殖具有促进作用。 相似文献
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本研究旨在建立鸭胚成纤维细胞离体培养、传代以及冷冻保存体系,为鸭胚胎或生殖干细胞的离体培养奠定基础。利用胰酶-EDTA消化鸭胚胎组织,10% FBS+DEME营养液培养,获得原代和传代成纤维细胞。分别用DMSO、甘油以及乙二醇作为冷冻保护剂对F1代鸭胚成纤维细胞进行冷冻保存,检测复苏后细胞的生长情况。利用此方法可以获得高活性的鸭胚成纤维细胞,并可成功传至第三代。其中F1代与F2代细胞活力最好,达到90%以上。3种冷冻剂保存的细胞复苏后均与F1代细胞的活力存在明显差异,均小于80%,其中用DMSO冷冻保存的细胞复苏后活力最强,生长密度保持较好。 相似文献
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奶牛耳成纤维细胞的分离培养及培养条件的研究 总被引：4，自引：0，他引：4
本试验研究了奶牛耳成纤维细胞的体外培养,培养液、消化液、胰岛素和血清饥饿对细胞的影响。采用组织块贴壁法培养。了奶牛耳成纤维细胞,采用0．25％胰酶差别消化获得了纯化的奶牛耳成纤维细胞。用三种不同的培养液即DMEM（低糖）、DMEM（高糖）、DMEM／F12,对细胞进行培养,结果细胞群体倍增时间均在2．3-2．5d,DMEM（高糖）的培养效果稍好。用三种不同的消化液（0．25％胰蛋白酶,0.02％EDTA,0．25％胰蛋白酶＋0．02％EDTA）对传代钿跑进行消化．结果表明0．25％胰蛋白酶＋0D2％EDTA所用时间短,消化后培养细胞贴壁率高。在培养液中添加姨岛素能促进细胞的生长。对指数生长期的细胞进行血清饥饿后,细胞仍保持着较高的活力。 相似文献
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J Risopatrón S Catalán W Miska W-B Schill R Sánchez 《Reproduction in domestic animals》2002,37(6):347-351
Sperm culture media used for in vitro fertilization (IVF) procedures are important factors concerning the viability, motility and acrosomal integrity of spermatozoa. The aim of this study was to investigate the effects of three different sperm diluting media, tissue culture medium (TCM‐199), sperm culture medium (Sp‐TALP) and human tubular fluid (HTF) supplemented with varying concentrations of bovine serum albumin (1, 4 and 6%) or polyvinyl alcohol (0.8%) on the acrosomal integrity, motility and viability of canine spermatozoa. Ejaculates collected from four dogs were diluted in all media and spermatozoa were separated from seminal plasma by the swim‐up technique. Sperm progressive motility was assessed using a phase contrast microscope. Viability and acrosomal integrity were evaluated using a dual stain technique (Giemsa–Trypan blue). The results demonstrated that the number of live canine spermatozoa was similar in culture media supplemented or not supplemented with macromolecules. A minimal concentration of albumin (1%) in the three media showed similar effects on vitality, motility and acrosomal integrity, as had higher concentrations (4 and 6%). The percentage of acrosome‐intact spermatozoa was markedly higher after HTF (94.1%) than after TCM‐199 (70.1%) or Sp‐TALP (71.0%) without supplementation. It is concluded that serum bovine albumin, irrespective of the concentration, preserved sperm viability and function, and HTF is the most suitable medium for preserving the acrosome in canine spermatozoa prepared for in vitro manipulation through short incubation. 相似文献
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This study was conducted to determine the adequate medium for a serum‐free culture system of domestic cat embryos produced by in vitro maturation (IVM) and fertilization (IVF). Cumulusoocyte complexes recovered from cat ovaries were matured in vitro for 24 h, and then inseminated in vitro for 12 h. After insemination, the oocytes were cultured in five media [Ham's F10, Waymouth 752/1 (Waymouth), TCM199, modified Earle's balanced salt solution (MK‐1) and CR1aa], each of which contained 0.4% bovine serum albumin. There were no significant differences among the rates of fertilization of oocytes cultured in five media following IVF. The rate of oocytes/embryos developed to at least the morula stage was significantly lower (p < 0.05) in Waymouth than in MK‐1, TCM199 and CR1aa. Moreover, none of the embryos cultured in Ham's F10 and Waymouth developed to the blastocyst stage. There were no differences among the rates of development to the blastocyst stage of oocytes/embryos cultured in MK‐1, TCM199 and CR1aa. These results indicate that the type of serum‐free medium has a major impact on in vitro development of domestic cat embryos derived from IVM/IVF, and MK‐1, TCM199 and CR1aa media are suitable for in vitro culture of cat embryos in a serum‐free culture system. 相似文献
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牛皮肤成纤维细胞的体外培养与冻存 总被引：10，自引：0，他引：10
利用牛皮肤组织块直接培养法，得到牛皮肤细胞的原代培养物，再用酶消化法和反复贴壁法处理，能够纯化成纤维细胞。成纤维细胞的冻存是通过选用6种分别含有二甲基亚砜（DMSO）、甘油（GL）及乙二醇（EG）的保护液，以相同的冻前处理方法，对牛皮肤成纤维细胞进行缓慢冷冻，冰箱预冷平衡1－2h，逐步投入液氮（－196℃）中保存，再经37℃水浴解冻，Hanks液脱保护剂，以贴壁率评价冻存效果。结果表明，20％DMSO保护液对牛皮肤成纤维细胞表现出较好且稳定的冷冻保护效果，其平均贴壁率达87．9％。 相似文献
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本研究旨在建立转双基因（pGH/IGF-Ⅰ）猪胎儿成纤维细胞系,保存转双基因猪的成纤维细胞以便于后续细胞水平研究。试验采用胰蛋白酶消化法对猪胎儿躯干组织进行原代培养,通过原代培养、细胞传代、冷冻保存等成功分离出转双基因猪胎儿成纤维细胞,并对细胞进行了形态学观察、冻存前和复苏后细胞活力检测、生长动力学分析、波形蛋白免疫组化及微生物污染检测等生物学特性分析。结果显示,原代细胞经过胰蛋白酶消化和差速离心分离培养出成纤维细胞,冻存前细胞活力为94.3%,冻存3个月后细胞复苏后活力为91.2%;细胞生长总趋势呈“S”型,经历了潜伏期、指数生长期和平台期3个阶段;细胞波形蛋白在成纤维细胞中呈阳性反应;细胞的细菌、真菌、病毒和支原体检测均为阴性。结果表明本试验成功建立了转双基因猪成纤维细胞系。 相似文献
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This study was aimed to establish a fetal fibroblast cell line of double transgenic (pGH/IGF-Ⅰ) pigs,and reserve fibroblast cells of double transgenic pigs for the follow-up study.A method of trypsin digestion was adopted to isolate and culture body tissues from pregnant porcine,and porcine fetal fibroblasts were isolated successfully through primary culture,subculturing and cryopreservation.The morphological observation,determination of viability before cryopreservation and after recovery,dynamic growth analysis,vimentin immunohistochemistry and microbial contamination detection were all done to study the biological characteristics of the cell line.The results showed that the fibroblasts were cultured and isolated successfully by trypsin digestion and differential centrifugation.The cell viability before cryopreservation and after recovery were 94.3% and 91.2%,respectively.The growth curve was sigmoidal,and experienced the incubation period,exponential growth period and platform three stages.The vimentin immunohistochemistry was positive,the microbial contamination detection were all negative.The results indicated that a fibroblast cell line of double transgenic porcine was successfully established. 相似文献
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Effects of different media (TCM 199 + BSA, TCM 199 + FCS, TCM 199 + NBCS, Whitten's medium + BSA) supplemented with estradiol-17β and two isolated and everted follicle shells on MPF and MAP kinase activities and the sensitivity to parthenogenetic activation of pig oocytes were examined at the end of culture (48 h). Elevated ( P < 0.05) activities of MAP kinase were recorded in metaphase II oocytes following culture in Whitten's medium, whereas MPF levels were lowest ( P < 0.05) in MII oocytes matured in TCM 199 supplemented with BSA. Oocytes matured in TCM 199 based media showed higher ( P < 0.05) activation rates when compared to oocytes incubated in Whitten's medium. Whitten's medium supplemented with different protein sources (amino acids, FCS, BSA) was used to study the effects of different exposure periods to eCG/hCG stimulation on MPF and MAP kinase activities and in vivo fertilisability following culture for 48 h. MPF and MAP kinase activities were significantly increased by eCG/hCG stimulation of COCs during maturation. Further, the continuous presence of eCG/hCG during culture (48 h) significantly increased the levels of both kinases in comparison to stimulation by gonadotrophins alone during the first 24 h of incubation. In vivo fertilisation of oocytes matured in Whitten's medium supplemented with eCG/hCG for 24 or 48 h led to a significant retardation of early embryonic development compared to ovulated oocytes. In conclusion, media composition and gonadotrophin stimulation affect MPF/MAP kinase activities and the susceptibility to parthenogenetic activation of IVM oocytes. However, elevated kinase levels in pig oocytes following culture do not indicate complete cytoplasmic maturation. 相似文献
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Primary cultures and cryopreservation procedure of bovine brain cells were established as in vitro experimental systems to study the responses of bovine brain cells to neuropathogenic agents. Brain cells were dissociated by papain from the cerebellum of a bovine fetus at 90 to 120 days old, and were cultured in different media. In a medium containing 1 per cent fetal bovine serum (FBS), neuronal cells were maintained and they formed clusters on glial and fibroblastic cell sheets. In a medium containing 10 per cent FBS, the proportion of neurones decreased, and fibroblastic and microglial cells dominated. In a serum-free medium containing epidermal growth factor, the highest neuronal proportion was obtained. Optimal cryopreservation condition for the brain tissues was investigated by changing the concentrations of DMSO and FBS. Brain cells could be cultured from cryopreserved tissue with only slightly reduced growth profiles and varying cell proportions in comparison to those prepared from fresh tissue. 相似文献
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RH Alvarez M Meneghel AC Martinez RML Pires EA Schammass 《Reproduction in domestic animals》2008,43(3):257-260
The aim of this study was to evaluate if blastocysts arising from in vitro culture of Grade 3 bovine morulae produced in vivo can promote acceptable pregnancy rates when transferred into recipients. Embryos of different stages and qualities were recovered from superovulated Bos taurus and B. indicus donors. Grade 3 morulae were cultured in either Holding Plus® or TCM‐199 (supplemented with 10% bovine fetal serum) media for 24 h at 38.5°C. After this culture period, the resulting blastocysts were morphologically classified (Grades 1, 2 and 3) and transferred into recipients previously synchronized with the donors. Non‐cultured Grades 1 and 3 morulae were used as control. Pregnancy diagnosis was carried out 60 days after embryo transfer and the data were analysed by logistic regression, considering variables, such as embryo quality (Grade), donor breed, culture medium, donor‐recipient synchrony and seasonality. Embryo quality was the only variable, showing significant effect on the pregnancy rate. Pregnancy rates for non‐cultured Grade 1 and 3 morulae, and blastocysts arising from cultured Grade 3 morulae were 58.1% (n = 31), 17.1% (n = 35) and 51.1% (n = 47), respectively (p < 0.05). There were no statistical differences between non‐cultured Grade 1 morulae and cultured blastocysts. Pregnancy rates for Grades 1 (65.0%) and 2 (60.0%) were higher than Grade 3 (29.4%) cultured blastocysts (p < 0.05). It was concluded that short‐term in vitro culture is a very convenient method of identifying morphologically low quality morulae with higher chances of continuing development after the transfer into recipients. 相似文献