黄牛表皮细胞分离与体外培养最适条件的探索

为了建立黄牛表皮细胞分离与体外培养的最适条件,比较了组织块法与单细胞悬液法、不同蛋白酶(胰蛋白酶和分离酶)的消化以及有无血清培养基对细胞生长的影响,以克隆形成率来检测细胞生长和存活情况。结果证明：采用分离酶分离表皮细胞进行无血清培养是黄牛表皮细胞体外培养的最适条件。     

人正常食管鳞状上皮不同原代培养方法的比较

该研究通过比较人正常食管鳞状上皮不同的原代培养方法,以期为不同的实验目的提供不同的培养方法。实验用到的正常食管粘膜上皮来源于食管癌患者手术切除的标本,采用组织块法和酶消化法,分别用DMEM/F12混合培养基和K-SFM无血清培养基进行培养。通过直接观察、细胞形态学观察和免疫细胞化学方法观察细胞的生长情况、细胞形态学特征及鉴定所得到的细胞,比较不同方法与不同培养基组合中原代培养细胞的生长状况。用组织块法,在DMEM/F12混合培养基中人正常食管上皮细胞生长较好,细胞融合较快,成纤维细胞污染较少,15～17天上皮细胞铺满瓶底的70%～80%,获得的细胞数量大,但细胞传代后成纤维细胞污染严重。用酶消化法,在K-SFM无血清培养基中人正常食管上皮细胞生长好,细胞融合快,成纤维细胞污染基本消除,细胞纯度高,10～12天细胞便可以铺满瓶底的70%～80%,这种方法培养的细胞可以冻存、复苏和传代。其余各种培养方法所得细胞无论在生长状态、培养周期、成纤维细胞污染和传代方面均较前两种方法差。以上各种方法培养的细胞经免疫细胞化学染色鉴定证实细胞呈广谱细胞角蛋白阳性,确定是食管上皮来源的细胞。酶消化法加K-SFM无血清培养基是本实...     

培养基血清浓度对人表皮干细胞增殖分化的影响

目的:比较不同血清浓度培养体系对表皮干细胞增殖分化的影响.方法:采用两步酶消化法和IV型胶原差速贴壁相结合的方法获得人原代表皮干细胞,分别以0%、5%、10%、15%和20%血清浓度的培养基在96孔板中进行培养.观察表皮干细胞形态,克隆形成及增殖的情况,应用四甲基偶氯唑蓝(MTT)比色法检测各组细胞存活和生长情况,分析量效和时效关系;持续传代培养细胞,每次传代的同时取适量细胞,用免疫细胞化学的方法进行表皮干细胞和表皮细胞相应标志物(K19、K14和K10)的测定.结果:表皮干细胞在各种血清浓度的培养基内均能形成克隆,增殖良好.用四甲基偶氮唑蓝(MTT)比色法测定,所得相同时间点各组OD值在统计学上没有差异(P>0.05),表皮干细胞生长速度各组间无差异.第1代表皮干细胞K19均有表达,而K14和K10表达均为阴性;其后高血清浓度(15%、20%)培养基中细胞较低血清浓度(0%、5%)先出现K14、K10蛋白的表达;培养至第10代是各组细胞均出现K10高表达,而K19、K14表达阴性.结论:在低血清浓度(0%、5%)的培养基中表皮干细胞生长良好,且能够相对较好保持表皮干细胞的特性.     

小鼠表皮干细胞的分离与培养

目的:探讨体外分离和培养小鼠表皮干细胞和分析表皮干细胞克隆形成能力的方法。方法:采用中性蛋白酶和胰酶消化新生小鼠表皮基底层细胞,将细胞直接接种在细胞瓶中,在无滋养层条件下培育;利用表皮干细胞标记物K15和α6整联蛋白进行免疫荧光鉴定;以小鼠胚胎成纤维细胞作为滋养层与成年小鼠角质细胞共培养,进而分析表皮干细胞的克隆形成能力。结果:新生小鼠表皮干细胞克隆在培养2～3 d后开始形成,细胞核质较小,细胞呈小而圆的形态特征;传代后的细胞可以被K15和α6整联蛋白特异性标记。结论:利用该方法能够实现对小鼠表皮干细胞的体外培养和传代。     

成人宫颈上皮细胞的原代培养及鉴定

目的:探索采用无血清培养基原代培养成人宫颈上皮细胞的方法。方法:以成人的宫颈上皮组织为研究对象,采用胰蛋白酶-EDTA消化法获得宫颈上皮细胞悬液,于上皮细胞专用无血清培养基中培养,采用免疫细胞化学法测定细胞中角蛋白及波形蛋白的表达,对细胞纯度进行鉴定。结果:原代培养10-15天细胞融合达60%,传代至4-6代,细胞出现生长衰退。早期细胞生长状态良好,细胞纯度在90%以上。结论:采用酶消化法及K-SFM无血清培养基培养可获得纯度高的成人宫颈上皮细胞。     

小鼠胚胎干细胞在单层粘附培养中向神经细胞的分化

目的 :探讨小鼠胚胎干 (ES)细胞在无血清培养基中以单层粘附培养方式向神经分化的方法。方法 :比较ES细胞在不同培养基中的生长情况 ,分析ES细胞在不同时间分化形成神经细胞的比例。结果 :( 1 )DMEM F1 2和Neurobasal B2 7的 1∶1混合培养基最适合ES的生长。 ( 2 )单层粘附的ES细胞表达神经细胞粘附分子 (NCAM)的比例随时间增长而增加 ,而nestin的表达先增加后下降。 ( 3)ES细胞可在两周分化为神经胶质及神经元 ,形成神经网络。结论 :小鼠ES细胞可在单层粘附培养中获得向神经的高效分化。     

小鼠角膜上皮祖细胞系TKE2的表型研究

目的：观察小鼠角膜上皮祖细胞系TKE2在扩增以及分化状态下的角蛋白及干细胞标志物的表达情况。方法小鼠角膜上皮祖细胞系TKE2在无血清培养基Keratinocyte-SFM （KSFM）以及含10﹪胎牛血清（FBS）的DMEM培养基中培养,约70﹪融合时进行角蛋白10、12、14、15、16（K10、K12、K14、K15、K16）以及Connexin43、ABCG2的免疫荧光染色,以及Ki67、P63、PCNA的免疫细胞化学染色。结果无血清培养状态下的TKE2细胞呈克隆样生长,克隆内所有细胞呈ABCG2、K14、Ki67、PCNA以及P63阳性,K15阳性细胞散在分布,K16阳性细胞呈片状分布于克隆中央区,K10、K12以及Connexin43染色为阴性。在含有10﹪胎牛血清的DMEM中培养2 d后,细胞明显增大, ABCG2、K15、P63、Ki67以及PCNA转为阴性,克隆内只有少量细胞呈K16、K14阳性染色, K10、K12、Connexin43仍为阴性。结论 TKE2细胞具有角膜上皮干细胞特性,可以作为角膜缘上皮干细胞表型维持和分化诱导研究的良好工具。     

CHO工程细胞株低血清培养的初探

以DMEM: F12(1: 1)为基础培养基,对CHO细胞的低血清培养进行了初步探索,降低培养基中血清含量,观察了细胞在10%、5%和1%等不同浓度低血清培养条件下生长状态和外源蛋白表达活性的变化.试验结果表明:在含1%血清的F12: DMEM(1: 1)培养基中,细胞仍能保持良好的生长状态,且培养上清中的目的蛋白活性与常规血清浓度10% DMEM培养条件下的活性接近.从而达到了既可以降低生产成本,又可以降低纯化难度的目的.     

大鼠膀胱Cajal间质细胞的分离、培养和鉴定

目的:探索大鼠膀胱Cajal间质细胞(ICC)的分离和培养方法,为进一步研究其在膀胱中的作用提供条件.方法:取大鼠的膀胱组织,采用Ⅱ型胶原酶酶解法分离细胞,将细胞悬液接种于含50ng/ml SCF、15%(v/v)FBS的DMEM培养基中,进行培养.用c-kit特异性杭体标记细胞,免疫荧光鉴定ICC细胞.结果:培养8小时后的ICC贴壁良好,并保持其固有特征:两个长的突起,多个短的侧突.胞体小,核大,c-kit抗体荧光染色阳性.结论:酶解法分离大鼠膀胱ICC并培养成功.     

微载体浓度与细胞接种密度对ST细胞生长的影响

选择合适的微载体浓度、细胞接种密度以提高微载体利用率,优化微载体培养体系猪睾丸细胞(Swine testicle cells)的贴附生长与维持。使用DMEM补加10%血清、LSM(Low serum medium)两种培养基考察微载体浓度、细胞接种密度对细胞生长维持的影响,进而比较ST细胞在不同条件下对Cytodex1微载体的利用率。结果显示,使用LSM在T150方瓶中连续传代培养30d,平均比生长速率为0.626d~(0-1),是DMEM补加10%FBS培养基的1.15倍。选择10×10~5cells/mL细胞接种3g/LCytodex1搅拌瓶体系,最大细胞密度为38.3×10~5cells/mL,微载体利用率上升到58.8%。在灌注培养体系中培养ST细胞15d,最终细胞密度达到36.6×10~5cells/mL,扩增了13.6倍。微载体悬浮培养的使用一方面有利于ST细胞的贴附与生长,实现高密度生长,另一方面增加了微载体的使用成本,选择合适的微载体浓度、细胞接种密度,能够最大化利用微载体与培养基中的营养物质实现细胞的最优生长。     

A method for the isolation and serial propagation of keratinocytes,endothelial cells,and fibroblasts from a single punch biopsy of human skin

Summary When multiple types of cells from normal and diseased human skin are required, techniques to isolate cells from small skin biopsies would facilitate experimental studies. The purpose of this investigation was to develop a method for the isolation and propagation of three major cell types (keratinocytes, microvascular endothelial cells, and fibroblasts) from a 4-mm punch biopsy of human skin. To isolate and propagate keratinocytes from a punch biopsy, the epidermis was separated from the dermis by treatment with dispase. Keratinocytes were dissociated from the epidermis by trypsin and plated on a collagen-coated tissue culture petri dish. A combination of two commercial media (Serum-Free Medium and Medium 154) provided optimal growth conditions. To isolate and propagate microvascular endothelial cells from the dermis, cells were released following dispase incubation and plated on a gelatin-coated tissue culture dish. Supplementation of a standard growth medium with a medium conditioned by mouse 3T3 cells was required for the establishment and growth of these cells. Epithelioid endothelial cells were separated from spindle-shaped endothelial cells and from dendritic cells by selective attachment toUlex europeus agglutinin I-coated paramagnetic beads. To establish fibroblasts, dermal explants depleted of keratinocytes and endothelial cells were attached to plastic by centrifugation, and fibroblasts were obtained by explant culture and grown in Dulbecco’s modified Eagle’s medium (DMEM) containing fetal bovine serum (FBS). Using these isolation methods and growth conditions, two confluent T-75 flasks of keratinocytes, one confluent T-25 flask of purified endothelial cells, and one confluent T-25 flask of fibroblasts could be routinely obtained from a 4-mm punch biopsy of human skin. This method should prove useful in studies of human skin where three cell types must be grown in sufficient quantities for molecular and biochemical analysis.     

Isolation and culture of amelanotic melanocytes from human hair follicles

We report a method to establish amelanotic melanocytes (AMMC) in culture and we investigate the effects of various components in the culture medium. Normal human scalp from cadaver donors was transected 1 mm below the epidermis, and hair follicles in the remaining dermis were isolated by a two-step enzyme treatment. The individual hair follicles were washed exhaustively and suspensions of hair follicle cells were prepared and cultured in Eagle's minimum essential medium supplemented with 12-o-tetradecanoyl-phorbol-13-acetate (TPA), cholera toxin and keratinocyte serum-free medium (K-SFM). Geneticin was used to eliminate contaminating fibroblasts. Proliferation of AMMC was observed after addition of TPA and K-SFM including bovine pituitary extract (BPE) into the culture medium. Cell type was determined by staining with monoclonal antibodies, NKI/beteb and HMB-45, which recognize premelanosomal and melanosomal antigens, respectively. The AMMC were also examined using transmission electron microscopy. Treatment with geneticin eliminates the majority of fibroblasts and does not impair the growth of keratinocytes or AMMC. After contaminating fibroblasts and keratinocytes were removed, two distinct cell morphologies remained: (1) large, dendritic and deeply pigmented cells, which did not proliferate and which disappeared by the third passage, and (2) small bipolar cells, which initially were unpigmented and proliferated very rapidly. We observed that TPA at various concentrations stimulated the proliferation of the cells, and at high concentrations could induce the formation of multiple dendrites. K-SFM including BPE accelerated the proliferation of the cells in a dose-dependent manner. After passage 3, almost all cells expressed premelanosomal and melanosomal antigens, recognized by NKI/beteb and HMB-45, respectively. Active mitochondria, abundant rough endoplasmic reticulum, Golgi complexes, ribosomes and melannosomes (predominantly in stages I, II or III with some at stage IV in some AMMC) were observed ultrastructurally in the cytoplasm of the cultured cells.     

胰酶对皮肤角质细胞分离和传代的影响

考察不同的胰酶浓度及其作用时间对角质细胞分离和传代的影响。实验发现在胰酶浓度 0.25%时作用5min可获得的总活细胞量和有克隆形成能力的细胞量均优于其它培养条件 ;而在胰酶浓度0.05%时作用 5min可获得最大的原代角质细胞贴壁率。随着胰酶浓度的提高 ,传代角质细胞的贴壁率和贴壁速率常数以及克隆形成率均随之增加 ,因此在传代培养时使用 0.25%胰酶浓度进行消化较为适宜.     

分步消化法原代培养鼠阴道黏膜上皮细胞的研究

目的探讨大鼠阴道黏膜上皮细胞的体外培养和扩增技术,为构建组织工程化阴道动物模型提供种子细胞。方法取大鼠阴道全层组织,经Dispase酶和胰酶分步消化后,接种于无血清角化细胞培养液中连续培养,观察细胞形态、体外生长特性和超微结构,绘制生长曲线,免疫组化鉴定。结果原代细胞培养24-36 h后开始贴壁,7-10d约80%融合,呈铺路石样外观,可连续传5-6代;扫描电镜下细胞表面可见微绒毛嵴;角蛋白染色阳性,细胞纯度98%;第五代细胞为正常二倍体核型。结论该方法培养的阴道上皮细胞增殖状态良好,细胞纯度高,扩增迅速,可在较短时间内获得大量细胞用于组织工程学研究。     

人卵巢颗粒细胞的体外培养方法探讨

目的建立人卵巢颗粒细胞分离纯化、体外培养的有效方法。方法收集体外受精—胚胎移植(IVF-ET)穿卵时的卵泡液,用胰蛋白酶消化法及密度梯度离心法分离纯化颗粒细胞并用不同培养基进行培养。结果用体积分数为50%的Percoll细胞分离液分离,DMEM/F12或McCoy’5a液体培养基进行培养,细胞纯度高,存活率高,后续生长良好。结论建立了人卵巢颗粒细胞体外培养的稳定模型,为颗粒细胞的体外研究奠定良好的基础。     

Comparison of the effects between animal-derived trypsin and recombinant trypsin on human skin cells proliferation,gene and protein expression

Animal-derivative free reagents are preferred in skin cell culture for clinical applications. The aim of this study was to compare the performance and effects between animal-derived trypsin and recombinant trypsin for skin cells culture and expansion. Full thickness human skin was digested in 0.6 % collagenase for 6 h to liberate the fibroblasts, followed by treatment with either animal-derived trypsin; Trypsin EDTA (TE) or recombinant trypsin; TrypLE Select (TS) to liberate the keratinocytes. Both keratinocytes and fibroblasts were then culture-expanded until passage 2. Trypsinization for both cell types during culture-expansion was performed using either TE or TS. Total cells yield was determined using a haemocytometer. Expression of collagen type I, collagen type III (Col-III), cytokeratin 10, and cytokeratin 14 genes were quantified via RT-PCR and further confirmed with immunocytochemical staining. The results of our study showed that the total cell yield for both keratinocytes and fibroblasts treated with TE or TS were comparable. RT-PCR showed that expression of skin-specific genes except Col-III was higher in the TS treated group compared to that in the TE group. Expression of proteins specific to the two cell types were confirmed by immunocytochemical staining in both TE and TS groups. In conclusion, the performance of the recombinant trypsin is comparable with the well-established animal-derived trypsin for human skin cell culture expansion in terms of cell yield and expression of specific cellular markers.     

人胚皮层神经干细胞培养方法的探讨(简报)

神经干细胞是一类具有多向分化潜能、自我复制能力,在特定条件诱因下,能够向特定类型神经元或神经胶质细胞分化的未分化或低分化细胞。神经干细胞的研究对于我们阐明神经发生机制,进行神经变性疾病的细胞替代治疗都有重要意义。目前,从人胚组织分离神经干细胞的方法不尽相同,对于大脑皮层神经干细胞的分离,一些学者认为机     

东方田鼠皮肤成纤维细胞的分离培养及生物学特性

目的探索和建立东方田鼠皮肤成纤维细胞体外分离、培养的技术方法并观察其生物学特性。方法采用含10%小牛血清的Dulbecco改良Eagle培养液(DMEM)和1640两种培养体系,运用组织块贴壁法和胰酶消化法,分别对出生后1、3 d和5 d的东方田鼠乳鼠皮肤成纤维细胞进行原代分离、培养。苏木素-伊红染色及倒置相差显微镜下观察成纤维细胞形态和生长特性。结果 0.25%胰酶消化分离出生后1 d和3 d东方田鼠乳鼠皮肤较出生后5 d组织可获得较多数量细胞,成纤维细胞在体外快速贴壁生长,一般6～7 d长满培养瓶,细胞纯度高,HE染色细胞呈长梭形,胞核明显;DMEM和1640两种培养液均可用于东方田鼠皮肤成纤维细胞的培养,但细胞传代后生长趋缓,只可传代2～3次。本实验运用组织块贴壁法未能培养出皮肤成纤维细胞。结论确定了有效分离东方田鼠皮肤成纤维细胞的日龄和方法,为进一步深入研究提供了技术方法和操作依据。     

Optimization of porcine urothelial cell cultures: Best practices,recommendations, and threats

Many experimental approaches have been conducted in order to isolate urothelial cells from bladder tissue biopsies, but each method described has utilized different protocols and sources of bladder tissue. In this study, we compared the different methods of urothelial cell isolation available in literature together with standardized methods in order to obtain more unified results. Five methods for primary porcine urothelial culture establishment were compared: tissue explants and four enzymatic methods utilizing collagenase II, dispase II, combination of dispase II and trypsin, and trypsin alone. The average number of isolated cells, cell morphology, success of established culture, average number of cells from the first passage, expression of p63 and pancytokeratin and the characterization of urothelial cell growth, and aging were analyzed during the in vitro culture. The method utilizing dispase II was the most efficient and reproducible method for the isolation and culture of porcine urothelial cells when compared to the other tested methods. Urothelial cells obtained by this method grew considerably well and the cultures were established with high efficiency, which enabled us in obtaining a large quantity of cells with normal morphology. Contamination with fibroblasts in this method was the lowest. The utilization of a proper method for urothelial cell isolation is a critical step in the urinary tract regeneration when using tissue engineering techniques. In summary, this study demonstrated that by utilizing the described method with dispase II, a suitable number of cells was achieved, proving the method useful for tissue regeneration.