人早孕绒毛滋养层细胞原代培养:差异贴壁法与消化排除法联合应用的可行性

背景:国内外有关人绒毛膜滋养层细胞的体外培养方法,大多步骤繁琐,细胞纯度低而且成本很高,不适于普通实验室推广应用。目的:拟求建立一种人妊娠5～10周绒毛滋养层细胞简便有效的分离培养及鉴定方法。方法:采用改进的胰酶消化法分离培养妊娠5～10周人绒毛滋养层细胞,加0.0625%胰酶,37℃消化25～40min;以差速贴壁法和消化排除法纯化细胞,倒置显微镜观察细胞形态,细胞免疫化学方法鉴定细胞来源和纯度。结果与结论:倒置显微镜下原代培养滋养层细胞接种后见大量圆形细胞悬浮存在,1h后可见部分细胞贴壁,24h后70%～80%细胞贴壁,五六天细胞数量明显增多,细胞呈三角形、多边形平铺片状生长,核大卵圆形居中,部分细胞连接成片,部分细胞呈长梭形。七八天长满瓶壁的80%～90%可传代。9～10d铺满培养瓶底部。细胞碎屑不贴壁。传代接种后1.5h贴壁,迅速增长,三四天爬满瓶底,各代细胞形态基本一致。可见细胞为上皮样细胞形态,呈片状铺展生长。细胞角蛋白染色阳性,波形蛋白染色阴性细胞达70%～80%。采用低浓度胰酶进行长时间消化分离培养人早孕绒毛滋养层细胞,利用差异贴壁法和消化排除法以及反复换液法进行纯化,可简单、快捷地获得较高纯度的人早孕绒毛滋养层细胞。     

分散人绒毛膜滋养层细胞及细胞培养

目的获取人绒毛膜滋养层细胞.方法应用胰酶/DNA酶法进行消化后进行培养.经光镜和电镜观察.结果我们获取了人绒毛滋养层细胞并成功培养.结论取人妊娠45～55D刮宫术后的绒毛组织,经胰酶、DNA酶消化可得绒毛滋养层细胞.     

平滑肌细胞对共培养体系滋养细胞侵袭力与MMP-2及MMP-9表达的影响

目的模拟体内环境,建立可保持平滑肌细胞与绒毛外细胞滋养层细胞(EVCT)生物学特性的共培养细胞模型,应用于研究平滑肌细胞与滋养细胞理化特性与滋养细胞的侵袭行为。方法利用组织块培养法培养脐动脉平滑肌细胞,组织块培养、胰酶消化和Percoll梯度沉降,收集纯化人早孕绒毛组织的滋养细胞,免疫组化检测细胞的纯度。将滋养细胞与平滑肌细胞分别放入Transwell的上下小室,观察该模型下滋养细胞形态变化、细胞活力、侵袭力改变与分泌功能等特性。结果免疫组化显示EVCT的细胞角蛋白7阳性表达的细胞数占95%以上,SMC a-actin阳性表达的细胞数也超过95%,证实共培养系统中EVCT和SMC纯度均在95%以上,且生物学特性得以维持。上室中的EVCT保持了其侵袭能力,且平滑肌细胞能促进滋养细胞增殖活性、侵蚀能力及MMP2、MMP9的表达。结论成功地建立了平滑肌细胞与滋养细胞原代共培养系统模型,便于研究滋养细胞侵袭和子宫螺旋动脉重铸障碍的分子机制。     

预损伤与改良传代体外培养许旺细胞的比较

背景：通过组织工程方法构建神经桥接体修复神经损伤,需要大量纯化体外培养的许旺细胞。 目的：对比观察预损伤法和改良传代法获取许旺细胞的纯度与质量。 方法：①预损伤法：预损伤SD乳鼠坐骨神经,3 d后取出坐骨神经,分离神经外膜,用胰酶、胶原酶消化,差速贴壁除去成纤维细胞,接种培养。②改良传代法：直接获取SD乳鼠坐骨神经,分离神经外膜,运用双酶消化法结合单酶消化法进行许旺细胞原代培养,5~7 d后采用单酶快速消化离心法行传代培养,同时纯化许旺细胞。 结果与结论：预损伤法和改良传代法体外培养的许旺细胞纯度均达95%以上,两种方法获得的许旺细胞纯度差异无显著性意义(P > 0.05)。两种方法获取的许旺细胞形态正常,数量及纯度高,增殖旺盛,说明预损伤法和改良传代法都是体外获取高质量与高纯度许旺细胞的理想方法。     

骨组织块法和酶消化法联合培养人成骨细胞

背景：成骨细胞培养难度大,不同培养方法得到的成骨细胞数量、纯度、增殖及分化活性各有区别。 目的：探讨理想的人成骨细胞体外培养的方法。 方法：取人髂骨松质骨,同时结合骨组织块法和酶消化法分离培养人成骨细胞。 结果与结论：分离培养的人成骨细胞生长状态良好,纯度较高。细胞贴壁生长,形态多样化,多呈多边形、纺锤型、梭形、三角形,胞浆丰富向外伸展出生长突,具有典型的成骨细胞形态特征;细胞增殖良好,碱性磷酸酶染色及矿化结节茜素红染色阳性。说明联合骨组织块法和酶消化法可成功分离培养人成骨细胞,是进行人成骨细胞体外培养较为理想的方法。 关键词：成骨细胞;原代培养;碱性磷酸酶;组织块法;酶消化法 doi:10.3969/j.issn.1673-8225.2012.07.002     

成人真皮成纤维细胞原代培养及鉴定

目的探讨建立成人真皮成纤维细胞原代培养方法。方法新鲜皮肤组织用分散酶Ⅱ(dispaseⅡ)消化,分离上皮和真皮层,分别采用组织块法和Ⅰ型胶原蛋白酶消化法分离真皮层成纤维细胞。用倒置相差显微镜观察细胞生长情况,波形蛋白免疫荧光细胞化学染色法鉴定细胞类型及纯度;噻唑蓝(MTT)法检测细胞增殖活性绘制生长曲线,流式细胞术检测细胞周期分布。结果培养48 h,经胶原蛋白酶分离得到的成纤维细胞95%以上贴壁,同时细胞开始从组织块边缘贴壁延伸生长。培养5 d时,组织块周围有大量细胞贴壁增殖。细胞生长迅速,第7天,增殖细胞层铺满培养皿。组织块法分离的成纤维细胞中可混杂少量上皮细胞团,而胶原蛋白酶消化法分离的细胞波形蛋白阳性率接近100%,几乎均为成纤维细胞。结论胶原蛋白酶消化和组织块法均是快速、经济、有效获取真皮成纤维细胞的方法。胶原蛋白酶消化法可以更有效避免上皮细胞污染,分离的真皮成纤维细胞有较好的增殖能力。     

雪旺细胞分步消化培养及其纯化实验研究

目的：探讨双酶分步消化法从乳兔周围神经中分离培养雪旺细胞，以及纯化获得高纯度的雪旺细胞的方法。方法：采用出生1周内乳兔的坐骨神经，先用胰蛋白酶消化分离出一部分细胞，移出后，再予胶原酶消化分离其余的细胞，计算上两步消化后的活细胞的百分率，另外采用：(1)无任何处理；(2)单纯双30min差速贴壁；(3)Ara—c；(4)Geniticin纯化法，以及后3种方法联合应用进行纯化，计算雪旺细胞的纯度。结果：采用分步消化法所得到的活细胞百分率分别为96．5％以上和95％以上，无任何处理的雪旺细胞纯度为85％，单纯双30min差速贴壁纯度为90％，Ara—c纯化的纯度为91％，Geniticin纯化的纯度为95％，(2)、(3)、(4)方法的联合应用纯化的纯度为98％。结论：双酶消化法和联合应用双30min差速贴壁、Ara—c、Geniticin纯化是获取高纯度雪旺细胞的理想方法。     

一种高效实用的人卵巢表面上皮细胞培养方法

背景：体外分离培养纯度高、活力强且生物学特性稳定的卵巢表面上皮难度很大,目前原代培养主要采用组织块贴壁法和酶消化法,但这两种方法在取材、细胞活力及细胞纯度上都存在一定的问题。 目的：建立一种高效实用的人卵巢表面上皮分离、培养和鉴定方法。  方法：创新性地运用细胞刷刷取人卵巢表面上皮,以红细胞裂解法、差速贴壁法对细胞进行分离纯化,并向无血清DMEM-F12培养基中添加人表皮生长因子进行细胞培养。在倒置显微镜下观察细胞形态,应用苏木  精-伊红染色和免疫细胞化学染色法对细胞进行鉴定,并绘制生长曲线。  结果与结论：卵巢表面上皮培养24 h开始贴壁生长,7-12 d后基本达到融合,细胞呈多角形或扁平型,透光性及折光性强。细胞形态符合正常上皮细胞特性,所分离的细胞几乎完全表达上皮细胞表面标志物CK19。细胞生长良好,可以传6-8代,细胞生长曲线呈“S”形,纯度达95%以上。结果提示细胞刷取培养法操作简单,能够快速分离获得大量卵巢表面上皮,所获得的细胞经红细胞裂解法和差速贴壁法处理后纯度达95%以上,且细胞生长稳定。     

乳鼠雪旺氏细胞的纯化培养研究

目的：获得高纯度的乳鼠雪旺氏细胞。方法：采用出生后３～５天乳鼠的坐骨、臂丛神经，植块法培养乳鼠雪旺氏细胞；并通过精细剥除神经外膜、组织块反复再植、低浓度胰酶快速消化、差速贴壁法的有机结合对雪旺氏细胞进行纯化；继用抗Ｓ－１００蛋白单抗通过间接免疫荧光法及ＡＢＣ法进行细胞鉴定。结果：首次植块获得的细胞经纯化后雪旺氏细胞可高达８５％以上，反复植块者可达９５％，甚至高达９８％。结论：本方法简单、稳定，所获得的雪旺氏细胞纯度高、数量大、受非实验因素影响小     

酶消化结合差时贴壁的方法体外分离培养人子宫内膜细胞

目的探讨酶消化结合差时贴壁培养的方法对人子宫内膜细胞进行体外培养的培养效果，以寻找一种简单高效的人子宫内膜细胞体外分离培养的方法。方法采用胶原酶消化结合差时贴壁的方法，对人正常子宫内膜细胞进行体外分离培养，并传代、冻存及复苏。光镜下观察其培养效果，通过免疫荧光法对腺上皮细胞及间质细胞进行鉴定，并分析其纯度。结果共培养人子宫内膜38份，成功培养35份，培养成功率92％，冻存后成功复苏率达97％；子宫内膜腺上皮细胞和间质细胞分别经角蛋白单抗和波形蛋白单抗免疫荧光显色为阳性，腺上皮细胞和间质细胞原代纯度均达90％以上。结论酶消化结合差时贴壁的培养方法，操作简单、培养成功率高、污染机会少、省时、维持细胞活性好，是子宫内膜细胞体外分离培养的简单高效方法。     

改良型混合酶消化法培养小鼠乳腺上皮细胞

背景：目前采用的A型胶原酶和胰蛋白酶混合酶分离乳腺细胞团的方法操作复杂,实验条件要求高。 目的：观察改良型混合酶消化法能否成功进行乳腺上皮细胞的体外培养。 方法：采用Ⅰ型、Ⅱ型胶原酶和胰蛋白酶(1∶1∶1)混合消化直接用眼科剪剪碎的小鼠乳腺组织,37 ℃振荡消化获取乳腺细胞团,并采用差速贴壁法去除成纤维细胞,将细胞团接种于细胞培养瓶进行培养。 结果与结论：倒置显微镜下观察显示,改良型混合酶消化法能获得较多的细胞团,且培养12 h后细胞团绝大多数贴在细胞瓶壁,培养72 h后细胞团完全铺开融合成片,细胞呈典型的铺路石样。免疫荧光细胞化学染色结果显示,培养细胞角蛋白18呈阳性反应。说明应用改良型混合酶消化法能在短时间内获得大量较纯的乳腺上皮细胞。     

Evaluation of Cytokeratin 7 as an accurate intracellular marker with which to assess the purity of human placental villous trophoblast cells by flow cytometry

Villous trophoblast cells (TC) obtained from first trimester and term human placentae after trypsin/Percoll gradient isolation were immunodepleted of contaminant cells. The level of purity was assessed by the intracellular expression of the pan trophoblast marker cytokeratin-7 (CK7) and comparisons were made with the GB25 trophoblast-specific (cytotrophoblast+syncytiotrophoblast) cell surface marker. The presence of contaminating cells was traced with intracellular vimentin, or cell surface CD2, CD36, and CD163 markers and evaluated by flow cytometric analysis. The pattern of CK7 expression by trophoblast cells was also analyzed by immunofluorescence microscopy. Most batches of TC from first trimester or term placentae (92+/-3% and 96+/-2%, respectively) showed a high percentage of CK7 expressing cells, with less than 2% contaminating vimentin positive cells. In some batches of TC with a lower percentage (65+/-4%) of CK7-expressing cells, no vimentin was found, but a low percentage of CD36-expressing cells was evidenced, with no presence of CD2, and/or CD163-expressing cells. The intracellular CK7 signal correlated significantly with that of GB25 (p<0.05) cell surface expression in TC of term placentae. The choriocarcinoma BeWo and Jar cell lines also showed high levels (>92%) of CK7-expressing cells. Conversely, the control U87 astrocytoma cell line showed a high percentage (>90%) of vimentin but no CK7-expressing cells. These results provide evidence that the mutually exclusive pattern of intracellular CK7/vimentin expression of human TC can be used for evaluation by flow cytometry of the purity of primary human trophoblast cells.     

兔主动脉血管内皮细胞原代改良提取法

BACKGROUND: Primary vascular endothelial cells are mostly harvested through aorta endothelial cell cultures and micro-artery endothelial cell cultures using enzyme digestion and tissue adhesion methods, and the quality and purity of harvested cells cannot meet the need for current scientific research. OBJECTIVE: To investigate an improved extraction of primary vascular endothelial cells and the relevant identification method. METHODS: A segment of rabbit aorta was cut to culture vascular endothelial cells using the improved extraction method in group A or using adhesion method in group B. In the group A, the vascular intima was striped out with microsurgical instruments, and digested enzymatically to acquire single primary cells followed by culture in endothelial cell culture medium. In the group B, the whole vascular intima was adhered to the culture dish that was incubated in a 5%CO2, 37 ℃ incubator for 1 hour. Cell pellets in the two groups were cultured in vitro. Cell morphology was observed using a microscope; immunohistochemical staining was used to detect CD31, VIII factor and Vimentin protein for identification of vascular endothelial cells. RESULTS AND CONCLUSION: The purity and number of vascular endothelial cells extracted by the improved method were higher than those by the adhesion method. Immunohistochemical findings showed positive expression of CD31 and VIII factor, but negative expression of Vimentin. These findings indicate that the improved extraction method can obtain more vascular endothelial cells with higher purity, which is of strong operability and practicality.       

小鼠乳鼠原代心肌细胞的改良培养

背景：大鼠心肌细胞原代培养技术日渐成熟,但小鼠心肌细胞在同样实验条件下不易获得,而小鼠基因组与人类基因组有更多相似之处,具有更高的研究价值。目的：改进小鼠乳鼠原代心肌细胞的培养方法,得到纯度高、活力强、保持心肌细胞原有结构和功能的心肌细胞。方法：将小鼠心肌组织利用酶消方法分离为单个的心肌细胞,实验步骤中将经典的胰酶、胶原蛋白酶混合酶消法分解开,先用胰酶消化心肌组织,使心肌组织变得松散,再用胶原蛋白酶,作用于细胞间质的胶原纤维,使心肌组织分离为单个的心肌细胞。调整胰酶和Ⅱ型胶原蛋白酶的浓度和作用时间,严格控制试剂pH值和各步骤的温度。结果与结论：心肌细胞在接种24 h后开始贴壁,48 h后可观察到部分心肌细胞出现自主搏动,72 h后彼此形成交联,96 h后可观察到心肌细胞形成细胞簇,并出现一致性搏动。心肌细胞的存活率和纯度均达95%以上。结果证实,实验采用改良方法成功培养出小鼠乳鼠心肌细胞,并且保留心肌细胞的结构和功能,纯度和成活率高,是可行的培养方法。中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

Ex vivo detection of apoptotic trophoblast cells applying flow cytofluorometry and immunocytochemistry using M30 antibody directed to the cytokeratin 18 neo-epitope

Apoptosis of placental trophoblast cells has become the subject of intensive research. Recently, a monoclonal antibody (M30) directed against a neo-epitope of cytokeratin 18, that is formed after cleavage of this cytoskeletal protein by caspases, was shown to be of advantage over other tests for the detection of trophoblast cell apoptosis. In the present study, we describe a method for the enrichment of highly pure villous trophoblast cells based on the proteolytic digestion of placental tissue, density gradient separation of dissected cells, and immunoelimination of contaminating, non-trophoblast cells employing an antibody to the HLA class I antigen. The high purity (94-99%) of the trophoblast cell preparation was shown by antibody staining for cytokeratin 7 and absence of vimentin. Furthermore, we demonstrate that after a simple permeabilization and fixation step with 90% methanol and using the M30 CytoDeath, FITC-conjugated antibody, apoptotic trophoblast cells could be distinguished from non-apoptotic cells by flow cytofluorometry in a highly quantitative and sensitive fashion. Our protocol is an improvement over previously used methods such as immunocytochemistry as it allows to differentiate rapidly between competent and apoptotic trophoblast cells by the quantitative method of flow cytofluorometry.     

Establishment and identification of human primary lung cancer cell culture in vitro

Objective: To explore a simple and practical method for human primary lung cancer cells culture in vitro. Methods: Tumor specimens from 6 lung cancer patients were isolated with collagenase digestion cultured in vitro. Then the characteristics of these cells were analyzed and identified by optical microscope observation, hematoxylin-eosin staining, immunocytochemistry, immunohistochemistry and tumor nude mice inoculation experiments, respectively. Results: Except for the small cell lung cancer, the other 5 samples were successfully isolated and cultured. The cultured cells showed typical characteristics of malignant cells and positive for cytokeratin 7 and 19. Moreover, the cancer cells readily formed subcutaneous tumors in nude mice and the pathological images of the transplanted tumor were consistent with its tumor origin. Conclusion: The primary culture for human lung cancer cells can be successfully achieved with the method of collagenase digestion.     

A novel model of human implantation: 3D endometrium-like culture system to study attachment of human trophoblast (Jar) cell spheroids

There is an urgent need to develop optimized experimental models to examine human implantation. These studies aimed to (i) establish a human endometrium-like three-dimensional (3D) culture system, and (ii) examine the attachment of trophoblast-like Jar spheroids to the culture. In the present work, 3D endometrial cultures were constructed with fibrin-agarose as matrix scaffold, and using epithelial and stromal cells from both human primary cultures and established cell lines. An attachment assay between trophoblast cells and the 3D culture was developed. Epithelial cells (cytokeratin(+)) concentrated on top of the matrix forming a monolayer, and stromal cells (vimentin(+)) resided within the matrix, resembling the normal endometrial structure. The capability of primary epithelial cells to form glands spontaneously was observed. Human trophoblast cells (Jar cells) were hCG(+) by immunostaining, allowed to form spheroids, and confirmed to secrete hCG into the medium. Time-dependent experiments demonstrated a high rate of attachment of Jar spheroids to the epithelium, and adhesion was strongly related to the various cell types present in the 3D culture. An architecturally and functionally competent 3D endometrial culture system was established, that coupled with Jar spheroids mimicking trophoblast cells, provides a unique in vitro model for the study of certain aspects of human implantation.     

新生大鼠心肌细胞和心肌成纤维细胞的同时分离

背景：利用心肌组织中不同细胞的黏附特性差异,通过优化心肌细胞的培养条件,一次分离同时获得心肌和心肌成纤维细胞。 目的：探讨新生大鼠心肌和心肌成纤维细胞同时分离的有效方法。 方法：采用低浓度胰蛋白酶冷消化结合胶原酶复合消化,差速分离心肌和心肌成纤维细胞。通过使用不同种类的血清改良心肌细胞培养条件,免疫荧光鉴定心肌细胞和心肌成纤维细胞纯度。 结果与结论：心肌培养48 h时Troponin T阳性细胞率为89.3%。第2代的心肌成纤维细胞培养波形蛋白阳性细胞率为93.6%。含马血清的培养基组心肌细胞阳性率明显高于牛血清培养组(P < 0.05)。实验应用的新生大鼠心肌细胞分离培养方法,心肌细胞和心肌成纤维细胞纯度高、活性好,且能同时获得心肌细胞和心肌成纤维细胞。