在铺有鼠尾胶原的TransWell培养板套皿气-液交界面培养角质形成细胞形成皮肤样器官

背景:浸没式体外培养角质形成细胞的技术已得较普遍的应用,但角质形成细胞的体外器官样培养技术尚存在不足.目的:以铺有鼠尾胶原的TransWell培养板套皿,提供的气-液交界面体外培养角质形成细胞,以改进角质形成细胞的体外器官样培养技术.设计、时间及地点:体外人工皮肤器官实验,于2008-07/2009-02在河北医科大学第一医院实验室完成.材料:角质形成细胞由解放军第二军医大学王教授馈赠.方法:将浸没式培养的角质形成细胞接种到铺有鼠尾胶原的Transwell培养板套皿当中,Transwell培养板套皿每板有6个孔,每孔当中有一插件,插件悬挂于孔的边缘上,插件底面为一孔径为0.4 μm的高分子膜,距离培养板孔的底面有1.5 mm的高度,使细胞处于气-液交界面上并培养4周,然后将不同培养时段的培养物进行苏木精-伊红染色、免疫组织化学、流式细胞仪检测,进而观察细胞分层和分化的状况.主要观察指标:观察细胞的生长状态及分层和分化状态.结果:培养2周的角质形成细胞大部分为单层结构,细胞彼此衔接成铺路石状结构,多数细胞为三角形或多角形结构,细胞核清晰可见,细胞比较饱满.部分区域细胞出现双层结构,表现为单层的角质形成细胞上面铺有连接成条索状的细胞.3周的角质形成细胞大部分为双层结构,异层及同层细胞之间彼此衔接,呈索形结构,细胞核仍然清晰可见,细胞不如2周时饱满.4周的角质形成细胞部分区域为三层结构,且细胞较前变得萎缩,表现为细胞浆减少,细胞核变小.角质形成细胞进行气-液交界面培养至4周,细胞会分为3层,表层的细胞会表达终末分化的标志物角蛋白10.结论:在Transwell培养板套皿膜上,角质形成细胞会出现分层和分化,形成体外的皮肤样器官.

Organotypic culture (air-liquid interface) of keratinocytes skin-like structure using Transwell plates paved with rat tail collagen

BACKGROUND: The technology of the immerse culture of the Keratinocytes in vitro has become more and more common.However, the technology of the organotypic culture of Keratinocytes in vitro has not been established successfully.OBJECTIVE: To improve the technology of the organotypic culture of keratinocytes in vitro and to culture the Keratinocytes on the air-liquid interface suffered from the Transwell plates.DESIGN, TIME AND SETTING: The experiment of the artificial skin organ in vitro was performed at the Department of the Otolaryngology Laboratory, The First Hospital of Hebei Medical University from July 2008 to February 2009.MATERIALS: Keratinocytes was presented by Professor Wang of the Second Military Medical University.METHODS:Transplant the keratinocytes which have been subcultured for certain time onto the membrane of the Transwell plate,which contained six holes, and every edge of the hole hanging an insert. The bottom of the insert was made up of polymer materials with diameter of 0.4 urn. And the bottom of the insert is 1.5 mm from the bottom of the Transwell culture plate. Keep the cells on the air-liquid interface for 4 weeks. Then haematoxylin-eosin staining, immunohistochemistrical staining, and the flow cytometer were used to detect the differentiation and the stratification of cells.MAIN OUTCOME MEASURES: The growth state, stratification and differentiation of cells.RESULTS: At 2 weeks after culture, most of keratinocytes displayed monolayer structure with obblestone-like morphologic characteristics. The nuclei were clear and the cell was well-stacked. Double layer structure could be seen in part of areas,displayed as monostratal keratinocytes linked with string-like cells. Most of keratinocytes differentiated into double layers, and cells linked with others with cable-like structure. The nuclei still could be seen. After the keratinocytes have been cultured on the air-liquid interface for four weeks, the cells grew into three layers, and the cells on the upper layer expressed cytokeratin 10, the maker of the differentiation and the stratification of the cells.CONCLUSION: Transplanted keratinocytes on the membrances of the Transwell plate can differentiation and the stratification,which formed skin organ in vitro.