Cooke B M, Usami S, Perry I, Nash G B
Department of Hematology, Medical School, University of Birmingham, United Kingdom.
Microvasc Res. 1993 Jan;45(1):33-45. doi: 10.1006/mvre.1993.1004.
We have developed a simplified technique for culturing human umbilical vein endothelial cells under shear flow conditions, using prefabricated glass microcapillary tubes ("microslides") with a well-defined rectangular cross section and good optical quality. These microslides have been incorporated into a controlled flow system for quantitative video-microscopic analysis of the adhesion of blood cells to endothelial cells. Microslides were pretreated with 3-aminopropyltriethoxy-silane, or gelatin, and then loaded with a suspension of endothelial cells. After the cells had settled and attached to the substrate, the microslides were inserted into a flow-based culture system. Medium was drawn through them at intervals or continuously until confluency was reached (approximately 24 hr). Cells were cultured at wall shear stresses over a range 0.06 to 2.2 Pa. For adhesion assays, the endothelialized microslides were attached to microscope slides, and suspensions of blood cells were drawn through at desired wall shear stresses (0.02-0.5 Pa). Adhesion of malarial-infected red blood cells and of neutrophilic granulocytes was quantitated by direct microscopic observation. The adhesive behavior of both cell types closely resembled that previously described by ourselves and others using flow chambers incorporating endothelial-coated glass coverslips. The use of microslides represents a significant simplification of methodology for endothelial cell growth and adhesion studies under flow conditions.
我们开发了一种简化技术，用于在剪切流条件下培养人脐静脉内皮细胞，该技术使用预制的具有明确矩形横截面和良好光学质量的玻璃微毛细管（“微载玻片”）。这些微载玻片已被整合到一个可控流动系统中，用于对血细胞与内皮细胞的黏附进行定量视频显微镜分析。微载玻片先用3-氨丙基三乙氧基硅烷或明胶进行预处理，然后接种内皮细胞悬液。细胞沉降并附着于基质后，将微载玻片插入基于流动的培养系统。每隔一段时间或持续抽吸培养基，直至达到汇合状态（约24小时）。细胞在0.06至2.2 Pa范围内的壁面剪应力下培养。对于黏附试验，将内皮化的微载玻片附着于显微镜载玻片上，并在所需的壁面剪应力（0.02 - 0.5 Pa）下抽吸血细胞悬液。通过直接显微镜观察对感染疟疾的红细胞和嗜中性粒细胞的黏附进行定量。两种细胞类型的黏附行为与我们自己以及其他人先前使用包含内皮涂层玻璃盖玻片的流动腔所描述的行为非常相似。微载玻片的使用代表了在流动条件下进行内皮细胞生长和黏附研究的方法的显著简化。
