Dyer Laura A, Patterson Cam
McAllister Heart Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
J Vis Exp. 2013 Jul 20(77):50463. doi: 10.3791/50463.
Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.
细胞培养极大地增强了我们在多种培养条件下评估单个细胞群体的能力。虽然永生化细胞系因其易于使用而具有显著优势，但并非所有潜在的细胞类型都能获得这些细胞系。通过从特定感兴趣区域，特别是从转基因小鼠中分离原代细胞，可以进行更细致入微的研究。基本技术包括解剖感兴趣的器官或部分器官（如心脏或心脏的特定区域），并将器官解离成单个细胞。然后将这些细胞与结合了识别感兴趣细胞类型抗体的磁珠一起孵育。然后可以使用磁铁分离出感兴趣的细胞，通过短暂的胰蛋白酶孵育将细胞与磁珠解离。然后可以根据需要对这些分离的细胞进行培养和分析。该技术最初是为成年小鼠器官设计的，但如本文所示，也可以很容易地缩小规模用于胚胎器官。由于我们关注的是发育中的冠状动脉血管系统，我们想在特定的胚胎阶段研究这群细胞。因此，必须对原始方案进行修改，以适应胚胎心室的小尺寸以及这些发育阶段内皮细胞的低潜在产量。利用这种缩小规模的方法，我们评估了转基因胚胎心室内皮细胞中的冠状动脉丛重塑。
