GROWING AND FREEZING CELLS IN 3-D MATRICES

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 用电子显微镜(EM)准确地观察细胞内的生物事件依赖于在固定之前保持细胞的生理状态。出于这个原因，目前细胞生长在蓝宝石圆盘上，以避免在化学/冷冻固定-冷冻替代之前分离。2-D衬底在玻璃化状态下极难切割，而且它们在每一节中产生的细胞材料非常少。在玻璃化切片上，这些区域很难找到。事实上，我们不知道任何一个成功执行任务的案例。因此，CEMOVIS(玻璃体切片冷冻电子显微镜)的超微结构研究在过去是使用再悬浮细胞培养完成的(除了类似组织的样本)。 在这里，我们建议使用三维基质来培养、高压冷冻和冷冻细胞。这种环境将为细胞的三维排列提供空间条件，就像在组织中一样，它们将消除在冷冻和切片之前将细胞从二维基质中分离的需要。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The accurate observation of biological events in cells by electron microscopy (EM) relies on the preservation of their physiological state prior to fixation. For this reason, cells are currently grown on sapphire discs to avoid detaching prior to chemical/cryofixation-cryosubstitution. 2-D substrates are extremely difficult to cut in a vitrified state, and they yield very little cellular material in each section. On vitrified sections, these areas are difficult to find. Indeed, we do not know of a single case where the task has been successfully executed. Thus, ultrastructural studies by CEMOVIS (Cryo Electron Microscopy of VItreous Sections) have been completed in the past using resuspended cell cultures (except for tissue like samples). Here we propose to Use 3-D matrices to grow, high pressure freeze and cryosection cells. This environment will provide the spatial conditions for the 3-D arrangement of cells, as in a tissue, and they will obviate the need to detach cells from a 2-D substrate prior to freezing and sectioning.