GROWING AND FREEZING CELLS IN 3-D MATRICES

在 3D 基质中培养和冷冻细胞

• 批准号: 8362555
• 负责人: Robert L Garcea
• 金额: $ 3.19万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-05-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8362555
• 关键词: 3-Dimensional; Area; Biological; Biological Preservation; Cell Culture Techniques; Cell physiology; Cells; Cellular Structures; Chemicals; Cryoelectron Microscopy; Cryopreservation; Cryoultramicrotomy; Electron Microscopy; Environment; Event; Freezing; Frozen Sections; Funding; Grant; National Center for Research Resources; Physiological; Principal Investigator; Research; Research Infrastructure; Resources; Sampling; Sapphire; Source; Tissues; United States National Institutes of Health; cost; pressure; sample fixation

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.

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 通过电子显微镜（EM）准确观察细胞中的生物学事件依赖于在固定之前保持其生理状态。出于这个原因，细胞目前生长在蓝宝石盘上，以避免在化学/冷冻固定-冷冻置换之前分离。2-D基质在玻璃化状态下非常难以切割，并且它们在每个切片中产生非常少的细胞材料。在玻璃化切片上，这些区域很难找到。事实上，我们不知道有任何一个案例成功地执行了这项任务。因此，过去已使用重悬细胞培养物（组织样样品除外）完成了CEMOVIS（Vitreous切片的冷冻电子显微镜检查）的超微结构研究。 在这里，我们建议使用三维矩阵生长，高压冷冻和冷冻切片细胞。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，并且它们将消除在冷冻和切片之前将细胞从二维基底上分离的需要。