GROWING AND FREEZING CELLS IN 3-D MATRICES

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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.