Nanoporous Membranes for cellular microarrays and in vitro assays

描述（由申请人提供）：蛋白质和DNA微阵列的进步使通量显著增加，设备标准化使这些技术更加普遍。高密度、高通量微阵列通过减少试剂体积和增加每个板的实验数量来降低药物发现和基础科学中的研究和开发成本。然而，这种小型化缺少细胞培养微阵列。现有的低孔计数细胞培养板需要更大体积的珍贵药物制剂用于渗透性测定，并且需要更多的板来完成一系列实验。这些相同的因素增加了基础科学中的平行细胞实验的成本，例如筛选干细胞培养分化条件。在本提案中，我们将测试使用新型纳米多孔膜的可行性，以实现细胞培养筛选的小型化，用于高通量药物渗透性和共培养研究。在极限情况下，我们将能够进行单细胞筛选，以研究细胞群体对刺激、药物治疗或共培养环境的反应中的表型和行为变化。在这项工作的第一个目标，我们将制作微阵列规模的细胞培养阵列使用多孔纳米晶硅（pnc-Si）。我们将通过比较细胞毒性和生长曲线测量值与较大的常规细胞插入物，确认这些器械和尺寸规格促进原代人脐静脉内皮细胞的健康生长。为了测试作为用于单细胞和共培养筛选的高通量平台的可行性，我们将在pnc-Si上开发威尔斯微阵列。我们的方法是新颖的，因为我们将是第一个提供膜支持的微阵列的人，该微阵列能够在共培养环境中研究癌细胞、干细胞以及原代细胞对药物治疗的反应。在第二阶段，我们将专注于药物筛选和干细胞分化，目标是开发自动细胞分配和荧光图像分析系统。在这两种情况下，我们也将寻求扩大微阵列（>100微米）与可降解的膜支持，这将允许生长一个小岛的分层组织。第一阶段的成功完成将使小规模细胞共培养的实时成像研究工具得以推出。在完成第二阶段的6个月内，我们将引入一个384窗口的微阵列系统，该系统具有> 10 × 5个威尔斯孔。

DESCRIPTION (provided by applicant): Advances in protein and DNA microarrays have enabled dramatic increases in throughput and equipment standardization has made these techniques more commonplace. High density, high throughput microarrays reduce the cost of research and development in drug discovery and basic science by decreasing reagent volumes and increasing the number of experiments per plate. Missing from this miniaturization, however, are cell culture microarrays. Existing low well count cell culture plates require greater volumes of precious drug formulations for permeability assays and more plates are required to complete a series of experiments. These same factors increase the cost of parallelized cellular experimentation in basic science such as screening stem cell culture differentiation conditions. In this proposal we will test the feasibility of using a new class of ultrathin nanoporous membrane to enable miniaturization of cell culture screening for high throughout drug permeability and co-culture studies. At the limit we will enable single cell screening to study phenotypic and behavioral variations in cell populations in response to stimuli, drug treatments or co-culture environments. In the first Aim of this work, we will fabricate microarray-scale cell culture arrays using porous nanocrystalline silicon (pnc-Si). We will confirm these devices and size format promote healthy growth of primary human umbilical vein endothelial cells by comparing cytotoxicity and growth curve measurements against larger conventional cell inserts. To test feasibility as a high throughout platform for single cell and co-culture screening, we will develop a microarray of wells on pnc-Si. Our approach is novel because we will be the first to offer a membranesupported microarray that enables study variations in populations of cancer cells, stem cells as well as primary cell response to drug treatment in a co-culture environment. In Phase II we will focus on drug screening and stem cell differentiation with the goal of developing an automated cell dispensing and fluorescent image analysis system. In both cases we will also pursue enlarged microarrays (>100 microns) with degradable membrane supports, which will permit the growth a small islands of stratified tissue. Successful completion of Phase I will enable the launch of a live imaging research tool for small-scale cell co-culture. Within 6 months of completing Phase II, we will introduce a 384-window microarray system with >10x5 wells.