Parallel Capillary Bioreactors for Leukocyte Transendothelial Migration Analysis

描述(申请人提供)：肿瘤微环境，特别是肿瘤相关炎症，是肿瘤进展的驱动力。免疫细胞(即白细胞)渗入炎症部位需要多个步骤的协调调节，包括内皮细胞的停滞、通过内皮屏障的迁移和通过结缔组织的定向迁移。一个潜在的白细胞渗透的关键调节因子是基质金属蛋白酶家族的成员MMP3。MMP3缺失的小鼠在各种模型系统中表现出白细胞渗透减少，一些已确定的基质和非基质基质包括那些已知影响免疫细胞功能的基质。我们假设，MMP3和其他MMPs在肿瘤进展过程中直接促进白细胞外渗，通过分析从单个家庭成员基因消融的小鼠中分离出的原代细胞，可以直接分析MMPs的作用。然而，目前检测白细胞外渗的技术要么没有概括关键的生理参数，如内皮的微流体剪切和顶底侧向组织，要么需要大量的组织样本，排除了它们与从小鼠分离的原代细胞的使用。这项建议的目的是利用我们由癌症生物学家和工程师组成的合作团队的专业知识，将软光刻微制造技术应用于研究肿瘤相关白细胞体外渗透的生物学挑战。我们提出了一种多学科的方法来开发平面和多层并行毛细管灌流生物反应器(PCPB)，1)更接近血管的空间约束和结构，2)可以提供可调节的剪切流，3)高通量的设计需要最少的细胞样本来分析条件。下面详细介绍平面和多层PCPBs在研究白细胞跨内皮细胞迁移方面的应用进展。完成这些目标将产生新的装置，将提供一个更接近生理血管的体外系统，促进对白细胞从循环中招募的新见解。 具体目标1：研制平面平行式毛细管灌流生物反应器 A)设计和制造具有循环营养供应系统的平面型PCPB B)将平面型PCPB应用于小鼠来源的白细胞与内皮单层的黏附实验。 具体目的2.开发一种多层平行毛细管灌流生物反应器(PCPB)，它将支持内皮细胞极化进入根尖和基底外侧表面 A)设计和制造包含过滤系统的多层PCPB；b)建立允许跨多层PCPB过滤器的内皮极化的培养条件；以及C)与传统的透射电子显微镜检测相比，确定多层PCPB中白细胞附着、滚动和跨内皮迁移的参数。

DESCRIPTION (provided by applicant): The tumor microenvironment, and in particular tumor associated inflammation, is a driving force of tumor progression. Immune cell (i.e. leukocyte) infiltration into sites of inflammation requires the coordinate regulation of multiple steps including arrest on endothelium, migration through the endothelial barrier and directed migration through connective tissue. A potential key regulator of leukocyte infiltration is a member of the matrix metalloproteinase family, MMP3. Mice which are null for MMP3 demonstrate reduced infiltration of leukocytes in a variety of model systems, and a number of matrix and non-matrix MMP substrates identified include those known to affect immune cell function. We hypothesize that MMP3, as well as other MMPs, directly enhances leukocyte extravasation during tumor progression Direct analysis of the roles of MMPs is possible through analysis of primary cells isolated from mice with genetic ablation of individual family members. However, current technologies to assay leukocyte extravasation either do not recapitulate key physiological parameters such as the microfluidic shear and apical-basolateral organization of endothelium, or they require extensive tissue samples that excludes their use with primary cells isolated from mice. The goal of this proposal is to exploit the expertise of our collaborative team of Cancer Biologists and Engineers in applying soft-lithography microfabrication technology to the biological challenge of the study of tumor- associated leukocyte infiltration ex vivo. We propose a multidisciplinary approach in the development of planar and multilayer Parallel Capillary Perfused Bioreactors (PCPB) that 1) better approximate the spatial constraints and architecture of blood vasculature, 2) can provide regulated shear flow and 3) are high- throughput in design requiring minimal cell samples for assay conditions. The development of the planar and multi-layer PCPBs for application in studying leukocyte transendothelial migration are detailed below. Completion of these aims will generate novel devices that will provide an ex vivo system that more closely approximates physiological vasculature facilitating novel insights into leukocyte recruitment from circulation. Specific Aim 1: To develop a planar parallel capillary perfused bioreactor (PCPB) A) Design and fabricate planar PCPB with a recirculating nutrient supply system B) Apply the planar PCPB to the assay murine derived leukocyte attachment to endothelial monolayers. Specific Aim 2. To develop a multilayer parallel capillary perfused bioreactor (PCPB) that will support endothelial polarization into apical and basolateral surfaces A) Design and fabricate a multilayer PCPB that will incorporate a filter system; B) establish culture conditions that allow for endothelial polarization across filter of multilayer PCPB and C) define parameters for leukocyte attachment, rolling and transendothelial migration in multilayer PCPB as compared to traditional TEM assay.