Parallel Capillary Bioreactors for Leukocyte Transendothelial Migration Analysis

描述（由申请人提供）：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）浸润到炎症部位需要多个步骤的协调调节，包括对内皮细胞的阻滞、通过内皮屏障的迁移和通过结缔组织的定向迁移。基质金属蛋白酶家族（MMP3）是白细胞浸润的一个潜在关键调节因子。在多种模型系统中，无MMP3的小鼠表现出白细胞浸润减少，并且确定了许多基质和非基质MMP底物，包括那些已知影响免疫细胞功能的底物。我们假设MMP3以及其他MMPs在肿瘤进展过程中直接促进白细胞外渗。通过分析从个体家族成员基因消融的小鼠中分离的原代细胞，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不能重现关键的生理参数，如微流体剪切和内皮的尖基底外侧组织，要么需要大量的组织样本，这就排除了它们与小鼠分离的原代细胞的使用。本提案的目标是利用我们的癌症生物学家和工程师合作团队的专业知识，将软光刻微加工技术应用于肿瘤相关白细胞体外浸润研究的生物学挑战。我们提出了一种多学科的方法来开发平面和多层平行毛细血管灌注生物反应器（PCPB）， 1)更好地接近血管的空间限制和结构，2)可以提供可调节的剪切流量，3)在设计上需要最少的细胞样品进行分析条件，具有高通量。下面详细介绍了用于研究白细胞跨内皮迁移的平面和多层PCPBs的进展。这些目标的完成将产生新的设备，这些设备将提供一个更接近生理血管系统的离体系统，促进对循环中白细胞募集的新见解。

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.