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协调调节多个步骤，包括内皮上的阻滞、通过内皮屏障的迁移和通过结缔组织的定向迁移。 白细胞浸润的潜在关键调节因子是基质金属蛋白酶家族MMP 3的成员。 MMP 3无效的小鼠在多种模型系统中表现出白细胞浸润减少，并且鉴定的许多基质和非基质MMP底物包括已知影响免疫细胞功能的那些。我们假设MMP 3以及其他MMPs在肿瘤进展期间直接增强白细胞外渗。通过分析从具有个体家族成员的遗传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，目前的技术来测定白细胞外渗要么不概括关键的生理参数，如微流体剪切和内皮的顶-基底外侧组织，或者它们需要大量的组织样品，这排除了它们与分离自小鼠的原代细胞一起使用。 该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，将软光刻微细加工技术应用于肿瘤相关白细胞浸润离体研究的生物学挑战。 我们提出了一种多学科方法来开发平面和多层平行毛细管灌注生物反应器（PCPB），其1）更好地近似血液脉管系统的空间约束和结构，2）可以提供调节的剪切流，以及3）在设计中是高通量的，需要最少的细胞样品用于测定条件。 下文详述了用于研究白细胞跨内皮迁移的平面和多层PCPB的开发。这些目标的完成将产生新的装置，其将提供离体系统，该离体系统更接近生理脉管系统，促进对来自循环的白细胞募集的新见解。 具体目标1：研制平面平行毛细管灌注生物反应器（PCPB） A）设计并制作具有循环营养供应系统的平面PCPB B）将平面PCPB应用于测定鼠源性白细胞与内皮单层的粘附。 具体目标2。开发一种多层平行毛细血管灌注生物反应器（PCPB），支持内皮极化至顶侧和基底侧表面 A）设计和制造将结合过滤系统的多层PCPB; B）建立培养物 与传统的TEM测定相比，允许内皮极化穿过多层PCPB的过滤器的条件和C）定义了多层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.