DataSheet1_Self-loading microfluidic platform with ultra-thin nanoporous membrane for organ-on-chip by wafer-level processing.pdf

Embedded porous membranes are a key element of various organ-on-chip systems. The widely used commercial polymer membranes impose limits with regard to chip integration and thinness. We report a microfluidic chip platform with the key element of a monolithically integrated, ultra-thin (700 nm) nanoporous membrane made of ultra-low-stress (<35 MPa) SixNy for culturing and testing reconstructed tissue. The membrane is designed to support various in vitro tissues including co-cultures and to allow passage of molecules but not of cells. A digital laser write method was used to produce nanopores with deterministic but highly flexible positioning within the membrane. A thin layer of photoresist was exposed by accumulation of femtosecond pulses for local two-photon polymerization, which allowed nanopores as small as 350 nm in diameter to be generated through the membranes in a subsequent plasma etch process. The fabricated membranes were used to separate a microfluidic chip into two compartments, which are connected to the outside by microchannel structures. With unique side inlets for fluids, all cells are exposed to identical flow velocities and shear stresses. With the hydrophilic nature of chip materials the self-loading seeding is controlled bottom-up by capillary forces, which makes the seeding procedure homogeneous and less dependent on the operator. The chip is designed to allow fabrication by wafer-level MEMS manufacturing technologies without critical assembly steps, thereby promoting reproducibility and scale-up of fabrication. In order to establish a fully functional test system to be used in a lab incubator, a holder for the bare chip was designed and 3D-printed with additional elements for gravity driven pumping. In order to mimic physiological conditions, the holder was designed to provide not only media delivery but also appropriate shear stress to the tissue. To prove usability, murine microvascular endothelial cells (muMEC) were seeded on the membrane within the chip. Cell compatibility was confirmed after 3 days of dynamic cultivation using fluorescence live/dead assays. Cultivation proved to be reproducible and led to confluent layers with cells preferentially grown on nanoporous areas. The system can in future be cost effectively manufactured in larger quantities in MEMS foundries and can be used for a wide variety of in vitro tissues and test scenarios including pumpless operation within cell incubator cabinets.

嵌入式多孔膜（embedded porous membranes）是各类器官芯片（organ-on-chip）系统的核心组件。当前广泛使用的商用聚合物膜在芯片集成性与厚度方面存在局限。本研究报道了一款微流控芯片平台，其核心组件为单片集成的超薄（700 nm）纳米多孔膜，该膜由超低应力（<35 MPa）的SixNy制成，可用于重构组织的培养与检测。该膜可支撑包括共培养体系在内的多种体外组织，并允许分子透过但阻挡细胞。研究采用数字激光直写技术制备纳米孔，孔径位置可确定且具备高度灵活的可调性。通过飞秒脉冲累积实现薄层光刻胶的局域双光子聚合，后续结合等离子体刻蚀工艺，可在膜上制备直径仅350 nm的纳米孔。所制备的膜将微流控芯片分隔为两个腔室，两腔均通过微通道结构与外部连通。该芯片配备独特的流体侧向入口，可使所有细胞承受一致的流速与剪切应力。依托芯片材料的亲水特性，接种过程可通过毛细管力实现自下而上的自动加载，使得接种过程均匀且对操作人员依赖度大幅降低。该芯片采用晶圆级微机电系统（MEMS）制造工艺即可完成制备，无需关键装配工序，有助于提升制备的可重复性与规模化能力。为构建可在实验室培养箱中使用的全功能测试系统，研究人员设计了裸芯片支架，并通过3D打印制作了带有重力驱动泵送附加结构的支架。该支架不仅可实现培养基输送，还可为组织提供适宜的剪切应力，以模拟生理环境。为验证系统可用性，研究人员将小鼠微血管内皮细胞（muMEC）接种至芯片内的膜上。经3天动态培养后，通过荧光死活检测证实了细胞相容性。培养过程具备可重复性，细胞最终形成致密融合层，且优先定植于纳米多孔区域。本系统未来可在微机电系统代工厂中实现低成本大批量生产，可适配多种体外组织与测试场景，包括在细胞培养箱内实现无泵运行。