cell seeding in the membrane-based cell culture platform and reconfiguring it to microfluidic mode

A Reconfigurable Barrier Tissue Platform with Open-Well and Microfluidic Modes

Vascular barriers serve as a critical interface that separates the blood compartment from the surrounding tissue. They play a critical role in preserving homeostasis by attracting immune cells, controlling molecular permeability, and shielding against the intrusion of pathogens into the tissue1,2. In vitro culture models have been developed to mimic the in vivo microenvironment, enabling systematic investigations into the factors and conditions that impact barrier properties in both healthy and diseased states3,4.
The most widely used approach for such culture models is the Transwell-like &#34;open-well&#34; configuration5, where a porous, track-etched culture membrane separates media-filled compartments (Figure 1A). In this format, cells can be seeded on either side of the membrane, and a wide range of experimental protocols has been developed. However, these systems are limited in their ability to provide the fluid flows essential for supporting barrier maturation and mimicking immune cell circulation seen in vivo5,6. Consequently, they cannot be used for studies requiring dynamic flows that introduce drug doses, mechanical stimulation, or fluid-induced shear stresses6,7,8.
To overcome the limitations of open-well systems, microfluidic platforms that combine porous culture membranes with individually addressable fluidic channels have been developed9. These platforms offer precise control over fluid routing, perfusion, and the introduction of chemical compounds, controlled shear stimulation, and dynamic cell addition capabilities7,10,11,12,13. Despite the advanced capabilities provided by microfluidic platforms, they have not seen widespread adoption in bioscience laboratories due to complex microfluidic protocols and their incompatibility with established experimental workflows4,10,14.
To bridge the gap between these technologies, we present a protocol that employs a magnetically reconfigurable, module-based system. This system can be easily switched between open-well and microfluidic modes based on the specific needs of the experiment. The platform features an open-well device, known as m-&#181;SiM (modular microphysiological system enabled by a silicon membrane), with a 100 nm thick culture membrane (nanomembrane). This nanomembrane possesses high porosity (15%) and glass-like transparency, as illustrated in Figure 1B. It physically separates the top compartment from a bottom channel, allowing for molecular transport across physiological length scales15. Unlike conventional track-etched membranes, which have known challenges in imaging live cells with bright-field imaging, the nanomembrane&#39;s favorable optical and physical properties enable clear visualization of cells on either side of the membrane surface15,16,17.
The present protocol outlines the fabrication of specialized seeding and flow modules and explains the magnetic reconfiguration of the platform. It demonstrates how the platform can be employed to establish endothelial barriers under both static and dynamic conditions. This demonstration reveals that endothelial cells align along the flow direction, with an upregulation of shear-sensitive gene targets under shear stimulation.

Author Spotlight: Developing a Unique Modular Microphysiological System to Mimic Human Barrier Tissue

Transforming Static Barrier Tissue Models into Dynamic Microphysiological Systems

We're developing modular microphysiological systems that mimic human barrier tissues. Our system is unique because it can be reconfigured from a static open well culture to a flow-enhanced system. This flexibility allows the platform to be used in both bioscience and engineering-focused laboratories.We present a protocol for creating a reconfigurable open well platform with flow-enhanced culture and lifestyle imaging. It is compatible with conventional bioscience protocols. Our reconfigurable design allows users to switch between the open well and microfluidic modes during an experiment, and allows the user to conduct each step using a format they are comfortable with.

Watch this Scientific Journal Video about Cell Seeding in the Membrane-Based Cell Culture Platform and Reconfiguring it to Microfluidic Mode at JoVE.com

Cell Seeding in the Membrane-Based Cell Culture Platform and Reconfiguring it to Microfluidic Mode

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Research

JoVE Journal

Bioengineering

113 次观看.  Rochester Institute of Technology. 首先，在室温下用每平方厘米5微克的纤连蛋白涂覆组装的可重构细胞培养平台的膜一小时，以改善细胞附着。然后，使用p200移液管吸出涂层溶液。将图案模板很好地放入核心模块中。将细胞介质添加到设备的孔和底部通道。将人脐静脉内皮细胞接种到孔中。将设备放入培养皿中。在培养皿中加入含有去离子水的 15 毫升锥形管盖以保持局部湿度。将培养皿装置转移...

视频: 在基于膜的细胞培养平台中进行细胞接种并将其重新配置为微流控模式

Microphysiological systems are miniaturized cell culture platforms used to mimic the structure and function of human tissues in a laboratory setting. However, these platforms have not gained widespread adoption in bioscience laboratories where open-well, membrane-based approaches serve as the gold standard for mimicking tissue barriers, despite lacking fluid flow capabilities. This issue can be primarily attributed to the incompatibility of existing microphysiological systems with standard protocols and tools developed for open-well systems.
Here, we present a protocol for creating a reconfigurable membrane-based platform with an open-well structure, flow enhancement capability, and compatibility with conventional protocols. This system utilizes a magnetic assembly approach that enables reversible switching between open-well and microfluidic modes. With this approach, users have the flexibility to begin an experiment in the open-well format using standard protocols and add or remove flow capabilities as needed. To demonstrate the practical usage of this system and its compatibility with standard techniques, an endothelial cell monolayer was established in an open-well format. The system was reconfigured to introduce fluid flow and then switched to the open-well format to conduct immunostaining and RNA extraction. Due to its compatibility with conventional open-well protocols and flow enhancement capability, this reconfigurable design is expected to be adopted by both engineering and bioscience laboratories.

This protocol describes a reconfigurable membrane-based cell culture platform that integrates the open-well format with fluid flow capabilities. This platform is compatible with standard protocols and allows for reversible transitions between open-well and microfluidic culture modes, accommodating the needs of both engineering and bioscience laboratories.

Microphysiological systems are miniaturized cell culture platforms used to mimic the structure and function of human tissues in a laboratory setting. ...