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.

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

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Research

JoVE Journal

Bioengineering

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