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Microfluidic transwell inserts for generation of tissue culture-friendly gradients in well plates.

ABSTRACT: Gradients of biochemical molecules play a key role in many physiological processes such as axon growth, tissue morphogenesis, and trans-epithelium nutrient transport, as well as in pathophysiological phenomena such as wound healing, immune response, bacterial invasion, and cancer metastasis. In this paper, we report a microfluidic transwell insert for generating quantifiable concentration gradients in a user-friendly and modular format that is compatible with conventional cell cultures and with tissue explant cultures. The device is simply inserted into a standard 6-well plate, where it hangs self-supported at a distance of ~250 ?m above the cell culture surface. The gradient is created by small microflows from the device, through an integrated track-etched porous membrane, into the cell culture well. The microfluidic transwell can deliver stable, quantifiable gradients over a large area with extremely low fluid shear stress to dissociated cells or tissue explants cultured independently on the surface of a 6-well plate. We used finite-element modeling to describe the porous membrane flow and molecular transport and to predict gradients generated by the device. Using the device, we applied a gradient of the chemotactic peptide N-formyl-met-leu-phe (fMLP) to a large population of HL-60 cells (a neutrophil cell line) and directly observed the migration with time-lapse microscopy. On quantification of the chemotactic response with an automated tracking algorithm, we found 74% of the cells moving towards the gradient. Additionally, the modular design and low fluid shear stress made it possible to apply gradients of growth factors and second messengers to mouse retinal explant cultures. With a simplified interface and well-defined gradients, the microfluidic transwell device has potential for broad applications to gradient-sensing biology.

SUBMITTER: Sip CG

PROVIDER: S-EPMC4362725 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

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Microfluidic transwell inserts for generation of tissue culture-friendly gradients in well plates.

Sip Christopher G CG Bhattacharjee Nirveek N Folch Albert A

Lab on a chip 20131113 2

Gradients of biochemical molecules play a key role in many physiological processes such as axon growth, tissue morphogenesis, and trans-epithelium nutrient transport, as well as in pathophysiological phenomena such as wound healing, immune response, bacterial invasion, and cancer metastasis. In this paper, we report a microfluidic transwell insert for generating quantifiable concentration gradients in a user-friendly and modular format that is compatible with conventional cell cultures and with ...[more]

PMID: 24225908

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Project description:Semipermeable cell culture membranes are commonly used in multilayered microfluidic devices to mimic the basement membrane in vivo and to create compartmentalized microenvironments for physiological cell growth and differentiation. However, existing membranes are predominantly made up of synthetic polymers, providing limited capacity to replicate cellular interactions with native extracellular matrices that play a crucial role in the induction of physiological phenotypes. Here we describe a new type of cell culture membranes engineered from native extracellular matrix (ECM) materials that are thin, semipermeable, optically transparent, and amenable to integration into microfluidic cell culture devices. Facile and cost-effective fabrication of these membranes was achieved by controlled sequential steps of vitrification that transformed three-dimensional (3D) ECM hydrogels into structurally stable thin films. By modulating the composition of the ECM, our technique provided a means to tune key membrane properties such as optical transparency, stiffness, and porosity. For microfluidic cell culture, we constructed a multilayered microdevice consisting of two parallel chambers separated by a thin membrane insert derived from different types of ECM. This study showed that our ECM membranes supported attachment and growth of various types of cells (epithelial, endothelial, and mesenchymal cells) under perfusion culture conditions. Our data also revealed the promotive effects of the membranes on adhesion-associated intracellular signaling that mediates cell-ECM interactions. Moreover, we demonstrated the use of these membranes for constructing compartmentalized microfluidic cell culture systems to induce physiological tissue differentiation or to replicate interfaces between different tissue types. Our approach provides a robust platform to produce and engineer biologically active cell culture substrates that serve as promising alternatives to conventional synthetic membrane inserts. This strategy may contribute to the development of physiologically relevant in vitro cell culture models for a wide range of applications.

Dataset Information

Microfluidic transwell inserts for generation of tissue culture-friendly gradients in well plates.

Publications

Microfluidic transwell inserts for generation of tissue culture-friendly gradients in well plates.

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