Microfluidic assay for simultaneous culture of multiple cell types on surfaces or within hydrogels

Shin, Yoojin; Han, Sewoon; Jeon, Jessie S.; Yamamoto, K.; Zervantonakis, Ioannis K.; Sudo, Ryo; Kamm, Roger D.; Chung, Seok

doi:10.1038/nprot.2012.051

Cited by 517 publications

(502 citation statements)

References 28 publications

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“…Microfluidics overcomes some of the technical limitations of traditional assays (16), allowing the study of cancer metastases under biochemically and biophysically controlled 3D microenvironments coupled with high-resolution real-time imaging (17). Various microfluidic models have been developed for studying tumor angiogenesis (18), transition to invasion (19), intravasation (20), the role of interstitial flow (21) and matrix stiffness (22) on…”

mentioning

confidence: 99%

Human 3D vascularized organotypic microfluidic assays to study breast cancer cell extravasation

Jeon

Bersini

Gilardi

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

617

549

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A key aspect of cancer metastases is the tendency for specific cancer cells to home to defined subsets of secondary organs. Despite these known tendencies, the underlying mechanisms remain poorly understood. Here we develop a microfluidic 3D in vitro model to analyze organ-specific human breast cancer cell extravasation into bone- and muscle-mimicking microenvironments through a microvascular network concentrically wrapped with mural cells. Extravasation rates and microvasculature permeabilities were significantly different in the bone-mimicking microenvironment compared with unconditioned or myoblast containing matrices. Blocking breast cancer cell A3 adenosine receptors resulted in higher extravasation rates of cancer cells into themyoblast-containingmatrices compared with untreated cells, suggesting a role for adenosine in reducing extravasation. These results demonstrate the efficacy of our model as a drug screening platform and a promising tool to investigate specific molecular pathways involved in cancer biology, with potential applications to personalized medicine

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mentioning

confidence: 99%

Human 3D vascularized organotypic microfluidic assays to study breast cancer cell extravasation

Jeon

Bersini

Gilardi

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

617

549

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“…Microfluidic devices were fabricated via soft lithography as described previously (47). MDA-MB 231 cells derived from pleural effusion were purchased from the American Type Culture Collection and cultured in growth medium comprised of DMEM (Invitrogen) with 10% (vol/vol) FBS (Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

Mechanotransduction of fluid stresses governs 3D cell migration

Polacheck

German

Mammoto

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

219

215

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Solid tumors are characterized by high interstitial fluid pressure, which drives fluid efflux from the tumor core. Tumor-associated interstitial flow (IF) at a rate of ∼3 μm/s has been shown to induce cell migration in the upstream direction (rheotaxis). However, the molecular biophysical mechanism that underlies upstream cell polarization and rheotaxis remains unclear. We developed a microfluidic platform to investigate the effects of IF fluid stresses imparted on cells embedded within a collagen type I hydrogel, and we demonstrate that IF stresses result in a transcellular gradient in β1-integrin activation with vinculin, focal adhesion kinase (FAK), FAK PY397 , F actin, and paxillindependent protrusion formation localizing to the upstream side of the cell, where matrix adhesions are under maximum tension. This previously unknown mechanism is the result of a force balance between fluid drag on the cell and matrix adhesion tension and is therefore a fundamental, but previously unknown, stimulus for directing cell movement within porous extracellular matrix.mechanobiology | breast cancer | metastasis I ntegrins and associated focal adhesion (FA) proteins form a tension-sensitive mechanical link between the extracellular matrix (ECM) and the cytoskeleton, and serve as key components in the signaling cascade by which cells transduce mechanical signals into biological responses (mechanotransduction) (1, 2). Contractile stresses generated by the cell are balanced by tractions at cell-substrate adhesions, and the FA protein vinculin accumulates at regions of high substrate stress (3, 4). The FA protein paxillin colocalizes with vinculin (4) and mediates β1-integrin FA turnover through interaction with FA kinase (FAK) (5). The FAK-paxillin signaling axis recruits vinculin to β1 integrins at regions of high matrix adhesion tension (6), and paxillin-a key mechanosensor (7)-mediates protrusion formation at regions of high stress on 2D substrates (8), and FAK-paxillin-vinculin signaling is required for mechanosensing and durotaxis (9).The tumor microenvironment imparts mechanical and chemical signals on tumor and stromal cells (10), and advanced breast carcinomas are characterized by high interstitial fluid pressure (11), an indicator of poor prognosis (12). This elevated fluid pressure drives interstitial flow (IF) and alters chemical transport within the tumor (13), and IF influences tumor cell migration through the generation of autocrine chemokine gradients (14). Equally important, although not as well understood, is the physical drag imparted on the ECM and constitutive cells (15) by IF, which is analogous to the FA-activating shear stresses generated on endothelial cells by hemodynamic forces (16). With endothelial cells, shear stress can be the dominant mechanical stimulus that induces FAK activation and cytoskeletal remodeling; however, for cells embedded within a porous matrix scaffold, the ratio of the force due to the pressure drop across the cell to the total shear force is inversely proportional to hydrogel p...

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“…Below, we describe briefly how to fabricate a suitable silicon master (Step 2.1-2.3) and PDMS stamp (Step 2.4). However, there are multiple protocols for microcontact printing described in the literature [19][20][21][22][23] , which are suitable for printing protein patterns onto glass coverslips. Masters can also be custom made and purchased from commercial sources.…”

Section: Fabrication Of Stamps For Microcontact Printingmentioning

confidence: 99%

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Ngalim

Magenau

Zhu

et al. 2013

JoVE

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Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues. Video LinkThe video component of this article can be found at

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Microfluidic assay for simultaneous culture of multiple cell types on surfaces or within hydrogels

Cited by 517 publications

References 28 publications

Human 3D vascularized organotypic microfluidic assays to study breast cancer cell extravasation

Human 3D vascularized organotypic microfluidic assays to study breast cancer cell extravasation

Mechanotransduction of fluid stresses governs 3D cell migration

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

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