MICROFLUIDIC PLATFORM TO STUDY THREE DIMENSIONAL CELL MIGRATION & CAPILLARY

MORPHOGENESIS S. Chung1, R. Sudo1, I. Zervantonakis1, T. Rimchala1, P.J. Mack1,

C.-R. Wan1, V. Vickerman1 and R.D. Kamm1 1Massachusetts Institute of Technology (MIT), CAMBRIDGE, USA

ABSTRACT

Here we present a new microfluidic platform that allows for the simultaneous imaging of cells migrating through a 3D matrix, production of a stable, concentration gradient of chemoattractant, and side-by-side comparisons of control and test condi-tions to normalize for sample-to-sample variability. We demonstrate the system here in a study of the migration of two tumor cell lines and the sprouting of endothelial cells into the matrix from a monolayer. We also show results using the same system to investigate the transmigration of a tumor cell across an endothelial monolayer. These experiments demonstrate the wide range of capabilities of the new system.

KEYWORDS: Microfluidic, Cell Migration, Capillary Morphogenesis,

INTRODUCTION

Capillary morphogenesis and cell migration are complex cellular processes that occur in response to external stimuli being essential to the formation of new blood vessels, cancer metastasis, would healing, and inflammation [1]. In the past decade or more, much attention by the biological communities has been focused on creating experimental systems that enable the visualization of cell migration under a con-trolled chemoattractant gradient. These studies are critical to the understanding of, for example, tumor cell migration in metastatic disease or the migration of inflam-matory cells into tissue. For years, these studies have been conducted on two-dimensional surfaces, since it was easier both to track the cells and to produce a sta-ble concentration gradient in two dimension than in the more physiological three-dimensional environment. With the recent advent of microfluidic systems that in-corporate regions of gel or matrix material (simulating extracellular matrix), these limitations can now be overcome [2].

EXPERIMENTAL

Figure 1 shows the newly developed platform to evaluate and quantify capillary morphogenesis and endothelial cell migration from an intact cell monolayer. Endo-thelial cells were seeded and cultured in a central microfluidic channel to form a confluent cell monolayer. The central channel is surrounded on two sides by colla-gen gel scaffolds that cells can migrate into under precisely controlled conditions of mechanical and chemical angiogenic factors (e.g., fluid shear stress, interstitial flow, scaffold stiffness and fixed gradients of growth factors (VEGF)). To demonstrate the capabilities of this assay, we applied VEGF to the right-hand side channel, gen-erating a VEGF gradient across the scaffold. The opposite side channel was filled

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Twelfth International Conference on Miniaturized Systems for Chemistry and Life SciencesOctober 12 - 16, 2008, San Diego, California, USA

with cell culture medium as a control. During several days of culture, the length and area of migrating cells into the scaffolds were observed and quantified.

Figure 1: (a) Schematic drawing of 3 channel microfluidic device. Chemoattrac-tant, growth factors or co-culturing cells are added in the right-hand condition channel to generate a chemical gradient in the gel region. (b) The gradient was

confirmed by experiments with fluorescent dextran.

RESULTS AND DISCUSSION Microvascular cells in contact with the VEGF gradient were highly active and

rapidly migrated into the scaffold, but cells in contact with the control scaffold were more restrained and demonstrated markedly less migration (Figure 2 & 3). We also developed a new surface coating technique to minimize the preference for surface migration of the cells which could dominate in microfluidic channels with large sur-face area. Confocal microscopic images confirmed three dimensional migration and also the cross-sectional distribution of the cells in the scaffolds.

Figure 2: Proof-of-principle utility of the microfluidic cell migration platform with other cell types and in co-culture. (a) Endothelial cell (HMVEC) migration under

VEGF gradient. Note large migration only on condition side. (b) Co-culture of HMVEC and rat mammary adenocarcinoma cell line (MTLn3) at ~ 1,000

cells/mm2. Migration of HMVEC is faster on condition side than control side. (b) Co-culture of HMVEC and smooth muscle cells (10T 1/2). Note HMVEC only

formed capillary structures into the control side. (c) Co-culture of HMVEC and human glioblastoma cells (U87MG). Note similar migration characteristics on

condition and control sides. We also evaluated the endothelial cell response in co-culture with physiologi-

cally relevant cell types, including cancer cells and smooth muscle cells. This re-

(a)

(b)

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Twelfth International Conference on Miniaturized Systems for Chemistry and Life SciencesOctober 12 - 16, 2008, San Diego, California, USA

sulted in the following observations: Cancer cells can either attract (MTLn3 cancer cell line) endothelial cells and induce capillary formation or have minimal effect (U87MG cancer cell line) while smooth muscle cells (10T 1/2) suppress endothelial activity. Results presented demonstrate that our newly developed platform provides a novel method for studying cellular morphogenesis both qualitatively and quantita-tively while having the advantage of enhanced imaging and internal biological con-trols.

Figure 3: (a) Quantification of the different re-sponses of HMVEC to time-dependent VEGF pres-entation. Note VEGF added only 1 day or 2 days af-ter cell seeding attracted capillary formation.

Figure 4 shows an example for using the assay in other application of intravasa-tion. A cancer cell (U87MG) can be co-cultured and monitored approaching and penetrating HMVEC monolayer. The developed platform has been applied to nu-merous applications producing interesting findings in the study of angiogenesis, or migration of other cell types including neurons, stem cells, yeast or bacteria, smooth muscle cells, cancer cells and leukocytes, into a three-dimensional scaffold or across an endothelial monolayer under conditions of precisely controlled mechanical, bio-chemical and co-culturing environment.

Figure 4: Cancer cells (U87MG) penetrating into the HMVEC mono-layer (intravasation).

ACKNOWLEDGEMENTS

We would like to thank Douglas Lauffenburger for providing the cancer cells (MTLn3, U87MG), Guillermo Garcia-Cardena for providing the smooth muscle cells (10T 1/2) and Jose Antonio Sanz-Hererrea for analyzing diffusion experiments. REFERENCES [1] "Quantitative angiogenesis assays: progress and problems," R.K.Jain,

K.Schlenger, M.Hockel, F.Yuan, Nature Medicine, 2, 1203-1208 (1997). [2] "Design, Fabrication and Implementation of a Novel Multi Parameter Control

Microfluidic Platform for Three-Dimensional Cell Culture and Real-Time Im-aging," V.Vickerman, J.Blundo, S.Chung, R.Kamm, Lab Chip, in press (2008).

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Twelfth International Conference on Miniaturized Systems for Chemistry and Life SciencesOctober 12 - 16, 2008, San Diego, California, USA