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

Abstract

This protocol describes a simple but robust microfluidic assay combining three-dimensional (3D) and two-dimensional (2D) cell culture. The microfluidic platform comprises hydrogel-incorporating chambers between surface-accessible microchannels. By using this platform, well-defined biochemical and biophysical stimuli can be applied to multiple cell types interacting over distances of <1 mm, thereby replicating many aspects of the in vivo microenvironment. Capabilities exist for time-dependent manipulation of flow and concentration gradients as well as high-resolution real-time imaging for observing spatial-temporal single-cell behavior, cell-cell communication, cell-matrix interactions and cell population dynamics. These heterotypic cell type assays can be used to study cell survival, proliferation, migration, morphogenesis and differentiation under controlled conditions. Applications include the study of previously unexplored cellular interactions, and they have already provided new insights into how biochemical and biophysical factors regulate interactions between populations of different cell types. It takes 3 d to fabricate the system and experiments can run for up to several weeks.

Figure 1

(a) Schematic and (b) photograph of the microfluidic cell culture assay incorporating hydrogel and colored liquids in the side channels (red) and central channel (blue). All scales are microns.

Figure 2

Schematic of the photolithography (a-c) and soft lithography (d-f) procedure. (a) SU-8 is spin-coated and pre-baked on a bare wafer. (b) Use a transparency photomask (black), UV light is exposed on the SU-8. (c) Exposed SU-8 is then post-exposure baked and developed to define channel patterns. (d) PDMS mixed solution is poured on the wafer and cured. (e) Cured PDMS is then peeled from the wafer. (f) Device is trimmed, punched and autoclaved ready for assembly.

Figure 3

Procedure of hydrogel incorporating microfluidic assay preparation. (a) Autoclaved PDMS device and coverslip are assembled with plasma treatment (b) to close the microfluidic channels. (c) PDL solution is filled and device is placed in an incubator. (d) After washing and aspiration, coated device is stored in dry oven for 24 hours to render the microfluidic channel surface hydrophobic. (e) Hydrogel is filled into the hydrogel region, and (f) medium is added into the microfluidic channel. Device is ready for cell seeding in incubator.

Figure 4

(a) Color of type 1 collagen solution (before gellation) with different pH; about 5.0 (left), 7.4 (center) and 11.0 (right). The prepared type 1 collagen solution was then filled into the hydrogel region. (b) SEM image of gelled filbers of type 1 collagen gel (after gellation) with different initial pH; about 5.0 (left), 7.4 (center) and 11.0 (right). Scale bars indicate 1 microns. (c) In hydrogel filling, use 10 ul pipette and slowly inject hydrogel into the gel region. (d) Injected hydrogel perfectly filled into the hydrogel region (red) and stoped by surface tension between post and PDMS wall.

Figure 5

Cell culture in microfluidic assay. (a) After aspiration of medium in the reservoir, 50~60 μl of the hMVEC suspension is added into one reservoir connected to the center channel. (b) Hydrostatic pressure difference in the reservoirs pushes the suspended hMVECs onto the collagen gel to facilitate attachment. hMVECs right after seeding (right top) and tight monolayer at 1 day (right bottom). Scale bar indicates 250 microns. (c) To add cell suspension or media into the reservoir, use a pipette and gently inject medium into one reservoir after aspiration. The filled liquid automatically fills the channel and replaces the contained medium. (d) Cell seeded assays can be stored in incubator or 6 well plate with various experimental conditions. (e) In 4 days of culture, hMVECs form intact monolayer in channel and on type 1 collagen wall. Actin filaments and nuclei were then stained with rhodamine-phalloidin (yellow; Sigma-Aldrich) and 4',6-diamidino-2-phenylindole (DAPI; blue; Sigma-Aldrich), respectively. Scale bar indicates 50 microns. EC monolayer was imaged by confocal microscope and its diffusion coefficient was measured by fluorescent dextran diffusion^,,.

Figure 6

Characterization of diffusion profile of applied VEGF. (a) 4 hours of VEGF diffusion into type 1 collagen is simulated (left). Diffusion coefficient of VEGF in type 1 collagen in the simulation was 5.8×10^-11 m²/sec. Arrows indicate diffusive flux. Steady-state concentration profile is shown along the black line (right). Blue color corresponds to 0 ng/ml VEGF while red corresponds to 50 ng/ml VEGF. (b) Gradient (left) and normalized intensity plot from 0 to 30 minutes (center) and from 30 minutes to 10 hours (right). Intensity was measured from right to left sides of type 1 collagen (2.0mg/ml, polymerized at pH 7.4), and then normalized to unity in each figure. (c) Angiogenic response from ECs monolayer was induced by the VEGF diffusion gradient from the right channel (red arrowhead). (d) Perimeter, area and length of angiogenic response were measured (segmented in pale red) and quantified in the captured images.

Figure 7

Confocal image of 3D angiogenic response into the type 1 collagen (2.0 mg/ml, polymerized at pH 7.4) induced by VEGF diffused from the right channel. (left) 3D projection image of the capillary-like structures formed by hMVEC on day 12 of culture, which was reconstructed from confocal z-stack images. hMVECs formed 3D capillary-like networks extending into the collagen gel, which were stained with CD-146 (hMVEC, red) and DAPI (nucleus, blue). (right) A single z-plane image and x-z and y-z cross-sectional images of the capillary-like networks. Image field corresponds to the left image. Arrowheads indicate a capillary-like structure which is continuous from the monolayer in the channel. An arrow in the x-z image indicates a lumen of the capillary-like structure.

Figure 8

3D interaction of various types of cells with incorporated hydrogel. (a) Cells are seeded onto hydrogel, and then migrate in individually. (top) Migration of mouse smooth muscle precursor cells (10T 1/2) into type 1 collagen (2.0 mg/ml, initial pH 9.0), on day 6 co-cultured with hMVECs (right channel). (center) Sheet like migration of mouse breast cancer cells (MTLn3) into type 1 collagen (2.0 mg/ml, initial pH 7.4) in day 3 co-cultured with hMVECs (right channel). (bottom) Migration of human breast cancer cells (MDA-MB-231) into type 1 collagen (2.0 mg/ml, initial pH 7.4) on day 3 under 5 % FBS gradient. (b) Cells formed 3D tissue-like structures on hydrogel. (top) Axonal growth into type 1 collagen (1.0 mg/ml, initial pH 7.4) from primary mouse cortical neurons cultured 2 days under 50 μM Forskolin gradient. (center) Primary hepatocyte cultured on type 1 collagen (2.0 mg/ml, initial pH 7.4) in 7 days under interstitial flow condition. Cells were fixed at day 7 and stained for actin filaments (rhodamine-phalloidin; red), nuclei (DAPI; blue) and SEC fraction (GFP; green). Tight tissue-like hepatocyte structure can be found, without migration. (bottom) Human breast cancer cells (MCF-7) cultured on type 1 collagen (2.0 mg/ml, initial pH 9.0) for 3 days under 5 % FBS gradient formed tight tumor colony without migration. (c) Endothelial cells form intact monolayer on hydrogel, showing capillary morphogenesis into hydrogel. hMVEC cells form complex capillary structure in type 1 collagen (2.0 mg/ml, initial pH 7.4) for 5 days under VEGF gradient and ANG-1 supplement. (d) Floating cells in the media migrate into hydrogel under chemoattractant gradient. T lymphocyte cells (Jurkat) migrating into type 1 collagen (2.0 mg/ml, initial pH 7.4) on day 3 under SDF-1 gradient. (e) Co-culture of neutrophil (HL60) in microfluidic channel covered by hMVEC monolayer cultured 3 days. HL60 migrated through the nMVEC monolayer and then into type 1 collagen (2.0 mg/ml, initial pH 10). Cells were fixed at day 4 and stained for actin filaments (rhodaminephalloidin; red), nuclei (DAPI; blue) and neutrophil (CFDA-SE cell tracker; green). In all other figures not mentioned, actin filaments and nuclei were stained with rhodamine-phalloidin (yellow) and DAPI (blue), respectively. White lines indicate outline of incorporated hydrogel. Scale bars indicate 100 μm.

Figure 9

Various experimental modes of cell seeding and morphogenesis in hydrogel incorporating microfluidic assay.

Figure 10

Examples of cell-cell 3D interaction in hydrogel. (a) Co-culture of SMCs (10T 1/2; right channel) and ECs (hMVEC; center channel). Active 3D sprouting angiogenesis into type 1 collagen is monitored (into left hydrogels), while the hMVEC monolayer is stabilized by the presence of SMCs (right hydrogels). 10T 1/2 migration was also activated by the existence of hMVEC monolayer in the center channel. Actin filaments and nuclei were stained with rhodamine-phalloidin (yellow) and DAPI (blue), respectively. (b) GFP transfected U87MG (white arrowheads) transmigrated through the hMVEC monolayer. Colors indicate actin (white), nucleus (blue) and GFP (green). Scale bars 50 μm. (c) Interaction between SMC (white arrowhead) and hMVEC (blue arrowheads). A SMC touches the hMVEC monolayer with its filopodia, and becomes adhered to the monolayer. Colors indicate actin (white), nucleus (blue) and EC-specific primary antibody (green). Scale bars 50 μm. (d) GFP transfected breast cancer cell MDA-MB-231 (white arrowhead) interacting with hMVEC (blue arrowheads) sprouts in type 1 collagen scaffold. Colors indicate actin (white), nucleus (blue) and GFP (green). Scale bars 100 μm. (e) Primary neurons generating axons towards GFP transfected glioblastoma cells U87MG (white arrowheads). Colors indicate actin (white), nucleus (blue) and GFP (green). Scale bars 100 μm.