This protocol describes how to form a 3D cell culture with explicit, endothelialized microvessels. The approach leads to fully enclosed, perfusable vessels in a bioremodelable hydrogel (type I collagen). The protocol uses microfabrication to enable user-defined geometries of the vascular network and microfluidic perfusion to control mass transfer and hemodynamic forces. These microvascular networks (μVNs) allow for multiweek cultures of endothelial cells or cocultures with parenchymal or tissue cells in the extra-lumen space. The platform enables real-time fluorescence imaging of living engineered tissues, in situ confocal fluorescence of fixed cultures and transmission electron microscopy (TEM) imaging of histological sections. This protocol enables studies of basic vascular and blood biology, provides a model for diseases such as tumor angiogenesis or thrombosis and serves as a starting point for constructing prevascularized tissues for regenerative medicine. After one-time microfabrication steps, the system can be assembled in less than 1 d and experiments can run for weeks.
At a glance
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- Video 5: Supplementary Video 5 (1.44 MB, Download)
- Supplementary video for time course shown in Figure 3. GFP-expressing endothelial cells exhibit dynamic migration within the endothelium in live fluorescence imaging experiments under physiological shear flow (∼11 mL/min, 17 dyne/cm2). Time course = 48-72 hours after onset of perfusion; scale bar = 50 μm.
- Supplementary Figure 1 (141 KB)
Technical drawings of the aluminum jig for casting the PDMS stamp. Technical drawings, including critical dimensions, for the three aluminum jig components for molding the PDMS stamp. A machine shop should be able to fabricate all of the necessary pieces based on these schematics.
- Supplementary Figure 2 (68 KB)
Technical drawings for the microfluidic polycarbonate culture device. Technical drawings, including critical dimensions, for the top and bottom pieces of the microfluidic culture device. A machine shop should be able to fabricate all of the necessary pieces based on these schematics.
- Supplementary Figure 3 (10,641 KB)
Photographs for detail steps not presented in Figure 2, including the process for device assembly (steps 24-48), seeding (steps 49-53) and gravity-driven perfusion culture (steps 54A and 54B) processes. (i) Place the top piece of the microfluidic culture device onto the PDMS stamp, aligning the Plexiglas reservoir ports with the PDMS channel inlets. (ii) Insert the 16 gauge stainless steel dowel pins into the reservoir ports (to prevent collagen from entering the reservoir ports during injection) on the top piece of the microfluidic device and inject collagen gel into the cavity via the injection port with a 1 ml taper tip syringe. (iii) Use a micropipette to dispense ∼ 170 μL collagen onto the glass microscope coverslip housed in the bottom piece of the microfluidic culture device. (iv) Gently place the flat square of PDMS on top of the collagen-coated coverslip. (v) Homogeneously distribute the collagen across the coverslip by gently depressing and scraping across the back of the PDMS square with a tweezers to spread the collagen. Allow both layers of collagen to gel to cure for 1 hour. (vi) Remove the PDMS stamp by injecting 250 μL of PBS around the interface of stamp with Plexiglas and carefully lift off the stamp. (vii) Dispense PBS around the perimeter of the flat PDMS square and gently remove the 10square from the bottom Plexiglas piece. (viii) Carefully assemble the top and bottom pieces of the microfluidic device, keeping both collagen surfaces wetted with PBS. (ix) Gently tighten all four screws in a rotating manner to ensure a level and uniform join. (x) Turn the device over, aspirate excess PBS from the reservoirs, and add a small volume of culture medium. (xi) After equilibration for one hour in an incubator at 37 °C and 5% CO2, aspirate the medium and add a 10 μL cell suspension into the reservoir inlet port using a gel loading pipette. (xii) Allow cells to adhere for 30 minutes in an incubator. (xiii) Add cell culture media to the inlet reservoir and inspect the device fabrication for channel integrity and absence of air bubbles.
- Supplementary Figure 4 (341 KB)
Successful seeding of cells into channels of the microfluidic device. Supplementary image showing successful seeding of cells into channels of microfluidic culture device, at an appropriate density (Steps 49-53). Scale bar 200 μm.
- Supplementary Data (64 KB)
Electronic CAD file (AutoCAD, *.dwg format) for creating the lithographic mask to microfabricate a silicon master via photolithography (Step 2).