Formation of microvascular networks in vitro

Journal name:
Nature Protocols
Volume:
8,
Pages:
1820–1836
Year published:
DOI:
doi:10.1038/nprot.2013.110
Published online

Abstract

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

Figures

  1. Examples of vessel configuration.
    Figure 1: Examples of vessel configuration.

    (ae) Diverse vessel configurations have been adapted for various applications, including μVNs (a–c), steady-state morphogen gradients (d) and live imaging under controlled flow regimes (e). (a,b) Illustration of an endothelial cell–coated microfluidic network in a cell-laden collagen construct; inset highlights the endothelial confluence, pericyte-endothelial cell interactions and angiogenic sprouting from the vessel. (c) Appropriate endothelial cell health, integrity and confluence are demonstrated by uniform CD31 (also known as PECAM-1) (red) staining of cell-cell junctions in a quiescent vascular network; such networks provide nutrient and waste transport to sustain cells within the contiguous biological matrix. Scale bar, 100 μm. (d) The incorporation of parallel source (C1) and sink (C2) channels generates a stable biochemical gradient to mimic the heterogeneous distribution of potent morphogens, such as VEGF, and to stimulate endothelial cell sprouting in the study of invasion angiogenesis. (e) μVNs are used to study responsiveness of vessels to hemodynamic forces with live imaging of GFP-expressing endothelial cells. Under physiological shear stress and flow, the endothelial cells align in the direction of the flow. Diagrams in a,b are reproduced with permission from Franco and Gerhardt47. Micrograph in c is adapted with permission from Zheng et al.2.

  2. Summary of microfluidic device fabrication (Steps 1–54), assembly (Steps 24–48), seeding (Steps 49–53) and culture (Steps 54A and 54B) processes.
    Figure 2: Summary of microfluidic device fabrication (Steps 1–54), assembly (Steps 24–48), seeding (Steps 49–53) and culture (Steps 54A and 54B) processes.

    (a) Photograph of all the components for casting the PDMS stamp and assembling the microfluidic culture device. (ae) Individual components are cross-referenced between the photograph in a and the diagrams in be using Roman numerals. (i) Machine screws for the aluminum casting jig; (ii, iii) top and middle pieces, respectively of the aluminum casting jig; (iv, v) bottom and top pieces, respectively of the microfluidic culture device; (vi) bottom piece of the aluminum casting jig; (vii) lithographically-patterned silicon wafer master mold; (viii) PDMS stamp; (ix) flat PDMS slab; (x) stainless steel dowel pins; (xi) stainless steel machine screws (4–40 thread size) for microfluidic culture device; (xii) glass microscope coverslip. Technical drawings for the aluminum casting jig and microfluidic culture device can be found in Supplementary Figures 1 and 2, respectively. (b) Schematic of the aluminum jig assembly for casting the PDMS stamp using the lithographically-patterned silicon wafer master mold. (c) (Top) 3D micropatterned vessels are formed by injection molding of native collagen gel against the PDMS stamp through the injection ports on the top piece of the microfluidic culture device. Stainless steel dowel pins are used to preserve the connection between the cell culture medium reservoirs and the microfluidic channels. (Bottom) Collagen is injected onto the glass coverslip in the bottom piece of the microfluidic culture device and molded into a thin layer by sealing the gel cavity with a flat slab (∼3 mm thick) of PDMS. (d) After the collagen gels, the top and bottom pieces of the microfluidic culture device are assembled to form the micropatterned, 3D microfluidic vessels, fully enclosed in collagen. The microvessels are then seeded with cells by pipetting a small (10 μl) cell suspension into the inlet reservoir. (e) The microvascular network is perfused with gravity-driven or pump-driven culture medium or whole blood. Photographs of detailed device assembly steps that are not depicted are available in Supplementary Figure 3. A video showing the detailed preparation and assembly of the microfluidic culture device (Steps 21–53) is available as Supplementary Video 2.

  3. Live fluorescence imaging of GFP-expressing HUVECs in μVNs, as described in Step 55A.
    Figure 3: Live fluorescence imaging of GFP-expressing HUVECs in μVNs, as described in Step 55A.

    (ac) The culture was run under physiological shear flow (∼11 μl min−1, 17 dyne cm−2) with a feedback-controlled peristaltic pump (Step 54B). The flow direction is from left to right. The snapshots reveal dynamic cell motility throughout the vessel wall. Cell tracking (red dots) traces an individual cell's path (yellow lines) as it migrates upstream and downstream within the endothelium. Yellow intensity corresponds to instantaneous velocity along the path length, with dark zones representing faster motion. Scale bars, 50 μm. Time stamps show hours:minutes:seconds after onset of flow. See Supplementary Video 1 for the full image sequence.

  4. Characterization of vessel structure by confocal fluorescence microscopy (Step 55B) and transmission electron microscopy (Step 55C).
    Figure 4: Characterization of vessel structure by confocal fluorescence microscopy (Step 55B) and transmission electron microscopy (Step 55C).

    (a,b) Complex geometrical features such as corners, junctions and bifurcations are readily visualized by confocal fluorescence imaging, and cross-sections of microchannels reveal rounded vessel morphology. Immunohistochemistry of CD31 (a, red) and VE-cadherin (b, red) are used to demonstrate confluent and healthy endothelium throughout the network. Blue, nuclei; scale bars, 100 μm. (c) Transmission electron micrographs enable imaging of cell-cell junctions, including focal contacts (arrow, top) and overlapping junctions (arrow, bottom); scale bars, 1 μm. EC, endothelial cell. Adapted with permission from Zheng et al.2.

  5. Heterotypic cell culture.
    Figure 5: Heterotypic cell culture.

    (a) Endothelial cells (HUVECs) respond to stimulation in the presence of cells (human brain vascular pericytes, HBVPCs) seeded in the matrix (Step 23) by sprouting new branches, as visualized by confocal microscopy (Step 55B). (b) Smooth muscle cells seeded in the matrix (Step 23) associate with the endothelium, as visualized by confocal microscopy (Step 55B). (c) Ultrastructure of the cellular interfaces formed between HUVECs and HBVPCs, including a deposited layer of basal lamina, can be visualized by transmission electron microscopy (Step 55C). In a,b: CD31, red; DAPI, blue; α-SMA, green; scale bars, 100 μm. In c, scale bar, 1 μm. Adapted with permission from Zheng et al.2.

  6. Characterization of the permeability of matrix and live endothelium (Box 1).
    Figure 6: Characterization of the permeability of matrix and live endothelium (Box 1).

    (a,b) Fluorescence micrographs show the distribution of 70-kDa FITC-dextran after injection into a network of channels in collagen with no endothelium (a) and with a live endothelium (b). Time evolution of the fluorescence intensity profiles (bottom) can be used to calculate the diffusivity of molecules in the matrix (acellular, a) and the permeability of the vessel membrane (cellular, b). For the complete method, see Zheng et al.2. Figure adapted with permission from Zheng et al.2. Scale bars, 100 μm.

  7. Interaction with whole blood.
    Figure 7: Interaction with whole blood.

    (a) Time sequences of whole-blood perfusion through a μVN, either quiescent (control vessels, top images) or stimulated (bottom images), at a flow rate of 10 μl min−1 at time points of 5, 50, 100, 150 and 250 s after initiation of perfusion. The platelets are in green, labeled with CD41a to platelet-specific glycoprotein IIb (integrin αIIb); flow direction is indicated with arrows (scale bars, 100 μm). (b) vWF fibers were either coated on the walls of the activated vessel or traveled through the lumens. The locations of vWF fibers in the vessel are color coded: bottom in blue, center in light green and top in red. Adapted with permission from Zheng et al.2. See Box 2.

Videos

  1. Supplementary Video 1
    Video 1: Supplementary Video 1
    Supplementary video showing detail preparation and assembly of microfluidic culture device (Steps 21-53).
  2. Supplementary Video 2
    Video 2: Supplementary Video 2
    Supplementary video showing successful seeding of cells into channels of microfluidic culture device, at an appropriate density (Steps 49-53).
  3. Supplementary Video 3
    Video 3: Supplementary Video 3
    Supplementary video showing unsuccessful seeding of cells into channels of microfluidic culture device (Steps 49-53), due to an inadequate density.
  4. Supplementary Video 4
    Video 4: Supplementary Video 4
    Supplementary video showing unsuccessful seeding of cells into channels of microfluidic culture device (Steps 49-53), due to an excessive flow rate.
  5. Supplementary Video 5
    Video 5: Supplementary Video 5
    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.

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Author information

  1. These authors contributed equally to this work.

    • John P Morgan &
    • Peter F Delnero

Affiliations

  1. School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York, USA.

    • John P Morgan,
    • Michael Craven,
    • Nak Won Choi,
    • Anthony Diaz-Santana &
    • Abraham D Stroock
  2. Department of Biomedical Engineering, Cornell University, Ithaca, New York, USA.

    • Peter F Delnero,
    • Scott S Verbridge &
    • Claudia Fischbach
  3. Department of Bioengineering, Institute of Stem Cell and Regenerative Medicine, University of Washington, Seattle, Washington, USA.

    • Ying Zheng
  4. Puget Sound Blood Center, Seattle, Washington, USA.

    • Junmei Chen &
    • José A López
  5. Department of Medicine, Weill Cornell Medical College, New York, New York, USA.

    • Pouneh Kermani &
    • Barbara Hempstead
  6. Department of Medicine (Hematology), University of Washington, Seattle, Washington, USA.

    • José A López
  7. CorSolutions, LLC, Ithaca, New York, USA.

    • Thomas N Corso
  8. Kavli Institute at Cornell for Nanoscale Science, Cornell University, Ithaca, New York, USA.

    • Abraham D Stroock
  9. Present addresses: Department of Biomedical Engineering, Virginia Polytechnic Institute, Blacksburg, Virginia, USA (S.S.V.); Ifyber, LLC, Ithaca, New York, USA (M.C.); Center for BioMicrosystems, Brain Science Institute, Korea Institute of Science and Technology (KIST), Seoul, Korea (N.W.C.); L'Oreal USA, Clark, New Jersey, USA (A.D.-S.).

    • Scott S Verbridge,
    • Michael Craven,
    • Nak Won Choi &
    • Anthony Diaz-Santana

Contributions

J.P.M., P.F.D., Y.Z., S.S.V., J.C, N.W.C., A.D.-S., J.A.L., T.N.C., C.F., and A.D.S. designed the research; J.P.M., P.F.D., Y.Z., S.S.V., J.C., M.C., P.K., and B.H. performed research; J.P.M., P.F.D., Y.Z., J.C., M.C., J.A.L., and A.D.S. analyzed data; and J.P.M., P.F.D., Y.Z. and A.D.S. wrote the paper.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

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Author details

Supplementary information

Video

  1. Video 1: Supplementary Video 1 (46.21 MB, Download)
    Supplementary video showing detail preparation and assembly of microfluidic culture device (Steps 21-53).
  2. Video 2: Supplementary Video 2 (2.29 MB, Download)
    Supplementary video showing successful seeding of cells into channels of microfluidic culture device, at an appropriate density (Steps 49-53).
  3. Video 3: Supplementary Video 3 (500 KB, Download)
    Supplementary video showing unsuccessful seeding of cells into channels of microfluidic culture device (Steps 49-53), due to an inadequate density.
  4. Video 4: Supplementary Video 4 (273 KB, Download)
    Supplementary video showing unsuccessful seeding of cells into channels of microfluidic culture device (Steps 49-53), due to an excessive flow rate.
  5. 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.

PDF files

  1. 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.

  2. 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.

  3. 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.

  4. 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.

Zip files

  1. Supplementary Data (64 KB)

    Electronic CAD file (AutoCAD, *.dwg format) for creating the lithographic mask to microfabricate a silicon master via photolithography (Step 2).

Additional data