lunes, 9 de septiembre de 2013

Formation of microvascular networks in vitro

29 August 2013

Nature Protocols 8,1820–1836(2013)doi:10.1038/nprot.2013.110 
Published online
 
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Abstract
Abstract•
Introduction
Materials
Procedure
Troubleshooting
Timing
Anticipated results
References
Acknowledgments
Author information
Supplementary information
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
(a–e) 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.
(a) Photograph of all the components for casting the PDMS stamp and assembling the microfluidic culture device. (a–e) Individual components are cross-referenced between the photograph in a and the diagrams in b–e 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. Aculture device (Steps 21–53) is available as Supplementary Video 2.


(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.

Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7 right
Videos
Compounds
Genes and Proteins
Introduction
Abstract
Introduction•
Materials
Procedure
Troubleshooting
Timing
Anticipated results
References
Acknowledgments
Author information
Supplementary information
The microvasculature is a pervasive organ system that mediates the transfer of solutes (for example, metabolites, waste products and signals) and cells (for example, leukocytes) throughout the body. These living pipes have a central role in the regulation of metabolic activity, development, healing, immune response and the progression of many diseases. This diversity of function places stringent constraints on the physical (network architecture) and biological (cellular composition) properties of the microvasculature. These constraints limit the size, complexity and physiological relevance of tissues grown in vitro for applications in regenerative medicine, pharmaco-toxicological studies and basic research. The development of methods to incorporate appropriate microvascular infrastructure into scaffolds for tissue engineering is indispensable for the progression of the field. Microfluidic control of mass transfer within biological scaffolds provides one solution to this crucial challenge in tissue engineering.
This protocol describes a platform (Fig. 1) that recapitulates the structure and function of microvascular vessels to serve in studies of basic vascular and blood biology, as models of diseases such as tumor angiogenesis or thrombosis, and as a starting point for engineering prevascularized tissues for regenerative medicine1, 2, 3, 4, 5. In contrast to other uses of microfluidic endothelial cell cultures by confined gels or 2D models6, 7, 8, 9, our approach1, 2, 3, 4, 5 leads to fully enclosed vessels in a bioremodelable hydrogel, suitable for biological questions that require a fully 3D extracellular environment with a contiguous parenchymal or stromal space. Our model represents a complementary extension of simpler, 3D assays of vasculogenesis and invasion angiogenesis10; this extension is necessary where explicit vessel structure and perfusion of lumens have important roles in the study or application of interest.
Figure 1: Examples of vessel configuration.

(a–e) 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.
Full size image (423 KB)
Figures/tables index
Next
Development of the protocol
Tissue engineers have long appreciated the need to incorporate vascular functionality in the design and fabrication of biological scaffolds11, 12, 13, 14. Diverse efforts have been made toward the incorporation of endothelial cells15, 16, 17 and explicit vessel structures18, 19, 20, 21 within 3D biomaterials. The approach presented in this protocol builds on work over the past decade to bring microfluidic structure—sub-millimeter channels formed by microfabrication—into biomaterial scaffolds. The design and fabrication of micropatterned biomaterials was pioneered by Whitesides and colleagues22, 23 with the development of replica molding of soft polymer microfluidic systems, also known as soft lithography. These techniques were later adapted to the molding of hydrogel scaffolds24, 25. Alternatives to soft lithography such as bioprinting, sacrificial elements21, 26 or modular assembly27 have emerged; Gauvin et al.28 have reviewed the distinct advantages and limitations of these methods. Soft lithography was first exploited to form vascular-like structure by Borenstein and colleagues29, 30, 31 within poly(lactic-co-glycolic acid) films, poly(glycerol-sebacate) and silk fibroin; however, such materials prohibit cell encapsulation during the fabrication process. Building on this work, our group and others used micropatterning of natural hydrogels to provide convective transport to cells embedded within the bulk of physiologically relevant biological scaffolds19, 1, 32. The Tien laboratory was the first to generate perfusable, endothelialized microvascular tubes and networks in such materials using lithographic methods and sacrificial elements20, 33, 34. Our current protocol extends this progress with the incorporation of cells in the perivascular space, perfusion with whole blood and functional angiogenic and thrombotic response to appropriate biochemical stimulation2. Here we present the methods for the design, fabrication and application of such µVNs.
Application of the method
In our first report, confocal fluorescence and TEM of the in vitro µVNs demonstrated the formation of a confluent, functional endothelium on the walls of the microfluidic channels and the viability of cells within the collagen bulk (Fig. 1c; ref. 2) . In addition, we demonstrated appropriate morphology, barrier function, angiogenic remodeling and appropriate cell-cell junctions. We further examined pericyte-endothelial cell interactions in defining barrier function and angiogenesis, as well as blood-endothelium interactions, including thrombosis. We have also exploited the microfluidic control of flows to study angiogenesis in the presence of well-defined gradients of vascular endothelial growth factor (VEGF) and doses of anti-VEGF (Avastin)3.
Moving forward, this assay presents opportunities to address questions in vascular biology that are inaccessible in planar cultures, such as the effects of geometry, hydrodynamic stresses and convective mass transfer on vessel stability, angiogenesis and development35. µVNs also provide a basis for in vitro models of clinical conditions that implicate the vasculature in tissue-scale processes, such as wound healing, solid tumor cancers and diabetes. Within such models, the explicit vasculature and perivascular space could, for example, allow for the study of mechanisms of intra- and extravasation4, 5, the capture and incorporation of circulating endothelial progenitor cells36 and the interplay of stroma, matrix and endothelium in defining health and disease37. In technological contexts, the ability to form vascularized scaffolds in vitro also opens new possibilities. For example, scaffolds with functional vasculature will have a central role in the engineering of any macroscopic and highly metabolically active tissue38; tissue models with explicit vasculature could markedly improve the effectiveness of in vitro screens of drugs and of strategies of drug delivery39. Further examples of the use of µVNs in models of solid tumors are reviewed in Stroock et al.4. Wong et al.40 present a comprehensive review of the opportunities for microfluidic models of vascular physiology.
Experimental design
This protocol describes a vascularization strategy to sustain 3D cell-laden biological scaffolds by convective mass transfer through endothelialized microfluidic networks. By combining a simple biomaterial and replica molding, artificial µVNs fully enclosed within cell-remodelable hydrogel constructs can be constructed. Our protocol includes photolithography (Steps 1–11), biomaterial-based soft lithography (Steps 12–47), 3D microfluidic culture (Steps 48–54), live fluorescence imaging (Step 55A), in situ immunofluorescence staining and confocal imaging and analysis (Step 55B), and TEM imaging (Step 55C), as well as assays for the characterization of endothelium diffusivity and permeability (Box 1) and interactions with whole blood (Box 2). For a detailed overview of lithographic processes (Steps 1–6), see reference23. Alternately, Steps 1–6 can be contracted to a custom third-party service, such as http://www.creatvmicrotech.com/intromicro.html, http://www.micralyne.com/novel-material-processing/ or http://www.nilt.com/default.asp?Action=Details&Item=500. Videos are provided that show detailed assembly of the microfluidic culture device (Supplementary Video 1); seeding of cells into the channels of the microfludic culture device, including both successful (Supplementary Video 2) and unsuccessful (Supplementary Videos 3 and 4) examples; and live fluorescence imaging (Supplementary Video 5).
Box 1: Characterization of the permeability of matrix and endothelium • TIMING 30 min
Full box
Box 2: Characterization of blood-endothelial interactions • TIMING 1 h
Full box
Limitations
Because this assay emphasizes physiological accuracy over simplicity, it introduces challenges with regard to throughput and multiplexing. This µVN assay is most appropriate for applications that require explicit vessel structures and network architectures and the ability to control perfusion of lumens. In some cases, simpler angiogenesis assays10 may be more appropriate, such as to perform screens of cell types, reagents and biomaterials41. These simpler platforms can be used to establish parameters for the µVN assay presented here. In our experience, the soft lithographic manipulation of native collagen used in our protocol fails to provide good fidelity of channel structures for lateral dimensions below 50 µm. In addition, microfabrication by SU-8 soft lithography described here necessarily generates unphysiological rectangular channel cross-sections, although remodeling of the matrix by endothelial cells yields a rounded vessel morphology after about 2 d. Alternatively, fabrication of molds with isotropic etching could produce hemispherical cross-sections without altering the rest of the protocol. We have not attempted to create µVNs with multiple layers of channels; such extensions of the microfluidic approach have been shown in other materials42. Chemical adhesion of the matrix to the boundaries of the jig and the use of higher densities of collagen mitigate contraction of microvessels as the density of cells embedded within the collagen bulk increases, as we have demonstrated in previous publications5, 41. To date, human umbilical vein endothelial cells (HUVECs) are the only endothelial cells that have been cultured in this system; however, we believe that the assay will be broadly applicable to diverse endothelial cell types that have been successfully cultured on collagen substrates for angiogenesis and vasculogenesis assays, if used with the appropriate culture medium20, 43.
Materials
Abstract
Introduction
Materials•
Procedure
Troubleshooting
Timing
Anticipated results
References
Acknowledgments
Author information
Supplementary information
REAGENTS
Photoresist SU-8 2000 series (Permanent epoxy resist for photolithography, Microchem)
Caution: Wear protective goggles and gloves and suitable protective clothing.
Silicon wafers for master mold (100-mm SSP silicon wafers, 500 µm in thickness, undoped; University Wafer)
Photoresist developer (Microchem)
Silane for passivation of master mold: tridecafluoro-1,1,2,2-tetrahydrooctyl-1-trichlorosilane (Gelest, cat. no. SIT8174.0)
Isopropyl alcohol (for photolithography; Sigma-Aldrich)
Sylgard 184 silicone elastomer base and curing agent (for soft lithography, Dow Corning)
(poly)-dimethylsiloxane (PDMS) (Sylgard 184, Dow Corning)
Sterile 1× PBS, pH 7.4 (Invitrogen, cat. no. 10010-023)
Ethanol, 70% (vol/vol) in sterile deionized water (VWR, cat. no. BDH1162-4LP)
Poly(ethyleneimine) (PEI, see Reagent Setup; Fluka Analytical, cat. no. P3143-500ML)
Glutaraldehyde (GA, see Reagent Setup; Fluka Chemika, cat. no. 49629)
Caution: Use GA in a chemical hood and wear protective goggles and gloves and suitable protective clothing.
Lyophilized type I collagen isolated from rat tails (Pel-Freez, cat. no. 5654-1) according to the procedures described by Bornstein44 or Rajan et al.45
HEPES buffer (Cambrex, cat. no. CC-5024)
Sodium hydroxide (NaOH) solution, 2N (BDH, cat. no. 3223-1)
Caution: Wear protective goggles and gloves and suitable protective clothing.
Cells of interest, e.g., human umbilical vein endothelial cells (HUVECs, Lonza, cat. no. CC-2519) or human brain perivascular cells (ScienCell, cat. no. 1200)
DMSO (Sigma Life Science, cat. no. D8418-100ML)
Medium appropriate for the cells being cultured, e.g., HUVEC culture medium EGM-2 (Lonza Clonetics, cat. no. CC-4176) or Lonza M199 (Lonza, cat. no. 12-117F)
Endothelial cell growth factor (Millipore, cat. no. 02-102)
FBS (Tissue Culture Biologicals, cat. no. 101)
Penicillin-streptomycin (Lonza, cat. no. 17-602F)
Heparin sodium (Acros, cat. no. 41121-0010, 9041-08-1)
Trypsin-EDTA, 0.025% (wt/vo


(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.

l) (Invitrogen, cat. no. R-001-100)
L-Glutamine (Cambrex, cat. no. 17-605C)
L(+)-Ascorbic acid, reagent ACS (Acros Organics, Code 401471000, cat. no. CAS:50-81-7, EC:200-066-2)
Human vascular endothelial growth factor (VEGF; Millipore, cat. no. 01-185, GF315)
Human basic fibroblast growth factor (bFGF-2; Millipore, cat. no. 01-106)
Phorbol 12-myristate 13-acetate (PMA) also known as 12-O-tetradecanoylphorbol-13-acetate (Sigma-Aldrich, product no. P 1585)
Caution: Wear protective goggles and gloves and suitable protective clothing.
BSA (Calbiochem, cat. no. 126609)
Rabbit polyclonal antibody (Rb pAb) to CD31, also known as platelet endothelial cell adhesion molecule (PECAM-1) (Abcam, cat. no. ab28364)
Rabbit polyclonal antibody (Rb pAb) to vascular endothelial (VE)-cadherin (Abcam, cat. no. ab33168)
Mouse monoclonal antibody to a-smooth muscle actin (a-SMA; Abcam, cat. no. ab54723)
Anti-von Willebrand factor antibody conjugated with fluorescein isothiocyanate (FITC) (Abcam, cat. no. ab8822)
APC anti-human CD41a (BD Pharmingen)
PE anti-human CD45 (BD Pharmingen)
Goat anti-rabbit IgG conjugated with Alexa Fluor 568 or Alexa Fluor 647 (Invitrogen and Molecular Probes, cat. nos. A11011 and A21244, respectively)
Goat anti-mouse IgG conjugated with Alexa Fluor 488, Alexa Fluor 568 or Alexa Fluor 647 (Invitrogen and Molecular Probes, cat. nos. A11001, A21124 and A21240, respectively)
DAPI, dilactate (Invitrogen, cat. no. D3571)
Alexa Fluor phalloidin 488 (Invitrogen and Molecular Probes, cat. nos. A12379 and 1023568 300U, respectively)
Formaldehyde, 16% (wt/vol) methanol-free, ultrapure electron microscopy grade (Polysciences, cat. no. 18814)
Triton X-100 solution (see Reagent Setup; MP Biomedicals, cat. no. 807426)
FITC protein label (Invitrogen, cat. no. F6434)
Fluorescein (Sigma-Aldrich)
TEM epoxy (Sigma-Aldrich)
Cacodylate buffer (Na(CH3)2AsO2·3H2O; 0.1M)
Ruthenium red (0.05%, wt/vol)
Caution: Use this reagent in a chemical hood; wear protective goggles, gloves and other suitable protective clothing.
Osmium tetroxide (OsO4, 1.0% wt/vol)
Caution: Use this reagent in a chemical hood; wear protective goggles, gloves and other suitable protective clothing.
Uranyl acetate (UO2(CH3COO)2·2H2O; 2%, wt/vol)
Caution: Use this reagent in a chemical hood; wear protective goggles, gloves and other suitable protective clothing.
Acetone
Bleach

EQUIPMENT
Plasma cleaner (for surface modification, Harrick, PDC-001, 115V)
Disposable biopsy punches (4 mm and 1 mm diameter, Miltex)
Autoclave (Market Force Industries, Sterilmatic)
Incubator at 37 °C, 5% CO2, (Thermo Electron Forma Series II water-jacketed CO2 incubator HEPA Class100, (also NAPCO series 8000WJ)
Water bath at 37 °C (VWR International, Sheldon Manufacturing, model no. 1212)
Inverted microscope (for imaging cells, Nikon Eclipse TS100)
Confocal microscope (for imaging cells, Zeiss 710)
Transmission electron microscope (Technai F20)
ImageJ software (or similar)
Microtome (Leica Ultracut UCT ultramicrotome)
Sterile syringe filters (PALL, Acrodisc 13-mm syringe filter with 0.2-µm HT Tufryn membrane, cat. no. PN 4454)
Filter system, 500 ml, 0.22 µm (Fisher Scientific, cat. no. 09-761-102)
Flask, 25 cm2 with membrane cap (Fisher Scientific, cat. no. 10-126-28)
Flask, 75 cm2 with membrane cap (Fisher Scientific, cat. no. 10-126-37)
Petri dish, small, 100 × 15 mm, 500/CS (Fisher Scientific, cat. no. 08-757-11Z)
Petri dish, large, 150 × 15 mm, 100/CS (Fisher Scientific, cat. no. 08-757-14)
Aluminum foil
Syringe, 1 ml (VWR, BD Syringe, cat. no. BD329650)
Syringe, 3 ml (VWR, BD Syringe, cat. no. BD09656)
Pipette tips, 1,000 µl (VWR, cat. no. BD329650)
Stainless steel machine screws, Phillips head, 4–40 (thread size) for microfluidic culture device
Machine screws for aluminum casting jig
Stainless steel dowel pins, 1 inch, 16-gauge diameter (Small Parts, cat. no. B001OBPJY6)
PDMS flat slabs (30 mm × 30 mm; 3–5 mm in thickness)
Self-standing, sterile conical tubes, 50 ml and 30 ml (VWR, cat. nos. 21008-777, 89012-778)
Sterile conical vial, 15 ml (Fisher Scientific, cat. no. 14-959-49B)
Tweezers
Scalpel handle and blades)
Spatula
Phillips head screwdriver
Glass crystallizing dish and cover (125 × 65 mm) (Corning, cat no. 3140-125)
Paper towels
Autoclavable, medical-grade tubing (CorSolutions)
Pump (CorSolutions) and pump connections (CorSolutions))

REAGENT SETUP
Caution: Be sure to consult the relevant MSDS for safety information and use appropriate protective equipment when handling reagents.
PEI solution
Dilute PEI to 1.0% (wt/vol) with sterile deionized water. Filter-sterilize the solution with a 0.22-µm sterile syringe filter. It can be stored for up to 6 months if it is sealed and refrigerated.

GA solution
Dilute GA to 0.1% (wt/vol) with sterile deionized water. Filter-sterilize the solution with a 0.22-µm sterile syringe filter. It can be stored for up to 6 months if it is sealed and refrigerated at 4 °C.

Triton X-100 solution
Dilute Triton X-100 to 1.0% (vol/vol) Triton X-100 in sterile deionized water and store it at room temperature (~20–25 °C). It can be stored for up to 6 months if it is sealed and refrigerated at 4 °C.

Formaldehyde solution
Dilute formaldehyde to 3.7% (wt/vol) formaldehyde in PBS and store it at room temperature in the dark. It can be stored for up to 1 year if it is sealed.

Endothelial cell growth medium (GM)
GM is a rich medium for expanding cells. To conduct rigorous biochemistry experiments, use a defined medium, such as EGM-2 (Lonza; see Reagents). To make GM, mix the components given in the following table:
ComponentsSupplier and cat. no.Volume
Growth serum M199 Lonza, cat. no. 12-117F 500 ml
L-Glutamine (for a 2-mM working concentration) Cambrex, cat. no. 17-605C 6.5 ml
Endothelial cell growth supplement (for a working concentration of 20 µg ml-1) Millipore, cat. no. 02-102 15 mg (one vial)
Heparin (10,000 U ml-1 stock solution) Acros, cat. no. 41121-0010, 9041-08-1 250 µl
FBS (for a working concentration of 18% (vol/vol)) Tissue Culture Biologicals, cat. no. 101 100 ml
Penicillin-streptomycin (for a working concentration of 150 U ml-1) (BioWhittaker, 10,000 U ml-1) 7.5 ml
Cap and mix the contents gently without creating bubbles (the FBS enhances bubble formation). Connect a filter system (see Equipment) and attach it to a vacuum pump. Filter-sterilize the solution. It may be stored for up to 1 month if refrigerated at 4 °C.

Vasculogenesis medium (VM)
VM is a rich medium used to activate endothelial cells to spontaneously form lumens and tubes in bulk collagen and undergo sprouting angiogenesis by invading from a monolayer. Determine the volume of VM needed (typically VM is only prepared as needed) on the basis of the volume of the channels, the flow rate and the duration of the experiment. Calculate the volumes to be added into the base GM to form the VM, and mix these reagents (below) into GM that has been preheated to 37 °C. The following quantities should be added per the final volume of the VM: PMA at 50 ng ml-1; VEGF165 at 40 ng ml-1 (keep on ice while handling it); bFGF at 40 ng ml-1 (keep on ice while handling it) and ascorbic acid at 50 µg ml-1.

The difference between the added volumes of these substances and the final VM volume desired is the volume of GM to add. For example, 1 ml of VM, 2 µl of VEGF (from a 20 µg ml-1 stock), 1.6 µl of bFGF (from a 25 µg ml-1 stock), 1.6 µl of PMA (from a 50 µM stock) and 10 µl of ascorbic acid (from a 5,000 µg ml-1 stock) would be added to 985 µl of GM.

Critical: Owing to protein degradation, keep VEGF165 and bFGF on ice while handling them, and only prepare VM as needed, typically 5 ml per day for gravity-driven flow or 10 ml for pump setup. The exact volume depends on device configuration and flow rates.

Collagen stock
Collagen stock takes ~2 d to prepare. Resuspend lyophilized type I rat tail collagen in 0.1% (vol/vol) acetic acid to 1.5 mg ml-1 in a conical tube, to create a working stock solution. Shake the conical tube vigorously once a day to mix the contents, and keep it refrigerated at 4 °C. The collagen will dissolve over an ~2-d period. When it has dissolved, centrifuge the mixture at 1,950g for 5 min at 4 °C to remove air bubbles. The stock collagen can be maintained refrigerated at 4 °C for up to 3 months.

Procedure
Abstract
Introduction
Materials
Procedure•
Troubleshooting
Timing
Anticipated results
References
Acknowledgments
Author information
Supplementary information Expand All
Steps 1 - 6: Fabrication of a master mold
Steps 7 - 8: Fabrication of machined parts of the molding and culture jig
Steps 9 - 11: Fabrication of the PDMS stamp
Steps 12 - 16: Sterilization of materials for device assembly
Steps 17 - 20: Coating of devices with sterile PEI and GA
Steps 21 - 23: Preparation of 1% (wt/wt) collagen gel
Steps 24 - 28: Injection-molding microstructured collagen
Steps 29 - 31: Molding collagen coating onto a glass coverslip
Steps 32 - 33: Collagen gelation
Steps 34 - 47: Assembling the device (top and bottom pieces)
Step 48: Device incubation in preparation for seeding channels
Steps 49 - 53: Seeding channels with endothelial cells
Step 54: Perfusion culture
Step 55: Imaging of cells
Troubleshooting
Abstract
Introduction
Materials
Procedure
Troubleshooting•
Timing
Anticipated results
References
Acknowledgments
Author information
Supplementary information
Troubleshooting advice can be found in Table 1.
Table 1: Troubleshooting table.
Full table
Figures/tables index
Timing
Abstract
Introduction
Materials
Procedure
Troubleshooting
Timing•
Anticipated results
References
Acknowledgments
Author information
Supplementary information
Steps 1–6, fabrication of a master mold of microfluidic channels by photolithography: 3 h
Steps 7 and 8, fabrication of machined parts of the molding and culture jig: 5 h
Steps 9–11, fabrication of the PDMS stamp: 10 h
Steps 12–16, sterilization of materials for device assembly: 30 min
Steps 17–20, coating of devices with sterile PEI and GA to enable adhesion to collagen: 45 min
Steps 21–23, preparation of collagen gel: 10 min
Steps 24–28, injection-molding microstructured collagen: 15 min
Steps 29–31, molding collagen coating onto a glass coverslip: 5 min
Steps 32 and 33, collagen gelation: 30 min
Steps 34–47, device assembly: 15 min
Step 48, device incubation in preparation for seeding channels: 1 h
Steps 49–53, seeding channels with endothelial cells: 40 min
Step 54A, gravity-driven perfusion culture: 5 min
Step 54B, pump-driven perfusion culture: 15 min
Step 55A, live fluorescence microscopy: variable, 1 h or 2–3 d
Step 55B, confocal fluorescence microscopy on fixed and stained samples: 14 h
Steps 55C, transmission electron microscopy: 3 d
Box 1, characterization of permeability of matrix and endothelium: 30 min
Box 2, characterization of blood-endothelial interactions: 1 h

Anticipated results

Abstract
Introduction
Materials
Procedure
Troubleshooting
Timing

Anticipated results•
References
Acknowledgments
Author information
Supplementary information
The fundamental platform comprises an endothelialized network of microchannels embedded within a bioremodelable hydrogel scaffold (Fig. 1a). This protocol allows for diverse experimental design and analysis for the study of microvascular phenomena with precise control of geometry, coculture seeding, distributions of soluble signals and mechanical stresses. The assay is amenable to in situ fluorescence confocal microscopy, histological analysis or TEM for high-resolution imaging. Furthermore, media or cell extracts can be used for proteomic or genomic biochemical analysis such as ELISA.
Without stimulation, the culture yields a confluent monolayer of endothelial cells on the walls of the microchannels with appropriate morphology and cell-cell junctions. Via live imaging (Step 55A), the dynamics of the endothelial cells within the endothelium can be tracked in the presence of well-defined luminal flow (Fig. 3). Immunohistochemically stained cultures (Step 55B) show that the endothelium remodels the walls to yield rounded vessels (Fig. 4a), expresses CD31 (Fig. 4a) and VE-cadherin (Fig. 4b) with appropriate localization and presents low nonspecific permeability (Fig. 6). One advantage of this platform is the opportunity to increase biological complexity incrementally, including the incorporation of additional cell types, the control of hemodynamic fluid forces or the generation of biochemical gradients (Fig. 1d). In cocultures with perivascular cells seeded in the bulk of the matrix (Step 23), one sees endothelial cell–perivascular cell interactions with the induction of sprouting (Fig. 5a), recruitment of perivascular cells to the abluminal side (Fig. 5b) and deposition of basement membrane (Fig. 5c). Upon exposure to tumor-like proangiogenic signals, robust sprouting angiogenesis occurs and the barrier properties of the endothelium are compromised2. Notably, perfusion of an unstimulated microvessel with citrate-stabilized whole blood leads to minimal adhesion of platelets and leukocytes to the vessel wall (Fig. 7a). Upon proinflammatory stimulation, the endothelium shows a strong response in the form of secreted von Willebrand factor (vWF), self-assembling of fibers of vWF and formation of platelet-vWF–derived thrombi in a manner that depends on the vessel architecture (Fig. 7b).
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.
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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 aIIb); 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.
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One advantage of this platform is the assimilation of increasing biological complexity, including incorporation of additional cell types, control of hemodynamic fluid forces or generation of biochemical gradients. Perivascular cells embedded within the collagen bulk migrated to and associated with the vascular network, and they stabilized vessel permeability under inflammatory assault. The device has been coupled to a sensitive pump apparatus for precise control of fluid dynamic forces, including various flow regimes, resulting in endothelial cell alignment. Finally, the inclusion of source and sink channels within the scaffold enables the steady-state generation of defined gradients to explore heterogeneous signals in the tissue microenvironment3. Taken together, these efforts establish a novel assay for the study of physiological phenomena in a fully 3D context in vitro, which not only has considerable implications for the study of vasculature and vascular tissues in health, disease and therapy, but also has appealing potential for other emerging research areas such as brain (neuroglial) science and engineering.
References
Abstract
Introduction
Materials
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Troubleshooting
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References•
Acknowledgments
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Acknowledgments
Abstract
Introduction
Materials
Procedure
Troubleshooting
Timing
Anticipated results
References

Acknowledgments•
Author information
Supplementary information
We acknowledge the technical assistance of G. Swan. We thank C. Murry and S. Schwartz for helpful discussions. We acknowledge the Life Sciences Core Laboratories Center at Cornell University and the Lynn and Mike Garvey Imaging Laboratory in the Institute of Stem Cell and Regenerative Medicine at University of Washington. We acknowledge the financial support from an American Heart Association Scientist Development Grant (Y.Z.); the US National Institutes of Health (NIH) (grant no. R01HL091153 to J.A.L.; and NIH grant no. RC1 CA146065); the Cornell Center on the Microenvironment and Metastasis (no. NCI-U54 CA143876); the Human Frontiers in Science Program; the Cornell Nanobiotechnology Center (no. NSF-STC; ECS-9876771); the Cornell Center for Nanoscale Science and Technology (no. NSF-NNIN ECS 03-35765); an Empire State Development Division of Science, Technology and Innovation (NYSTAR) Center for Advanced Technology (CAT) award; a New York State J.D. Watson Award (A.D.S.); and an Arnold and Mabel Beckman Foundation Young Investigator Award (A.D.S.) P.F.D. acknowledges a National Science Foundation Graduate Fellowship.

Author information
Abstract
Introduction
Materials
Procedure
Troubleshooting
Timing
Anticipated results
References
Acknowledgments

Author information•
Supplementary information

Primary authors
These authors contributed equally to this work.
John P Morgan &
Peter F Delnero
Affiliations
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
Department of Biomedical Engineering, Cornell University, Ithaca, New York, USA.
Peter F Delnero,
Scott S Verbridge &
Claudia Fischbach
Department of Bioengineering, Institute of Stem Cell and Regenerative Medicine, University of Washington, Seattle, Washington, USA.
Ying Zheng
Puget Sound Blood Center, Seattle, Washington, USA.
Junmei Chen &
José A López
Department of Medicine, Weill Cornell Medical College, New York, New York, USA.
Pouneh Kermani &
Barbara Hempstead
Department of Medicine (Hematology), University of Washington, Seattle, Washington, USA.
José A López
CorSolutions, LLC, Ithaca, New York, USA.
Thomas N Corso
Kavli Institute at Cornell for Nanoscale Science, Cornell University, Ithaca, New York, USA.
Abraham D Stroock
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
Correspondence to:
Abraham D Stroock

Supplementary information
Abstract
Introduction
Materials
Procedure
Troubleshooting
Timing
Anticipated results
References
Acknowledgments
Author information

Supplementary information•
Video
Video 1: Supplementary Video 1 (46.21 MB, Download) Supplementary video showing detail preparation and assembly of microfluidic culture device (Steps 21-53).
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).
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.
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.
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
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.
Zip files
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

1 comentario:

  1. This tutorial is indeed useful. I have just downloaded it. Thanks again.sigma antibody

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