In situ Fabrication and Perfusion of Tissue Engineered Blood Vessel Microphysiological System

Abstract

Human tissue engineered blood vessels (TEBVs) that exhibit vasoactivity can be used to test drug toxicity, modulate pro-inflammatory cytokines, and model disease states in vitro. We developed a novel device to fabricate arteriole-scale human endothelialized TEBVs in situ with smaller volumes and higher throughput than previously reported. Both primary and induced pluripotent stem cell (iPSC)-derived cells can be used. Four collagen TEBVs with 600 μm inner diameter and 2.9 mm outer diameter are fabricated by pipetting a solution of collagen and medial cells into a three-layer acrylic mold. After gelation, the TEBVs are released from the mold and dehydrated. After suturing the TEBVs in place and changing the mold parts to form a perfusion chamber, the TEBVs are endothelialized in situ and then media is perfused through the lumen. By removing 90% of the water after gelation, the TEBVs become mechanically strong enough for perfusion at the physiological shear stress of 0.4 Pa within 24 hours of fabrication and maintain function for at least 5 weeks.

1. Introduction

Vascular microphysiological systems (MPS) represent small-scale models of blood vessels in vitro. The MPS are comprised of either human primary cells or cells derived from induced pluripotent stem cells (iPSCs). Human-sourced vascular MPS can serve as models to study vessel disease or tools to evaluate therapeutics [1]. Tissue engineered blood vessels (TEBVs) are three-dimensional models of blood vessels consisting of a natural or synthetic scaffold with cells added prior to implantation or following ingrowth from blood or surrounding tissue [2, 3]. Human vascular MPS can be used to model diseases in vitro which cannot always be replicated accurately in animals [4–6]. The scale ranges from models of the microvasculature [7–9] to medium size arterioles [1, 4, 5, 10, 11].

Considerations for in vitro human vascular MPS, also known as “vessels on a chip”, include the ability to easily fabricate, sufficient mechanical strength to resist physiological pressures, a nonthrombogenic surface, and vasoactivity. A wide range of materials have been used to make arterial- and arteriole-scale tissue-engineered vascular grafts for implantation [2, 3]. While the mechanical strength of these bioengineered grafts is very good, there are challenges matching the stress-strain curve of arteries and ensuring a non-thrombogenic surface that facilitates endothelialization [12–14]. Further, relative to drug screens using cells in two-dimensional culture, TEBVs are limited by low production efficiency and a complex fabrication process. While these TEBVs can model large arteries and arterioles, other systems have been developed to model the microvasculature through either self-assembly of capillary networks [7, 8] or creation of endothelialized channels in compatible biomaterials [8, 15].

To address the challenges limiting TEBV throughput, we developed a novel method to fabricate arteriole-scale endothelialized TEBVs in situ while maximizing throughput and ease of fabrication. The device we describe can be easily assembled first into a seeding chamber, then a perfusion chamber by switching out two components. To begin fabrication, the seeding chamber is assembled and includes molds for four parallel TEBVs. A mixture of collagen gel and medial cells such as neonatal human dermal fibroblasts (hNDFs), primary smooth muscle cells (SMCs), mesenchymal stem cells (MSCs), or iPSC-derived SMCs is injected into the molds to fabricate four collagen TEBVs in a single chamber. After gelation, the TEBVs undergo a process of plastic compression [16], removing over 90% of the water, and are then sutured in place. Vessels are endothelialized by injecting endothelial cells such as blood-derived endothelial colony forming cells (ECFCs) [17], primary vascular endothelial cells, or iPSC-derived endothelial cells. Following endothelialization, the perfusion chamber is assembled and the four TEBVs are perfused in situ (Fig. 1). These TEBVs can resist physiological pressures, exhibit vasoactivity levels found in arteries, and exhibit endothelial release of nitric oxide (NO) throughout the duration of an experiment.

Figure 1.

a. Chip housing 4 TEBVs. b. Flow loops showing position of TEBV chip, pump, media reservoir and tubing.

2. Materials

2.1. Cells

The TEBVs can be fabricated using either primary human cells or cells differentiated from iPSCs.

1. Primary human neonatal dermal fibroblasts (hNDFs, ThermoFisher #C0045C), passages 5–11.

2. Primary human mesenchymal stem cells (MSC, ATCC #PCS-500–010, or Lonza #PT-2501).

3. Primary human endothelial cells (ECs) (ThermoFisher, aortic endothelial cells, #C0065C; coronary artery endothelial cells, Lonza #CC-2585) (see Note 1).

4. iPSCs can be obtained from the Corriell Institute for Medical Research (www.corriell.org) or WiCell (www.wicell.org) (see Note 2).

2.2. Cell Culture Media

1. hNDF growth media, comprised of Dulbecco’s Modified Eagle’s Medium (DMEM) with 4.5 g/L glucose (Gibco, #11960–044) supplemented with 10% HI-FBS (Gibco, #16140–071), 1 × Non-essential amino acids (NEAA, Gibco, #11140–076), 1% Penicillin/Streptomycin (Gibco, #15140–122), 1 × Glutamax (Gibco, #35050–061), 1 × Sodium Pyruvate (Gibco, #11360–070) and 0.1% β -mercaptoethanol (Gibco, #21985023).

2. hMSC media, comprised of 500 mL MEM Alpha Base Media (Gibco, #12561–056), 100 mL Heat-inactivated fetal bovine serum (HI-FBS), 10 mL L-Glutamine, and Penicillin/Streptomycin, 5 mL.

3. viSMC media, comprised of 250 mL DMEM/F12, HEPES medium (Gibco, #11330032) combined with 250 mL Neurobasal medium (Gibco, #21103049) and supplemented with 10 mL (2% v/v) B27 without vitamin A (Gibco, #12587010), 5 mL (1% v/v) N2 supplement (Gibco 17502048), 500 μl (0.1% v/v) β-mercaptoethanol (Gibco 21985–023), 5 mL Penicillin/Streptomycin (Gibco, #15140–122), 2 ng/mL Activin A (Peprotech, #120–14E), and 2 μg/mL heparin (Sigma, #H3149).

4. Endothelial cell media (Cell Applications, #211–500) containing 1% (v/v) Antibiotic-Antimycotic (Gibco, #15240–062).

5. viEC media, comprised of 500 mL StemPro-34 Basal Medium plus supplement (ThermoFisher 10639011), 10% heat inactivated fetal bovine serum (Gibco #10082147), 5 mL GlutaMAX (Gibco #35050–061), 5 mL Penicillin/Streptomycin (Gibco 315140–122), 50 ng/mL VEGF165 (GenScript #Z02689), and 2 μg/mL heparin (Sigma #H3149) (see Note 3).

2.3. Cell Culture Supplies

1. T75 flasks (VWR, #BD353136).

2. Phosphate-buffered saline (PBS), (Sigma-Aldrich, #P4593).

3. Trypsin (Gibco #25300062), 0.05% diluted 1:1 in sterile PBS without Ca⁺⁺ and Mg⁺⁺.

4. Accutase (Innovative Cell Technologies #AT104).

2.4. Materials for Mold Assembly

1. The molds are created using acrylic or polycarbonate and composed of 5 parts: part A-E (Fig. 2).

2. Part A (Fig. 2) (40 mm × 42 mm × 8 mm) houses the TEBVs and connects them to the perfusion loop via metal tubing with inner diameter 0.65 mm and outer diameter 0.95 mm (New England Small Tube).

3. Parts A, B, C are assembled for the seeding chip and the parts A, D, and E can be assembled for the perfusion chip (see Note 4).

4. The holes for the tubing and TEBVs are spaced 4.84 mm apart. The inner opening is 26.5 mm × 26.5 mm.

5. There are four holes placed at the corners of parts A-E for 6/32 screws and nuts. O-rings (70 BUNA, Size 24) are needed to provide a good seal. The side ports fit #10–32 UNF straight hole adapters to connect to tubing.

6. Parts B and C are 40 mm × 42 mm × 6 mm (the raised area is 3.9 mm) and the opening dimensions are the same as for part A.

7. The channels in Parts B and C (each forms a hemicylindrical region of diameter 2.2 mm, length 24 mm) serve as molds for the TEBVs.

8. Parts D and E are 40 mm × 42 mm × 4.5 mm (the raised area is 2 mm) and the opening dimensions are the same as for part A.

9. Eight stainless steel mandrels with outer diameter 0.63 mm and inner diameter 0.33 mm (New England Small Tube).

10. PDMS (10:1 mixed, Sylgard 184 PDMS Kit, Dow, Inc.).

11. Two barbed tubes with screws (match the size on part A, McMaster Carr, Elmhurst, IL).

12. Two O-rings (match the sealing grooves on part A).

Figure 2.

The design of the mold. a. The drafts of all the parts to assemble the mold. b. The assembled seeding chip (TEBV fabrication mold) and perfusion chip (TEBV culture chamber).

2.5. Materials for TEBV fabrication

1. Rat Tail Collagen I, High Concentration (8–10 mg/mL), 100 mg (VWR/Corning, #47747–218)

2. Acetic acid, (0.6%), diluted from glacial acetic acid in deionized water and sterile filtered.

3. Dulbecco’s Modified Eagle’s Medium, Low Glucose, 10X (Sigma, #D2429).

4. 1M NaOH, sterile, Prepared by dissolving NaOH pellets in deionized water and sterile filtering after cooling.

5. 50 mL Falcon tube.

6. Two 1-mL sterile disposable syringe, BD Biosciences.

7. One 3-mL sterile disposable syringe, BD Biosciences.

8. Sterile sutures (REDISILK, #SK683-SI).

9. 5–6 Kimwipes, autoclaved

10. 15–16, 2cm × 2cm Kimwipes (cut), autoclaved.

11. Two BD Adapters, LUER-STUB 23GA (BD, #427565).

12. Two stainless steel tweezers, sharp tip, autoclaved.

13. One small scissor, autoclaved.

14. Four screws with 4 nuts (6/32 Screw 3/4”, McMaster-Carr, #99607A124), autoclaved.

2.6. Materials for TEBV perfusion

1. 1/16” barb fitting (Y-shape connector of syringe to tubing).

2. Small Tygon tubing (inner diameter match to the mandrel), autoclaved.

3. Large silicon tubing (inner diameter match to the 1/16” Y shape connector), autoclaved.

4. Pump tubing (depends on the model of pump).

5. Two-way straight connectors.

6. Masterflex digital modular drive pump (Cole-Parmer, #EW-07557-10) with a multi-channel pump head (Cole-Parmer, #EW-07623-10).

2.7. Materials for vasoactivity testing

1. Phenylephrine hydrochloride (Sigma, #P6126), 100 mM stock solution in PBS.

2. Acetylcholine chloride (Sigma, #A6625), 10 mM stock solution in PBS.

3. Stereo microscope (AmScope, #SM-3BZ-80S) with video camera 10 MP USB 2.0 Microscope (Amscope, #MA1000).

3. Methods

3.1. Set up mandrels and side connectors.

1. Fix the mandrels on part A of the mold using PDMS (Fig 3).

2. Screw the 2 barbed tubes into part A of the mold.

3. Set the O-rings in the sealing grooves on the part A of the mold.

Figure 3.

Set up the mandrels and the side connectors on the part A of the chip.

3.2. Passage primary cells (ECs, hNDFs, and hMSCs)

1. Aspirate the media from the culture flask.

2. Wash the flask with PBS 1–2 times.

3. Add 5 mL 0.025% trypsin (0.05% for hNDFs) into the T-75 flask and incubate at 37°C for 5 minutes.

4. Add 5 ml of culture media to inhibit trypsin activity.

5. Transfer the cell suspension into centrifuge tubes.

6. Centrifuge cells at 1000 rpm (150 g) for 5 minutes.

7. After centrifugation, remove media and re-suspend 10⁶ cells in 60 μL fresh culture media.

3.3. Passage viSMCs and viECs

1. Aspirate the media from the culture flask.

2. Wash the flask once with warm PBS.

3. Add 5 ml Accutase to T-75 flask and incubate at 37°C for 3 minutes.

4. Dilute Accutase with 5 ml culture media.

5. Transfer cell suspension to centrifuge tube.

6. Centrifuge cells at 1000 rpm (150 g) for 5 minutes.

7. Remove supernatant and resuspend cells in desired amount of fresh culture media.

3.4. TEBV fabrication

1. Insert four mandrels into the chamber at each side of part A. Make sure each pair of mandrels at opposite positions of the chamber are touching each other at the middle to form one long tube.

2. Assemble part A, part B and part C to form the seeding chip (Fig. 3).

3. Passage and resuspend 10⁶ hNDFs in 60 μl hNDF media (or 3 × 10⁶ viSMCs in 60 μl viSMC media).

4. Make 7 mg/ml solution of rat-tail collagen I in 1.5 ml Eppendorf tube, total volume 1 mL (See Note 5).

5. The formula for 1 mL collagen and cell mixture for the four TEBV system is (see Note 6):

   • Volume of 10X DMEM = 94 μl
   • Volume of collagen solution (V_c)= 1 ml × (7 mg/ml)/collagen stock concentration)
   • Volume 1M NaOH (V_NaOH) = Volume of collagen solution × 0.023
   • Volume of hDNF suspension = 60 μl
   • Volume of hNDF media (V_hNDF) = 1 ml - 94 μl - V_c - V_NaOH - 60 μl (if negative then do not add this extra media)

6. Add the solutions in the following sequence: 10×DMEM→NaOH→Collagen→ hDNF suspension→ hNDF media.

7. Using a 200 μl pipette, add collagen mixture into each mold chamber (four chambers per chip, the volume of each chamber depends on design).

8. Allow the collagen solution with cells to gel in the chip for 30 minutes in a cell culture incubator at 37°C.

3.5. Plastic Compression and Dehydration of TEBVs in situ

1. Move the mold from the incubator to the biosafety cabinet.

2. Remove part B and part C of the mold (Fig. 5a).

3. Dehydrate the TEBVs with 2 cm × 2 cm wipes 5 times on each side (Fig. 4b–d).

4. Allow Kimwipes to remain in place until water absorption is visible (see Notes 7, 8).

5. Attach part D to the bottom of part A.

6. Add 2 mL warm hNDF media (or viSMC media for TEBVs made with iPS-derived cells) into the chamber using a serological pipette.

7. Soak the dehydrated TEBVs for at least 1 minute.

8. Remove the media from chamber.

9. Draw out all the mandrels such that there is 5 mm remaining in the interior of the chip on each side.

10. Firmly attach TEBVs to the mandrels with sutures, tying in double knots if needed (Fig. 5a).

11. Cover the top layer of the chamber with part E, screw in place, and fill the chamber with warm EGM media (or viSMC media for iPSC-derived TEBVs) using a syringe and tubing through the barbed side port tube (Fig. 5b).

Figure 5.

Suture the vessels and set up the culture chamber. a. Part A of vessel chamber with drawn-out mandrels and sutured TEBVs. b. Fully assembled TEBV chip filled with media.

Figure 4.

Dehydration of the TEBVs. a. Release the TEBVs from the mold. b. The cut and autoclaved wipes. c. Dehydrate the TEBVs using wipes. d. The dehydrated TEBVs.

3.6. Endothelial Seeding of TEBVs

1. Passage EPCs (or viECs).

2. Prepare a suspension of 6×10⁶ cells/mL in 0.45 mL EC media (or 4×10⁶ cells in 0.45 mL viEC media for iPSC-derived TEBVs).

3. Connect one empty syringe (1 mL syringe with BD adapter needle, 22 gauge or greater) to one mandrel (link the adapter needle and mandrel using Tygon tubing).

4. Fill another 1 mL syringe with EC suspension and connect it to the mandrel at opposite position of the chamber.

5. Slowly push suspension through, counteracting by pulling on the other syringe to act as a pressure balance. Ensure that suspension is going through the TEBV and filling the whole lumen.

6. Move the syringes to the next vessel and repeat the filling step. Seed the four vessels one by one.

7. Place the perfusion chip with ECs for at least 30 minutes in incubator and flip it after 15 minutes.

8. Keep the perfusion chip in the incubator overnight. Setup the perfusion 24 hours after fabrication.

3.7. TEBV perfusion

There are two perfusion loops for the chip: the vessel loop and the side loop (Fig. 1). The perfusion tubing is composed of silicon tubing and pump tubing. The tubing is linked by two-way straight connectors. The length of each loop depends on the experiment.

1. Set up the side loop and fill the loop using EC media (viSMC media for iPSC-derived TEBVs).

2. Set up the vessel loop and fill the loop using EC media (viSMC media for iPSC-derived TEBVs). The four TEBVs in one chamber are linked to one perfusion tubing using 1/16” Y-shape connectors (Fig. 6).

3. Set up the pump tubing on the pump and perfuse. The flow rate depends on the desired wall shear stress (usually 0.1–1.5 Pa). The relationship between wall shear stress (τ_w, Pa) and volumetric flow rate (Q, m³/s) for a tube of radius R (m) and Newtonian fluid of viscosity μ (Pa s) is:

   $${\tau }_w=\frac{4\mu Q}{\pi R^3}$$ (1)

   For culture media at 37°C, μ = 0.087 Pa s (see Note 9).

Figure 6.

Set up perfusion tubing. a. The Y connecter. b. The two loops of perfusion.

3.8. Vasoactivity testing

1. Place perfusion chamber with TEBVs beneath stereo microscope and image TEBVs at 9x magnification.

2. Start video recording of the TEBVs being perfused at flow rate that meets the desired shear stress (equation 1) to acquire vessel baseline diameter.

3. After at least 30 seconds of baseline, add phenylephrine at 1 μM (final concentration) to the media stock vial of the vessel loop.

4. Record phenylephrine response for 6 minutes.

5. Add acetylcholine at 1 μM (final concentration) to the media stock vial of the vessel loop.

6. Record acetylcholine response for 6 minutes.

7. Obtain three frames of images from the video: (1) before adding phenylephrine as base line, (2) after phenylephrine treatment for 6 min, and (3) after acetylcholine treatment for 6 minutes.

8. Measure diameter of each vessel at 3–5 different locations under baseline conditions. For example, for each TEBV divide the vessel into 4 equal parts and measure the diameter in the middle of each region.

9. Average the values for each vessel to obtain the average TEBV diameter before treatment. Then average the diameter of each vessel.

10. Repeat the process for the images after 6 minutes of treatment with 2 μM phenylephrine (PE) and 6 minutes of treatment with 2 μM acetylcholine (ACh). Then average the diameter of each vessel.

11. Divide the average diameter of phenylephrine-treated TEBV by the diameter of TEBV at baseline, subtract from 1 and multiply by 100 to obtain percent vasoconstriction: (1-ave Diameter after PE/Initial Diameter)×100% (see Note 10).

12. Use the average diameter of acetylcholine-treated TEBV divided by the diameter of phenylephrine-treated TEBV and subtract 1 to get vasodilation: (Diameter after ACh) /Diameter after PE −1)×100%.

3.9. En Face Immunofluorescence Imaging of TEBV Endothelium

1. At the end of an experiment, perfuse the TEBVs with 4% Paraformaldehyde (PFA) solution for one hour at room temperature.

2. Following fixation, remove the sample from the samples from the chamber and transfer to a well plate containing PBS.

3. Wash samples in PBS three times to remove excess PFA.

4. If imaging intracellular proteins (e.g. von Willebrand Factor, vWF), samples should be permeabilized with 0.1% Triton-X for 5 minutes.

5. After rinsing 3 times with Dulbecco’s Phosphate Buffered Saline DPBS (Sigma Aldrich, D8662) (following permeabilization), incubate in blocking buffer (PBS+10% goat serum+5% BSA) for 2 hours at room temperature.

6. Incubate the lumen with one of the following antibodies overnight at 4 °C: CD31 (BD #550389, 1:200 dilution), VCAM-1 (Santa Cruz Biotechnology (SCBT), #sc-13160, 1:200 dilution), ICAM-1 (Santa Cruz Biotechnology, #sc-107,1:200 dilution), E-selectin (SCBT, #sc-14011, 1:200 dilution), VE-Cadherin (ThermoFisher, Clone 16B1, #14-1449-82), vWF (ThermoFisher, Clone F8/86, #MA5–14029, 1:100 dilution).

7. Rinse the samples three times with DPBS.

8. Incubate the lumen with the following secondary antibodies for 160 minutes at room temperature: Alexa Fluor 488-goat anti-mouse IgG (ThermoFisher, #A-32723), Alexa Fluor 488-goat anti rabbit IgG (ThermoFisher, #A-32731), Alexa Fluor 546-goat anti-mouse IgG (ThermoFisher, #A-11030), Alexa Fluor 546-goat anti-rabbit IgG (ThermoFisher, #A-11035), Alexa Fluor 633-goat anti-mouse (ThermoFisher, #A21052) each at 1:200 dilution, and Hoechst dye (ThermoFisher, #H1398, 1:1000 dilution) to stain nuclei

9. Cut the vessel longitudinally to open, with the endothelial surface facing up. Avoid contact with endothelial layer and place flat between two coverslips. Acquire images on a confocal microscope at 20X or 40X magnification (see Note 11).

4. Notes

1. Alternatively, blood-derived endothelial cells (also known as endothelial colony forming cells, ECFCs) derived as described by Ingram et al.[18] and used through passages 4–6.

2. Protocols for differentiating iPS cells to vascular smooth muscle cells (viSMCs) and endothelial cells (viECs)[4] were adapted from Patsch et al.[19].

3. Warm media to 37°C prior to use.

4. Acrylic components can be sterilized with ethylene oxide (ETO) treatment. Alternatively, clear polycarbonate can be autoclaved.

5. All solutions need to be placed on ice. The operation of mixing collagen needs to be performed very carefully to avoid air bubbles.

6. Use NaOH and acetic acid to regulate the collagen pH at 8.5. Solution should change from yellow to pink.

7. Kimwipe applications 1–2: double layer wipe on each side

   Kimwipe applications 3–5: single layer wipe on each side

8. To ensure that at least 90% of the water is removed, weigh the TEBV cell suspension mixture before adding Kimwipes and weigh each Kimwipe before and after contacting TEBV. Calculate weight change and divide by the initial amount of water in TEBV to determine water lost from TEBV.

9. Viscosity is measured at 37°C by a fall ball viscometer (Gilmont, Cole-Parmer, #EW-08701–00) or a rheometer that can provide results in shear rate ranges of 150–2500 s⁻¹ (e.g. Brookfield Engineering Laboratories DV-III rheometer).

10. Vasoactivity measurements are performed to identify the functional behavior of the TEBVs over time and after treatment with drugs, cytokines, or other molecules that may modulate function.

11. Portions of the vessels overlapping with the chamber grips should not be imaged, as endothelial cells are unable to adhere well in these areas, and shear stresses will be inconsistent.