Scaling of Engineered Vascular Grafts Using 3D Printed Guides and the Ring Stacking Method

Summary

Scalable engineered blood vessels would improve clinical applicability. Using easily sizable 3D-printed guides, rings of vascular smooth muscle were created and stacked into a tubular form, forming a vascular graft. Grafts can be sized to meet the range of human coronary artery dimensions by simply changing the 3D-printed guide size.

Abstract

Coronary artery disease remains a leading cause of death, affecting millions of Americans. With the lack of autologous vascular grafts available, engineered grafts offer great potential for patient treatment. However, engineered vascular grafts are generally not easily scalable, requiring manufacture of custom molds or polymer tubes in order to customize to different sizes, constituting a time-consuming and costly practice. Human arteries range in lumen diameter from about 2.0-38 mm and in wall thickness from about 0.5-2.5 mm. We have created a method, termed the "Ring Stacking Method," in which variable size rings of tissue of the desired cell type, demonstrated here with vascular smooth muscle cells (SMCs), can be created using guides of center posts to control lumen diameter and outer shells to dictate vessel wall thickness. These tissue rings are then stacked to create a tubular construct, mimicking the natural form of a blood vessel. The vessel length can be tailored by simply stacking the number of rings required to constitute the length needed. With our technique, tissues of tubular forms, similar to a blood vessel, can be readily manufactured in a variety of dimensions and lengths to meet the needs of the clinic and patient.

Introduction

In treatment of coronary artery disease (CAD), a patient's own blood vessels are harvested as graft material for bypass surgery. However, oftentimes, ill patients do not have viable vessels to donate to themselves, and in cases where they do, the donor site causes considerable additional harm and has a serious risk for infection.¹ Engineered vascular grafts could fill this need. Scalability is of utmost importance for engineering vessels in order to meet the wide range of patient vessel size requirements. However, present methods for engineering vessels are not easily scalable, and typically require remanufacture of complex molds or polymer scaffolds. Most engineered grafts either utilize a polymer tubular scaffold that is seeded with vascular fibroblasts, smooth muscle, or endothelial cells; or rolling a cell sheet around a mandrel to create a tissue tube. Two engineered vascular grafts in clinical trials are based on a decellularized polymer-ECM platform.^2,3,4 Polymer grafts available for use in vascular repair are already known to have issues with patency, which could arise as a major issue with long-term application of a graft with a sustained polymer presence. Tubular molds have been used to fabricate completely cellular vessels,^{5,6,7,8,9,10,11,12,13} which procedures would require additional design and tool manufacturing for custom molds in order to produce vessels in a variety of sizes.

The method described herein encompasses a novel technique for creating easily scalable engineered vascular grafts using customizable 3D printed inserts and traditional culture plates.¹⁴ Cells are seeded into plates with inserts of a central post and outer shell. The post controls lumen diameter and allows the cell monolayer to self-assemble into a ring of tissue. The outer shell controls thickness of the ring, and thus wall thickness of the final vessel. Completed tissue rings are then stacked to form a tubular, vascular graft. The advantage to this method, termed the "Ring Stacking Method," is that any adherent cell type can be seeded into the plate setup and tissue rings or tubes of any size needed for the desired application can be generated by simply modifying guide inserts. Comparative techniques in tissue engineering creating rings of tissue remain difficult to scale,^15,16 requiring remanufacture of molds for each desired size. Additionally, vascular grafts made using this method can be produced in 2-3 weeks, several weeks faster compared to other engineered vessels.⁶ For the clinic, this time discrepancy can make a significant difference in the treatment of a deteriorating patient.

Protocol

1. Cell Culture Preparation

1. Utilize human aortic smooth muscle cells purchased commercially.
2. Maintain cells in smooth muscle cell growth media composed of 88.6% 231 media, 0.1% each of recombinant human insulin (rH-insulin), recombinant human fibroblast growth factor (rH-FGF), recombinant human epidermal growth factor (rH-FGF), and ascorbic acid; and 5% each of fetal bovine serum (FBS) and L-glutamine; and 1% antibiotic/antimycotic.
   NOTE: Each growth factor, FBS and L-glutamine are purchased as a vascular media growth kit.
3. Change media every 48 hours until the cells are about 90% confluent and ready for tissue seeding.
4. Store cells in an incubator in between media changes for expansion.

2. Preparation of 3D Printed Inserts and Custom Silicone Molded Plates

1. Use a commercial 3D printer (e.g., Replicator Mini) for 3D printing the plate inserts.
   1. Use open source 3D design software such as Blender to create the 3D models of the printed outer-shells and posts.
   2. Export the model's driver file via a .stl format allowing for portability to the 3D printer's software.
   3. Produce printed posts and outer shells using poly(lactic acid) filament (PLA) loaded into the 3D printer.
   4. Following printing, perform a 30-minute soak in a 70%-100% ethanol solution to sterilize each insert.
2. Prepare a 1:10 curing agent to base polymer mixture of poly(dimethylsiloxane) (PDMS) silicone polymer and allow the mixture to degas at room temperature for 10 min.
   1. Define petri dish sizes used as small (35 mm), intermediate (60 mm), and large (100 mm).
   2. Add 2 mL, 4 mL and 6 mL of uncured silicone to each small, intermediate and large plate, respectively, and create a thin layer across the entire bottom of the petri dish.
   3. Create posts for the small plate by pouring PDMS into a 100 mm plate to a height of 7 mm and allow to cure on a hot plate at 60 °C for about 2-3 hours. Then use a 5 mm biopsy punch to punch out cylindrical posts. Use a small amount of uncured PDMS to secure each PDMS cylinder and to the center of each small plate.
   4. For the intermediate and large plates, prior to complete curing of the PDMS at the bottom of the plate, place the 3D printed posts 10 mm and 20 mm in diameter centrally into each intermediate and large plates, respectively. For the large plates, additionally place a 3D printed outer shell about 66.7 mm in diameter equidistance from the post.
      1. Allow each dish to cure in open air on a hot plate at 60 °C for about 2-3 hours, allowing 18 hours for degassing of the polymer. Fix printed components to the plate in the proper region and orientation as seen in Figure 1.
   5. Add a solution of 70% ethanol with 30% distilled water for 30 minutes to the inside of all plates for sterilization and then cover each plate.
   6. Carefully aspirate the ethanol from each plate and allow to air dry.
   7. Arrange each plate in a biological safety cabinet (BSC) next to its lid, face-up. Expose plates and lids to UV light under the BSC for 30 minutes to complete sterilization. Sterile technique is performed with every step after UV exposure.

3. Preparation of Fibrin Hydrogel, Seeding with Smooth Muscle Cells and Maintenance of Plates

1. Mix a solution of fibrin gel containing growth media + 0.01% TGF-β1 in the amounts of 0.5 mL, 1.1 mL and 1.81 mL for small, intermediate and large plate sizes, respectively.
   1. Add 40 µL, 88.4 µL and 145 μL of thrombin, from a stock of 100 U/mL, to the media of each small, intermediate and large plate, respectively. Gently swirl each plate by hand to ensure that thrombin is evenly mixed within the media.
   2. Next, add 160 µL, 354 µL and 580 μL fibrinogen, from a stock of 20 mg/mL, drop-wise circularly to the thrombin-media mixture to each small, intermediate and large plate, respectively. Gently swirl by hand to ensure mixing and distribution of the hydrogel into an even layer.
   3. Allow hydrogel to cure for 10-15 minutes at room temperature.
2. Trypsinize smooth muscle cells expanded in 150 mm cell culture plates and centrifuge according to standard protocols. The resulting pellet should be resuspended in 3 mL of differentiation media consisting of 98% - 231 media, 1% FBS and 1% antibiotic/antimycotic.
   1. Vigorously mix cells by titrating up and down with a 2 mL pipette to break up any cell clumps. Count cells with a hemocytometer and create a cell suspension of 2 x 10⁶ cells/mL, 1.0 x 10⁷ cells/mL and 1.4 x 10⁷ cells/mL for small, intermediate and large plates, respectively.
   2. Add 1 mL of each cell suspension into a corresponding 50 mL conical labelled small, intermediate and large. Set up an additional 50 mL conical in this manner for each additional tissue ring desired.
   3. Add differentiation media to each conical to obtain final seeding volumes of 2 mL, 4 mL and 5 mL for each small, intermediate and large vessel, respectively. Then, carefully pipet the cell solution drop-wise on top of the prepared hydrogel in each corresponding plate.
   4. Place plates into the incubator at 37 °C and 5% CO₂.
3. Change differentiation media every 48-72 hours for each plate. In the case of the larger plate, change media initially after 24 hours, then change it every 48-24 hours to compensate for the large cell seeding density.
   1. After 2-4 days as the rings will have completely contracted in towards the post, add 10 µL, 20 µL and 35 μL of TGF-β1 to each small, intermediate and large ring, respectively. After exposure to TGF-β1 for at least 24 hours, rings are ready to be handled.

4. Assembly of Vascular Construct and Maintenance

1. Before the fabrication of the final vascular construct, a specialized container is created to hold the completed vessel.
   1. For the small vessel, create a tall plate for ring stacking by cutting a 2-inch section off the top of a 50 mL polycarbonate conical tube, and then PDMS glue the cut edge into a 35 mm plate. Use the conical lid as the plate lid.
   2. For the intermediate and large vessel tall ring stacking plates, cut a 1.75-inch diameter polycarbonate tube into 2.5 inch sections lengthwise to serve as the tall plate walls. For the tall plate bottoms, cut a 0.125-inch-thick polycarbonate sheet into 2-inch diameter circle pieces. Using acrylic solvent cement, bind the polycarbonate tube section to the circular cut piece. Use the lid from a 60 mm petri dish as the lid for the tall plate.
   3. 3D print posts 5, 10 and 20 mm in diameter and 50 mm in length.
   4. Add 10 mL of uncured silicone to each container. Prior to the complete curing of the PDMS, centrally place each post created in step 4.1.3 into each small, intermediate, and large container.
   5. Allow to cure on a hot plate set to 60 °C for 2-3 hours.
   6. Sterilize with a solution of 70% ethanol with 30% distilled water for 30 minutes.
   7. Carefully aspirate the ethanol from each container and then allow to air dry in the BSC. Next, arrange the containers in the hood with each plate placed next to its lid, face-up. Expose containers to UV light under the BSC for an additional 30 minutes for further sterilization. Use sterile technique with every step after UV exposure.
2. Using very fine forceps, carefully remove each tightly rolled smooth muscle hydrogel ring from its post and transfer to its corresponding larger container with the tall posts.
   1. Use a pair of forceps in each hand and lift one side of the ring from the post, and then the other. Be careful to protect and maintain the lumen.
   2. Perform the transfer with this two handed method, sliding first one side, then the other side of the ring onto the tall post. Using gentle, gradual movements, and working circumferentially, slowly push the ring down onto the tall post. Subsequently stack tissue rings until the desired vessel length has been obtained, with each ring adding approximately 1-2 mm of length to the completed construct.
3. With the ring stack positioned on the tall 3D printed posts, turn the plate so that the post is parallel with the working surface.
   1. Using a micropipette, add 40, 80 and 160 μL of thrombin at a concentration of 100 U/mL gently to the outer surface of each small, intermediate and large vessel, respectively. While adding the thrombin, slowly rotate the plate to ensure even coverage of all surfaces of the construct. This will be the base for the fibrin glue utilized to secure the ring stack construct in the initial days after construction.
   2. Next, add 40, 80 and 160 μL of fibrinogen at a concentration of 20 mg/mL to each small, intermediate, and large construct, respectively, using a micropipette while rotating the construct quickly. The thrombin and fibrinogen quickly set into a firm gel once mixed. Due to the short curing time, apply the fibrinogen as quickly and evenly as possible.
4. Add 20 mL of differentiation media to each container holding the construct. Place vessels into a 37 °C incubator until needed. Change media every 3-5 days.

Results

Demonstrated here is fabrication of 3 different engineered vascular graft sizes (Figure 1), showing that the Ring Stacking Method (RSM) is scalable. To prove applicability, the 3 different vessel sizes chosen correlate to actual human vessel size for the left anterior descending artery (small; lumen diameter = 4 mm)¹⁷, descending aorta (intermediate; lumen diameter = 10 mm) and ascending aorta (large; lumen diameter = 20 mm)¹⁸

Discussion

The Ring Stacking Method presents multiple advantages over current vascular tissue engineered construct techniques. The RSM can be adapted to create human vessels of any size by simply customizing the post and outer shell dimensions. Our method allows for development of polymer-free engineered vessels composed solely of human cells and rapidly degrading support material found in the body's natural wound healing process. Polymer grafts are known to cause restenosis in the clinic and could become problematic if contain...

Materials

Name	Company	Catalog Number	Comments
Human Aortic Smooth Muscle Cells	ATCC	PCS-100-012	vascular smooth muscle cells
Medium 231	Gibco (Life Technologies	M-231-500	media specific to vascular smooth muscle cells
Human Aortic Smooth Muscle Cell Growth Kit	ATCC	PSC-100-042	growth factors for maintaining vascular smooth muscle cell viability
Replicator Mini 3D printer	MakerBot	N/A	3D printer
Poly(lactic acid) 3D ink (PLA)	MakerBot	N/A	3D printer filament
Poly(dimethlysiloxane) (PDMS)	Ellworth Adhesives	3097358-1004	polymer for gluing plate parts
Fibrinogen	Hyclone Labratories, Inc.	SH30256.01	fibrin gel component
Thrombin	Sigma Life Sciences	F3879-5G	fibrin gel component
Tranforming Growth Factor-Beta 1	PeproTech	100-21	growth factor for stimulating collagen production
Hemocytometer	Hausser Scientific Co.	3200	for cell counting
Polycarbonate tubing	US Plastics	PCTUB1.750X1.625	material for making tall, ring stacking plates
Polycarbonate sheet	Home Depot	409497	material for making tall, ring stacking plates
Adhesive polymer solvent	SCIGRIP	10799	material for making tall, ring stacking plates
Instron 5940	Instron	N/A	tensile testing machine
U-Stretch	Cell Scale	N/A	tensile testing machine
Smooth Muscle Actin	MA5-11547	Thermo Fisher	antibody
Tropomyosin	MA5-11783	Thermo Fisher	antibody