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Abstract

Introduction

Protocol

Representative Results

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Acknowledgements

Materials

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Bioengineering

Ex Vivo Perfusion Culture of Large Blood Vessels in a 3D Printed Bioreactor

Published: July 28th, 2023

DOI:

10.3791/65465

1Department of Biochemical Sciences, School of Biosciences, University of Surrey, 2Department of Clinical and Experimental Medicine, School of Biosciences, University of Surrey
* These authors contributed equally

This protocol presents the setup and operation of a newly developed, 3D printed bioreactor for the ex vivo culture of blood vessels in perfusion. The system is designed to be easily adopted by other users, practical, affordable, and adaptable to different experimental applications, such as basic biology and pharmacological studies.

Vascular disease forms the basis of most cardiovascular diseases (CVDs), which remain the primary cause of mortality and morbidity worldwide. Efficacious surgical and pharmacological interventions to prevent and treat vascular disease are urgently needed. In part, the shortage of translational models limits the understanding of the cellular and molecular processes involved in vascular disease. Ex vivo perfusion culture bioreactors provide an ideal platform for the study of large animal vessels (including humans) in a controlled dynamic environment, combining the ease of in vitro culture and the complexity of the live tissue. Most bioreactors are, however, custom manufactured and therefore difficult to adopt, limiting the reproducibility of the results. This paper presents a 3D printed system that can be easily produced and applied in any biological lab, and provides a detailed protocol for its setup, enabling users' operation. This innovative and reproducible ex vivo perfusion culture system enables the culture of blood vessels for up to 7 days in physiological conditions. We expect that adopting a standardized perfusion bioreactor will support a better understanding of physiological and pathological processes in large animal blood vessels and accelerate the discovery of new therapeutics.

The vascular wall exists in a reactive steady state, which ensures both responsivities to external stimuli (i.e., change of pressure, vasoconstrictors) and a consistent non-activating surface preventing blood coagulation and inflammatory cell infiltration1. In response to aging- and lifestyle-dependent stimuli and upon direct damage, the vascular wall activates remodeling processes such as restenosis and atherosclerosis, which are known contributors to common cardiovascular diseases (CVDs), such as ischemic stroke and myocardial infarction2. While interventional approaches such as percutaneous revascularisation and stent....

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This protocol describes the assembly and use of a system composed of two EasyFlow (bioreactor) inserts: one representing the reaction chamber (C), containing the perfused artery sample, and one functioning as a medium reservoir (R) (Figure 1 and Figure 2A). Carotid arteries were obtained from 4-6-week-old male and female piglets (6-12 kg) at The Pirbright Institute, UK. Animal procedures were carried out under the Home Office Animals (Scientific Procedures) Act .......

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This study has established a versatile and affordable perfusion system (EasyFlow)13. The 3D printed design of the system facilitates the adoption of the system by other labs and therefore encourages reproducibility.

The fabricated perfusion insert is housed in a 50 mL centrifuge tube, creating an isolated environment. Using two perfusion inserts, a perfusion loop can be established containing a reservoir and a reaction chamber, where the biological sample is incubated. .......

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Ex vivo vascular perfusion systems constitute a unique platform to study the function and behavior of vascular cells within their native tissues under controlled conditions, which enables the dissection of complex processes such as post-injury vascular remodeling22. However, most reported bioreactors are in-house made systems based on custom-made components and are often difficult to replicate by others23. Alternative commercial solutions exist, but lack flexibilit.......

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The authors wish to thank the Veterinary Pathology Centre at the University of Surrey School of Veterinary Medicine for histology services. We also thank Drs L. Dixon, A. Reis, and M. Henstock from The Pirbright Institute (Pirbright, UK) for their support in procuring the animal tissues, and the Department of Biochemical Sciences at the University of Surrey, especially the technical team, for their continuing support. RSM was supported by the Doctoral College studentship award (University of Surrey), DM and PC were supported by the National Centre for the Replacement, Refinement & Reduction of Animals in Research (grant numbers: NC/R001006/1 and NC/T001216/1).

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Name Company Catalog Number Comments
EasyFlow - - 3D printed by MultiJet Fusion by Protolabs
PA12 - 3D printing Protolabs - -
Peristaltic pump Heidolph  PD5201
Culture media components:
Amphotericin B solution, 250 mug/mL in deionized water Sigma-Aldrich A2942-20ML
Dextran  from Leuconostoc spp. Sigma-Aldrich D8802-25ML
Dulbecco's Modified Eagle's Medium - high glucose, w/ 4500 mg/L glucose, L-glutamine, sodium pyruvate, and sodium bicarbonate Sigma-Aldrich D6429-6X500ML
Fetal Bovine Serum Sigma-Aldrich F9665
Penicillin-Streptomycin Sigma-Aldrich P4333-100ML
Immunostaining materials:
Cryostat LEICA CM3050 S
DAPI Sigma-Aldrich D9542-10MG
Goat serum Sigma-Aldrich G9023-10ML
Goat α-Rabbit Alexa Fluor 488 Thermo Fisher Scientific A11008
Invitrogen eBioscience Fluoromount G Thermo Fisher Scientific 50-187-88
MX35 Premier + Microtome Blade Thermo Scientific 3052835
Optimal Cooling Tempearure Compound - OCT Agar Scientific AGR1180
Rabbit α-CD31 antibody Abcam ab28364
Sudan Black B Santa Cruz Biotechnology SC-203760
X72 SuperFrost Plus Adhesion slide, 25x75x1mm, White, 90° Ground Edges, Frosted Area 20mm, 72/box Fisher Scientific J1800AMNZ
α-Smooth Muscle Actin (SMA) Alexa Fluor® 647-conjugated antibody R&D Systems IC1420R
Material for laser cutting of components:
Clear Plastic Sheet, 1250 mm x 610 mm x 1 mm (for laser cutting of  washers) RS Components 258-6590
RS PRO Translucent Rubber Sponge Sheet, 600 mm x 600 mm x 1.5 mm (for laser cutting of  silicone seals) RS Components 840-5541
Optional pressure monitors:
Pressure sensor Parker Hannifin 080-699PSX-3P-5
SciPres Pressure Monitor Parker Hannifin 206-200-M
Pre-sterilized single use plasticware:
0.2 um filter Sarstedt 70.1114.210
20 mL Sterile syringe IMS Euro 40004
50 mL Centrifuge Tube Thermo Fisher Scientific Sarstedt - 62.547.254
Small components:
Cable ties - -
Masterflex Adapter Fittings, Female Luer to Hose Barb Cole-Parmer WZ-30800-10 Barb Adaptor
Masterflex Polycarbonate Luer Fittings Cole-Parmer AU-45504-84
Nylon Miniature Check Valve Cole-Parmer 98553-00
RS PRO Translucent Rubber Sponge Sheet, 600 mm x 600 mm x 1.5 mm (for laser cutting of  silicone seals) RS Components 840-5541
Stainless Steel M2 Hex Nuts RS Components 527-218
Stainless Steel M2 x 6 mm Screws RS Components 418-7426
Stainless Steel M5 Hex Nuts RS Components 189-585
Surgical vessel loop Vascular Silicone Ties,International Medical Supplies  10-1003
Three-way valves IMS Euro  91000
Surgical Equipment
Anatomical Forceps, GRAEFE, Curved, 10 cm SKU: BD-07 International Medical Supplies SKU: BD-07
Micro Forceps, Angled, 0.3 mm, 11 cm International Medical Supplies SKU: BD-361
Micro Scissors Noyes, Curved, 12 cm International Medical Supplies SKU: FD-12
Troge Surgical Scalpels - Size 23 - Box of 100 International Medical Supplies 63114
Tubing:
Eppendorf silicone tubing (I.D.1.6 mm, O.D.4.7 mm) Eppendorf M0740-2396 System tubing
Masterflex PharMed BPT 3-Stop Tubing ISMATEC 95714-48 Soft wall tubing (for clamp)
RS PRO Transparent Hose Pipe, 0.8 mm ID, Silicone RS Components 667-8432 Resistance tubing (small inner diameter)
Tygon for food (I.D. 4.8 mm, W.T. 1.6 mm) Heidolph 525-30027-00-0 One way valve tube
Verderflex Yellow Hose Pipe, 6.4 mm ID, Verderprene RS Components 125-4042 Pump Tubing

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