Name: EasyFlow Â– An Economical and Adaptable Perfusion Bioreactor for Large Blood Vessel Culture (Video) | JoVE
Uploaded: 2023-07-28T14:30:00
Description: Watch this Scientific Journal Video about EasyFlow Â– An Economical and Adaptable Perfusion Bioreactor for Large Blood Vessel Culture at JoVE.com

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 &#38; Reduction of Animals in Research (grant numbers: NC/R001006/1 and NC/T001216/1).

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 ...

A Novel 3D Printed Bioreactor for Ex Vivo Culture of Blood Vessels in Perfusion

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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...

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 stenting are available to tackle advanced manifestations of vascular disease, these are known to provoke further vascular damage, often leading to recurrence. In addition, only limited preventative and early-stage solutions are available. Understanding the mechanisms maintaining vascular wall homeostasis and driving its dysfunction is at the heart of developing new cures3.
Despite the constant development and advances in molecular biology and tissue engineering, animal studies remain a crucial component of vascular biology studies. In vivo animal studies have provided enormous insight into the mechanisms of vascular homeostasis and pathology; however, these procedures are costly, have relatively low throughput, and pose substantial ethical issues. In addition, small animals are poorly representative of human vascular physiology, and larger animal experiments are vastly more expensive and create further ethical considerations4,5. With the increasing demand for pharmaceutical and medical solutions for a rapidly aging population, the downsides of animal use are magnified, impacting the reproducibility, reliability, and transferability of results to patient care6.
In vitro systems offer a simplified platform to study basic mechanisms but fail to recapitulate the complexity of the whole tissue, the interactions between cells and the extracellular matrix, and the mechanical forces, which are critical determinants in the development of vascular diseases7.
Ex vivo studies performed on whole tissues maintained in artificially controlled environments mimic the in vivo complexity while enabling relatively high-throughput investigations8. Given the ability to closely control the culture conditions and environment, ex vivo models allow a broad range of complex studies and provide a suitable alternative to reduce the use of animal procedures in vascular biology. Static vascular ring cultures offered interesting insights but failed to incorporate the crucial hemodynamic element9. Indeed, the study of the vascular system ex vivo poses specific challenges related to the many dynamic forces which apply to the cells within the blood vessel wall. Stimuli such as luminal flow, turbulence, shear stress, pressure, and wall deformation significantly impact tissue pathophysiology10,11,12.
Perfusion bioreactors are essential for studying vascular homeostasis and remodeling in response to injury or hemodynamic changes13. Furthermore, perfusion culture can be used to improve the maturation and durability of tissue-engineered blood vessels (TEBVs), providing suitable alternatives for vascular grafts14.
Commercially available perfusion bioreactors are limited in flexibility and adaptability and are costly. Many of the existing in-house developed bioreactors are instead difficult to replicate in other labs, due to the limited descriptions and unavailability of specially made components7,8,9,10,11,12. To overcome these limitations, we have recently developed a new bioreactor (EasyFlow), which is economical to produce, accommodates a range of tissues, and enables relatively simple modifications to adapt to different research demands13. The insert is 3D printed and fits as in a lid of a standard 50 mL centrifuge tube. Its modular design and 3D printing manufacture make it accessible and reproducible across different labs, as well as easily modifiable to adapt to different scientific needs. This protocol describes the assembly and basic operation of the bioreactor system in an arterial perfusion setting.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>EasyFlow</td>
			<td>-</td>
			<td>-</td>
			<td>3D printed by MultiJet Fusion by Protolabs</td>
		</tr>
		<tr>
			<td>PA12 - 3D printing</td>
			<td>Protolabs</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Heidolph</td>
			<td>&#160;PD5201</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture media components:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B solution, 250 mug/mL in deionized water</td>
			<td>Sigma-Aldrich</td>
			<td>A2942-20ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran&#160; from Leuconostoc spp.</td>
			<td>Sigma-Aldrich</td>
			<td>D8802-25ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium - high glucose, w/ 4500 mg/L glucose, L-glutamine, sodium pyruvate, and sodium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>D6429-6X500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma-Aldrich</td>
			<td>F9665</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P4333-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Immunostaining materials:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>LEICA</td>
			<td>CM3050 S</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9542-10MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Sigma-Aldrich</td>
			<td>G9023-10ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat &#945;-Rabbit Alexa Fluor 488</td>
			<td>Thermo Fisher Scientific</td>
			<td>A11008</td>
			<td></td>
		</tr>
		<tr>
			<td>Invitrogen&#160;eBioscience Fluoromount G</td>
			<td>Thermo Fisher Scientific</td>
			<td>50-187-88</td>
			<td></td>
		</tr>
		<tr>
			<td>MX35 Premier + Microtome Blade</td>
			<td>Thermo Scientific</td>
			<td>3052835</td>
			<td></td>
		</tr>
		<tr>
			<td>Optimal Cooling Tempearure Compound - OCT</td>
			<td>Agar Scientific</td>
			<td>AGR1180</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit &#945;-CD31 antibody</td>
			<td>Abcam</td>
			<td>ab28364</td>
			<td></td>
		</tr>
		<tr>
			<td>Sudan Black B</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-203760</td>
			<td></td>
		</tr>
		<tr>
			<td>X72 SuperFrost Plus Adhesion slide, 25x75x1mm, White, 90&#176; Ground Edges, Frosted Area 20mm, 72/box</td>
			<td>Fisher Scientific</td>
			<td>J1800AMNZ</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-Smooth Muscle Actin (SMA) Alexa Fluor&#174; 647-conjugated antibody</td>
			<td>R&#38;D Systems</td>
			<td>IC1420R</td>
			<td></td>
		</tr>
		<tr>
			<td>Material for laser cutting of components:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Clear Plastic Sheet, 1250 mm x 610 mm x 1 mm (for laser cutting of&#160; washers)</td>
			<td>RS Components</td>
			<td>258-6590</td>
			<td></td>
		</tr>
		<tr>
			<td>RS PRO Translucent Rubber Sponge Sheet, 600 mm x 600 mm x 1.5 mm (for laser cutting of&#160; silicone seals)</td>
			<td>RS Components</td>
			<td>840-5541</td>
			<td></td>
		</tr>
		<tr>
			<td>Optional pressure monitors:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pressure sensor</td>
			<td>Parker Hannifin</td>
			<td>080-699PSX-3P-5</td>
			<td></td>
		</tr>
		<tr>
			<td>SciPres Pressure Monitor</td>
			<td>Parker Hannifin</td>
			<td>206-200-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Pre-sterilized single use plasticware:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.2 um filter</td>
			<td>Sarstedt</td>
			<td>70.1114.210</td>
			<td></td>
		</tr>
		<tr>
			<td>20 mL Sterile syringe</td>
			<td>IMS Euro</td>
			<td>40004</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Centrifuge Tube</td>
			<td>Thermo Fisher Scientific</td>
			<td>Sarstedt - 62.547.254</td>
			<td></td>
		</tr>
		<tr>
			<td>Small components:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cable ties</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Masterflex Adapter Fittings, Female Luer to Hose Barb</td>
			<td>Cole-Parmer</td>
			<td>WZ-30800-10</td>
			<td>Barb Adaptor</td>
		</tr>
		<tr>
			<td>Masterflex&#160;Polycarbonate Luer Fittings</td>
			<td>Cole-Parmer</td>
			<td>AU-45504-84</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon Miniature Check Valve</td>
			<td>Cole-Parmer</td>
			<td>98553-00</td>
			<td></td>
		</tr>
		<tr>
			<td>RS PRO Translucent Rubber Sponge Sheet, 600 mm x 600 mm x 1.5 mm (for laser cutting of&#160; silicone seals)</td>
			<td>RS Components</td>
			<td>840-5541</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless Steel M2 Hex Nuts</td>
			<td>RS Components</td>
			<td>527-218</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless Steel M2 x 6 mm Screws</td>
			<td>RS Components</td>
			<td>418-7426</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless Steel M5 Hex Nuts</td>
			<td>RS Components</td>
			<td>189-585</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical vessel loop</td>
			<td>Vascular Silicone Ties,International Medical Supplies</td>
			<td>&#160;10-1003</td>
			<td></td>
		</tr>
		<tr>
			<td>Three-way valves</td>
			<td>IMS Euro</td>
			<td>&#160;91000</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anatomical Forceps, GRAEFE, Curved, 10 cm SKU: BD-07</td>
			<td>International Medical Supplies</td>
			<td>SKU: BD-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Forceps, Angled, 0.3 mm, 11 cm</td>
			<td>International Medical Supplies</td>
			<td>SKU: BD-361</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Scissors Noyes, Curved, 12 cm</td>
			<td>International Medical Supplies</td>
			<td>SKU: FD-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Troge Surgical Scalpels - Size 23 - Box of 100</td>
			<td>International Medical Supplies</td>
			<td>63114</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubing:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf silicone tubing (I.D.1.6 mm, O.D.4.7 mm)</td>
			<td>Eppendorf</td>
			<td>M0740-2396</td>
			<td>System tubing</td>
		</tr>
		<tr>
			<td>Masterflex&#160;PharMed BPT 3-Stop Tubing</td>
			<td>ISMATEC</td>
			<td>95714-48</td>
			<td>Soft wall tubing (for clamp)</td>
		</tr>
		<tr>
			<td>RS PRO Transparent Hose Pipe, 0.8 mm ID, Silicone</td>
			<td>RS Components</td>
			<td>667-8432</td>
			<td>Resistance tubing&#160;(small inner diameter)</td>
		</tr>
		<tr>
			<td>Tygon for food (I.D. 4.8 mm, W.T. 1.6 mm)</td>
			<td>Heidolph</td>
			<td>525-30027-00-0</td>
			<td>One way valve tube</td>
		</tr>
		<tr>
			<td>Verderflex Yellow Hose Pipe, 6.4 mm ID, Verderprene</td>
			<td>RS Components</td>
			<td>125-4042</td>
			<td>Pump Tubing</td>
		</tr>
	</tbody>
</table>

ex vivo perfusion culture of large blood vessels in a 3d printed bioreactor

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&#39; 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.

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

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. ...

Watch this Scientific Journal Video about EasyFlow Â– An Economical and Adaptable Perfusion Bioreactor for Large Blood Vessel Culture at JoVE.com

EasyFlow Â– An Economical and Adaptable Perfusion Bioreactor for Large Blood Vessel Culture (Video) | JoVE

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 ...