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 (1986) (ASPA) and approved by the Animal Welfare and Ethical Review Board (AWERB) at The Pirbright Institute. The animals were housed in accordance with the Code of Practice for the Housing and Care of Animals Bred. All procedures were conducted by Personal License holders who were trained and competent under Project License PPL70/8852. Piglets were culled according to the schedule one method under the ASPA.
1. Insert manufacturing
<ol>
	<li>Fabricate the insert by 3D printing, using the provided 3D model (Supplementary File 1). 
	NOTE: 3D modeling enables easy changes to the design to adapt to new applications. Alternative materials and alternative manufacturing techniques can also be used to produce the insert. Due to the complex inner structure, selective laser sintering and stereolithography are appropriate alternatives15. Polyamide 12 (PA12; see Table of Materials) is a good material candidate due to its superior performance in terms of liquid retention and resistance to repeated heat sterilization cycles16.</li>
	<li>Fabricate the silicon gaskets and polycarbonate washers by laser cutting17, using the designs provided in Supplementary File 2. 
	&#8203;NOTE: Laser cutting is easily outsourced and a cheap manufacturing method. Washers can be manufactured from stainless steel, providing a more resistant component for repeated use. All other components are commercially available items. A complete list of the required materials is provided in Table 1. The commercial details of the items are included in the Table of Materials.</li>
</ol>
2. Device sterilization, assembly, and priming
<ol>
	<li>Sterilize all components following the instructions in Table 1.</li>
	<li>Under laminar flow conditions, assemble two fabricated inserts (step 1), as shown in Figure 1A.</li>
	<li>Assemble the perfusion system as in Figure 2, following the steps below:
	<ol>
		<li>Attach two connected three-way valves to the reservoir&#39;s R1 port (media exchange port).</li>
		<li>Connect the resulting outlet to the peristaltic pump&#39;s head using a tube equipped with a one-way valve (one-way valve tube).</li>
		<li>Connect the pump head to the reaction chamber&#39;s C4 port using system tubing equipped with a one-way valve. 
		NOTE: This branch can optionally be equipped with a pressure sensor enabling constant monitoring.</li>
		<li>Connect the reaction chamber port C1 to soft wall tubing, and this to the resistance channel (small inner diameter). 
		NOTE: The length of the resistance tubing will greatly influence the pressure present in the system. This should be further investigated to ensure adequate perfusion conditions.</li>
		<li>Extend the resistance channel with the system tubing, creating a return channel and closing the luminal circulation loop by connecting to the reservoir at R5 (Figure 2C).</li>
		<li>Create an overflow channel by connecting the reaction chamber C3 to the reservoir R3 with system tubing.</li>
		<li>Attach a ventilation filter through R2.</li>
		<li>Create a pressure dampener by connecting a syringe containing air and media to the reaction chamber C6. 
		NOTE: The correct air-to-media ratio will depend on the required pressure dampening.</li>
	</ol>
	</li>
	<li>Prime the system with perfusion medium (Dulbecco&#39;s modified eagle medium [DMEM] + 10% [v/v] fetal bovine serum [FBS] + 1% [v/v] penicillin-streptomycin + 1% [v/v] amphotericin B + 30% [v/v] dextran; see Table of Materials) through the media exchange ports and the reservoir. 
	&#8203;NOTE: Priming the system reduces the risk of trapped bubbles in the system and identifies any potential leaks. The recommended volume of media is about 100-120 mL. The volume used will depend on the dead volume of the tubing used for experimentation.</li>
</ol>
3. Sample harvesting and preparation
<ol>
	<li>Collect the left and right common carotid arteries, minimizing direct handling of the arterial tissue13.</li>
	<li>Place the tissue in cold transport media (DMEM+ 20% [v/v] FBS + 2% [v/v] penicillin-streptomycin + 1% [v/v] amphotericin B, see Table of Materials) for transfer.</li>
	<li>In a laminar flow cabinet, remove the excess connective tissue and trim the ends of the tissue using a scalpel blade. Wash the tissue twice in cold transport media.</li>
	<li>Place tissue on an orbital shaker in transport media for at least 30 min for thorough washing.</li>
	<li>Connect a non-branching segment of the artery to the bioreactor system using two barbed luer connectors and fix it in place using a vessel bond (vascular silicone ties; Table 1). 
	NOTE: The vessel bond provides the proper tension and retraction to secure the vessel. The oval section prevents tissue damage.</li>
	<li>Gently flow media through the artery to verify patency.</li>
	<li>After securing the artery to the fabricated insert, fill the reaction space with perfusion media (step 2.4). Finally, gently fill the luminal circulation loop with media to eliminate any remaining air from the system.</li>
	<li>Connect the reaction space with the previously assembled perfusion system (section 2), completing the circulation. 
	&#8203;NOTE: It is recommended to perform a quality check by immunohistochemistry of processed tissue. This identifies any damage due to excessive handling during preparation.</li>
</ol>
4. Perfusion culture and media change
<ol>
	<li>Place the perfusion system in a 37 &#176;C incubator with 5% CO2 and then connect it to a peristaltic pump (see Table of Materials)</li>
	<li>Connect any additional (facultative) acquisition systems, such as pressure sensors (see Table of Materials).</li>
	<li>Leave the system to equilibrate overnight with a low media flow rate of ~10-15 mL/min. 
	NOTE: Initial experiments to determine pump settings, media flow, system pressure, and optimal pressure dampeners/clamps settings are required to ensure appropriate conditions are met.</li>
	<li>The next day, increase the flow incrementally (+1 rpm every hour, equivalent to a ~2.5 mL/min increase every hour, in the current system) until the final flow rate (35 mL/min) is achieved. Monitor the system periodically for leaks. 
	NOTE: To calculate the accurate flowrate based on the peristaltic pump speed, users must first perform adequate pump calibration18. Using the formula (1), volumetric flow rate (Q) can be calculated based on the volume dispensed (V) within a given time (t). To calculate the estimated flow velocity (<img alt="Equation 3" src="/files/ftp_upload/65465/65465eq03.jpg" style="margin: auto;" />) we can use the previously calculated flow rate (Q) and the area of vessel cross-section (A), described in equation (2). 
	<img alt="Equation 1" src="/files/ftp_upload/65465/65465eq01.jpg" style="margin: auto;" />&#160; &#160; (1) 
	<img alt="Equation 2" src="/files/ftp_upload/65465/65465eq02.jpg" style="margin: auto;" />&#160; &#160; (2)</li>
	<li>Every 3 days, exchange 50% of the media (~50 mL) by connecting one syringe filled with fresh media to the media exchange port closer to the pump and an empty syringe to the port closer to the reservoir to collect the spent medium (Figure 2). 
	NOTE: Using two three-way valves as media exchange ports facilitates the continuous operation of the perfusion during the medium exchange.</li>
	<li>At the end of the experiment, harvest the tissue from the reaction chamber by trimming the ends connected to the bioreactor using sterile surgical scissors.</li>
	<li>Dismantle, clean, and sterilize the system for further use.</li>
</ol>
5. Sample analysis
<ol>
	<li>Fix the harvested sample in 4% paraformaldehyde (PFA) overnight at 4 &#176;C.</li>
	<li>Embed the tissue in optimal cutting temperature (OCT; see Table of Materials)19 and freeze by immersion in iso-pentane cooled in liquid nitrogen.</li>
	<li>Obtain cryo-sections of 3-5 mm thickness using a cryostat.</li>
	<li>Perform immunohistochemistry (haematoxylin and eosin [H&#38;E]) and/or immunofluorescence. Follow the typical immunofluorescence protocol:
	<ol>
		<li>Block the slices for 1 h at room temperature with 20% [v/v] goat serum in phosphate-buffered saline (PBS; see Table of Materials).</li>
		<li>Incubate the slices overnight at 4 &#176;C with primary rabbit antibody against CD31 diluted 1:50 in PBS (see Table of Materials).</li>
		<li>Wash three times with PBS for 5 min.</li>
		<li>Incubate for 1 h at 37 &#176;C with fluorescently labeled goat anti-rabbit secondary antibody diluted 1:200 in PBS and &#945;-smooth muscle actin (SMA) directly conjugated fluorescent antibody diluted 1:200 in PBS (see Table of Materials).</li>
		<li>Wash three times with PBS for 5 min.</li>
		<li>Stain the nuclei with DAPI diluted 1:1,000 in PBS for 10 min at room temperature.</li>
		<li>Incubate with 0.1% (w/v) Sudan Black (see Table of Materials) in 70% (v/v) ethanol for 10 min at room temperature to reduce tissue autofluorescence.</li>
		<li>Wash the tissue with abundant deionized water. Mount the slides in the mounting medium.</li>
		<li>Image the samples with a confocal laser scanning microscope.</li>
	</ol>
	</li>
</ol>

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

<ol>
	<li>Davies, P. F., Civelek, M., Fang, Y., Fleming, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+atherosusceptible+endothelium:+Endothelial+phenotypes+in+complex+haemodynamic+shear+stress+regions+in+vivo.">The atherosusceptible endothelium: Endothelial phenotypes in complex haemodynamic shear stress regions in vivo.</a> Cardiovascular Research. 99 (2), 315-327 (2013).</li><li>Gugliandolo, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Palmitoylethanolamide+and+Polydatin+combination+reduces+inflammation+and+oxidative+stress+in+vascular+injury.">Palmitoylethanolamide and Polydatin combination reduces inflammation and oxidative stress in vascular injury.</a> Pharmacological Research. 123, 83-92 (2017).</li><li>Anselmino, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Catheter+ablation+of+atrial+fibrillation+in+patients+with+left+ventricular+systolic+dysfunction:+A+systematic+review+and+meta-analysis.">Catheter ablation of atrial fibrillation in patients with left ventricular systolic dysfunction: A systematic review and meta-analysis.</a> Circulation, Arrhythmia, and Electrophysiology. 7 (6), 1011-1018 (2014).</li><li>Viola, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcutaneous+delivery+of+monoclonal+antibodies:+How+do+we+get+there.">Subcutaneous delivery of monoclonal antibodies: How do we get there.</a> Journal of Controlled Release. 286, 301-314 (2018).</li><li>Kim, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+cellular+models+for+nasal+drug+absorption+studies.">In vitro cellular models for nasal drug absorption studies.</a> Drug Absorption Studies: In Situ, In Vitro and In Silico Models. , 216-234 (2008).</li><li>Lewis, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+experimentation:+Implementation+and+application+of+the+3Rs.">Animal experimentation: Implementation and application of the 3Rs.</a> Emerging Topics in Life Sciences. 3 (6), 675-679 (2019).</li><li>Rouwkema, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+platforms+for+tissue+engineering:+Implications+for+basic+research+and+clinical+translation.">In vitro platforms for tissue engineering: Implications for basic research and clinical translation.</a> Journal of Tissue Engineering and Regenerative Medicine. 5 (8), e164-167 (2011).</li><li>Xu, Y., Shrestha, N., Pr&#233;at, V., Beloqui, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+in+vitro,+ex+vivo+and+in+vivo+models+for+studying+the+transport+of+drugs+across+intestinal+barriers.">An overview of in vitro, ex vivo and in vivo models for studying the transport of drugs across intestinal barriers.</a> Advanced Drug Delivery Reviews. 175, 113795 (2021).</li><li>Vaghela, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vessel+grafts+for+tissue+engineering+revisited-Vessel+segments+show+location-specific+vascularization+patterns+in+ex+vivo+ring+assay.">Vessel grafts for tissue engineering revisited-Vessel segments show location-specific vascularization patterns in ex vivo ring assay.</a> Microcirculation. 29 (2), e12742 (2022).</li><li>H&#229;kansson, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Individualized+tissue-engineered+veins+as+vascular+grafts:+A+proof+of+concept+study+in+pig.">Individualized tissue-engineered veins as vascular grafts: A proof of concept study in pig.</a> Journal of Tissue Engineering and Regenerative Medicine. 15 (10), 818-830 (2021).</li><li>Saucy, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+pulsatile+perfusion+of+human+saphenous+veins+induces+intimal+hyperplasia+and+increased+levels+of+the+plasminogen+activator+inhibitor+1.">Ex vivo pulsatile perfusion of human saphenous veins induces intimal hyperplasia and increased levels of the plasminogen activator inhibitor 1.</a> European Surgical Research. 45 (1), 50-59 (2010).</li><li>Tosun, Z., McFetridge, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variation+in+cardiac+pulse+frequencies+modulates+vSMC+phenotype+switching+during+vascular+remodeling.">Variation in cardiac pulse frequencies modulates vSMC phenotype switching during vascular remodeling.</a> Cardiovascular Engineering and Technology. 6 (1), 59-70 (2015).</li><li>Matos, R. S., Maselli, D., McVey, J. H., Heiss, C., Campagnolo, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+printed+bioreactor+enabling+the+pulsatile+culture+of+native+and+angioplastied+large+arteries.">3D printed bioreactor enabling the pulsatile culture of native and angioplastied large arteries.</a> Frontiers in Cardiovascular Medicine. 9, 864580 (2022).</li><li>Neff, L. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+smooth+muscle+enhances+functionality+of+tissue-engineered+blood+vessels+in+vivo.">Vascular smooth muscle enhances functionality of tissue-engineered blood vessels in vivo.</a> Journal of Vascular Surgery. 53 (2), 426-434 (2011).</li><li>Boparai, K. S., Singh, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+Fused+Deposition+Modeling.+In:+Module.">Advances in Fused Deposition Modeling. In: Module.</a> Refrence in Materials Science and Materials Engineering. , (2017).</li><li>McKeen, L. W., McKeen, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+6+-+Polyamides+(Nylons).">Chapter 6 - Polyamides (Nylons).</a> The Effect of Creep and Other Time Related Factors on Plastics and Elastomers (Second Edition). , 197-262 (2012).</li><li>Moradi, M., Mehrabi, O., Azdast, T., Benyounis, K. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+low+power+CO2+laser+cutting+process+for+injection+molded+polycarbonate).">Enhancement of low power CO2 laser cutting process for injection molded polycarbonate).</a> Optics &amp; Laser Technology. 96, 208-218 (2017).</li><li>Ghasem, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Computer Methods in Chemical Engineering. , (2021).</li><li>Lying, F., Gazi, F., Gardner, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+tissues+and+cells+for+infrared+and+raman+spectroscopy+and+imaging.">Preparation of tissues and cells for infrared and raman spectroscopy and imaging.</a> Biomedical Applications of Synchrotron Infrared Microspectroscopy.RSC Analytical Spectroscopy Monographs. (11), 147-185 (2011).</li><li>Sassi, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+perfusion+bioreactor+for+longitudinal+monitoring+of+bioengineered+liver+constructs.">A perfusion bioreactor for longitudinal monitoring of bioengineered liver constructs.</a> Nanomaterials. 11 (2), 275 (2021).</li><li>Haykal, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double-chamber+rotating+bioreactor+for+dynamic+perfusion+cell+seeding+of+large-segment+tracheal+allografts:+Comparison+to+conventional+static+methods.">Double-chamber rotating bioreactor for dynamic perfusion cell seeding of large-segment tracheal allografts: Comparison to conventional static methods.</a> Tissue Engineering. Part C, Methods. 20 (8), 681-692 (2014).</li><li>Kural, M. H., Dai, G., Niklason, L. E., Gui, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ex+vivo+vessel+injury+model+to+study+remodeling.">An ex vivo vessel injury model to study remodeling.</a> Cell Transplant. 27 (9), 1375-1389 (2018).</li><li>Wong, M. M., Hong, X., Karamariti, E., Hu, Y., Xu, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+grafting+of+tissue-engineered+vessels+in+a+mouse+model.">Generation and grafting of tissue-engineered vessels in a mouse model.</a> Journal of Visualized Experiments. (97), 52565 (2015).</li><li>Alvino, V. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+in+vivo+preclinical+testing+of+pericyte-engineered+grafts+for+the+correction+of+congenital+heart+defects.">In vitro and in vivo preclinical testing of pericyte-engineered grafts for the correction of congenital heart defects.</a> Journal of the American Heart Association. 9 (4), e014214 (2020).</li><li>Nerurkar, N. L., Sen, S., Baker, B. M., Elliott, D. M., Mauck, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+culture+enhances+stem+cell+infiltration+and+modulates+extracellular+matrix+production+on+aligned+electrospun+nanofibrous+scaffolds.">Dynamic culture enhances stem cell infiltration and modulates extracellular matrix production on aligned electrospun nanofibrous scaffolds.</a> Acta Biomaterialia. 7 (2), 485-491 (2011).</li><li>Engebretson, B., Mussett, Z. R., Sikavitsas, V. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+varying+frequency+and+duration+of+mechanical+stimulation+on+a+tissue-engineered+tendon+construct.">The effects of varying frequency and duration of mechanical stimulation on a tissue-engineered tendon construct.</a> Connective Tissue Research. 59 (2), 167-177 (2018).</li><li>Saunders, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+perfusion-driven+cell+seeding+of+small+diameter+engineered+tissue+vascular+grafts+with+a+custom-designed+seed-and-culture+bioreactor.">Evaluation of perfusion-driven cell seeding of small diameter engineered tissue vascular grafts with a custom-designed seed-and-culture bioreactor.</a> PLoS One. 17 (6), e0269499 (2022).</li><li>Stephenson, M., Grayson, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+bioreactors+for+cell-based+therapies.">Recent advances in bioreactors for cell-based therapies.</a> F1000Research. 7, (2018).</li></ol>

The authors do not have any conflicts of interest to disclose.

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 flexibility in the design and can be relatively costly24.
We have developed an alternative system that provides an easy, cheap, and reproducible platform that can be manufactured using open-source 3D printing techniques13. The present article describes the system&#39;s setup to enable reproducible applications by end-users. This setup enables the application of physiological and pathological conditions of pressure (40-180 mmHg), flow rate (6-30 mL/min), and in combination with media mimicking the blood viscosity, with varying degrees of shear stress.
Reproducibility is an essential aspect of the scientific process, as it allows researchers to validate the findings of others and build upon them to progress our understanding of vascular diseases. Furthermore, tools that enable and promote collaborations between groups are essential for advancing scientific knowledge. EasyFlow represents an example of such open-source, accessible solutions that can be easily produced and adopted by labs working on a wide range of projects within the field of vascular sciences and beyond.
We report that this device culture maintains arterial tissue viability for at least 7 days, and can be used to model specific steps of vascular disease. Using this, one could model physiological flow rates and pressure conditions13. Importantly, this perfusion culture is cost-effective due to the low production costs and the low volume of medium necessary to operate the system.
The 3D design can also be adapted to new applications, and new materials for printing can be tested. Even in its current format, the sample lodging space can be easily adapted to samples of different sizes by changing the length of the fittings or the bore of the luer connectors. It is important to note that, given the modular nature of the device and its small dimensions, this bioreactor can be used in several setups (Figure 5) and can be applied to multiplex cultures, where several samples can be exposed to different conditions at the same time in separate bioreactors.
It is envisioned that the use of the system be expanded in the future to support the culture of blood vessels from different origins (e.g., different species) and of different natures (e.g., veins, lymphatic), and perhaps applied to the culture of other hollow tissues (e.g., trachea, intestine). In particular, research shows that culturing tissue-engineered scaffolds in constant perfusion helps the homogenous distribution of cells within the construct and maturation of the resulting tissue25,26. In addition, seeding vascular grafts in perfusion contributes to achieving a more uniformly cellularized vascular lumen, as compared to static methods27. For this reason, we envision the system being applied to tissue engineering to help address the current challenges, allowing for the future development of reproducible synthetic blood vessel substitutes28.
The protocol described here presents some important steps critical to the success of the flow culture. Establishing and monitoring appropriate flow conditions is not trivial and needs to be performed on each system when it is set up for the first time, to ensure that the culture conditions are physiological. The flow and pressure were monitored using pressure sensors and ultrasound imaging. Another critical point is ensuring that the tissue is viable and intact at the beginning of the culture. This requires a fresh source, careful handling and can be verified by histological analysis. Additionally, troubleshooting must be performed at the start of every experiment to identify any potential bacterial contamination or media leakage source.
It is important to highlight that the perfusion system described, while providing physiological pressure and flow conditions, is unable to entirely mimic the complex pressure wave patterns recorded in vivo. This limitation is ascribable to using a peristaltic pump and can be resolved using more specialized equipment to reproduce advanced hemodynamic conditions. The culture of blood vessels in a bioreactor is also unable to address studies where the immune system or the interaction with other organs is critical.
In conclusion, a simple 3D printed perfusion system that can mimic the physiological hemodynamic environment is presented, which is expected to contribute to the standardization of ex vivo blood vessel cultures. Its potential for customization and application to long-term culture makes it an essential tool for advancing the understanding of these complex biological systems in physiology and pathological conditions.

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><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>&amp;nbsp;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&amp;nbsp; from Leuconostoc spp.</td><td>Sigma-Aldrich</td><td>D8802-25ML</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;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 &amp;alpha;-Rabbit Alexa Fluor 488</td><td>Thermo Fisher Scientific</td><td>A11008</td><td></td></tr><tr><td>Invitrogen&amp;nbsp;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 &amp;alpha;-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&amp;deg; Ground Edges, Frosted Area 20mm, 72/box</td><td>Fisher Scientific</td><td>J1800AMNZ</td><td></td></tr><tr><td>&amp;alpha;-Smooth Muscle Actin (SMA) Alexa Fluor&amp;reg; 647-conjugated antibody</td><td>R&amp;amp;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&amp;nbsp; 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&amp;nbsp; 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&amp;nbsp;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&amp;nbsp; 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>&amp;nbsp;10-1003</td><td></td></tr><tr><td>Three-way valves</td><td>IMS Euro</td><td>&amp;nbsp;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&amp;nbsp;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&amp;nbsp;(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.

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. The perfusion system is then connected to a peristaltic pump and optional acquisition systems, such as pressure sensors and ultrasound devices, to monitor culture conditions (Figure 2).
Porcine carotid artery samples were collected and processed before being cultured in perfusion for 7 days. To ensure the quality of the tissue before culture, preliminary experiments were performed where the samples were fixed at the time of excision, after tissue preparation, and after perfusion. Fluorescent staining for endothelial (CD31) and smooth muscle (&#945;SMA) markers was used to assess the maintenance of tissue integrity. Examples of well-preserved and damaged tissues are presented in Figure 3 for comparison. The images show the importance of gentle handling of the tissue at the time of excision, as incorrect processing (excessive stretch, crushing, etc.) may result in loss of the endothelium ahead of the culture. Results also show the importance of the gradual establishment of perfusion to avoid luminal damage.
H&#38;E staining was performed alongside immunofluorescence (IF) staining to show maintenance of the morphology and overall distribution of the cells in the vessel wall after 7 days of culture (Figure 4). The application of physiological culture conditions in the device ensures maintenance of the endothelial coverage of the lumen, alignment of the smooth muscle cells in the media, and preservation of the vasa vasorum in the adventitia.
The compact and modular design of the bioreactor system also allows for a wide range of system setups. The smaller setup comprises a single bioreactor and is ideal for pharmacological and low-volume studies (recirculating perfusion20, total volume of 50-70 mL). To increase the length of culture and reduce the number of media changes, a single reservoir circulation system, like the one described in this protocol, or a constant feeding system is more ideal, as they have a larger volume of medium in circulation. A double circulation21 setup can be established to explore experimental settings where the localization of the stimuli is critical. The current device can also be combined with more sophisticated control systems to enable precise feedback control of parameters such as pH and dissolved oxygen (Figure 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65465/65465fig01.jpg" /> 
Figure 1: EasyFlow assembly schematics.&#160;(A) 3D rendered schematics aiding the assembly of the perfusion insert. (B) Detailed schematics are provided to facilitate assembly of the connection sites. (C) A cross-section view of the reaction space highlights the essential components of the EasyFlow insert and the connection of the tissue to the insert. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65465/65465fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65465/65465fig02.jpg" /> 
Figure 2: Schematic representation of the perfusion system assembly.&#160;(A) Assembled perfusion system containing all important components, highlighting their relative position in a 3D-rendered environment. Not all the components are to scale. (B) Individual isometric views of components are also presented. (C) A top view of the assembled perfusion system is shown to aid the assembly and connection of the different components. The ports have been labeled and numbered counterclockwise to navigate the various connection sites across the perfusion system. This principle has been applied to the reservoir (R) and reaction chamber (C). The various channels connecting the two chambers have also been assigned names for reference. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65465/65465fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65465/65465fig03.jpg" /> 
Figure 3: Tissue maintenance during processing. (A) Confocal images show the normal structure of an artery at the time of harvest, (B) after cleaning and processing, and (C) after perfusion culture, demonstrating the maintenance of a well-preserved morphology. (D) Examples of damaged tissue due to excessive or incorrect handling during processing or (E) due to the application of non-physiological culture conditions (i.e., abrupt initiation of high flow) show depletion of the luminal coverage and disruption of the media. (F) Negative control indicates the specificity of the staining. CD31: green; &#945;SMA: red; DAPI: blue. Scale bars: 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65465/65465fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65465/65465fig04.jpg" /> 
Figure 4: Histological evaluation of tissue before and after perfusion culture.&#160;(A and B) Arterial tissue was evaluated by histology at the time of harvest and (C and D) after 7 days of culture with the bioreactor system. (A and C) H&#38;E staining reveals preservation of the arterial wall structure and organization. (B and D) Immunofluorescence staining of the same tissue evidence endothelial coverage, smooth muscle cell alignment, and vasa vasorum in the adventitia. CD31: green; SMA: red; DAPI: blue. Scale bars: 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65465/65465fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65465/65465fig05.jpg" /> 
Figure 5: Schematic representation of possible perfusion setups. Various alternative setups can be accommodated with the perfusion insert to enable different experimental studies. (A) The recirculating perfusion setup minimizes the volume of medium required. (B) The constant feeding setup provides a steady supply of medium to the tissue. (C) Single reservoir circulation (as described in this paper) provides a larger volume of media for long-term incubations and includes a buffer zone for air exchange and pressure equilibration. (D) The double circulation setup provides two distinct loops feeding the inner (inside the artery) and outer (reaction space) circulations independently. (E) The dynamic perfusion setup includes continuous gasses and pH control by a proportional-integral-derivative (PID) controller. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65465/65465fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Components for fabricating the inserts. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65465/65465_Table 1.xlsx" target="_blank">Please click here to download this Table.</a>
Supplementary File 1:&#160;3D model of the EasyFlow insert for 3D printing manufacture. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65465/Supplementary File 1.stl" target="_blank">Please click here to download this File.</a>
Supplementary File 2: Design schematics of the constructed device, section view, printout, and sealing components. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65465/Supplementary File 2.pdf" target="_blank">Please click here to download this File.</a>

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

Author Spotlight: EasyFlow - An Economical and Adaptable Perfusion Bioreactor for Large Blood Vessel Culture 

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.

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 ...

ex-vivo-perfusion-culture-large-blood-vessels-3d-printed

Research

JoVE Journal

Bioengineering

3.1K Views.  University of Surrey. 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. 

Video: Author Spotlight: EasyFlow - An Economical and Adaptable Perfusion Bioreactor for Large Blood Vessel Culture 

Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological environment, both mechanical and chemical stimulation are required to maintain the proper development and functionality of arterial vessels1,2.
Bioreactor culture systems developed by our group are designed to support vessel regeneration within a precisely controlled chemo-mechanical environment mimicking that of native vessels. Our bioreactor assembly and maintenance procedures are fairly simple and highly repeatable3,4. Smooth muscle cells (SMCs) are seeded onto a tubular polyglycolic acid (PGA) mesh that is threaded over compliant silicone tubing and cultured in the bioreactor with or without pulsatile stimulation for up to 12 weeks. There are four main attributes that distinguish our bioreactor from some predecessors. 1) Unlike other culture systems that simulate only the biochemical surrounding of native blood vessels, our bioreactor also creates a physiological pulsatile environment by applying cyclic radial strain to the vessels in culture. 2) Multiple engineered vessels can be cultured simultaneously under different mechanical conditions within a controlled chemical environment. 3) The bioreactor allows a mono layer of endothelial cells (EC) to be easily coated onto the luminal side of engineered vessels for animal implantation models. 4) Our bioreactor can also culture engineered vessels with different diameter size ranged from 1 mm to 3 mm, saving the effort to tailor each individual bioreactor to fit a specific diameter size. 
The engineered vessels cultured in our bioreactor resemble native blood vessels histologically to some degree. Cells in the vessel walls express mature SMC contractile markers such as smooth muscle myosin heavy chain (SMMHC)3. A substantial amount of collagen is deposited within the extracellular matrix, which is responsible for ultimate mechanical strength of the engineered vessels5. Biochemical analysis also indicates that collagen content of engineered vessels is comparable to that of native arteries6. Importantly, the pulsatile bioreactor has consistently regenerated vessels that exhibit mechanical properties that permit successful implantation experiments in animal models3,7. Additionally, this bioreactor can be further modified to allow real-time assessment and tracking of collagen remodeling over time, non-invasively, using a non-linear optical microscopy (NLOM)8. To conclude, this bioreactor should serve as an excellent platform to study the fundamental mechanisms that regulate the regeneration of functional small-diameter vascular grafts.

Our group has developed a bioreactor culture system that mimics the physiological pulsatile stresses of the cardiovascular system to regenerate implantable small-diameter vascular grafts.

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Enhanced Viability for Ex vivo 3D Hydrogel Cultures of Patient-Derived Xenografts in a Perfused Microfluidic Platform

Patient-derived xenografts (PDX), generated when resected patient tumor tissue is engrafted directly into immunocompromised mice, remain biologically stable, thereby preserving molecular, genetic, and histological features, as well as heterogeneity of the original tumor. However, using these models to perform a multitude of experiments, including drug screening, is prohibitive both in terms of cost and time. Three-dimensional (3D) culture systems are widely viewed as platforms in which cancer cells retain their biological integrity through biochemical interactions, morphology, and architecture. Our team has extensive experience culturing PDX cells in vitro using 3D matrices composed of hyaluronic acid (HA). In order to separate mouse fibroblast stromal cells associated with PDXs, we use rotation culture, where stromal cells adhere to the surface of tissue culture-treated plates while dissociated PDX tumor cells float and self-associate into multicellular clusters. Also floating in the supernatant are single, often dead cells, which present a challenge in collecting viable PDX clusters for downstream encapsulation into hydrogels for 3D cell culture. In order to separate these single cells from live cell clusters, we have employed density step gradient centrifugation. The protocol described here allows for the depletion of non-viable single cells from the healthy population of cell clusters that will be used for further in vitro experimentation. In our studies, we incorporate the 3D cultures in microfluidic plates which allow for media perfusion during culture. After assessing the resultant cultures using a fluorescent image-based viability assay of purified versus non-purified cells, our results show that this additional separation step substantially reduced the number of non-viable cells from our cultures.

This protocol demonstrates methods to enable extended in vitro culture of patient-derived xenografts (PDX). One step enhances overall viability of multicellular cluster cultures in 3D hydrogels, through straightforward removal of non-viable single cells. A secondary step demonstrates best practices for PDX culture in a perfused microfluidic platform.

Patient-derived xenografts (PDX), generated when resected patient tumor tissue is engrafted directly into immunocompromised mice, remain biologically ...

Cancer Research

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage dependent cells on the 2-dimensional surface of tissue culture plastic. More recently, there is a growing body of data demonstrating more in vivo-like behaviors of cells grown in 3-dimensional culture systems. This manuscript describes in detail the set-up and operation of a hollow fiber bioreactor system for the in vivo-like culture of mammalian cells. The hollow fiber bioreactor system delivers media to the cells in a manner akin to the delivery of blood through the capillary networks in vivo. The system is designed to fit onto the shelf of a standard CO2 incubator and is simple enough to be set-up by any competent cell biologist with a good understanding of aseptic technique. The systems utility is demonstrated by culturing the hepatocarcinoma cell line HepG2/C3A for 7 days. Further to this and in line with other published reports on the functionality of cells grown in 3-dimensional culture systems the cells are shown to possess increased albumin production (an important hepatic function) when compared to standard 2-dimensional tissue culture.

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

The functional behavior of cells in culture can be improved by culturing in more in vivo-like 3-dimensional culture environments16-21. This manuscript describes the set-up and operation of a hollow fiber bioreactor system for in vivo-like mammalian tissue culture.

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the spheroid cultures, a perfusable vascular network in the spheroids remains a critical challenge for long-term culture required to maintain and develop their functions, such as protein expressions and morphogenesis. The protocol presents a novel method to integrate a perfusable vascular network within the spheroid in a microfluidic device. To induce a perfusable vascular network in the spheroid, angiogenic sprouts connected to microchannels were guided to the spheroid by utilizing angiogenic factors from human lung fibroblasts cultured in the spheroid. The angiogenic sprouts reached the spheroid, merged with the endothelial cells co-cultured in the spheroid, and formed a continuous vascular network. The vascular network could perfuse the interior of the spheroid without any leakage. The constructed vascular network may be further used as a route for supply of nutrients and removal of waste products, mimicking blood circulation in vivo. The method provides a new platform in spheroid culture toward better recapitulation of living tissues.

The protocol describes how to engineer a perfusable vascular network in a spheroid. The spheroid's surrounding microenvironment is devised to induce angiogenesis and connect the spheroid to the microchannels in a microfluidic device. The method allows the perfusion of the spheroid, which is a long-awaited technique in three-dimensional cultures.

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the ...

Standardized and Scalable Assay to Study Perfused 3D Angiogenic Sprouting of iPSC-derived Endothelial Cells In Vitro

Pre-clinical drug research of vascular diseases requires in vitro models of vasculature that are amendable to high-throughput screening. However, current in vitro screening models that have sufficient throughput only have limited physiological relevance, which hinders the translation of findings from in vitro to in vivo. On the other hand, microfluidic cell culture platforms have shown unparalleled physiological relevancy in vitro, but often lack the required throughput, scalability and standardization. We demonstrate a robust platform to study angiogenesis of endothelial cells derived from human induced pluripotent stem cells (iPSC-ECs) in a physiological relevant cellular microenvironment, including perfusion and gradients. The iPSC-ECs are cultured as 40 perfused 3D microvessels against a patterned collagen-1 scaffold. Upon the application of a gradient of angiogenic factors, important hallmarks of angiogenesis can be studied, including the differentiation into tip- and stalk cell and the formation of perfusable lumen. Perfusion with fluorescent tracer dyes enables the study of permeability during and after anastomosis of the angiogenic sprouts. In conclusion, this method shows the feasibility of iPSC-derived ECs in a standardized and scalable 3D angiogenic assay that combines physiological relevant culture conditions in a platform that has the required robustness and scalability to be integrated within the drug screening infrastructure.

This method describes the culture of iPSC-derived endothelial cells as 40 perfused 3D microvessels in a standardized microfluidic platform. This platform enables the study of gradient-driven angiogenic sprouting in 3D, including anastomosis and stabilization of the angiogenic sprouts in a scalable and high-throughput manner.

Pre-clinical drug research of vascular diseases requires in vitro models of vasculature that are amendable to high-throughput screening. However, current ...

In Vitro 3D Cell-Cultured Arterial Models for Studying Vascular Drug Targeting Under Flow

The use of three-dimensional (3D) models of human arteries, which are designed with the correct dimensions and anatomy, enables the proper modeling of various important processes in the cardiovascular system. Recently, although several biological studies have been performed using such 3D models of human arteries, they have not been applied to study vascular targeting. This paper presents a new method to fabricate real-sized, reconstructed human arterial models using a 3D printing technique, line them with human endothelial cells (ECs), and study particle targeting under physiological flow. These models have the advantage of replicating the physiological size and conditions of blood vessels in the human body using low-cost components. This technique may serve as a new platform for studying and understanding drug targeting in the cardiovascular system and may improve the design of new injectable nanomedicines. Moreover, the presented approach may provide significant tools for the study of targeted delivery of different agents for cardiovascular diseases under patient-specific flow and physiological conditions.

In Vitro 3D Cell-Cultured Arterial Models for Studying Vascular Drug Targeting Under Flow

Here, we present a new protocol to study and map the targeted deposition of drug carriers to endothelial cells in fabricated, real-sized, three-dimensional human artery models under physiological flow. The presented method may serve as a new platform for targeting drug carriers within the vascular system.

The use of three-dimensional (3D) models of human arteries, which are designed with the correct dimensions and anatomy, enables the proper modeling of ...

Fabrication of Engineered Vascular Flaps Using 3D Printing Technologies

Engineering implantable, functional, thick tissues requires designing a hierarchical vascular network. 3D bioprinting is a technology used to create tissues by adding layer upon layer of printable biomaterials, termed bioinks, and cells in an orderly and automatic manner, which allows for creating highly intricate structures that traditional tissue engineering techniques cannot achieve. Thus, 3D bioprinting is an appealing in vitro approach to mimic the native vasculature complex structure, ranging from millimetric vessels to microvascular networks. 
Advances in 3D bioprinting in granular hydrogels enabled the high-resolution extrusion of low-viscosity extracellular matrix-based bioinks. This work presents a combined 3D bioprinting and sacrificial mold-based 3D printing approach for fabricating engineered vascularized tissue flaps. 3D bioprinting of endothelial and support cells using recombinant collagen-methacrylate bioink within a gelatin support bath is utilized for the fabrication of a self-assembled capillary network. This printed microvasculature is assembled around a mesoscale vessel-like porous scaffold, fabricated using a sacrificial 3D printed mold, and is seeded with endothelial cells. 
This assembly induces the endothelium of the mesoscale vessel to anastomose with the surrounding capillary network, establishing a hierarchical vascular network within an engineered tissue flap. The engineered flap is then directly implanted by surgical anastomosis to a rat femoral artery using a cuff technique. The described methods can be expanded for the fabrication of various vascularized tissue flaps for use in reconstruction surgery and vascularization studies.

Engineered flaps require an incorporated functional vascular network. In this protocol, we present a method of fabricating a 3D printed tissue flap containing a hierarchical vascular network and its direct microsurgical anastomoses to rat femoral artery.

Engineering implantable, functional, thick tissues requires designing a hierarchical vascular network. 3D bioprinting is a technology used to create ...

Multi-Stream Perfusion Bioreactor Integrated with Outlet Fractionation for Dynamic Cell Culture

Certain cell and tissue functions operate within the dynamic time scale of minutes to hours that are poorly resolved by conventional culture systems. This work has developed a low-cost perfusion bioreactor system that allows culture medium to be continuously perfused into a cell culture module and fractionated in a downstream module to measure dynamics on this scale. The system is constructed almost entirely from commercially available parts and can be parallelized to conduct independent experiments in conventional multi-well cell culture plates simultaneously. This video article demonstrates how to assemble the base setup, which requires only a single multichannel syringe pump and a modified fraction collector to perfuse up to six cultures in parallel. Useful variants on the modular design are also presented that allow for controlled stimulation dynamics, such as solute pulses or pharmacokinetic-like profiles. Importantly, as solute signals travel through the system, they are distorted due to solute dispersion. Furthermore, a method for measuring the residence time distributions (RTDs) of the components of the perfusion setup with a tracer using MATLAB is described. RTDs are useful to calculate how solute signals are distorted by the flow in the multi-compartment system. This system is highly robust and reproducible, so basic researchers can easily adopt it without the need for specialized fabrication facilities.

This paper presents a method to construct and operate a low-cost, multichannel perfusion cell culture system for measuring the dynamics of secretion and absorption rates of solutes in cellular processes. The system can also expose cells to dynamic stimulus profiles.

Certain cell and tissue functions operate within the dynamic time scale of minutes to hours that are poorly resolved by conventional culture systems. This ...

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

Summary

Explore More Videos

Chapters in this video

Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor

Tissue Engineering of a Human 3D in vitro Tumor Test System

Enhanced Viability for Ex vivo 3D Hydrogel Cultures of Patient-Derived Xenografts in a Perfused Microfluidic Platform

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

Micropatterning and Assembly of 3D Microvessels

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

Standardized and Scalable Assay to Study Perfused 3D Angiogenic Sprouting of iPSC-derived Endothelial Cells In Vitro

In Vitro 3D Cell-Cultured Arterial Models for Studying Vascular Drug Targeting Under Flow

Fabrication of Engineered Vascular Flaps Using 3D Printing Technologies

Multi-Stream Perfusion Bioreactor Integrated with Outlet Fractionation for Dynamic Cell Culture