Name: Author Spotlight: Automated Bioprinting for High-Throughput Vascular Model Fabrication
Uploaded: 2024-08-16T12:30:00
Description: Vascular permeability is a key factor in developing therapies for disorders associated with compromised endothelium, such as endothelial dysfunction in ...

This work was supported by the National Research Foundation of Korea (NRF) grants funded by the Korea government (Ministry of Science and ICT, MSIT) [No. NRF-2019R1C1C1009606; No. 2020R1A5A8018367; and No. RS-2024-00423107]. This research was supported by the Bio and Medical Technology Development Program of the NRF grants funded by the MSIT [No. NRF-2022M3A9E4017151 and No. NRF-2022M3A9E4082654]. This work was supported by the Technology Innovation Program [No. 20015148] and the Alchemist Project [No. 20012378] funded By the Ministry of Trade, Industry and Energy (MOTIE, Korea). This work was also supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through the Agriculture and Food Convergence Technologies Program for Research Manpower development, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) [No. RS-2024-00397026].

1. Generation of G-code for the bioprinter
<ol>
	<li>To generate and visualize the printing path, visit an online G-code simulation tool (e.g., NCviewer).</li>
	<li>Click the New File icon on the interface to create a new G-code file.</li>
	<li>Generate a printing path by manually writing the G-code commands for the sacrificial channel and the silicon chamber. Use the dimensions of a standard six-well plate as a reference for creating the geometry. 
	NOTE: G-code used here is base...

3D Bioprinting for Rapid Vascular Network Fabrication Using Vessels-on-a-Plate

Taking advantage of the precision, automation, and computer-controlled nature of 3D bioprinting technology, we established a streamlined method for fabricating vascular channels in standard six-well plates, which were chosen for their compatibility with commercial microplate readers and microscope imaging setups. The plate&#39;s design can accommodate multi-size channels and a sufficient volume of media for the growth of larger channels while decreasing the necessary frequency of media changes. Future adaptations of this...

The vascular network throughout the human body functions as a crucial transport barrier by dynamically regulating the exchange of molecules and cells between the blood and surrounding tissues. This regulation is essential for preventing tissue edema and enabling selective nutrient and cell exchange, thus supporting tissue metabolism and homeostasis1. Altered endothelial permeability, a factor in many health conditions, affects both disease severity and treatment efficacy2. Vascular endothelium acts as a selective barrier, facilitating the transfer between vessels, tissues, and organs. This regulation involves several mechanisms, such as the basic filtering of solutes and small molecules, intentional disruption of the vascular barrier, and the influence of molecules such as prostaglandins and growth factors on permeability levels3.
Key factors in this regulation include endothelial cell junctions, the migration of leukocytes, and the functionality of the blood-brain barrier4. Given its complexity, the process varies across different environments, involving various blood vessel types and utilizing distinct anatomical pathways. Comprehending the biological underpinnings of vascular permeability is crucial for devising therapeutic approaches to treat conditions associated with abnormal vascular permeability. Maintaining vascular permeability is crucial for the health of the vascular system and surrounding tissues; consequently, impairment of this function leads to endothelial dysfunction, a state in which the endothelium loses its normal functionality.
Endothelial dysfunction is a precursor to several prevalent human diseases, including hypertension, coronary artery disease, diabetes, and cancer5,6,7. This condition can present in several ways, including decreased vasodilation, increased vessel permeability, and a tendency toward a pro-inflammatory state. This pathological state is the earliest stage of several critical cardiovascular issues, such as coronary artery disease, stroke, and peripheral artery disease8, which continue to be the leading causes of mortality in the United States1. Endothelial dysfunction affects cardiovascular health as well as the blood-brain barrier (BBB) and plays a major role in the progression of various neurological disorders. Dysfunction can increase BBB permeability, thus allowing toxins, pathogens, and immune cells to infiltrate into the central nervous system and contributing to neurological disorders such as stroke, Alzheimer&#39;s disease, multiple sclerosis, and brain infections9.
Endothelial dysfunction in diabetes is marked by the compromised ability of the endothelium to regulate vascular tone and produce vasodilator mediators, such as nitric oxide, leading to impaired vasodilation10. This condition is exacerbated by hyperglycemia-induced pathways such as protein kinase C activation and oxidative stress, contributing significantly to the progression of diabetic vascular disease11. Moreover, an inflammatory environment has been found to enhance tumor cell adhesion to brain microvascular endothelial cells while a leaky endothelium has been reported to be a major factor in cancer metastasis12,13. The geometry of blood vessels has been found to directly influence brain cancer metastasis. Tumor cells preferentially attach to areas of greater blood vessel curvature7. This finding underscores the importance of vascular geometry in cancer metastasis. More importantly, in conditions such as fibrosis and cancer, disrupted endothelial barrier function not only plays a role in disease development but also hinders treatment effectiveness by hindering adequate drug delivery14. Research on vascular permeability is crucial for advancing cardiovascular disease treatment and offering insights into managing other diseases involving compromised vascular function.
Given the crucial role of vascular permeability in health and disease, considerable research has focused on examining the selective nature of the endothelial barrier for therapeutic development by using animal models, alongside traditional 2D and 3D in vitro testing platforms. However, animal models have limitations because of species-specific differences and ethical issues, as well as high costs15,16. For instance, Pfizer, in 2004, stated that over the previous 10 years, it had spent over $2 billion on drug developments that showed promising effects in animal models but eventually failed in advanced human testing stages17. Moreover, traditional 2D models do not accurately mimic the three-dimensional (3D) architecture and the complex geometric structure of vascular channels.
With advancements in biofabrication technologies, extensive efforts have been aimed at fabricating vascular channels while recapitulating 3D architecture. Microscale vascular channels can be effectively fabricated within microfluidic chips by using soft lithography, thus offering an advantage of real-time analysis18,19. Alternative methods, such as hydrogel casting or wrapping cell sheets around a mold or mandrel, can be used to create freestanding tubular structures with the desired diameter20,21. However, these methods have limitations; for example, microfluidic chips are restricted to microchannel configurations, and hydrogel casting around a mold does not effectively replicate multiple geometries.
With the emergence of 3D bioprinting technology22, replicating complex geometries by precisely depositing various extracellular matrix (ECM)-based hydrogel materials has become possible23,24. Some bioprinting methods, such as those using concentrically arranged nozzles, e.g., coaxial and triaxial25,26, cannot create bifurcated tubes; however, complex structures can be achieved with sacrificial patterning methods27. None of these bioprinting methods have been demonstrated to enable high-throughput in vitro modeling-a crucial requirement for pharmacological research in drug discovery. Herein, we present a method for efficiently fabricating endothelialized vascular channels with efficient control over dimensions.
We established a straightforward approach using commercially available six-well plates, combined with a sacrificial patterning method in which a bioprinter fabricates vascular channels of desired sizes and patterns within an ECM hydrogel. Human umbilical vein endothelial cells (HUVECs) were seeded to endothelialize these channels and evaluate the functionality of endothelium through a permeability assay. This design enables pumpless perfusion by creating media reservoirs on both sides of the channel and uses gravity-driven flow with the help of a commonly used 2D rocker to mimic the dynamic culture. This approach eliminates the need for peristaltic pumps and facilitates the scalability of this platform for high-throughput applications. The computer-controlled nature of 3D bioprinting technology also streamlines the fabrication process, thus decreasing the likelihood of errors during manufacturing. The VOP model shows promise as a valuable tool for pharmacological testing in drug discovery.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 mL Serological Pipette</td>
			<td>SPL</td>
			<td>SPL 91010</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL syringe&#160;</td>
			<td>Shinchang Medical</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tube</td>
			<td>SPL</td>
			<td>50015</td>
			<td></td>
		</tr>
		<tr>
			<td>3D Bioprinter&#160;</td>
			<td>T&#38;R Biofab</td>
			<td>3DX-Printer</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate&#160;</td>
			<td>SPL</td>
			<td>37206</td>
			<td></td>
		</tr>
		<tr>
			<td>Biological Safety Cabinets</td>
			<td>CHC LAB</td>
			<td>PCHC-777A2-04,&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Brightfield Inverted Microscopes</td>
			<td>Leica</td>
			<td>DMi1</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Counting Kit (CCK8)</td>
			<td>GlpBio</td>
			<td>GK10001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Counting Kit (CCK8)</td>
			<td>GlpBio</td>
			<td>GK10001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Flask 75T</td>
			<td>SPL</td>
			<td>70075</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix, LDEV-free, 10 mL</td>
			<td>Corning</td>
			<td>354230</td>
			<td></td>
		</tr>
		<tr>
			<td>Distilled water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco</td>
			<td>11320033</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO, Cell Culture Grade</td>
			<td>Sigma aldrich</td>
			<td>D2438</td>
			<td></td>
		</tr>
		<tr>
			<td>Dow-Corning, PDMS-Sylgard 184a Kit</td>
			<td>DOW</td>
			<td>DC-184</td>
			<td></td>
		</tr>
		<tr>
			<td>DOWSIL SE 1700 Clear W/C 1.1 KG Kit&#160;</td>
			<td>DOW</td>
			<td>2924404</td>
			<td></td>
		</tr>
		<tr>
			<td>D-PBS - 1x</td>
			<td>Welgene</td>
			<td>LB001-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial Cell Growth Medium MV 2 (Ready to use)</td>
			<td>Promocell</td>
			<td>C-22022</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Micro pipette(1000,200,100,20,10)</td>
			<td>eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl Alcohol 99.9%</td>
			<td>Duksan</td>
			<td>D5</td>
			<td></td>
		</tr>
		<tr>
			<td>Excel</td>
			<td>Microsoft</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fibrinogen from bovine plasma</td>
			<td>Sigma Aldrich</td>
			<td>F8630-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC Dextran 70 kDa</td>
			<td>Sigma Aldrich</td>
			<td>46945-100MG-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent beads (1.0 &#956;m, green)</td>
			<td>Sigma Aldrich</td>
			<td>L1030-1ML</td>
			<td></td>
		</tr>
		<tr>
			<td>GelMA-powder (Gelatin methacrylate) 50 g</td>
			<td>3D Materials&#160;</td>
			<td>20JT29</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco, Recovery Cell Culture Freezing Medium, 50 mL</td>
			<td>Gibco</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HUVECs (Human Umbillical Vein Endothelial Cells)</td>
			<td>Promocell</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ software</td>
			<td>NIH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Thermo SCIENTIFIC</td>
			<td>Forma STERI-CYCLE i160 CO2 Incubator</td>
			<td></td>
		</tr>
		<tr>
			<td>Invitrogen, Live/dead viability/cytotoxicity Kit (for mammalian cells)</td>
			<td>Thermo Fisher</td>
			<td>L3224</td>
			<td></td>
		</tr>
		<tr>
			<td>Lithium Phenyl (2,4,6-trimethylbenzoyl) phosphate powder&#160;</td>
			<td>Tokoyo Chemical Industry CO.</td>
			<td>85073-19-4&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Marienfeld Superior, Counting chamber cover</td>
			<td>Marienfeld Superior</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Marienfeld Superior, Hemocytometer, cell counting chamber</td>
			<td>Marienfeld Superior</td>
			<td>HSU-0650030</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>eppendorf</td>
			<td>Centrifuge 5920 R</td>
			<td></td>
		</tr>
		<tr>
			<td>NCViewer.com</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrogen tank</td>
			<td>WORTHINGTON INDUSTRIES</td>
			<td>LS750</td>
			<td></td>
		</tr>
		<tr>
			<td>Omnicure UV Laser</td>
			<td>EXCELITAS</td>
			<td>SERIES 1500</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm M</td>
			<td>amcor</td>
			<td>PM-996</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Solution (100x)</td>
			<td>GenDEPOT</td>
			<td>CA005-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Planetary Mixer</td>
			<td>THINKY CORPORATION, japan</td>
			<td>ARE-310</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma treatment machine</td>
			<td>FEMTO SCIENCE</td>
			<td>CUTE-1MPR</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F-127</td>
			<td>Sigma aldrich</td>
			<td>P2443-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>Pre-made buffer, (P2007-1) 10x PBS</td>
			<td>Biosesang</td>
			<td>PR4007-100-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagent storage cabinet</td>
			<td>ZIO FILTER TECH</td>
			<td>SC2-30F-1306D1-BC</td>
			<td></td>
		</tr>
		<tr>
			<td>Real time Live cell Imaging Microscope</td>
			<td>Carl ZEISS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerator</td>
			<td>SAMSUNG</td>
			<td>RT50K6035SL</td>
			<td></td>
		</tr>
		<tr>
			<td>ROCKER 2D digital</td>
			<td>IKA</td>
			<td>4003000</td>
			<td></td>
		</tr>
		<tr>
			<td>Scoop-Spatula</td>
			<td>CacheBy</td>
			<td>SL-SCO7001-EA</td>
			<td></td>
		</tr>
		<tr>
			<td>sigma,Trypsin-EDTA solition, 0.25%</td>
			<td>Sigma aldrich</td>
			<td>T4049-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Dodecyl Sulfate (SDS)</td>
			<td>Thermo Fisher scientific</td>
			<td>151-21-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Barrel Tip Cap</td>
			<td>FISNAR</td>
			<td>3051806</td>
			<td></td>
		</tr>
		<tr>
			<td>Tally counter</td>
			<td>Control Company</td>
			<td>&#160;C23-147-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Tapered Nozzle (18 G)</td>
			<td>Mushashi</td>
			<td>TPND-18G-U</td>
			<td></td>
		</tr>
		<tr>
			<td>Tapered Nozzle (22 G)</td>
			<td>Mushashi</td>
			<td>TPND-22G-U</td>
			<td></td>
		</tr>
		<tr>
			<td>Tapered nozzle 20 G</td>
			<td>Musashi</td>
			<td>TPND-20G-U</td>
			<td></td>
		</tr>
		<tr>
			<td>Thrombin from bovine plasma</td>
			<td>Sigma Aldrich</td>
			<td>T7326-1KU</td>
			<td></td>
		</tr>
		<tr>
			<td>Timer, 4-channel</td>
			<td>ETL</td>
			<td>SL.Tim3005</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Solution 0.4%</td>
			<td>Gibco</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin Neutralizing Solution</td>
			<td>Promocell</td>
			<td>C-41120</td>
			<td></td>
		</tr>
		<tr>
			<td>UG 24 mL UG ointment jar</td>
			<td>Yamayu</td>
			<td>No. 3-53</td>
			<td></td>
		</tr>
		<tr>
			<td>UG 58 mL UG ointment jar</td>
			<td>Yamayu</td>
			<td>No. 3-55</td>
			<td></td>
		</tr>
		<tr>
			<td>Water Bath</td>
			<td>DAIHAN Scientific</td>
			<td>WB-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Weight machine</td>
			<td>Sartorius</td>
			<td>bce2241-1skr</td>
			<td></td>
		</tr>
	</tbody>
</table>

high-throughput bioprinting method for modeling vascular permeability in standard six-well plates with size and pattern flexibility

Vascular permeability is a key factor in developing therapies for disorders associated with compromised endothelium, such as endothelial dysfunction in coronary arteries and impaired function of the blood-brain barrier. Existing fabrication techniques do not adequately replicate the geometrical variation in vascular networks in the human body, which substantially influences disease progression; moreover, these techniques often involve multi-step fabrication procedures that hinder the high-throughput production necessary for pharmacological testing. This paper presents a bioprinting protocol for creating multiple vascular tissues with desired patterns and sizes directly on standard six-well plates, overcoming existing resolution and productivity challenges in bioprinting technology. A simplified fabrication approach was established to construct six hollow, perfusable channels within a hydrogel, which were subsequently lined with human umbilical vein endothelial cells to form a functional and mature endothelium. The computer-controlled nature of 3D bioprinting ensures high reproducibility and requires fewer manual fabrication steps than traditional methods. This highlights VOP's potential as an efficient high-throughput platform for modeling vascular permeability and advancing drug discovery.

Author Spotlight: Automated Bioprinting for High-Throughput Vascular Model Fabrication

High-Throughput Bioprinting Method for Modeling Vascular Permeability in Standard Six-well Plates with Size and Pattern Flexibility

Developing the novel therapies targeting vascular permeability, a precursor to prevalent diseases like atherosclerosis requires rigorous preclinical testing. However, complex fabrication methods often hinder the reproducibility and high-throughput production of the in vitro models, which is a prerequisite for pharmacological testing. Here, we harness the automation of multi-material bioprinting technology to address this issue.By integrating the conventional well plate system with computer-controlled 3D printing, we streamlined the fabrication process, simplified the culture process, and enabled the real-time analysis due to its compatibility with microplate readers and microscopic imaging setups. However, existing methods often involve multi-step and manual fabrication processes, which hinder their reproducibility. Our research introduces software-related production of in vitro human blood vessel models with complex geometries.Bioprinting on multi-well plates allows for on-demand, high-throughput production. These advancements suggest that in vitro tissue models will be crucial for future medical discoveries.

The VOP platform, featuring flexibility in size and pattern, was fabricated with a multi-head bioprinting system. Channels, both hollow and capable of perfusion, were seeded with HUVECs to facilitate endothelialization and were subsequently assessed with a permeability assay (Figure 1A). To demonstrate the multiscale manufacturing capability of this method, we printed three distinct configurations: straight, bifurcated, and convoluted (Figure 1B). Through a stra...

We present a protocol for high-throughput production of vascular channels with flexible sizes and desired patterns on a standard six-well plate using 3D bioprinting technology, referred to as vessels-on-a-plate (VOP). This platform has the potential to advance the development of therapeutics for the disorders associated with compromised endothelium.

Vascular permeability is a key factor in developing therapies for disorders associated with compromised endothelium, such as endothelial dysfunction in ...

high-throughput-bioprinting-method-for-modeling-vascular-permeability

Research

JoVE Journal

Bioengineering

Programming and Preparation of a 3D Bioprinter to Produce Vessels-on-a-Plate (VOP) for Cell Culture

To begin with, visit an online G-Code simulation tool such as NCViewer to generate and visualize the printing path. Click the new file icon on the interface to create a new G-Code file. Manually write the G-Code commands for the sacrificial channel and the silicon chamber.Once the G-Code is completed, click the save file icon on the interface to download the file with a nc extension. Combine the silicone polymers SE1700 and PDMS in a 10 to two ratio. Then add the curing agent for each polymer in a 10 to one ratio, polymer to curing agent.With a planetary mixer, thoroughly mix and degas the polymer mixture at 2, 000 RPM. Now use a spatula to transfer the mixed silicone polymer into a 10 milliliter disposable syringe. Centrifuge the loaded syringe at 400 G for three minutes at five degree Celsius to ensure uniform consistency and avoid bubbles during printing.Next, add a weight amount of Pluronic F127 to distilled water and create a 40%stock solution. Mix the Pluronic F127 solution in a planetary mixer for three minutes at 400 G.Keep the homogenized mixture at four degrees Celsius for the complete dissolution of the solid. Before printing, mix 100 microliters of thrombin with 900 microliters of the Pluronic F127 to prepare one milliliter of sacrificial ink.

Fabrication of Vessels-on-a-Plate (VOP) Platform Using a 3D Bioprinter to Facilitate Cell Culture

To begin, prior to fabrication, treat a six-well plate with oxygen plasma at a strength of 100 watts for one minute. Set the temperature of the bioprinter's head to 37 degrees Celsius for the sacrificial ink and five degrees Celsius for the silicone ink. Next, attach a 22-gauge double-screw thread tapered nozzle to the silicone syringe.Load the silicone and sacrificial inks into the print head of the bio-printer. Load the desired G-code and press the SERVO READY function on the bioprinter software interface for three seconds. Position the plate on the stage at the starting point of the G-code and click START to initiate the printing process.Now place a lid over the plate, then cure the silicone chambers in a humidified carbon dioxide incubator at 37 degrees Celsius for 72 hours. The sacrificial pattern on the VOP remained free from desiccation.

Hydrogel Preparation and Channel Embedding of a Vessels-on-a-Plate (VOP) Platform for the Culture of Human Umbilical Vein Endothelial Cells (HUVEC)

To begin dissolve, 0.01 grams of LAP into a 50 milliliter conical tube containing 20 milliliters of PBS. Add three grams of gelatin methacrylate and 0.2 grams of fibrinogen to the LAP solution. Place the mixture in a 37 degree Celsius water bath.Add 300 microliters of the pre warmed gel fib into each hydrogel chamber of the VOP platform to embed the sacrificial pattern. Use UV light to rapidly cross-link the gelatin methacrylate. After repeating the UV cross-linking for all other wells, pipette one milliliter of DPBS into each side of the vascular channel of each well.Place the plate at four degrees Celsius for 15 minutes to liquefy the Pluronic F127. Before introducing cells into the channels profuse DMEMF12 containing 1%matcha gel through the channels for 30 minutes.

Human Umbilical Vein Endothelial Cell (HUVEC) Endothelization of a Vessels-on-a-Plate (VOP) Platform

To begin, submerge a vial of cryopreserved HUVECs in a 37 degree water bath for two minutes. Then rinse the vial with 70%ethanol to prevent contamination. Pipette five milliliters of pre-warmed ECGM into a 15 milliliter conical tube.With a micropipette, carefully transfer the thawed cells from the vial into the conical tube. Then add one milliliter of fresh pre-warmed medium to the cryovial to rinse the inside and transfer any remaining cells into the conical tube. After discarding the supernatant, resuspend the cells in 10 milliliters of fresh medium.Then transfer the cells to a T75 flask. Incubate the flask in a humidified carbon dioxide incubator at 37 degrees Celsius. Rinse the 90%confluent HUVEC cells in a T75 flask with 10 milliliters of DPBS.Then add one milliliter of 0.25%TE to the flask. After incubation, gently tap the sides of the flask and add five milliliters of trypsin neutralizing solution. Then transfer the cells to a 15 milliliter conical tube.Collect the remaining cells with five milliliters of fresh ECGM and transfer them into the conical tube. Now centrifuge the conical tube at 250 x g for three minutes to collect the cell pellet. After discarding the supernatant, resuspend the cell pellet in one milliliter of fresh ECGM and count the cells.After centrifuging again, discard the supernatant and resuspend the cells in 90 microliters of fresh ECGM. Next, briefly vacuum suction the channels of the printed vessels-on-a-plate to clear the lumen. With a micropipette, gently load the channel with 15 microliters of the HUVEC cell suspension.Place the plate flat inside an incubator for two hours. Subsequently, invert the plate to 180 degrees, maintaining it in a flat position for the next two hours. After four hours, wash the channel with DPBS to remove non-adherent and dead cells.Then pipette two milliliters of fresh ECGM into each well. Place the dynamic culture on a rocker at a 10 degree tilt angle and five RPM. The HUVEC cells rapidly attached and proliferated to cover the luminal surface of the vascular channels of the VOP.The maturation of the endothelium was verified by immunostaining for CD31 5 days post cell seeding, which demonstrated the localization of CD31 at the tight junctions.