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

Summary

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.

Abstract

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.

Introduction

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 cancer^5,6,7. 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'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 metastasis^12,13. 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 costs^15,16. 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 analysis^18,19. 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 diameter^20,21. 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 possible^23,24. Some bioprinting methods, such as those using concentrically arranged nozzles, e.g., coaxial and triaxial^25,26, 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.

Protocol

1. Generation of G-code for the bioprinter To generate and visualize the printing path, visit an online G-code simulation tool (e.g., NCviewer). Click the New File icon on the interface to create a new G-code file. 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…

Representative Results

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…

Discussion

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'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…

Materials

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