Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

Summary

We provide a generalized protocol based on a microfluidic bioprinting strategy for engineering a microfibrous vascular bed, where a secondary cell type could be further seeded into the interstitial space of this microfibrous structure to generate vascularized tissues and organoids.

Abstract

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic bioprinting to generate a scaffold with multilayer interlacing hydrogel microfibers. To achieve smooth bioprinting, a core-sheath microfluidic printhead containing a composite bioink formulation extruded from the core flow and the crosslinking solution carried by the sheath flow, was designed and fitted onto the bioprinter. By blending gelatin methacryloyl (GelMA) with alginate, a polysaccharide that undergoes instantaneous ionic crosslinking in the presence of select divalent ions, followed by a secondary photocrosslinking of the GelMA component to achieve permanent stabilization, a microfibrous scaffold could be obtained using this bioprinting strategy. Importantly, the endothelial cells encapsulated inside the bioprinted microfibers can form the lumen-like structures resembling the vasculature over the course of culture for 16 days. The endothelialized microfibrous scaffold may be further used as a vascular bed to construct a vascularized tissue through subsequent seeding of the secondary cell type into the interstitial space of the microfibers. Microfluidic bioprinting provides a generalized strategy in convenient engineering of vascularized tissues at high fidelity.

Introduction

Tissue engineering targets to generate functional tissue substitutes that can be used to replace, restore, or augment those injured or diseased in the human body^1,2,3,4, often through a combination of desired cell types, bioactive molecules^5,6, and biomaterials^7,8,9,10. More recently, tissue engineering technologies have also been increasingly adopted to ge....

Protocol

The neonatal rat cardiomyocytes used in this protocol were isolated from 2-day-old Sprague-Dawley rats following a well-established procedure⁵⁶ approved by the Institutional Animal Care and Use Committee at the Brigham and Women's Hospital.

1. Instrumentation of the Bioprinter

1. Insert a smaller blunt needle (e.g., 27G, 1 inch) as the core into the center of a larger blunt needle (e.g., 18G, ½ inch) as the sheath to construct the dual-layer, concentric microfluidic printhead; make sure that the core needle is protruding slightly (~1 mm) longer than the outer shell (

Results

The microfluidic bioprinting strategy allows for direct extrusion bioprinting of microfibrous scaffolds using low-viscosity bioinks^54,55. As illustrated in Figure 2A, a scaffold with a size of 6 × 6 × 6 mm³ containing >30 layers of microfibers could be bioprinted within 10 min. The immediate ionic crosslinking of the alginate component with CaCl₂ allowed for e.......

Discussion

Construction of the co-axial printhead represents a critical step towards successful microfluidic bioprinting to allow for simultaneous delivery of both the bioink from the core and the crosslinking agent from the sheath. While in this protocol an example printhead was created using a 27G needle as the core and an 18G needle as the shell, it may be readily extended to a variety of combinations using different sizes of needles. However, the alteration in the needle sizes, which results in the change in the amount of flow .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Alginic acid sodium salt from brown algae	Sigma-Aldrich	A0682	BioReagent, plant cell culture tested, low viscosity, powder
Gelatin type A from porcine skin	Sigma-Aldrich	G2500	Gel strength 300
Irgacure 2959 (2-Hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone)	Sigma-Aldrich	410896	98%
HEPES buffer	Sigma-Aldrich	H0887	1 M, pH 7.0 - 7.6, sterile-filtered, BioReagent, suitable for cell culture
Fetal bovine serum	Thermo Fisher Scientific	10438026	Qualified, heat-inactivated, USDA-approved regions
Calcium chloride dihydrate	Sigma-Aldrich	C5080	BioXtra, ≥99.0%
Phosphate buffered saline	Thermo Fisher Scientific	10010023	pH 7.4
Human umbilical vein endothelial cells	Angio-Proteomie	cAP-0001	Human Umbilical Vein Endothelial Cells (HUVECs)
GFP-expressing human umbilical vein endothelial cells	Angio-Proteomie	cAP-0001GFP	GFP-Expressing Human Umbilical Vein Endothelial Cells (GFPHUVECs)
Endothelial cell growth medium	Lonza	CC-3162	EGM-2 BulletKit
Dulbecco’s Modified Eagle Medium	Thermo Fisher Scientific	12430054	High glucose, HEPES
Sylgard 184 silicone elastomer kit	Ellsworth Adhesives	184 SIL ELAST KIT 0.5KG	Clear 0.5 kg Kit
UV curing lamp system	Excelitas Technologies	OmniCure S2000	Spot UV Light Curing System with Intelligent UV Sensor