DOI:

10.3791/55957

⸱

August 11th, 2017

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.

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.

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....

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 (

The microfluidic bioprinting strategy allows for direct extrusion bioprinting of microfibrous scaffolds using low-viscosity bioinks^54,55. As illustrated inFigure 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 al.......

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 .......

The authors declare that they have no competing financial interests.

The authors acknowledge the National Cancer Institute of the National Institutes of Health Pathway to Independence Award (K99CA201603).

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