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 generate in vitro tissue and organ models that mimic the important functions of their in vivo counterparts, for applications such as drug development, in replacement of the conventional over-simplified planar cell cultures^{11,12,13,14,15,16,17,18,19}. In both situations, the ability to recapitulate the complex microarchitecture and hierarchical structure of the human tissues is critical in enabling functionality of the engineered tissues¹⁰, and in particular, ways to integrate a vascular network into the engineered tissues are of demand since vascularization presents one of the greatest challenges to the field^20,21,22,23.

To date, a variety of approaches have been developed in this regard in an attempt to build blood vessel structures into engineered tissue constructs with various degrees of success⁸. For example, self-assembly of endothelial cells allows for generation of microvascular networks²⁴; delivery of angiogenic growth factors induces sustained neovascularization^25,26; use of vascular progenitor cells and pericytes facilitates endothelial cell growth and assembly^24,27; designing scaffold properties enables precise modulation of vascularization^28,29; and cell sheet technology allows for convenient manipulation of vascular layering³⁰. Nevertheless, these strategies do not endow the capability of controlling the spatial patterning of the vasculature, often leading to random distribution of blood vessels within an engineered tissue construct and thus limited reproducibility. During the past few years bioprinting has emerged as a class of enabling technologies towards the solution of such a challenge, due to their unparalleled versatility of depositing complex tissue patterns at high fidelity and reproducibility in an automated or semi-automated manner^31,32,33. Sacrificial bioprinting^{34,35,36,37,38}, embedded bioprinting^39,40,41, and hollow structure bioprinting/biofabrication^{42,43,44,45,46,47,48,49,50,51,52,53} have all demonstrated the feasibility of generating vascular or vascularized tissues.

Alternatively, a microfluidic bioprinting strategy to fabricate microfibrous scaffolds have been recently developed, where a hybrid bioink composed of alginate and gelatin methacryloyl (GelMA) was delivered through the core of a concentric printhead and a calcium chloride (CaCl₂) solution was carried through the outer sheath flow of the printhead^54,55. The co-extrusion of the two flows allowed for immediate physical crosslinking of the alginate component to enable microfiber formation, while subsequent photocrosslinking ensured permanent stabilization of the multi-layer microfibrous scaffold. Of note, endothelial cells encapsulated within the bioprinted microfibers were found to proliferate and migrate towards the peripheries of the microfibers assuming lumen-like structures that mimicked the vascular bed^54,55. These bioprinted, endothelialized vascular beds could be subsequently populated with desired secondary cell types to further construct vascularized tissues⁵⁵. This protocol thus provides a detailed procedure of such a microfluidic bioprinting strategy enabled by the concentric nozzle design, which ensures convenient fabrication of vascularized tissues for potential applications in both tissue engineering and organoid modeling.

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 (Figure 1A). The alignment is usually adjusted manually, but if necessary spacers of proper sizes can be temporally sandwiched in between the inner/outer needles at both the tip and the barrel sides to assist concentric alignment. Seal the junction of the barrels with epoxy glue and remove the alignment spacers from the tip side when applicable.
2. Insert another needle (23G) in the barrel of the central needle in the reverse direction.  Then generate a hole on the side of the barrel of the outer needle and insert a metal connector of matching size into the hole, followed by sealing with epoxy glue.
3. Connect the inlets of the printhead to a dual-channel syringe pump for injection of the bioink and the crosslinking solution, individually, through two PVC tubes. Mount the extruder onto the head of a bioprinter using a plastic holder made of poly(methyl methacrylate) (PMMA).
   NOTE: Bioprinter selection depends on availability. In our case, we have successfully tested this setup on several commercially available bioprinters. However, any bioprinter that features an x-y-z motorized stage should, in principle, enable integration of this microfluidic printhead.

2. Bioprinting the Microfibrous Vascular Bed

1. Make the bioink using a mixture of alginate (4 w/v%, low viscosity), gelatin methacryloyl (GelMA, 1-2 w/v%)^57,58, and photoinitiator Irgacure 2959 (0.2 - 0.5 wt.%) dissolved in 25 mM 2-[4-(2-hydroxyethyl) piperazin-1-yl] ethane sulfonic acid (HEPES buffer, pH 7.4) containing 10 vol.% fetal bovine serum (FBS).
2. Make a solution of 0.3-M CaCl₂ in HEPES buffer containing 10 vol.% FBS as the crosslinking carrier fluid.
3. Immediately before bioprinting, dissociate human umbilical vein endothelial cells (HUVECs) from the flasks using treatment by 0.05 w/v% trypsin for 5 - 10 min, and resuspend the cells in the bioink at a concentration of 5 - 10 × 10⁶ cells/mL. Pipette the suspension slowly 5 to 10 times to ensure homogenous distribution.
4. Start the injection of the bioink/crosslinking fluid using a dual-channel syringe pump at the same flow rates of 5 µL/mL. The flows can be allowed to continuously run for up to 1 min until they stabilize. Subsequently, initiate the movement of the printhead by controlling the bioprinter at a deposition speed of approximately 4 mm/s (Figure 1B). These speeds may need fine tuning with each new setup to ensure optimal bioprinting. The bioprinting process is usually conducted at room temperature (21 - 25 °C) but this temperature may be altered. The bioprinting process should allow for fast ionic gelation of the alginate component and deposition of a microfibrous scaffold (Figure 1B).
5. After the scaffold is bioprinted, achieve chemical gelation by further photocrosslinking the GelMA component, at approximately 5 - 10 mW/cm² of UV light (360 - 480 nm) for 20 - 30 s (Figure 1C).
6. Following the bioprinting and crosslinking, gently rinse the scaffold with phosphate-buffered saline (PBS) to remove the excess CaCl₂. Culture the HUVECs-laden microfibrous scaffold in endothelial cell growth medium (EGM) in an incubator at 37 °C and 5 vol.% CO₂ for up to 16 days with medium changed at least every 2 days. Monitor the morphologies of the HUVECs under a microscope during the culture period.

3. Constructing the Vascularized Tissues

1. Once the HUVECs have migrated to the peripheries of the microfibers in the scaffold to form the lumen-like structures (Figure 1D), retrieve the scaffold and gently place it on the surface of a hydrophobic surface (e.g., a slab of polymethylsiloxane [PDMS]). Use a piece of sterile filter paper to carefully remove all the medium from the interstitial space of the scaffold with capillary force.
2. Immediately add a drop (approximately 20 - 40 µL) of suspension of a secondary cell type (e.g., cardiomyocytes) in medium at a density of 1 - 10 × 10⁶ cells/mL on top of the scaffold, which should infiltrate the entire interstitial space of the scaffold (Figure 1E). Incubate such a configuration in an incubator (37 °C, 5 vol.% CO₂, 95% relative humidity) for 0.5 - 2 h to allow the cells to adhere onto the individual microfibers. Monitor the droplet size over the period to ensure that no noticeable evaporation is observed.
3. Gently wash the scaffolds by shaking in a PBS bath to remove any non-adherent cells and culture the construct in relevant medium until the desired vascularized tissue is formed.

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