The overall goal of this microfluidic bioprinting methodology, is to generate a vascularized tissue construct. This method can solve key questions in biofabrication of vascularized tissues. The main advantage of this technique is that it is versatile in generating a three dimensional shape controlled vascular vat for engineering vascularized tissues through a secondary cell seeding process.
Though this protocol provides insight into engineering vascularized cardiac tissues, it can also be applied to many other tissue types such as liver, skin, and even cancers. General individuals new to this method may struggle because setting up the bioprinter may not be simple. To start this procedure, construct a dual layer concentric microfluidic print head by inserting a smaller blunt needle, serving as the core, into the center of the larger blunt needle, serving as the sheath.
Make sure that the core needle is protruding from the outer shell approximately one millimeter. After that insert a 23 gauge needle in the barrel of the central needle in the reverse direction. Make a hole on the side of the barrel of the outer needle and insert a matching size metal connector.
Seal with epoxy glue. Mount the extruder onto the head of a bioprinter, using a polymethyl methacrylate or PMMA holder. Next for injection of the bioink and the crosslinking solution through two PVC tubes individually, connect the inlets of the print head to a dual channel syringe pump.
Make the bioink using a mixture of alginate, gelMA, and photoinitiator. Dissolved in 25 millimolar HEPES buffer, containing 10%fetal bovine serum or FBS. Then make a solution of 0.3 molar calcium chloride in HEPES buffer containing 10%FBS to serve as the crosslinking carrier fluid.
Right before bioprinting, trypsinize human umbilical vein endothelial cells or HUVECs for five to 10 minutes. Centrifuge the cells at 800 RPM for five minutes in a 15 milliliter tube. Resuspend the cells in the bioink at a concentration of five to 10 times 10 to the six cells per milliliter by slowly pipetting five to 10 times.
Next use a dual channel syringe pump to start the injection of the HUVECs laiden bioink through one, and the crosslinking fluid through the other channel at a flow rate of five microliters per minute. Allow the flows to continuously run for up to one minute until they stabilize. After that start the print head movement by maintaining the bioprinter deposition speed of approximately four millimeters per second.
This bioprinting should result in fast ionic gelation of the alginate component and deposition of a microfiber scaffold. After the scaffold has been printed, photo crosslink the gelMA component with five to 10 milliwatts per square centimeter of UV light for 20 to 30 seconds to accomplish chemical gelation. Then remove the excess calcium chloride from the scaffold by gently rinsing it with 37 degrees Celsius warm PBS.
Culture this scaffold in endothelial cell growth medium at 37 degrees Celsius in five volume percent CO2, for up to 16 days. Change the medium at least every two days. During the culture period, monitor the HUVECs under a microscope until they migrate to the peripheries of the scaffold microfibers and form lumen like structures.
Then carefully remove the entire medium from the intrastitial space of the scaffold with capillary force using a piece of sterile filter paper. Instantly add a drop of suspension of a secondary cell type such as cardiomyocytes on top of the scaffold, allowing the cells to infiltrate the entire intrastitial space. After that incubate this scaffold in an incubator for 30 minutes to two hours, allowing the cells to adhere onto the individual microfibers.
Remove non adherent cells by gently washing the scaffold with PBS. Culture this scaffold in appropriate medium until the desired vascularized tissue is formed. Microfluidic bioprinting described here, allows for direct bioprinting of microfibrous scaffolds using low viscosity bioinks.
A scaffold that is six by six by six square millimeters in size
After microfluidic extrusion of the bioink, ionic crosslinking and photo crosslinking, HUVECs maintained a relatively high viability. The cells proliferated and migrated from the initially random distribution at day zero, to the peripheries of the microfibers at day 16. Neonatal rat cardiomyocytes that were seeded on the scaffold matured and populated the scaffold.
They showed strong expression of functional cardiac biomarkers. Such as sarcomeric alpha actinin, and connexin 43. Confocal microscopy of a bioprinted microfibrous scaffold, populated with cardiomyocytes, revealed coexistence of both HUVECs and cardiomyocytes.
HUVECs are mainly present in the boundaries of the mircofibers, whereas the cardiomyocytes surround the exterior of the microfibers. The cells were able to maintain their spontaneous and synchronized beating for up to nine to 28 days. Depending on the cell source and configuration of the scaffolds.
After watching this video you should have a good understanding of how to produce vascularized tissues using the microfluidic bioprinting technique as well as print head fabrication and bioprinter operations. Visual demonstration of this method is critical as the fabrication of the print head and the operations of the bioprinter may be tricky for people who have not used one before. While attempting this procedure, it is important to remember to make the two needles concentric in the print head and let the flow stabilize prior to starting the bioprinting.
With the development of this technique researchers in the field of tissue engineering and biofabrication now have another enabling tool to generate vascularized tissue constructs for either regeneration purposes in vivo or for modeling tissues in vitro.
