The overall goal of this procedure is to facilitate the rapid and reproducible pre-cellularization of bioinks for bioprinting applications while preserving cell viability. This method can help streamline and improve the reproducibility when blending a cell suspension with a bioink to ensure the printing of viable cells. The main advantage of this technique is that it eliminates our ability in mixing by blending cells with a bioink in an enclosed system with minimal handling and risk of sample loss.

Visual demonstration of this method is critical, as there is a risk of improper mixing if the assembly is performed incorrectly. To begin, obtain two syringes. One syringe is for the cell suspension while the other syringe is for the bioink.

The mixing ratio utilized in this study is 10 to one. Therefore, use a 12 milliliter syringe and a one milliliter syringe, as required by the 10 to one mixing unit. Also, obtain a sterile passive mixing unit that can be coupled with dual syringes via a luer lock connection and a sterile female-female luer lock connector.

Retrieve a dispensing unit that can extrude a volume from two sterile syringes simultaneously at a controlled rate. Finally, obtain a sterile cartridge to directly mix the bioink and cell suspension into. In this protocol, a nanocellulose alginate based bio ink is utilized, which is crosslinked through the addition of a calcium chloride solution post printing.

To prepare the cells, detach them using a 0.5%trypsin EDTA solution. In this protocol, human fibroblasts are used. Then, count the total number of cells using the trypan blue exclusion method.

Determine what cell density is desired in the final printed construct. Calculate the concentration of the harvested cells that must be diluted to achieve this target final cell density. In this protocol, a final cell density of five million cells per milliliter was utilized.

Transfer the cell suspension into the cell suspension syringe. Also transfer the bioink to the other syringe. Next, pull the bioink syringe plunger back and insert the syringe into the dispensing unit.

Ensure at least half the volume in the pull back syringes is sterile air from within the bio safety cabinet. It is important that the positions of the syringe plungers are maintained during the insertion into the dispensing unit. Position the unit vertically with the luer lock connector upwards.

Then pull the plunger of the cell syringe back to a similar length as the bioink syringe and insert it into the dispensing unit. Make sure there is no accidental discharge of the cell suspension or the bioink by making sure that the mixing unit is attached evenly to the two syringes. Double check before mixing.

Attach both syringes to the mixing unit by twisting the luer lock connectors. Then prime the mixing system by pushing on the dispensing unit to extrude the air in the syringe. Stop the priming prior to the solution reaching the luer lock.

After priming, attach the filling cartridge to the end of the mixing unit via the luer lock connector. Ensure that the plunger in the filling cartridge is at the bottom prior to attachment. Slowly compress the dispensing unit to mix the bio ink and cell suspension together into the cartridge.

Push the plunger in the filling cartridge downward with a sterile pipette tip to contact the bioink cell mixture after mixing. Keep the dispensing unit compressed to ensure the cell bioink mixture is not extruded back into the mixing unit. Cap the cartridge and gently tap on the work surface to move any air bubbles to the top of the cartridge.

At this point, the cell bioink mixture is ready for printing. In this protocol, a square structure with dimensions 4.8 by 4.8 by 0.9 cubic millimeters was exported as a stereolithography file. A g-code file was generated of the lattice structure using the settings found in the text protocol.

Isolate and cryopreserve primary human nasal chondrocytes, or HNCs, from patients, following the referenced protocol. Thaw and expand cryopreserved HNCs and expand once in a monolayer culture using standard culture medium at 37 degrees celsius. Detach the cells at 80%to 90%confluence with a 0.5%trypsin EDTA solution and count using a trypan blue exclusion protocol.

All experiments were conducted using HNCs at passage two. We suspend the HNCs at 100 million cells per milliliter within 300 microliters of culture medium, supplemented with 10%fetal bovine serum, 1%penicillin streptomycin, and 50 micrograms per milliliter of ascorbic acid in preparation for blending with the bioink. Blend the HNC cell suspension into a nanocellulose alginate based bioink, following the passive mixing unit protocol at a 10 to one bioink to cell suspension ratio, to obtain a final cell concentration of nine million cells per milliliter.

Ensure that the bioprinter is sterilized via UV exposure and wipe it down with 70%ethanol. Maintain sterility by placing the bioprinter in the laminar flow cabinet. Then attach sterile printing nozzles to the cartridges containing the bioink cell suspension blends and insert them into the bioprinter.

After calibrating the bioprinter, bioprint the lattice structured cell laden constructs using the 25 gauge conical nozzle at a pressure of 25 kilopascals. Bioprint cell-free constructs as a control. Crosslink the constructs by adding an ionic solution of 100 milimer or calcium chloride.

After five minutes, rinse the constructs. Then incubate the constructs in culture medium under standard culture conditions, changing the media every second or third day. Collect samples for histological analysis at weeks two and four.

Stain the samples for glycosaminoglycans, or GAG production, using an alcian blue stain. Shown here is the cell viability 24 hours after mixing with the passive mixing unit or a manual mixing technique. The passive mixing unit showed better viability, as the extent of mixing was increased.

This is important because longer mixing times may be necessary when preparing large batches of precellularized bioinks. Cell viability at day 14 and day 28 of culture are shown here. Good cell viability is visualized, showing long term cell survival when this technique is used.

Glycosaminoglycans are visualized in bioprinted cartilage constructs, stained using alcian blue, at days zero, day 14, and day 28. These images demonstrate the formation of neocartilage within these bioprinted constructs. Once mastered, this technique can be done in 30 minutes if performed properly.

After watching this video, you should have a good understanding of how to utilize a passive mixing unit system to cellularize bioinks. After its development, this technique paved the way for researchers in the field of bioprinting to rapidly and uniformly cellularize bioinks with a gentle mixing technique.