Fabrication of a Crystalline Nanocellulose Embedded Agarose Biomaterial Ink for Bone Marrow-Derived Mast Cell Culture

This protocol offers an easy and rapid approach to screen candidate biomaterial inks for potential cytotoxic effects against sensitive primary and immune cells prior to complex 3D bioprinting experiments. After incubation of the immune cells on the biomaterial ink constructs, the cells can be easily retrieved from the constructs by gentle washing to perform downstream cytotoxicity or biomarker analyses. This protocol is directly relevant to tissue engineering in which bioprinted tissues that are destined for transplantation must be constructed from primary cells and biocompatible biomaterial inks.

To begin, prepare a bio ink cartridge for installation onto the INKREDIBLE+3D bioprinter. First, remove the blue end caps from the three milliliter biomaterial ink cartridge and affix a sterile 22 gauge conical bioprinting nozzle to the Luer lock end of bio ink cartridge. Connect the air pressure supply tubing for printhead one or PH1 to the opposite end of the cartridge and insert the cartridge with its attached conical bioprinting nozzle into the vertical slot of PH1.

Next, firmly seat the cartridge in PH1 with a conical bioprinting nozzle extending below the printhead. Tighten the screw on PH1 clockwise until finger tight to lock the bio ink cartridge in place. Note that the 3D bioprinting can be performed with PH2 left empty.

Power on the bioprinter and launch the bioprinter software on a PC connected to the 3D bioprinter via a USB 2.0 cable. In the software, click the connect button to sync it with the 3D bioprinter. Observe three options in the main control menu of the 3D bioprinter.

First, prepare bioprint. Second, utilities menu. And third, status screen.

Select the prepare bioprint option, then scroll down to and select the home axes option. After successful calibration of the XYZ axes, the height of the print bed will lower slightly and the print heads will relocate to above the center point of the print bed. To proceed with the starting point calibration for bioprinting in 24-well plates, remove a sterile 24-well culture plate from its sealed plastic wrapping and mark a dot on the center point of well D1 on the underside of the plate with a permanent marker.

Remove the plate cover and place the 24-well culture plate on the print bed with well D1 located at the front left corner of the print bed. On the main control menu, select utilities menu and then move axis. Move the print heads in one millimeter increments along the X and Y axes until the conical bioprinting nozzle of PH1 is directly over the dot marked under well D1.If necessary, fine-tune the position of the conical bioprinting nozzle over the dot by moving the print heads in 0.1 millimeter increments.

Record the X and Y coordinates of the conical bioprinting nozzle when directly over the center of well D1 as indicated in the control panel screen of the 3D bioprinter and mark down the coordinates. Next, raise the print bed in one millimeter increments until the bottom of well D1 is almost touching the conical bioprinting nozzle installed in printhead one. Fine-tune the movement of the print bed in 0.1 millimeter increments if necessary.

From the utilities menu, select the Z axis calibration option and further select and confirm the store Z calibration option. Return to the main menu and select the prepare bioprint option. Scroll down to and select the calibrate Z option.

Update the 24-well plate geometric code or G-code with the correct starting point coordinates. Open the provided 24-well G-code file on the bioprint software. Update the X and Y coordinates on line one with the values obtained in previous steps and save the file under a new name.

Ensure that the pneumatic pump is tightly connected to the rear air intake port of the INKREDIBLE+3D bioprinter and turn it on. Pull out the forward control knob located on the right side of the INKREDIBLE+3D bioprinter. Ensure that the digital pressure gauges for PH1 and PH2 located on the front of the bioprinter each read close to zero kilopascal.

Slowly rotate the forward control knob clockwise until the pressure indicated on the left gauge for PH1 reaches 12 kilopascals. Place a folded tissue paper or a piece of waterproof sealing film under the print nozzle of the installed cartridge, being careful not to touch the print nozzle. From the main control menu on the 3D bioprinter, select prepare bioprint.

Navigate to and select turn on PH1. Note that the bio ink starts to extrude from the print nozzle. If necessary, increase the extrusion pressure by rotating the control knob clockwise until the bio ink is extruded in a continuous filament and record the new pressure setting.

Select turn off PH1 to stop extrusion of the bio ink. Remove tissue paper or film containing the extruded bio ink from the print bed and close the bioprinter door. To perform 3D bioprinting of rectilinear hydrogel substrates in a 24-well plate format, select the utilities menu from the main control menu, then select disable SD print which will allow the bioprinter software to transmit G-code files to the 3D bioprinter for printing.

Click on the load button in the bioprinter software and select the updated 24-well plate G-code file. In the right-hand control panel in the software, select the print preview tab and click on the print button to commence bioprinting in well D1.Upon completion of bioprinting of the rectilinear constructs, cover the 24-well plate with its lid and move it to a class II biosafety cabinet. Immerse each rectilinear construct in two drops of sterile 50 millimolar calcium chloride solution and incubate at room temperature for five minutes.

Carefully aspirate the calcium chloride solution from each well construct and rinse once in one milliliter of 1X PBS to remove excess calcium chloride. To prevent dehydration of the bioprinted hydrogel constructs, maintain the 3D bioprinted rectilinear constructs in fresh 1X PBS until the bone marrow-derived mast cells are ready to be seeded onto the constructs. Based on the forward and side scatter parameters of the BMMCs analyzed by flow cytometry, it was observed that the hydrogel substrate with the highest CNC concentration did not alter the native size or granularity of the BMMCs as compared to the BMMCs cultured in the absence of the CNC agarose D-mannitol hydrogel substrates.

Flow cytometric analysis of the BMMCs'permeability to propidium iodide demonstrated that none of the CNC concentrations tested elicited any adverse effect on BMMC viability. The CNC agarose substrate increased the expression of the mast cell biomarkers at CNC concentrations greater than or equal to 2.5%However, the expression levels of these receptors remained relatively consistent between 2.5%and 12.5%CNC which suggests an effect that is independent of the concentration of CNC. The XTT assay demonstrated that the metabolic activity of the BMMCs cultured on the 3D bioprinted hydrogel scaffolds remained relatively consistent at about 100%across all tested time points.

Similarly, the LDH release and PI dye exclusion assays revealed no significant changes in viability of the BMMCs. Careful attention to the selection of the printing nozzle gauge, calibration of the 3D bioprinter's axes, and the extrusion pressure setting will ensure reproducible printing of high-quality 3D constructs. The ease with which the mast cells can be retrieved allows them to be probed by a broad range of biological assays to further study any aspect of their cellular functions.