Design and Validation of a Volumetric-extrusion Bioprinter for Bioprinting of Soluble Basement Membrane Extract for Translational Research

The scope of this research is designing and validating a low-cost volumetric control bioprinting system and developing a specific protocol for bioprinting soluble basement membrane extracts. SBMEs are characterized by unique and complex serology that pose many challenges in their handling, requiring meticulous control during their preparation and severely limiting their use as pure materials. Standard pragmatic-driven bioprinting systems fail to properly control SBME dispensing, often requiring bio-ink mixtures that introduce variability. Volumetric control bioprinting system can address these problems, but are limited at constant solutions. Compared to the techniques, our custom volumetric control bioprinting system offers a local cost solution for bioprinting pure or diluted constructs using SBMEs with good shape fidelity.

[Instructor] To begin, load the STL design file into the slicing software. Locate the piece on a flat surface and move it to coordinates 65, 42.5, 0 millimeters in X, Y, and Z axes. Create two copies by clicking on the STL file name in the object list and selecting multiply selected. Move them to the coordinates: 16, 42.5, 0 millimeters and -33, 42.5, 0 millimeters. Navigate to print settings in the stage menu. In the wall section, set wall line count to zero. Set top and bottom thickness to zero in the top and bottom section. In the infill panel, set infill pattern to grid; infill line distance to five millimeters and infill line directions to zero. Disable retraction in the travel section and then disable print cooling in the cooling section. In the build plate adhesion section, select none as the build plate adhesion type. In the special mode section, select one at a time option as the print sequence. Click on the slice button to generate the G-code. Open the G-code file using Notepad. Remove the lines M104 S200, M105, and M109 S200 in line 12 after the phrase, Generated with Cura_SteamEngine 4.9.0. Add G1 Z25 before the line, ;MESH column file name.STL. Split the next line into two lines. Modify the Z value to Z25 in every line present after TIME_ELAPSED comment for the last layer instruction of each piece. Place the flask containing commercial murine prostate cancer cells under a biosafety cabinet. Using a pipette, remove the cell culture medium from the flask. Wash the cells once with two milliliters of PBS and then aspirate it. Add 500 microliters of trypsin into the flask. Place the flask in an incubator for five minutes to facilitate cell detachment from the plate. Check cells for trypsinization. Next, add 4.5 milliliters of culture medium to the flask. Carefully wash the plate to ensure all detached cells are floating in the medium. Aspirate the medium and transfer it to a 15 milliliter centrifuge tube. Take a 20 microliter aliquot of the suspension and mix it with trypan blue. Pipette 10 microliters of this mixture into a cell counting chamber to count the live cells. Centrifuge the remaining cell suspension at 345 g for five minutes at four degrees Celsius. Remove the supernatant and resuspend the pellet in cold soluble basement membrane extract, or SBME matrix at the desired cell density. Using a cold syringe, aspirate 800 microliters of the matrix suspension containing cells. After removing any trapped air bubbles from the syringe, place the syringe in a sterile box and incubate for 15 minutes to allow gelation. After incubation, insert the syringe into the Bioprinter print head. Ensure the syringe plunger is in contact with the pusher block. Secure the plunger and the syringe using the syringe piston retainer. Use two screws and respective nuts to fix. Use M4 nuts at the bottom part of the extruder to adjust the spring pressure and lock the syringe in place. Calibrate the system by selecting auto home setting in the bio-printer menu to automatically detect the origin of the axes. Place a six well plate on the printing plane. Move the extruder using the move axes command until the needle tip touches the bottom of the well to manually define Z zero. select the G-code file from the menu option in the bio-printer and start printing to deposit a square six layer grid with a 2.6 centimeter side in each well. After printing, use an inverted microscope to ensure that the printing process is successful. Place the multi-well plate in the incubator for two minutes to allow the SBME to settle after extrusion. Finally, pipette two milliliters of cell culture medium into each well. This figure presents the evaluation of printability in serpentine patterns focusing on fiber homogeneity and spreading ratio at varying feed rates. As the feed rate increased from E100 to E200, the spreading ratio also increased, indicating a wider fiber deposition at higher flow rates. When the printing speed was varied from five millimeters per second to 20 millimeters per second, the spreading ratio remained relatively stable across all tested conditions suggesting that printing speed had minimal impact on fiber width. This figure demonstrates the printability evaluation of single layer and multi-layer grids. In the single layer structures, variations in pore size led to noticeable differences in grid uniformity. When printing multi-layer grids, the constructs largely maintained their shaped fidelity. The printed structures mean fiber diameter was consistent with CAD models and single layer constructs. This shows that the bioprinting process maintains reliability across multi-layer structures and varying geometries. This figure evaluates cell viability in 3D bio-printed constructs compared to control samples. Viability remained above 80%, similar to control samples indicating that the printing process had minimal impact. Microscopy images at 10X and 20X magnification showed no significant morphological differences confirming similar cell distribution and morphology in both groups.