Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells

This protocol describes a bioprinting methodology using an automated robotic depositing system that incorporates etched topographical guidance cues with the precision deposition of a cell bearing hydrogel bioink. The printed cells are directly delivered to the etched features and are able to sense and orientate with them.

The overall goal of this automated robotic dispensing technique is to create a surface for topographical cell guidance and then deliver cells to these features following a programmed pattern, allowing control over cell behavior and distribution. This method can help answer key questions in the field of biomedical engineering, such as how surface topologies can affect cell behavior, both in monoculture and in co-culture. The main advantage of this technique is that it is less time consuming to program and print cell guidance pattern compared to more established methods.

It also includes cell delivery step for controlled dispensing. We first had the idea for this method when we realized that 2D patterns of cells deposited in hydrogels could benefit from surface guidance. As such, we developed this technique to print hydrogels in a manner that matched the surface features.

This protocol describes the use of back pressure-assisted robotic dispensing system as a surface etching and extrusion-based bioprinter. To prepare a polystyrene surface for the etching and printing, select a one millimeter thick polystyrene sheet. Thicker sheets will bow more.

Then, treat the sheet with oxygen plasma. Set the oxygen gas regulator to two bars. Then, switch on the plasma machine and turn on the vacuum pump.

Proceed to program the machine for 150 watts and 30 standard cubic centimeters per minute of oxygen gas flow, and expose the sheet to these conditions for 10 minutes. Then, evacuate the chamber and seal the door and start the cycle. Next, submerge the treated sheet in pure fetal bovine serum and incubate it at 37 degrees Celsius for two hours with agitation.

After the serum treatment, wash the sheet three times with 1X PBS and sterilize the sheet. After the final wash, leave the sheet in a 37 degree Celsius oven to dry overnight for approximately 12 hours. First prepare a printing stylus for etching.

With great care, insert a 1.5 millimeter diameter textile needle into the nozzle of a dispensing syringe until it becomes wedged and secured. When first attempting to create a bioprinted arrangement, sketch the desired pattern on graph paper with numbered axes to generate the x, y coordinates. Then input the coordinates of the pattern into a spreadsheet.

Next, in the printing software, select Program, Add Program, followed by Edit, Add Point to establish the program. Then, copy the x and y coordinate values from the spreadsheet into the print dispensing software. Before each run, calibrate the z height of the robot to set the stylus contact position.

First, select the Robot option. Then, click on Changing Mode and enable the Teaching Mode option. This enables the JOG function of the robot.

To JOG the robot, first put the robot in its default position by selecting Robot, Meca Initialize. Then, Robot, JOG. Next, in the x and y slots, input the distance in millimeters needed to place the stylus exactly on the origin of the pattern.

Then, also in millimeters, input a numerical value in the z slot to place the stylus or nozzle in contact with the surface without flexing or indenting the surface. This is designated as z's starting value. The depth of each groove can be varied using the z height.

For example, the cut grooves could be 40, 80, or 170 microns deep. It takes concentration and close observation to find a point of contact such that there's no flexing of the stylus or noticeable indent on the surface. To ensure success, we recommend preparing several sheets and running the program at different z height to find the ideal starting position.

The next step is to define the print instruction for each of the coordinate points. Use Start of Line Dispense to define the first point and print initiation. Use Line Passing to designate the intermediate points, and finally, use End of Line Dispense to signal to the robot to terminate the print run.

To communicate the program to the robot, select Robot, Send C&T Data. Then, initiate the run by selecting Robot, Changing Mode, Switch Run Mode and switching the setting to Run. Finally, start the printing.

To make the bioink, dissolve 2%gelatin in Alpha MEM supplemented with 10%FBS and 2%antibiotic antimycotic. Place the medium at 60 degrees Celsius for two hours to allow the gelatin to dissolve in the medium. Culture the cells for the bioink to 70%confluence in 10-centimeter dishes.

Any cells responding to surface guidance features may be used and they should express a fluorescent label so they can be seen during the embedding process. Release the attached cells into the suspension by removing the medium, washing the cells with PBS, and coating the cells with a 1X Trypsin-EDTA solution for five minutes at 37 degrees Celsius. After neutralizing the Trypsin with medium, collect the cells in a suspension and pellet them at 1, 000 g's for five minutes.

Describe the supernatant and re-suspend the cells in 0.5 milliliters of medium. After measuring the cell density, mix the suspension into the cooled bioink solution to make a solution with five million cells per millimeter. Then, pour the cell-bearing bioink into a prepared printing syringe blocked by a lure cap.

Chill the loaded syringe to four degrees Celsius in order to attain a printable viscosity. Then, take the loaded and chilled syringe out, remove the syringe cap, and attach the print nozzle. Then, attach the loaded syringe into the dispensing system and connect it to the air pressure lines.

In order to print the bioink onto the pre-designed pattern, the edges and lines need to be distinct. Precise printing is actually obtained by optimizing the back pressure of the dispenser and print nozzle's needle gauge. Set the dispenser's back pressure to 0.05 megapascals for a 10 milliliter syringe with a 34-gauge tapered needle.

Then, in the software, set the writing speed to five millimeters per second onto a polystyrene film surface. Now, following the programmed pattern, deposit the cellularized bioink onto the pre-etched grooves. After depositing the cells, let the sheet incubate for 20 minutes at room temperature.

Later, cover the printed cell scaffolding in the appropriate growth media and incubate the cells for 24 hours so the cells attach prior to further experimentation. Stem cells seeded by bioprinting within the bioink eventually sedimented to the surface and sensed and elongated along a direction of the discreetly etched lines. Cells seeded in culture medium without bioprinting aligned in the direction of the features, as well.

However, they also covered the entire surface, thus, the bioink constrained the cells to the printed trace. When seeded onto sheets without the etched features, the cells showed no orientation or alignment. After watching this video, you should have a good understanding of how to precisely etch grooves on the polystyrene sheet and then precisely bioprint cells into the grooves.

Once mastered, this technique can be done in approximately two to three hours. It is very useful for researchers in the field of bioengineering to expose cell surface interactions in models where cell anisotropy and positioning is required. Don't forget that the bioink contains live cells and it's very important to use sterile technique when printing the sheets.