The overall goal of this procedure is to construct a three dimensional human cartilage tissue to test the feasibility of 3D tissue printing, using a modified thermal inkjet printer with simultaneous photo polymerization. This is accomplished by first modifying a commercially available Hewlett Packard desk jet 500 thermal ink jet printer and 5 1 16 2 6, a mono color ink cartridge to a bio printer. The second step is to combine a simple 3D printing stage and a long wavelength UV lamp to provide instant polymerization of injected bio ink during the manufacturing process.
Next, the bio ink is prepared by suspending expanded human articular chondrocytes in 10%polyethylene glycol solution at 5 million cells per milliliter. The final step is to print the cartilage tissue in a layer by layer fashion with simultaneous photo polymerization. Ultimately confocal microscopy and histology.
Staining is used to show the even distribution of printed chondrocytes in a 3D hydrogel and excellent cartilage extracellular matrix production in the homogeneous neo cartilage during the culture. The main advantage of this technology over existing measures like polymerization after manual fabrication or sitting cells after scan for the fabrication is that the printed cells are precisely delivered to the tactile location and stay in position in the matrix. Instead of thinking to the bottom of the scaffold due to gravity, so it forms a homogeneous cartridge.
The printer modification demonstrated in this video is based on a Hewlett Packard desk jet 500 thermal ink jet printer and a 5 1 6 2 6 a black ink cartridge. Start by removing the top plastic cover of the printer and carefully detaching the control panel from the cover. Detach the three cable connections between the printer top portion and base.
Then remove the printer top portion from the base. Next, the small plastic and rubber accessories that comprise the printhead cleaning system are removed from the printer top portion. Remove the base of the paper tray with springs.
The metallic plate covering the plastic paper feeding bar must also be removed. In addition, the plastic paper feeding bar must be cut off at the middle feeding wheel position and the two paper feeding wheels removed. For the purpose of this video, these components have already been removed.
After cleaning the dust and debris using canned air and ethanol wipes, attach the printer top portion to the base prior to use uv. Sterilized the modified printer for at least two hours in a laminar flow hood. To modify the ink cartridge first cut off its cap.
Using a hand saw, empty the ink and remove the filter that covers the bottom well reservoir of the cartridge. Rinse the cartridge thoroughly with running tap water to remove the residual ink ultras. Sonicate the cartridge in deionized water for 10 minutes.
Examine the cartridge to make sure all the ink has been removed. Spray the cartridge thoroughly with 70%ethanol for sterilization, followed by sterilized deionized water. Lastly, set up a long wavelength UV lamp over the printing platform to provide simultaneous photo polymerization capacity.
Measure UV intensity at the printing platform using a UV light meter. Adjust the distance between the UV lamp and the printer platform, so the intensity at the printing subject is between four to eight milliwatts per square centimeter. This distance between the lamp and the printer platform should be approximately 25 centimeters.
The bio ink is prepared from human chondrocytes to expand chondrocytes in a monolayer plate. 5 million human chondrocytes into each T 1 75 tissue culture flask for cell expansion In DMEM supplemented with 10%calf serum and one x penicillin streptomycin glutamine culture cells at 37 degrees Celsius with humidified air containing 5%carbon dioxide. Change the culture medium every three days until the cells are 85%confluent.
Use cells from the same passage. Prepare the polyethylene glycol DL or peda solution. Dissolve peda in PBS to a final concentration of 10%weight per volume.
Add photo initiator I 2 9 5 9 to a final concentration of 0.05%weight per volume filter. Sterilize the solution when the cultured human chondrocytes are 85%confluent. Collect the cells after calculating the cell concentration.
Transfer the volume of cell suspension needed to prepare bio in at 5 million cells per milliliter into a conical tube centrifuge at 1200 RPM for five minutes. To pellet the cells, aspirate the supernatant and suspend the cells in the prepared Pega solution to form the bio ink. To begin this procedure, turn on the printer and laptop using Microsoft Word, create a printing pattern of a solid circle with a diameter of four millimeters.
Place the plastic mold into the printer. Adjust the position of the pattern and make sure it will print exactly into the plastic mold. Calculate the number of prints needed to reach the desired thickness of scaffold.
For a height of four millimeters, 220 prints are required to create the desired scaffold. Load the bio ink into the ink cartridge. Cover the cartridge with aluminum foil to protect from direct uuv exposure.
During printing, send the printing command to the printer. When the printer starts to print, pull the paper sensor in order to continue printing. In the absence of paper passing through the printer, the whole printing process should take less than four minutes for a scaffold of four millimeters in diameter and four millimeters in height.
Transfer printed neo cartilage to a 24 well plate and add 1.5 milliliters of culture medium to each well. To evaluate cell viability, incubate the printed neo cartilage in live dead viability. Cytotoxicity working solution at room temperature for 15 minutes in the dark, cut the cell laden hydrogel in half After acquiring fluorescent images of the cutting area, have a blinded observer count live and dead cells in five randomly taken images to calculate cell viability.
A representative printed neo cartilage tissue with a diameter of four millimeters and a height of four millimeters is shown in this photo based on the properties of the modified thermal inkjet printer. For a construct of this size, the volume and thickness of each printed layer during layer by layer construction were 0.23 microliters and 18 microns respectively. The entire printing process to construct the neo cartilage tissue took less than four minutes.
These images are of chondrocytes labeled with green and orange fluorescent dyes. Panel A shows an even cell distribution of printed chondrocytes in the 3D scaffold. Due to simultaneous photo polymerization of the surrounding scaffold during cell deposition, the printing and photo polymerization process was completed in four minutes with a cell viability of 90%By contrast, panel B shows that without simultaneous photo polymerization, instead of remaining at their initially deposited locations, the cell sank to the bottom horizonal interface due to gravity.
It took 10 minutes of UV exposure to gel, a construct of the same size as an A with a cell viability of 63%Saffron and o staining of printed chondrocytes in the PEG hydrogel shows gradually increased prot glycan production during the culture indicating of the congenic phenotype of printed chondrocytes in 3D and the formation of a homogeneous neo cartilage. After watching this video, you should have a good understanding of how to utilize a modified thermal ink jet printer to create three dimensional calculated tissue with even cell distribution in a 3D scaffold.
