Large-Scale, Automated Production of Adipose-Derived Stem Cell Spheroids for 3D Bioprinting

Here, we describe the large-scale production of adipose-derived stromal/stem cell (ASC) spheroids using an automated pipetting system to seed the cell suspension, thus ensuring homogeneity of spheroid size and shape. These ASC spheroids can be used as building blocks for 3D bioprinting approaches.

The significance of the protocol relies reducing human errors and microbiology contamination. In addition, responsible to produce thousands of spheroids for 3D bioprinting approaches. The main advantage of this matter, though, is that it's optimized the labor hours.

The software parameters are crucial because they can influence the final quality of the spheroids. And the best advice would be to perform tests only with cell culture media. The visual demonstration of this method is critical because it is an old traditional 3D cell culture method based on Begin by harvesting the stem cell suspension with a 10 milliliter serological pipette and transferring it to a 50 milliliter centrifuge tube.

Then, centrifuge at 400 RCF for 5 minutes to obtain the ASC pellet. Resuspend the cell pellet using DMEM-Low containing 10%FBS. After performing cell count, take ASCs in separate 15 milliliter centrifuge tubes to seed the 81 and 256 recessions micromolded with non-adherent hydrogel.

Refer to the text manuscript for details of the number of cells used. Add 500 microliters of sterile 2%ultra pure agarose solution in the center of a silicone mold containing 81 or 256 circular recesses. After 40 minutes, unmold the ultra pure agarose from the silicone mold and place it in a well of a 12-well plastic plate.

Add two milliliters of DMEM-Low in the well with the micromolded agarose and incubate at 37 degrees Celsius with 5%atmospheric carbon dioxide supply for at least 12 hours before seeding the ASCs. Ensure that the laminar flow is on and the cabinet's airflow is working properly. Confirm that the equipment is connected to the correct voltage and the tablet is connected to the equipment.

Check whether the height of the cabinet protection glass is at the same level as the sensor marking of the equipment. Press the on or off button on the left side of the equipment. Then, wait for the tablet and the equipment to start.

Position the tip boxes, the rack for centrifuge tubes, and the plate containing the micromolded agarose hydrogel in the workspace of the equipment. In the software opened in the tablet, click LabWare Editor. Set up a virtual work table and choose the positions of the pipettes, tip boxes, the rack for the plastic tubes, and the plates.

Click on the switch to procedure"button to include all the parameters and commands carried out by the equipment throughout the experiment. Wait for a toolbar and a procedure list to open in the software. Initially, to indicate the commands, incorporate the number of samples to be pipetted and drag it to the procedure list.

Then, click on the reagent transfer"command button on the software to transfer the ASC suspension to the wells of the 12-well plate containing the micro molds and drag it to the procedure list. Click on properties"to set up the start"and end"positions for the equipment to transfer the sample. Wait for the software to return to the Work Table page.

Set up the parameters for automated seeding of the ASCs as described in the text manuscript. Select water as the standard liquid type. Click on the options"button to set up the parameters to create a homogeneous suspension of the ASCs as described in the text manuscript.

Click on the button with a check"symbol on the top bar of the software to ensure there is no programming error. Click on the play"button on the top bar of the software to start the program. Let the equipment start as programmed.

And wait for it to make a sound indicating it has finished. Collect the 12-well plate then incubate it at 37 degrees Celsius with a 5%atmospheric carbon dioxide supply for at least 18 hours, to allow complete and compact spheroids formation. Manually harvest the spheroids after one, three, and seven days of culture for analysis.

Flush the medium with a micro pipette to release the spheroids from the micromolded non-adherent agarose hydrogel. Collect the spheroids manually using a micro pipette and transfer them to a 15 milliliter centrifuge tube. A total of 85 and 160 spheroids were measured from micromolded non-adhesive hydrogels with 81 and 256 circular recessions, respectively.

The automatic pipette system seeded the ASC cell suspension into 12 wells of a single 12-well plate in 15 minutes. Using the 81 micromolded non-adherent hydrogel, produced 972 spheroids in this study. While the 256 micromolded non-adherent hydrogel, produced 3072 spheroids.

ASC spheroids were analyzed for the homogeneity of their size and shape. ASC steroids from micro molds with 81 recessions showed homogeneous diameter during the culture period. In contrast to ASC steroids from micro molds with 256 recessions, the smallest and largest diameters ratio was close to one in ASC spheroids from micro molds with 81 and 256 recessions.

The viability, morphology and force analyses provided evidence for the successful, large scale production of ASC spheroids. The most case of is to ensure that the problem was solved abruptly without errors.