Our protocol is significant because its enables the efficient large-scale production of homogeneous tissue spheroids, which are critical for our advanced tissue engineering, drug development, and disease modeling applications. The major advantage of this technique is its ability to produce large quantities of uniform tissue spheroid quickly and cost effectively, ensures the high cell viability, and the consist spheroid quality, which is crucial our various biomedical applications By producing patient-specific steroids, this technique offers a physiologically accurate model, enabling precise disease modeling, and the understanding of molecular and behavioral mechanisms, including cancer. This method can provide insights into cancer research, neurodegenerative disease, and tissue engineering, and can be applied to drug development and personalized medicine, enhancing our understanding of various biological systems.
Our vision demonstration is critical as it clearly illustrates intricate steps, such as device insertion and cell seeding, helping prevent common errors, and ensuring proper technique is a cushion for consistent results. Begin by adding the agarose powder into one x PBS to prepare 2%agarose gel in a glass container. Homogenize the suspension with circular movements.
Place the glass container in a microwave oven and set the time to 30 seconds. Stop the microwave every five seconds, remove the glass bottle, and manually homogenize the solution with circular movements. Perform the heating process, until the solution reaches a liquid limpid state.
Next, add six milliliters of the agarose solution to each well of a six-well plate and wait for 15 minutes or until the agarose solidifies. Then gently insert the 3D-printed bio device above the liquid agarose and wait for 30 minutes or until the agarose solidifies. Then gently remove the device from the agarose and add two milliliters of DMEM media.
Wait for 10 minutes before discarding the media and replacing it with fresh DMEM. Repeat the wash three times. Once done, add two milliliters of DMEM and place the six-well plate for cell seeding at 37 degrees Celsius in an incubator with 5%carbon dioxide and 80%humidity.
Grow the mouse fibroblast cells in cell culture flasks and maintain them at 37 degrees Celsius in an incubator with 5%carbon dioxide, until 80%confluency is achieved. Then wash the cells with one x PBS and add the dissociation enzyme. Incubate the cells for two to five minutes at 37 degrees Celsius in 5%carbon dioxide and 80%humidity.
Once the cells are detached from the cell culture flask, add a growth medium to neutralize the cell dissociation enzyme. Centrifuge the cell suspension at 400 G for five minutes at room temperature and count the cells. To 50 times 10 to the power of five cells taken in a tube, add five milliliters of one x PBS.
Next, centrifuge the cell suspension at 400 G for five minutes at room temperature. Remove the supernatant using a pipette before adding one milliliter of the cell culture medium and homogenizing the solution. From the six-well plate prepared earlier, remove two milliliters of medium and add one milliliter of the cell suspension to the center of the agarose mold formed by the 3D-printed bio device.
Wait 20 to 30 minutes for the cells to sediment in the micro resections before adding one milliliter of cell culture medium into the well. Place the six-well plate in the incubator at 37 degrees Celsius for approximately 24 to 48 hours for tissue spheroid formation. The 3D-printed stamp-like device composed of cylindrical micro pins was successfully manufactured by the stereolithography method using a photo curable resin.
The device was simple, easy to sterilize, reusable, and tuneable for different sizes of well plates and Petri dishes. It generated 750 homogeneous micro resections well or 4, 716 per six-well plates. The early withdrawal of the device from the plate disrupted the non-adherent mold and deformed the micro resection geometry.
The cells seeded onto the non-adherent agarose molds sedimented and formed the tissue spheroids approximately after 24 hours. This methodology successfully demonstrated the large-scale production of the spheroids by maintaining their shape, size, and viability, and supported the steroid culture for months. The most important thing to remember when I think to this procedure is to carefully insert the device to prevent air bubbles and gently add culture medium to avoid disturbing the cells.
Following this procedure, drug testing and gene expression analysis can be performed. Answering questions about drug efficacy, savory, and genetical response to treatments. This technique will enable new research in tissue engineering and regenerative medicine by allowing large-scale, high-quality spheroid production critical for 3D bio-printing, and enhance on cell quality, and quantity for pharmaceutical, and cosmetic toxicity testing.
