Generation of Three-Dimensional Spheroids/Organoids from Two-Dimensional Cell Cultures Using a Novel Stamp Device

The scope of our research is to establish an efficient and reproducible protocol for generation three-dimensional cell cultures using innovative step-based microwell system in agarose molds. We aim to answer how to create simplified, controlled environment that promote uniform, high-quality 3D cultures for drug testing, gene engineering, and related applications. Recent advances in 3D cell culture systems improve cell interactions and create more in vivo-like environments.

This approach enhances spheroid organoid formation, rendering it a promising tool for biomedical research. Current technologies for 3D cell culture includes hanging drops, rotating cell culture, low attachment plastics, plates with conical wells, microporous scaffolds, magnetic beads, and scaffold-free hydrogels. These methods enable the formation of 3D cell structures, each with its own advantages and limitations.

Our protocol address the challenge of generating uniform 3D spheroids and organoids with consistent size and quality on a large scale. It solves issues of viability and cell density, handling difficulties and instability during the process, proving a cost-effective, reproducible solution for reliable 3D cell cultures research. We are committed to pursuing improvement in research tools, generation of spheroids organoids of insulin-producing beta cells for in vitro analysis and encapsulation in biopolymers, aiming at reversion of Type I diabetes and generation of spheroids organoids from triple-negative human breast cancer cells as a drug screening platform prior to patient treatment.

Design the stamp device using the specified software. Wash the custom-made stamp with a soft bristle sponge and distilled water to remove any residues. Expose the clean stamp to ultraviolet light for five to 10 minutes to ensure sterility.

To prepare 1 to 2%agarose solution, weigh the required amount of pure agarose powder and dissolve it in PBS. Heat the solution in a microwave until the agarose is completely dissolved. Next, pipette approximately three milliliters of the 40 degrees Celsius agarose solution into each well of a six-well plate.

Place the stamp in the center of the well and allow the agarose to solidify for 5 to 10 minutes. After solidification, remove the stamp with gentle back and forth motions to release the vacuum without damaging the microwells. Wash the microwells three times with PBS to remove residues.

Expose the plate to ultraviolet light for 10 minutes to eliminate microbial contamination. Add three milliliters of culture medium to each well of the six well plate and incubate the plate overnight at 37 degrees Celsius in a carbon dioxide incubator. To obtain a homogenous suspension of porcine pancreatic islets, add trypsin to a bi-dimensional cell culture to dissociate the cells.

Then, count the cells and adjust the concentration to achieve the desired number of cells per microwell of a six-well plate. Pipette three milliliters of the cell suspension over the agarose microwells and swirl the plate to ensure even distribution. Allow the cells to settle by gravity for 10 to 15 minutes.

Then, observe the settled cells under the microscope. Incubate the plate at 37 degrees Celsius in a 5%carbon dioxide incubator. Every two to three days, replace 50%of the old or spent medium with fresh medium.

Continue culturing until compact, three-dimensional structures are fully formed. To collect spheroids or organoids, remove the culture medium and using a cut pipette tip, collect the three-dimensional structures via vigorous pipetting. Compact three-dimensional structures were successfully formed by 48 hours of culturing in agarose microwells as observed through time-dependent cell aggregation.

Viable three-dimensional structures were retained upon removal from agarose microwells with green staining indicating live cells within the structures, confirming cell viability and stability.