3D and virtual culture models, like cell spheroids, have been widely used in the fields of gene engineering, delayed modeling, and general screening. Our research focuses on developing an advanced engineering method to fabricate spheroids or other 3D and virtual culture models with high quality for diverse biological studies. In the current protocol, the spheroids grew within the JAMA scaffold and are achieved finally by dissolving the scaffold, causing additional procedures.
We will try to acoustically assemble cell aggregates in the culture medium to enable them to grow within spheroids without scaffold. Compared to magnetic assembly techniques, acoustic waves can directly assemble cells in a labor-free manner without potential toxic effect on cells. Besides, our protocol enables cell spheroids to be assembled in a three-dimensional array, substantially increasing throughput.
To begin, sterilize the chamber of the acoustic device with 75%alcohol for five minutes, then clean the device with sterile PBS and irradiate it with UV light for one hour. Mount the sterile acoustic device onto a microscope stage for top view observation of the chamber's interior. Position a digital microscope on the side of the device that is free from PZT transducers, enabling side view observation of the chamber's interior.
Independently connect the wires from the three PZT transducers in series to three power amplifiers and three output channels of function generators. Program the settings on the function generators for each PZT transducer, specifying parameters such as sinusoidal waveforms, frequency, and amplitude. Culture 3CA cells in DMEM supplemented with 10%FBS and 1%penicillin streptomycin in a T25 cell culture flask.
When the cells become 80%confluent, wash the culture twice with PBS. After removing the PBS, add two milliliters of 0.05%Trypsin-EDTA to the flask and incubate at 37 degrees Celsius for cell detachment. Add two milliliters of complete cell culture medium into the flask to stop the trypsinization.
Transfer the cell suspension to a 15-milliliter tube and centrifuge at 200 G for five minutes. To prepare the bioink solution, mix an appropriate amount of C3A cell suspension with the sterilized GelMA solution. Prestain the C3A cell spheroids with two micromolar cell tracker solution at 37 degrees for 20 minutes.
To wash the cells, first, remove the supernatant by centrifuging and then add fresh cell culture medium. Add at least two milliliters of bioink into the sterilized acoustic device chamber. Turn on the function generator and power amplifier to initiate the actuation of each PZT transducer to generate a 3D array of cell aggregates.
Cross-link the bio ink using blue light for 30 seconds to create 3D hydrogel scaffolds, encapsulating the assembled cell aggregates acoustically. Next, carefully transfer the 3D hydrogel scaffold from the chamber into a Petri dish. Cut it into small pieces using a clean razor, and then add cell culture medium into the Petri dish.
Incubate the C3A cell spheroids at different culture time points in one milliliter of PBS containing one microliter of calcium AM and two microliters of propidium iodide for 15 minutes at 37 degrees Celsius. Rinse the sample embedded in hydrogel scaffolds twice with PBS. For retrieved spheroids, perform centrifugation at 200 G for five minutes and wash twice with PBS.
Using a fluorescence microscope, observe the spheroids and capture the images. The cell aggregates were arranged in the regular 3D dot array pattern with a green fluorescent signal. The assembled C3A GelMA aggregates gradually integrated and formed tight spheroids by day three, accompanied by an increase in spheroid diameter.
The viability of the cell spheroids remained good before day three, but slightly decreased after a week of culture. The released spheroids maintained a good spherical morphology, with a narrow size distribution along with desirable viability and expression of albumin.
