Semi-Automated Phenotypic Analysis of Functional 3D Spheroid Cell Cultures

This protocol demonstrates how to grow highly reproducible spheroids and shows how proteomic experiments can be conducted to characterize the biological function of 3D structures. Our 3D culture can simultaneously produce hundreds of biological replicates in the same bioreactor, and the LC-MS approach can measure the abundance of thousands of proteins in a single experiment. Demonstrating the procedure will be Stephanie Stransky, a postdoc from the lab.

To begin, take the suspension of HepG2/C3A and THLE-3 cells. Count the number of cells and dilute the cell suspension in complete growth media to obtain 1 million cells in a maximum volume of 1.5 milliliters. Wash the wells of an ultra-low attachment, 24-well round bottom plate with 0.5 milliliters of growth media and centrifuge at 3, 000g for five minutes.

Transfer the cell suspension to the plate and centrifuge it for three minutes at 120g. Then, incubate the plate to initiate spheroid formation. Next, equilibrate the bioreactor 24 hours before transferring the spheroids by filling the humidity chamber with 25 milliliters of sterile water and the cell chamber with nine milliliters of growth media using a 10-milliliter syringe with a long needle.

Then, place the bioreactor in a 3D incubator for 24 hours at 37 degrees Celsius with 5%carbon dioxide. Gently pipette the spheroids up and down using one-milliliter wide bore tips to detach them from the ultra-low attachment plate and transfer them to a tissue culture dish. To capture any remaining spheroids, wash the plate with 0.5 milliliters of prewarmed growth media and transfer them to the culture dish.

Using a light microscope, evaluate the size, compactness, and roundness of the spheroids to select the ones that are adequately formed. Transfer the selected spheroid into the equilibrating bioreactor filled with five milliliters of fresh growth media. Then, fill the entire bioreactor with fresh growth media.

Position the bioreactor in the 3D incubator and adjust the rotation speed using the control unit. Replace 10 milliliters of old growth media with 10 milliliters of fresh media every two to three days. Change the rotation speed every time after replacing the media.

Culture the spheroids for 15 days and divide them between two fresh bioreactors. Open the 3D app installed on the tablet, select the bioreactor, and capture the image. As an alternative, place a black background behind the bioreactor and ensure no light is reflected on the cell chamber.

Capture the image as close to the bioreactor as feasible. Then, collect spheroids by removing five milliliters of media from the bioreactor using a syringe attached to a long needle through the top port. Ensure the spheroids descend to the bioreactor's bottom center.

Now, open the front port and collect the spheroids using a one-milliliter wide bore tip. Transfer these spheroids into micro centrifuge tubes, centrifuge at 500g for five minutes, and dispose of the media. Wash the spheroid by adding 200 microliters of HBSS to remove the FBS.

After this, perform centrifugation at 500g for five minutes and discard the supernatant. Then, immerse the spheroid pellet rapidly in liquid nitrogen to freeze it and store this frozen pellet at minus 80 degrees Celsius till further processing. To lyse the cells, take a tube of collected spheroids and resuspend them in 25 microliters of 5%SDS.

Homogenize the pellet by pipetting up and down. Next, dissolve one microgram of samples and 10 microliters of 0.1%trichloroacetic acid. After extracting the protein and cleaning the samples, set up the MS acquisition method to generate the MS/MS spectra from the identified peptide signals.

Then, transfer the plate into the nanoliter liquid chromatography autosampler. Set the full MS scan to 300 to 1, 100 mass-to-charge ratio in the Orbitrap. Adjust the resolution to 120000 and set the automatic gain control target to 125.

Then, set MS/MS in the Orbitrap with a sequential isolation window of 50 mass-to-charge ratio. Aim the automatic gain control target at 400 and set a higher energy collisional dissociation energy of 30. According to viability analysis of spheroids, the levels of adenylate kinase increase until day 17, resulting in around 7%of cell death.

Afterward, the death rate decreases to below 5%Furthermore, an analysis of the first principal component reveals a clear distinction between spheroid samples and flat cell cultures. The proteomic analysis indicated that THLE-3 and HepG2/C3A spheroids have similar profiles that reflect liver function. Additionally, the spheroids showed overexpression of two isoforms of metallothioneins compared to flat cells.

Functionally grouped networks showed gene ontology enrichment for the carbohydrate metabolic process in HepG2/C3A and THLE-3 spheres. To avoid growth media spilling, do not open or close the front port when the cell chamber is fully filled with liquid. The sample preparation and analysis described in this protocol can be used to explore the proteome of cells culture using 2D and 3D methods, as well as tissue samples.