Growing Desmoplastic Three-Dimensional Pancreatic Cancer Spheroids from Co-Culture

3D spheroid cultures are increasingly used to answer mechanistic questions about tumor growth and serve to screen new therapeutics. Therefore, it is critical that these models are clinically representative. We focus on replicating the desmoplasia seen in many cancers, including pancreatic cancer, using this technique.

Spheroid cultures have evolved to incorporate patient-derived cells, complex co-cultures with immune cells, and physiological structures like vasculature. Additionally, microfluidic technology is employed for high-throughput screening and creating organ on-a-chip systems. Multi-omic profiling in spheroids is now utilized to gain mechanistic insights.

The described method can be used to create reproducible 3D co-cultured spheroids with a robust ECM, free of synthetic matrix materials. The advantage is that the ECM closely replicates clinical tumors without the relevant synthetic matrix interfering with therapeutic screening applications. This model will enable the valuation of ECM response to therapeutics and answer important mechanistic questions on the dynamic barrier that the ECM presents in numerous cancers.

We plan to expand this model to include immune cell and macrophage components, in addition to expanding the model used to understand ECM as a barrier to therapeutics and diseases outside of cancer, such as tuberculosis granulomas. To begin, obtain PANC-1 cell culture, and wash the cells with nine milliliters of Hanks'Balanced Salt Solution in three milliliter increments. Incubate the cells with two milliliters of trypsin for about 10 minutes.

To neutralize the suspension, add three milliliters of DMEM supplemented with 10%FBS. After washing the cells seven times with one-milliliter media increments, collect each neutralized aliquot in a 15-milliliter tube. Similarly, obtain trypsinized human pancreatic stellate cells, or HPaSteC, using trypsin neutralization solution and stellate cell media.

Mix the required amount of both the cell suspensions in 11 milliliters of DMEM with 10%FBS, avoiding froth formation. Gently dispense 100 microliters of cell suspension mix into the culture plate, and incubate it at 37 degrees Celsius with 5%carbon dioxide. On day three, add 50 microliters of DMEM with 10%FBS to each well along the side of the well.

On day 10 or 11, aspirate the spent media without disturbing the solution near the halo, and add 100 microliters of fresh media along the side of each well. On day 14 or 17, using a one-milliliter pipette, remove media from each well until the halo is reached. Align the plate against the background of the hood to locate the spheroids at the bottom.

Gently pipette around 50 microliters of media near the spheroid to disturb it for pooling. The spheroid obtained on day 14 were found to grow up to an average volume of 0.048 cubic millimeters.