Microfluidic Co-culture of Renal Healthy and Tumor Epithelium to Model Kidney Cancer Progression

Our research uses a kidney cancer on chip model to investigate the interactions between healthy and cancer kidney cells. This system help us to replicate an in viva condition that enables us to study tumor-driven changes in gene expression, inflammation, and metabolism. It also provides very clear insights into kidney cancer progression and also its effects on surrounding healthy tissues.

The advanced kidney cancer on chip system utilizes many approaches like microfluidics, 3D bioprinting, high resolution imaging, real-time biosensors, RNA sequencing, and CRISPR screening. These technologies enable us to create a really precise simulation of in viva kidney conditions that allow us to study the cellular responses and gene expression changes. Achieving reproducibility in microfluidic experiments, it's very challenging.

Technical issues like precise liquid handling can lead to a variability in results. Additionally, the complex protocols fluctuations in flow rates and environmental conditions further influence consistent outcomes. Our upcoming research will focus on identifying micro RNA signatures associated with the kidney cancer progression and evaluating their potential as disease biomarkers.

To begin, design the required chamber comprising four parallel compartments using computer-assisted design software. The chamber must contain two cell compartments, one matrix compartment, and a connecting canal. For 3D printing the chamber, melt a bioplastic filament, specifically polypropylene, using a heating nozzle and deposit it layer by layer.

After printing, mix 1, 000 microliters of collagen type I with the genipin and sodium hydroxide solution while keeping the mixture on ice. Carefully add the agar and the collagen solution into the respective matrix compartments of the 3D-printed chamber. Place the chamber in a carbon dioxide incubator at 37 degrees Celsius for 60-90 minutes to let the matrix polymerize.

Then add 150 microliters of culture medium on top of the matrix and incubate it overnight. For caki-1 cell embedding, add 25 microliters of cell suspension, followed by 75 microliters of collagen type I into a 1.5 milliliter microcentrifuge tube. Vortex the mixture thoroughly.

Now, carefully add 100 microliters of molten 2%agarose gel to the tube and continue vortexing until the gel becomes homogeneous. Place the chamber in the incubator and maintain conditions at 37 degrees Celsius with 5%carbon dioxide. After one hour, add 150 microliters of cell media on top of the matrix to prevent drying.

On the following day, use a spatula to remove the matrix from the chamber. Place the 3D matrix with cells into a 24-well plate containing 1, 000 microliters of culture medium and incubate at 37 degrees Celsius with 5%carbon dioxide for 24 hours. For injection of RPTEC/TERT1 cells, load 10 microliters of the cell suspension into a micro pipette with a 10 microliter tip.

Carefully inject the cell suspension into the previously prepared solidified collagen matrix through the cell compartment within the chamber. Place the chamber in a 37 degree Celsius 5%carbon dioxide incubator. After 60 minutes, add 150 microliters of cell media on top of the matrix to prevent drying and incubate the cells for 24 hours.

Next, remove the matrix from the chamber using a spatula and place it into a 24-well plate containing 1, 000 microliters of culture medium and incubate at 37 degrees Celsius with 5%carbon dioxide. Now add the gels to the chip. Connect the chip with the 3D cell matrix to the perfusion system using tubing.

Set the system parameters and put the chip into the incubator. After completion of perfusion, remove the gels from the perfusion system and rinse them with PBS. Immunofluorescence staining confirmed the typical morphology of renal tubules as continuous with a tight monolayer located centrally in the gel.

The renal cell carcinoma spheroid appeared rounded and homogeneously sized. Cell viability remained consistent over the culture period without treatments, as shown by stable LDH leakage between days one to five. Increased caspase activity in the presence of immune cells indicated enhanced apoptosis.

Under the influence of the renal cell carcinoma spheroid, renal tubules exhibited an enriched expression of genes associated with the regulation of the immune system. TNF-alpha secretion was elevated in the renal tubules in the co-culture with the renal cell carcinoma spheroids.