The overall goal of this methodology is to co-culture three dimensional tumor strumous spheroids from pancreatic cancer cells and fibroblasts for their metabolic assessment, using an extra-cellular flux analyser. This method can help answer questions about the effects of the tumor myco on cell metabolism and the drug sensitivity. The main advantages of these techniques are that it is fast, relatively easy and highly reproducible, and that it can be easily adapted to other cases of cell aligns.
Demonstrating the procedure will be, Pawan Noel and Ruben Munoz. Using standard aseptic tissue culture technique, culture the cancer cells and fibroblasts of interest in T75 flasks in the appropriate growth medium. When the cells reach 70%to 80%confluency, wash the cultures one time, in five milliliters of PBS and detach the cells from the flask surfaces, with two milliliters of 0.05%trypsin EDTA per flask for five minutes at 37 degrees celsius.
After confirming detachment under a microscope, deactivate the trypsin with eight milliliters of growth medium per flask, and pipette the cells a few times, to generate single cell suspensions for counting. Collect the cells by centrifugation and re-suspend the pellets in 1 x 10 to the sixth cells per milliliter concentrations in fresh growth medium. To magnetize the cells, add 10 microliters of nanoshuttle in growth medium, pe 100 microliters of cells and agitate the solutions gently.
Gently invert the tubes a few times and transfer the cell nanoshuttle mixes into individual wells of a 24 well plate. Incubate the cells at room temperature for two hours with gentle shaking, to facilitate the nanoshuttle binding to the cell surface. At the end of the binding incubation, gently mix each magnetized cell suspension with pipetting and adjust the concentration to 1.5 x 10 to the fourth magnetized cancer cells or fibroblasts per 150 microliters of medium.
Mix the fibroblasts and cancer cells in a two to one ratio, for a total of 150 microliters of seeded cells per well. Place the cell repellent 96 well plate on top of the 96 well magnetic spheroid drive with thinner magnets and seed 150 microliters of cell mixture into each well. When all of the cells have been plated, incubate the plate overnight at 37 degrees celsius and 5%CO2, with the magnetic spheroid drive still attached.
The next morning, remove the magnetic drive and return the cells to the incubator for up to seven more days. The day before the metabolic assay, hydrate the probe cartridge with 200 microliters of assay caliber per well in the provided utility plate and incubate the utility plate overnight, in a humidified 37 degree celsius non-CO2 incubator. The next morning, observe the spheroids in the growth plate under a light microscope, to check their morphology and overall uniformity.
Transfer the growth plate onto a magnetic holding drive and carefully aspirate approximately, 120 microliters of growth medium per well. Gently wash the spheroids with 120 microliters of warmed assay medium three times. After the last wash, inspect the spheroids under the microscope again to confirm that the spheroids were not washed away and add 180 microliters of warmed assay medium to each well.
Use a wide bore tip to carefully aspirate a single pre-washed spheroid from the cell repellent growth plate and gently transfer the spheroid directly into the center of one well of a spheroid assay plate, allowing the spheroid to fall into the central micro chamber of the well by gravity. When all of the spheroids have been transferred, place the assay plate in the non-CO2 37 degree celsius humidified air incubator for one hour. During the incubation, orient the sensor cartridge so that rows A through H are on the left side and place a loading guide on top of the cartridge, such that the letter corresponding to the port to be loaded, is in the upper left hand corner.
If the plate has been evenly loaded, open the analysis software and click templates. Double click on blank template and click generate groups. Under the plate map tab, assign groups to the assay plate, corresponding to the experimental design and select the four corner wells as the background wells.
Under the instrument protocol tab, check calibrate, equilibrate and baseline measurement cycle. Click injections and define the compound for each port. Change the measurement cycles to six, review the protocol and group summary, then, save the assay design template.
Based on the assay design as a guide, use a multi-channel pipette to dispense each reagent directly into the injection ports, holding the loading guides in place with finger tips. When all the reagents have been loaded, remove the loading guide and hold the cartridge at eye level to visually inspect the injection ports for even loading. Transfer the cartridge at the utility plate to the extracellular flux analyser and initiate the pre-assay calibration.
Once calibrated, replace the utility plate with the pre-warmed assay plate containing the 3D spheroids and begin the assay. When the assay is finished, export the data into the appropriate data analysis software. In a typical experiment, the diameter of the spheroids increases from around 400 micrometers on day two to almost 600 micrometers on day seven, post printing, with no significant increase observed after day seven.
All three types of spheroid cultures exhibit a biologically functional response to the glycolysis stress assay in response to an injection of a saturated concentration of glucose as evidenced by an increase in their extracellular assertification rates. In general, a higher extracellular assertification rate signal from tumor cell derived spheroids, compared to the signal from relatively smaller PS1 fibroblast derived spheroids, maybe attributed to the inherent metabolic inclination towards glycolysis of the cancer cells. In this representative mitochondria stress test of tumor fibroblast spheroids, a decrease in mitochondria respiration was observed in response to an inhibitor of ATP synthase, correlating to the fraction of mitochondria respiration used for cellular ATP production.
A second injection with an uncoupling agent, stimulated the maximal oxygen consumption of the spheroids with the corresponding increase in mitochondria respiration. A third injection of electron transport chain complex one and three inhibitors, blocked mitochondria respiration observed as a sharp decrease in the oxygen consumption rate. These parameters and basal respiration rates, can be used to calculate the proton leak and spare respiratory capacities of the spheroids, providing a multi parametric metabolic analysis of the magnetized spheroids.
Once mastered, this technique can be completed in three to four hours, if it is planned well. While attempting this procedure, it's important to remember to hydrate the probe in a 37 degree incubator four to 16 hours before performing the metabolic assay. Following the spheroid culturing procedure, other assays like dose response experiments can be performed to answer additional questions about drug efficacy, toxicity, penetrants and susceptibility.
After its development, this technique has paved the way for researchers in the field of pancreatic cancer to explore the tumor stroma interactions within the tumor microenvironment in a three dimensional model that mimics the in-vivo tumor biology and its characteristics. After watching this video, you should have a good understanding of how to culture three dimensional spheroids, starting from cultured pancreatic tumor and fibroblast cells to downstream analysis of cellular metabolic functions.
