Our protocol allows researchers to probe the mitochondrial energy metabolism and seamless spheroids to allow comparisons to be made between several different cell lines, cell numbers and phenotypes, as well as drug treatments in a of field drug discovery and cancer research. Our work presents a detailed protocol for exploring mitochondrial energy metabolism using 3D spheroids providing the superior physiological model compared to cell monolayer experiments. Biological observations could be extrapolating from a vitreous spheroids to in vivo tumors and identify pathways that may target spheroid tumor metabolism.
For example, carbohydrate utility during spheroids growth. All data seahorse extracellular flux 96 instrument is very sensitive. Positioning in vitro spheroids within the assay plate is paramount to obtaining accurate data at the single spheroid level.
Check spheroid viability using an inverted light microscope with phase contrast at 4x magnification to ensure intact spheroid structure, morphology, and overall uniformity between samples. To hydrate the sensor cartridge, aliquot approximately 20 milliliters of the calibrant into a conical tube. Place the conical tube containing the calibrant in a non-carbon dioxide 37 degrees Celsius incubator overnight.
Take out the contents of the assay kit. Place the sensor cartridge upside down next to the utility plate. Pipette 200 microliters of sterile double distilled water into each well of the utility plate using a multi-channel P300 pipette.
Place the sensor cartridge on top of the utility plate. Check if the water level in each well is high enough to submerge the sensor probes. Transfer the assembled sensor cartridge to a non-carbon dioxide 37 degree Celsius incubator and leave it overnight.
To coat the spheroid assay microplate, add 30 microliters of sterile 0.1 milligrams per milliliter Poly-D-Lysine solution per well of spheroid microplate using a septic technique. Aspirate the solution from each well of the spheroid microplate, invert the plate, and tap it firmly onto tissue paper to remove any residual solution. Wash each well of the plate with 200 microliters of sterile double distilled water.
After the final wash, invert the microplate and tap it firmly onto tissue paper to remove any residual water. Allow the plate to air dry for 30 minutes before future use. Prepare XF RPMI medium as described in the manuscript.
Prewarm the supplemented XF RPMI assay medium to 37 degrees Celsius one hour prior to assay. And prewarm the coated spheroid assay microplate in a non-carbon dioxide 37 degree Celsius incubator. Take out the conical tube containing the calibrant and the sensor cartridge from the air incubator.
Remove the sensor cartridge from the utility plate and place it upside down on the work surface. Using a P300 multi-channel pipette, aspirate the water from the utility plate and discard it. Add 200 microliters of the prewarmed calibrant to each well of the utility plate.
Place the sensor cartridge on the utility plate ensuring the sensors are well submerged in the calibrant. Transfer the assembled sensor cartridge into the non-carbon dioxide, 37 degree Celsius incubator until ready to load the port injection solutions. Take out the spheroid culture plate from the incubator and observe the spheroids under the microscope to ensure their integrity before the spheroid transfer steps.
Load 180 microliters of prewarm assay medium into each well of the spheroid plate including any background correction wells. Partially fill a seven centimeter Petri dish with three milliliters of the assay medium. Transfer the spheroids from the 96 well culture plate into a Petri dish, using a multichannel pipette loaded with wide orifice pipette tips, and volume set to 10 to 50 microliters.
Set the volume of a single channel pipettor fitted with a wide orifice pipette tip to 20 microliters and carefully aspirate a single spheroid. Place the tip directly in the center of each well of this spheroid assay microplate and allow capillary action to withdraw the spheroid from the pipette tip. Confirm the evolution under the microscope by pipetting back the contents of the pipettor into the Petri dish.
Once all this spheroids have been transferred to the spheroid assay microplate, transfer the plate to a non-carbon dioxide 37 degree Celsius incubator for a minimum of one hour. Prepare the working stock concentrations of each compound as described in the manuscript, using a fully supplemented prewarmed XF RPMI assay medium. Dispensed 20 microliters of the working solution of each compound into all corresponding ports using a calibrated P100 multi-channel pipette.
Prepare the analyzer for loading the sensor cartridge. To obtain well-formed compact spheroids, each cell line was optimized individually for seeding density and duration of cultivation. At optimized seeding strategies, spheroid volume for SK-OV-3 was around 5.46 x 10 to the seven micrometers cubed and for A549, it was 1.45 x 10 to eight micrometers cubed.
All spheroid types had a linear correlation between the initial seeding density and the spheroid volume. Perfect spheroid symmetry had a circularity of 1.0 and a deviation from 1.0 indicated a loss of circularity. For all spheroid types, oxygen consumption rate increased with spheroid size and was linearly correlated to spheroid volume with the highest in MCF-7 spheroids and the lowest in SK-OV-3 spheroids.
MCF-7 spheroids did not respond to the vehicle control throughout the experiment. Oxygen consumption rate in MCF-7 spheroids was lowered with oligomycin after five measurement cycles following the first injection of 0.5 micrograms per milliliter. In response to BAM15, the oxygen consumption rate was increased before the second injection.
Whereas the combination of Rotenone plus Antimycin A lowered it. A protocol for accurately probing basal mitochondrial respiration. ADP phosphorylation respiration, Leak respiration, coupling efficiency, maximal respiratory capacity, and spare respiratory capacity was developed using cancer derived 3D spheroids.
All respiratory compounds yielded the expected kinetic oxygen consumption rate profiles for the selected compounds revealing an average steady basal respiration rate of 20 to 30 picomoles of oxygen per minute, per well. Location of spheroids within the microplate and measurement cycle number following compound injection to ensure respiration rates are stable between compound additions. When exploring mitochondrial respiratory capacity with uncouplers, not only is the concentration key but also its ability to uncouple respiration in the presence of oligomycin, which is typically added before uncoupler in experiments probing mitochondrial energy metabolism.
