Exploring Mitochondrial Energy Metabolism of Single 3D Microtissue Spheroids Using Extracellular Flux Analysis

Summary

These protocols will help users probe mitochondrial energy metabolism in 3D cancer cell-line-derived spheroids using Seahorse extracellular flux analysis.

Abstract

Three-dimensional (3D) cellular aggregates, termed spheroids, have become the forefront of in vitro cell culture in recent years. In contrast to culturing cells as two-dimensional, single-cell monolayers (2D culture), spheroid cell culture promotes, regulates, and supports physiological cellular architecture and characteristics that exist in vivo, including the expression of extracellular matrix proteins, cell signaling, gene expression, protein production, differentiation, and proliferation. The importance of 3D culture has been recognized in many research fields, including oncology, diabetes, stem cell biology, and tissue engineering. Over the last decade, improved methods have been developed to produce spheroids and assess their metabolic function and fate.

Extracellular flux (XF) analyzers have been used to explore mitochondrial function in 3D microtissues such as spheroids using either an XF24 islet capture plate or an XFe96 spheroid microplate. However, distinct protocols and the optimization of probing mitochondrial energy metabolism in spheroids using XF technology have not been described in detail. This paper provides detailed protocols for probing mitochondrial energy metabolism in single 3D spheroids using spheroid microplates with the XFe96 XF analyzer. Using different cancer cell lines, XF technology is demonstrated to be capable of distinguishing between cellular respiration in 3D spheroids of not only different sizes but also different volumes, cell numbers, DNA content and type.

The optimal mitochondrial effector compound concentrations of oligomycin, BAM15, rotenone, and antimycin A are used to probe specific parameters of mitochondrial energy metabolism in 3D spheroids. This paper also discusses methods to normalize data obtained from spheroids and addresses many considerations that should be considered when exploring spheroid metabolism using XF technology. This protocol will help drive research in advanced in vitro spheroid models.

Introduction

Advances in in vitro models in biological research have rapidly progressed over the last 20 years. Such models now include organ-on-a-chip modalities, organoids, and 3D microtissue spheroids, all of which have become a common focus to improve the translation between in vitro and in vivo studies. The use of advanced in vitro models, particularly spheroids, spans several research fields, including tissue engineering, stem cell research, cancer, and disease biology^{1,2,3,4,5,6,7}, and safety testing, including genetic toxicology^8,9,10, nanomaterials toxicology^11,12,13,14, and drug safety and efficacy testing^{8,15,16,17,18,19}.

Normal cell morphology is critical to biological phenotype and activity. Culturing cells into 3D microtissue spheroids allows cells to adopt a morphology, phenotypic function, and architecture, more akin to that observed in vivo but difficult to capture with classical monolayer cell culture techniques. Both in vivo and in vitro, cellular function is directly impacted by the cellular microenvironment, which is not limited to cellular communication and programming (e.g., cell-cell junction formations, opportunities to form cell niches); cell exposure to hormones and growth factors in the immediate environments (e.g., cellular cytokine exposure as part of an inflammatory response); composition of physical and chemical matrices (e.g., whether cells are grown in stiff tissue culture plastic or an elastic tissue environment); and most importantly, how cellular metabolism is impacted by nutrition and access to oxygen as well as the processing of metabolic waste products such as lactic acid.

Metabolic flux analysis is a powerful way to examine cellular metabolism within defined in vitro systems. Specifically, XF technology allows for the analysis of live, real-time changes in cellular bioenergetics of intact cells and tissues. Given that many intracellular metabolic events occur within the order of seconds to minutes, real-time functional approaches are paramount for understanding real-time changes in cellular metabolic flux in intact cells and tissues in vitro.

This paper provides protocols for cultivating cancer-derived cell lines A549 (lung adenocarcinoma), HepG2/C3A (hepatocellular carcinoma), MCF-7 (breast adenocarcinoma), and SK-OV-3 (ovarian adenocarcinoma) as in vitro 3D spheroid models using forced-aggregation approaches (Figure 1). It also (i) describes in detail how to probe mitochondrial energy metabolism of single 3D spheroids using the Agilent XFe96 XF analyzer, (ii) highlights ways to optimize XF assays using single 3D spheroids, and (iii) discusses important considerations and limitations of probing 3D spheroid metabolism using this approach. Most importantly, this paper describes how datasets are collected that allow the calculation of oxygen consumption rate (OCR) to determine oxidative phosphorylation and thus mitochondrial function in cellular spheroids. Though not analyzed for this protocol, extracellular acidification rate (ECAR) is another parameter that is measured alongside OCR data in XF experiments. However, ECAR is often poorly or incorrectly interpreted from XF datasets. We provide a commentary as to the limitations of calculating ECAR following basic approaches from the technology manufacturer.

Protocol

Figure 1: Graphical workflow for the generation of cellular spheroids, extracellular flux analysis and downstream assays. Four cancer cell lines were selectively cultured as monolayers (A), detached from tissue culture flasks, and seeded into ultralow attachment 96-well microplates to form spheroids (B). A549 lung carcinoma, HepG2/C3A liver carcinoma, SK-OV-3 ovarian adenocarcinoma, and MCF-7 breast carcinoma cells were seeded at 1 × 10³-8 × 10³ cells/well and grown up to 7 days to form single spheroids and optimize spheroid seeding density and cultivation time by continuous observation and planimetric measurements. Once formed, single spheroids were washed into a serum-free XF medium and carefully seeded into spheroid assay microplates, precoated with poly-D-lysine (C). Spheroids were subjected to extracellular flux analysis using the XFe96 analyzer using several protocols to address: (1) optimal spheroid size for basal mitochondrial respiration response; (2) optimized titration of mitochondrial respiratory inhibitors; (3) optimization of spheroid placement within microplate wells. (D) Post XF analyses, phase contrast microscopy, and spheroid DNA quantification were used for data normalization and other downstream in vitro assays. Please click here to view a larger version of this figure.

1. Cultivation of cancer cell lines as 3D in vitro spheroids

Cell line	Description	Culture medium	Source
A549	Lung carcinoma cell line	RPMI 1640	European Collection of Authenticated Cell Cultures (ECACC)
		Sodium pyruvate (1 mM)
		Penicillin- Streptomycin - (100 U/mL – 100 mg/mL)
		10 % (v/v) FBS
HepG2/C3A	Hepatic carcinoma cell line, a clonal derivative of the parent HepG2 cell line	DMEM	American Tissue Culture Collection (ATCC)
		Penicillin- Streptomycin - (100 U/mL – 100 mg/mL)
		10 % (v/v) FBS
MCF7	Breast adenocarcinoma cell line	RPMI 1640	European Collection of Authenticated Cell Cultures (ECACC)
		Sodium pyruvate (1 mM)
		Penicillin- Streptomycin - (100 U/mL – 100 mg/mL)
		10 % (v/v) FBS
SK-OV-3	Ovarian adenocarcinoma cell line	RPMI 1640	European Collection of Authenticated Cell Cultures (ECACC)
		Sodium pyruvate (1 mM)
		Penicillin- Streptomycin - (100 U/mL – 100 mg/mL)
		10 % (v/v) FBS
Component	RPMI assay medium (50 mL final volume)
Base Medium	Agilent Seahorse XF RPMI, pH 7.4
Glucose (1 M sterile stock)	11 mM (0.55 mL stock solution)
L-glutamine (200 mM sterile stock)	2 mM (0.5 mL of stock solution)
Sodium pyruvate (100 mM sterile stock)	1 mM (0.5 mL of stock solution)

Table 1: Cancer cell line media and XF media compositions.

1. Culture all cell lines using standard aseptic tissue culture technique and confirm that they are free of mycoplasma using a suitable assay kit.
2. Culture the cell lines in T75 tissue culture flasks or equivalent, using the recommended medium (Table 1). Culture the cell lines to 65-80% confluency and passage them regularly up to a maximum of 25 passages.
3. Rinse the cell culture flasks twice in Dulbecco's modified phosphate-buffered saline (DBPS).
4. Detach the cells from the flasks with 3 mL of the cell dissociation reagent (see the Table of Materials) for 5 min at 37 °C and confirm the detachment by microscopy.
5. Aspirate the detached cell suspension gently to ensure a single-cell suspension and deactivate the cell dissociation reagent with 7 mL of complete tissue culture medium.
6. Collect the cells by centrifugation at 300 × g for 5 min, discard the supernatant, and resuspend the cells in complete medium.
7. Count the cells using a hemocytometer or an automated cell counter and titrate to the desired cell density required for seeding.
   NOTE: To seed an entire 96-well plate at 100 µL/well at 4 × 10³ cells/well, cells should be titrated to 4 × 10⁴ cells/mL in a recommended volume of 12 mL.
8. Decant the cell suspension into a sterile reservoir and dispense 100 µL of the cell suspension into each well of a cell-repellent microplate using a multichannel pipettor.
   NOTE: Only the inner 60 wells of a microplate should be seeded and the remainder filled with DPBS. This will form an evaporation barrier, ensure spheroid homogeneity across the plate, and minimize plate edge effects.
9. Centrifuge spheroid microplates at 300 × g for 15 min to force the cells into loose aggregates.
10. Incubate the plates at 37 °C, 5% CO₂ for a minimum of 3 days to ensure spheroid formation.
11. Perform phase-contrast microscopy using standardized laboratory practices to monitor the growth of spheroids. Replenish the cell culture medium every 3 days or twice weekly by performing a half-volume medium exchange.

2. Probing mitochondrial energy metabolism of single spheroids using Extracellular Flux (XF) Technology

1. Assay preparation (one day prior)
   1. 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.
   2. Hydrate the sensor cartridge.
      1. Aliquot ~20 mL of the calibrant into a conical tube.
      2. Place the conical tube containing the calibrant in a non-CO₂ 37 °C incubator overnight.
      3. Remove the contents from the assay kit.
      4. Remove the sensor cartridge from the utility plate and place it upside down on the worktop next to the utility plate.
      5. Pipette 200 µL of sterile ddH₂O into each well of the sensor cartridge utility plate using a multichannel P300 pipette.
      6. Place the sensor cartridge on top of the utility plate.
      7. Check that the water level in each well is high enough to submerge the sensor probes.
      8. Transfer the assembled sensor cartridge to a non-CO2 37 °C incubator and leave it overnight.
         NOTE: This step can be performed 12-72 h prior to assay commencement.
2. Coat spheroid assay microplate
   1. Using aseptic techniques, add 30 µL/well of sterile Poly-D-Lysine (0.1 mg/mL) solution to the spheroid microplate and incubate it for 30 min at room temperature.
   2. 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.
   3. Wash the plate twice with 200 µL/well of sterile ddH₂O.
   4. After the final wash, invert the microplate and tap it firmly onto tissue paper to remove any residual water.
   5. Allow the plate to air-dry for 30 min before using or storing it at 4 °C for future use.
      NOTE: The spheroid assay microplate should be coated with a molecular adhesive to ensure that the spheroids are fixed at the bottom of the microplate. Without a molecular adhesive, spheroids can become dislodged and interfere with assay results. Other molecular adhesives can also be used as an alternative to Poly-D-Lysine for precoating plates. Precoated plates can be stored at 4 °C but should be left to equilibrate to room temperature before assay commencement.
3. Prepare XF Assay medium
   1. Prepare XF RPMI medium, as detailed in Table 1, and sterile-filter with a 0.22 μm syringe filter
4. Assay preparation (1 h prior to assay)
   1. Prewarm the supplemented XF RPMI assay medium to 37 °C.
   2. Prewarm the coated spheroid assay microplate in a non-CO₂ 37 °C incubator or dry bath.
   3. Prepare the sensor cartridge.
      1. Take out the conical tube containing the calibrant and the sensor cartridge from the air incubator.
      2. Remove the sensor cartridge from the utility plate and place it upside down on the work surface.
      3. Using a P300 multichannel pipette, aspirate the water from the utility plate and discard it.
      4. Pour the calibrant solution into a sterile reagent reservoir and add 200 µL/well of the prewarmed calibrant to the utility plate using a P300 multichannel pipette.
      5. Pick up the sensor cartridge and place it back on top of the utility plate, ensuring the sensors are well submerged in the calibrant.
      6. Transfer the assembled sensor cartridge back into the non-CO₂ 37 °C incubator until ready to load the port injection solutions.
5. Wash the spheroids with the assay medium.
   1. Remove the spheroid culture plate from the 37 °C, 5% CO₂ incubator and observe the spheroids under the microscope to ensure their integrity prior to the spheroid transfer steps.
   2. Load all wells of the spheroid plate with 180 µL/well of prewarmed assay medium, including any background correction wells.
   3. Partially fill a 7 cm Petri dish with 3 mL of the assay medium.
   4. Using a multichannel pipette loaded with wide orifice pipette tips, transfer the spheroids from the 96-well culture plate into 7 cm Petri dishes by setting the pipettor at an aspiration volume of 10-50 µL.
6. Seed spheroids into the pre-coated spheroid assay microplate.
   1. Using a dissection microscope and a lightbox apparatus, transfer the spheroids from the Petri dish to the spheroid assay microplate as detailed below.
      1. Set the volume of a single-channel pipettor fitted with a wide orifice pipette tip to 20 µL and carefully aspirate a single spheroid. Place the tip directly in the center of each well of the spheroid assay microplate and allow gravity to elute a single spheroid into the center of each well, i.e., do not expel any medium from the pipette tip and allow capillary action to withdraw the spheroid from the pipette tip. To confirm elution, the contents of the pipettor can be pipetted back into the 7 cm Petri dish under the microscope.
         NOTE: Gravity elution of a single spheroid typically takes 15-30 s depending on spheroid size/density. During this time, the pipettor should not be removed. Any background correction wells should be free of spheroids and only contain assay medium. Under the microscope, confirm the position of each spheroid. Each spheroid should ideally be positioned within the center of each well.
      2. Once all the spheroids have been transferred to the spheroid assay microplate, transfer the plate to a non-CO₂ incubator at 37 °C for a minimum of 1 h prior to the assay.

3. Preparation and loading of compounds into the sensor cartridge for XF assays

Injection Strategy	Compound (Port)	XFe96 microwell starting volume (µL)	Desired final well concentration	Port Volume (µL)	Final XFe96 microwell volume post injection (µL)	Working stock concentration
1	Oligomycin (A)	180	3 ug/mL	20	200	30 µg/mL
	Rotenone (B)	200	2 µM	20	220	22 µM
	Antimycin A (B)	200	2 µM	20	220	22 µM
2	BAM15 (A)	180	5 µM	20	200	50 µM
	Rotenone (B)	200	2 µM	20	220	22 µM
	Antimycin A (B)	200	2 µM	20	220	22 µM

Table 2: Mitochondrial compound concentrations for probing mitochondrial energy metabolism of single 3D spheroids using the XFe96 Analyzer.

1. Prepare working stock concentrations of each compound as noted in Table 2 using fully supplemented, prewarmed XF RPMI assay medium.
2. Orient the cartridge plate (coupled to the utility plate) column-wise, 1-12 from left to right.
3. If using a loading guide, place it atop the cartridge plate according to the well-loading procedure, e.g., if port A is be loaded first, ensure that A is visible in the upper-left corner of the guide.
4. Transfer the working solution of each compound into a suitable reservoir and, using a calibrated P100 multichannel pipette, dispense 20 µL into all corresponding ports. Repeat for each compound into the remaining ports.
   NOTE: If any ports are not used on the sensor cartridge plate, these can be left empty or filled with assay medium. If only a selection of a specific port letter is being used, ensure that the other ports corresponding to that letter are loaded with assay medium; otherwise, air will be injected into the well, compromising the results in those wells.
5. After port loading, remove the plate-loading guides (if used) and prepare the analyzer for loading the sensor cartridge.
   NOTE: If the assay is not being run immediately after loading the ports, place the lid back on the sensor cartridge and put the plate back in the 37 °C air incubator until ready to load into the machine.

4. Assay design, injection strategies, and data acquisition

1. Running the assay
   1. Power on the analyzer and connect to controller (computer).
      NOTE: This can be verified by the instrument connection status in the widget panel of the Wave Controller software.
   2. Navigate to the templates page in the WAVE software, find the assay template file for the experiment and double-click to open it.
      NOTE: If the assay template does not appear on the Templates view, import the template file into the template folder from a shared network drive or USB flash drive.
   3. To start the assay, click the Run Assay tab.
      NOTE: If the group definitions have been correctly allocated within the plate map, the assay will be ready to run as indicated by the green tick on the right-hand side of the page. At this stage, any additional information can be input on the assay summary page or the page left blank; proceed to the next step. Due to the delayed penetration of mitochondrial modulators in 3D microtissue spheroids (Figure 2), use the measurement protocol information described in Table 3.

Measurement Period	Injection Number and Port	Measurement Details	Period Duration (h:min:s)
Calibration	Not applicable	XF analysers always perform this calibration to make sure measurements are accurate	00:20:00 (this is an average and can vary between machines)
Equillibration	Not applicable	Equilibration occurs after Calibration and it is recommended.	00:10:00
Basal	Not applicable	Cycles = 5	00:30:00
		Mix = 3:00
		Wait = 0:00
		Measure = 3:00
Oligomycin / BAM15	Injection 1 (Port A)	Cycles = 10	01:00:00
		Mix = 3:00
		Wait = 0:00
		Measure = 3:00
Rotenone + antimycin A	Injection 2 (Port B)	Cycles = 10	01:00:00
		Mix = 3:00
		Wait = 0:00
		Measure = 3:00
Total Time:			03:00:00

Table 3: Protocol setup for probing mitochondrial energy metabolism of single 3D spheroids using the XFe96 Analyzer.

4. Click start run to bring up the save location dialog box.
5. Enter the save location for the result file, and place the assembled sensor cartridge onto the thermal tray that appears from the door on the side of the analyzer. Wait for the thermal tray to open automatically and the screen to display the Load Calibrant Utility Plate message. Before following the on-screen prompts, ensure i) proper fit of the sensor cartridge on the Utility plate, ii) the lid is removed from the sensor cartridge, and iii) correct orientation of the sensor cartridge on the utility plate.
6. Follow the on-screen commands to initiate sensor cartridge calibration.
   NOTE: The time taken to complete calibration is approximately 10-20 min (for assays at 37 °C).
7. After sensor cartridge calibration, load the spheroid microplate into the analyzer by following the on-screen instructions on the Wave Controller to initiate the 12 min equilibration step.
   NOTE: Green boxes with white ticks indicate a 'good' calibration for that well. If any wells fail to provide a 'good' calibration, they will be indicated with a red box and white cross. Such wells should be noted and excluded from any analysis after the assay is completed using the modification assay tab.
8. Wait for the analyzer to automatically begin acquiring baseline measurements after the machine has completed the equilibration step (as outlined in the instrument protocol).
9. To complete the experiment, follow the on-screen commands on the WAVE controller.
   NOTE: Once the spheroid microplate has been removed from the analyzer, discard the sensor cartridge and set aside the spheroid plate for further analysis if necessary (e.g., double-stranded (ds) DNA quantification). If the microplate is not required for further analysis, it can be discarded along with the sensor cartridge.
10. Wait for the assay dialog to appear and view the results or return to the templates view.

5. Data normalization and analysis strategies - post assay normalization and downstream assays (optional steps)

1. Data normalization
   1. To normalize spheroid data, refer to the series of protocols pertinent to data normalization strategies for calculating spheroid size and volume and quantifying dsDNA in spheroid assays. These have been included as supplemental files; see Supplemental file 1 and Supplemental file 2.
2. Data analysis
   1. To export data into one of the automated analysis generators, follow the data export commands on the WAVE controller and select the export generator that matches the assay type. Alternatively, export the data file and upload it into Seahorse analytics.
      NOTE: The downside of report generators and Seahorse analytics is that data analysis is limited to how the XF assay is designed and does not allow for averages to be taken across measurement cycles. Manual export of datasets from the instrument software allows for user preference in this regard. Given that the injection strategy for assessing mitochondrial respiration of 3D spheroids will likely differ from that of a typical 'MitoStress' test, a series of spreadsheet templates have been developed to help analyze these datasets, specific to 3D cell cultures and will be provided upon request. These data template files will provide data on the key mitochondrial respiratory parameters detailed and explained in Figure 2.
   2. To analyze the data, export the data as a spreadsheet report from the WAVE controller software and use an independent spreadsheet template for analysis.

Figure 2: Schematic descriptors for parameters derived from extracellular flux data analyses. Abbreviation: OCR = oxygen consumption rate. Please click here to view a larger version of this figure.

Results

To obtain well-formed, compact spheroids, each cell line was optimized individually for seeding density and duration of cultivation (Figure 3). A549, HepG2/C3A, and SK-OV-3 cell lines initially formed loose aggregates that did not progress to round spheroids with clearly defined perimeters until after 7 days in culture. Conversely, MCF-7 cells could form spheroids within 3 days. There was a clear correlation between the initial cell seeding density and spheroid volume after the culture perio...

Discussion

Main findings and outputs
This paper provides a detailed protocol to probe mitochondrial energy metabolism of single 3D spheroids using a series of cancer-derived cell lines with the XFe96 XF Analyzer. A method is developed and described for the rapid cultivation of A549, HepG2/C3A, MCF7, and SK-OV-3 cellular spheroids using cell-repellent technologies for forced aggregation. This protocol addresses many considerations of probing spheroid metabolism with XF technology, including (1) optimizati...

Materials

Name	Company	Catalog Number	Comments
A549	ECACC	#86012804	Lung carcinoma cell line
Agilent Seahorse XF RPMI Medium, pH 7.4	Agilent Technologies Inc.	103576-100	XF assay medium with 1 mM HEPES, without phenol red, sodium bicarbonate, glucose, L-glutamine, and sodium pyruvate
Agilent Seahorse XFe96 Extracellular Flux Analyzer	Agilent Technologies Inc.	-	Instrument for measuring rates of spheroid oxygen uptake in single spheroids
Antimycin A	Merck Life Science	A8674	Mitochondrial respiratory complex III inhibitor
BAM15	TOCRIS bio-techne	5737	Mitochondrial protnophore uncoupler
Black-walled microplate	Greiner Bio-One	655076	For fluorescence-based assays
CELLSTAR cell-repellent surface 96 U well microplates	Greiner Bio-One	650970	Microplates for generating spheroids
CellTiter-Glo 3D Cell Viability Assay	Promega	G9681	Assay for the determination of cell viability in 3D microtissue spheroids
Cultrex Poly-D-Lysine	R&D Systems a biotechne brand	3439-100-01	Molecular cell adhesive for coating XFe96 spheroid microplates to facillitate attachment of spheroids
D-(+)-Glucose	Merck Life Sciences	G8270	Supplement for cell culture growth and XF assay medium
Dulbecco’s Modified Eagle Medium (DMEM)	Gibco	11885084	Culture medium for HepG2/C3A spheroids
EVOS XL Core Imaging System	Thermo Fisher Scientific	AMEX1000	Phase-contrast imaging microscope
EZ-PCR Mycoplasma test kit	Biological Industries	20-700-20	Mycoplasma screening in cell cultures
FIJI Is Just Image J			Analysis of collated images
Foetal bovine serum	Merck Life Science	F7524	Supplement for cell culture medium
HepG2/C3A	ATCC	#CRL-10741	Hepatic carcinoma cell line, a clonal derivative of the parent HepG2 cell line
Lactate-Glo	Promega	J5021	Assay for measurement of lactate within spheorid culture medium
L-glutamine (200 mM solution)	Merk Life Sciences	G7513	Supplement for cell culture growth and XF assay medium
M50 Stereo microscope	Leica Microsytems	LEICAM50	Stereo dissection micrscope; used for spheorid handling
MCF-7	ECACC	#86012803	Breast adenocarcinoma cell line
Oligomycin from Streptomyces diastatochromogenes	Merck Life Science	O4876	ATP Synthase Inhibitor
Penicilin-Streptomycin	Gibco	15140122	Antibiotics added to cell culture medium
Quant-iT PicoGreen dsDNA Assay Kit	Initrogen	P7589	Analysis of dsDNA in spehroids
Rotenone	Merck Life Science	R8875	Mitochondrial Respiratory Complex I Inhibitor
RPMI 1640	Gibco	21875091	Culture medium for A549, MCF7, and SK-OV-3 spheroids
Seahorse Analytics	Agilent Technologies Inc.	Build 421	https://seahorseanalytics.agilent.com
Seahorse XFe96 Spheroid FluxPak	Agilent Technologies Inc.	102905-100	Each Seahorse XFe96 Spheroid FluxPak contains: 6 Seahorse XFe96 Spheroid Microplates (102978-100), 6 XFe96 sensor cartridges, and 1 bottle of Seahorse XF Calibrant Solution 500 mL (100840-000)
Serological pipette: 5, 10, and 25 mL	Greiner Bio-One	606107; 607107; 760107	Consumables for cell culture
SK-OV-3	ECACC	#HTB-77	Ovarian adenocarcinoma cell line
Sodium pyruvate (100 mM solution)	Merck Life Science	S8636	Supplement for cell culture growth and XF assay medium
T75 cm2 cell culture flask	Greiner Bio-One	658175	Tissue culture treated flasks for maintaining cell cultures
TrypLExpress	Gibco	12604-021	Cell dissociation reagent
Wave controller software	Agilent Technologies Inc.	-
Wide orifice tip	STARLAB International GmbH	E1011-8400	Pipette tips with wide opening for spheroid handling