Name: Exploring Mitochondrial Energy Metabolism of Single 3D Microtissue Spheroids Using Extracellular Flux Analysis (Video) | JoVE Journal
Uploaded: 2022-02-03T14:00:00.000+00:00
Duration: 8 min 15 s
Description: Explore mitochondrial energy metabolism in 3D cancer spheroids using Seahorse extracellular flux analysis. This protocol enables single spheroid measurements for drug discovery and cancer research applications.

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5 minutes

N.J.C was supported by a BBSRC MIBTP CASE Award with Sygnature Discovery Ltd (BB/M01116X/1, 1940003)

The authors have no conflicts of interest to declare.

Main findings and outputs 
This paper provides a detailed protocol to&#160;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...

An erratum was issued for: <a href="https://www.jove.com/t/63346">Exploring Mitochondrial Energy Metabolism of Single 3D Microtissue Spheroids using Extracellular Flux Analysis</a>. The Representative Results section was updated.
Figure 5 was updated from:
<img alt="Oxygen consumption rate (OCR) graphs with oligomycin effects on A549, HepG2/C3A, MCF-7, SK-OV-3 cells." class="xfigimg" src="/files/ftp_upload/63346/63346fig05.jpg" data-altupdatedat="1761830439000" data-alt="Figure 5"> 
Figure 5: Single or sequential injection of mitochondrial respiratory compounds. Cancer-cell-derived spheroids of MCF-7, HEPG2/C3A, SK-OV-3, and A549 were placed into wells of an XFe96 spheroid microplate in XF RPMI and probed for OCR using the Agilent Seahorse XFe96 analyzer. OCR was measured 5x, after which 2 µg/mL oligomycin (injection Port A: green trace) or 5 µM BAM15 (injection Port A: blue trace or injection port B: green trace) to inhibit the mitochondrial ATP synthase and determine maximal respiratory capacity, respectively. Kinetic OCR data are expressed as % basal (A-D). Maximal respiratory capacity (OCRmax) was calculated as a factor of basal OCR by the equation: OCRmax = OCRBAM15 / OCRbasal. OCRmax was obtained from OCR averages across measurement cycles 8-10 post BAM15 injection with (green bars) and without (blue bars) oligomycin. Data are averages ± SEM from 3-8 individual well replicates across the spheroid assay microplate. Abbreviations: OCR = oxygen consumption rate. <a href="https://www.jove.com/files/ftp_upload/63346/63346fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
to:
<img alt="Oligomycin effect on OCR in cancer cell lines; comparison graph; metabolic activity analysis." class="xfigimg" src="/files/ftp_upload/63346/63346fig5v2.jpg" data-altupdatedat="1761830439000" data-alt="Figure 5"> 
Figure 5: Single or sequential injection of mitochondrial respiratory compounds. Cancer-cell-derived spheroids of MCF-7, HEPG2/C3A, SK-OV-3, and A549 were placed into wells of an XFe96 spheroid microplate in XF RPMI and probed for OCR using the Agilent Seahorse XFe96 analyzer. OCR was measured 5x, after which 2 µg/mL oligomycin (injection Port A: green trace) or 5 µM BAM15 (injection Port A: blue trace or injection port B: green trace) to inhibit the mitochondrial ATP synthase and determine maximal respiratory capacity, respectively. Kinetic OCR data are expressed as % basal (A-D). Maximal respiratory capacity (OCRmax) was calculated as a factor of basal OCR by the equation: OCRmax = OCRBAM15 / OCRbasal. OCRmax was obtained from OCR averages across measurement cycles 8-10 post BAM15 injection with (green bars) and without (blue bars) oligomycin. Data are averages ± SEM from 3-8 individual well replicates across the spheroid assay microplate. Abbreviations: OCR = oxygen consumption rate. <a href="https://www.jove.com/files/ftp_upload/63346/63346fig5v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

An erratum was issued for:&#160;<a href="https://www.jove.com/t/63346">Exploring Mitochondrial Energy Metabolism of Single 3D Microtissue Spheroids using Extracellular Flux Analysis</a>. The Representative Results&#160;section was&#160;updated.

Erratum: Exploring Mitochondrial Energy Metabolism of Single 3D Microtissue Spheroids using Extracellular Flux Analysis

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 biology1,2,3,4,5,6,7, and safety testing, including genetic toxicology8,9,10, nanomaterials toxicology11,12,13,14, and drug safety and efficacy testing8,15,16,17,18,19.
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&#160;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.

<table><tbody><tr><td>A549</td><td>ECACC</td><td>&amp;nbsp;#86012804</td><td>Lung carcinoma cell line</td></tr><tr><td>Agilent Seahorse XF RPMI Medium, pH 7.4</td><td>Agilent Technologies Inc.</td><td>103576-100</td><td>XF assay medium with 1 mM HEPES, without phenol red, sodium bicarbonate, glucose, L-glutamine, and sodium pyruvate</td></tr><tr><td>Agilent Seahorse XFe96 Extracellular Flux Analyzer</td><td>Agilent Technologies Inc.</td><td>-</td><td>Instrument for measuring rates of spheroid oxygen uptake in single spheroids</td></tr><tr><td>Antimycin A</td><td>Merck Life Science</td><td>A8674</td><td>Mitochondrial respiratory complex III inhibitor</td></tr><tr><td>BAM15</td><td>TOCRIS bio-techne</td><td>5737</td><td>Mitochondrial protnophore uncoupler</td></tr><tr><td>Black-walled microplate</td><td>Greiner Bio-One</td><td>655076</td><td>For fluorescence-based assays</td></tr><tr><td>CELLSTAR cell-repellent surface 96 U well microplates</td><td>Greiner Bio-One</td><td>650970</td><td>Microplates for generating spheroids</td></tr><tr><td>CellTiter-Glo 3D Cell Viability Assay</td><td>Promega</td><td>G9681</td><td>Assay for the determination of cell viability in 3D microtissue spheroids</td></tr><tr><td>Cultrex Poly-D-Lysine</td><td>R&amp;amp;D Systems a biotechne brand</td><td>3439-100-01</td><td>Molecular cell adhesive for coating XFe96 spheroid microplates to facillitate attachment of spheroids</td></tr><tr><td>D-(+)-Glucose</td><td>Merck Life Sciences</td><td>G8270</td><td>Supplement for cell culture growth and XF assay medium</td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium (DMEM)</td><td>Gibco</td><td>11885084</td><td>Culture medium for HepG2/C3A spheroids</td></tr><tr><td>EVOS XL Core Imaging System</td><td>Thermo Fisher Scientific</td><td>AMEX1000</td><td>Phase-contrast imaging microscope</td></tr><tr><td>EZ-PCR Mycoplasma test kit</td><td>Biological Industries</td><td>20-700-20</td><td>Mycoplasma screening in cell cultures</td></tr><tr><td>FIJI Is Just Image J</td><td></td><td></td><td>Analysis of collated images</td></tr><tr><td>Foetal bovine serum</td><td>Merck Life Science</td><td>F7524</td><td>Supplement for cell culture medium</td></tr><tr><td>HepG2/C3A</td><td>ATCC</td><td>&amp;nbsp;#CRL-10741</td><td>Hepatic carcinoma cell line, a clonal derivative of the parent HepG2 cell line</td></tr><tr><td>Lactate-Glo</td><td>Promega</td><td>J5021</td><td>Assay for measurement of lactate within spheorid culture medium</td></tr><tr><td>L-glutamine (200 mM solution)</td><td>Merk Life Sciences</td><td>G7513</td><td>Supplement for cell culture growth and XF assay medium</td></tr><tr><td>M50 Stereo microscope</td><td>Leica Microsytems</td><td>LEICAM50</td><td>Stereo dissection micrscope; used for spheorid handling</td></tr><tr><td>MCF-7</td><td>ECACC</td><td>#86012803</td><td>Breast adenocarcinoma cell line</td></tr><tr><td>Oligomycin from &lt;em&gt;Streptomyces diastatochromogenes&lt;/em&gt;</td><td>Merck Life Science</td><td>O4876</td><td>ATP Synthase Inhibitor</td></tr><tr><td>Penicilin-Streptomycin</td><td>Gibco</td><td>15140122</td><td>Antibiotics added to cell culture medium</td></tr><tr><td>Quant-iT PicoGreen dsDNA Assay Kit</td><td>Initrogen</td><td>P7589</td><td>Analysis of dsDNA in spehroids</td></tr><tr><td>Rotenone</td><td>Merck Life Science</td><td>R8875</td><td>Mitochondrial Respiratory Complex I Inhibitor</td></tr><tr><td>RPMI 1640</td><td>Gibco</td><td>21875091</td><td>Culture medium for A549, MCF7, and SK-OV-3 spheroids</td></tr><tr><td>Seahorse Analytics</td><td>Agilent Technologies Inc.</td><td>Build 421</td><td>https://seahorseanalytics.agilent.com</td></tr><tr><td>Seahorse XFe96 Spheroid FluxPak</td><td>Agilent Technologies Inc.</td><td>102905-100</td><td>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)</td></tr><tr><td>Serological pipette: 5, 10, and 25 mL</td><td>Greiner Bio-One</td><td>606107; 607107; 760107</td><td>Consumables for cell culture</td></tr><tr><td>SK-OV-3</td><td>ECACC</td><td>&amp;nbsp;#HTB-77</td><td>Ovarian adenocarcinoma cell line</td></tr><tr><td>Sodium pyruvate (100 mM solution)</td><td>Merck Life Science</td><td>S8636</td><td>Supplement for cell culture growth and XF assay medium</td></tr><tr><td>T75 cm&lt;sup&gt;2&lt;/sup&gt; cell culture flask</td><td>Greiner Bio-One</td><td>658175</td><td>Tissue culture treated flasks for maintaining cell cultures</td></tr><tr><td>TrypLExpress</td><td>Gibco</td><td>12604-021</td><td>Cell dissociation reagent</td></tr><tr><td>Wave controller software</td><td>Agilent Technologies Inc.</td><td>-</td><td></td></tr><tr><td>Wide orifice tip</td><td>STARLAB International GmbH</td><td>E1011-8400</td><td>Pipette tips with wide opening for spheroid handling</td></tr></tbody></table>

exploring mitochondrial energy metabolism of single 3d microtissue spheroids using extracellular flux analysis

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

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,&#160;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.

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...

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

Three-dimensional (3D) cellular aggregates, termed spheroids, have become the forefront of in vitro cell culture in recent years. In contrast to culturing ...

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Research

JoVE Journal

Cancer Research

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

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