Method Article

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

DOI:

10.3791/63346

⸱

February 3rd, 2022

 ,  ,  ,  , 
1School of Biosciences, University of Birmingham, 2Nanomedicine, Drug Delivery & Nanotoxicology Lab, School of Pharmacy, University of Birmingham, 3Mitochondrial Profiling Centre, University of Birmingham

In This Article

Erratum Notice

Important: There has been an erratum issued for this article. View Erratum Notice

Summary

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

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

Abstract

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

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

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

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<....

Access restricted. Please log in or start a trial to view this content.

Protocol

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

Monolayer cell culture diagram; spheroid cultivation; extracellular flux analysis; data assays.
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

Access restricted. Please log in or start a trial to view this content.

Results

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

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.......

Access restricted. Please log in or start a trial to view this content.

Discussion

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

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.......

Access restricted. Please log in or start a trial to view this content.

Disclosures

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

The authors have no conflicts of interest to declare.

Acknowledgements

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

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

....

Access restricted. Please log in or start a trial to view this content.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
A549ECACC #86012804Lung carcinoma cell line
Agilent Seahorse XF RPMI Medium, pH 7.4Agilent Technologies Inc.103576-100XF assay medium with 1 mM HEPES, without phenol red, sodium bicarbonate, glucose, L-glutamine, and sodium pyruvate
Agilent Seahorse XFe96 Extracellular Flux AnalyzerAgilent Technologies Inc.-Instrument for measuring rates of spheroid oxygen uptake in single spheroids
Antimycin AMerck Life ScienceA8674Mitochondrial respiratory complex III inhibitor
BAM15TOCRIS bio-techne5737Mitochondrial protnophore uncoupler
Black-walled microplateGreiner Bio-One655076For fluorescence-based assays
CELLSTAR cell-repellent surface 96 U well microplatesGreiner Bio-One650970Microplates for generating spheroids
CellTiter-Glo 3D Cell Viability AssayPromegaG9681Assay for the determination of cell viability in 3D microtissue spheroids
Cultrex Poly-D-LysineR&D Systems a biotechne brand3439-100-01Molecular cell adhesive for coating XFe96 spheroid microplates to facillitate attachment of spheroids
D-(+)-GlucoseMerck Life SciencesG8270Supplement for cell culture growth and XF assay medium
Dulbecco’s Modified Eagle Medium (DMEM)Gibco11885084Culture medium for HepG2/C3A spheroids
EVOS XL Core Imaging SystemThermo Fisher ScientificAMEX1000Phase-contrast imaging microscope
EZ-PCR Mycoplasma test kitBiological Industries20-700-20Mycoplasma screening in cell cultures
FIJI Is Just Image JAnalysis of collated images
Foetal bovine serumMerck Life ScienceF7524Supplement for cell culture medium
HepG2/C3AATCC #CRL-10741Hepatic carcinoma cell line, a clonal derivative of the parent HepG2 cell line
Lactate-GloPromegaJ5021Assay for measurement of lactate within spheorid culture medium
L-glutamine (200 mM solution)Merk Life SciencesG7513Supplement for cell culture growth and XF assay medium
M50 Stereo microscopeLeica MicrosytemsLEICAM50Stereo dissection micrscope; used for spheorid handling
MCF-7ECACC#86012803Breast adenocarcinoma cell line
Oligomycin from Streptomyces diastatochromogenesMerck Life ScienceO4876ATP Synthase Inhibitor
Penicilin-StreptomycinGibco15140122Antibiotics added to cell culture medium
Quant-iT PicoGreen dsDNA Assay KitInitrogenP7589Analysis of dsDNA in spehroids
RotenoneMerck Life ScienceR8875Mitochondrial Respiratory Complex I Inhibitor
RPMI 1640Gibco21875091Culture medium for A549, MCF7, and SK-OV-3 spheroids
Seahorse AnalyticsAgilent Technologies Inc.Build 421https://seahorseanalytics.agilent.com
Seahorse XFe96 Spheroid FluxPakAgilent Technologies Inc.102905-100Each 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 mLGreiner Bio-One606107; 607107; 760107Consumables for cell culture
SK-OV-3ECACC #HTB-77Ovarian adenocarcinoma cell line
Sodium pyruvate (100 mM solution)Merck Life ScienceS8636Supplement for cell culture growth and XF assay medium
T75 cm2 cell culture flaskGreiner Bio-One658175Tissue culture treated flasks for maintaining cell cultures
TrypLExpressGibco12604-021Cell dissociation reagent
Wave controller softwareAgilent Technologies Inc.-
Wide orifice tipSTARLAB International GmbHE1011-8400Pipette tips with wide opening for spheroid handling

References

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,
  1. Correa de Sampaio, P., et al. A heterogeneous in vitro three dimensional model of tumour-stroma interactions regulating sprouting angiogenesis. PLoS One. 7 (2), 30753(2012).
  2. Amann, A., et al.

Access restricted. Please log in or start a trial to view this content.

Reprints and Permissions

Request permission to reuse the text or figures of this JoVE article

Request Permission

Erratum

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

Formal Correction: Erratum: Exploring Mitochondrial Energy Metabolism of Single 3D Microtissue Spheroids using Extracellular Flux Analysis
Posted by JoVE Editors on 3/11/2022. Citeable Link.

An erratum was issued for: Exploring Mitochondrial Energy Metabolism of Single 3D Microtissue Spheroids using Extracellular Flux Analysis. The Representative Results section was updated.

Figure 5 was updated from:

Oxygen consumption rate (OCR) graphs with oligomycin effects on A549, HepG2/C3A, MCF-7, SK-OV-3 cells.
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. Please click here to view a larger version of this figure.

to:

Oligomycin effect on OCR in cancer cell lines; comparison graph; metabolic activity analysis.
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. Please click here to view a larger version of this figure.

Tags

Mitochondrial Energy Metabolism3D Spheroid CultureExtracellular Flux AnalysisSpheroid MicroplatesOxygen Consumption RateCancer Cell SpheroidsXF AnalyzerBasal Mitochondrial RespirationSpheroid ViabilityPoly D Lysine Coating

Related Articles