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<....

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 l.......

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