JoVE Journal > Cancer Research
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
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Cancer Research

Udforskning af mitokondrieenergimetabolisme af enkelt 3D-mikrotissu-sfroider ved hjælp af ekstracellulær fluxanalyse

Published: February 03, 2022
doi:
10.3791/63346
, , , ,
1School of Biosciences,University of Birmingham, 2Nanomedicine, Drug Delivery & Nanotoxicology Lab, School of Pharmacy,University of Birmingham, 3Mitochondrial Profiling Centre,University of Birmingham

Summary

Disse protokoller vil hjælpe brugerne med at undersøge mitokondrie energimetabolisme i 3D-kræftcellelinjeafledte sfæroider ved hjælp af Seahorse ekstracellulær fluxanalyse.

Abstract

Tredimensionelle (3D) cellulære aggregater, kaldet sfæroider, er blevet forkant med in vitro-cellekultur i de senere år. I modsætning til dyrkning af celler som todimensionale, encellede monolag (2D-kultur) fremmer, regulerer og understøtter sfæroid cellekultur fysiologisk cellulær arkitektur og egenskaber, der findes in vivo, herunder ekspression af ekstracellulære matrixproteiner, cellesignalering, genekspression, proteinproduktion, differentiering og proliferation. Betydningen af 3D-kultur er blevet anerkendt inden for mange forskningsområder, herunder onkologi, diabetes, stamcellebiologi og vævsteknik. I løbet af det sidste årti er der udviklet forbedrede metoder til at producere sfæroider og vurdere deres metaboliske funktion og skæbne.

Ekstracellulære flux (XF) analysatorer er blevet brugt til at udforske mitokondriefunktion i 3D-mikrotissuer såsom sfæroider ved hjælp af enten en XF24-ø-indfangningsplade eller en XFe96-sfæroid mikroplade. Imidlertid er forskellige protokoller og optimering af sonderende mitokondrieenergimetabolisme i sfæroider ved hjælp af XF-teknologi ikke beskrevet detaljeret. Dette papir indeholder detaljerede protokoller til sondering af mitokondrieenergimetabolisme i enkelt 3D-sfæroider ved hjælp af sfæroidmikroplader med XFe96 XF-analysatoren. Ved hjælp af forskellige kræftcellelinjer er XF-teknologi påvist at være i stand til at skelne mellem cellulær respiration i 3D-sfæroider af ikke kun forskellige størrelser, men også forskellige volumener, cellenumre, DNA-indhold og type.

De optimale mitokondrie effektor sammensatte koncentrationer af oligomycin, BAM15, rotenon og antimycin A anvendes til at undersøge specifikke parametre for mitokondrie energimetabolisme i 3D sfæroider. Dette papir diskuterer også metoder til normalisering af data opnået fra sfæroider og behandler mange overvejelser, der bør overvejes, når man udforsker sfæroid metabolisme ved hjælp af XF-teknologi. Denne protokol vil hjælpe med at drive forskning i avancerede in vitro sfæroidmodeller.

Introduction

Fremskridt inden for in vitro-modeller inden for biologisk forskning har udviklet sig hurtigt i løbet af de sidste 20 år. Sådanne modeller omfatter nu organ-on-a-chip-modaliteter, organoider og 3D-mikrotissu-sfæroider, som alle er blevet et fælles fokus for at forbedre translationen mellem in vitro- og in vivo-undersøgelser. Anvendelsen af avancerede in vitro-modeller, navnlig sfæroider, spænder over flere forskningsområder, herunder vævsteknik, stamcelleforskning, kræft og sygdomsbiologi 1,2,3,4,5,6,7 og sikkerhedstest, herunder genetisk toksikologi 8,9,10, nanomaterialer toksikologi11 12,13,14 og lægemiddelsikkerhed og effektivitetstest 8,15,16,17,18,19.

Normal cellemorfologi er afgørende for biologisk fænotype og aktivitet. Dyrkning af celler i 3D-mikrotissue-sfæroider gør det muligt for celler at vedtage en morfologi, fænotypisk funktion og arkitektur, mere beslægtet med den, der observeres in vivo , men vanskelig at fange med klassiske monolagscellekulturteknikker. Både in vivo og in vitro påvirkes cellulær funktion direkte af det cellulære mikromiljø, som ikke er begrænset til cellulær kommunikation og programmering (f.eks. Celle-celle-krydsdannelser, muligheder for at danne cellenicher); celleeksponering for hormoner og vækstfaktorer i de umiddelbare miljøer (f.eks. cellulær cytokineksponering som en del af en inflammatorisk reaktion); sammensætning af fysiske og kemiske matricer (f.eks. om celler dyrkes i stiv vævskulturplast eller et elastisk vævsmiljø) og vigtigst af alt, hvordan cellulær metabolisme påvirkes af ernæring og adgang til ilt samt behandling af metaboliske affaldsprodukter såsom mælkesyre.

Metabolisk fluxanalyse er en kraftfuld måde at undersøge cellulær metabolisme inden for definerede in vitro-systemer . Specifikt giver XF-teknologi mulighed for analyse af levende ændringer i realtid i cellulær bioenergetik af intakte celler og væv. I betragtning af at mange intracellulære metaboliske hændelser forekommer inden for få sekunder til minutter, er funktionelle tilgange i realtid afgørende for at forstå realtidsændringer i cellulær metabolisk flux i intakte celler og væv in vitro.

Dette papir indeholder protokoller til dyrkning af kræftafledte cellelinjer A549 (lungeadenocarcinom), HepG2 / C3A (hepatocellulært carcinom), MCF-7 (brystadenocarcinom) og SK-OV-3 (ovarieadenocarcinom) som in vitro 3D-sfæroidmodeller ved hjælp af tvungen aggregeringsmetoder (figur 1). Det beskriver også (i) detaljeret, hvordan man undersøger mitokondrieenergimetabolisme af enkelt 3D-sfæroider ved hjælp af Agilent XFe96 XF-analysatoren, (ii) fremhæver måder at optimere XF-assays ved hjælp af enkelt 3D-sfæroider og (iii) diskuterer vigtige overvejelser og begrænsninger ved sondering af 3D-sfæroidmetabolisme ved hjælp af denne tilgang. Vigtigst af alt beskriver dette papir, hvordan datasæt indsamles, der gør det muligt at beregne iltforbrugshastighed (OCR) for at bestemme oxidativ phosphorylering og dermed mitokondriefunktion i cellulære sfæroider. Selvom det ikke analyseres for denne protokol, er ekstracellulær forsuringshastighed (ECAR) en anden parameter, der måles sammen med OCR-data i XF-eksperimenter. ECAR fortolkes dog ofte dårligt eller forkert fra XF-datasæt. Vi giver en kommentar til begrænsningerne ved beregning af ECAR efter grundlæggende tilgange fra teknologiproducenten.

Protocol

Figur 1: Grafisk arbejdsgang til generering af cellulære sfæroider, ekstracellulær fluxanalyse og downstream-assays. Fire kræftcellelinjer blev selektivt dyrket som monolag (A), løsrevet fra vævskulturkolber og podet i ultralave vedhæftede 96-brønd mikroplader til dannelse af sfæroider (B). A549 lungecarcin…

Representative Results

For at opnå velformede, kompakte sfæroider blev hver cellelinje optimeret individuelt til såningstæthed og dyrkningsvarighed (figur 3). A549, HepG2/C3A og SK-OV-3 cellelinjer dannede oprindeligt løse aggregater, der ikke gik videre til runde sfæroider med klart definerede perimeter før efter 7 dage i kultur. Omvendt kan MCF-7-celler danne sfæroider inden for 3 dage. Der var en klar sammenhæng mellem den indledende cellesåningstæthed og sfæroidvolumen efter dyrkningsperioden for a…

Discussion

Vigtigste resultater og resultater
Dette papir giver en detaljeret protokol til sonde mitokondrie energimetabolisme af enkelt 3D-sfæroider ved hjælp af en række kræftafledte cellelinjer med XFe96 XF Analyzer. En metode er udviklet og beskrevet til hurtig dyrkning af A549, HepG2 / C3A, MCF7 og SK-OV-3 cellulære sfæroider ved hjælp af celleafvisende teknologier til tvungen aggregering. Denne protokol omhandler mange overvejelser om sondering af sfæroidmetabolisme med XF-teknologi, herunder (1) o…

Disclosures

The authors have nothing to disclose.

Acknowledgements

N.J.C blev støttet af en BBSRC MIBTP CASE Award med Sygnature Discovery Ltd (BB/M01116X/1, 1940003)

Materials

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
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Tags

Mitochondrial Energy MetabolismExtracellular Flux AnalysisCell LinesCell NumbersPhenotypesDrug TreatmentsDrug DiscoveryCancer ResearchIn Vitro SpheroidsSpheroid ViabilitySensor CartridgeCalibrantAssay KitPoly-D-LysineXF RPMI Medium
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Coltman, N. J., Rochford, G., Hodges, N. J., Ali-Boucetta, H., Barlow, J. P. Exploring Mitochondrial Energy Metabolism of Single 3D Microtissue Spheroids Using Extracellular Flux Analysis. J. Vis. Exp. (180), e63346, doi:10.3791/63346 (2022).
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