Method Article

Beeldverwerking en flowcytometrie-gebaseerde analyse van de Cel standpunt en de celcyclus in 3D Melanoom Sferoïden

DOI:

10.3791/53486

⸱

December 28th, 2015

 ,  ,  ,  , 
1The Centenary Institute, 2Sydney Medical School, University of Sydney, 3The University of Queensland Diamantina Institute, Translational Research Institute, The University of Queensland, 4Department of Dermatology, Royal Prince Alfred Hospital, 5Discipline of Dermatology, University of Sydney

In This Article

Summary

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We describe two complementary methods using the fluorescence ubiquitination cell cycle indicator (FUCCI) and image analysis or flow cytometry to identify and isolate cells in the inner G1 arrested and outer proliferating regions of 3D spheroids.

Abstract

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Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to traditional two-dimensional (2D) cell culture. The spheroid model is able to mimic the effects of cell-cell interaction, hypoxia and nutrient deprivation, and drug penetration. One characteristic of this model is the development of a necrotic core, surrounded by a ring of G1 arrested cells, with proliferating cells on the outer layers of the spheroid. Of interest in the cancer field is how different regions of the spheroid respond to drug therapies as well as genetic or environmental manipulation. We describe here the use of the fluorescence ubiquitination cell cycle indicator (FUCCI) system along with cytometry and image analysis using commercial software to characterize the cell cycle status of cells with respect to their position inside melanoma spheroids. These methods may be used to track changes in cell cycle status, gene/protein expression or cell viability in different sub-regions of tumor spheroids over time and under different conditions.

Introduction

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Multicellulaire sferoïden 3D zijn bekend als een tumormodel decennia, maar het is pas onlangs aan dat zij vaker gebruik als een in vitro model voor vele vaste kankers zijn gekomen. Ze worden steeds vaker gebruikt in high-throughput drug discovery schermen als intermediair tussen complexe, kostbare en tijdrovende in vivo modellen en de eenvoudige, goedkope 2D monolaag model 1-6. Studies in 2D cultuur zijn vaak niet in staat om te worden gerepliceerd in vivo. Bolvormige modellen van vele soorten kanker kunnen de groeikenmerken, drug gevoeligheid, geneesmiddelpenetratie, cel-cel interacties, beperkte beschikbaarheid van zuu....

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Protocol

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1. Fucci Transductie en Cultuur van de Cel

  1. Fucci transductie
    1. Maak cellijnen stabiel tot expressie de Fucci construeert mKO2-hCdt1 (30-120) en MAG-hGem (1-100) 15 met behulp van lentivirus co-transductie zoals eerder beschreven 7.
      Opmerking: Het Fucci systeem is nu in de handel verkrijgbaar.
    2. Genereren sub-klonen met heldere fluorescentie door eencellige sorteren. Sorteer enkele cellen positief voor zowel AG en KO (geel) van fluorescentie-geactiveerde celsortering in een 96-putjes plaat zoals hierboven beschreven 7,16.
  2. Melanoom celcultuur
    1. Culture C8161 humane melanoomcell....

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Results

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Er zijn verschillende methoden voor het produceren van bolvormige tumoren, dit protocol gebruikt de niet-hechtende groei methode, waarbij cellen gekweekt op agar of agarose 3,7,9. Figuur 1 toont een voorbeeld van een melanoom C8161 sferoïde na 3 dagen op agar. C8161 sferoïden vormen normaal formaat bolvormige deeltjes met een diameter van 500 - 600 urn (gemiddelde = 565, SD = 19, n = 3) na 3 dagen. Andere melanoom cellijnen die sferoïden zullen vormen, omvatten: WM793, WM983C, WM983B, WM164, .......

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Discussion

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Semi-automatische beeldanalyse geïdentificeerd de bolvormige innerlijke G1 gearresteerd regio en uitdijende buitenste lagen. Deze werkwijze kan worden toegepast voor levende sferoïden behulp van een optische sectie of in vaste bolvormige gedeelten, veranderingen in niet alleen de celcyclus maar merkerexpressie (via immunofluorescentie), celdood of celmorfologie in de verschillende regio's te identificeren. Cel beweeglijkheid binnen de verschillende bolvormige regio kan ook worden gekwantificeerd - als live-confocale.......

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Disclosures

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PerkinElmer contributed to the costs of publication.

Acknowledgements

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We thank Ms. Danae Sharp and Ms. Sheena Daignault for technical assistance. We thank Dr. Atsushi Miyawaki, RIKEN, Wako-city, Japan, for providing the FUCCI constructs, Dr. Meenhard Herlyn and Ms. Patricia Brafford, The Wistar Institute, Philadelphia, for providing cell lines, the Imaging and Flow Cytometry Facility at the Centenary Institute for outstanding technical support. We thank Mr. Chris Johnson and Dr. Andrew Barlow for Volocity software technical support. N.K.H. is a Cameron fellow of the Melanoma and Skin Cancer Research Institute, Australia. K.A.B. is a fellow of the Cancer Institute New South Wales (13/ECF/1-39). W.W. is a fellow of the Cancer Institute Ne....

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Hoechst 33342Life TechnologiesH3570
agarose low melting pointLife Technologies16520-050For sectioning
noble agar SigmaA5431For making spheroids
agarose for spheroidsFisher ScientificBP1356-100For making spheroids
0.05% trypsin/EDTALife Technologies25300-054
HBSSLife Technologies14175-103
10% formalinSigmaHT5014-1CSCAUTION: Harmful, corrosive. Use Personal Protective Equipment, do  not breath fumes (open in a fume cupboard).
live/dead near IRLife TechnologiesL10119
vibratomeTechnical Products International, Inc
coulture cupThermo-Fisher ScientificSIE936Mold for sectioning spheroids
hemocytometerSigmaZ359629
96-well tissue culture plateInvitroFAL353072
collagenaseSigmaC5138 
confocal microscopeLeicaTCS SP5
Flow cytometer analyserBecton DickinsonLSRFortessa
volocityPerkinElmerImaging software
flowjoTree StarFlow cytometry software
Vaccuum greaseSigmaZ273554
Mounting mediaVector LaboratoriesH1000
FUCCI (commercial constructs)Life TechnologiesP36238Transient transfection only
Cell strainer 70 μmIn VitroFAL352350
Round bottom 5 ml tubes (sterile)In VitroFAL352003
Round bottom 5 ml tubes (non-sterile)In VitroFAL352008

References

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  1. LaBarbera, D. V., Reid, B. G., Yoo, B. H. The multicellular tumor spheroid model for high-throughput cancer drug discovery. Expert opinion on drug discovery. 7, 819-830 (2012).
  2. Beaumont, K. A., Mohana-Kumaran, N., Haass, N. K. Modeling Melanoma In Vitro and In Vivo. Health....

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Tags

3D Melanoma SpheroidsCell Cycle AnalysisFlow CytometryFUCCI SystemConfocal ImagingImage AnalysisCell Position TrackingSpheroid SectioningNecrotic CoreProliferating Cells

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