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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Optical coherence tomography (OCT), a three-dimensional imaging technology, was used to monitor and characterize the growth kinetics of multicellular tumor spheroids. Precise volumetric quantification of tumor spheroids using a voxel counting approach, and label-free dead tissue detection in the spheroids based on intrinsic optical attenuation contrast, were demonstrated.

Abstract

Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, high-throughput imaging modalities utilizing bright field or fluorescence detection, are unable to resolve the overall 3D structure of the tumor spheroid due to limited light penetration, diffusion of fluorescent dyes and depth-resolvability. Recently, our lab demonstrated the use of optical coherence tomography (OCT), a label-free and non-destructive 3D imaging modality, to perform longitudinal characterization of multicellular tumor spheroids in a 96-well plate. OCT was capable of obtaining 3D morphological and physiological information of tumor spheroids growing up to about 600 µm in height. In this article, we demonstrate a high-throughput OCT (HT-OCT) imaging system that scans the whole multi-well plate and obtains 3D OCT data of tumor spheroids automatically. We describe the details of the HT-OCT system and construction guidelines in the protocol. From the 3D OCT data, one can visualize the overall structure of the spheroid with 3D rendered and orthogonal slices, characterize the longitudinal growth curve of the tumor spheroid based on the morphological information of size and volume, and monitor the growth of the dead-cell regions in the tumor spheroid based on optical intrinsic attenuation contrast. We show that HT-OCT can be used as a high-throughput imaging modality for drug screening as well as characterizing biofabricated samples.

Introduction

Cancer is the second leading cause of death in the world1. Developing drugs targeting cancer is of crucial importance for patients. However, it is estimated that more than 90% of new anti-cancer drugs fail in the development phase because of a lack of efficacy and unexpected toxicity in clinical trials2. Part of the reason can be attributed to the use of simple two-dimensional (2D) cell culture models for compound screening, which provide results with limited predictive values of compound efficacy and toxicity for the following stages of drug discovery2,3,

Protocol

1. Preparation of Cells

  1. Obtain cell lines from a qualified supplier.
    NOTE: Verify that cells from the cell lines of interest can form spheroid in the culture media or with the help of a substrate (basement membrane matrix like Matrigel). Look into the literature9 or perform one round of a pre-experiment for a check.
  2. Thaw the frozen cells following the specific procedure provided by the cell-line supplier. A general procedure can be found elsewhere43

Representative Results

High Throughput Optical Coherence Tomography Imaging of Spheroids in a 96-well Plate

Figure 3 exhibits the result of HT-OCT scanning of a 96-well plate with HCT 116 tumor spheroids on Day 3. The sequential scan of the whole plate starts from the bottom-right well (H12). Figure 3B shows the flow chart of the software implementation of the HT-OCT system. After one spheroid d.......

Discussion

Tumor activity is highly relevant to its morphological structure. Similar to monitoring characteristic growth curve for 2D cell cultures, tracking the growth curve for 3D tumor spheroids is also a conventional approach to characterize the long-term spheroid growth behavior for different cell lines. Notably, we can characterize the drug response by analyzing tumor degradation or tumor regrowth directly reflected in the growth curve. Therefore, quantitative assessment of 3D tumor spheroids, including the size and volume, t.......

Acknowledgements

This work was supported by NSF grants IDBR (DBI-1455613), PFI:AIR-TT (IIP-1640707), NIH grants R21EY026380, R15EB019704 and R01EB025209, and Lehigh University startup fund.

....

Materials

NameCompanyCatalog NumberComments
Custom Spectral Domain OCT imaging systemDeveloped in our lab
Superluminescent Diode (SLD)ThorlabsSLD1325light source
2×2 single mode fused fiber coupler, 50:50 splitting ratioAC PhotonicsWP13500202B201
Reference Arm
Lens TubeThorlabs
AdapterThorlabs
Collimating LensThorlabsAC080-020-C
Focusing LensThorlabs
Kinematic Mirror MountThorlabs
MirrorThorlabs
1D Translational StageThorlabs
Continuous neutral density filterThorlabs
Pedestrial PostThorlabs
Clamping ForkThorlabs
Sample Arm
Lens TubeThorlabs
AdapterThorlabs
Collimating LensThorlabsAC080-020-C
GalvanometerThorlabs
Relay LensThorlabsAC254-100-Ctwo Relay lens to make a telescope setup
Triangle Mirror MountThorlabs
MirrorThorlabs
ObjectiveMitutoyo
Pedestrial PostThorlabs
Clamping ForkThorlabs
Polarization ControllerThorlabs
30mm Cage MountThorlabs
Cage RodThorlabs
Stage
3D motorized translation stageBeijing Mao Feng Optoelectronics Technology Co., Ltd.JTH360XY
2D Tilting Stage
Rotation Stage
Plate Holder3D printed
Spectrometer
Lens TubeThorlabs
AdapterThorlabs
Collimating LensThorlabsAC080-020-C
GratingWasatchG = 1145 lpmm
F-theta LensThorlabsFTH-1064-100
InGaAs Line-scan CameraSensor UnlimitedSU1024-LDH2
NameCompanyCatalog NumberComments
Cell Culture Component
HCT 116 Cell lineATCCCCL-247
Cell Culture FlaskSPL Life Sciences70025
PipetteFisherbrand14388100
Pipette tipsSorenson Bioscience10340
Gibco GlutaMax DMEMThermo Fisher Scientific10569044
Fetal Bovine Serum, certified, US originThermo Fisher Scientific16000044
Antibiotic-Antimycotic (100X)Thermo Fisher Scientific15240062
Corning 96-well Clear Round Bottom Ultra-Low Attachment MicroplateCorning7007
Gibco PBS, pH 7.4Thermo Fisher Scientific10010023
Gibco Trypsin-EDTA (0.5%)Thermo Fisher Scientific15400054
Forma Series II 3110 Water-Jacketed CO2 IncubatorsThermo Fisher Scientific3120
GlovesVWR89428-750
ParafilmSigma-AldrichP7793
Transfer pipetsGlobe Scientific138080
CentrifugeEppendorf5702 RTo centrifuge the 15 mL tube
CentrifugeNUAIREAWEL CF 48-RTo centrifuge the 96-well plate
MicroscopeOlympus
NameCompanyCatalog NumberComments
Histology & IHC
Digital slide scannerLeicaAperio AT2Obtain high-resolution histological images
Histology ServiceHistowizRequest service for histological and immunohistological staining of tumor spheroid
NameCompanyCatalog NumberComments
List of Commerical OCTs
SD-OCT systemThorlabsTelesto Series
SD-OCT systemWasatch PhotonicsWP OCT 1300 nm
NameCompanyCatalog NumberComments
Software for Data Analyses
Basic Image AnalysisNIHImageJFiji also works.
3D RenderingThermo Fisher ScientificAmiraCommercial software. Option 1
3D RenderingBitplaneImarisCommercial software. Option 2. Used in the protocol
OCT acquisition softwarecustom developed in C++.
Stage ControlBeijing Mao Feng Optoelectronics Technology Co., Ltd.MRC_3Incorporated into the custom OCT acquisition code
OCT processing softwarecustom developed in C++. Utilize GPU. Incorporated into the custom OCT acquisition code.
Morphological and Physiological Analysiscustom developed in MATLAB

References

  1. Kola, I., Landis, J. Can the pharmaceutical industry reduce attrition rates?. Nature Reviews Drug Discovery. 3 (8), 711-716 (2004).
  2. Breslin, S., O'Driscoll, L. Three-dimensional cell culture: the missing link in drug discovery.

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3D Tumor SpheroidsOptical Coherence Tomography3D Cell CultureAnti cancer Drug DiscoveryCell SeedingCell SuspensionUltra low Attachment PlateCentrifugationCell ConcentrationCell MonitoringIncubationMedium RefreshOCT System Construction

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