Longitudinal Morphological and Physiological Monitoring of Three-dimensional Tumor Spheroids Using Optical Coherence Tomography

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 discovery^2,3,

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.
3. Culture the cells for 1-2 passages in 25 cm² culture flasks. The cells are then ready to use for 3D cell culture.

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.......

Materials

Name	Company	Catalog Number	Comments
Custom Spectral Domain OCT imaging system			Developed in our lab
Superluminescent Diode (SLD)	Thorlabs	SLD1325	light source
2×2 single mode fused fiber coupler, 50:50 splitting ratio	AC Photonics	WP13500202B201
Reference Arm
Lens Tube	Thorlabs
Adapter	Thorlabs
Collimating Lens	Thorlabs	AC080-020-C
Focusing Lens	Thorlabs
Kinematic Mirror Mount	Thorlabs
Mirror	Thorlabs
1D Translational Stage	Thorlabs
Continuous neutral density filter	Thorlabs
Pedestrial Post	Thorlabs
Clamping Fork	Thorlabs
Sample Arm
Lens Tube	Thorlabs
Adapter	Thorlabs
Collimating Lens	Thorlabs	AC080-020-C
Galvanometer	Thorlabs
Relay Lens	Thorlabs	AC254-100-C	two Relay lens to make a telescope setup
Triangle Mirror Mount	Thorlabs
Mirror	Thorlabs
Objective	Mitutoyo
Pedestrial Post	Thorlabs
Clamping Fork	Thorlabs
Polarization Controller	Thorlabs
30mm Cage Mount	Thorlabs
Cage Rod	Thorlabs
Stage
3D motorized translation stage	Beijing Mao Feng Optoelectronics Technology Co., Ltd.	JTH360XY
2D Tilting Stage
Rotation Stage
Plate Holder			3D printed
Spectrometer
Lens Tube	Thorlabs
Adapter	Thorlabs
Collimating Lens	Thorlabs	AC080-020-C
Grating	Wasatch		G = 1145 lpmm
F-theta Lens	Thorlabs	FTH-1064-100
InGaAs Line-scan Camera	Sensor Unlimited	SU1024-LDH2
Name	Company	Catalog Number	Comments
Cell Culture Component
HCT 116 Cell line	ATCC	CCL-247
Cell Culture Flask	SPL Life Sciences	70025
Pipette	Fisherbrand	14388100
Pipette tips	Sorenson Bioscience	10340
Gibco GlutaMax DMEM	Thermo Fisher Scientific	10569044
Fetal Bovine Serum, certified, US origin	Thermo Fisher Scientific	16000044
Antibiotic-Antimycotic (100X)	Thermo Fisher Scientific	15240062
Corning 96-well Clear Round Bottom Ultra-Low Attachment Microplate	Corning	7007
Gibco PBS, pH 7.4	Thermo Fisher Scientific	10010023
Gibco Trypsin-EDTA (0.5%)	Thermo Fisher Scientific	15400054
Forma Series II 3110 Water-Jacketed CO2 Incubators	Thermo Fisher Scientific	3120
Gloves	VWR	89428-750
Parafilm	Sigma-Aldrich	P7793
Transfer pipets	Globe Scientific	138080
Centrifuge	Eppendorf	5702 R	To centrifuge the 15 mL tube
Centrifuge	NUAIRE	AWEL CF 48-R	To centrifuge the 96-well plate
Microscope	Olympus
Name	Company	Catalog Number	Comments
Histology & IHC
Digital slide scanner	Leica	Aperio AT2	Obtain high-resolution histological images
Histology Service	Histowiz		Request service for histological and immunohistological staining of tumor spheroid
Name	Company	Catalog Number	Comments
List of Commerical OCTs
SD-OCT system	Thorlabs	Telesto Series
SD-OCT system	Wasatch Photonics	WP OCT 1300 nm
Name	Company	Catalog Number	Comments
Software for Data Analyses
Basic Image Analysis	NIH	ImageJ	Fiji also works.
3D Rendering	Thermo Fisher Scientific	Amira	Commercial software. Option 1
3D Rendering	Bitplane	Imaris	Commercial software. Option 2. Used in the protocol
OCT acquisition software			custom developed in C++.
Stage Control	Beijing Mao Feng Optoelectronics Technology Co., Ltd.	MRC_3	Incorporated into the custom OCT acquisition code
OCT processing software			custom developed in C++. Utilize GPU. Incorporated into the custom OCT acquisition code.
Morphological and Physiological Analysis			custom developed in MATLAB