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.

1. Preparation of Cells
<ol>
	<li>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.</li>
	<li>Thaw the frozen cells following the specific procedure provided by the cell-line supplier. A general procedure can be found elsewhere43</...

The authors disclose no competing interest.

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...

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 discovery2,3,4. Recently, three-dimensional (3D) tumor spheroid models have been developed to provide clinically relevant physiological and pharmacological data for anti-cancer drug discovery3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25. Since these spheroids can mimic tissue-specific properties of tumors in vivo, such as nutrient and oxygen gradient, hypoxic core as well as drug resistance19, the use of these models can potentially shorten drug discovery timelines, reduce costs of investment, and bring new medicines to patients more effectively. One critical approach to evaluating compound efficacy in 3D tumor spheroid development is to monitor the spheroid growth and recurrence under treatments9,26. To do this, quantitative characterizations of the tumor morphology, involving its diameter and volume, with high-resolution imaging modalities, are imperative. 
Conventional imaging modalities, such as bright-field, phase contrast7,9,22,24, and fluorescence microscopy8,9,16,18,22 can provide a measurement of the spheroid's diameter but cannot resolve the overall structure of the spheroid in 3D space. Many factors contribute to these limitations, including penetration of the probing light in the spheroid; diffusion of the fluorescent dyes into the spheroid; emitting fluorescent signals from excited fluorescent dyes inside or on the opposite surface of the spheroid due to strong absorption and scattering; and depth-resolvability of these imaging modalities. This often leads to an inaccurate volume measurement. Development of the necrotic core in spheroids mimics necrosis in in vivo tumors6,10,15,19,25. This pathological feature is unlikely reproduced in 2D cell cultures19,25,27,28. With a spheroid size larger than 500 &#181;m in diameter, a three-layer concentric structure, including an outer layer of proliferating cells, a middle layer of quiescent cells, and a necrotic core, can be observed in the spheroid6,10,15,19,25, due to lack of oxygen and nutrients. Live and dead cell fluorescence imaging is the standard approach to label the boundary of the necrotic core. However, again, penetrations of both these fluorescent dyes and visible light hinder the potential to probe into the necrotic core to monitor its development in its actual shape.
An alternative 3D imaging modality, optical coherence tomography (OCT) is introduced to characterize the tumor spheroids. OCT is a biomedical imaging technique that is capable of acquiring label-free, non-destructive 3D data from up to 1-2 mm depths in biological tissues29,30,31,32,33,34. OCT employs low-coherence interferometry to detect back-scattered signals from different depths of the sample and provides reconstructed depth-resolved images at micron-level spatial resolutions in both lateral and vertical directions. OCT has been widely adopted in ophthalmology35,36,37 and angiography38,39. Previous studies have used OCT to observe the morphology of in vitro tumor spheroids in basement membrane matrix (e.g., Matrigel) and evaluate their responses to photodynamic therapy40,41. Recently, our group established a high-throughput OCT imaging platform to systematically monitor and quantify the growth kinetics of 3D tumor spheroids in multi-well plates42. Precise volumetric quantification of 3D tumor spheroids using a voxel counting approach and label-free necrotic tissue detection in the spheroids based on intrinsic optical attenuation contrast were demonstrated. This paper describes the details of how the OCT imaging platform was constructed and employed to obtain high-resolution 3D images of tumor spheroids. The step-by-step quantitative analyses of the growth kinetics of 3D tumor spheroids, including accurate measurements of spheroid diameter and volumes, is described. Also, the method of the non-destructive detection of necrotic tissue regions using OCT, based on the intrinsic optical attenuation contrast is presented.

<table>
	<tbody>
		<tr>
			<td>Custom Spectral Domain OCT imaging system</td>
			<td></td>
			<td></td>
			<td>Developed in our lab</td>
		</tr>
		<tr>
			<td>Superluminescent Diode (SLD)</td>
			<td>Thorlabs</td>
			<td>SLD1325</td>
			<td>light source</td>
		</tr>
		<tr>
			<td>2&#215;2 single mode fused fiber coupler, 50:50 splitting ratio</td>
			<td>AC Photonics</td>
			<td>WP13500202B201</td>
			<td></td>
		</tr>
		<tr>
			<td>Reference Arm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lens Tube</td>
			<td>Thorlabs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adapter</td>
			<td>Thorlabs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collimating Lens</td>
			<td>Thorlabs</td>
			<td>AC080-020-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Focusing Lens</td>
			<td>Thorlabs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Kinematic Mirror Mount</td>
			<td>Thorlabs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mirror</td>
			<td>Thorlabs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1D Translational Stage</td>
			<td>Thorlabs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Continuous neutral density filter</td>
			<td>Thorlabs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pedestrial Post</td>
			<td>Thorlabs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Clamping Fork</td>
			<td>Thorlabs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sample Arm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lens Tube</td>
			<td>Thorlabs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adapter</td>
			<td>Thorlabs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collimating Lens</td>
			<td>Thorlabs</td>
			<td>AC080-020-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Galvanometer</td>
			<td>Thorlabs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Relay Lens</td>
			<td>Thorlabs</td>
			<td>AC254-100-C</td>
			<td>two Relay lens to make a telescope setup</td>
		</tr>
		<tr>
			<td>Triangle Mirror Mount</td>
			<td>Thorlabs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mirror</td>
			<td>Thorlabs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Objective</td>
			<td>Mitutoyo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pedestrial Post</td>
			<td>Thorlabs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Clamping Fork</td>
			<td>Thorlabs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polarization Controller</td>
			<td>Thorlabs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>30mm Cage Mount</td>
			<td>Thorlabs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cage Rod</td>
			<td>Thorlabs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stage</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3D motorized translation stage</td>
			<td>Beijing Mao Feng Optoelectronics Technology Co., Ltd.</td>
			<td>JTH360XY</td>
			<td></td>
		</tr>
		<tr>
			<td>2D Tilting Stage</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rotation Stage</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plate Holder</td>
			<td></td>
			<td></td>
			<td>3D printed</td>
		</tr>
		<tr>
			<td>Spectrometer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lens Tube</td>
			<td>Thorlabs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adapter</td>
			<td>Thorlabs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collimating Lens</td>
			<td>Thorlabs</td>
			<td>AC080-020-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Grating</td>
			<td>Wasatch</td>
			<td></td>
			<td>G = 1145 lpmm</td>
		</tr>
		<tr>
			<td>F-theta Lens</td>
			<td>Thorlabs</td>
			<td>FTH-1064-100</td>
			<td></td>
		</tr>
		<tr>
			<td>InGaAs Line-scan Camera</td>
			<td>Sensor Unlimited</td>
			<td>SU1024-LDH2</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Cell Culture Component</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HCT 116 Cell line</td>
			<td>ATCC</td>
			<td>CCL-247</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Flask</td>
			<td>SPL Life Sciences</td>
			<td>70025</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette</td>
			<td>Fisherbrand</td>
			<td>14388100</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips</td>
			<td>Sorenson Bioscience</td>
			<td>10340</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco GlutaMax DMEM</td>
			<td>Thermo Fisher Scientific</td>
			<td>10569044</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, certified, US origin</td>
			<td>Thermo Fisher Scientific</td>
			<td>16000044</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic (100X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 96-well Clear Round Bottom Ultra-Low Attachment Microplate</td>
			<td>Corning</td>
			<td>7007</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco PBS, pH 7.4</td>
			<td>Thermo Fisher Scientific</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco Trypsin-EDTA (0.5%)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15400054</td>
			<td></td>
		</tr>
		<tr>
			<td>Forma Series II 3110 Water-Jacketed CO2 Incubators</td>
			<td>Thermo Fisher Scientific</td>
			<td>3120</td>
			<td></td>
		</tr>
		<tr>
			<td>Gloves</td>
			<td>VWR</td>
			<td>89428-750</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Sigma-Aldrich</td>
			<td>P7793</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer pipets</td>
			<td>Globe Scientific</td>
			<td>138080</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5702 R</td>
			<td>To centrifuge the 15 mL tube</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>NUAIRE</td>
			<td>AWEL CF 48-R</td>
			<td>To centrifuge the 96-well plate</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Histology &#38; IHC</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Digital slide scanner</td>
			<td>Leica</td>
			<td>Aperio AT2</td>
			<td>Obtain high-resolution histological images</td>
		</tr>
		<tr>
			<td>Histology Service</td>
			<td>Histowiz</td>
			<td></td>
			<td>Request service for histological and immunohistological staining of tumor spheroid</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>List of Commerical OCTs</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SD-OCT system</td>
			<td>Thorlabs</td>
			<td>Telesto Series</td>
			<td></td>
		</tr>
		<tr>
			<td>SD-OCT system</td>
			<td>Wasatch Photonics</td>
			<td>WP OCT 1300 nm</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software for Data Analyses</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Basic Image Analysis</td>
			<td>NIH</td>
			<td>ImageJ</td>
			<td>Fiji also works.</td>
		</tr>
		<tr>
			<td>3D Rendering</td>
			<td>Thermo Fisher Scientific</td>
			<td>Amira</td>
			<td>Commercial software. Option 1</td>
		</tr>
		<tr>
			<td>3D Rendering</td>
			<td>Bitplane</td>
			<td>Imaris</td>
			<td>Commercial software. Option 2. Used in the protocol</td>
		</tr>
		<tr>
			<td>OCT acquisition software</td>
			<td></td>
			<td></td>
			<td>custom developed in C++.</td>
		</tr>
		<tr>
			<td>Stage Control</td>
			<td>Beijing Mao Feng Optoelectronics Technology Co., Ltd.</td>
			<td>MRC_3</td>
			<td>Incorporated into the custom OCT acquisition code</td>
		</tr>
		<tr>
			<td>OCT processing software</td>
			<td></td>
			<td></td>
			<td>custom developed in C++. Utilize GPU. Incorporated into the custom OCT acquisition code.</td>
		</tr>
		<tr>
			<td>Morphological and Physiological Analysis</td>
			<td></td>
			<td></td>
			<td>custom developed in MATLAB</td>
		</tr>
	</tbody>
</table>

longitudinal morphological and physiological monitoring of three-dimensional tumor spheroids using optical coherence tomography

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 &#181;m 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.

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...

Watch this Scientific Journal Video about Longitudinal Morphological and Physiological Monitoring of Three-dimensional Tumor Spheroids Using Optical Coherence Tomography at JoVE.com

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

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.

Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, ...

Three-dimensional tumor spheroids can mimic tissue-specific properties of tumors in vivo, providing more clinically-relevant data for anti-cancer drug discovery than simple 2D cell culture. Our imaging technique, optical coherence tomography, can easily visualize the 3D structure of a single tumorous nerves, with a size of several hundred microns, and provide more accurate characterization of its morphology and job infest, often within a few seconds. Using 3D spherical model we can potentially shorten the drug discovery timeline, reduce cost and bring new medicine to patients more effectively.Forming the spherical shape of tumor cluster is one critical step in our experiment. It is important to choose the right ultra-low attachment plate with the round bottom, and the proper centrifugation speed after cell seeding. Start this experiment by growing cells of interesting in a culture flask as described in the manuscript.Maintain the cells in an incubator under standard conditions, and monitor their health status every day. Refresh the media as needed. To perform 3D cell culture in multi-well plates, first remove the medium from the cell culture flask, and wash the cells with sterilized, 33 degree Celsius pre-warmed PBS.Then add one milliliter of Trypsin-EDTA to resuspend the cells and incubate for three minutes at 37 degrees Celsius. Add three milliliters of culture medium to dilute the Trypsin. Transfer this cell suspension into a 15 milliliter centrifuge tube, and centrifuge at 500G and room temperature for five minutes.Then remove the supernatant and resuspend the cell pellet with four milliliters of pre-warmed culture medium. To determine cell concentration, hypat one drop of sample onto a hemocytometer, and count the cells. Dilute to desired seeding concentration.Seed 200 microliters of cell suspension into each well of an ultra-low attachment, round-bottom multi-well plate, at a concentration of 3, 000 cells per milliliter, to achieve about 600 cells per well. Immediately after seeding, centrifuge the whole plate at the lowest speed available, at room temperature, for seven minutes. Place the plate in the incubator at 37 degrees Celsius and 5%Co2, making sure to refresh the medium every three days.First, construct the reference arm and sample arm of the OCT system, following the schematics in this manuscript. Then, construct the spectrometer, including a kilometer, a grading, an F-Theta lens and a line scan camera. Use a plate adapter to hold the multi-well plate in a fixed position.Before imaging, correct the tilting and rotation of the multi-well plate using a 2D tilting stage, and a rotation stage mounted on the transition stage to minimize variation of the focal plane from different wells. Then adjust the rotation to ensure the edges of the plate are parallel with the direction of the stage movement, so that the wells remain at the same horizontal positions in the OCT images. Then adjust the tilting stage to ensure the plate is parallel to the optical table, so that the wells remain at the same vertical locations for the imaging.On the day of the tumor spheroids imaging, remove the multi-well plate from the incubator. Transfer it under the OCT imaging system, and place it on the plate adapter. To adjust the height of the plate, move it along the Z direction of the translation stage.In the custom imaging software, set a desired OCT scanning range to cover the whole tumor spheroid, depending on its developmental stages, and click save parameters to save the setting. Then show the optimized XZ and YZ OCT previews of tumor spheroids. Acquire 3D OCT images of tumor spheroids one-by-one for all the wells of the plate containing spheroids.To view the preview image, click the preview button. And to acquire the OCT image, click the acquire button. Record the overall stage moving process of OCT data acquisition.To ensure the optimal image quality for all the tumor spheroids, the plate adapter need to be tuned accurately and the median volume need to be the same. Use a custom C+processing code to process 3D OCT data sets of tumor spheroids to generate OCT structural images. Use 2D OCT images in three cross-sectional, XY, XZ, and YZ planes across the centroid of the spheroid to generate the collage of spheroid images.To obtain 3D rendering of the spheroid using a desired software, first load the SD OCT data into the software. Click the surpass panel, then add new volume, and choose the blend mode to use for 3D rendering. To adjust the viewing angle, use the mouse pointer to drag the image and proceed with quantification as described in the manuscript.A collage of unfazed OCT images of HCT116 cell line spheroids was generated from the processed data, with results comparable with images from other 2D high-throughput imaging systems. Furthermore, the collage of 2D cross-sectional spheroid images from 96 wells was generated to monitor spheroid heights, and visualize spheroid inhomogeneity in the vertical direction. A collage of 3D-rendered spheroid images can be generated from any pre-defined angle to visualize the overall 3D shape and evaluate the roundness of the spheroid.After general OCT post-processing, 3D OCT structural images of a tumor spheroid have been obtained. From the OCT data, a 3D surface post and XZ, YZ, and XY orthogonal slices were generated to visualize the structure of the tumor spheroid in any direction. Longitudinal monitoring of a single tumor spheroid was performed to characterize its diameter, height, and voxel-based volume, generating the growth curves in size and volume during the 21-day development.Here, the spheroid became disrupted on day 11, and fully collapsed on day 21. Longitudinal tracking showed the increase of dead cell areas in the tumor spheroid. 3D-rendered images of the tumor spheroid showed the appearance and growth of dead cell regions from day seven to day 14, as shown by increase of red highlighted necrotic areas.As the percentage of the necrotic areas increased, the tumor spheroid could not maintain its perfect shape, and hence collapsed. With this imaging platform we can further explore other complex tumor sphere models, such as 3D imaging model, vast creature model and co-culture models to better simulate in vivo tumors. Our high-throughput OCT imaging system can provide an alterative approach for drug screening in cancer drug discovery.This is something also characterized, 3D biofabricated samples for various biomedical applications.

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JoVE Journal

Cancer Research

7.4K 조회수.  Lehigh University. 3차원 종양 스페로이드는 생체 내에서 종양의 조직 별 특성을 모방할 수 있으며, 간단한 2D 세포 배양보다 항암 약물 발견을 위한 임상적으로 관련된 데이터를 제공할 수 있습니다. 우리의 이미징 기술, 광학 일관성 단층 촬영, 쉽게 수백 미크론의 크기와 단일 종양 신경의 3D 구조를 시각화 할 수 있습니다, 종종 몇 초 이내에, 그 형태와 작업 감염의 보다 정확한 특성을 제공 ?...