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

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.