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3D tumour spheroid models for in vitro therapeutic screening3D tumour spheroid models for in vitro therapeutic screening: a systematic approach to enhance the biological relevance of data obtained.3D tumour spheroid models for in vitro therapeutic screening

By Ian Kriel, Adam Nahor, Kaelyn Coates, and Resa Nelson

A map of the earth fails to accurately describe the shape of the planet when compared to a globe. Similarly, 2D cancer cultures fail to capture the scope, morphology, and organisation present in a tumor. Using 3D spheroids to replace 2D cell cultures, dynamics of oxygen exchange, cell-cell interactions, and drug penetration can be more faithfully represented. One challenge faced in 3D spheroid preparation is that results vary depending on preparation method. This paper demonstrated that proper spheroid preparation leads to consistent results.

The search for a cure to cancer has engaged researchers across the world in developing lifesaving technology that is invaluable to the lives of patients. One of the biggest tools used to combat this disease is the creation of models to determine treatments that work, and to do this, spheroids are most often used. While spheroids work very well as models, the major challenge current research faces is variability in spheroid preparation. Using several different methods and a screening algorithm, the most effective spheroid preparation procedures can be identified and optimal spheroids are selected for further testing.

This study investigated four methods of spheroid preparation, evaluated and selected the optimal spheroids and modelled the ability of drugs to enter a tumour using different types of dyes to penetrate the spheroids. First, to identify the best method for spheroid generation, a line of A549 cells – cancer cells isolated from a lung cancer patient – were prepared using four preparation methods: Magnetic Levitation, Hanging Drop, Pellet Cultures, and Rotating Wall Vessel. Of the 4 methods used in the study, the pellet culture method had a higher output of homogenous spheroids in the shortest amount of time. The rotating wall vessel also generated optimal spheroids, however required a longer period of time. Spheroids were screened using the Reconstruction and Visualization from a Single Projection (ReViSP) tool coded in MATLAB©. This program considers spheroid volume and sphericity index in the selection process of optimal spheroids.

Next, the researchers used light sheet fluorescence microscopy (LSF) to determine if the spheroids accurately reflect tumour anatomy. Using LSF, the researchers found that necrotic cells were localised in the core of the spheroid surrounded by a layer of quiescent cells and an outer layer of prolific cells, all of which are seen in patients with cancer.
Finally, the researchers used three cytotoxicity assays, CellTiter-Glo®3D, Perfecta3D (luminescent), and trypan blue (fluorescent) to evaluate structural disruption. The results found that after exposure to a cytotoxic drug for 72 hours, the structure of the spheroid was disrupted and observed using all three assays. When exposed to a radiation schedule similar to those in clinical practice, the spheroids began to deteriorate 25 days after treatment. Despite this, the different cytotoxicity assays had variability, the CellTiterGlo®3D assay had the lowest variability due to an increased ability to penetrate the spheroids. Overall this experiment shows how models can be used to predict how things will behave, a lesson important in all fields of science.

Citations

Zanoni, M., Piccinini, F., Arienti, C. et al. 3D tumour spheroid models for in vitro therapeutic screening: a systematic approach to enhance the biological relevance of data obtained. Sci Rep 6, 19103 (2016). https://doi.org/10.1038/srep19103.