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Assessment of Chemosensitivity | Formation of Co-Culture Spheroids | Assay Performance

Assessment of Chemosensitivity using Perfecta3D™ Hanging Drop Culture Plates

Experimental results obtained using the patent-pending Perfecta3D™ Hanging Drop Plates were first published in 2011. In this study, which was featured on the cover of the journal Analyst, Takayama and his team of researchers at the University of Michigan demonstrated that drugs with different modes of action produce distinct responses in the physiological 3D spheroids compared to conventional 2D cell monolayers.

Spheroid formation

COS7 African green monkey kidney fibroblasts, ES-D3 murine embryonic stem (mES) cells, and A431.H9 human epithelial carcinoma cells were cultured in hanging drops using the Perfecta3D™ Hanging Drop Plates to form spheroids. While mES cells readily formed spheroids on Day 1, the COS7 cells formed smaller aggregates initially that later assembled into a larger spheroid. Live/dead assay indicated that more than 90% of the cells remained alive after 12 days of culture, demonstrating that the Perfecta3D™ Hanging Drop Plates are suitable for long-term spheroid cultures.

A431.H9 cells were seeded at various cell densities and the size of the spheroids was recorded over a 7-day culture period. The data shows that cells remained proliferative by the end of the culture period. Moreover, it shows that spheroid size can be fine-tuned by altering the cell seeding density and the length of culture period. This is important since the size of cell colonies has been reported to affect the differentiation of stem cells.

2D versus 3D

Two anti-cancer drugs with distinctly different activity profiles were used to assess chemosensitivity: 5-fluorouracil (5-FU) is a conventional compound that inhibits cellular proliferation, and tirapazamine (TPZ) is a hypoxia-trigger cytotoxin that causes DNA damage. Our data shows that A431.H9 cells are more resistant to 5-FU under 3D spheroid than 2D culture conditions. However, the opposite is true for treatment with TPZ. Our data indicates that A431.H9 cells are more resistant to TPZ when cultured under 2D than 3D spheroid culture conditions.

5-FU primarily targets proliferating cells. Therefore, it is more effective against the rapidly proliferating cells in 2D monolayer culture and would not kill the quiescent cells in the spheroids. In contrast, TPZ is a hypoxia-activated cytotoxin, so it is more effective in the spheroids, where active oxygen consumption by cells and limits in diffusive oxygen transport create a hypoxic core similar to that of solid tumors.

Chemosensitivity testing

The IC50 of A431.H9 cells treated with FU-5 is about 0.1 μM in 2D condition and 1 to 100 μM, depending on cell density, in 3D spheroid culture. For the TPZ treated cells, the IC50 is about 50 μM when cultured in 2D and 8 μM for all spheroid sizes. Such distinctly and dramatically different responses from the same cells to the same drugs tested under different culture conditions illustrates the importance and unmistaken need of using 3D models in drug screening and testing.

Combination drug treatment

The combined treatment of 5-FU and TPZ is more effective at killing the A431.h9 cells. At a drug concentration of 10 μM, cell viability decreased from 75% and 40% when treated with 5-FU and TPZ, respectively, to only 20% when the spheroids received the combined treatment. By combining two drugs of distinctly different mechanisms, both the proliferating cells in the peripheral layer and cells in the hypoxic core of the spheroids can be targeted effectively.

Data and images adapted from: Tung et al. High-throughput 3D spheroid culture and drug testing using 384 hanging drop array. Analyst, 2011, 136, 473-478.
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