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| - Traditionally, two-dimensional (2D) monolayer cell culture models have been used to study in vitro conditions for their ease of use, simplicity and low cost. However, recently, three-dimensional (3D) cell culture models have been heavily investigated as they provide better physiological relevance for studying various disease behaviors, cellular activity and pharmaceutical interactions. Typically, small-sized tumor spheroid models (100–500 μm) are used to study various biological and physicochemical activities. Larger, millimetric spheroid models are becoming more desirable for simulating native tumor microenvironments (TMEs). Here, we assess the use of ultra-large spheroid models (~2000 μm) generated from scaffolds made from a nozzle-free, ultra-high resolution printer; these models are explored for assessing chemotherapeutic responses with molecular doxorubicin (DOX) and two analogues of Doxil(Ⓡ) (Dox-NP(Ⓡ), Doxoves(TM)) on MDA-MB-231 and MCF-7 breast cancer cell lines. To provide a comparative baseline, small spheroid models (~500 μm) were developed using a self-aggregation method of MCF-7 breast cancer cell lines, and underwent similar drug treatments. Analysis of both large and small MCF-7 spheroids revealed that Dox-NP tends to have the highest level of inhibition, followed by molecular doxorubicin and then Doxoves. The experimental advantages and drawbacks of using these types of ultra-large spheroids for cancer research are discussed.
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