Abstract
The use of three-dimensional (3D) cell culture systems is widely accepted as representing a more physiologically relevant means to propagate mammary epithelial and breast cancer cells. However, 3D cultures systems are plagued by several experimental and technical limitations as compared to their traditional 2D counterparts. For instance, quantifying the growth of mammary epithelial or breast cancer organoids longitudinally is particularly troublesome using standard [3H]thymidine or MTT assay systems, or using computer-assisted area calculations. Likewise, the nature of the multicellular aggregates and organoids formed by breast cancer cells under 3D conditions precludes efficient recovery of the cells from 3D matrices, an event that is time consuming and leads to spurious results. The assay described here utilizes stable expression of firefly luciferase as means to quantify the longitudinal outgrowth of cells propagated within a 3D matrices. The major advantages of this technique include its high-throughput nature and ability to longitudinally track single wells over a defined period of time, thereby decreasing the costs associated with assay performance. Finally, this technique can be readily combined with drug treatments and/or genetic manipulations to assay their effects on the growth of 3D organoids.