Three-dimensional spheroid culture

Abstract

Monolayer cultures have been the preferred method for testing the activity of anticancer drug candidates in vitro. However, cell based assays performed in monlayer culture provides data from cells in different state lacking many of the characteristics of the tumor environment such as the necrotic core, hypoxic region and low pH at core. A novel approach to overcome the drawbacks of monolayer cultures is the three-dimensional in vitro culture also knows as multi-cellular tumor spheroids or Spheroid culture or three-dimensional culture. Micro tumor tissues produced by spheroid culture helps to mimic many of the characteristics of tumor environment as well as helps to produce data more relevant to in vivo tumors. In this report, we optimized various techniques of culturing spheroids and worked towards making spheroid culture quick, accurate, and high-throughput. Two methods of spheroid culture were examined, non-adhesive liquid overlay with and without Reconstituted Basement Membrane (RBM / Matrigel) and hanging drop method. Non-adhesive liquid overlay without RBM was found to be the most effective. These spheroids were consistent with respect to size, shape and cell number. The spheroids generated by this method were utilized to evaluate cancer cell behavior in three dimension. To determine the cytotoxicity of paclitaxel in spheroids the spheroid cultures after treatment with a drug, the activity of phosphatase enzyme, using pNPP as the substrate, was measured. The cytotoxicity of paclitaxel in spheroids was then compared to the cytotoxicity in monolayer cultures of A549 cells. The results showed significant reduction in cytotoxicity of paclitaxel on A549 cells when treated in spheroid culture compared to monolayer cultures. Three-dimensional spheroids of A549 cells did demonstrate inherent drug resistance demonstrated by many in vivo tumors . The reduced cytotoxicity of paclitaxel could be due to inhibited drug penetration resulting from cell-cell interaction and presence of cells in different cell cycle stages. It is, therefore, cell cycle analysis was carried out on A549 cells grown as spheroids and compared with cells in monolayer culture using propidium iodide. Results indicated presence of significantly higher population in G0/G1 phase and reduced population in S phase in spheroids compared to monolayer culture. Based on the results obtained, we believe use of spheroids in cancer research could provide a unique platform to conduct previously unfeasible experiments and provide valuable insights for cancer cell behavior in tissue like structure.