Isolation and establishment of cancer cell lines from tumor tissues using membrane filtration method: Model experiments for isolation of cancer cell lines

Cancer-initiating cells (CICs) or cancer stem cells (CSCs) generally comprise a few percent of the total tumor cell population. However, it is difficult to identify and distinguish CICs or CSCs from ordinal tumor cells. Currently, no reliable surface marker for colon CICs and CSCs is available. Therefore, it is not useful to isolate CICs or CSCs using conventional separation methods, such as the magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS) methods, which rely on surface markers of CICs or CSCs. For isolation of CICs or CSCs, we will design a membrane filtration method to enrich CICs (CSCs) from several colon cancer cell lines (HT29 cells, CoLo205 and LoVo cells) and from ovarian cancer cell lines (ES-2, OVCAR3, and SKOV-3 cells) via nylon mesh filter membranes with 11 and 20 μm pore sizes and poly(lactide-co-glycolic acid)/silk screen (PLGA/silk screen) porous membranes (pore sizes of 20-30 μm). The cancer cell solution containing fibroblasts will be filtered through the membranes to obtain a permeate solution. Subsequently, the cell culture medium will be filtered through the membranes to collect the recovery solution where the cells attached to the membranes will be rinsed off into the recovery solution. Then, the membranes will be cultivated in the cultivation medium to collect the migrated cells from the membranes. We will evaluate which isolated cells, i.e., cells in permeate solution or recovery solution or the migrated cells will have (a) (a) higher expression of cell tracker-expressing cells, (b) higher expression of the CSC surface markers (CD44 and CD133), and (c) higher colony formation ability than the cancer cells cultivated on conventional tissue culture plates (control). The goal of this research is to establish a method to enrich the CICs (CSCs) of cancer cells by the membrane filtration method developed in this study.