Potential capacities of different intestinal epithelial cell lines to form enteroids in 3D culture system: comparison and application

Abstract

Objective To compare the potentiality and characteristics of different intestinal epithelial cell (IEC) lines to grow into enteroids when cultured in 3D system in order to provide experimental basis for the cultivation of enteroids by IECs. Methods Three IEC lines, IEC-6, CT26.WT and Caco-2, were cultured in a 3D system, and further characterized by morphology. Single Caco-2 cell was acquired by fluorescent activated cell sorting (FACS) and seeded in Matrigel. The growth speed of enteroids derived from single Caco-2 cell was determined. The expression levels of intestinal stem cell (ISC) genes, including Lgr5 and Olfm4, and proliferative marker, Ki67 were determined by qRT-PCR, and the results were compared between 2D and 3D cultured Caco-2 cells. The distribution of Ki67-positive cells and lamp1-positive lysosomes in 2D and 3D cultured Caco-2 cells were observed with aid of immunofluorescent staining. TUNEL staining was employed to analyze the apoptosis of Caco-2 cells after 5 and 15 Gy radiation, respectively. Results Among the 3 IEC lines, only Caco-2 cells could grow into enteroids when cultured in 3D system. Single Caco-2 cell acquired by FACS could also form enteroids. Compared to 2D culture, Caco-2 derived enteroids expressed lower mRNA levels for ISC markers Lgr5 and Olfm4, and also lower proliferative marker Ki67 (P < 0.05). There were less Ki67-positive cells in 3D than 2D cultured Caco-2 cells. Lysosomes in 3D cultured Caco-2 cells exhibited a polar distribution, which was similar to them in in vivo pattern. Radiation exposure caused apoptosis of IECs in Caco-2 derived enteroids, and the apoptotic rate was positively correlated to the dose of radiation. Conclusion Caco-2 cells could form enteroids when cultured in 3D system, which is similar to the structure of in vivo intestine in structure and function. This study provides reliable in vitro model for gastrointestinal radiation and pharmaceutical evaluation in the future.

Keywords

• intestinal epithelial cells
• caco-2 cells
• enteroid
• stem cells
• ionizing radiation