Immunocytochemical Analysis of a Three-dimensional Spheroidal Culture System

Abstract

Three-dimensional (3D) cell cultures are expected to mimic in vivo environments. The NanoCulture plate (Scivax Corporation, Kawasaki) allows the formation of 3D spheroids. The cytoskeletal proteins are considered to be important regulators of cellular morphology and structure. Among the cytoskeletal proteins, fibrillar actin (F-actin) is a main component of microfilament proteins, and α-tubulin is the major protein forming microtubules in cells. Therefore, in this study, we used NanoCulture plates to examine the expression patterns of F-actin and α-tubulin in PANC-1, a human pancreatic cancer cell line. The cells were stained with polyclonal antibodies against α-tubulin antibody and with Alexa 568-labeled phalloidin to detect F-actin. Eighty-eight horizontal images (Fig. 1) were obtained at 0.25-μm intervals with a confocal laser microscope (TE2000-E, Nikon Instech Co., Ltd., Tokyo). In horizontal images, the expression of F-actin was observed at the periphery of cells (Fig. 2A― C, red), and strong F-actin expression was observed on the grids of the NanoCulture plate at the levels of cells nearest the plate (Fig. 2C, arrows and inset). The expression of α-tubulin was observed in the cytoplasm of cells in 3D cultures (Fig. 2A―C, green). Vertical images revealed that the cells piled up in 3D culture (Fig. 2D) and that cells formed actin-rich cell projections that attached to the surface (Fig. 2D arrows). The 3D images Correspondence to Zenya Naito, MD, PhD, Departments of Pathology, Integrative Oncological Pathology, Nippon Medical School, 1―1―5 Sendagi, Bunkyo-ku, Tokyo 113―8602, Japan