In experiments with colon cancer cell lines, luminal-like structures were observed in horizontal sections of 3D cultures of DLD-1 cells (Fig. SqCC cells cultured in Cellbed coated with collagen IV showed enhanced invasive and proliferative abilities. Conclusion Because the morphology of cancer cells cultured in this 3D culture system is similar to that in living organisms, we called the system a tissueoid cell culture system. Coating with collagen IV enables the modification of cell-matrix interactions as well as recapitulation of the in vivo microenvironment. test was used for statistical analysis to evaluate the effects of collagens I, III, and IV on invasion depth and proliferative ability. Results Observation of Adherent Adenocarcinoma Cells Clear ductal luminal formations were observed in horizontal and vertical cross sections of 3D cultures of OE-19 cells (Fig. 2a, b). Additionally, immunostaining was successfully performed using the first antibody of MUC1 (Fig. ?(Fig.2c).2c). In experiments with colon cancer cell lines, luminal-like structures were observed in horizontal sections of 3D cultures of DLD-1 cells (Fig. ?(Fig.2d).2d). In the vertical sections of 3D cultures, DLD-1 cells partly exhibited polarity and were regularly aligned on the surface of Cellbed (Fig. ?(Fig.2e2e). Open in a separate windows Fig. 2 Adenocarcinoma cells (adherent cells). a A horizontal section of OE-19 cells cultured for 4 weeks. Scale bar, 100 m. HE. A luminal structure was detected in the gland. b A vertical section from the 4-week culture. HE. c MUC1 was positive around the luminal surface. Scale bar, 200 m. d A horizontal section of DLD-1 cells cultured for 3 weeks. Lumina-like structures of the glandular cavity were visible (arrow). Scale bar, 100 m. HE. e A vertical section of DLD-1 cells cultured for 4 weeks. Scale bar, 200 m. HE. We partly detected that columnar cells exhibited polarity and were regularly aligned around the Cellbed surface. Observation of Tongue SqCC Cells Abnormal keratinization and cell stratification, which are characteristics of well-differentiated SqCC cells, were observed in horizontal and vertical cross-sections TAK-632 of HSC-4 and SCC15 cells produced in 3D culture (Fig. 3aCc). Staining positivity was confirmed upon immunostaining using CK17 (Fig. ?(Fig.3d)3d) and fluorescence immunostaining using ezrin (green) and cortactin (red; Fig. 3eCh). HSC-4 scanning electron micrographs showed that cancer cells were present among the Cellbed fibers with cytoplasm (Fig. 4a, b). Desmosomes were observed between cells by TEM of vertical cross-sections of SCC-15 cells produced for 4 weeks in 3D culture (Fig. 4c, d). Open in a separate windows Fig. 3 Squamous cell carcinoma cells. Abnormal keratinization in HSC-4 cells (a) and SCC-15 cells (b) in 3D culture. c Three-week culture of HSC-4 IL1A cells in vertical cross-sections. A layered structure and surface-lying differentiated cells were observed. d CK17-positive cells were detected. eCh Cells cultured for 4 weeks. Fluorescence immunostaining of vertical sections (DAPI, blue; ezrin, green; cortactin, red). Scale bar, 100 m. Open in a separate windows Fig. TAK-632 4 Electron microscopy image of 3D culture of squamous cell carcinoma cells. Scanning electron micrograph of HSC-4 cells cultured for 2 weeks. a A proliferating cell entangled in Cellbed fibers. Scale bar, 20 m. b Confirmation of cellular extension into Cellbed. Scale bar, 5 m. c, d Transmission electron microscopy image of SCC-15 cells cultured for 4 weeks. d A magnified image of (c). Desmosomes were observed even in the vertical section (arrows) Scale bar, 1 m. Morphological Observation of Nonadherent Cells SNU-1 and KATOIII cells proliferated in the 3D culture system (Fig. 5a, b). These cancer cells were partly clustered, but no luminal structure was detected. Immunostaining experiments were successfully performed using formaldehyde-fixed paraffin-embedded sections of growing cells in Cellbed (Fig. 5c, d). Most SNU-1 cells were positive for Ki67 (nuclear staining; Fig. ?Fig.5c)5c) and all KATOIII cells were positive for CK (AE1/AE3; membranous and cytoplasmic staining; Fig. ?Fig.5d5d). Open in a separate windows Fig. 5 Adenocarcinoma cells (nonadherent cells). a SNU-1 cells cultured for 3 weeks. Scale bar, 50 m. HE. b KATOIII cells cultured for 3 weeks. Scale bar, 50 m. HE. SNU-1 and KATOIII cells partly clustered, but no obvious luminal structure was TAK-632 detected. c Ki67 immunostaining of SNU-1 cells cultured for 3 weeks. Scale bar, 50 m. Most SNU-1 cells were positive for Ki67. d CK (AE1/AE3) immunostaining of KATOIII cells cultured for 3 weeks. Scale bar, 50 m. All KATOIII cells were positive for CK (AE1/AE3). Collagen IV Coating Increases Invasion Depth We evaluated the invasion depth of cancer cells in the vertical section of Cellbed, the results of which are shown in Physique ?Physique6.6. We had reported.