Transparent 3-Layered Bacterial Nanocellulose as a Multicompartment and Biomimetic Scaffold for Co-Culturing Cells

Abstract

Three-dimensional (3D) cell culture models are widely used to provide a more physiologically relevant microenvironment in which to host and study desired cell types. These models vary in complexity and cost, ranging from simple and inexpensive to highly sophisticated and costly systems. In this study, we introduce a novel translucent multi-compartmentalized stacked multilayered nanocellulose scaffold and describe its fabrication, characterization, and potential application for co-culturing multiple cell types. The scaffold consists of bacterial nanocellulose (BNC) layers separated by interlayers of a lower density of nanocellulose fibers. Using this system, we co-cultured the MDA-MB-231 cell line with two tumor-associated cell types, namely BC-CAFs and M2 macrophages, to simulate the tumor microenvironment (TME). Cells remained viable and metabolically active for up to 15 days. Confocal microscopy showed no signs of cell invasion. However, BC-CAFs and MDA-MB-231 cells were frequently observed within the same layer. The expression of breast cancer-related genes was analyzed to assess the downstream functionality of the cells. We found that the E-cadherin expression was 20% lower in cancer cells co-cultured in the multi-compartmentalized scaffold than in those cultured in 2D plates. Since E-cadherin plays a critical role in preventing the initial dissociation of epithelial cells from the primary tumor mass and is often downregulated in the tumor microenvironment in vivo, this finding suggests that our scaffold more effectively recapitulates the complexity of a tumor microenvironment.

Keywords: 3D cell culture model; bacterial nanocellulose; triple-cell co-culture.

Figure 1

Schematic of ^3LBNC scaffold fabrication and triple-cell co-culture. (A) Three-layer structure after 21 days of incubation in DMCM. (B) Cell injection/seeding protocol into each interfacial layer of ^3LBNC scaffold for confocal microscopy analysis. A total of 9.4 × 10⁴ BC-CAFs were seeded under the first layer, then 2 × 10⁶ MDA-MB-231 and 9.3 × 10⁴ M2 macrophages were injected into the first and the second interlayer, respectively.

Figure 2

Microstructure and pore size of ^3LBNC scaffolds. (A) SEM micrographs of individual layers of ^3LBNC compared to the ^SLBNC control showing a more porous structure for the bottom layer (5000×). (B) Cross-section of ^3LBNC showing layers (arrows) and interlayers (*) at 100×, 500×, and 5000× magnification. (C) Feret diameter of the ^3LBNC-derived individual layers compared to ^SLBNC and Pearson’s correlation matrix of individual layers’ surfaces: P: porous; D: dense. n = 1 for ^SLBNC, and n = 3 for layers. * 0.01 < p < 0.05, *** p < 0.001, and **** p < 0.0001.

Figure 3

Evaluation of cell culturing into layered scaffolds. (A) Diffusion assay in ^2LBNC compared to ^SLBNC. DMEM tested and compared to glucose solution in both scaffolds. (B) Cell proliferation (MTS) assay with MDA-MB-231 in ^2LBNC samples and in 2D cell culture plate (2 × 10⁶ cells/scaffold 3D culture and 8 × 10⁴ cells/well 2D culture). One-way ANOVA (p < 0.05) with n = 9. (C) Live/dead assay with EOMA cells after one-week cell culture in the ^2LBNC and ^3LBNC scaffolds (live cells in green, dead cells in red, merged images in last column, scale bars: 100 µm). (D) DAPI assay of EA.hy926 cultured in second interlayer of ^3LBNC scaffolds for up to 15 days. Samples remained inside inserts to avoid lateral cell migration; from left to right: top view of entire scaffold, bottom view of third and second layers. **** p < 0.0001.

Figure 4

Triple-cell co-culture in ^3LBNC scaffold. (A) Three-dimensional confocal microscopy of cells seeded/injected into ^3LBNC at different time points: MDA-MB-231 in green, BC-CAFs in red, M2 macrophages in blue. (B) CDH1, JUNB, and DUSP5 gene expression relative to GAPDH. The 15-day time point plate-grown cells was not tested. Asterisks refer to statistical significance of cells cultured in the ^3LBNC versus cells cultured on the plates (* 0.01 < p < 0.05, ** 0.001 < p < 0.01).