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.

Cultured meat is considered a sustainable protein alternative. To produce chicken meat in a controlled environment, bacterial nanocellulose (BNC) was used to provide structure similar to traditional chicken meat. BNC was synthesized as a hydrogel from Novacetimonas hansenii in vitro culture. Chicken mesenchymal stem cells (MSCs) from pathogen-free eggs were cultivated in both 2D (monolayers) and 3D (spheroids) forms and introduced into BNC. Results showed that the shape, viability, and stemness of 3D spheroids and 2D monolayers were maintained. Notably, 3D platforms better replicate natural cellular environments, enhancing differentiation potential. Differentiated spheroids and monolayers were successfully cultured on both modified and unmodified BNC hydrogels, with tissue-like organization observed mainly in modified BNC. The cultured chicken prototype using nanocellulose proved promising for developing cultured meat products with co-cultivation of 2D and 3D cells. Visual analyses revealed significant similarities between cultured and farmed chicken.