## 3 Results  
The primary culture of human RECs and fibroblast were successfully established from nasal turbinate samples (Figure 1). In our previous studies, we successfully characterized the RECs isolated from nasal turbinate via gene expression (_CK18_ and _14_, _MUC5B_, and _Ki67_) [32] and immunocytochemical analysis (acetylated \(\beta\)-tubulin, CK14, MUC5 AC, and Ki67) [32,33].  
The overall appearance of the collagen scaffold was spongy and white in color, and the irregular-sized pores on the scaffold were observable with the naked eye (Figure 2A). The SEM analysis showed that the scaffold had interconnected pores with size range of 100 to 200 \(\upmu\)m (Figure 2B,C).  
The hematoxylin and eosin (H&E) staining of the 3D human RE construct cross-section showed that the collagen scaffold contained interconnected pores and the cells (RECs and fibroblasts) were able to migrate into the collagen scaffold upon 4 weeks of culture (Figure 3). However, the cell attachment and migration were not uniform, as some parts of the 3D human RE construct showed patchy cell organization while there were no cells present on other parts.  
The presence of distinct RECs and fibroblasts layers on the collagen scaffold was confirmed by the immunostaining analysis. As the anti-collagen type I antibody bound to the collagen scaffold as well as the seeded fibroblasts, it was difficult to identify the presence of fibroblasts based on collagen type I expression. So, the cells with a nucleus stained with DAPI and negative expression of CK 14 were assumed to be the seeded fibroblasts on the collagen scaffold. We found that the RECs and fibroblast co-existed in the same area and the RECs had not yet migrated to form a separate layer (Figure 4).  
The ultrastructural study of the 3D human RE construct revealed that 4 weeks post cell seeding, the seeded cells had formed a confluent layer on top of the scaffold and were actively secreting extracellular matrix (Figure 5). The SEM results showed collagen degradation, which indicated the biodegradability of the scaffold. However, no cilia were found on the cells, which showed that the RE layer was not yet functional.  
Figure 1: Monolayer cells cultured in 6-well plate: (**A**) Co-culture of respiratory epithelial cells (RECs), and fibroblasts, (**B**) fibroblasts, and (**C**) RECs. The co-cultured cells became confluent in a week’s time and fibroblasts were separated by differential trypsinization before seeding into a new flask.  
Figure 2: The freeze-dried collagen type I (Col I) scaffold at \(-80\)\({}^{\circ}\)C: (**A**) overall view, (**B**) view under scanning electron microscope (SEM) at 300\(\times\), and (**C**) view under SEM at 1650\(\times\).  
Figure 4: The top view of the cell-seeded collagen scaffold immunostained with (**A**) anti-Col type I antibody (green) as the fibroblast marker and (**B**) anti-cytokeratin 14 antibody (red) as the marker of RECs. (**C**). The cell nuclei were counter-stained with 4\({}^{\prime}\),6-diamidino-2-phenylindole (DAPI) (blue). (**D**) Merged image 10\(\times\) and (**E**) 20\(\times\). (**A**). The anti-Col type I antibody was found to bind to the scaffold in addition to the fibroblasts. The intense green color located almost centrally (**A**) formed due to the accumulation of green fluorescence in unknown debris.  
Figure 3: Cross-section view of the hematoxylin and eosin (H&E)-stained 3D human RE construct: (**A**) top layer of collagen construct with RECs and (**B**) bottom layer of collagen scaffold with fibroblasts. The cells were found to enter and migrate into the collagen scaffold at 4 weeks post cell seeding.