Multifaceted Lung Tissue Model for Tuberculosis

The lung tissue models were prepared as previously described (Braian et al., 2015 (link)). In brief, 16HBE cells and human primary macrophages (uninfected/infected at MOI 10 with Mtb expressing GFP), PKH26 red dye-labeled monocytes (stained according to manufacturer's instructions) were seeded into a matrix of collagen-embedded MRC-5 cells. During tissue development, the models were air-exposed at day 5 post infection allowing mucus secretion and stratification (Nguyen Hoang et al., 2012 (link); Parasa et al., 2014 (link)). At day 7 after infection, the models were harvested and fixed with 4% paraformaldehyde for 30 min. Supernatants were collected and stored at −80°C for cytokine and chemokine analysis. For MMP inhibition experiments, primary macrophages treated with a global MMP inhibitor, 200 nM marimastat (Merck Millipore), 16 h prior to Mtb infection were introduced in the lung tissue model. After the addition of macrophages, the tissue models were cultured in the presence of the inhibitor until tissue harvest.