Precision cut lung slices (PCLS)
8-12-weeks-old C57-Bl/6 mice are administered via oropharyngeal administration with BLM (1, 2). On day 11, mice are sacrificed and perfused lungs are inflated with low melting agarose in saline. Lungs are then incubated with RPMI medium supplemented with 10% FBS and 1% penicillin-streptomycin for 30 min at 4OC to allow for agarose polymerization. The left lobe of the agarose filled lung is then cut into 200μm slices (PCLS) with Vibratome. PCLS are then cultured at standard conditions and incubated for 3 days with novel compounds to be tested. PCLS are then fixed with PFA, embedded in paraffin, sectioned (5μm) and stained accordingly. Using PCLS it was recently shown that an inhibitor of src kinase (A-419259) attenuated BLM-induced fibrosis )(3).
3D primary fibroblast cultures
The efficacy of novel compounds is usually first tested in vitro in TGFβ-exposed lung fibroblasts, assessing the inhibition of the expression of major TGFβ-induced pro-fibrotic genes, such as Collagen1a1 and aSMA. In BSRC Fleming, primary lung fibroblasts are cultured in autologous acellular extracellular matrix (aECM) isolated from the same mice. The autologous substrate bypasses the spontaneous activation and differentiation of lung fibroblasts upon culture to plastic and allows for a more accurate determination of the effects of the growth factors and pharmaceutical compounds under examination. Moreover, the use of aECM in invasion (Boyden) chambers is the ideal 3D system for monitoring migration and invasion, significantly upgrading the in vitro cell model for the study of fibrinogenic mechanisms (3).
References
1. Barbayianni I, Ninou I, Tzouvelekis A, Aidinis V. Bleomycin Revisited: A Direct Comparison of the Intratracheal Micro-Spraying and the Oropharyngeal Aspiration Routes of Bleomycin Administration in Mice. Frontiers in medicine. 2018;5:269.
2. Zannikou M, Barbayianni I, Fanidis D, Grigorakaki T, Vlachopoulou E, Konstantopoulos D, et al. MAP3K8 Regulates Cox-2-Mediated Prostaglandin E(2) Production in the Lung and Suppresses Pulmonary Inflammation and Fibrosis. J Immunol. 2021;206(3):607-20.
3. Barbayianni I, Kanellopoulou P, Fanidis D, Magkrioti C, Tsitoura E, Antoniou K, et al. SRC-mediated and TKS5-enabled podosome formation is an inherent property of IPF fibroblasts, promoting ECM invasion and pulmonary fibrosis. Biorxiv. 2023.