Optimization of Human Lung-Chip cultures to investigate effects of biomechanics on primary airway epithelial cell biology

Abstract

With advances brought on by Organ-on-Chip technology, a new level of complexity was introduced into cell systems that allows research into human cellular cross-talk combined with mechanical cues. This added complexity is especially relevant in the lungs where biomechanics play a prominent role. Here we leveraged the commercial (EmulateTM) Lung-Chip and optimized its use for primary bronchial epithelial cell (PBEC) culturing to investigate the impact of airflow and mild stretch on epithelial biology. For this, PBEC culturing had to be optimized on a flexible PDMS, 7 µm pore-size membrane that allows application of stretch and leukocyte migration. To obtain a fully differentiated airway epithelium, cell media selection, coating strategy, and prevention of PBEC migration to the bottom channel was successfully achieved. Inter-chip variability (baseline IL-8 levels (ELISA)) was low (coefficient of variation <0.11 (n=3 chips)) and was comparable between donors (n=2). Furthermore, cells in the chips showed presence of relevant epithelial cell markers (qPCR), e.g. TP63 (basal cells), pIgR (luminal cells), FOXj1 (ciliated cells), MUC5AC (goblet cells). Pilot studies investigated effects of 1 week automatized mild (5%, 0.25 Hz) cyclic stretch and airflow during differentiation (week 2 at air-liquid interface) on survival, morphology, and cellular composition as assessed by qPCR. Results showed no macroscopic changes in morphology, and similar cell marker gene expression as control chips (n=1). To date, we have optimized human PBEC culturing on a flexible 7 µm pore-size membrane chip that allows studies on the impact of biomechanics on lung biology.