"Breathing exposes lung cells to continual mechanical stimuli, which is part of the microenvironmental signals directing cellular functions. Therefore, developing in vitro model systems that incorporate physiomimetic mechanical stimuli is urgent to fully understand cell behavior. This study aims to introduce a novel in vitro culture methodology combining mechanical stimuli that simulates in vivo breathing in 3D cell culture platforms in the form of recellularized human lung extracellular matrix (ECM) scaffolds and precision cut lung slices (PCLS) from rats.
To this end, we have constructed a device for controlled cyclic stretch, mimicking amplitudes and frequencies of distensions seen in the breathing human lung. For its validation, we cultured H441 lung epithelial cells in decellularized human lung slices exposed to 16 stretch cycles per minute with a 10% stretch amplitude. Cell viability (resazurin reduction), proliferation (Ki-67) and YAP1 activation were evaluated at 24 and 96 hours by immunohistochemistry, while the expression of SFTPB, COL3A1, COL4A3 and LAMA5 was evaluated by qPCR.
Cyclic stretch induced an increase in SFTPB expression after 24 hours without a concomitant increase in the stretch responsive gene YAP1. The ECM milieu lowered the expression of the basement membrane protein genes COL4A3 and LAMA5 compared to tissue culture plastic control cultures, without any additional effect induced by the mechanical stimuli. Additionally, we show compatibility of the device with PCLS culture showing preserved morphology and metabolism in rat PCLS after 72 hours of mechanical stretch. Thus, we present a novel device and methodology for the study of lung tissue slice cultures subjected to physiomimetic mechanical stimuli, which shows promise for future studies of cell and tissue function in a lung ECM milieu with physiological or pathological mechanical stimuli."