Acoustically detectable cellular-level lung injury induced by fluid mechanical stresses in microfluidic airway systems

Abstract

We describe a microfabricated airway system integrated with computerized air-liquid two-phase microfluidics that enables on-chip engineering of human airway epithelia and precise reproduction of physiologic or pathologic liquid plug flows found in the respiratory system. Using this device, we demonstrate cellular-level lung injury under flow conditions that cause symptoms characteristic of a wide range of pulmonary diseases. Specifically, propagation and rupture of liquid plugs that simulate surfactant-deficient reopening of closed airways lead to significant injury of small airway epithelial cells by generating deleterious fluid mechanical stresses. We also show that the explosive pressure waves produced by plug rupture enable detection of the mechanical cellular injury as crackling sounds.

Fig. 1.

Compartmentalized microfluidic airway systems. (A) The microfabricated small airways are comprised of PDMS upper and lower chambers sandwiching a porous membrane. (B) SAECs are grown on the membrane with perfusion of culture media in both upper and lower chambers until they become confluent. (C) Once confluence is achieved, media are removed from the upper chamber, forming an air–liquid interface over the cells. During air–liquid interface (ALI) culture, the cells are fed basally and undergo cellular differentiation. (D) Physiologic airway closure is recreated in the microfluidic system by exposing the differentiated cells to plug flows. (E) Liquid plugs created in a plug generator progress over a monolayer of the epithelial cells and rupture in the downstream region, reopening the in vitro small airways. (F) SAECs seeded into the upper chamber attach to a membrane within 5 hr after seeding in the absence of fluid flows. Continuous perfusion of media supports cell growth into monolayers with typical epithelial appearance. Confluence is reached ≈6 days after seeding, at which time ≈95% of the membrane surface inside the microchannel is uniformly covered with SAECs. (Scale bars: 150 μm.)

Fig. 2.

Microfluidic production of in vitro small airway tissue. Liquid perfusion culture over 6 days generates a confluent monolayer of SAECs with high viability (mean ± SD = 90.4 ± 5.17%), as illustrated in A. Green and red represent live and dead cells, respectively. (B) During ALI culture lasting 3 weeks, the steady flow of culture media in the lower chamber sustains the cells on the membrane, maintaining high viability (87.6 ± 3.77%). (C) The cells in the areas without basal feeding die from starvation, leading to no detection of live cells. (Scale bars: 150 μm.) (D) ALI culture induces differentiation of SAECs as indicated by the production of CC10. The level of protein secretion is not measurable during liquid perfusion culture and increases considerably within 9 days after the formation of an air–liquid interface. Data represent mean ± SD of samples collected from three independent experiments (n = 3).

Fig. 3.

Formation, propagation, and rupture of liquid plugs generated by dynamic fluidic switching in a microfabricated plug generator. (A) In the plug generator, injected liquid is stably focused by air to form stratified air–liquid two-phase flows. Liquid exits to a waste outlet. (B) Blockage of air causes liquid to enter the culture chamber. (C) Subsequently, air flow is resumed and original two-phase stratification is recovered, resulting in the formation of a liquid plug in the culture chamber. (D) As liquid plugs move through the upper chamber, they become shorter because of volume loss and ultimately rupture in the downstream region. (Scale bars: 1 mm.) (E) Plug rupture in microchannels produces pressure waves resembling transient acoustic waves of respiratory crackles. Note that the time scale in the pressure plot is expanded over the period of 15 ms to emphasize the dynamics of rapid pressure fluctuations caused by plug rupture.

Fig. 4.

Cellular injury caused by propagation and rupture of liquid plugs. (A) Single-phase liquid flows do not damage differentiated SAECs. Exposure of the cells to propagation and subsequent rupture of liquid plugs results in progressively larger numbers of injured cells, as shown in B–D. PR represents the number of plug propagations and subsequent ruptures over a period of 10 min. “Upstream and midstream” and “downstream” areas range from x = 0 to 2.9 mm and from x = 2.9 mm to 3.7 mm, respectively. (Scale bars: 150 μm.) (E) The extent of cellular damage is elevated with the increasing number of reopening events. (F) Numerical simulation reveals that propagating liquid plugs form recirculation in the core region and generate large gradients of wall pressure and wall shear stress in the precursor film where the film thickness is the smallest. (G) Downstream viability normalized by the percent viability of cells in the upstream and midstream regions decreases significantly in the presence of reopening flows, suggesting that more deleterious mechanical stresses are generated in the vicinity of the site of plug rupture.