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Biomechanics

Biomechanics - Poster Session A

Poster D7 - Altered Cell Contractility of Aerosol Exposed Epithelium Cells

Thursday, October 24, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Aerosols such as combustion cigarette aerosols (CCA) have been linked to diseases such as chronic obstructive pulmonary disease (COPD). One of the mechanical characteristics of COPD is altered levels of epithelial cell contractility. While the connection of CCA to COPD is known, how these aerosols differ in their impacts on epithelial contractility has not yet been reported. This study aims to quantify differences in epithelial cell contractility after exposure to CCA aerosol-conditioned media. Cell contractility is, in part, controlled by the actin cytoskeleton. These contractile forces are transmitted to the extracellular matrix through focal adhesions. Using adenocarcinoma human alveolar basal epithelial cells (A549) seeded onto collagen type 1 coated elastic polyacrylamide hydrogels (PAH), we quantified the contractility via traction force microscopy (TFM). We find that A549 contractility increases with the number of smoking puffs for CCA.

Materials and

Methods:  Stiff (Young’s modulus E = 32 kPa and Poisson’s ratio ν = 0.37) polyacrylamide hydrogels (PAH) containing 0.5 µm red fluorescent beads were fabricated on top of glass bottom dishes. Such dishes were flipped upside-down during polymerization to ensure fluorescent beads were near the surface. Then, using sulfo-SANPAH as a crosslinker, collagen type 1 was immobilized on the PAH surface. Afterward, A549 cells were seeded onto the PAH and left to attach for 24 hours. Using a CH Technologies Cigarette Smoking Machine (CSM-STEP), 25 mL of media (DMEM + 10% FBS) was preconditioned with 2, 4, or 6 “puffs” of CCA. Next, seeded cells were exposed to 2 mL of conditioned media for 18 hours. Upcoming, we imaged CCA-exposed cells by taking a phase contrast image of an isolated cell, a fluorescent image of the fluorescent beads with the cell attached to the substrate, and a fluorescent image of the same beads after detaching the cell. Cells were detached with a 10% SDS solution. Images were post-processed with a TFM script generating quantitative stress heat maps of the cellular tractions. We report the root mean squared (RMS) tractions and the projected area of the cell.

Results, Conclusions, and Discussions: Figure 1 is a superplot of the projected cell area (Fig. 1a), RMS tractions (Fig. 1b), and RMS tractions normalized by the projected cell area (Fig. 1c). Each small dot represents an individual data point. The color code represents the experiment in which it was collected. A larger circle with a black outline represents the averages of all the individual experiments. These averages were used to create a box and whisker plot for each condition, with the center line representing the median of such averages.
For the RMS tractions, TRMS, there appears to be no clear trend as a function of puff exposure, as seen in Figure 1a. The projected cell area, Acell, appears to decrease significantly for the 6-puff condition only, as shown in Figure 1b. However, when normalizing the TRMS by Acell for control, 2, 4, and 6 puffs of CCA, we observe a normalized increase in TRMS/Acell with increasing puff exposure, Figure 1c. Using the averages of the experimental means, preliminary data shows how the TRMS/Acell for control, 2, 4, and 6 puffs is 0.03016 ± 0.008786 Pa/m2, 0.03067 ± 0.007863 Pa/m2, 0.03475 ± 0.01066 Pa/m2, and 0.03936 ± 0.009539 Pa/m2, respectively. In other words, there was a 1.66% increase between control and 2-puffs, 11.74% increase between 2- and 4-puffs, and 11.70% increase between 4- and 6-puffs. This pattern shows that with increased concentration of CCA, cellular contractile forces of A549s on stiff col-1 coated PAH increase.
Increasing contractility, specifically of the alveolar epithelium, changes how cellular monolayers function. We hypothesize that with increased cellular contractility due to CCA aerosols, the alveolar epithelium’s permeability will be disturbed, potentially causing fluid to leak into the alveolus. However, further research on a monolayer must be conducted to prove such hypothesis.

Acknowledgements (Optional):  P.K.P, M.M., and R.C.A.E. acknowledge funding from the NSF- CREST: Center for Cellular and Biomolecular Machines through the support of the National Science Foundation (NSF) Grant No. NSF-HRD-1547848. R.C.A.E. acknowledges funding from the Tobacco-Related Disease Research Program through the support of the University of California Office of the President Grant No. T31KT1583 awarded to R.C.A.E. P.K.P. acknowledges funding from the CIRM-funded COMPASS grant Training Undergraduates in Stem Cell Engineering and Biology (TUSCEB) award # EDUC5-13686.