Stiffness And Composition Of Diseased Lungs Modulates Cell Behavior: Novel In Vitro Model
Conference Year
January 2020
Abstract
Chronic obstructive pulmonary disease (COPD), is a leading cause of death in the United States and it is expected to be the third leading cause of death in westernized countries by 2020. Obstruction in the airways due to COPD is caused by a combination of factors, including airway remodeling, emphysema, and pulmonary inflammation in varying combinations and severities. Unfortunately, no current drug treatments modify the course of the disease. Therefore, understanding how the extracellular matrix (ECM) is being remodeled in the disease is particularly important. To conduct this research, both healthy and diseased human lungs from cadaveric donors were processed utilizing a detergent-based protocol, including Triton X-100 and sodium deoxycholate (SDC) in order to isolate the ECM. Decellularized lung tissue from both, healthy and emphysematous lungs were lyophilized, liquid nitrogen milled, and enzyme digested. Polyacrylamide gels (PAA) substrates were prepared using a mixture 40% acrylamide and 2% bis-acrylamide solution (Bio-rad). Both adenocarcinomic human alveolar basal epithelial (A549) and human lung epithelial (HLF) cells were seeded on thin collagen-coated polyacrylamide gel disks. Atomic force microscopy was performed with a silicon-nitred regular pyramid cantilever (MLCT-Bio, Bruker) with a nominal spring constant of 0.07 N/m to validate the stiffness of PAA. Immunohistochemistry staining for collagen I was performed on healthy and diseased lung ECMs and its amounts were quantified by mean fluorescence intensity using image J. AFM on PAA demonstrated that the stiffness could be modulated between different ranges. We were able to produce gels at 3.8 ± 0.1 kPa and 21.4 ± 2.5 kPa by varying the concentration of its components. Moreover, cells demonstrated regular morphology on PAA coated with collagen, confirming protein interaction. Finally, we observed a significant increment p>0.01 of collagen I deposition in diseased lungs, suggesting a possible remodeling of the extracellular matrix due to the disease. Overall, an in vitro model which utilizes healthy and diseased ECM was developed. The initial data suggests that the ECM suffers an important remodeling, mainly an increased deposition of a key proteins such as collagen I. This opens the possibility to focus on the combination of stiffness modulation and protein composition to determine the cell behavior in diseased lungs.
Primary Faculty Mentor Name
Daniel Weiss
Secondary Mentor Name
Juan Uriarte and Robert Pouliot
Status
Undergraduate
Student College
College of Engineering and Mathematical Sciences
Program/Major
Biomedical Engineering
Primary Research Category
Biological Sciences
Secondary Research Category
Engineering & Physical Sciences
Tertiary Research Category
Health Sciences
Stiffness And Composition Of Diseased Lungs Modulates Cell Behavior: Novel In Vitro Model
Chronic obstructive pulmonary disease (COPD), is a leading cause of death in the United States and it is expected to be the third leading cause of death in westernized countries by 2020. Obstruction in the airways due to COPD is caused by a combination of factors, including airway remodeling, emphysema, and pulmonary inflammation in varying combinations and severities. Unfortunately, no current drug treatments modify the course of the disease. Therefore, understanding how the extracellular matrix (ECM) is being remodeled in the disease is particularly important. To conduct this research, both healthy and diseased human lungs from cadaveric donors were processed utilizing a detergent-based protocol, including Triton X-100 and sodium deoxycholate (SDC) in order to isolate the ECM. Decellularized lung tissue from both, healthy and emphysematous lungs were lyophilized, liquid nitrogen milled, and enzyme digested. Polyacrylamide gels (PAA) substrates were prepared using a mixture 40% acrylamide and 2% bis-acrylamide solution (Bio-rad). Both adenocarcinomic human alveolar basal epithelial (A549) and human lung epithelial (HLF) cells were seeded on thin collagen-coated polyacrylamide gel disks. Atomic force microscopy was performed with a silicon-nitred regular pyramid cantilever (MLCT-Bio, Bruker) with a nominal spring constant of 0.07 N/m to validate the stiffness of PAA. Immunohistochemistry staining for collagen I was performed on healthy and diseased lung ECMs and its amounts were quantified by mean fluorescence intensity using image J. AFM on PAA demonstrated that the stiffness could be modulated between different ranges. We were able to produce gels at 3.8 ± 0.1 kPa and 21.4 ± 2.5 kPa by varying the concentration of its components. Moreover, cells demonstrated regular morphology on PAA coated with collagen, confirming protein interaction. Finally, we observed a significant increment p>0.01 of collagen I deposition in diseased lungs, suggesting a possible remodeling of the extracellular matrix due to the disease. Overall, an in vitro model which utilizes healthy and diseased ECM was developed. The initial data suggests that the ECM suffers an important remodeling, mainly an increased deposition of a key proteins such as collagen I. This opens the possibility to focus on the combination of stiffness modulation and protein composition to determine the cell behavior in diseased lungs.