Date of Completion

2022

Thesis Type

College of Arts and Science Honors

Department

Biochemistry

First Advisor

Daniel Weiss

Second Advisor

Matthias Brewer

Third Advisor

Jay Silveira

Keywords

hydrogels, alveolar hydrogels, protein composition, extracellular matrix (ECM), ECM hydrogels, decellularization

Abstract

Pulmonary diseases, including Chronic Obstructive Pulmonary Disease (COPD) and Idiopathic Pulmonary Fibrosis (IPF), remain common causes of death, yet still have no cure. Recently, research of lung tissue, both healthy and diseased, has begun utilizing hydrogels as 3-dimensional (3D) environments for cell culture studies as well as in vivo therapeutics to more closely mimic the in vivo tissue environment. Synthetic hydrogels, such as Matrigel, have been commonly utilized as a 3D scaffold in which stem cells can be cultured and their behavioral patterns can be observed. The origin of Matrigel, however, is tumorigenic mouse tissue and it is not an optimal model for studying human cells, including lung cells. Recently, hydrogels formed from human decellularized tissues are becoming increasingly utilized in cell culture studies as physiologically relevant extracellular matrices (ECM). These hydrogels are thought to closely resemble the respective tissue environment, therefore providing specific conditions to the cells and positively influencing their growth and behavior due to the presence of tissue-specific ECM proteins. However, the formation of such hydrogels requires harsh processing steps, including 72-hour ECM pepsin digestion. Importantly, while several studies have analyzed the impact of tissue decellularization on ECM protein composition, the impact of the other hydrogel processing steps have been overlooked, particularly the effects of pepsin digestion. Additionally, several studies have utilized these tissue-specific hydrogels to analyze cell proliferation and behavior. Yet, the protein composition of the final hydrogels remains unknown. As ECM composition has significant influence on cellular behavior, we here aimed to determine the impact of pepsin digestion on the final protein content of decellularized lung hydrogels. Utilizing mass spectrometry analysis, total protein staining, and western blotting, we identified an overall decrease in total protein and an overall concentrating effect of Collagen I protein subunits following pepsin digestion.

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