Presentation Title
A Novel Histological Method for Analyzing Cell Morphology within Decellularized Extracellular Matrix Hydrogels
Abstract
Hydrogels derived from decellularized tissues are utilized as innovative physiologically relevant matrices for advanced three-dimensional (3D) cell culture applications. More specifically, decellularization is a process of clearing tissues or whole organs of all cellular material using sequential detergent-based washes, resulting in a bare extracellular matrix (ECM) scaffold. Subsequently, the ECM scaffold can be utilized in a variety of applications, including further enzymatic digestion and the formation of hydrogels. Recently, we have formed hydrogels from decellularized human alveolar ECM (aECM). Further, we have shown that aECM hydrogels support the proliferation of induced pluripotent stem cell (iPSC)-derived induced type 2 alveolar epithelial cells (iAEC2s) in 3D cell culture. However, as aECM hydrogels are non-adherent to glass coverslips, which are typically necessary for high-resolution imaging, novel methods of cellular imaging within aECM hydrogels are necessary for further analysis of iAEC2 morphology. Here, we have adapted previously utilized histological protocols in order to derive microscopy sections of aECM hydrogel-embedded iAEC2s. In brief, iAEC2s were cultured in aECM hydrogels on tissue culture treated plastic using established protocols. Subsequently, aECM hydrogels were fixed using paraformaldehyde, mechanically removed from the tissue culture plastic, and embedded in commercially available HistogelÒ before standard paraffin embedding. Importantly, this method is capable of producing microscopy sections that are amenable to: (1) high-resolution microscopy, and (2) the utilization of dyes and immunohistochemistry staining. Using this method, we show two separate methods of identifying subcellular vesicles, called lamellar bodies, that are representative of an in vivo type 2 alveolar epithelial cell (AEC2) phenotype. Such analyzes are critical to determining the overall cellular morphology of iAEC2s. Additionally, this novel method for deriving histological sections from decellularized ECM hydrogels can be applied to a variety of cell types, and therefore, be used to determine the effects of physiologically relevant ECMs on cell behavior.
Primary Faculty Mentor Name
Daniel Weiss, Md, PhD
Faculty/Staff Collaborators
Evan Hoffman (Graduate Student Mentor)
Status
Undergraduate
Student College
College of Arts and Sciences
Program/Major
Biochemistry
Primary Research Category
Health Sciences
A Novel Histological Method for Analyzing Cell Morphology within Decellularized Extracellular Matrix Hydrogels
Hydrogels derived from decellularized tissues are utilized as innovative physiologically relevant matrices for advanced three-dimensional (3D) cell culture applications. More specifically, decellularization is a process of clearing tissues or whole organs of all cellular material using sequential detergent-based washes, resulting in a bare extracellular matrix (ECM) scaffold. Subsequently, the ECM scaffold can be utilized in a variety of applications, including further enzymatic digestion and the formation of hydrogels. Recently, we have formed hydrogels from decellularized human alveolar ECM (aECM). Further, we have shown that aECM hydrogels support the proliferation of induced pluripotent stem cell (iPSC)-derived induced type 2 alveolar epithelial cells (iAEC2s) in 3D cell culture. However, as aECM hydrogels are non-adherent to glass coverslips, which are typically necessary for high-resolution imaging, novel methods of cellular imaging within aECM hydrogels are necessary for further analysis of iAEC2 morphology. Here, we have adapted previously utilized histological protocols in order to derive microscopy sections of aECM hydrogel-embedded iAEC2s. In brief, iAEC2s were cultured in aECM hydrogels on tissue culture treated plastic using established protocols. Subsequently, aECM hydrogels were fixed using paraformaldehyde, mechanically removed from the tissue culture plastic, and embedded in commercially available HistogelÒ before standard paraffin embedding. Importantly, this method is capable of producing microscopy sections that are amenable to: (1) high-resolution microscopy, and (2) the utilization of dyes and immunohistochemistry staining. Using this method, we show two separate methods of identifying subcellular vesicles, called lamellar bodies, that are representative of an in vivo type 2 alveolar epithelial cell (AEC2) phenotype. Such analyzes are critical to determining the overall cellular morphology of iAEC2s. Additionally, this novel method for deriving histological sections from decellularized ECM hydrogels can be applied to a variety of cell types, and therefore, be used to determine the effects of physiologically relevant ECMs on cell behavior.