Title of Oral/Poster Presentation

Growing Retinal Pigment Epithelial Cells on a Recombinant Spider Silk Protein Membrane

Presenter Information

Chase PatersonFollow
Harsh SinghFollow

Class

Article

Graduation Year

2019

College

College of Engineering

Department

Biological Engineering Department

Faculty Mentor

Dr. Elizabeth Vargis

Presentation Type

Poster Presentation

Abstract

Age-related macular degeneration (AMD) is the leading cause of blindness in developed countries and accounts for nearly 10% of vision loss around the world. Retinal pigment epithelial (RPE) cells grow posterior to the photoreceptors in the eye on an acellular protein structure, called Bruch’s membrane. RPE cells transport nutrients, remove waste products, and provide support for the photoreceptor cells. As aging occurs, a lipid substance called drusen may form inside Bruch’s membrane and inhibit RPE cell growth and function. To better understand the relationship between the disruption of RPE cells and the onset of AMD, RPE cells must be grown and thoroughly characterized in vitro. Such research could lead to the development of an in vitro model of the human eye for evaluating diagnostic methods and treatments to counteract the effects of AMD. This model would incorporate a synthetic Bruch’s membrane that is also capable of mimicking its natural structure and function. In this project, recombinant spider silk proteins (rSSps) were used to develop a proteinaceous membrane and to support RPE growth.

First, freestanding semipermeable films were engineered using either spin coating or gravity spreading techniques to ensure an rSSp membrane that is biomimetic to Bruch’s membrane. Cell proliferation was analyzed using a PicoGreen dsDNA assay. Immunohistochemistry staining was also employed to assess the morphology of cells. Results from transepithelial electrical resistance (TEER) measurements, cell staining, and a permeability assay demonstrated that the rSSp analogue of major ampullate spidroin one or M4 promoted the optimal RPE cell growth. Furthermore, the incorporation of the integrin peptide arginyl-glycyl-aspartic acid (RGD) into the synthetic membranes also enhanced RPE cell growth. Based upon these initial studies and results, the application of rSSps may prove beneficial for in vitro modeling of Bruch’s membrane, RPE cells, and AMD. Ongoing work includes seeding RPE cells onto spider silk membranes of thicknesses ranging from 7 to 25 micrometers to determine the optimal membrane structure for cell growth and proliferation. Future work will focus on incorporating extracellular matrix proteins, such as fibronectin, laminin, vitronectin, or collagen into the synthetic membranes to promote cellular attachment and proliferation. These coatings would mimic the physiological structure of Bruch’s membrane and allow for models to be developed to study AMD.

Location

North Atrium

Start Date

13-4-2017 1:30 PM

End Date

13-4-2017 2:45 PM

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Apr 13th, 1:30 PM Apr 13th, 2:45 PM

Growing Retinal Pigment Epithelial Cells on a Recombinant Spider Silk Protein Membrane

North Atrium

Age-related macular degeneration (AMD) is the leading cause of blindness in developed countries and accounts for nearly 10% of vision loss around the world. Retinal pigment epithelial (RPE) cells grow posterior to the photoreceptors in the eye on an acellular protein structure, called Bruch’s membrane. RPE cells transport nutrients, remove waste products, and provide support for the photoreceptor cells. As aging occurs, a lipid substance called drusen may form inside Bruch’s membrane and inhibit RPE cell growth and function. To better understand the relationship between the disruption of RPE cells and the onset of AMD, RPE cells must be grown and thoroughly characterized in vitro. Such research could lead to the development of an in vitro model of the human eye for evaluating diagnostic methods and treatments to counteract the effects of AMD. This model would incorporate a synthetic Bruch’s membrane that is also capable of mimicking its natural structure and function. In this project, recombinant spider silk proteins (rSSps) were used to develop a proteinaceous membrane and to support RPE growth.

First, freestanding semipermeable films were engineered using either spin coating or gravity spreading techniques to ensure an rSSp membrane that is biomimetic to Bruch’s membrane. Cell proliferation was analyzed using a PicoGreen dsDNA assay. Immunohistochemistry staining was also employed to assess the morphology of cells. Results from transepithelial electrical resistance (TEER) measurements, cell staining, and a permeability assay demonstrated that the rSSp analogue of major ampullate spidroin one or M4 promoted the optimal RPE cell growth. Furthermore, the incorporation of the integrin peptide arginyl-glycyl-aspartic acid (RGD) into the synthetic membranes also enhanced RPE cell growth. Based upon these initial studies and results, the application of rSSps may prove beneficial for in vitro modeling of Bruch’s membrane, RPE cells, and AMD. Ongoing work includes seeding RPE cells onto spider silk membranes of thicknesses ranging from 7 to 25 micrometers to determine the optimal membrane structure for cell growth and proliferation. Future work will focus on incorporating extracellular matrix proteins, such as fibronectin, laminin, vitronectin, or collagen into the synthetic membranes to promote cellular attachment and proliferation. These coatings would mimic the physiological structure of Bruch’s membrane and allow for models to be developed to study AMD.