Z40 - Nanofibrous polyethylene glycol scaffolds to support retinal pigment epithelium adhesion and growth

Saturday, October 11, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

Grace Burns

Undergraduate Researcher
University of Virginia
Aiken, South Carolina, United States

Co-Author(s)

TB

Thomas H. Barker, PhD

Biomedical Engineering Professor
University of Virginia
Charlottesville, Virginia, United States

Primary Investigator(s)

TB

Thomas H. Barker, PhD

Biomedical Engineering Professor
University of Virginia
Charlottesville, Virginia, United States

Co-Author(s)

CH

Chris Highley, PhD

Assistant Professor, Biomedical Engineering and Chemical Engineering
University of Virginia, United States
EF

Emily Ferrarese, BS MS

PhD Student
University of Virginia, United States
JS

Jagathpala Shetty

Research Scientist
University of Virginia, United States
Ally O'Shea

Undergraduate Researcher
University of Virginia
Glen Allen, Virginia, United States
RB

Rishi Bhogaraju

Undergraduate Researcher
University of Virginia, United States

Introduction: : Estimates suggest that by 2040 ~300 million people will present with age-related macular degeneration, an irreversible blinding disease [1]. This does not include millions more with diabetic retinopathy, retinitis pigmentosa, choroideremia, and other retinal degenerative diseases. Despite the significant prevalence of these diseases, no approved treatments to prevent retinal degradation exist [2]. A key hallmark of these diseases is retinal pigment epithelial cell (RPE) dysfunction. RPEs provide critical support for photoreceptor cells and are part of the outer blood-retinal barrier (oBRB), composed of the RPE, Bruch’s membrane (BM), and the choriocapillaris. These components interact dynamically, making in vitro models of only RPE monocultures insufficient to mimic the in vivo phenotype. To best model RPEs in vitro, RPE cells should be cultured in conditions similar to their native environment [2]. In vivo, BM provides a permeable, flexible, but robust substrate composed of fibrous extracellular matrix [1]. One persistent gap in the field is optimizing a biomaterial that resembles the natural BM and instructs native RPE phenotypes.
Electrospinning, a technique suitable for creating fibrous substrates, may enable BM mimicry by producing fiber diameters on the nanometer scale, similar to ECM fibers [3]. It also permits tuning of scaffold parameters such as pore size, thickness, density, and material [1]. Polyethylene glycol (PEG) may be an attractive polymer for electrospinning; it is FDA approved, well understood, hydrophilic, and biocompatible [4]. Here, we explore PEG fiber parameters for culturing human RPE monolayers, assessing fiber dimensions and ECM-derived adhesive ligands using RPE phenotypic markers as outcomes.

Materials and

Methods: : A combination of norborene-modified polyethylene glycol (NorPEG) and thiol-modified polyethylene glycol (PEG-thiol) were integrated to create an electrospinning solution. This solution included 10 wt% NorPEG, 7 wt% 10 kDA PEG-thiol, I2959 photoinitiator, 2 mM RGD, and non-reactive polyethylene oxide. Glass coverslips (22 x 22 mm) were surface modified to covalently crosslink fibers deposited on them. NorPEG was electrospun onto coverslips for 15 minutes at 300 rpm (random alignment). The scaffolds were nitrogen purged and UV crosslinked. Coverslides were sterilized with 70% ethanol, transferred to a new 6 well plate, and stored at 37 °C, 5% CO2 until use.
Human retinal pigment epithelial cells (ARPE-19; ATCC) were seeded at 6.69 x 10^5 cells/mL on electrospun fibers or control coverslips in growth media containing MEM alpha with GlutaMAX, 10% FBS, 1% Penicillin/Streptomycin, 1% N1 supplement, taurine (0.25 mg/ml), hydrocortisone (20 ng/ml), triiodo-thyronin (0.013 ng/ml), and 10 mM nicotinamide [5]. Each well received 1 mL of media and was left in the incubator overnight.
The following day, media was removed and 1 mL of ARPE-19 cell suspension was added to each well. After 3 hours of incubation, 1 mL of fresh media was added. Cell growth was imaged over the next week to monitor proliferation and morphology. The media was changed every 3 days. To further validate RPE behavior, cells will be stained for key phenotypic markers, such as ZO-1 and Ezrin, and cytokine quantification will be done to assess PEDF and VEGF secretion [6].

Results, Conclusions, and Discussions:: Though the cells were seeded at the same density, initially the control coverslide appeared to have more adhered cells than the PEG-RGD scaffold (Table 1). After additional days of growth, more cells proliferated on the scaffold, but were consistently less dense than the control. The control exhibits dense and uniform cell coverage. Therefore, cells are proliferating on the PEG-RGD scaffolds but more slowly than the rate of the control. Some of the seeded cells found their way under the coverslides, however, this is not believed to impact the results.
Despite apparent lower proliferation rates, the results show that this PEG-RGD scaffold did successfully support adhesion and allowed cells to proliferate which holds promise for future BM biomimetic designs. Though the growth rate was not optimal, cell density did increase, indicating that these scaffolds may require a longer culture period before they become fully confluent and exhibit characteristics of natural RPE cells. We are currently exploring phenotypic markers of RPE cells and anticipate reporting results on the poster.
This scaffold system lays a foundation for development of in vitro oBRB disease models for retinal degeneration. Building upon this system to create a physiologically more accurate model is critical for nurturing more advanced medical treatments. However, there are many future steps that need to be taken before such a model exists. Laminin and fibronectin are different adhesion motifs that may be more favorable for RPE adhesion and growth. Therefore a PEG scaffold with one or both of these components may be a better alternative to RGD. Furthermore, there is likely an opportunity to optimize the base material for the scaffold. Polycaprolactone (PCL) presents advantageous capabilities such as biocompatibility, biodegradability, cost efficacy, and its ability to maintain its properties throughout physical, chemical, and mechanical stresses [7]. Electrospinning parameters such as fiber diameter, fiber alignment, and scaffold thickness could also be adjusted to further optimize cell adherence and growth. Furthermore, to biologically ensure that the RPE cells are displaying native quality function staining for RPE biomarkers such as ZO-1 and ezrin is essential.

Acknowledgements and/or References (Optional): : [1] B. Molins, A. Rodríguez, V. Llorenç, and A. Adán, “Biomaterial engineering strategies for modeling the Bruch’s membrane in age-related macular degeneration,” Neural Regen. Res., vol. 19, no. 12, pp. 2626–2636, Mar. 2024, doi: 10.4103/NRR.NRR-D-23-01789.
[2] J. Kim, J. Y. Park, J. S. Kong, H. Lee, J. Y. Won, and D. W. Cho, “Development of 3D Printed Bruch’s Membrane-Mimetic Substance for the Maturation of Retinal Pigment Epithelial Cells,” Int. J. Mol. Sci., vol. 22, no. 3, p. 1095, Jan. 2021, doi: 10.3390/ijms22031095.
[3] A. N and O. H, “Cytocompatibility of electrospun poly-L-lactic acid membranes for Bruch’s membrane regeneration using human embryonic stem cell-derived retinal pigment epithelial cells,” PubMed, Accessed: Jul. 18, 2025. [Online]. Available: https://pubmed.ncbi.nlm.nih.gov/38726752/
[4] T. Serra, M. Ortiz-Hernandez, E. Engel, J. A. Planell, and M. Navarro, “Relevance of PEG in PLA-based blends for tissue engineering 3D-printed scaffolds,” Mater. Sci. Eng. C, vol. 38, pp. 55–62, May 2014, doi: 10.1016/j.msec.2014.01.003.
[5] R. A. Hazim, S. Volland, A. Yen, B. L. Burgess, and D. S. Williams, “Rapid differentiation of the human RPE cell line, ARPE-19, induced by nicotinamide,” Exp. Eye Res., vol. 179, pp. 18–24, Feb. 2019, doi: 10.1016/j.exer.2018.10.009.
[6] C. Dujardin, W. Habeler, C. Monville, D. Letourneur, and T. Simon-Yarza, “Advances in the engineering of the outer blood-retina barrier: From in-vitro modelling to cellular therapy,” Bioact. Mater., vol. 31, pp. 151–177, Aug. 2023, doi: 10.1016/j.bioactmat.2023.08.003.
[7] R. Dwivedi et al., “Polycaprolactone as biomaterial for bone scaffolds: Review of literature,” J. Oral Biol. Craniofacial Res., vol. 10, no. 1, pp. 381–388, 2020, doi: 10.1016/j.jobcr.2019.10.003.