Abstract
Purpose :
Retinal pigment epithelium (RPE) and retina interact closely, with RPE performing multiple tasks ensuring proper retinal physiology and forming a tight, interlocked structure with photoreceptor outer segments. However, current RPE cell culture systems lack the contacts between RPE and retina, possibly limiting not only RPE maturation but also hindering the studies addressing the RPE-retina interface and its biophysical characteristics. We hypothesized that a retina-mimicking structure can be constructed from a hydrogel and introduced to RPE cells’ apical side during culturing, and used to model the RPE-retina interface in vitro.
Methods :
Soft polyacrylamide hydrogels were coated with Matrigel and placed on the apical side of near-mature human stem cell derived RPE that were cultured on semipermeable membranes using protocols established before. RPE cells adhered to the hydrogels on their apical side during culture, enabling further studies of the biophysical properties of RPE with methods developed from rheology and traction force microscopy. Force generation in RPE during phagocytosis was followed by using a hydrogel coated with purified photoreceptor outer segment particles.
Results :
During culture, the hydrogel did not affect RPE cell morphology or functionality. After 14 days of culture, RPE-hydrogel adhesion force was 1.8 nN/µm2 (± 0.4, n=14 biological replicates) quantified with a rheometric tack adhesion test. Force transmission was characterized during phagocytosis with the hydrogel. During phagocytosis, RPE cells generated traction forces on average 400 pN/µm2 (± 200, n=21 images, 8 biological replicates) within a 20-minute time interval. Cytochalasin D, an actin polymerization inhibitor, reduced the average traction forces to 100 pN/µm2 (±70, n= 10 images, 5 biological replicates, p<0.001).
Conclusions :
A retina-mimicking structure was constructed from soft hydrogel and cultured on RPE cells’ apical side without affecting morphology or functionality of the cells. Thus, this enabled biophysical studies of the RPE-hydrogel interface in vitro, including characterization of forces exerted by RPE during phagocytosis. These results support the view of RPE as a mechanically active partner for retina. In addition, our retina-mimicking structure offers a tool for further biophysical modeling of the RPE-retina interface.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.