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Peter Heiduschka, Julian Alexander Zimmermann, Tanja Plagemann, Piotr Stafiej, Marcus Himmler, Thomas Armin Fuchsluger, Nicole Eter; Cultivation and characterisation of primary retinal pigment epithelium cells on nanofibre scaffolds. Invest. Ophthalmol. Vis. Sci. 2019;60(9):3937.
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© ARVO (1962-2015); The Authors (2016-present)
Replacement of RPE is a therapeutic option considered in diseases with degenerating RPE. One way to replace defective RPE is to cultivate RPE cells on a polymeric scaffold, thus enabling a controlled insertion into the subretinal space. We examined scaffolds made of nanofibres on their usefulness for the cultivation of primary RPE cells, and we checked behaviour of RPE cells on the scaffolds.
Polymeric scaffolds were prepared from of networks of nanofibres made from polycaprolactone (PCL) or a PCL-chitosan co-polymer by electrospinning. Primary RPE cells were isolated from pig eyes and cultivated. After reaching a stable state, the RPE cells were given onto small pieces of the scaffolds in a 24-well plate. Expression of various genes (PEDF, VEGF, ZO-1, IL-1β, IL-6, TNF-α, CDH1) was checked by quantitative PCR two and ten days later. IL-6 and TNF-α protein release was measured by ELISA after two, six and ten days of cultivation. RPE cells cultivated on the bottom of empty wells served as a control. RPE cells were identified by RPE65 and ZO-1 immunocytochemistry.
Almost all cells on the scaffolds could be identified as RPE cells. Within the observation time, RPE cells did not reach confluency. In general, adhesion of RPE cells on the scaffolds was weaker than on the bottom of the culture plate wells. Number of RPE cells on the scaffolds decreased during time of experiment. After two days of cultivation on the scaffolds, a clear increase in expression of inflammatory genes (IL-1β 400×, IL-6 14×, and TNF-α 47×) was found compared to controls, whereas expression of the factors PEDF and VEGF as well as of the adhesion proteins CDH1 and ZO-1 showed only a slight change (1.5× increase). After ten days, only IL-6 and TNF-α genes showed an increased expression (1.6× and 30×, respectively). Increased release of IL-6 and TNF-α proteins compared to controls was also found by ELISA.
RPE cells can be cultivated on the PCL nanofibre scaffolds. However, cell number was smaller and adhesion weaker than under control conditions. RPE cells showed a clearly increased expression of inflammatory cytokines that declined during the time of observation. Further optimisation of the nanofibres is necessary to increase survival and adhesion of RPE cells on the scaffold, e.g. by polymer modification, changed weaving pattern or coating with proteins, such as collagen, fibronectin or laminin.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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