Preparation of Human Choroidal ECM Scaffolds to Study Cell Replacement Strategies

Abstract

Purpose : Endothelial cells (ECs) of the choriocapillaris are lost very early during the pathogenesis of age-related macular degeneration (AMD), and cell replacement therapy is currently the most promising option for patients with advanced AMD. Here, we sought to develop a reliable method for the production of human choroidal extracellular matrix (ECM) scaffolds, which will allow for the study of choroidal EC (CEC) replacement therapies in an environment that closely resembles the native tissue.

Methods : Using unfixed RPE/choroid punches from human donor eyes, four different protocols were evaluated for their ability to remove cells from the tissue. After decellularization, immunohistochemistry (IHC) was employed to compare treated to untreated punches, confirm cell removal, and assess the localization and relative abundance of the remaining ECM proteins. To further validate CEC removal, decellularized tissue ultrastructure was visualized using transmission electron microscopy (TEM), and mass spectrometry (MS) was performed to evaluate protein composition. Finally, the acellular choroid scaffold was co-cultured with RF/6A CECs or human endothelial cells to assess the ability to recellularize the tissue.

Results : Sequential treatment with 1% Triton X-100, 0.1% SDS, and DNase solutions is most effective at removing all native cells. Decellularized RPE/choroid tissue shows a loss of CD31 immunolabeling and endothelial cell-specific lectin labeling (UEA-I), as well as complete absence of the nuclear DAPI stain compared to native RPE/choroid. In contrast, the decellularized tissue exhibits preserved collagen IV, elastin, and laminin in both IHC and MS analyses. TEM micrographs reveal complete removal of CECs within the choriocapillaris, with retained basement membrane surrounding the vessel walls. Both RF/6A and human ECs are able to successfully migrate into the choriocapillary tubes.

Conclusions : Our data demonstrate the ability to remove all cells from human RPE/choroid punches while keeping the ECM largely intact. Furthermore, intact vascular walls with basal laminae are visible in the remaining ECM, suggesting that these methods are gentle enough to preserve tissue structure and allow for the optimization of cell replacement strategies.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

 

RF/6A cells (phalloidin; green) are shown recellularizing the choroid scaffold (collagen IV; red). Cell nuclei are shown in blue. Scale bar = 50µm.