Abstract
Purpose :
The ex vivo retina is useful to study Müller glia (MG). Survival of MG in ex vivo tissue can be maintained long term by culturing tissue on a transwell membrane. Electroporation to this system is difficult. Submergence in liquid damages tissue due to fragility of adherence to the membrane. We have developed a novel electroporation method for gene delivery to ex vivo retinae using an agarose disk for electric flow which avoids submergence. We have found an increase in the ability to transfect MG at 14DIV vs 1DIV. I hypothesize that this is due to increased survival of MG over other cell types and increased MG surface area accessibility at the time of electroporation.
Methods :
For electroporation of ex vivo tissue, a 0.5% agarose disk is attached to an electrode. Surface tension between tissue and agarose disk via DNA allows for current flow. Expression vectors were delivered using this method to tissue from MG and retinal ganglion cell reporter mice at 1DIV and 14DIV. Electroporated tissues were immunostained at 1DIV and 14DIV with markers to identify various cell types and states.
Bulk RNA-seq of 0, 7, and 14DIV ex vivo tissue was used to compare transcriptomic changes over time in culture. Ingenuity pathway analysis was used to compare differential gene expression at these timepoints to cell type specific gene lists to determine what cell-type specific markers are enriched at these timepoints.
Results :
Cell type-specific reporter mice reveal loss of retinal ganglion cells by 14DIV, and persistence of MG. Changes in MG morphology are observed and increases in GFAP labeling indicate reactivity of MG. An increase in surface area of MG due to hypertrophy and morphology changes appears to make MG easier to transfect at 14DIV vs 1DIV in the central retina.
Ingenuity pathway analysis of differentially expressed genes in cultures at 0DIV and 14DIV reveals significant upregulation of MG-specific genes at 14DIV and downregulation of neuronal genes. This indicates increased survival of MG compared to other cell types.
Conclusions :
MG are more accessible to transfection at 14DIV vs 1DIV. At 14DIV, there is loss of retinal ganglion cells and remodeling of MG cells. Transcriptomic changes in ex vivo tissue at 14DIV vs 1DIV indicates survival of MG over other cell types. The agarose disk electroporation method is a robust system to deliver genetic material to ex vivo retinal tissue and could be used for other explant tissue types.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.