Work

Protocol for generation of immortalized retinal, choroid, and brain endothelial cell lines

Year: 2025

Type: article

Abstract: The integrity and functionality of endothelial cells (ECs) play a crucial role in the homeostasis of different tissues; however, ECs show tissue-specific morphology and gene expression profile. Here, we present a protocol for the production of stable EC lines from mouse retinal, choroidal, and brain tissue. We outline the procedures for preparing the three specified tissues and generating single-cell suspensions through enzymatic digestion. Subsequently, we describe the methods used to selectively immortalize ECs and maintain their cultures for downstream experiments. For complete details on the use and execution of this protocol, please refer to Heiden et al. 1 • Protocol for dissection of murine brain, retina, and choroid • Description of optimal conditions for enzymatic digestion to generate living single cells • Generation of stable endothelial cell lines from brain, retina, or choroid Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. The integrity and functionality of endothelial cells (ECs) play a crucial role in the homeostasis of different tissues; however, ECs show tissue-specific morphology and gene expression profile. Here, we present a protocol for the production of stable EC lines from mouse retinal, choroidal, and brain tissue. We outline the procedures for preparing the three specified tissues and generating single-cell suspensions through enzymatic digestion. Subsequently, we describe the methods used to selectively immortalize ECs and maintain their cultures for downstream experiments.

Source: STAR Protocols

DOI: 10.1016/j.xpro.2025.104089