Choroid Sprouting Assay to Study Ocular Microvascular Proliferation Ex Vivo

Abstract

Source: Tomita, Y., et al. An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis. J. Vis. Exp. (2020)

This video demonstrates the development of an ex vivo model of choroidal sprouting to study ocular microvascular angiogenesis. The assay involves culturing mouse eye explants containing the choroid tissue attached to the sclera and retinal pigment epithelium under specific conditions that promote choroidal microvascular sprouting.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Preparation

1. Add 5 mL of Penicillin/Streptomycin (10000 U/mL) and 5 mL and 10 mL of commercially available supplements to 500 mL of complete classic medium with serum. Aliquot 50 mL of the medium initially.
   NOTE: Do not return any medium back to the stock to avoid contamination.
2. Put an aliquot of the complete classic medium on ice.
3. Use 70% ethanol to clean the dissecting microscope, forceps, and scissors.
4. Prepare two cell culture d....

Results

Figure 1: Schematic illustration showing choroid sprouting assay.
Eyes were first enucleated and cut circumferentially about 0.5 mm posterior to the limbus. The cornea, iris, lens, and vitreous were removed. Then a 1 mm cut was made from the edge of the eye cup towards the optic n.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
AnaSed (Xylazine)	AKORN	59339-110-20
Basal membrane extract (BME) Matrigel	BD Biosciences	354230
Cell culture dish	NEST	704001	10cm
Complete classic medium with serum and CultureBoost	Cell systems	4Z0-500
Ethyl alcohol 200 Proof	Pharmco	111000200	use for 70%
Kimwipes	Kimberly-Clark	06-666
Microscope	ZEISS	Axio Observer Z1
Penicillin/Streptomycin	GIBCO	15140	10000 U/mL
Tissue culture plate (24-well)	Olympus	25-107
VetaKet CIII (Ketamine)	AKORN	59399-114-10