Prostate Organoid Assay: A Matrix Gel Ring-based Ex Vivo Culture Technique to Study the Differentiation Capacity of Prostate Epithelial Cells

Abstract

Source: Crowell, P. D. et al. Evaluating the Differentiation Capacity of Mouse Prostate Epithelial Cells Using Organoid Culture. J. Vis. Exp. (2019)

In this video, we demonstrate the culturing of isolated mouse prostate epithelial cells using a matrix gel-ring based culture technique. This approach can provide complementary information about the differentiation capacity of prostate epithelial cells to form 3D glandular structures in response to genetic or pharmacological manipulation.

Protocol

1. Plating Sorted Prostate Epithelial Cells into Primary Mouse Organoid Culture —  TIMING: 2-3 H (Excluding Poly-HEMA-coated Plate Preparation)

NOTE: Plates are coated with Poly-HEMA to prevent 2D colony formation on the surface of the well beneath the matrix gel. Prepare Poly-HEMA-coated plates 1 day prior to plating sorted basal or luminal prostate epithelial cells into mouse organoid culture. Thaw 1 mL aliquots of reduced growth factor matrix gel, hereafter referred to as matrix gel, on ice 2 h prior to step 1.1. Y-27632 (ROCK inhibitor) should be added to mouse organoid media immediately p....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fetal Bovine Serum (FBS)	Sigma	F8667
B-27 Supplement (50x), Serum Free	Thermo Fisher Scientific	17504044
Advanced DMEM/F-12	Thermo Fisher Scientific	12634010
µ-Dish 35 mm, high	Ibidi	81156
Matrigel GFR Membrane Matrix	Corning	CB-40230C
Penicillin-Streptomycin (10,000 U/ mL)	Thermo Fisher Scientific	15-140-122
Poly(2-hydroxyethyl methacrylate) (Poly-HEMA)	Sigma	P3932-25G
Y-27632 dihydrochloride (ROCK inhibitor)	Selleck Chemical	S1049-50MG
A83-01	Tocris	2939
(DiHydro)testosterone (5αAndrostan-17β-ol-3-one)	Sigma	A-8380
GlutaMAX	Thermo Fisher Scientific	35050061
Normocin	Thermo Fisher Scientific	ant-nr-1
Recombinant Human EGF, Animal Free	PeproTech	AF-100-15
Recombinant Human Noggin	PeproTech	120-10C
RPMI 1640 Medium, HEPES (cs of 10)	Thermo Fisher Scientific	22400105
Halt Phosphatase Inhibitor	Thermo Fisher Scientific	78428
Complete Protease Inhibitor Cocktail	Sigma	11836145001
Triton X-100	Sigma	X100-5ML
Sonic Dismembrator	Thermo Fisher Scientific	FB120