Prostate Organoid Assay: A Matrix Gel Ring-based Ex Vivo Culture Technique to Study the Differentiation Capacity of Prostate Epithelial Cells
Published: April 30, 2023
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 …
Açıklamalar
The authors have nothing to disclose.
Materials
| 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 |
Bu Makaleden Alıntı Yapın
Prostate Organoid Assay: A Matrix Gel Ring-based Ex Vivo Culture Technique to Study the Differentiation Capacity of Prostate Epithelial Cells. J. Vis. Exp. (Pending Publication), e20389, doi: (2023).