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

Samenvatting

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 prior to step 1.1. Perform steps 1.1-1.8 on ice.

1. Pellet the cells in 5 mL round-bottom tubes by centrifugation at 800 x g for 5 min at 4 °C and aspirate the supernatant.
2. Wash the cell pellet in 500 µL of mouse organoid media (Table 1).
3. Pellet the cells by centrifugation at 800 x g for 5 min at 4 °C and aspirate the supernatant.
4. Resuspend in mouse organoid media at a cell density of 1,000 cells/µL.
5. To prepare master mixes, mix epithelial cells suspended in mouse organoid media with matrix gel to generate a final mixture that contains 25% cells/media and 75% matrix gel. Basal cells are typically plated at a concentration of 100-2,000 cells/80 µL, whereas luminal cells are typically plated at a concentration of 2,000-10,000 cells/80 µL. The density of cells plated varies depending upon the day of anticipated material collection and the desired downstream application.
   NOTE: Chill appropriately sized tube(s) for expected master mix volume 5 min prior to master mix preparation. To ensure the matrix gel does not harden while handling, it is critical to chill the pipette tip by pipetting the matrix gel 3-4 times prior to transferring it to a new tube.
6. Add 80 µL of the matrix gel/cell mixture per well of a 24-well plate. Pipetting a droplet onto the lower half of the wall of the well, while avoiding direct contact with the Poly-HEMA coating is recommended. After adding the matrix gel, swirl the plate to allow the matrix gel/cell mixture to form a ring around the rim of the well.
7. Place the 24-well plate into a 37 °C 5% CO₂ incubator right side up for 10 min to allow the matrix gel to partially harden.
   NOTE: Begin warming mouse organoid media at 37 °C immediately after placing the 24-well plate in the incubator.
8. After incubating for 10 min, flip the 24-well plate upside-down and incubate for an additional 50 min to allow the matrix gel to completely harden.
9. Add 350 µL of pre-warmed mouse organoid media dropwise to the center of each well.
   NOTE: To maintain the integrity of the matrix gel, it is critical to avoid the matrix gel ring while adding media.
10. After adding the media, return the 24-well plate to the 37 °C 5% CO₂ incubator.

2. Replenishing Mouse Organoid Media — TIMING: 10-15 Min Per 24-well Plate

NOTE: Existing media should be replaced with fresh media every 48 h. Before each media change, pre-warm mouse organoid media. It is not necessary to add ROCK inhibitor to the media used for replenishing.

1. Tilt the 24-well plate at a 45° angle and gently remove existing media from the center of each well using a P1000 pipette while avoiding the matrix gel ring.
2. Add 350 µL of pre-warmed mouse organoid media as in step 1.9. It is recommended to add a larger volume of media (up to 1 mL) to organoids cultured for longer than 5 days to prevent the rapid depletion of key nutrients and growth factors.

Table 1: Instructions for the preparation of mouse organoid media

Component	Concentration
B-27	1x (dilute from 50x concentrate)
GlutaMAX	1x (dilute from 100x concentrate)
N-acetyl-L-cysteine	1.25 mM
Normocin	50 µg/mL
Recombinant Human EGF, Animal-Free	50 ng/mL
Recombinant Human Noggin	100 ng/mL
R-spondin 1-conditioned media	10% conditioned media
A83-01	200 nM
DHT	1 nM
Y-27632 dihydrochloride (ROCK inhibitor)	10 µM
Advanced DMEM/F-12	Base media
R-spondin 1-conditioned media is generated as described in Drost et al. After addition of all components, filter sterilize mouse organoid media using 0.22 µm filter. ROCK inhibitor is only added during establishment of culture and passaging of organoids.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
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