Mouse Enteric Organoid Culture Protocol

This protocol is intended as a guide only, for full experimental details please read the reference provided.

Introduction

Organoid cultures represent the next generation of tissue culture models. These cultures are extracted directly from living tissues. Stem cell populations are maintained using a feeder layer-free extracellular matrix environment under non-differentiating conditions. When subjected to differentiating conditions, these organoids exhibit expression of tissue-specific genes and differentiation of stem cells into tissue-specific architecture and cell types. The protocol provided below is intended to culture organoid progenitor cells from normal and healthy mouse gastric, small intestine, or colon tissues using Cultrex™ UltiMatrix RGF Basement Membrane Extract as a scaffold.

This protocol provides a procedure for subculturing normal mouse enteric organoids, modified from the original submerged method published by Yin, X. et al. (2014) Niche-independent high-purity cultures of Lgr5+ intestinal stem cells and their progeny. Nat. Methods 11:106. This protocol includes a series of tips on how to prepare culture media for each type of enteric organoid culture (gastric, small intestine, and colon) as well as a general guide to start and passage these organoids.

The majority of reagents used in this protocol were sourced from the Bio-Techne brands of R&D Systems™ and Tocris Bioscience™.

Equipment

  1. Cell culture incubator (37 °C, 5% CO2)
  2. Cell culture hood with laminar flow
  3. Centrifuge with refrigeration and swinging bucket rotor
  4. 37 °C water bath
  5. Ice bucket
  6. Laboratory refrigerator
  7. Pipet aid and serological pipettes (5 mL)
  8. Micropipettes and tips (2–200 μL)
  9. Conical tubes, 15 mL and 50 mL, sterile
  10. 24-well plate, tissue-culture treated, sterile
  11. Vacuum pump
  12. Medium filtration unit, 0.1 μm, 500 mL, sterile
  13. Syringe, 50 mL, sterile
  14. Syringe filter, 0.2 μm, sterile
  15. Cell culture waste container
  16. 20-gauge needle, sterile

Other Required Reagents

  1. Distilled (DW) or deionized water (DI)
  2. Phosphate buffered saline (PBS)
  3. 1% Ammonium
  4. 1% BSA/PBS
  5. DMSO

Materials

TABLE 1. Materials Needed for Mouse Enteric Organoid Culture

Product Name

Supplier

Catalog #
Cultrex Organoid Harvesting Solution R&D Systems 3700-100-01
Cultrex UltiMatrix Reduced Growth Factor Basement Membrane Extract  R&D Systems BME001-05
Advanced DMEM/F-12 Cell Culture Medium Thermo Fisher 12634010
Fetal Bovine Serum (FBS) - Premium R&D Systems S11150
GlutaminePlus R&D Systems B90210
HEPES  Tocris Bioscience 3173
Penicillin-Streptomycin R&D Systems B21210
N21-MAX Supplement  R&D Systems AR008
N-2 MAX Supplement R&D Systems AR009
N-Acetylcysteine  Tocris Bioscience 7874
Valproic Acid Tocris Bioscience 2815
Y-27632 dihydrochloride (Rho Kinase inhibitor) Tocris Bioscience 1254
CHIR 99021 (GSK-3 inhibitor) Tocris Bioscience 4423
Recombinant Human EGF R&D Systems 236-EG
Recombinant Human R-Spondin 1  R&D Systems 4645-RS
Recombinant Human Noggin  R&D Systems 6057-NG
Recombinant Human FGF-10 R&D Systems 345-FG
Recombinant Human Wnt-3a R&D Systems 5036-WN

Reagent Preparation

Use aseptic technique at all times during this protocol. 

1. Prepare stock solutions for mouse enteric organoid culture, as indicated in TABLE 2.

TABLE 2. Preparation of Stock Solutions for Enteric Organoid Culture Medium

Reagent Name

Solvent

Stock Solution

Preparation

Storage
N-Acetylcysteine DI water 500 mM = 81.6 mg/mL 200 mg in 2.4 mL 4 °C
GlutaminePlus PBS 100 mM = 14.6 mg/mL 0.73 g in 50 mL 4 °C
Recombinant Human EGF 1% BSA/PBS 500 µg/mL 200 µg in 400 mL -80 °C
Recombinant Human R-Spondin 1 1% BSA/PBS 1 mg/mL 1 mg in 1 mL -80 °C
Recombinant Human Noggin 1% BSA/PBS 100 µg/mL 100 µg in 1 mL -80 °C
Recombinant Human Wnt-3a 1% BSA/PBS 600 µg/mL 500 µg in 833 µL -80 °C
Valproic Acid DI water 200 mM = 33 mg/mL 100 mg in 3 mL -20 °C
CHIR 99021 DMSO 20 mM = 9.3 mg/mL 10 mg in 1.08 mL -20 °C
Y-27632 dihydrochloride PBS 10 mM = 3.2 mg/mL 10 mg in 3.1 mL 4 °C
HEPES DI water 1 M = 238.3 mg/mL 11.9 g in 50 mL 4 °C

 

2. Prepare 10X Solution M1, as indicated in TABLE 3.

TABLE 3. Preparation of 10X Solution M1

Reagent Name

[STOCK]

[FINAL]

Volume
Advanced DMEM/F12 Cell Culture Medium NA NA 28 mL
N21-MAX Supplement 50X 10X 20 mL
GlutaminePlus 100 mM 20 mM 20 mL
HEPES 1 M 100 mM 10 mL
Penicillin-Streptomycin 100X 10X 10 mL
N-2 MAX Supplement 100X 10X 10 mL
N-Acetylcysteine 500 mM 10 mM 2 mL
   

Total

100 mL
Dispense 4.5 mL per tube into sterile 15 mL conical tubes, label, and store at ≤ -20 °C.

 

3. Prepare 10X Solution M2, as indicated in TABLE 4. 

TABLE 4. Preparation of 10X Solution M2

Reagent Name

[STOCK]

[FINAL]

Volume
Advanced DMEM/F12 Cell Culture Medium NA NA 98.9 mL
Recombinant Human Noggin 100 µg/mL 1 µg/mL 1 mL
Recombinant Human EGF 500 µg/mL 50 ng/mL 100 µL
   

Total

100 mL
Dispense 4.5 mL per tube into sterile 15 mL conical tubes, label, and store at ≤ -70 °C.

 

4. Prepare 1000X Solution M3 by dissolving 500 µg of Wnt-3a in 833 µL of 1% BSA/PBS, and store at ≤ -70 °C.

5. Prepare 1000X Solution M4 by dissolving 1 mg of R-Spondin 1 in 1 mL of 1% BSA/PBS, and store at ≤ -70 °C.

6. Prepare 10X Solution M5 as indicated in TABLE 5.

TABLE 5. Preparation of 10X Solution M5

Reagent Name

[STOCK]

[FINAL]

Volume
Valproic acid 200 mM 20 mM 10 mL
CHIR 99021 20 mM 25 µM 125 µL
Advanced DMEM/F-12 Cell Culture Medium NA NA 89.9 mL
   

Total

100 mL
Dispense 4.5 mL per tube into sterile 15 mL conical tubes, label, and store at ≤ -20 °C.

 

7. Prepare 500X Solution M6 by suspending FGF-10 at a concentration of 100 μg/mL in 1% BSA/PBS, and store at ≤ –70 °C.

8. Prepare Organoid Culture Medium - To prepare each type of Mouse Enteric Organoid Culture Medium, add the appropriate Stock Solution to a 50 mL conical tube, and complete with Advanced DMEM/F12 medium to a final volume of 45 mL. Filter sterilize and keep medium stored at 4 °C for no longer than 2 weeks, as growth factors and supplements lose activity after prolonged storage. Use TABLE 6 as a reference.

TABLE 6. Summary of Stock Solution Volumes Needed to Prepare Mouse Enteric Organoid Culture Medium

Solution

Name

Storage

Concentration

Gastric

Small Intestine

Colon

Aliquot Volume
M1 Supplements -20 °C 10X X X X 4.5 mL
M2 EGF/Noggin -80 °C 10X X X X 4.5 mL
M3 Wnt-3a -80 °C 1000X X   X 45 µL
M4 R-Spondin 1 -80 °C 1000X X X X 45 µL
M5 Valproic Acid -80 °C 10X X X   4.5 mL
M6 FGF-10 -80 °C 500X X     9 µL

 

Methods for Culturing Mouse Enteric Organoids

1. Starting Organoids from a Cryovial

a. Thaw Cultrex UltiMatrix RGF Basement Membrane Extract on ice for four hours or overnight at 2 - 8 °C (on ice in the refrigerator).

b. Thaw the cryovial containing organoids in a 37 °C water bath. NOTE: The contents should thaw in 2–3 minutes; do not allow the cryovial to remain at 37 °C any longer than is necessary.

c. Transfer the contents of the cryovial to a 15 mL conical tube and add 9 mL of Advanced DMEM/F12 with 10% FBS. Gently pipet up and down three times using a serological pipette to resuspend the organoids. NOTE: Organoids may be counted at this time if needed to determine seeding volumes.

d. Centrifuge the vial at 500 × g for 3 minutes to pellet the organoids and aspirate the medium.

e. Resuspend the organoids in Cultrex UltiMatrix RGF Basement Membrane Extract, at 10,000 organoids per mL (500 organoids per well). Pipet up and down three times using a serological pipette to disperse the organoids in the Cultrex UltiMatrix RGF Basement Membrane Extract. Dispense 50 μL of the Cultrex UltiMatrix RGF Basement Membrane Extract/organoid mixture in the center of each well of a 24-well plate or arrange domes placing 6 to 8 domes in a well of a 6-well plate (FIGURE 1). NOTE: The Cultrex UltiMatrix RGF BME-contained organoids should not touch the sides of the well.

Placement of Cultrex UltiMatrix RGF BME Organoid Mixture in a 24-well and 6-well Plate.

FIGURE 1. Placement of Cultrex UltiMatrix RGF BME/Organoid Mixture in a 24-well or 6-well Plate. (A) Placement of Cultrex UltiMatrix RGF BME/organoid mixture in the center of a well in a 24-well plate or (B) placement of multiple domes within a well of a 6-well plate.

f. Incubate the plate in the cell culture incubator for 25 minutes to polymerize the Cultrex UltiMatrix RGF Basement Membrane Extract.

g. Calculate the volume of Organoid Starting/Passaging Medium needed. Each well of a 24-well plate requires 500 uL of Organoid Starting/Passaging Medium, while 3 mL per well is required for a 6-well plate.

h. Prepare Organoid Starting/Passaging Medium by adding Y-27632 dihydrochloride to the Organoid Culture Medium at a final concentration of 10 µM.

i. Add 500 µL of Organoid Starting/Passaging Medium per well of a 24-well plate or 3 mL per well of a 6-well plate. NOTE: Medium should be gently pipetted into the corner of the well away from the Cultrex UltiMatrix RGF Basement Membrane Extract domes to prevent their disruption.

j. Return the plate containing the organoid cultures to the cell culture incubator to promote organoid growth.

 

2. Enteric Organoid Culture Maintenance

The culture medium should be aspirated from each well and replaced with fresh Organoid Culture Medium every other day (i.e., Monday, Wednesday, and Friday). Mouse enteric organoids can be cultured for up to two weeks before passaging, depending on the cell seeding density (see the protocol below). NOTE: Medium should be gently aspirated from and pipetted into the corner of the well away from the Cultrex UltiMatrix RGF BME/organoids to prevent their disruption.

 

Microscopic brightfield images showing the progression of undifferentiated (A) to differentiated mouse small intestine organoids (B,C)

FIGURE 2. Mouse Small Intestine Organoids. Representative brightfield images of (A) undifferentiated and (B, C) differentiated mouse small intestine organoids that were cultured using Cultrex UltiMatrix RGF Basement Membrane Extract (R&D Systems, Catalog # BME001-05) and the other Bio-Techne reagents listed in this protocol.

3. Passaging Organoids

a. View organoids under the microscope. Each well of a 24-well plate should contain approximately 100 to 500 organoids for optimal growth. Organoid cultures exhibiting rapid growth may be split 1:4 during passaging, while slow growing cultures may benefit from a 1:1 split. Make this determination prior to harvesting to estimate reagent needs before starting. NOTE: Organoid density is important for optimal growth; too many organoids will strain culture resources, while too few organoids lack paracrine signaling necessary to sustain growth.

b. Aspirate the medium without disturbing the organoids at the bottom of the wells.

c. Wash each well with 10 volumes of cold (4 °C) PBS, and aspirate without disturbing the Cultrex UltiMatrix RGF Basement Membrane Extract dome (e.g. if the well has 1 x 50 μL dome, 500 μL of wash solution must be used).

d. Add 10 volumes of cold (4 °C) Cultrex Organoid Harvesting Solution to each well to depolymerize the Cultrex UltiMatrix RGF Basement Membrane Extract. If each well contains 50 μL of Cultrex UltiMatrix RGF Basement Membrane Extract, 500 μL of Organoid Harvesting Solution will be needed per well in the plate. 

e. Place the plate in a 4 °C cooler with moderate shaking for one hour to depolymerize the Cultrex UltiMatrix RGF Basement Membrane Extract. NOTE: Most of the Cultrex UltiMatrix RGF Basement Membrane Extract should be visibly depolymerized during this incubation; however, some small amount may remain.

f. Pipet up and down three times with a serological pipette across the well to solubilize any remaining gel.

g. Pass the organoid solution through a 20-gauge needle into a conical tube to fragment the organoids.

h. Centrifuge the tube at 500 × g at 4 °C for 5 minutes.

i. Aspirate the supernatant but be careful not to disturb the organoid pellet.

j. Resuspend the pellet in 10 volumes of cold (4 °C) PBS.

k. Centrifuge the tube at 500 × g at 4 °C for 5 minutes.

l. Aspirate the supernatant but be careful not to disturb the organoid pellet.

m. Repeat the centrifugation and aspiration to remove all of the liquid to prevent dilution of the Cultrex UltiMatrix RGF Basement Membrane Extract.

n. Resuspend the segmented organoids in Cultrex UltiMatrix RGF Basement Membrane Extract and dispense 50 μL of the Cultrex UltiMatrix RGF Basement Membrane Extract/organoid mixture into the center of each well of a 24-well plate to form a dome. NOTE: The Cultrex UltiMatrix RGF Basement Membrane Extract-contained organoids should not touch the sides of the wells.

o. Incubate the plate in the cell culture incubator for 25 minutes to polymerize the Cultrex RGF Basement Membrane Extract.

p. Add 500 µL of Organoid Starting/Passaging Medium per well of a 24-well plate, or 3 mL per well of a 6-well plate. NOTE: Medium should be gently pipetted into the corner of the well away from the Cultrex UltiMatrix RGF Basement Membrane Extract to prevent its disruption.

q. Return the plate containing the organoid cultures to the cell culture incubator to promote organoid growth.

 

4. Cryobanking Organoids

a. View organoids under the microscope. Each well of a 24-well plate should contain approximately 100-500 organoids.

b. Aspirate the medium without disturbing the organoids at the bottom of the wells.

c. Wash each well with 10 volumes of cold (4 °C) PBS, and aspirate without disturbing the Cultrex UltiMatrix RGF Basement Membrane Extract dome.

d. Add 10–20 volumes of cold (4 °C) Organoid Harvesting Solution to each well to depolymerize the Cultrex UltiMatrix RGF Basement Membrane Extract.

e. Place the plate(s) in a 4 °C cooler with moderate shaking for one hour to depolymerize the Cultrex UltiMatrix RGF Basement Membrane Extract. NOTE: Most of the Cultrex UltiMatrix RGF Basement Membrane Extract should be visibly depolymerized during this incubation; however, some small amount may remain.

f. Pipet up and down three times with a serological pipette across the well to solubilize any remaining gel.

g. Pass the organoid solution through a 20-gauge needle into a conical tube to fragment the organoids.

h. Centrifuge the tube at 500 × g at 4 °C for 5 minutes.

i. Aspirate the supernatant but be careful not to disturb the organoid pellet.

j. Resuspend the pellet in 10 volumes of cold (4 °C) PBS.

k. Centrifuge the tube at 500 × g at 4 °C for 5 minutes.

l. Aspirate the supernatant but be careful not to disturb the organoid pellet.

m. Repeat the centrifugation and aspiration to remove all the liquid to prevent dilution of the cryopreservation medium.

n. Resuspend the segmented organoids in 90% FBS, 10% DMSO, and 10 μM Y-27632 dihydrochloride, and dispense 500 μL of the organoid mixture into each labeled cryovial.

o. Place the cryovials in a freezing container, and store at  -80 °C for 24 hours.

p. Transfer the cryovials to a liquid nitrogen tank for long-term storage.

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