Materials and Reagents

Disposable scalpels (Swan Morton, catalog number: 0510 )
Petri dishes 92 x 16 mm with cams (SARSTEDT, catalog number: 82.1473 )
Tubes 50 ml, 30 x 115 mm, PP (Corning, Falcon^®, catalog number: 352070 )
20 ml eccentric tip syringe (BD, catalog number: 300613 )
Needle 21 G x 1 ½ (BD, catalog number: 305167 )
Tips refill (VWR, catalog numbers: 89079-464 ; 89079-470 ; 89079-478 )
70 µm nylon filters (Corning, Falcon^®, catalog number: 352350 )
40 µm nylon filters (Corning, Falcon^®, catalog number: 352340 )
P6 well plate (VWR, catalog number: 734-2323 )
P12 well plate (VWR, catalog number: 734-2324 )
Microcentrifuge tubes, 1.5 ml (VWR, catalog number: 212-0198 )
Tubes 10 ml, 100 x 16 mm, PP (SARSTEDT, catalog number: 62.9924.284 )
Cryotubes 1 ml (Greiner Bio One, catalog number: 123263 )
Syringe filter 0.2 µm (VWR, catalog number: 28145-477 )
Serological pipets 5 ml, 10 ml and 25 ml (Corning, Falcon^®, catalog numbers: 357543 ; 357551 ; 357535 )
Mice (RjOrl:SWISS and C57BL/6JRj backgrounds-6 to 8 weeks old-males and females)
Dulbecco’s phosphate-buffered saline (DPBS), CaCl₂ free, MgCl₂ free (Thermo Fisher Scientific, Gibco^TM, catalog number: 14190-094 )
Fetal bovine serum (FBS) (Thermo Fisher Scientific, Gibco^TM, catalog number: 10270 )
Stem Pro Accutase cell dissociation reagent (Thermo Fisher Scientific, Gibco^TM, catalog number: A1110501 )
Matrigel^® basement membrane matrix (Corning, catalog number: 354234 )
Liquid nitrogen (supplied from Air liquide)
500 mM EDTA (pH 8.0) (Thermo Fisher Scientific, Invitrogen^TM, catalog number: 15575-038 )
Albumin from bovine serum (BSA) (Sigma-Aldrich, catalog number: A3294 )
Advanced DMEM/F12 (Thermo Fisher Scientific, Gibco^TM, catalog number: 12634-010 )
Gentamycin 50 mg/ml (Thermo Fisher Scientific, Gibco^TM, catalog number: 15750-037 )
Penicillin-streptomycin cocktail 100x (Thermo Fisher Scientific, Gibco^TM, catalog number: 15140-122 )
Amphotericin B 250 µg/ml (Thermo Fisher Scientific, Gibco^TM, catalog number: 15290-026 )
L-glutamine (Thermo Fisher Scientific, Gibco^TM, catalog number: 25030-081 )
N-2 supplement 100x (Thermo Fisher Scientific, Gibco^TM, catalog number: 17502-048 )
B-27 w/o vit. A 50x (Thermo Fisher Scientific, Gibco^TM, catalog number: 12587-010 )
1 M HEPES (Thermo Fisher Scientific, Gibco^TM, catalog number: 15630-056 )
N-acetyl cysteine (Sigma-Aldrich, catalog number: A7250 )
Growth factors:
Recombinant murine EGF (Peprotech, catalog number: 315-09 )
Recombinant murine Noggin (Peprotech, catalog number: 250-38 )
Recombinant murine CHO-derived R-spondin 1 (R&D Systems, catalog number: 7150-RS/CF )
Recombinant murine Fgf10 (R&D Systems, catalog number: 6224-FG )
Recombinant murine Wnt3a (R&D Systems, catalog number: 1324-WN/CF )
Gastrin I (Sigma-Aldrich, catalog number: SCP0152 )
Rho kinase inhibitor Y27632 (Sigma-Aldrich, catalog number: Y0503 )
DMSO (Sigma-Aldrich, catalog number: D8418 )
Propanol-2 (VWR, catalog number: 1.09634.9900 )
Ethanol 95-97% (VWR, TechniSolv^®, catalog number: 84857.360 )
70% ethanol (see Recipes)
DPBS-EDTA (10 mM) (see Recipes)
DPBS-BSA 2%-EDTA (2 mM) (see Recipes)
Basal crypt medium (BCM) (see Recipes)
ENRGWF medium for initial seeding (see Recipes)
ENRGWF medium for maintenance (see Recipes)
Freezing medium (see Recipes)
De-freezing medium (see Recipes)

Equipment

Binocular (Motic, model: SMZ-168 )
Cold light source (SCHOTT, model: KL1500 LCD )
Scissors: straight sharp tip (Fine Science Tool, catalog numbers: 14090-09 and 14084-08 )
Angled serrated tip forceps (Fine Science Tool, catalog number: 11080-02 )
Standard (fine) tip forceps (Fine Science Tool, catalog number: 11251-20 )
Micro-dissecting scissors (Fine Science Tool, catalog number: 15018-10 )
Refrigerated centrifuge (Beckman Coulter, model: Allegra X-15R )
MaxQ^TM 4000 shaker with adaptable temperature (Thermo Fisher Scientific, Thermo Scientific^TM, model: MaxQ^TM 4000)
Biological safety cabinet (Esco Micro Pte, model: Class II Type A2 )
Pipettors with Tip Ejector 20-200 µl and 100-1,000 µl (VWR, catalog numbers: 89079-970 and 89079-974 )
Cell culture incubator (37 °C, 5% CO₂) (BINDER, model: C150 )
Inverted bright field microscope (Motic, model: AE31 )
Nalgene Cryo ‘Mr Frosty’ freezing container (Thermo Fisher Scientific, Thermo Scientific^TM, model: 5100-0050 )
Ultra-low temperature upright freezer (Thermo Fisher Scientific, model: Thermo scientific Queue Basic)
Cryostorage system K Series (Taylor-Wharton, model: 24K )

Procedure

Notes:

General considerations regarding mice: Animals should be housed in a temperature (21 ± 1 °C) and humidity (55 ± 10%) - controlled room with a 12 h light/12 h dark cycle.
Unless specified, steps are carried out at room temperature (RT).

Dissection procedures and preparation of samples
1. Isolation of antral glands for culture (Figure 1)
  
  Figure 1. Overall procedure schematic of step A1

Note: These steps allow the isolation of adult antral glands that can be used to give rise to gastric organoids in culture (Figure 4). However, single cell isolation from antral glands might be the best option to perform culture after FACS for example. In this case, the user of this protocol should follow the modification described in step A2 (see below).

Figure 4. Adult glands isolation. Example of mouse antral glands suspension before plating or single cell dissociation. Scale bars = 100 µm.

Single cell isolation from antral glands (Figure 5)

Figure 5. Overall procedure schematic of step A2

Re-suspend the pellets from step A1z.iii in 4 ml Stem Pro Accutase cell dissociation reagent.
Transfer each suspension to a P6 well plate and incubate it at 37 °C with 75 rpm agitation for 40 min. It is recommended to check the dissociation every 10 min under the inverted microscope and to help this process with mechanical up and down pipetting (micropipette P1000).
When single cell suspension is reached, pass it through a 40 µm filter into a new tube.
Centrifuge the tubes at 300 x g for 5 min, 4 °C.
Remove supernatant and re-suspend the pellets in 2 ml ice cold 2 mM DPBS-BSA 2%-EDTA solution to wash.
Repeat the wash twice.
Finally re-suspend the single cell preparation in 1 ml 2 mM DPBS-BSA 2%-EDTA. Maintain the cell suspension on ice and proceed with staining steps or direct sorting in case of fluorescent protein expression. It is recommended to pass the suspension through a 40 µm filter once again before sorting.
Single cells of interest can finally be centrifuged at 300 x g for 5 min, 4 °C. Remove supernatant and if desired, proceed with sample seeding for ex vivo culture.

Ex vivo culture – 3D
1. General preparations before starting
  Defreeze Matrigel aliquots on ice and keep them always on ice, small changes in the temperature might accelerate undesired polymerization.
2. Choose the surface of plating and amount of Matrigel for each sample according to the size of the pellet obtained in step A. For individual samples of antral glands suspension, it is recommended to use 1 well of a 6 well plate and 240 µl Matrigel per pellet, however for sorted samples, 1 well of a 12 well plate and 100 µl of Matrigel per pellet might be preferred. All these estimations may change according to the user’s plans and needs.
3. Re-suspend the pellet in the tube with the adequate amount of Matrigel and homogenize the suspension on ice.
4. Transfer the mix sample/Matrigel suspension to the plate as a drop. Stretch the drop from the center until the bottom of the well is covered without touching the walls (use a tip for this purpose) (Figure 6a).
5. Place the plate in an incubator at 37 °C for 10 min until the mix polymerizes.
6. Distribute the ENRGWF medium for initial seeding (see Recipes section): 700 µl per well (12 well plate) and 1.4 ml per well (6 well plate).
7. Place the plate in the incubator.
8. Medium should be fully changed every other day with ENRGWF medium for maintenance (see Recipes section).

Note: All steps, including the decision of plating according to density of sample should be followed by observation under inverted bright field microscope.

Figure 6. Antral organoids at day 5 of the initial seeding: scheme showing side view of the 3D culture (a) and representative picture of culture at day 5 (b). Scale bars = 100 µm.

Maintenance of organoids
1. General preparations before starting
  Defreeze Matrigel aliquots on ice and keep them always on ice, small changes in the temperature might accelerate undesired polymerization.
  After 7-10 days in culture (depending on the rate of growth) organoids should be replated (Figure 6b). The replating can be done by ‘picking’ selected elements (step C2) or by harvesting a complete part of the well (step C3). See below details for each case.
2. Replating by element selection
3. Replating without selection
Organoids cryopreservation
1. Freezing protocol
2. De-freezing protoco

Data analysis

Details of replicates are provided in the original research paper published in free access (Fernandez Vallone et al., 2016).

Notes

In order to optimize reproducibility, it is suggested to use age-matched animals.
In order to improve cell viability after cell sorting, single sorted cells are collected in the BCM medium containing 10 µM Y27632.

Recipes

70% ethanol
70% ethanol (v/v) in dI water
DPBS-EDTA (10 mM)
1 ml 500 mM EDTA in DPBS
Final volume: 50 ml
DPBS-BSA 2%-EDTA (2 mM)
1 g BSA
0.1 ml 500 mM EDTA in DPBS
Final volume: 50 ml
Pass the solution through a 0.22 µm filter
Basal crypt medium (BCM)
500 ml Advanced DMEM/F12 supplemented with:
0.4 ml gentamycin
5 ml penicillin-streptomycin cocktail stock
5 ml amphotericin B
5 ml L-glutamine (final concentration: 2 mM)
ENRGWF medium for initial seeding
BCM supplemented with:
0.5 ml N-2
1 ml B-27 w/o vit. A
0.5 ml 10 mM HEPES
0.1 ml 1 mM N-acetyl cysteine
Growth factors at a final concentration of: 50 ng/ml for EGF, 100 ng/ml for Noggin, 200 ng/ml for CHO-derived R-spondin 1, 100 ng/ml Fgf10, 100 ng/ml Wnt3a, 10 nM Gastrin and 10 µM Rho kinase inhibitor (Y-27632)
Final volume: 50 ml
ENRGWF medium for maintenance
BCM supplemented with:
0.5 ml N-2
1 ml B-27 w/o vit. A
0.5 ml 10 mM HEPES
0.1 ml 1 mM N-acetyl cysteine
Growth factors at a final concentration of: 50 ng/ml for EGF, 100 ng/ml for Noggin, 200 ng/ml for CHO-derived R-spondin 1, 100 ng/ml Fgf10, 100 ng/ml Wnt3a and 10 nM Gastrin
Final volume: 50 ml
Freezing medium
BCM
1 ml FBS
1 ml DMSO
Final volume: 10 ml
De-freezing medium
BCM
1 ml FBS
Final volume: 10 ml

Acknowledgments

This work was supported by the Interuniversity Attraction Poles Programme-Belgian State-Belgian Science Policy (6/14), the Fonds de la Recherche Scientifique Médicale of Belgium, the Walloon Region (program CIBLES) and the non-for-profit Association Recherche Biomédicale et Diagnostic. This protocol was adapted from the initial report of Barker et al. (2010) for ex vivo culture conditions.

References

Barker, N., Huch, M., Kujala, P., van de Wetering, M., Snippert, H. J., van Es, J. H., Sato, T., Stange, D. E., Begthel, H., van den Born, M., Danenberg, E., van den Brink, S., Korving, J., Abo, A., Peters, P. J., Wright, N., Poulsom, R. and Clevers, H. (2010). Lgr5^+vestem cells drive self-renewal in the stomach and build long-lived gastric units in vitro. Cell Stem Cell 6(1): 25-36.
Fernandez Vallone, V., Leprovots, M., Strollo, S., Vasile, G., Lefort, A., Libert, F., Vassart, G. and Garcia, M. I. (2016). Trop2 marks transient gastric fetal epithelium and adult regenerating cells after epithelial damage. Development 143(9): 1452-1463.
Sato, T., Vries, R. G., Snippert, H. J., van de Wetering, M., Barker, N., Stange, D. E., van Es, J. H., Abo, A., Kujala, P., Peters, P. J. and Clevers, H. (2009). Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459(7244): 262-265.