Materials and Reagents

Disposable scalpels (Swan Morton, catalog number: 0510 )
Petri dishes 92 x 16 mm with cams (SARSTEDT, catalog number: 82.1473 )
Microcentrifuge tubes, 1.5 ml (VWR, catalog number: 212-0198 )
Tubes 10 ml, 100 x 16 mm, PP (SARSTEDT, catalog number: 62.9924.284 )
Tubes 50 ml, 30 x 115 mm, PP (Corning, Falcon^®, catalog number: 352070 )
70 µm nylon filters (Corning, Falcon^®, catalog number: 352350 )
P6 well plate (VWR, catalog number: 734-2323 )
40 µm nylon filters (Corning, Falcon^®, catalog number: 352340 )
P12 well plate (VWR, catalog number: 734-2324 )
Tips refill (VWR, catalog numbers: 89079-464 ; 89079-470 ; 89079-478 )
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 (tested on RjOrl:SWISS and C57BL/6JRj backgrounds)
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)
Ethanol 95-97% (VWR, TechniSolv^®, catalog number: 84857.360 )
Glucose (Merck Millipore, catalog number: 1083371000 )
Leibovitz’s L-15 medium (Thermo Fisher Scientific, catalog number: 11415-049 )
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-spondin1 (R&D Systems, catalog number: 7150-RS/CF )
Rho kinase inhibitor Y27632 (Sigma-Aldrich, catalog number: Y0503 )
DMSO (Sigma-Aldrich, catalog number: D8418 )
Propanol-2 (VWR, catalog number: 1.09634.9900 )
70% ethanol (see Recipes)
1 M glucose (see Recipes)
Embryo’s medium (see Recipes)
DPBS-EDTA 5 mM (see Recipes)
DPBS-BSA 2%-EDTA 2 mM (see Recipes)
Basal crypt medium (BCM) (see Recipes)
ENR medium for initial seeding (see Recipes)
ENR 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 )
Pipettors with Tip Ejector 20-200 µl and 100-1,000 µl (VWR, catalog numbers: 89079-970 and 89079-974 )
Refrigerated centrifuge 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 )
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 and pregnancies: 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. Embryonic stage is calculated as embryonic day 0.5 (E0.5) the day the gestational plug is observed. It is important to remove the male from the mating cage the morning the plug is observed. Pregnancies can be followed by daily weighing of the females.
Unless specified, steps are carried out at room temperature (RT).

Dissection procedures and preparation of samples
1. Isolation of gastric fetal epithelium for culture (Figure 1)
  
  Figure 1. Overall procedure schematic of step A1

Note: These steps allow isolating gastric fetal epithelium as clumps or group of cells that will ultimate give rise to gastric fetal spheroids in culture. However, gastric fetal spheroids can also be obtained from isolated single cells after FACS for example. In that case the user of this protocol should follow step A2 instead of step A1 (see below).

Isolation of gastric fetal epithelium as single cells (Figure 4)

Figure 4. Overall procedure schematic of step A2
1. Follow protocol described in section A from step 1a to step 1o.
2. Re-suspend the pellet in Stem Pro Accutase cell dissociation reagent, using volumes as described in Table 1.
3. Transfer each suspension to a P6 well plate and incubate it at 37 °C with 75 rpm agitation. Time of incubation varies according to the quantity of initial material (between 30 min to 2 h). It is recommended to check the dissociation every 15 min under the inverted microscope and to help this process with mechanical up and down pipetting (micropipette P1000).
4. When single cell suspension is reached, pass it through a 40 µm filter into a new tube.
5. Centrifuge the tubes at 300 x g for 5 min, 4 °C.
6. Remove supernatant and re-suspend the pellets in 2 ml ice cold 2 mM DPBS-BSA 2%-EDTA solution to wash.
7. Repeat step A2f twice.
8. 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.
9. 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 always keep them on ice, small changes in the temperature might accelerate undesired polymerization.
2. The surface of plating and amount of Matrigel will be chosen for each sample based on the size of the pellet obtained in step A. For individual samples obtained from embryos at E14-18 or sorted samples, it is recommended to use 1 well of 12 wells plate and 100 µl of Matrigel per pellet. For pooled samples obtained after step A1, 1 well of 6 wells plate and 240 µl Matrigel per pellet will 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 5a).
5. Place the plate in an incubator at 37 °C for 10 min until the mix polymerizes.
6. Distribute the Spheroids-ENR 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 ENR 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 5. Spheroids initial seeding. a. Scheme showing side and upper view of the 3D culture; b. Representative pictures of gastric spheroids culture at day 6, arrows show dead cells inside the spheroids (b). Scale bars = 100 µm.
Maintenance of gastric spheroids
1. General preparations before starting
  After 5 to 7 days in culture (depending on the rate of growth), when few dead cells start to accumulate inside the spheroids, they should be replated (Figure 5b). This can be done by ‘picking’ selected elements (amplify selected population) (step C2) or by harvesting a complete part of the well (just culture maintenance) (step C3). See below details (Figure 6) for each case.
  
  Figure 6. Procedure for maintenance of gastric spheroids. Fully grown spheroids are isolated from the Matrigel, mechanically dissociated into small groups of cells, which are then mixed with a new Matrigel aliquot and seeded. The ENR seeding medium is added after Matrigel polymerization.
2. Replating by element selection
  1. Select elements to be picked under the inverted microscope.
  2. Prepare one tube per sample with 1 ml DPBS at room temperature, pick the elements from the well (1 ml DPBS for 12 well plate) with a micropipette (P200) and transfer them to the tube with DPBS. Repeat the process for all the desired elements.
  3. Centrifuge the tube at 300 x g for 5 min.
  4. Remove supernatant and re-suspend the elements in 0.3 ml DPBS.
  5. Disrupt the elements mechanically by pipetting up and down with a micropipette (P200) until visual disappearance of big pieces.
  6. Add 1 ml DPBS to further wash the suspension
  7. Centrifuge the tube at 300 x g for 5 min.
  8. Remove supernatant and re-suspend the pellet in the adequate volume of Matrigel as described in step B.
  9. Repeat steps B3-B7.
3. Replating without selection
Cryopreservation of gastric spheroids
1. Freezing protocol
2. De-freezing protocol

Data analysis

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

Notes

The yield of spheroid production obtained upon initial seeding is highly dependent on the developmental stage, with higher efficiency at E14-E15, and progressive decrease over time. At later developmental stages, a low proportion of clear spheroids can be obtained among the grown elements. In this case, replating of gastric spheroids will require selective picking of the clearest elements from the initial plate, the rest of the elements (usually dark spheroid-like structures) correspond to adult-type stem cells, which cannot efficiently grow in the ENR medium.
In order to improve cell viability after cell sorting, single sorted cells are collected in the BCM medium containing 10 µM Y27632.
This protocol can be similarly used to efficiently grow mouse fetal progenitors of the small intestine, a protocol initially described by Mustata et al. (2013).

Recipes

70% ethanol
70% ethanol (v/v) in distilled water
1 M glucose
Dissolve 180 g glucose in 1 L distilled water
Pass the solution through a 0.22 µm filter
Embryo’s medium
Add 17.5 ml 1 M glucose (sterile) to 500 ml Leibovitz’s l-15 medium
DPBS-EDTA (5 mM)
0.5 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)
ENR 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 and 100 ng/ml for CHO-derived R-spondin 1, 10 µM Rho kinase inhibitor (Y-27632)
Final volume: 50 ml
ENR 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 and 100 ng/ml for CHO-derived R-spondin1
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 previous work for sample preparation (Mustata et al., 2013) and the initial report of Sato et al. (2009) 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^+ve stem 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.
Mustata, R. C., Vasile, G., Fernandez-Vallone, V., Strollo, S., Lefort, A., Libert, F., Monteyne, D., Perez-Morga, D., Vassart, G. and Garcia, M. I. (2013). Identification of Lgr5-independent spheroid-generating progenitors of the mouse fetal intestinal epithelium. Cell Rep 5(2): 421-432.
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.