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Ex vivo Culture of Adult Mouse Antral Glands
成年鼠胃窦腺的离体培养

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Abstract

The tri-dimensional culture, initially described by Sato et al. (2009) in order to isolate and characterize epithelial stem cells of the adult small intestine, has been subsequently adapted to many different organs. One of the first examples was the isolation and culture of antral stem cells by Barker et al. (2010), who efficiently generated organoids that recapitulate the mature pyloric epithelium in vitro. This ex vivo approach is suitable and promising to study gastric function in homeostasis as well as in disease. We have adapted Barker’s protocol to compare homeostatic and regenerating tissues and here, we meticulously describe, step by step, the isolation and culture of antral glands as well as the isolation of single cells from antral glands that might be useful for culture after cell sorting as an example (Fernandez Vallone et al., 2016).

Keywords: ex vivo(离体), Murine gastric epithelium(鼠胃上皮), Antral glands(胃窦腺), Gastric organoids(胃类器官), 3D(3D), Single cell(单细胞)

Background

Mouse adult stem cells from the glandular stomach can be grown ex vivo in a 3D matrigel as ‘mini-glands’ for indefinite periods of time (Barker et al., 2010). As compared to stem cells from the mouse adult small intestine growing in presence of EGF, Noggin and R-spondin 1, gastric stem cells need to be further supplemented with Fgf10, Gastrin, Wnt3a and a higher concentration of R-spondin 1 (referred to as ENRFGW) to get productive cultures. Till recently, whether, and if so how, adult regenerating antral glands grow in the ex vivo culture system following stem cell ablation, remained unknown. Using the present protocol, it was demonstrated that homeostatic and regenerating antral glands do not grow similarly upon seeding and exhibit different growth culture requirements.

Materials and Reagents

  1. Disposable scalpels (Swan Morton, catalog number: 0510 )
  2. Petri dishes 92 x 16 mm with cams (SARSTEDT, catalog number: 82.1473 )
  3. Tubes 50 ml, 30 x 115 mm, PP (Corning, Falcon®, catalog number: 352070 )
  4. 20 ml eccentric tip syringe (BD, catalog number: 300613 )
  5. Needle 21 G x 1 ½ (BD, catalog number: 305167 )
  6. Tips refill (VWR, catalog numbers: 89079-464 ; 89079-470 ; 89079-478 )
  7. 70 µm nylon filters (Corning, Falcon®, catalog number: 352350 )
  8. 40 µm nylon filters (Corning, Falcon®, catalog number: 352340 )
  9. P6 well plate (VWR, catalog number: 734-2323 )
  10. P12 well plate (VWR, catalog number: 734-2324 )
  11. Microcentrifuge tubes, 1.5 ml (VWR, catalog number: 212-0198 )
  12. Tubes 10 ml, 100 x 16 mm, PP (SARSTEDT, catalog number: 62.9924.284 )
  13. Cryotubes 1 ml (Greiner Bio One, catalog number: 123263 )
  14. Syringe filter 0.2 µm (VWR, catalog number: 28145-477 )
  15. Serological pipets 5 ml, 10 ml and 25 ml (Corning, Falcon®, catalog numbers: 357543 ; 357551 ; 357535 )
  16. Mice (RjOrl:SWISS and C57BL/6JRj backgrounds-6 to 8 weeks old-males and females)
  17. Dulbecco’s phosphate-buffered saline (DPBS), CaCl2 free, MgCl2 free (Thermo Fisher Scientific, GibcoTM, catalog number: 14190-094 )
  18. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10270 )
  19. Stem Pro Accutase cell dissociation reagent (Thermo Fisher Scientific, GibcoTM, catalog number: A1110501 )
  20. Matrigel® basement membrane matrix (Corning, catalog number: 354234 )
  21. Liquid nitrogen (supplied from Air liquide)
  22. 500 mM EDTA (pH 8.0) (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15575-038 )
  23. Albumin from bovine serum (BSA) (Sigma-Aldrich, catalog number: A3294 )
  24. Advanced DMEM/F12 (Thermo Fisher Scientific, GibcoTM, catalog number: 12634-010 )
  25. Gentamycin 50 mg/ml (Thermo Fisher Scientific, GibcoTM, catalog number: 15750-037 )
  26. Penicillin-streptomycin cocktail 100x (Thermo Fisher Scientific, GibcoTM, catalog number: 15140-122 )
  27. Amphotericin B 250 µg/ml (Thermo Fisher Scientific, GibcoTM, catalog number: 15290-026 )
  28. L-glutamine (Thermo Fisher Scientific, GibcoTM, catalog number: 25030-081 )
  29. N-2 supplement 100x (Thermo Fisher Scientific, GibcoTM, catalog number: 17502-048 )
  30. B-27 w/o vit. A 50x (Thermo Fisher Scientific, GibcoTM, catalog number: 12587-010 )
  31. 1 M HEPES (Thermo Fisher Scientific, GibcoTM, catalog number: 15630-056 )
  32. N-acetyl cysteine (Sigma-Aldrich, catalog number: A7250 )
  33. 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 )
  34. DMSO (Sigma-Aldrich, catalog number: D8418 )
  35. Propanol-2 (VWR, catalog number: 1.09634.9900 )
  36. Ethanol 95-97% (VWR, TechniSolv®, catalog number: 84857.36 0)
  37. 70% ethanol (see Recipes)
  38. DPBS-EDTA (10 mM) (see Recipes)
  39. DPBS-BSA 2%-EDTA (2 mM) (see Recipes)
  40. Basal crypt medium (BCM) (see Recipes)
  41. ENRGWF medium for initial seeding (see Recipes)
  42. ENRGWF medium for maintenance (see Recipes)
  43. Freezing medium (see Recipes)
  44. De-freezing medium (see Recipes)

Equipment

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

Procedure

Notes:

  1. 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.
  2. Unless specified, steps are carried out at room temperature (RT).
  1. Dissection procedures and preparation of samples
    1. Isolation of antral glands for culture (Figure 1)


      Figure 1. Overall procedure schematic of step A1

      1. General preparations before starting the dissection
        Sterilize working area and dissection tools with 70% ethanol. It is not necessary to work under laminar flow, meanwhile all the steps are carried out carefully and in a clean disinfected area. It is recommended to maintain dissection tools in a glass with 70% ethanol during intermediate steps of the dissection protocol to minimize contamination.
        Equipment: binocular and cold light source (Figure 2a).
        Dissection tools: scissors, curved forceps, standard tip forceps, scalpel, micro-dissection scissors, dissection bed (Figure 2b).
        Box with ice, plastic Petri dishes, ice cold DPBS and 50 ml tubes according to the number of mice that will be processed.



        Figure 2. Examples of equipment (a) and dissection tools (b). a. Binocular and cold light source; b. Dissection tools: micro-dissection scissors, scalpel, straight thin tip forceps, curved serrated forceps and different size scissors.

      2. Euthanize the mouse according to the local institutional guidelines.
      3. Lay the mouse on its back and fix it to the dissection bed. Spray the abdominal area with 70% ethanol in order to sterilize the area and minimize further contamination.
      4. Grasp the skin with an angled forceps and cut transversally through the skin and peritoneum at the level of the lower abdomen. Perform another 2 cuts on each side in order to be able to lift up the skin and visualize internal organs until the diaphragm (Figure 3a).
      5. Dissect out with curved forceps and scissors the whole stomach and clean it from rests of other organs (pancreas, duodenum, esophagus, fascias, etc.) and connective tissue (Figures 3b-3d).
      6. Place the stomach on a Petri dish filled with ice-cold DPBS and cut off the squamous area (Figure 3e).
      7. Wash the inside stomach with DPBS to remove the rests of partially digested food. This cleaning process may be helped by flushing cold DPBS into the stomach through the open squamous area towards the pylorus and vice-versa with a syringe plus needle.
      8. With the stomach cleaned and flattened, make a cut with the scalpel to separate corpus from antral area. There is a clear delineation and separation between both areas. Keep antral area (Figures 3e-3f).
         

        Figure 3. Representative pictures of stomach dissection. Consecutive steps are detailed (a-l). Ant: antral area, Cor: corpus area, Col: colon, Duo: duodenum, Dia: diaphragm, Eso: esophagus, Fas: fascias, GC: greater curvature, Liv: liver, Mu: mucosa, Ms: muscle, SC: small curvature, SI: small intestine, Sq: squamous area, Sto: stomach. Scale bars = 0.5 cm.

      9. Open the antral stomach along the small curvature and wash once more with ice-cold DPBS.
      10. Place the open antral tissue on a dry Petri dish with the muscle layer facing up and the mucosa down to the Petri (Figure 3g).
      11. Separate serosal muscle from antral mucosa under the binocular using micro-dissecting scissors and fine forceps. During separation of the serosal muscle, the mucosa becomes more fragile and relaxed (Figures 3h-3j).
      12. When whole muscle tissue is separated, cut the mucosa into small pieces (< 5 mm2) with the scalpel (Figures 3k-3l).
      13. Transfer the tissue to a 50 ml tube containing 10 ml sterile ice cold DPBS and place it on ice.
      14. Continue the dissection of the rest of the mice always keeping the tissue already processed on ice.
      15. Centrifuge the tubes at 230 x g for 5 min, 4 °C.
      16. Re-suspend the pellet of each sample in 10 ml of sterile 10 mM DPBS-EDTA.
      17. Place the tubes laid down on ice and incubate them for 2 h with 75 rpm agitation. Incubation with DPBS-EDTA detaches the epithelial layer from the mesenchymal one.
      18. Centrifuge the tubes at 230 x g for 5 min, 4 °C.
      19. From this step onward, the protocol should be carried out under a tissue culture hood.
      20. Remove supernatant by aspiration and re-suspend each pellet in 10 ml of sterile DPBS.
      21. Prepare FBS-coated pipette by pipetting up and down once FBS from 50 ml tube containing complete FBS. Empty the pipette so that it is ready to use.
      22. Pipet 40 times up and down samples using FBS-coated pipette of 10 ml to disrupt the tissue and further separate the antral glands from the basal layer in contact with mesenchyme. Process each tube at a time maintaining the rest on ice.
      23. Centrifuge the tubes at 300 x g for 5 min, 4 °C.
      24. Remove supernatant by aspiration and re-suspend each pellet in 5 ml of sterile DPBS.
      25. Pipet 40 times up and down samples using FBS-coated pipette of 5 ml to disrupt the tissue and further separate the antral glands from the basal layer in contact with mesenchyme. Process each tube at a time maintaining the rest on ice.
      26. Centrifuge the tubes at 300 x g for 5 min, 4 °C.
        1. Pass each suspension through a 70 µm filter into a new tube.
        2. Centrifuge the tubes at 300 x g for 5 min, 4 °C.
        3. Remove supernatant. Pellet is ready to be seeded in culture (see step B).

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.

  1. Single cell isolation from antral glands (Figure 5)


    Figure 5. Overall procedure schematic of step A2

    1. Re-suspend the pellets from step A1z.iii in 4 ml Stem Pro Accutase cell dissociation reagent.
    2. 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).
    3. When single cell suspension is reached, pass it through a 40 µm filter into a new tube.
    4. Centrifuge the tubes at 300 x g for 5 min, 4 °C.
    5. Remove supernatant and re-suspend the pellets in 2 ml ice cold 2 mM DPBS-BSA 2%-EDTA solution to wash.
    6. Repeat the wash twice.
    7. 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. 
    8. 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.

  1. 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 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.

  1. 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
      1. Select elements to be picked up under the inverted microscope.
      2. Prepare one microcentrifuge tube per sample with 1 ml DPBS, pick up the elements from the well with a (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 mechanically the elements 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
      1. Aspirate the medium from the well.
      2. Add 1 ml DPBS to the well (P6 or P12 well plate) and detach the whole Matrigel with the elements embedded from the well by pipetting up and down with a micropipette (P1000) until homogeneous suspension is reached.
      3. Take ¼ from the suspension and transfer it to a 10 ml tube. It is not recommended to replate the whole well into the same size well as it might be too much material and debris, ending with a bad quality of replating. Depending on user’s needs, the rest of the material can be frozen, replated if culture needs to be amplified or discarded.
      4. Wash with 1 ml DPBS and centrifuge it at 300 x g for 5 min.
      5. Remove supernatant and re-suspend the elements in 0.3 ml DPBS.
      6. Disrupt mechanically the elements by pipetting up and down with a micropipette (P200) until visual disappearance of big pieces.
      7. Add 1 ml DPBS to further wash the suspension
      8. Centrifuge the tube at 300 x g for 5 min.
      9. Remove supernatant and re-suspend the pellet in the adequate volume of Matrigel as described in step B.
      10. Repeat steps B3-B7.

  2. Organoids cryopreservation
    1. Freezing protocol
      1. Aspirate the medium from the well.
      2. Add 1 ml DPBS to the well (P6 or P12 well plate) and harvest the whole Matrigel with the embedded elements by pipetting up and down with a micropipette (P1000) until homogeneous suspension is reached. If some material is left in the well, harvest it with extra 1 ml DPBS to the same collection tube.
      3. Centrifuge the tube at 300 x g for 5 min.
      4. Remove supernatant and wash the pellet in 2 ml DPBS.
      5. Centrifuge the tube at 300 x g for 5 min.
      6. Remove supernatant and re-suspend the pellet in 1 ml of freezing medium (see Recipes section).
      7. Transfer the suspension to a labeled cryotube and place it in the Cryo freezing container (filled with isopropanol at RT). Put the container in a -80 °C freezer.
      8. After 48 h, cryotubes can be stored in liquid nitrogen.
    2. De-freezing protoco
      1. Prepare a tube with 2 ml of de-freezing medium (see Recipes section) and warm it at 37 °C.
      2. Take out from liquid nitrogen the selected cryotube and de-freeze the sample by pipetting up and down with pre-warmed de-freezing medium.
      3. Transfer all the organoid suspension to the pre-warmed tube and centrifuge it at 300 x g for 5 min.
      4. Remove supernatant and wash the pellet in 2 ml of BCM medium twice and centrifuge it at 300 x g for 5 min.
      5. Remove supernatant and re-suspend the elements in 0.3 ml DPBS.
      6. Disrupt mechanically the elements by pipetting up and down 20 times with a micropipette (P200).
      7. Add 1 ml DPBS to further wash the suspension.
      8. Centrifuge the tube at 300 x g for 5 min.
      9. Remove supernatant and re-suspend the pellet in the adequate volume of Matrigel as described in step B.

Data analysis

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

Notes

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

Recipes

  1. 70% ethanol
    70% ethanol (v/v) in dI water
  2. DPBS-EDTA (10 mM)
    1 ml 500 mM EDTA in DPBS
    Final volume: 50 ml
  3. 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
  4. 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)
  5. 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
  6. 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
  7. Freezing medium
    BCM
    1 ml FBS
    1 ml DMSO
    Final volume: 10 ml
  8. 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

  1. 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.
  2. 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.
  3. 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.

简介

为了分离和表征成年小肠的上皮干细胞,最初由Sato等人(2009)描述的三维培养物已经随后适应于许多不同的器官。其中一个例子是Barker等人(2010)分离和培养窦性干细胞,他们有效地产生了在体外重现成熟幽门上皮的组织细胞。这种“离体”方法是适合的,并且有希望研究体内平衡和疾病中的胃功能。我们已经调整了Barker的方案来比较稳态和再生组织,这里,我们一步一步地仔细地描述了窦腺的分离和培养,以及从细胞分选后可能用于培养的窦腺中分离单细胞一个例子(Fernandez Vallone等人,2016)。

背景来自腺体的小鼠成体干细胞可以在3D matrigel中离体生长,作为“迷你腺体”无限期(Barker等人,2010) 。与EGF,Noggin和R-spondin 1存在下生长的小鼠成年小肠的干细胞相比,胃干细胞需要进一步补充Fgf10,胃泌素,Wnt3a和更高浓度的R-螺旋菌素1(称为作为ENRFGW)获得生产性文化。直到最近,在干细胞消融后,在离体培养系统中成体再生窦腺是否生长,如果是这样,仍然是未知的。使用本方案,证明了内源性和再生的腺体在接种时不会类似地生长并且表现出不同的生长培养要求。

关键字:离体, 鼠胃上皮, 胃窦腺, 胃类器官, 3D, 单细胞

材料和试剂

  1. 一次性手术刀(Swan Morton,目录号:0510)
  2. 带凸轮的培养皿92 x 16毫米(SARSTEDT,目录号:82.1473)
  3. 管50毫升,30×115毫米,PP(康宁,猎鹰,目录号:352070)
  4. 20毫升偏心尖端注射器(BD,目录号:300613)
  5. 针21 G x 1½(BD,目录号:305167)
  6. 提示补充(VWR,目录号:89079-464; 89079-470; 89079-478)
  7. 70μm尼龙过滤器(Corning,Falcon ®,目录号:352350)
  8. 40μm尼龙过滤器(Corning,Falcon ®,目录号:352340)
  9. P6孔板(VWR,目录号:734-2323)
  10. P12孔板(VWR,目录号:734-2324)
  11. 微量离心管,1.5毫升(VWR,目录号:212-0198)
  12. 管10 ml,100 x 16 mm,PP(SARSTEDT,目录号:62.9924.284)
  13. 冷冻筒1ml(Greiner Bio One,目录号:123263)
  14. 注射器过滤器0.2μm(VWR,目录号:28145-477)
  15. 血清学移液管5ml,10ml和25ml(Corning,Falcon ,目录号:357543; 357551; 357535)
  16. 小鼠(RjOrl:SWISS和C57BL/6JRj背景 - 6至8周龄男性和女性)
  17. Dulbecco的磷酸盐缓冲盐水(DPBS),游离CaCl 2,不含MgCl 2(Thermo Fisher Scientific,Gibco< sup>,目录号:14190- 094)
  18. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:10270)
  19. Stem Pro Accutase细胞解离试剂(Thermo Fisher Scientific,Gibco TM,目录号:A1110501)
  20. Matrigel ®基底膜基质(Corning,目录号:354234)
  21. 液氮(由液化气供应)
  22. 500mM EDTA(pH8.0)(Thermo Fisher Scientific,Invitrogen TM,目录号:15575-038)
  23. 来自牛血清(BSA)的白蛋白(Sigma-Aldrich,目录号:A3294)
  24. 高级DMEM/F12(Thermo Fisher Scientific,Gibco TM ,目录号:12634-010)
  25. 庆大霉素50mg/ml(Thermo Fisher Scientific,Gibco TM,目录号:15750-037)
  26. 100x(Thermo Fisher Scientific,Gibco TM,目录号:15140-122)的青霉素 - 链霉素混合物
  27. 两性霉素B250μg/ml(Thermo Fisher Scientific,Gibco TM,目录号:15290-026)
  28. L-谷氨酰胺(Thermo Fisher Scientific,Gibco TM,目录号:25030-081)
  29. N-2补充100x(Thermo Fisher Scientific,Gibco TM,目录号:17502-048)
  30. B-27 w/o vit 50x(Thermo Fisher Scientific,Gibco TM ,目录号:12587-010)
  31. 1 M HEPES(Thermo Fisher Scientific,Gibco TM ,目录号:15630-056)
  32. 乙酰半胱氨酸(Sigma-Aldrich,目录号:A7250)
  33. 生长因子:
    重组鼠EGF(Peprotech,目录号:315-09)
    重组鼠Noggin(Peprotech,目录号:250-38)
    重组鼠CHO衍生的R-spondin 1(R& D Systems,目录号:7150-RS/CF)
    重组鼠Fgf10(R& D Systems,目录号:6224-FG)
    重组鼠Wnt3a(R& D Systems,目录号:1324-WN/CF)
    胃泌素I(Sigma-Aldrich,目录号:SCP0152)
    Rho激酶抑制剂Y27632(Sigma-Aldrich,目录号:Y0503)
  34. DMSO(Sigma-Aldrich,目录号:D8418)
  35. 丙醇-2(VWR,目录号:1.09634.9900)
  36. 乙醇95-97%(VWR,TechniSolv ,目录号:84857.360)
  37. 70%乙醇(见食谱)
  38. DPBS-EDTA(10mM)(参见食谱)
  39. DPBS-BSA 2%-EDTA(2mM)(参见食谱)
  40. 基础隐窝培养基(BCM)(见食谱)
  41. ENRGWF用于初始播种的培养基(参见食谱)
  42. ENRGWF维护介质(见配方)
  43. 冷冻介质(参见食谱)
  44. 脱冻介质(参见食谱)

设备

  1. 双目(Motic,型号:SMZ-168)
  2. 冷光源(肖特,型号:KL1500 LCD)
  3. 剪刀:直尖尖(Fine Science Tool,目录号:14090-09和14084-08)
  4. 斜角锯齿镊子(精细科学工具,目录号:11080-02)
  5. 标准(精细)尖端镊子(精细科学工具,目录号:11251-20)
  6. 微解剖刀(精细科学工具,目录号:15018-10)
  7. 冷冻离心机(Beckman Coulter,型号:Allegra X-15R)
  8. MaxQ TM 具有适应温度的摇床(Thermo Fisher Scientific,Thermo Scientific TM,型号:MaxQ TM 4000)
  9. 生物安全柜(Esco Micro Pte,型号:Class II Type A2)
  10. 带喷嘴的移液器20-200μl和100-1,000μl(VWR,目录号:89079-970和89079-974)
  11. 细胞培养箱(37℃,5%CO 2)(BINDER,型号:C150)
  12. 倒立式明视野显微镜(Motic,型号:AE31)
  13. Nalgene Cryo先生Frosty先生的冷冻容器(Thermo Fisher Scientific,Thermo Scientific TM ,型号:5100-0050)
  14. 超低温立式冷冻机(Thermo Fisher Scientific,型号:Thermo Scientificifc Queue Basic)
  15. 冷冻系统K系列(泰勒沃顿,型号:24K)

程序

注意:

  1. 关于小鼠的一般考虑:动物应在12小时光/12小时黑暗循环的温度(21±1℃)和湿度(55±10%)的控制室内饲养。
  2. 除非另有说明,步骤在室温(RT)下进行。
  1. 解剖程序和样品的制备
    1. 肛门腺分离培养(图1)


      图1.步骤A1的总体程序原理图

      1. 开始解剖前的一般准备工作
        用70%乙醇灭菌工作区域和解剖工具。不需要在层流下工作,同时所有的步骤都要在干净的消毒区进行。建议在解剖方案的中间步骤时,将70%乙醇玻璃中的夹层工具保持在最低限度。
        设备:双目和冷光源(图2a)。
        解剖工具:剪刀,弯钳,标准镊子,解剖刀,微解剖剪刀,解剖床(图2b)。
        根据要处理的小鼠数量,装有冰块,塑料培养皿,冰冷DPBS和50ml管。



        图2.设备(a)和解剖工具(b)的示例。 a。双目冷光源; b。解剖工具:微分解剪刀,手术刀,直薄尖镊子,弯曲锯齿镊子和不同尺寸的剪刀。

      2. 根据当地的制度指导,对小鼠进行安乐死。
      3. 将鼠标放在其背面并将其固定在解剖床上。用70%乙醇喷洒腹部以消毒该区域,并减少进一步的污染。
      4. 用镊子抓住皮肤,并通过下腹部的皮肤和腹膜横向切割。在每侧进行另外2个切口,以便能够抬起皮肤并将内部器官直视到隔膜(图3a)。
      5. 用弯曲的镊子剪切整个胃,并从其他器官(胰腺,十二指肠,食管,fascias,等等)的其余部分清洁它)和结缔组织(图3b-3d)。 >
      6. 将胃放在装有冰冷DPBS的培养皿中,切断鳞状区域(图3e)。
      7. 用DPBS清洗内部胃,以除去其余部分消化的食物。这种清洁过程可以通过将冷DPBS通过朝向幽门的开放的鳞状区域进入胃来帮助,反之亦然,用注射器加针。
      8. 用胃清洁并平整后,用手术刀切开分离语料库与肛门区域。这两个领域之间有明确的划分和分离。保留肛门区域(图3e-3f)  

        图3.胃解剖的代表性图片。详细的连续步骤(a-l)。肛门:肛门区域,Cor:区域,Col:结肠,Duo:十二指肠,Dia:隔膜,Eso:食管,Fas:fascias,GC:更大的曲率,Liv:肝脏,Mu:粘膜,Ms:肌肉,SC:曲率,SI:小肠,Sq:鳞状区域,Sto:胃。比例尺= 0.5厘米。

      9. 沿着小曲率打开窦胃,再用冰冷的DPBS清洗一次。
      10. 将开放的窦组织置于干燥的陪替氏培养皿上,肌肉层面朝上,粘膜下降至佩特里(图3g)。
      11. 使用微剪切剪刀和精细镊子,在双眼下使用肛门粘膜分离浆膜肌肉。在浆膜分离期间,粘膜变得更加脆弱和松弛(图3h-3j)。
      12. 当整个肌肉组织分离时,用手术刀将粘膜切成小块(<5 mm 2)(图3k-31)。
      13. 将组织转移到含有10ml无菌冰冷DPBS的50ml管中,并将其置于冰上
      14. 继续解剖剩下的小鼠,始终保持组织已经在冰上处理。
      15. 将管以230×g离心5分钟,4℃。
      16. 将每个样品的沉淀重新悬浮在10ml无菌的10mM DPBS-EDTA中
      17. 将管放置在冰上,并用75rpm搅拌孵育2小时。用DPBS-EDTA培养,从间充质中分离上皮层
      18. 将管以230×g离心5分钟,4℃。
      19. 从这一步开始,协议应该在组织培养罩下进行。
      20. 通过抽吸去除上清液,并将每个沉淀重新悬浮在10ml无菌DPBS中
      21. 通过从含有完整FBS的50ml管中的FBS向上和向下移液来制备FBS涂布移液管。清空移液器,以便它可以使用。
      22. 使用10毫升的FBS涂层移液管吸取40次上下样品,以破坏组织,并进一步将基底层的窦腺与间质接触。一次处理每根管子,将其余部分保持在冰上。
      23. 将管以300×g离心5分钟,4℃。
      24. 通过抽吸去除上清液,并将每个沉淀重新悬浮在5ml无菌DPBS中
      25. 使用5毫升的FBS涂层移液管吸取40次上下样品,以破坏组织,并进一步将基底层的窦腺与间质接触进一步分离。一次处理每根管子,将其余部分保持在冰上。
      26. 将管以300×g离心5分钟,4℃。
        1. 将每个悬浮液通过70μm过滤器通入新管中
        2. 将管以300×g离心5分钟,4℃。
        3. 去除上清液。颗粒准备种植在文化中(见步骤B)。

注意:这些步骤允许隔离可用于在培养物中引起胃组织的成年窦腺(图4)。然而,例如,在FACS之后,单细胞与窦腺分离可能是进行培养的最佳选择。在这种情况下,此协议的用户应遵循步骤A2中所述的修改(见下文)。


图4.成人腺体分离。 电镀前或单细胞解离时小鼠窦腺悬液的实例。刻度棒=100μm。

  1. 单细胞与窦腺分离(图5)

    图5.步骤A2的总体程序原理图

    1. 将来自步骤A1z.iii的颗粒重新悬浮在4ml Stem Pro Accutase细胞解离试剂中。
    2. 将每个悬浮液转移到P6孔板,并在37℃下用75rpm搅拌孵育40分钟。建议在倒置显微镜下每10分钟检查解离,并通过机械上下移液(微量移液管P1000)帮助该过程。
    3. 当达到单细胞悬浮液时,将其通过40μm的过滤器通入新的管中
    4. 将管以300×g离心5分钟,4℃。
    5. 除去上清液并将沉淀重新悬浮在2ml冰冷的2mM DPBS-BSA 2%-EDTA溶液中以洗涤。
    6. 重复两次洗涤。
    7. 最后将单细胞制备物重新悬浮在1ml 2mM DPBS-BSA 2%-EDTA中。将细胞悬浮液保持在冰上,并进行染色步骤或在荧光蛋白表达的情况下进行直接分选。建议在分选前再次将悬浮液通过40μm过滤器。
    8. 感兴趣的单细胞最终可以在300×g下离心5分钟,4℃。去除上清液,如果需要,进行离体培养的样品种子。

  1. 文化 - 3D
    1. 开始前的一般准备工作
      在冰上分解Matrigel等分试样并将它们始终保持在冰上,温度的微小变化可能加速不期望的聚合。
    2. 根据步骤A中获得的颗粒的大小,选择每个样品的电镀表面和Matrigel的量。对于窦腺悬液的个别样品,建议使用6孔板的1孔和每粒240μl的Matrigel然而,对于分选的样品,可以优选12孔板的1孔和每粒子100μl的Matrigel。所有这些估算可能会根据用户的计划和需求而改变。
    3. 用足够量的基质胶重新悬浮在管中,并将悬浮液均匀化在冰上
    4. 将混合样品/Matrigel悬浮液以一滴水转移到板上。从中心向下拉伸,直到井底不覆盖墙壁(为此目的使用尖端)(图6a)。
    5. 将板放在37℃的培养箱中10分钟,直到混合物聚合。
    6. 分配ENRGWF培养基进行初次播种(参见食谱部分):每孔700μl(12孔板)和每孔1.4 ml(6孔板)。
    7. 将板放在培养箱中。
    8. 应使用ENRGWF介质进行维护,每隔一天进行一次全面更换(参见食谱部分)。

注意:所有步骤,包括根据样品密度进行电镀的决定,应在倒置的明场显微镜下观察。





图6.初始种子第5天的胭脂红有机物:显示3D培养物(a)的侧视图和第5天(b)的培养的代表性图。比例尺=100μm。

  1. 维持有机体
    1. 开始前的一般准备工作
      在冰上分解Matrigel等分试样并将它们始终保持在冰上,温度的微小变化可能加速不期望的聚合。
      培养7-10天后(取决于生长速率),应重新组织有机体(图6b)。可以通过"挑选"所选择的元素(步骤C2)或通过收获井的完整部分来完成重新填充(步骤C3)。请看下面每个案例的细节。
    2. 按元素选择进行替换
      1. 选择要在倒置显微镜下拾取的元素。
      2. 每个样品用1ml DPBS准备一个微量离心管,用(P200)从孔中取出元件,并用DPBS将其转移到管中。对所有需要的元素重复此过程。
      3. 以300×g离心管5分钟。
      4. 去除上清液并将元件重新悬浮在0.3ml DPBS中。
      5. 通过用微量吸管(P200)上下移动来机械地破碎元件,直到大块的视觉消失。
      6. 加入1ml DPBS以进一步洗涤悬浮液。
      7. 以300×g离心管5分钟。
      8. 除去上清液,并将沉淀物重新悬浮在足够体积的Matrigel中,如步骤B所述
      9. 重复步骤B3-B7。
    3. 无需选择即可
      1. 从井中吸出培养基。
      2. 向孔(P6或P12孔板)中加入1ml DPBS,并用微量移液管(P1000)上下移动,将整个Matrigel与孔中嵌入的元件分离,直到达到均匀悬浮液。
      3. 从悬浮液中取¼,并将其转移到10ml管中。不建议将整个井重新整合成相同的尺寸,因为它可能是太多的材料和碎片,以不良质量的重现结束。根据用户的需要,如果文化需要被放大或者丢弃,其余的材料可以被冻结,可以重新填写
      4. 用1ml DPBS洗涤,并以300×g离心5分钟。
      5. 去除上清液并将元件重新悬浮在0.3ml DPBS中。
      6. 通过用微量吸管(P200)上下移动来机械地破碎元件,直到大块的视觉消失。
      7. 加入1ml DPBS以进一步清洗悬浮液
      8. 以300×g离心管5分钟。
      9. 除去上清液,并将沉淀物重新悬浮在足够体积的Matrigel中,如步骤B所述
      10. 重复步骤B3-B7。

  2. 有机体冷冻保存
    1. 冻结协议
      1. 从井中吸出培养基。
      2. 向孔(P6或P12孔板)中加入1ml DPBS,并用微量移液管(P1000)上下移动,用嵌入元件收获整个Matrigel,直到达到均匀的悬浮液。如果有一些物质留在井中,用额外的1毫升DPBS收集到同一个收集管。
      3. 以300×g离心管5分钟。
      4. 去除上清液,并在2ml DPBS中洗涤沉淀。
      5. 以300×g离心管5分钟。
      6. 去除上清液,并将沉淀物重新悬浮在1ml冷冻培养基中(见食谱部分)
      7. 将悬浮液转移到标记的冷冻管中,并将其放入冷冻冷冻容器中(在室温下装满异丙醇)。将容器放在-80°C冰箱中。
      8. 48小时后,冷冻管可以储存在液氮中
    2. 去冻原型
      1. 准备一个带有2毫升冷冻介质的管子(见食谱部分),并在37°C温热
      2. 从液氮中取出选定的冷冻管,并用预先加热的冷冻介质上下移动来冻干样品。
      3. 将所有有机体悬浮液转移到预热管中,并以300 x g离心5分钟。
      4. 除去上清液,并将沉淀物在2ml BCM培养基中洗涤两次,并以300×g离心5分钟。
      5. 去除上清液并将元件重新悬浮在0.3ml DPBS中。
      6. 用微量移液管(P200)上下移动20次,从而机械地破碎元件。
      7. 加入1ml DPBS以进一步洗涤悬浮液。
      8. 以300×g离心管5分钟。
      9. 除去上清液,并将沉淀物重新悬浮在足够体积的Matrigel中,如步骤B所述

数据分析

在免费访问的原始研究论文(Fernandez Vallone等人,2016年)中提供了重复的细节。

笔记

  1. 为了优化再现性,建议使用年龄匹配的动物。
  2. 为了提高细胞分选后的细胞活力,将单个分选的细胞收集在含有10μMY27632的BCM培养基中。

食谱

  1. 70%乙醇
    70%乙醇(v/v)在dI水中
  2. DPBS-EDTA(10mM)
    1毫升500毫克EDTA在DPBS
    最终体积:50 ml
  3. DPBS-BSA 2%-EDTA(2mM)
    1克BSA
    在DPBS中0.1ml 500mM EDTA 最终体积:50 ml
    将溶液通过0.22μm过滤器
  4. 基底隐窝培养基(BCM)
    500 ml Advanced DMEM/F12补充:
    0.4 ml庆方霉素 5毫升青霉素 - 链霉素鸡尾酒库存
    5 ml两性霉素B
    5ml L-谷氨酰胺(终浓度:2mM)
  5. ENRGWF用于初始播种的培养基
    BCM补充:
    0.5 ml N-2
    1 ml B-27 w/o vit A
    0.5 ml 10 mM HEPES
    0.1ml 1mM N-乙酰半胱氨酸
    生长因子终浓度为50ng/ml,EGF为100ng/ml,Noggin为100ng/ml,CHO-衍生的R-spondin1,100ng/ml Fgf10,100ng/ml Wnt3a,10nM胃泌素和10μMRho激酶抑制剂(Y-27632)
    最终体积:50 ml
  6. ENRGWF维护介质
    BCM补充:
    0.5 ml N-2
    1 ml B-27 w/o vit A
    0.5 ml 10 mM HEPES
    0.1ml 1mM N-乙酰半胱氨酸
    生长因子终浓度为:EGF为50ng/ml,Noggin为100ng/ml,CHO衍生的R-spondin为100ng/ml,100ng/ml Fgf10,100ng/ml Wnt3a和10nM胃泌素
    最终体积:50 ml
  7. 冷冻介质
    BCM
    1 ml FBS
    1 ml DMSO
    最终体积:10 ml
  8. 脱冻介质
    BCM
    1 ml FBS
    最终体积:10 ml

致谢

这项工作得到了比利时国家比利时科学政策(6/14),比利时Fonds de la Recherche科学院,瓦隆地区(计划CIBLES)和非营利协会RechercheBiomédicale的支持et诊断。该协议从Barker等人的初步报告改编而来。 (2010)针对离体培养条件。

参考文献

  1. Barker,N.,Huch,M.,Kujala,P.,van de Wetering,M.,Snippert,HJ,van Es,JH,Sato,T.,Stange,DE,Begthel,H.,van den Born,M ,Danenberg,E.,van den Brink,S.,Korving,J.,Abo,A.,Peters,PJ,Wright,N.,Poulsom,R.and Clevers,H.(2010)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/20085740"target ="_ blank"> Lgr5 + ve 干细胞驱动自我更新在胃中并在体外构建长寿命胃单位。细胞干细胞 6(1):25-36。
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引用:Vallone, V. F., Leprovots, M., Vassart, G. and Garcia, M. (2017). Ex vivo Culture of Adult Mouse Antral Glands. Bio-protocol 7(1): e2088. DOI: 10.21769/BioProtoc.2088.
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