Chapter 3. Co-culture of Gastric Organoids and Immortalized Stomach Mesenchymal Cells

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1 Chapter 3 Co-culture of Gastric Organoids and Immortalized Stomach Mesenchymal Cells Nina Bertaux-Skeirik, Jomaris Centeno, Rui Feng, Michael A. Schumacher, Ramesh A. Shivdasani, and Yana Zavros Abstract Three-dimensional primary epithelial-derived gastric organoids have recently been established as an important tool to study gastric development, physiology, and disease. Specifically, mouse-derived fundic gastric organoids (mfgos) co-cultured with Immortalized Stomach Mesenchymal Cells (ISMCs) reflect expression patterns of mature fundic cell types seen in vivo, thus allowing for long-term in vitro studies of gastric epithelial cell physiology, regeneration, and bacterial-host interactions. Here, we describe the development and culture of mfgos, co-cultured with ISMCs. Key words Mouse fundic gastric organoids, Co-culture, Immortalized stomach mesenchymal cells 1 Introduction In vitro three-dimensional organoid culture systems have been developed to investigate the development, physiology, and disease of multiple organs including liver [ 1, 2 ], pancreas [ 3 ], Barrett s esophagus [ 4 ], stomach [ 5 9 ], small intestine [ 5, 10 ], and colon [ 4, 11 ]. In an effort to enhance our understanding of the development of gastric disease, our lab has established a protocol to generate primary cultured whole fundic gland-derived gastric organoids [ 5, 7, 8, 12, 13 ]. Cultures derived from whole dissected glands are distinct from similar corpus-derived cultures generated from singlecell preparations of Troy + ve populations [ 14 ]. Troy-positive cells are expressed at the corpus gland base in a subset of differentiated chief cells and generate long-lived gastric organoids that are differentiated toward the mucus-producing cell lineages of the neck and pit regions. Here we report a culture method of fundic-derived gastric organoids devised for the maintenance of mature cell lineages observed throughout the fundic epithelium in vivo. Maintained in matrigel and gastric organoid growth media, organoids are co- cultured with immortalized stomach mesenchymal cells (ISMCs) [ 15 ]. Andrei I. Ivanov (ed.), Gastrointestinal Physiology and Diseases: Methods and Protocols, Methods in Molecular Biology, vol. 1422, DOI / _3, Springer Science+Business Media New York

2 24 Nina Bertaux-Skeirik et al. Gastric organoids recapitulate the differentiated cell types normally found in the stomach and have a polarized epithelium with a defined lumen. These gastric organoids have been used as a model system for the study of Helicobacter pylori -induced disease [ 8, 12 ], signaling pathways involved in proliferation of the epithelium [ 13 ], and gastric physiology [ 7 ]. 2 Materials 2.1 Immortalized Stomach Mesenchymal Cells (ISMCs) 2.2 Mouse-Derived Fundic Gastric Organoids (mfgos) 1. Growth media: DMEM High Glucose, 10 % Fetal Calf Serum, and 1 % Penicillin/Streptomycin. 2. Freezing medium: Recovery Cell Culture Freezing Medium (Life Technologies). 3. Washing Solution: Dulbecco s phosphate buffered saline (DPBS) without calcium or magnesium. 4. Cell Releasing Solution: Trypsin-EDTA 0.25 % phenol red. 5. Corning 75 cm 2 tissue culture treated flasks. 1. Storage and Washing buffer for gastric glands and organoids: ice-cold Dulbecco s phosphate buffered saline (DPBS) without calcium or magnesium. 2. Conical tubes, 50 ml, 15 ml, and 5 ml volume. 3. Chelation buffer for isolation of mouse gastric glands: ice-cold Dulbecco s phosphate buffered saline (DPBS) without calcium or magnesium with 5 mm EDTA, ph Dissociation buffer for isolation of mouse gastric glands: icecold Dulbecco s phosphate buffered saline (DPBS) without calcium or magnesium with 43.4 mm Sucrose and 54.9 mm D-Sorbitol. 5. Basement membrane for growth of organoids: Growth Factor Reduced, Phenol Red-free Matrigel (Fisher). 6. Plates for growth of organoids: Costar Transwell Clear Polyester Membrane Inserts for 12-Well Plates. 7. Growth and culture medium: Advanced Dulbecco s modified Eagle medium/f12 medium (Life Technologies, ) supplemented with 2 mm Glutamax, 100 U/ml Penicillin/ Streptomycin, 10 mm HEPES Buffer, 1 mm nacetylcysteine (Sigma), 1 N2 (Life Technologies), 1 B27 (Life Technologies), 50 % Wnt-conditioned medium, 10 % R-spondin conditioned medium supplemented with gastric growth factors including 100 ng/ml bone morphogenetic protein inhibitor Noggin (PeproTech), 10 nm [Leu-15]- gastrin I (Sigma), 50 ng/ml epidermal grow factor (EGF) (PeproTech), and 100 ng/ml fibroblast growth factor 10 (FGF10) (PeproTech).

3 Co-culture of Gastric Organoids For staining: 4 % paraformaldehyde made up in regular 1 PBS, EdU proliferation assay kit (Life Technologies). 9. Hoechst (Life Technologies, H3570), 1:1000 solution in DPBS. 3 Methods 3.1 Isolation of Mouse Gastric Glands All mouse studies were approved by the University of Cincinnati Institutional Animal Care and Use Committee (IACUC) that maintains an American Association of Assessment and Accreditation of Laboratory Animal Care (AAALAC) facility. 1. Euthanize mouse of at least 6 weeks of age. Using a pair of sterile surgical scissors, with the mouse stomach facing up, make an incision into the lower right abdominal cavity. Grasp the stomach (underneath the liver) with forceps, and use a small pair of scissors to cut the stomach from the esophagus and the duodenum. Open the stomach along the greater curvature, all the way through both the opening to the esophagus and the duodenum, so that the stomach can be laid flat. Wash the stomach twice in a weigh-boat filled with ice-cold DPBS without calcium or magnesium (w/o Ca/Mg), and then place into a 50 ml conical tube with 30 ml of ice-cold DPBS w/o Ca/Mg for transport to the micro-dissecting table. 2. Remove the stomach carefully and pin the stomach (basolateral side upwards, and luminal side down) open on a silicon filled dish so that the forestomach is pointing toward the top of the dish ( see Note 1 ). Working quickly but carefully, use fine point curved forceps to lift the serosal muscle layer in segments and use micro-dissecting curved scissors to cut and remove the muscle from the epithelium ( see Note 2 ). See Fig. 1a for reference. For fundic-specific gastric organoids, cut and separate the fundic tissue with a wide margin from the forestomach and antrum to prevent gland contamination, and for antral specific organoids, isolate the antrum from any remaining pyloric sphincter tissue ( see Note 3 ). Using scissors cut the fundus or antrum into small (less than 5 mm 2 ) pieces, and scoop into a sterile 15 ml conical tube prefilled with 5 ml of chelation buffer. Place tube on gentle rocker at 4 C for 2 h. 3. Thaw desired volume of Matrigel on ice or at 4 C during this 2 h EDTA incubation step ( see Note 4 ). 4. After incubation with EDTA chelation buffer, remove EDTA buffer after allowing tissue pieces to settle to the bottom of the tube. Replace this solution with 5 ml of dissociation buffer. Shake the tube by hand, with the bottom of the tube pointed toward the ground for 2 min ( see Note 5 ). See Fig. 1b for picture of the gland isolation.

4 26 Nina Bertaux-Skeirik et al. Fig. 1 Development of Mouse Fundic Gastric Organoids. Mouse stomach is harvested, serosal muscle layer is removed ( a ) and sections of the desired portion of the stomach are collected. The stomach glands (fundic depicted) are harvested ( b ), embedded into Matrigel and provided with growth medium. Mouse organoids formation can be visualized by day six of culture ( c )

5 Co-culture of Gastric Organoids Set the tube back on ice briefly to let larger fragments of tissue settle to the bottom of the tube, but not long enough for gastric glands to settle. Using a 1000 μl pipet, remove the 5 ml of volume without disturbing the large tissue fragments at the bottom of the tube. Transfer this volume into a sterile 5 ml tube and centrifuge at 150 g for 5 min at 4 C. 6. Remove supernatant carefully, so as not to disturb the pellet of glands ( see Note 6 ). Keeping the tube on ice, transfer desired volume of Matrigel to the glands ( see Note 7 ) and mix gently to avoid air bubbles. 7. Using a 200 μl pipet, pipet 40 μl of the glands in Matrigel directly onto the Transwell membrane ( see Note 8 ). Incubate the plate at 37 C for 15 min to allow Matrigel to solidify. After this incubation add 0.5 ml of growth media to the top of the transwell, and 1 ml of growth media to the bottom of the transwell. Allow organoids to grow for 4 days ( see Note 9 ). See Subheading 3.3 for co-culture technique with ISMCs. 3.2 Growth of Immortalized Stomach Mesenchymal Cells 1. Prepare culture growth media with desired volume of DMEM Hi Glucose, 10 % Fetal Calf Serum, and 1 % Penicillin/ Streptomycin (PS). 2. Thaw the Immortalized Stomach Mesenchymal Cells (ISMCs) stock at 37 C. 3. In a 75 cm 2 cell culture flask, add 29 ml of the growth media and pipette 1 ml of thawed ISMC stock solution for a total volume of 30 ml ( see Note 10 ). 4. After cell addition, slide the flask in forward and backward motion to mix the cells and media. Incubate cells at 37 C. 5. Cell adhesion should be visible after 24 h under light microscope ( see Note 11 ). 6. After cells reach % confluence, to passage to 12-well plates for co-culture, wash cells with 5 ml of warm DPBS w/o Ca/Mg, remove by vacuum, and treat with 5 ml of 0.25 % trypsin at 30 C to release cells from flask. Add 10 ml of culture medium to inactive trypsin. Transfer to 50 ml conical tube, and spin at 1000 rpm for 5 min. Remove supernatant, and resuspend in 10 ml growth medium. 7. Count cells using a hemocytometer, and determine appropriate dilution factor to suspend ISMCs to seed 12,000 cells per well in the 12-well plate ( see Note 12 ). Let cells adhere overnight (or h) before culturing with organoids. 8. Passage the rest of the ISMCs onto the T75 cm 2 flasks for further growth to continue providing fresh ISMCs to organoids.

6 28 Nina Bertaux-Skeirik et al. 3.3 Co-culture of ISMCs with Mouse Gastric Organoids 3.4 Immunofluorescence and EdU Proliferation Assay 1. At day 4 of growth, transfer Transwell membrane inserts to the 12-well plate seeded with ISMCs ( see Note 13 ). Provide fresh organoid growth medium to both the top and bottom compartment of the Transwell, taking care to remove old media using a 1000 μl pipet, without disturbing the organoids. 2. Every other day (every 48 h) the organoid culture must be supplied with a fresh 12-well plate of ISMCs; otherwise the organoids will not have enough growth factors to continue growth ( see Note 14 ). 3. Allow organoids to continue growing up to 7 10 days (post gland embedding in Matrigel ), and harvest for experimentation, staining, or passaging to maintain growth. 1. To stain for proliferating cells, you must complete an EdU uptake step prior to fixation. Otherwise, proceed directly to fixation and staining ( step 3 ). Thaw EdU component at 37 C, and prepare solution in DMEM basic medium with a ratio of 1: For EdU uptake, remove half of the growth media volume in the Transwell, 250 mkl from upper compartment and 500 mkl from bottom compartment and replace with same volume of EdU-DMEM solution, incubate at 37 C for 1 h. 3. (Begin here if EdU uptake is not desired) For fixation, remove all the liquid from the Transwell, add prewarmed 4 % paraformaldehyde 500 μl to upper and 1 ml to bottom compartments and incubate for 15 min at room temperature ( see Note 15 ). 4. Remove the 4 % paraformaldehyde from upper and bottom compartments and replace with the same volume of DPBS. Wrap with Parafilm and store at 4 C until staining. 5. For EdU staining, follow manufacture s directions. 6. For permeabilization, incubate in 0.5 % Triton X-100 for 20 min at room temperature, adding 1 ml liquid to the bottom portion of the Transwell, and 500 μl liquid to the insert with organoids ( see Note 16 ). 7. Wash 2 times with DPBS, adding 1 ml liquid to the bottom portion of the Transwell, and 500 μl liquid to the insert with organoids, and resuspend in primary antibody solution (made up in DPBS) and incubate at 4 C overnight, again adding 1 ml of liquid to the bottom portion of the Transwell, and 500 μl of liquid to the insert with organoids. 8. Wash 2 times with 1 ml DPBS, and resuspend cells in secondary antibody at 4 C overnight, using same volumes as in step 7.

7 Co-culture of Gastric Organoids Wash two times DPBS, and Hoechst solution if desired, and incubate for 30 min in the dark at room temperature, using the same volumes as in step Wash two times with DPBS; leave membrane submerged in 1 ml of DPBS to prevent cells from drying on the top insert, and in 2 ml of DPBS in the bottom portion of the Transwell. 11. For imaging, place the insert (with the organoids still suspended in DPBS) on a glass coverslip. Store wrapped in Parafilm in DPBS (1 ml in the insert, 2 ml in the bottom compartment) at 4 C. 4 Notes 1. To prevent the tissue from drying out, you can pour 3 5 ml of ice-cold DPBS w/o Ca/Mg over the pinned down stomach. 2. Be careful not to let the curved micro-dissecting scissors cut into the epithelium. To prevent this from happening, hold the scissors in parallel to the stomach as you are cutting away the muscle layer. 3. The antrum will appear more translucent and thinner than the fundus, and thus this distinction will make it easier to delineate where to cut if making region-specific organoids. If making whole-stomach organoids, combine fundus and antrum tissue. 4. If thawing a full bottle of Matrigel, place in an ice bucket at 4 overnight. Mix gently with a 1000 μl pipet, and aliquot into desired volumes, keeping all materials on ice to prevent Matrigel from solidifying. 5. Shake up and down twice per one second for best results. If, after shaking, you do not see gastric glands under the microscope, you can shake again for 1 2 min to increase the yield. This can be done by removing 50 μl from freshly shaken tube to check the density of the gastric glands, underneath a 10 objective on a light microscope. Extended shaking (>5 10 min) will result in low organoid yield, as glands will fragment into single cells. 6. It is easiest to remove the supernatant by vacuum, and then pull off the remaining liquid with a 1000 μl pipet. 7. It is important to avoid air bubbles by first pipetting the desired volume, and then decreasing the volume on the pipet, making sure to completely dissolve the pellet of glands in the Matrigel evenly, while keeping the tube on ice.

8 30 Nina Bertaux-Skeirik et al. 8. Do not pipet more than 40 μl on to the membrane because the Matrigel will spread instead of forming a domed bubble shape, and organoids will not form. 9. Sphere formation should occur within 24 h of growth. 10. Must pipette slowly to prevent cell breakage. 11. Cells should be passaged after cells have adhered and reached % confluence. 12. To passage ISMCs onto the 12-well plate, add 1 ml of cell solution to each well. For example, to seed one plate of cells, create a suspension of 12 ml of volume, for 1 ml per well. 13. To transfer, first remove the DMEM growth medium from the 12-well plate with the ISMCs, then carefully transfer the transwell membrane to the 12-well plate containing the ISMCs, making sure to keep everything sterile in the hood. Make sure to provide fresh organoid growth medium on the top and bottom of the transwell. 14. This means that the organoid growth media will be fully replaced every other day, when the organoids on the Transwell inserts are switched to a fresh 12-well plate of ISMCs. 15. The paraformaldehyde must be prewarmed; otherwise the Matrigel will dissolve, and the organoids will be lost during the washes for staining. 16. Do not remove liquid by vacuum, instead take great care and use a 1000 μl pipet, always placing new volume down the side of the insert, not directly on top of the organoids. Acknowledgments This work was supported by NIH 1R01DK grant (Zavros), NIH 5T32GM grant (Bertaux-Skeirik), and the Albert J. Ryan Fellowship (Bertaux-Skeirik). References 1. Huch M, Dorrell C, Boj SF et al (2013) In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration. Nature 494: Skardal A, Devarasetty M, Rodman C, Atala A, Soker S et al (2015) Liver-tumor hybrid organoids for modeling tumor growth and drug response in vitro. Ann Biomed Eng 43: Huch M, Bonfanti P, Boj SF et al (2013) Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R- spondin axis. EMBO J 32: Sato T, Stange DE, Ferrante M et al (2011) Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett s epithelium. Gastroenterology 141:

9 Co-culture of Gastric Organoids Mahe MM, Aihara E, Schumacher MA et al (2013) Establishment of gastrointestinal epithelial organoids. Curr Protoc Mouse Biol 3: Barker N, Huch M, Kujala P et al (2010) Lgr5(+ve) stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro. Cell Stem Cell 6: Schumacher MA, Aihara E, Feng R et al (2015) The use of murine-derived fundic organoids in studies of gastric physiology. J Physiol 593: Bertaux-Skeirik N, Feng R, Schumacher MA et al (2015) CD44 plays a functional role in helicobacter pylori-induced epithelial cell proliferation. PLoS Pathog 11:e Wroblewski LE, Piazuelo MB, Chaturvedi R et al (2015) Helicobacter pylori targets cancerassociated apical-junctional constituents in gastroids and gastric epithelial cells. Gut 64: Sato T, Vries RG, Snippert HJ et al (2009) Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459: Jung P, Sato T, Barriga FM et al (2011) Isolation and in vitro expansion of human colonic stem cells. Nat Med 17: Schumacher MA, Feng R, Aihara E et al (2015) Helicobacter pylori-induced Sonic Hedgehog expression is regulated by NFκB pathway activation: the use of a novel in vitro model to study epithelial response to infection. Helicobacter 20: Feng R, Aihara E, Kenny S et al (2014) Indian Hedgehog mediates gastrin-induced proliferation in stomach of adult mice. Gastroenterology 147: Stange DE, Koo BK, Huch M et al (2013) Differentiated Troy + chief cells act as reserve stem cells to generate all lineages of the stomach epithelium. Cell 155: Jayewickreme CD, Shivdasani RA (2015) Control of stomach smooth muscle development and intestinal rotation by transcription factor BARX1. Dev Biol 405:21 32

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