Live imaging analysis of human gastric epithelial spheroids reveals spontaneous rupture, rotation and fusion events

Abstract

Three-dimensional cultures of primary epithelial cells including organoids, enteroids and epithelial spheroids have become increasingly popular for studies of gastrointestinal development, mucosal immunology and epithelial infection. However, little is known about the behavior of these complex cultures in their three-dimensional culture matrix. Therefore, we performed extended time-lapse imaging analysis (up to 4 days) of human gastric epithelial spheroids generated from adult tissue samples in order to visualize the dynamics of the spheroids in detail. Human gastric epithelial spheroids cultured in our laboratory grew to an average diameter of 443.9 ± 34.6 μm after 12 days, with the largest spheroids reaching diameters of >1000 μm. Live imaging analysis revealed that spheroid growth was associated with cyclic rupture of the epithelial shell at a frequency of 0.32 ± 0.1/day, which led to the release of luminal contents. Spheroid rupture usually resulted in an initial collapse, followed by spontaneous re-formation of the spheres. Moreover, spheroids frequently rotated around their axes within the Matrigel matrix, possibly propelled by basolateral pseudopodia-like formations of the epithelial cells. Interestingly, adjacent spheroids occasionally underwent luminal fusion, as visualized by injection of individual spheroids with FITC–Dextran (4 kDa). In summary, our analysis revealed unexpected dynamics in human gastric spheroids that challenge our current view of cultured epithelia as static entities and that may need to be considered when performing spheroid infection experiments.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

References

  1. Bartfeld S, Bayram T, van de Wetering M, Huch M, Begthel H, Kujala P, Vries R, Peters PJ, Clevers H (2015) In vitro expansion of human gastric epithelial stem cells and their responses to bacterial infection. Gastroenterology 148:126–136

    Article  PubMed  Google Scholar 

  2. Benton G, George J, Kleinman HK, Arnaoutova IP (2009) Advancing science and technology via 3D culture on basement membrane matrix. J Cell Physiol 221:18–25

    CAS  Article  PubMed  Google Scholar 

  3. Bertaux-Skeirik N, Feng R, Schumacher MA, Li J, Mahe MM, Engevik AC, Javier JE, Peek RM Jr, Ottemann K, Orian-Rousseau V, Boivin GP, Helmrath MA, Zavros Y (2015) CD44 plays a functional role in helicobacter pylori-induced epithelial cell proliferation. PLoS Pathog 11:e1004663

    Article  PubMed  PubMed Central  Google Scholar 

  4. Bimczok D, Smythies LE, Waites KB, Grams JM, Stahl RD, Mannon PJ, Peter S, Wilcox CM, Harris PR, Das S, Ernst PB, Smith PD (2013) Helicobacter pylori infection inhibits phagocyte clearance of apoptotic gastric epithelial cells. J Immunol 190(12):6626–6634

  5. Bimczok D, Kao JY, Zhang M, Cochrun S, Mannon P, Peter S, Wilcox CM, Mönkemüller KE, Harris PR, Grams JM, Stahl RD, Smith PD, Smythies LE (2014) Human gastric epithelial cells contribute to gastric immune regulation by providing retinoic acid to dendritic cells. Mucosal Immunol 8(3):533–544

  6. Bohorquez DV, Chandra R, Samsa LA, Vigna SR, Liddle RA (2011) Characterization of basal pseudopod-like processes in ileal and colonic PYY cells. J Mol Histol 42:3–13

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. Bradford EM, Ryu SH, Singh AP, Lee G, Goretsky T, Sinh P, Williams DB, Cloud AL, Gounaris E, Patel V, Lamping OF, Lynch EB, Moyer MP, De Plaen IG, Shealy DJ, Yang GY, Barrett TA (2017) Epithelial TNF receptor signaling promotes mucosal repair in inflammatory bowel disease. J Immunol 199:1886–1897

    CAS  Article  PubMed  Google Scholar 

  8. Brumfield SK, Ortmann AC, Ruigrok V, Suci P, Douglas T, Young MJ (2009) Particle assembly and ultrastructural features associated with replication of the lytic archaeal virus sulfolobus turreted icosahedral virus. J Virol 83:5964–5970

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  9. Burgess DR (1976) Structure of the epithelial - mesenchymal interface during early morphogenesis of the chick duodenum. Tissue Cell 8:147–158

    CAS  Article  PubMed  Google Scholar 

  10. Corpron RE (1966) The ultrastructure of the gastric mucosa in normal and hypophysectomized rats. Am J Anat 118:53–90

    CAS  Article  PubMed  Google Scholar 

  11. Creamer B, Shorter RG, Bamforth J (1961) The turnover and shedding of epithelial cells. I. The turnover in the gastro-intestinal tract. Gut 2:110–118

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  12. Dedhia PH, Bertaux-Skeirik N, Zavros Y, Spence JR (2016) Organoid models of human gastrointestinal development and disease. Gastroenterology 150:1098–1112

    Article  PubMed  PubMed Central  Google Scholar 

  13. Dekkers JF, Wiegerinck CL, de Jonge HR, Bronsveld I, Janssens HM, de Winter-de Groot KM, Brandsma AM, de Jong NW, Bijvelds MJ, Scholte BJ, Nieuwenhuis EE, van den Brink S, Clevers H, van der Ent CK, Middendorp S, Beekman JM (2013) A functional CFTR assay using primary cystic fibrosis intestinal organoids. Nat Med 19:939–945

    CAS  Article  PubMed  Google Scholar 

  14. Demitrack ES, Gifford GB, Keeley TM, Carulli AJ, VanDussen KL, Thomas D, Giordano TJ, Liu Z, Kopan R, Samuelson LC (2015) Notch signaling regulates gastric antral LGR5 stem cell function. EMBO J 34:2522–2536

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. Demitrack ES, Gifford GB, Keeley TM, Horita N, Todisco A, Turgeon DK, Siebel CW, Samuelson LC (2017) NOTCH1 and NOTCH2 regulate epithelial cell proliferation in mouse and human gastric corpus. Am J Physiol Gastrointest Liver Physiol 312:G133–G144

    Article  PubMed  Google Scholar 

  16. Diamond JM, Tormey JM (1966) Role of long extracellular channels in fluid transport across epithelia. Nature 210:817–820

    CAS  Article  PubMed  Google Scholar 

  17. Engevik AC, Feng R, Choi E, White S, Bertaux-Skeirik N, Li J, Mahe MM, Aihara E, Yang L, DiPasquale B, Oh S, Engevik KA, Giraud AS, Montrose MH, Medvedovic M, Helmrath MA, Goldenring JR, Zavros Y (2016) The development of spasmolytic polypeptide/TFF2-expressing metaplasia (SPEM) during gastric repair is absent in the aged stomach. Cell Mol Gastroenterol Hepatol 2:605–624

    Article  PubMed  PubMed Central  Google Scholar 

  18. Gifford GB, Demitrack ES, Keeley TM, Tam A, La Cunza N, Dedhia PH, Spence JR, Simeone DM, Saotome I, Louvi A, Siebel CW, Samuelson LC (2017) Notch1 and Notch2 receptors regulate mouse and human gastric antral epithelial cell homoeostasis. Gut 66:1001–1011

    Article  PubMed  Google Scholar 

  19. den Hartog G, Chattopadhyay R, Ablack A, Hall EH, Butcher LD, Bhattacharyya A, Eckmann L, Harris PR, Das S, Ernst PB, Crowe SE (2016) Regulation of Rac1 and reactive oxygen species production in response to infection of gastrointestinal epithelia. PLoS Pathog 12:e1005382

    Article  Google Scholar 

  20. Heath JP (1996) Epithelial cell migration in the intestine. Cell Biol Int 20:139–146

    CAS  Article  PubMed  Google Scholar 

  21. Howitt MR, Lavoie S, Michaud M, Blum AM, Tran SV, Weinstock JV, Gallini CA, Redding K, Margolskee RF, Osborne LC, Artis D, Garrett WS (2016) Tuft cells, taste-chemosensory cells, orchestrate parasite type 2 immunity in the gut. Science 351:1329–1333

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. Hynds RE, Giangreco A (2013) Concise review: the relevance of human stem cell-derived organoid models for epithelial translational medicine. Stem Cells 31:417–422

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. Iizuka M, Konno S (2011) Wound healing of intestinal epithelial cells. World J Gastroenterol 17:2161–2171

    Article  PubMed  PubMed Central  Google Scholar 

  24. Karam SM, Li Q, Gordon JI (1997) Gastric epithelial morphogenesis in normal and transgenic mice. Am J Phys 272:G1209–G1220

    CAS  Google Scholar 

  25. Larsen EH, Willumsen NJ, Mobjerg N, Sorensen JN (2009) The lateral intercellular space as osmotic coupling compartment in isotonic transport. Acta Physiol (Oxford) 195:171–186

    CAS  Article  Google Scholar 

  26. Leushacke M, Barker N (2014) Ex vivo culture of the intestinal epithelium: strategies and applications. Gut 63:1345–1354

    CAS  Article  PubMed  Google Scholar 

  27. Lipkin M (1965) Cell replication in the gastrointestinal tract of man. Gastroenterology 48:616–624

    CAS  PubMed  Google Scholar 

  28. Mahe MM, Aihara E, Schumacher MA, Zavros Y, Montrose MH, Helmrath MA, Sato T, Shroyer NF (2013) Establishment of gastrointestinal epithelial organoids. Curr Protoc Mouse Biol 3:217–240

    Article  PubMed  PubMed Central  Google Scholar 

  29. McCracken KW, Cata EM, Crawford CM, Sinagoga KL, Schumacher M, Rockich BE, Tsai YH, Mayhew CN, Spence JR, Zavros Y, Wells JM (2014) Modelling human development and disease in pluripotent stem-cell-derived gastric organoids. Nature 516:400–404

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  30. McNiven MA (2013) Breaking away: matrix remodeling from the leading edge. Trends Cell Biol 23:16–21

    CAS  Article  PubMed  Google Scholar 

  31. Miyoshi H (2017) Wnt-expressing cells in the intestines: guides for tissue remodeling. J Biochem 161:19–25

    Article  PubMed  Google Scholar 

  32. Miyoshi H, Stappenbeck TS (2013) In vitro expansion and genetic modification of gastrointestinal stem cells in spheroid culture. Nat Protoc 8:2471–2482

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. Miyoshi H, Ajima R, Luo CT, Yamaguchi TP, Stappenbeck TS (2012) Wnt5a potentiates TGF-beta signaling to promote colonic crypt regeneration after tissue injury. Science 338:108–113

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  34. Mollazade K, Omid M, Tab FA, Mohtasebi SS (2012) Principles and applications of light backscattering imaging in quality evaluation of agro-food products: a review. Food Bioprocess Technol 5:1465–1485

    Article  Google Scholar 

  35. Necchi V, Manca R, Ricci V, Solcia E (2009) Evidence for transepithelial dendritic cells in human H. Pylori active gastritis. Helicobacter 14:208–222

    CAS  Article  PubMed  Google Scholar 

  36. Ootani A, Li X, Sangiorgi E, Ho QT, Ueno H, Toda S, Sugihara H, Fujimoto K, Weissman IL, Capecchi MR, Kuo CJ (2009) Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche. Nat Med 15:701–706

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  37. Pechhold K, Pohl T, Kabelitz D (1994) Rapid quantification of lymphocyte subsets in heterogeneous cell populations by flow cytometry. Cytometry 16:152–159

    CAS  Article  PubMed  Google Scholar 

  38. Powell RH, Behnke MS (2017) WRN conditioned media is sufficient for in vitro propagation of intestinal organoids from large farm and small companion animals. Biol Open 6:698–705

    Article  PubMed  PubMed Central  Google Scholar 

  39. Riehl TE, Santhanam S, Foster L, Ciorba M, Stenson WF (2015) CD44 and TLR4 mediate hyaluronic acid regulation of Lgr5+ stem cell proliferation, crypt fission, and intestinal growth in postnatal and adult mice. Am J Physiol Gastrointest Liver Physiol 309:G874–G887

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  40. Rohrer GV, Scott JR, Joel W, Wolf S (1965) The fine structure of human gastric parietal cells. Am J Dig Dis 10:13–21

    CAS  Article  PubMed  Google Scholar 

  41. Sancak Y, Peterson TR, Shaul YD, Lindquist RA, Thoreen CC, Bar-Peled L, Sabatini DM (2008) The rag GTPases bind raptor and mediate amino acid signaling to mTORC1. Science 320:1496–1501

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  42. Sato T, Clevers H (2015) SnapShot: growing organoids from stem cells. Cell 161:1700-1700 e1701

    Article  Google Scholar 

  43. Sato T, Vries RG, Snippert HJ, van de Wetering M, Barker N, Stange DE, van Es JH, Abo A, Kujala P, Peters PJ, Clevers H (2009) Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459:262–265

    CAS  Article  PubMed  Google Scholar 

  44. Schlaermann P, Toelle B, Berger H, Schmidt SC, Glanemann M, Ordemann J, Bartfeld S, Mollenkopf HJ, Meyer TF (2016) A novel human gastric primary cell culture system for modelling helicobacter pylori infection in vitro. Gut 65:202–213

    CAS  Article  PubMed  Google Scholar 

  45. Schumacher MA, Aihara E, Feng R, Engevik A, Shroyer NF, Ottemann KM, Worrell RT, Montrose MH, Shivdasani RA, Zavros Y (2015) The use of murine-derived fundic organoids in studies of gastric physiology. J Physiol 593:1809–1827

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  46. Schwank G, Andersson-Rolf A, Koo BK, Sasaki N, Clevers H (2013) Generation of BAC transgenic epithelial organoids. PLoS ONE 8:e76871

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  47. Silen W, Ito S (1985) Mechanisms for rapid re-epithelialization of the gastric mucosal surface. Annu Rev Physiol 47:217–229

    CAS  Article  PubMed  Google Scholar 

  48. Smith JM, Johanesen PA, Wendt MK, Binion DG, Dwinell MB (2005) CXCL12 activation of CXCR4 regulates mucosal host defense through stimulation of epithelial cell migration and promotion of intestinal barrier integrity. Am J Physiol Gastrointest Liver Physiol 288:G316–G326

    CAS  Article  PubMed  Google Scholar 

  49. Stelzner M, Helmrath M, Dunn JC, Henning SJ, Houchen CW, Kuo C, Lynch J, Li L, Magness ST, Martin MG, Wong MH, Yu J, Consortium NIHISC (2012) A nomenclature for intestinal in vitro cultures. Am J Physiol Gastrointest Liver Physiol 302:G1359–G1363

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  50. Stewart SA, Dykxhoorn DM, Palliser D, Mizuno H, Yu EY, An DS, Sabatini DM, Chen IS, Hahn WC, Sharp PA, Weinberg RA, Novina CD (2003) Lentivirus-delivered stable gene silencing by RNAi in primary cells. RNA 9:493–501

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  51. VanDussen KL, Marinshaw JM, Shaikh N, Miyoshi H, Moon C, Tarr PI, Ciorba MA, Stappenbeck TS (2015) Development of an enhanced human gastrointestinal epithelial culture system to facilitate patient-based assays. Gut 64:911–920

    CAS  Article  PubMed  Google Scholar 

  52. van de Wetering M, Oosterwegel M, Dooijes D, Clevers H (1991) Identification and cloning of TCF-1, a T lymphocyte-specific transcription factor containing a sequence-specific HMG box. EMBO J 10:123–132

    PubMed  PubMed Central  Google Scholar 

  53. Williams JM, Duckworth CA, Burkitt MD, Watson AJ, Campbell BJ, Pritchard DM (2015) Epithelial cell shedding and barrier function: a matter of life and death at the small intestinal villus tip. Vet Pathol 52:445–455

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  54. Yui S, Nakamura T, Sato T, Nemoto Y, Mizutani T, Zheng X, Ichinose S, Nagaishi T, Okamoto R, Tsuchiya K, Clevers H, Watanabe M (2012) Functional engraftment of colon epithelium expanded in vitro from a single adult Lgr5(+) stem cell. Nat Med 18:618–623

    CAS  Article  PubMed  Google Scholar 

  55. Zeitoun P, Lambling A (1967) Ultrastructure of the gastric mucosa in human hemochromatosis. Scand J Gastroenterol 2:222–234

    CAS  Article  PubMed  Google Scholar 

Download references

Acknowledgements

Funding for our study was provided by the National Institutes of Health grants K01 DK097144 (DB); R03 DK107960 (DB), the National Science Foundation, DMR-1455247 (JW) and the Montana University System Research Initiative 51040-MUSRI2015-03 (DB). We greatly appreciate support from the National Institutes of Health IDeA Program grant GM110732, an equipment grant from the M.J. Murdock Charitable Trust and the Montana State University Agricultural Experimental Station for the Flow Cytometry Core Facility at Montana State University. Funding for shared facilities used in this work was also provided by the NSF under award number CBET-1039785. GeneSearch, Inc. development of the GeneSearch Embryo Cradle was funded by an SBIR grant from ORIP/NIH 5R44OD012083 (PJT). We would also like to thank Dr. K. Sasse (Sasse Surgical Associates, Reno, NV) for collecting human gastric tissue samples, Dr. T. Stappenbeck (Washington University, St. Louis) for sharing the L-WRN cell line with us and Dr. Seth Walk for helpful discussions.

Author information

Affiliations

Authors

Contributions

D.B., B.W., L.C.S. and J.W. planned and oversaw the experiments; T.A.S., B.S., R.B. and R.A.W. performed the experiments; P.J.T. developed microinjection equipment and protocols, B.A.P. provided human gastric tissue samples; D.B., T.A.S. and R.B. analyzed the data; T.A.S. and D.B. wrote the manuscript; all authors provided critical feedback on the manuscript.

Corresponding author

Correspondence to Diane Bimczok.

Ethics declarations

Conflict of interest statement

Dr. Paul Taylor has a potential conflict of interest, since he is the owner of GeneSearch, Inc., Bozeman, MT, which manufactures the EmbryoCradle microinjector that was used in this study. None of the other authors declare a conflict of interest.

Electronic supplementary material

441_2017_2726_MOESM4_ESM.mp4

Live imaging of a gastric epithelial spheroid microinjected with 4 kDa FITC dextran. At 13.20 h, the spheroid ruptures and FITC-dextran is released into the Matrigel matrix surrounding the spheroid and slowly disappears from the lumen. (MP4 2744 kb)

441_2017_2726_MOESM5_ESM.avi

Spontaneous rotation of gastric epithelial spheroids in Matrigel visualized by phase contrast live imaging. Note change in rotational direction of the spheroid. (AVI 3836 kb)

Supplementary Table 1

(DOCX 21 kb)

Supplemental movie 3

Live imaging of a gastric epithelial spheroid microinjected with 4 kDa FITC dextran. At 13.20 h, the spheroid ruptures and FITC-dextran is released into the Matrigel matrix surrounding the spheroid and slowly disappears from the lumen. (MP4 2744 kb)

Supplemental movie 4

Spontaneous rotation of gastric epithelial spheroids in Matrigel visualized by phase contrast live imaging. Note change in rotational direction of the spheroid. (AVI 3836 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Sebrell, T.A., Sidar, B., Bruns, R. et al. Live imaging analysis of human gastric epithelial spheroids reveals spontaneous rupture, rotation and fusion events. Cell Tissue Res 371, 293–307 (2018). https://doi.org/10.1007/s00441-017-2726-5

Download citation

Keywords

  • Stomach
  • Epithelium
  • Organoid
  • Live imaging
  • Human