Live imaging analysis of human gastric epithelial spheroids reveals spontaneous rupture, rotation and fusion events
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.
KeywordsStomach Epithelium Organoid Live imaging Human
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.
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.
Compliance with ethical standards
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.
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