Live imaging analysis of human gastric 
epithelial spheroids reveals spontaneous 
rupture, rotation and fusion events
Authors: T. Andrew Sebrell, Rachel Bruns, Royce A. 
Wilkinson, Blake Wiedenheft, Paul J. Taylor, Brian A. 
Perrino, Linda C. Samuelson, James N. Wilking, and 
Diane Bimczok. 
The final publication is available at Springer via https://doi.org/10.1007/s00441-017-2726-5
Sebrell TA, B Sidar, R Bruns, RA Wilkinson, B Wiedenheft, PJ Taylor, BA Perrino, LC Samuelson, 
JN Wilking, D Bimczok, “Live imaging analysis of human gastric epithelial spheroids reveals 
spontaneous rupture, rotation and fusion events,” Cell and Tissue Research. February 
2018;371(2):293-307. doi: 10.1007/s00441-017-2726-5.
Made available through Montana State University’s ScholarWorks 
scholarworks.montana.edu 
Live imaging analysis of human gastric epithelial 
spheroids reveals spontaneous rupture, rotation and 
fusion events
T. Andrew Sebrell , Barkan Sidar , Rachel Bruns , Royce A. Wilkinson ,  Blake Wiedenheft , Paul J. Taylor , Brian A. Perrino , 
Linda C. Samuelson ,  James N. Wilking , Diane Bimczok
Three-dimensional cultures of primary epithelial cells including organoids, enteroids and epithelial spheroids 
have become increasingly popular for studies of gastrointesti-nal development, mucosal immunology and 
epithelial infec-tion. However, little is known about the behavior of these complex cultures in their three-
dimensional culture matrix. Therefore, we performed extended time-lapse imaging analy-sis (up to 4 days) of 
human gastric epithelial spheroids gener-ated from adult tissue samples in order to visualize the dynam-ics of 
the spheroids in detail. Human gastric epithelial spher-oids 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 re-vealed 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 indi-vidual 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.
In recent years, epithelial organoids have become increasingly popular as a new and 
powerful tool to study gastrointestinal development and disease (Dedhia et al. 
2016; Hynds and Giangreco 2013; Leushacke and Barker 2014). Since the ini-tial 
description of epithelial organoids derived from murine intestine (Ootani et al. 
2009; Sato et al. 2009), organoid cul-ture systems have been adapted to multiple 
epithelial organ systems and species (Powell and Behnke 2017; Sato and  Clevers 
2015). For the first time, primary gastrointestinal ep-ithelial cells from various 
species can now be propagated for extended periods of time by culturing the cells 
in a three-dimensional collagen matrix and by supplementing Wnt3a, noggin and 
R-spondin to support maintenance of the stem cell compartment. Importantly, the 
utilization of adult stem cells enables the generation of primary epithelial cell lines 
from patient tissues with the potential of performing translational studies and 
patient-specific analyses (Dekkers et al. 2013; VanDussen et al. 2015). The 
availability of established proto-cols for primary epithelial cell cultures has led to 
exponential
growth in publications using these methods over the last
5 years, with the number of Borganoid^ publications per year
increasing from <10 in 2007 to ≥450 in 2016. Thus, various
forms of organoid cultures are starting to replace more tradi-
tional epithelial culture methods that involve the use of trans-
formed cell lines derived from gastrointestinal tumors.
However, little is known about the behavior of these com-
plex living spheres in their three-dimensional matrix, beyond
diametrical expansion due to cell proliferation. Several publi-
cations on gastrointestinal organoids or spheroids include vid-
eo supplements showing growth over time (Mahe et al. 2013;
Schlaermann et al. 2016; Schumacher et al. 2015; Schwank
et al. 2013). Some of these videos challenge our concept of
epithelial organoids as static entities that undergo diametrical
growth, though the dynamic behaviors observed were not
evaluated in detail.
In our study, we established primary gastric epithelial
cell lines from 13 healthy human adults. These lines were
maintained as spheroids following the protocol published by
Miyoshi and Stappenbeck (2013), which has been used by
multiple groups to culture organoids from intestinal
(Bradford et al. 2017; Howitt et al. 2016; Powell and
Behnke 2017; Riehl et al. 2015) and gastric sites (Demitrack
et al. 2017; den Hartog et al. 2016; Gifford et al. 2017;
VanDussen et al. 2015). With these cultures, we per-
formed live imaging analysis for up to 4 days. Under the
culture conditions used, rupture events occurred cyclically in
the majority of the cultures imaged and were associated with
the release of luminal material and subsequent healing of the
majority of spheroids. In addition, we also observed spheroids
that rotated in the Matrigel and spheroids that fused with other
spheroids. These observations indicate that human gastric
spheroid cultures are surprisingly dynamic, at least under the
specific culture conditions used here. Notably, the epithelial
barrier function of the spheroids may temporarily be compro-
mised because of relatively common spontaneous rupture
events that are not visible using routine monitoring
techniques.
Materials and methods
Human gastric epithelial spheroid culture
Thirteen gastric tissue specimens from sleeve gastrectomy
surgeries were obtained with Institutional Review Board
(IRB) approval by the National Disease Research
Interchange (NDRI; Philadelphia, PA, USA) or by Dr. Kent
Sasse (Sasse Surgical Associates, Reno, NV, USA). Donor
characteristics are listed in Table 1. Information on patholog-
ical alteration of the tissues was provided by the NDRI.
Gastric glands were isolated as described previously
(Bimczok 2013, 2014). Briefly, we dissected the gastric
mucosa off the muscle layer and cut the mucosa into <1-mm
pieces, which were placed into RPMI-1640 medium supple-
mented with 0.5 U/mL collagenase type IV, 0.2 mg/mL
DNAse (Sigma-Aldrich, St. Louis, MO, USA), 0.3% BSA,
250 μg/mL amphotericin B (Fisher Scientific, Fair Lawn,
NJ, USA), 100 U/mL penicillin/streptomycin, 2 mM L-gluta-
mine, 1 mM HEPES (GE Healthcare Life Sciences, Logan
UT, USA) and 50 μg/mL Gentamycin (IBI Scientific,
Peosta, IL, USA). The tissue was incubated at 200 rpm in a
37 °C water bath for 1 h. The tube was then vortexed for 30 s
to release glands from the tissue. To establish gastric spheroid
cultures, the glands and remaining tissue pieces were centri-
fuged at 200g at 4 °C for 5 min. The pellet was resuspended in
30mL ice-cold DPBS (Hyclone GEHealthcare Life Sciences)
and vortexed again for 30 s. Tissue pieces were allowed to
settle to the bottom of the tube and gastric glands in the
supernatant were transferred to a new 50-mL tube, pelleted
and transferred to Matrigel (Corning, Bedford, MA, USA).
Glands suspended in Matrigel were pipetted into a pre-
warmed 24-well plate. Gastric spheroid cultures were main-
tained following the protocol of Miyoshi and Stappenbeck
(2013), with minor modifications as described by Gifford
et al. (2017). After polymerization, Matrigel was overlaid with
500 μL of L-WRN medium composed of Advanced
DMEM/F12 (Gibco by Life Technologies, Grand Island,
NY, USA) supplemented with 10 mM HEPES, 1% Pen/
Strep, 50% L-WRN conditioned medium, 10 μM ROCK-
inhibitor Y-27632 (Tocris Biosciences, Bristol, UK),
Amphotericin B, Gentamycin, L-Glutamine, 10 μM TGF-β
inhibitor SB-431542 (Tocris), 10% FBS (Rocky Mountain
Bio, Missoula, MT, USA) and 50% of cell culture supernatant
from L-WRN cells, which constitutively secrete murine
Wnt3a, noggin and R-spondin 3 (Miyoshi et al. 2012). L-
WRN cells were kindly provided by Dr. T. Stappenbeck,
Washington University, St. Louis, USA. The final concentra-
tion of Wnt3a in the culture media was 6.2 ± 0.2 μg/mL as
determined by testing 3 individual supernatants from L-WRN
cells with the TopFlash assay (van de Wetering et al. 1991),
using the Firefly Luciferase Assay Kit 2.0 (Biotium, Fremont,
CA, USA) and recombinant murineWnt3a (Peprotech, Rocky
Hill, NJ, USA) as a standard. Formed spheroids were main-
tained in a 37 °C, 5% CO2 incubator, with fresh medium
added every 2–3 days and were passaged by trypsinization
and re-plating at 1:4 every 5–7 days. The conditions used in
this study allowed the continuous culture of human gastric
spheroids for at least 50 passages (approximately 11 months),
without apparent changes in growth, morphology or viability.
Immunohistochemistry and immunofluorescence analysis
For paraffin-embedded sections, Matrigel plugs containing
spheroids were fixed in 10% neutral buffered formalin
(Richard Allen Scientific, Kalamazoo, MI, USA). Paraffin-
embedded sections were prepared on a Sakura Tissue-Tek
VIP1000 tissue processor and embedding center and sec-
tioned at 5 μm on a Leica 2035 rotary microtome. Sections
were stained with hematoxlin-eosin (HE) reagent (Richard
Allen Scientific). For whole-mount staining, gastric epithelial
spheroids grown on 8-well chamber slides were fixed for
30 min at 4 °C in Cytofix (BD Biosciences, San Diego, CA,
USA). Samples were blocked in PBS containing 10% FBS
(Rocky Mountain Bio), 0.2% Triton X-100, 0.1% BSA and
0.05% Tween-20 (Fisher Scientific) and were incubated with
primary antibody overnight at 4 °C. The following primary
antibodies were used: mouse anti-human cytokeratin, clone
CAM5.2 (BD Biosciences, San Jose, CA, USA; recognizes
Moll’s cytokeratin peptide #8 and #7) and mouse anti-human
E-Cadherin, clone 67A4 (BioLegend, San Diego, CA, USA).
Wells were washed with DPBS and isotype-specific
Alexa488-labeled secondary antibody (SouthernBiotech,
Birmingham, AL, USA) was added for 2 h at room tempera-
ture. After a final wash step, samples were covered with
ProLong Gold with DAPI (Fisher Scientific).
Quantitative RT-PCR
Total RNAwas isolated from epithelial spheroids and human
gastric tissue samples using the Direct-zol RNAMiniPrep Kit
(ZymoResearch, Irvine, CA, USA). Complementary DNA
was generated using reverse transcription performed from
1 μg RNA using the iScript cDNA Synthesis Kit (Bio-Rad
Laboratories, Hercules, CA, USA). qPCR was performed
using iTaq Universal SYBR Green Supermix (Bio-Rad) on a
Roche LightCyler96 (Roche, Basel, Switzerland). PCR
primers sequences used are listed in Suppl. Table 1. PCR data
were analyzed using the 2-ΔΔCTmethod, with gastric spheroid
gene expression normalized to GAPDH and gene expression
in human gastric tissue.
Growth measurements and counting
To determine spheroid size, cultures were imaged by phase
contrast microscopy and spheroid diameters were measured
on composite images of entire culture wells. To obtain cell
counts, spheroid cultures were incubated with 0.25%
Trypsin EDTA solution (EMD Millipore, Billerica, MA,
USA) at 37 °C for 5–7 min and mixed with a pipet tip until
a single cell suspension was obtained. Cells were then counted
on a hemocytometer or on a flow cytometer using reference
cells (Pechhold et al. 1994).
Generation of EGFP- and mCherry-expressing spheroids
by lentiviral transduction
To produce lentiviral particles, HEK293 cells were plated at
40% confluency in DMEM/10% fetal calf serum/penicillin/
streptomycin the day before transfection. One hour before
transfection, the media was replaced with pre-warmed
OptiMEM (Gibco, Grand Island, NY, USA). Cells were co-
transfected with pCMV-VSV-G (a gift from Bob Weinberg;
Addgene plasmid # 8454; Stewart et al. 2003), psPAX2 (a gift
from Didier Trono; Addgene plasmid # 12260) and either
pLJM1-EGFP (a gift from David Sabatini; Addgene plasmid
# 19319; Sancak et al. 2008) or pLV-mCherry (a gift from
Pantelis Tsoulfas; Addgene plasmid # 36084) using
Lipofectamine 3000 (ThermoFisher) according to the manu-
facturer’s instructions. Six hours after transfection, the
OptiMEM was removed and replaced with DMEM/10% fetal
calf serum/1% BSA. The supernatant containing the lentivirus
was harvested 48 h and 60 h post-transfection. The combined
(48 h and 60 h) samples were centrifuged for 10 min at 1000g
at 4 °C, filtered through a low-protein binding 0.45-μm filter
and concentrated by ultracentrifugation (80,000g at 4 °C for
2 h). The virus was resuspended by overnight incubation at
Table 1 Organoid lines and
donor characteristics used in this
study
Organoid line Tissue Donor (age/sex) Pathology Experiments
hu001 Gastric body 45/F Polypoid fundic nodules Imaging, FACS
hu002 Gastric body 49/F Moderate gastritis Imaging, FACS
hu003 Gastric body 45/F Mild gastritis FACS
hu004 Gastric body 52/F Mild chronic inflammation Imaging, FACS
hu005 Gastric body 38/F No alterations observed FACS
hu006 Gastric antrum 42/F Not available Imaging, FACS
hu007 Gastric body 26/F Not available FACS
hu008 Gastric body 35/F No alterations observed Imaging, FACS
hu009 Gastric body 31/F No alterations observed FACS
hu010 Gastric body 43/F Mild gastritis FACS
hu011 Gastric body 52/M No alterations observed qRT-PCR
hu013 Gastric body 57/M Not available qRT-PCR
hu014 Gastric body 57/M No alterations observed qRT-PCR
4 °C in DMEM/10% FCS/1% BSA, aliquoted and stored at
−80 °C.
To perform lentiviral transduction of gastric spheroids, the
spheroids were harvested and trypsinized to obtain a single
cell suspension. Cells were suspended in spheroid L-WRN
media prepared with 10 μM ROCK Inhibitor Y-27632
(Tocris Biosciences) and hexadimethrine bromide (Sigma-
Aldrich). Cells were incubated with 30 μL of lentivirus in
250 μL L-WRN media and incubated at 37 °C in 5% CO2.
After 6 h, suspensions were transferred to Eppendorf tubes
and centrifuged at 500g for 5 min. Epithelial cells were trans-
ferred to Matrigel and plated into a warmed 24-well plate for
further culture and were maintained in the presence of puro-
mycin (Sigma) for EGFP. Spheroids with high EGFP or
mCherry expression were derived from FACS-purified sin-
gle-cell suspensions.
Microinjection of gastric spheroids with FITC–Dextran
A Fisher stereomicroscope fitted with a Genesearch Embryo
Cradle (Genesearch, Bozeman, MT, USA) and a 2 μL syringe
(Hamilton, Reno, NV, USA) was used for microinjection.
Injection needles with beveled tips were pulled from glass
capillaries to a size of 21–23 μm. For the injection, 10-day
plated spheroids in 35 mm MatTek dishes were injected with
0.2 μL of 25 μM FITC–Dextran 4 kDa (Sigma-Aldrich, St.
Louis, MO, USA). Spheroids were left in the Matrigel matrix
for injection.
Microscopic analysis
Epiflourescence and phase contrast images were acquired
using a Life Technologies EVOS FL Auto system equipped
with an onstage incubator. Live confocal imaging analysis
was performed on an inverted Leica SP5 Confocal Scanning
Laser Microscope with 405-, 488-, 561- and 633-nm laser
excitation lines and a heated stage with an environmental con-
trol chamber. Fluorescence, phase contrast and backscattered
light images were collected at 10-min intervals over 17–112 h
using a ×10 objective. Backscattered laser light images were
obtained by adjusting the wavelength range of the imaging
detector to capture the excitation laser wavelength. This tech-
nique allows the visualization of interfaces between materials
of different densities.
For transmission electron microscopy analysis (Brumfield
et al. 2009), epithelial spheroid cultures were fixed overnight
with 3% glutaraldehyde in 0.05 M potassium sodium phos-
phate buffer, pH 7.2, followed by postfixation in 2% osmium
tetroxide for 4 h. Samples were dehydrated using an ethanol
series (50–100%) and propylene oxide. After dehydration,
epithelial spheroids were gradually infiltrated with Spurr’s
resin and baked overnight at 70 °C.Then, 60- to 90-nm ultra-
thin sections were cut with a Diatome diamond knife on a
Reichert OM-U2 ultramicrotome, floated onto 300-mesh cop-
per grids and stained with uranyl acetate and Reynold’s lead
citrate. All sections were viewed with a LEO 912AB TEM
and photographed with a Proscan 2048 × 2048-pixel charge-
coupled-device camera.
Results
Phenotypic analysis of human gastric epithelial spheroids
To confirm the identity and differentiation stage of our gastric
epithelial spheroid lines maintained using the protocol of
Miyoshi and Stappenbeck (2013), we first performed pheno-
typic analyses using light microscopy, qRT-PCR and trans-
mission electron microscopy (TEM). As anticipated, the epi-
thelial cells formed spheroids with columnar to cuboidal epi-
thelium and expressed epithelial cytokeratin and E-cadherin
(Fig. 1a–c). Quantitative RT-PCR analysis of the gastric spher-
oids confirmed expression of genes specific for all five major
gastric epithelial cell subsets, i.e., parietal cells (ATP4B), chief
cells (pepsinogen C, PGC), surface mucus cells (MUC5ac),
mucus neck cells (MUC6) and enteroendocrine cells
(chromogranin A, CHGA; Fig. 1d). Notably, gene expression
for the enteroendocrine marker CHGA was very low.
Moreover, gene expression levels of all five genes analyzed
were decreased compared to the levels detected in the gastric
biopsy specimens that were used as positive controls for PCR
normalization.
Ultrastructural analysis by TEM revealed that the gastric
spheroids contained several morphologically distinct cell
types (Fig. 2). The predominant cell type was a secretory-
type cell with large electron-dense vesicles in the apical por-
tion of the cell (Fig. 2a, a′), comparable to a mucus pit cell
(Corpron 1966; Zeitoun and Lambling 1967). Cells with
electron-lucent vesicles near their luminal surface were also
present (Fig. 2a, a′′), similar to mucus neck cells. Some cells
had large amounts of rough ER and mitochondria, consistent
with active protein synthesis as typically found in chief cells
(Fig. 2b) (Zeitoun and Lambling 1967). Moreover, we also
detected cells with large intracellular canaliculi with
projecting microvilli, similar to those described for parietal
cells (Fig. 2c) (Rohrer et al. 1965). We did not detect any cells
with basal vesicles typical for enteroendocrine cells.
Consistent with the general morphology of the gastrointestinal
epithelium, spheroid cells had basal nuclei, short apical mi-
crovilli (Fig. 2d, d′) and distinct apical junctional complexes
(Fig. 2e). We also observed extensive formation of interdigi-
tating lateral processes (Fig. 2d, f), as shown by Necchi et al.
(2009) for human gastric mucosa. Overall, gastric spheroids
show typical features of the human gastric epithelium but do
not appear to be fully differentiated.
Fig. 2 Transmission electron microscopy of human gastric epithelial
spheroids. a Cross-section of a gastric spheroid shows a simple
cuboidal to columnar microvillated epithelium with vacuolated
secretory cells and basal nuclei. Lm lumen; bar 5 μm. a′ Epithelial cell
from (a) with large, electron-dense vacuoles (arrowheads). N nucleus;
bar 2 μm. a′′ Epithelial cell from (a) with large, electron-lucent
vacuoles (arrowheads). N nucleus; bar 2 μm. b Epithelial cell with
copious amounts of rough endoplasmic reticulum (arrowheads) and
multiple large mitochondria (Mi); bar 1 μm. c Large microvillated
intracellular canaliculus (Cn) in a gastric epithelial cell. Bar 1 μm. d
Columnar epithelial cells with luminal microvilli and lateral
interdigitating intercellular leaflets (arrowheads)b bar 2 μm. d′
Enlarged image from (d, dashed box) shows luminal microvilli
(arrowheads) and mucus (Mu); bar 500 nm. e Luminal-junctional
complex (arrowhead) between two epithelial cells; bar 500 nm. (f)
Lateral interdigitating intercellular leaflets; bar 1 μm
Fig. 1 Microscopic analysis and gene expression analysis of human
gastric epithelial spheroids. a Spheroids were fixed, paraffin-embedded
and sections stained with hematoxylin-eosin. Bar 50 μm. b, c E-Cadherin
(FITC) and cytokeratin (FITC) whole-mount immunofluorescence
staining of human gastric spheroids; nuclei were labeled with DAPI.
Bar 50 μm. d Quantitative RT-PCR analysis of gastric spheroids (n = 3;
hu011, hu013 and hu014). Samples were analyzed for expression of
MUC5A (mucous neck cells), PGC (pepsinogen C; chief cells), MUC6
(surface mucus cells), CHGA (chromogranin A, enteroendocrine cells)
and ATP4B (Potassium-transporting ATPase subunit beta, parietal cells).
Data were analyzed by the 2-ΔΔCT method, with GAPDH used as a
housekeeping gene and cDNA from human gastric tissue (geometric
mean of n = 3) used for normalization
Growth dynamics of human gastric epithelial spheroids
Next, we analyzed spheroid growth dynamics, size distribu-
tion and cellularity of the spheroids (Fig. 3). Spheroids ex-
panded in size over a period of around 10 days and then
plateaued, with a median diameter of 398 μm (average
443.9 ± 34.6 μm) after 12 days of culture (Fig. 3a–a′′′′, b).
Spheroids within one culture well varied significantly in size
(Fig. 3c). To determine the size distribution of gastric spher-
oids within a culture, spheroids from 4 different lines were
grown until the largest ones reached a size of >600 μm.
Cultures were then imaged and spheroid diameters measured
on composite digital images of culture wells (Fig. 3d). Size
distribution was remarkably consistent between individual
spheroid lines and followed a right-skewed bell curve, with
73 ± 8% of spheroids only reaching diameters of ≤300 μm. A
small subset of spheroids (0.4 ± 0.4%) reached diameters of
≥800 μm after 7–10 days of culture.
Using composite images of entire culture wells, digital im-
age measurements and manual cell counting of single cell
suspensions derived from the spheroids, we determined the
average relationship between spheroid size and cell numbers
in our cultures (Fig. 3e), with n being equivalent to the num-
bers of cells that can be recovered from a spheroid of a defined
radius (in μm):
n ¼ 0:00395 0:0007ð Þ 1
μm
4 π r
These cell counts correspond to a radius of 20 μm per single
epithelial cell. Similar results were obtained using FACS-
based cell counting with reference cells (Pechhold et al.
1994). The number of cells per spheroid can be used to assess
the recovery of DNA, RNA and protein from cultures with
organoids of certain sizes and to calculate the multiplicity of
infection for pathogen microinjection experiments.
Spontaneous rupture and healing events of human gastric
organoids
Based on published and anecdotal reports of organoid rupture
events (Mahe et al. 2013; Schlaermann et al. 2016; Schwank
et al. 2013), we performed live imaging analysis of six distinct
human gastric spheroid lines, with 9–83 spheroids per line im-
aged over 17–112 h. As shown in Fig. 4a–a′′′′ and Suppl.
Movie 1, spheroids frequently oscillated in diameter, consistent
with rupture and healing processes. We observed an average of
0.32 ± 0.1 ruptures per spheroid per 24-h period (Fig. 4b), with
significant variations between the three lines that were analyzed
(P ≤ 0.01).Within a given 24-h time period, some spheroids did
not rupture at all, whereas some spheroids ruptured more than
once. Overall, 43 ± 10% of spheroids in each line analyzed
showed rupture events. We did not observe any significant
difference in rupture frequency between low passage (<10)
and high passage (>10) spheroids (Fig. 4c). Interestingly, there
was a trend for an increased rupture frequency in larger epithe-
lial spheroids (Fig. 4d).
Imaging of EGFP-expressing epithelial spheroids with a
combination of fluorescence confocal imaging, backscatter
confocal imaging (Mollazade et al. 2012) and bright field
imaging revealed that rupture events were focal and as-
sociated with the release of material with increased scattering
properties—possibly mucus and cell debris—from the spher-
oid lumen (Fig. 5a–c′′′′; Suppl. Movie 2). To visualize loss of
material from the spheroid lumen, we performed live imaging
on gastric epithelial spheroids that were microinjected with 4-
kDa FITC–Dextran. As shown in Fig. 5d–e′′′′, injected FITC–
Dextran remained in the spheroid lumen for >12 h, indicating
that the epithelial barrier of the spheroids was intact. Upon
rupture, the FITC–Dextran completely disappeared from the
spheroid lumen (Fig. 5d–e′′′′; Suppl. Movie 3). Notably, epi-
thelial thickness was low prior to rupture events and increased
immediately following a rupture event. In summary, our data
show that spontaneous spheroid rupture is a common event
and involves release of luminal material.
Human gastric spheroids rotate within the Matrigel
matrix
Surprisingly, our live imaging analysis also revealed that, even
in the absence of rupture events, spheroid interactions with the
Matrigel matrix were not static. In particular, spheroids fre-
quently rotated around an axis, with an average of
53.6 ± 11.4% of spheroids in all cultures analyzed displaying
rotational movement within the matrix (Fig. 6a–a′′′′′, b; Suppl.
Movie 4). This behavior was predominantly observed in
smaller spheroids (< 100 μm in diameter, data not shown)
but showed a large variation between individual spheroid lines
and individual cultures. Again, there was no significant differ-
ence between low passage (≤ p 10) or high passage (>10)
spheroids. To further investigate the dynamic interactions be-
tween the spheroids and the Matrigel, we performed backscat-
ter light imaging (Fig. 6c-c′′; Suppl. Movie 5). This backscat-
ter light analysis revealed that spheroid epithelia form tempo-
rary, pseudopod-like basolateral projections that may enable
movement of the spheroids within the Matrigel capsule.
Adjacent spheroids may undergo membrane fusion
Live imaging analysis also revealed that, in some cases,
two adjacent spheroids fused to form one larger spheroid
(Fig. 7). These events were rare but seen in three out of
five different lines analyzed between passages 4 and 18
(Fig. 7a–a′′′′′, b). To confirm that the observed events rep-
resented true luminal fusions, spheroids that had been
injected with FITC–Dextran (4 kDa) were monitored by
live confocal imaging. As shown in Fig. 7c–d′′′′ and Suppl.
Movie 6, spheroid fusion resulted in the transfer of green
fluorescent FITC–Dextran from the lumen of one injected
spheroid to a non-injected spheroid. These observations
indicate that spheroid fusions lead to the formation of a
common luminal compartment.
Fig. 3 Gastric epithelial spheroid growth. a– a′′′′ Representative phase
contrast images of one gastric epithelial spheroid imaged repeatedly
between days 3 and 12 after plating (line hu001, p17). b Diameters of
65 randomly selected spheroids in one representative well (line hu001,
p17) were measured repeatedly on days 3, 5, 7, 10 and 12 after plating.
Box delineates 1st and 3rd quartile and median, whiskers show minimum
and maximum size. c Composite image of a representative culture well
containing mCherry-expressing human gastric spheroids; bar 500 μm. d
Size distribution of gastric epithelial spheroids in one representative well
each from 4 different lines, imaged on days 7 or 10 after seeding (see
label); graph shows mean (–) and individual values for the 4 spheroid
lines. e Relationship between spheroid diameter and cell count was
determined by measuring all spheroids within one well followed by
disruption of the spheroids using trypsin-EDTA and hemocytometer
cell counting. Averages ± SEM calculated from 3 independent cultures
are shown

Discussion
In this study, we used live imaging to demonstrate that human
gastric epithelial spheroids—or gastrospheres (Stelzner et al.
2012)—cultured under the conditions described (see
BMaterials and methods") display several dynamic behaviors
that may be overlooked when performing end-point analyses
and that may impact experimental results. Specifically, spher-
oids spontaneously ruptured and sometimes fused and
frequently rotated in the Matrigel matrix. Notably, there were
significant differences in the behavior of distinct gastric spher-
oid lines and in the behavior of individual spheroid lines
analyzed after a different number of passages.
Spontaneous rupture was a relatively common event, with
>40% of spheroids imaged showing rupture events. Videos
included in several previous publications on murine intestinal
organoids and human gastric organoids also showed organoid
ruptures, although these events were not addressed directly
(Mahe et al. 2013; Schlaermann et al. 2016; Schwank et al.
2013), which suggests that spontaneous ruptures are not spe-
cific to the specific culture conditions used here. In our study,
we quantified rupture events and demonstrated that ruptures
occurred in all gastric spheroid lines analyzed in our labora-
tory. We also showed that ruptures were preceded by thinning
of the epithelium, suggesting that the luminal pressure of the
spheroids increased over time. There was a trend for larger
spheroids to rupture more frequently but the reasons for this
increased rupture behavior are unclear. Since the gastric epi-
thelium normally lines a tubular structure rather than a closed
sphere, mechanisms that prevent rupture are not likely to exist.
Notably, our observation that spheroids rupture on a regular
basis could theoretically impact the outcome of experiments if
injected microorganisms or other luminally secreted sub-
stances were to be released into theMatrigel and unexpectedly
interacted with the basolateral side of the epithelium.
Gastric spheroids generally underwent additional growth
following rupture, indicating efficient and rapid epithelial res-
titution. As previously described (Bartfeld et al. 2015;
Miyoshi and Stappenbeck 2013), isolated glands spontane-
ously form spheres within several hours, which represents
another example of epithelial healing behavior. Moreover,
we observed that adjacent spheroids occasionally fused,
which may also represent a healing mechanism. The ability
of organoids to contribute to epithelial healing has been pre-
viously demonstrated. In a recent study by Engevik et al.
(2016), gastric organoids were transplanted into the gastric
submucosa of mice following chemical induction of gastric
ulcers. These transplanted organoids significantly improved
gastric wound healing, demonstrating that gastric organoids
efficiently regenerate defective epithelia (Engevik et al. 2016).
Similarly, colonic organoids improved the healing of epitheli-
al defects in the murine colon (Yui et al. 2012). Such wound-
healing behavior of gastrointestinal epithelia involves chemo-
tactic migration of epithelial cells and epithelial flattening and
lateral lamellipodia extension that lead to rapid closure of
epithelial gaps independent of cell proliferation (Iizuka and
Konno 2011; Silen and Ito 1985; Smith et al. 2005). In addi-
tion, the efficient reformation of spheroids after rupture and
the fusion of closely adjacent spheroids was possibly en-
hanced by the high medium concentrations of Wnt, which
promotes gastrointestinal epithelial wound healing (Miyoshi
2017).
Notably, gastrointestinal epithelial cells are highly dynamic
in vivo, with a high epithelial cell turnover rate and efficient
closure of gaps caused by apoptotic cell extrusion (Heath
1996; Williams et al. 2015). Gastric surface mucus cells mi-
grate from the isthmus, where stem cells are located, to the
surface mucosa within 1–3 days (Creamer et al. 1961; Karam
et al. 1997; Lipkin 1965). Intestinal epithelial cells move as
much as 5–10 μm/h from the base of the crypts towards the
tips of the villi and apical junctional complexes have to be
constantly re-organized to close gaps left by epithelial cells
that undergo apoptosis (Iizuka and Konno 2011). This con-
stant reformation of intracellular junctions due to epithelial
cell turnover may have played a role in the formation of
cell–cell junctions in the observed fusions of intact spheroids.
It remains unclear whether the formation of dynamic pseu-
dopods observed on the basolateral side of human gastric
spheroids was a normal process or specific to our culture con-
ditions. Basal pseudopodia or lamellipodia that may promote
epithelial cell movement have been identified in normal intes-
tinal epithelium but they occur at low frequencies (Heath
1996). In contrast, basal pseudopodia play a major role in
epithelial carcinogenesis, where pseudopodia can contribute
to invasion and matrix degradation by expanding tumors
(McNiven 2013). Basal pseudopod-like processes in the small
intestine also appear to be more frequent during early devel-
opment (Burgess 1976) and on certa in types of
enteroendocrine cells, where they are thought to allow direct-
ed secretion of hormones (Bohorquez et al. 2011). In our
spheroid cultures, increased basal pseudopod formation might
be associated with the low differentiation stage of the cells or
might have been provoked by the specific characteristics of
ƒFig. 4 Spontaneous spheroid rupture and healing. a– a′′′′Representative
image series from a 22-h time course experiment shows epithelial
spheroid expansion and rupture (indicated by arrows), followed by re-
forming and continued expansion over time. Phase contrast images;
bar 100 μm. b Frequency of rupture events in 3 different gastric
epithelial spheroid lines analyzed at passage ≤10. Graph shows average
number of rupture events per 24-h period for individual spheroids
(n = 33–59) and mean ± SD of each line. **Statistically significant
difference at P ≤ 0.01. c Average rupture frequency per 24 h in low
passage number cultures (P ≤ 10; hu002, n = 59; hu006, n = 38; hu008,
n = 33) and high passage number cultures (P > 11, hu001, n = 9; hu004,
n = 9; hu006, n = 45); individual values, mean ± SD; n.s. not significant. d
Relationship between rupture frequency and size. Spheroid lines hu001
(n = 33 spheroids), hu002 (n = 53) and hu006 (n = 31) were analyzed
the Matrigel matrix, which differs from normal basement
membrane and extracellular matrix in composition and struc-
ture (Benton et al. 2009). Since pseudopodia are generally
associated with cellular movement, these processes may have
enabled spheroid movement including the observed rotation
and fusion events within the Matrigel shell.
Our detailed analysis of gastric spheroid growth under pre-
viously published culture conditions (Demitrack et al. 2017;
Fig. 5 Release of luminal contents from ruptured gastric epithelial
spheroids. a– c′′′′ Confocal and backscatter light image series from a
14-h time course analysis shows rupture, release of optically dense
material (a– a′′′′) from the inside of the GFP-expressing spheroid (b– b
′′′′) and thickening of the epithelial layer upon healing. c– c′′′′
Brightfield images. Bar 50 μm. d– e′′′′ Release of injected 4-kDa
FITC–Dextran (d– d′′′′) from an mCherry-expressing gastric spheroid
(e– e′′′′) upon rupture. Images show disappearance of green FITC–
Dextran signal after rupture at 13:20 hours. Bar 200 μm

den Hartog et al. 2016; Gifford et al. 2017; Miyoshi and
Stappenbeck 2013) revealed several additional interesting
properties of this model system. Thus, although some spher-
oids grew to a size of >1000 μm in diameter, the percentage of
these large spheroids was <0.5%, while the majority of spher-
oids remained in the 100–300 μm diameter size range after 7–
10 days in culture. Also, we never observed spheroids that
grew larger than 1300 μm. Interestingly, TEM analysis of
the cultures revealed extensive formation of lateral interdigi-
tating folds, as also observed in some gastric tissue specimens
in previous studies (Necchi et al. 2009). Structurally similar
Fig. 7 Gastric epithelial spheroid fusion events. a– a′′′′′ Phase contrast
image series of two human gastric spheroids undergoing fusion. Spheroid
line hu004. Bar 200 μm. b Percentage of spheroids with observed fusion
events in 5 different human gastric spheroid lines (hu001, p10, n = 149;
hu002, p8–10, n = 59; hu004, p18, n = 9; hu006, p7–16, n = 83; and
hu008, p4, n = 33), normalized to a 24-h observation period. c– d′′′′ 4-
kDa FITC–Dextran injected into one gastric spheroid (arrowhead) is
transferred to an adjacent spheroid upon membrane fusion (arrow).
Bright field (c– c′′′′) and green fluorescent images (d– d′′′′). Spheroid
line hu006. Bar 500 μm
ƒFig. 6 Gastric epithelial spheroids rotate within the Matrigel. Spheroids
commonly rotate around their axis within the Matrigel matrix. a– a′′′′′
Representative image series of a small single spheroid observed rotating
counter-clockwise as indicated by the arrows over a time course of 16 h. b
Percentage of low passage (hu002, p8–10, n = 59; hu006, p7–10, n = 38;
hu008, p4, n = 33) and high passage gastric spheroids (hu001, p44–45,
n = 9; hu004, p18, n = 9; hu006, p11–16, n = 45) observed for 46 ± 1 h
that showed rotation. Bars mean ± SEM of individual cultures (n = 1–3
per line). The percentage of rotating epithelial spheroids did not differ
significantly between individual lines. c– c′′ Backscatter light imaging
reveals basolateral formation of pseudopod-like extensions. Arrows
indicate pseudopods. Bar 25 μm
lateral folds have been detected in the gall bladder and other
epithelial tissues with increased ion transport capacity and
have been implicated in contributing to water retention
(Diamond and Tormey 1966; Larsen et al. 2009).
Notably, we did not alter the composition of the culture
media during the experiments in order to induce gastric epi-
thelial cell differentiation and our spheroids did not develop
gland-like invaginations. Differentiation of primary gastric
epithelial cell cultures has previously been achieved by with-
drawal of Wnt and noggin (Sato and Clevers 2015) or Notch
inhibition (Demitrack et al. 2015, 2017; VanDussen et al.
2015). Since we detected expression of genes specific for all
five major epithelial cell subsets and the TEM analysis re-
vealed structures very similar to the intracellular canaliculi
that are considered typical for gastric parietal cells, we consid-
er the epithelial cells in our cultures partially differentiated. It
remains unknown whether the behaviors and growth dynam-
ics described here similarly occur under culture conditions
different from the ones used in our study. Ruptures and
organoid movement in the Matrigel have been shown for a
number of different epithelial and culture protocols (Mahe
et al. 2013; Schlaermann et al. 2016; Schwank et al. 2013)
but the frequency of these events had not previously been
evaluated. Several different protocols have been used to gen-
erate and maintain gastric organoids (Bartfeld et al. 2015;
Bertaux-Skeirik et al. 2015; McCracken et al. 2014;
Schlaermann et al. 2016; Schumacher et al. 2015) and varia-
tions in media composition were shown to affect organoid
morphology, differentiation and growth. Future studies will
evaluate whether altering culture conditions or adding drugs
to affect gastric secretion, e.g., omeprazole, cimetidine or
naproxen, may prevent dynamic events such as ruptures that
may be undesirable for certain experimental applications.
Overall, our study revealed several interesting features of
three-dimensional gastric spheroids, including spontaneous
ruptures, fusions and rotation events. Interestingly, human
gastric epithelial spheroid lines derived from different human
donors and spheroids analyzed after a different number of
passages showed marked, sometimes significant, differences
in many of the parameters analyzed. Consequently, for future
studies using human spheroids, gastroids and other organoid
culture systems, a general consensus within the scientific com-
munity on quality control parameters of organoids would be
beneficial, so that data obtained in different laboratories with
different cell lines can be compared.
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 shar-
ing the L-WRN cell line with us and Dr. Seth Walk for helpful
discussions.
Author contributions D.B., B.W., L.C.S. and J.W. planned and over-
saw the experiments; T.A.S., B.S., R.B. and R.A.W. performed the ex-
periments; 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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