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\begin{document}

\title{Intravenous injection of mesenchymal stem cell spheroids for efficient
pulmonary delivery and prolonged \emph{in vivo} survival}



\author[1]{Yoshihiko Shimazawa}%
\author[1]{Kosuke Kusamori}%
\author[1]{Mari Tsujimura}%
\author[2]{Kazunori Shimizu}%
\author[3]{Satoshi Konishi}%
\author[1]{Makiya Nishikawa}%
\affil[1]{Tokyo University of Science - Noda Campus}%
\affil[2]{Nagoya University Graduate School of Engineering School of Engineering}%
\affil[3]{Ritsumeikan University College of Science and Engineering Graduate School of Science and Engineering}%


\vspace{-1em}



  
  \date{May 25, 2024}


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\selectlanguage{english}
\begin{abstract}
Because of their excellent therapeutic potential, mesenchymal stem cells
(MSCs) have been used as cell therapeutics for various diseases.
However, the survival rate and duration of MSCs after transplantation
are extremely low and short, respectively. To solve these problems, in
this study, we prepared multicellular spheroids of MSCs and investigated
their survival and function after intravenous injection in mice. The
murine adipose-derived MSC line m17.ASC was cultured in agarose-based
microwell plates to obtain size-controlled m17.ASC spheroids of an
average diameter and cell number of approximately 170 \selectlanguage{greek}μ\selectlanguage{english}m and 1100
cells/spheroid, respectively. The intravenously injected m17.ASC
spheroids mainly accumulated in the lung and showed a higher survival
rate than suspended m17.ASC cells during the experimental period of 7
days. m17.ASC spheroids efficiently reduced the
lipopolysaccharide-induced increase in plasma concentrations of
interleukin-6 and tumor necrosis factor-\selectlanguage{greek}α. \selectlanguage{english}These results indicate that
spheroid formation improved the pulmonary delivery and survival of MSCs,
as well as their therapeutic potential against inflammatory pulmonary
diseases.%
\end{abstract}%



\sloppy


\textbf{1. Introduction}

Mesenchymal stem cells (MSCs) possess excellent therapeutic properties,
including the ability to exert anti-inflammatory and immunosuppressive
effects and to differentiate into tissue-specific
cells.\textsuperscript{{[}1{]}} In addition, MSCs are self-proliferating
and can be prepared on a large scale after isolation from a variety of
tissues.\textsuperscript{{[}2,3{]}} Based on these superior features,
MSCs have been used in the treatment of graft-versus-host
disease,\textsuperscript{{[}4{]}} autoimmune
diseases,\textsuperscript{{[}5{]}} and inflammatory
diseases.\textsuperscript{{[}6{]}} Although the therapeutic usefulness
of MSC transplantation in patients with these diseases has been
demonstrated in some cases, many studies have revealed that the survival
rate and duration of MSCs after transplantation are extremely low and
short, respectively.\textsuperscript{{[}7{]}} Therefore, it is necessary
to increase the survival of administered MSCs for more efficient
MSC-based therapy.

Multicellular spheroids, which are three-dimensional cultured cell
aggregates, are highly functionalized cells, because spheroid formation
generally increases cellular function through extensive cell--cell
interactions.\textsuperscript{{[}8{]}} It has been reported that
spheroid formation upregulates the expression of transporters in cancer
cells.\textsuperscript{{[}9{]}} Therefore, cancer cell spheroids have
been used to screen for bioactive compounds, including anticancer
agents. In addition, Bartosh et al. reported that multicellular
spheroids derived from MSCs secrete large amounts of anti-inflammatory
cytokines.\textsuperscript{{[}10{]}} Additionally, our previous studies
using poly(N-isopropylacrylamide)-coated polydimethylsiloxane-based
microwell plates showed that spheroid formation improved the survival of
mouse insulinoma NIT-1 cells after transplantation in
mice,\textsuperscript{{[}11{]}} enhanced albumin secretion from human
hepatoblastoma HepG2 cells,\textsuperscript{{[}12{]}} and forcibly
polarized mouse macrophage-like J774.1 cells to M1-type macrophages with
tumoricidal activity.\textsuperscript{{[}13{]}} Furthermore, we
demonstrated that compared to suspended mouse adenocarcinoma colon26
cells, multicellular spheroids of colon26 cells efficiently accumulated
in the lung after intravenous injection in mice and established
pulmonary tumor colonies.\textsuperscript{{[}14{]}} Therefore, we
hypothesized that spheroid formation could increase the survival of MSCs
after intravenous injection and improve their therapeutic effects in
lung diseases.

Here, we investigated the survival and function of MSC spheroids after
intravenous injection in mice. First, the murine adipose-derived MSC
line m17.ASC transfected with NanoLuc luciferase (NLuc) was cultured in
agarose-based microwell plates to obtain size-controlled m17.ASC
spheroids. Thereafter, tissue distribution was evaluated by measuring
NLuc activity after intravenous injection of m17.ASC spheroids or
suspended m17.ASC cells. Finally, the anti-inflammatory effect of
suspended m17.ASC cells or m17.ASC spheroids was evaluated in a
lipopolysaccharide (LPS)-induced inflammatory mouse model.

\textbf{2. Materials and methods}

2.1. Materials

Dulbecco's modified Eagle's medium (DMEM) was purchased from Nissui
Pharmaceutical (Tokyo, Japan). Trypsin-ethylenediaminetetraacetic acid
(EDTA) solution (0.25\% trypsin and 1 mM EDTA) and trypan blue were
purchased from Nacalai Tesque (Kyoto, Japan). SYBR Green PCR Master Mix
was obtained from Gibco Invitrogen (Carlsbad, CA, USA). Fetal bovine
serum (FBS) was purchased from Thermo Fisher Scientific (Waltham, MA,
USA). Agarose S was purchased from Nippon Gene (Toyama, Japan).
Isoflurane was purchased from Fujifilm Wako Pure Chemical Industries
(Osaka, Japan). Hygromycin B Gold was purchased from InvivoGen (San
Diego, CA, USA). Hank's Balanced Salt solution (HBSS) was purchased from
Sigma-Aldrich (St. Louis, MO, USA). All other chemicals were of the
highest grade commercially available.

2.2. Animals

Male FVB/N mice (6--8 weeks old) were purchased from CLEA Japan Inc.
(Tokyo, Japan). All animal experiments were conducted in accordance with
the principles and procedures outlined in the National Institutes of
Health Guide for the Care and Use of Laboratory Animals. The protocols
for the animal experiments were approved by the Animal Experimentation
Committee of the Tokyo University of Science.

2.3. Cell culture

The murine adipose-derived MSC line m17.ASC was purchased from DS Pharma
Biomedical (Osaka, Japan) and grown in DMEM supplemented with 20\%
inactivated FBS and penicillin-streptomycin-glutamine mixed solution.
Peritoneal macrophages were collected from male FVB/N mice. In detail,
cold phosphate-buffered saline (PBS, 5 mL) was injected into the
abdominal cavity of euthanized FVB/N mice using a 26G needle and
syringe, and the PBS containing peritoneal cells was collected after
rubbing the abdomen. The collected PBS was centrifuged at 300 \selectlanguage{ngerman}×
\emph{g}for 3 min to sediment and obtain the peritoneal cells. The cells
were grown in DMEM supplemented with 10\% inactivated FBS and
penicillin-streptomycin-glutamine mixed solution. After 24-h incubation,
adherent cells were used as peritoneal macrophages. All cells were
maintained in a humidified atmosphere containing 5\%
CO\textsubscript{2}at 37 °C.

2.4. Preparation of agarose-based microwell plates

Micropillar arrays fabricated using a 3D printing system (Objet
Geometries, Rehovot, Israel)\textsuperscript{{[}15{]}} were used as
molds for agarose-based microwell plates. Agarose was dissolved in
distilled water at a final concentration of 3\% (w/v), heated in a
microwave oven, and poured into the micropillar arrays. After gelation
of the agarose solution by cooling for 30 min at approximately 20 °C,
the micropillar arrays were removed from the agarose gel. The
agarose-based microwell plate was cut with a cutter to adjust the size
to fit a well in a six-well cell culture plate. Microwell plates in
culture plates were sterilized by ultraviolet irradiation prior to cell
seeding. The structures of the micropillar arrays and agarose-based
microwell plates were imaged using a digital camera (IXY 200F, Canon,
Tokyo, Japan) and BZ-9000 microscope (Keyence, Osaka, Japan).

2.5. Preparation of m17.ASC spheroids

m17.ASC cells (2 × 10\textsuperscript{6} cells) suspended in a cell
culture medium were added to an agarose-based microwell plate in a
six-well plate, and the culture plates were shaken for 2 h. The medium
was then replaced with fresh medium, and the cells were incubated at 37
°C with rotary shaking at 60 rpm. At 24, 48, and 72 h of incubation,
spheroids formed in microwells were collected using a micropipette.

2.6. Evaluation of m17.ASC spheroids

The diameter of the m17.ASC spheroids was measured by observation under
a BZ-9000 microscope (Keyence) using the BZ-9000 analyzer software. The
average diameter was calculated as the average diameter of 100
spheroids. The number and viability of cells in spheroids were evaluated
by trypan blue staining after dispersing 20 spheroids in trypsin-EDTA
solution.

2.7. Transforming growth factor (TGF)-\selectlanguage{greek}β \selectlanguage{english}release from m17.ASC spheroids

To evaluate cytokine release from m17.ASC spheroids, monolayered m17.ASC
cells (1.25 \selectlanguage{ngerman}× 10\textsuperscript{4} cells) or m17.ASC spheroids (11
spheroids: approximately 1.25 × 10\textsuperscript{4} cells, 24-h
incubation) were seeded into a 12-well culture plate. After 24 and 48 h
of incubation, the supernatant was collected and centrifuged at 2,500
rpm for 5 min to remove cell debris. The concentration of TGF-\selectlanguage{greek}β \selectlanguage{english}in the
supernatant was measured using a TGF beta-1 Human/Mouse Enzyme-Linked
Immunosorbent Assay kit (Thermo Fisher Scientific).

2.8. Polarization of macrophages by co-culture with m17.ASC spheroids

To evaluate the polarization of macrophages by co-culture with m17.ASC
spheroids, monolayered m17.ASC cells (1 \selectlanguage{ngerman}× 10\textsuperscript{5} cells)
or m17.ASC spheroids (90 spheroids: approximately 1 ×
10\textsuperscript{5} cells) were added to the upper wells of a 12-well
Transwell plate (Corning, NY, USA), and 1 ×
10\textsuperscript{5}peritoneal macrophages were seeded into the lower
wells. After 24-h incubation, total RNA was extracted from peritoneal
macrophages using Sepasol RNA I Super G (Nacalai Tesque). mRNA was
reverse-transcribed to cDNA using qPCR RT Master Mix with gDNA Remover
(Toyobo, Osaka, Japan). To quantify the gene of interest, cDNA samples
were amplified using Thunderbird SYBR qPCR Mix (Toyobo) and specific
primers with the CFX Connect Real-Time System (Bio-Rad Laboratories,
Hercules, CA, USA). The primer sequences used were as follows:
\emph{GAPDH} (F) 5'-AGGTCGGTGTGAACGGATTTG-3', \emph{GAPDH} (R)
5'-TGTAGACCATGTAGTTGAGGTCA-3'; \emph{Arg1} (F)
5'-TGGCTTGCGAGACGTAGAC-3', \emph{Arg1} (R)
5'-GCTCAGGTGAATCGGCCTTTT-3';\emph{Ym1} (F)
5'-TTATCCTGAGTGACCCTTCTAAG-3', and \emph{Ym1} (R)
5'-TCATTACCCTGATAGGCATAGG-3'. Fold changes in mRNA expression were
calculated using the \selectlanguage{greek}ΔΔ\selectlanguage{english}CT method.\textsuperscript{{[}16{]}} The results
were normalized to \emph{GAPDH} mRNA levels in the same samples.

2.9. Establishment of the \emph{Nluc} -expressing m17.ASC cells

The \emph{Nluc} sequence was inserted into the pEBMulti/Hyg vector to
construct a plasmid encoding \emph{Nluc} (pEBMulti-Nluc) as previously
reported.\textsuperscript{{[}17{]}} In brief, the cDNA fragment
of\emph{Nluc} was amplified by PCR and digested using the restriction
enzymes KpnI and EcoRV (New England Biolabs, Ipswich, MA, USA).
Subsequently, the product was inserted into the multiple cloning site of
the pEBMulti-Hyg vector using the same restriction enzymes and Ligation
High (Toyobo). m17.ASC cells (5 \selectlanguage{ngerman}× 10\textsuperscript{4} cells) were
seeded into a 48-well culture plate and cultured for 24 h. The
pEBMulti-Nluc plasmid was transfected into m17.ASC cells using
Lipofectamine 3000 (InvivoGen). The transfected m17.ASC cells were
cultured in DMEM containing 20\% FBS for 3 days. The cells were then
cultured in media containing 400 \selectlanguage{greek}μ\selectlanguage{english}g/mL hygromycin B to select\emph{Nluc}
-expressing m17.ASC (m17.ASC/Nluc) cells. \emph{Nluc}expression in cells
was confirmed using an Nluc substrate (Nano-Glo Luciferase Assay
Reagent, Promega, Madison, WI, USA). m17.ASC/Nluc cells were cultured in
a medium containing 400 \selectlanguage{greek}μ\selectlanguage{english}g/mL hygromycin.

2.10. Tissue distribution of m17.ASC/Nluc spheroids after intravenous
injection in mice

m17.ASC/Nluc spheroids (135 spheroids: approximately 1.5 \selectlanguage{ngerman}×
10\textsuperscript{5} cells) or suspended m17.ASC/Nluc cells (1.5 ×
10\textsuperscript{5} cells) were intravenously injected into FVB/N
mice. Blood and tissues (heart, lung, liver, kidney, and spleen) were
collected 24 h after intravenous injection. Tissues were homogenized in
a lysis buffer (20 mM Tris-HCl, 200 mM NaCl, 2.5 mM
MgCl\textsubscript{2}, 0.05\% (w/v) NP-40, pH 7.4) using a homogenizer
(Microtec, Chiba, Japan). Blood and homogenized tissues were centrifuged
at 10,000 × \emph{g} for 15 min at 4 °C to obtain plasma and the
supernatant of homogenized tissues, respectively. Subsequently, the
relative light units of the plasma and supernatants were measured using
the Nano-Glo Luciferase Assay Reagent with an Envision multilabel plate
reader (PerkinElmer, Waltham, MA, USA). In addition, m17.ASC/Nluc
spheroids (100 spheroids, 24-h incubation: approximately 1.5 ×
10\textsuperscript{5} cells) or suspended m17.ASC/Nluc cells (1.5 ×
10\textsuperscript{5} cells) were intravenously injected into FVB/N
mice. Lungs were collected at 6, 24, 48, 120, and 168 h after
intravenous injection and homogenized in the lysis buffer. The
homogenates were centrifuged at 10,000 × \emph{g} for 10 min at 4 °C to
obtain the supernatant of the homogenized lungs. The relative light
units of the supernatants were measured, as described above.

To image m17.ASC spheroids or suspended m17.ASC cells in the lung after
intravenous injection, suspended m17.ASC cells (1 ×
10\textsuperscript{5} cells) or m17.ASC spheroids (90 spheroids, 24-h
incubation: approximately 1 × 10\textsuperscript{5} cells) were labeled
with carboxyfluorescein diacetate succinimidyl ester (CFSE,
Sigma-Aldrich). Lungs were collected from euthanized FVB/N mice 24 h
after intravenous injection and fixed using O.C.T. compound (Sakura
Finetek Japan, Tokyo, Japan). Cryosections of the lung were prepared
using a Leica CM3050 S cryostat (Leica Microsystems, Nussloch, Germany)
and observed under a BZ-9000 microscope (Keyence).

2.11. Effect of m17.ASC spheroids on the LPS-induced inflammatory mouse
model

LPS (Sigma-Aldrich) was intraperitoneally administered to FVB/N mice at
a dose of 1 mg/kg. The mice were then bred on a hot plate (40 °C) for 1
h. Subsequently, m17.ASC spheroids (850 spheroids, 24-h incubation:
approximately 1 × 10\textsuperscript{6} cells) or suspended m17.ASC
cells (1 × 10\textsuperscript{6} cells) were intravenously injected in
the mice. Blood was collected from each mouse at 6 and 10 h after the
injection. Blood was centrifuged at 10,000 × \emph{g} for 5 min at 4 °C
to obtain plasma. The plasma levels of IL-6 and TNF-\selectlanguage{greek}α \selectlanguage{english}were measured
using ELISA kits (Thermo Fisher Scientific).

2.12. Statistical analysis

The statistical significance of differences was evaluated by one-way
analysis of variance (ANOVA) followed by Tukey's test for multiple
comparisons or Student's \emph{t} -test for two groups. Differences were
considered statistically significant at \emph{P} \textless{}0.05.

\textbf{3. Results}

3.1. Preparation of m17.ASC spheroids

Figure 1A and B show the appearance of the micropillar arrays and an
agarose-based microwell plate, respectively. Figure 1C and D show
microscopic images of the typical top and cross-sectional views of the
agarose-based microwell plate. m17.ASC spheroids were successfully
prepared using the agarose-based microwell plate 24 h after seeding the
cells (Figure 1E). Figure 2 shows the size histogram of m17.ASC
spheroids collected at 24, 48, and 72 h after incubation. The average
diameter of the m17.ASC spheroids was approximately 170 \selectlanguage{greek}μ\selectlanguage{english}m, irrespective
of the incubation period (Table 1). The number of m17.ASC cells ranged
from 1,100 to 1,300 cells/spheroid and remained almost unchanged during
the incubation period. The viability of the cells in spheroids was
\textgreater{}90\%. Based on these results, m17.ASC spheroids prepared
from 24-h incubation were used in subsequent experiments.

3.2. Characteristics of m17.ASC spheroids

To evaluate the function of m17.ASC spheroids, the secretion of TGF-\selectlanguage{greek}β,
\selectlanguage{english}an anti-inflammatory cytokine, from m17.ASC spheroids or monolayered
m17.ASC cells was measured. TGF-\selectlanguage{greek}β \selectlanguage{english}secretion in both groups increased
with time, and the amount of TGF-\selectlanguage{greek}β \selectlanguage{english}secreted from m17.ASC spheroids
tended to be higher than that from monolayered m17.ASC cells (Figure
3A). In addition, the effect of m17.ASC spheroids on the polarization of
macrophages was evaluated, because MSCs are reported to modulate
macrophages to the immunosuppressive M2-type and contribute to
suppressing inflammation.\textsuperscript{{[}18{]}} m17.ASC spheroids
and monolayered m17.ASC cells tended to increase the expression
of\emph{Arg1} and \emph{Ym1} , two M2-type macrophage markers, in
peritoneal macrophages (Figure 3B). The expression of \emph{Arg1} in
m17.ASC spheroids was higher than that in monolayered m17.ASC cells,
although the difference was not statistically significant. These results
indicate that spheroid formation enhanced the functions of m17.ASC
cells, as previously reported in MSC spheroid
formation.\textsuperscript{{[}19{]}}

3.3. Tissue distribution of m17.ASC/Nluc spheroids and suspended
m17.ASC/Nluc cells in mice

Figure 4A shows the tissue distribution of m17.ASC/Nluc spheroids and
suspended m17.ASC/Nluc cells 24 h after intravenous injection in mice.
m17.ASC/Nluc spheroids and suspended m17.ASC/Nluc cells showed high
accumulation in the lung compared to other tissues. m17.ASC/Nluc
spheroid accumulation in the lung was significantly higher than that in
suspended m17.ASC/Nluc cells. Figure 4B shows the m17.ASC/Nluc cells
remaining in the lung over time. At all the time points examined, the
number of remaining cells was higher in the m17.ASC/Nluc spheroid group
than that in the suspended m17.ASC/Nluc cell group. Furthermore,
m17.ASC/Nluc spheroids were detected in the lung even at 168 h after
injection, whereas suspended m17.ASC/Nluc cells almost completely
disappeared after 48 h. Figure 4C shows typical images of the
cryosections of the lung at 24 h after intravenous injection of
CFSE-labeled m17.ASC spheroids or suspended CFSE-labeled m17.ASC cells.
The fluorescence signal of CFSE-labeled m17.ASC spheroids was observed
in most sections examined, whereas the fluorescence signal of suspended
CFSE-labeled m.17ASC cells was hardly detected in any sections.

3.4. Effect of m17.ASC spheroids on LPS-induced inflammation in mice

Figure 5 shows the plasma concentrations of IL-6 and TNF-\selectlanguage{greek}α \selectlanguage{english}in the
LPS-induced inflammation mouse model. m17.ASC spheroids and suspended
m17.ASC cells significantly reduced the LPS-induced increase in IL-6 and
TNF-\selectlanguage{greek}α \selectlanguage{english}levels. Importantly, m17.ASC spheroids were more effective than
suspended m17.ASC cells in reducing TNF-\selectlanguage{greek}α \selectlanguage{english}levels at 6 h. These results
indicate that the therapeutic effect of m17.ASC spheroids was higher
than that of suspended m17.ASC cells.

\textbf{4. Discussion}

Multicellular spheroids are useful cell formations in cell-based therapy
because of the excellent cell--cell interactions, physical strength, and
avoidance of anoikis, which is an apoptotic response to the absence of
cell--matrix interactions. Although many reports have demonstrated
improved cellular functions by spheroid formation \emph{in vitro}
,\textsuperscript{{[}20-22{]}} there have been few reports on the
function or survival of multicellular spheroids after transplantation
into animals. Our previous studies demonstrated that the survival of
insulin-secreting cells was prolonged after transplantation into the
subrenal capsule in mice\textsuperscript{{[}11{]}} and that tumor colony
formation in the lungs after intravenous injection in mice was
accelerated by spheroid formation of tumor
cells.\textsuperscript{{[}14{]}} However, the function, survival, and
tissue distribution of multicellular spheroids of MSCs after intravenous
injection have been barely elucidated thus far. In particular, the
quantitative comparison of suspended cells and spheroids after
intravenous injection in terms of survival and tissue distribution has
not been evaluated. Here, we showed that spheroid formation of MSCs
improved their accumulation in the lungs and prolonged their survival
after intravenous injection.

Several studies have reported that spheroid formation significantly
increases cytokine release from MSCs.\textsuperscript{{[}10,23{]}} Here,
TGF-\selectlanguage{greek}β \selectlanguage{english}release from m17.ASC spheroids was slightly higher than that from
monolayered m17.ASC cells (Figure 3A). Additionally, the release of
other cytokines from m17.ASC spheroids may have been improved, because
the expression of \emph{Arg1} , an M2-type marker, by peritoneal
macrophages was greatly increased by co-culture with m17.ASC spheroids
(Figure 3B). As the MSC-induced polarization of macrophages to the
M2-type has been reported to be related to an anti-inflammatory
function,\textsuperscript{{[}24{]}} the results of the present study
strongly suggest the therapeutic potential of MSC spheroids for
inflammatory diseases. In addition to cytokine expression, spheroid
formation of MSCs can be an effective means of cell-based therapy.

The size of multicellular spheroids is an important parameter in
determining the tissue distribution of cells after intravenous
injection. This is because the diameter of microvessels in the lung, the
first-pass organ after intravenous injection, is approximately 10 \selectlanguage{greek}μ\selectlanguage{english}m,
and most cells transiently form the embolus in the lung after
intravenous injection.\textsuperscript{{[}25{]}} Our previous study
showed that intravenously injected multicellular spheroids of
adenocarcinoma cells with a diameter of approximately 170 \selectlanguage{greek}μ\selectlanguage{english}m effectively
formed tumor colonies in the lung compared to suspended
cells.\textsuperscript{{[}14{]}} Therefore, m17.ASC spheroids of
approximately 170 \selectlanguage{greek}μ\selectlanguage{english}m diameter were prepared in this study (Table 1), and
intravenously injected m17.ASC spheroids efficiently accumulated and
remained in the lung.

LPS-induced inflammatory mouse models are frequently used as systemic
inflammatory disease models through endotoxin shock, which causes an
increase in the release of inflammatory cytokines such as IL-6 in the
plasma and multiple organ failures.\textsuperscript{{[}26{]}} Many
reports have demonstrated that MSC administration effectively suppresses
inflammation and shows a therapeutic effect in LPS-induced inflammatory
mouse models.\textsuperscript{{[}27,28{]}} Compared to m17.ASC cells,
m17.ASC spheroids more efficiently suppressed the plasma levels of
inflammatory cytokines in LPS-induced inflammation mouse models (Figure
5). This result reflects the prolonged survival of the m17.ASC spheroids
after intravenous injection (Figure 4).

In conclusion, we demonstrated that spheroid formation of m17.ASC cells
improved the delivery efficiency of the cells to the lung and prolonged
their survival after intravenous injection. Additionally, compared to
suspended cells, the m17.ASC spheroids showed excellent therapeutic
effect in the inflammatory mouse models.

\textbf{Acknowledgments}

The authors are grateful to Editage (www.editage.com) for English
language editing. This work was supported in part by a GSK Japan
Research Grant 2017.

\textbf{Conflict of interest}

The authors declare no competing interests.

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\textbf{Figure 1.}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}



\textbf{Figure 2.}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}



\textbf{Figure 3.}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}



\textbf{Figure 4.}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}



\textbf{Figure 5.}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Table 1. Characteristics of m17.ASC spheroids}\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
Time (h) & Diameter (\selectlanguage{greek}μ\selectlanguage{english}m) & Cell number/spheroid & Viability
(\%)\tabularnewline
24 & 173 \selectlanguage{ngerman}± 16 & 1,110 \selectlanguage{ngerman}± 90 & 94.5 \selectlanguage{ngerman}± 0.7\tabularnewline
48 & 168 \selectlanguage{ngerman}± 23 & 1,300 \selectlanguage{ngerman}± 90 & 93.3 \selectlanguage{ngerman}± 2.7\tabularnewline
72 & 173 \selectlanguage{ngerman}± 21 & 1,170 \selectlanguage{ngerman}± 60 & 94.6 \selectlanguage{ngerman}± 0.8\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

\textbf{Figure legends}

\textbf{Figure 1.} Preparation of m17.ASC spheroids. Macroscopic images
of (A) micropillar arrays and (B) agarose-based microwell plate. Scale
bars indicate 500 \selectlanguage{greek}μ\selectlanguage{english}m. (C) Top and (D) cross-sectional images of
agarose-based microwell plate by microscopic observation. Scale bars
indicate 300 \selectlanguage{greek}μ\selectlanguage{english}m. (E) A typical image of m17.ASC spheroid after 24-h
incubation. Scale bar indicates 50 \selectlanguage{greek}μ\selectlanguage{english}m.

\textbf{Figure 2.} Size histogram of m17.ASC spheroids. m17.ASC
spheroids were incubated for (A) 24 h, (B) 48 h, and (C) 72 h after
seeding cells in an agarose-based microwell plate. The diameters of 100
spheroids were measured using a BZ-9000 microscope.

\textbf{Figure 3.} Characteristics of m17.ASC spheroids. (A) TGF-\selectlanguage{greek}β
\selectlanguage{english}secretion from m17.ASC spheroids. Monolayered m17.ASC cells (1.25 \selectlanguage{ngerman}×
10\textsuperscript{4} cells) or m17.ASC spheroids (11 spheroids: 1.25 ×
10\textsuperscript{4} cells) were seeded into a 12-well culture plate.
After 24- and 48-h incubation, the amount of TGF-\selectlanguage{greek}β \selectlanguage{english}in the supernatant
was measured using an ELISA kit. (B) \emph{Arg1} and
\emph{Ym1}expression in peritoneal macrophages after co-culture with
m17.ASC spheroids. Monolayered m17.ASC cells (1 \selectlanguage{ngerman}× 10\textsuperscript{5}
cells) or m17.ASC spheroids (90 spheroids: 1 × 10\textsuperscript{5}
cells) were seeded into the upper wells of a 12-well Transwell plate,
and 1 × 10\textsuperscript{5} peritoneal macrophages were seeded into
the lower wells. After 24-h incubation, mRNA (\emph{Arg1} and \emph{Ym1}
) expression in peritoneal macrophages was evaluated using real time
RT-PCR. Results are normalized to mRNA levels of \emph{GAPDH} . Data are
expressed as the mean ± SD of three to four experiments.

\textbf{Figure 4.} Biodistribution of m17.ASC/Nluc spheroids after
intravenous injection. (A) Relative light units of m17.ASC/Nluc
spheroids in the plasma and tissues. m17.ASC/Nluc spheroids (135
spheroids: 1.5 × 10\textsuperscript{5} cells/100 \selectlanguage{greek}μ\selectlanguage{english}L/mouse) or suspended
m17.ASC/Nluc cells (1.5 \selectlanguage{ngerman}× 10\textsuperscript{5}cells/100 \selectlanguage{greek}μ\selectlanguage{english}L/mouse) were
intravenously injected in mice. Blood and tissues were collected at 24 h
after intravenous injection, and relative light units of the plasma and
supernatants of homogenized tissues were measured. (B) The numbers of
m17.ASC/Nluc spheroids remaining in the lung. m17.ASC/Nluc spheroids
(100 spheroids: 1.5 \selectlanguage{ngerman}× 10\textsuperscript{5}cells/mouse) or suspended
m17.ASC/Nluc cells (1.5 × 10\textsuperscript{5} cells/mouse) were
intravenously injected in mice. Lungs were collected at 6, 24, 48, 120,
and 168 h after intravenous injection. The numbers of cells remaining in
lungs were calculated by measuring the relative light units. (C) Typical
images of CFSE-labeled m17.ASC spheroids in the lung. Suspended m17.ASC
cells (1 × 10\textsuperscript{5} cells) or m17.ASC spheroids (90
spheroids: 1 × 10\textsuperscript{5} cells) were labeled with CFSE.
Lungs were collected from euthanized mice 24 h after intravenous
injection of cells. Cryosections of the lung were observed using a
BZ-9000 microscope. Scale bar indicates 100 \selectlanguage{greek}μ\selectlanguage{english}m. Data are expressed as
the mean \selectlanguage{ngerman}± SD of three to four experiments. *\emph{P} \textless{} 0.05;
statistically significant differences compared with suspension group.

\textbf{Figure 5.} Effect of m17.ASC spheroids on LPS-induced
inflammatory mouse model. LPS was intraperitoneally administered to mice
at 1 mg/kg. The mice were then bred on a hot plate (40 °C) for 1 h.
m17.ASC spheroids (850 spheroids: 1 × 10\textsuperscript{6} cells/100
\selectlanguage{greek}μ\selectlanguage{english}L/mouse) and suspended m17.ASC cells (1 \selectlanguage{ngerman}×
10\textsuperscript{6}cells/100 \selectlanguage{greek}μ\selectlanguage{english}L/mouse) were intravenously injected.
Blood was collected at 6 and 10 h after injection of cells. The (A) IL-6
and (B) TNF-\selectlanguage{greek}α \selectlanguage{english}levels in plasma was measured using ELISA kits. Data are
expressed as the mean \selectlanguage{ngerman}± SD of three to four experiments. *\emph{P}
\textless{} 0.05; statistically significant differences compared with
the PBS group.

\selectlanguage{english}
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\end{document}

