micromachines
ArticleNovel Platform for Regulation of Extracellular Vesicles andMetabolites Secretion from Cells Using a Multi-Linkable
Horizontal Co-Culture Plate
Takeo Shimasaki 1,2,3, *, Satoko Yamamoto 1 , Risa Omura 1 , Kagenori Ito 4,5 , Yumiko Nishide 6 , Hideki Yamada 6 ,Kazumi Ohtomo 6 , Tomo Ishisaka 7 , Keiichiro Okano 7 , Takenori Ogawa 8 , Hiroyuki Tsuji 7 , Yoichi Matsuo 9 ,Toshinari Minamoto 2 , Naohisa Tomosugi 1 , Etienne Ferain 10 and Takahiro Ochiya 5,11
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5

6

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Citation: Shimasaki, T.; Yamamoto,

7

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S.; Omura, R.; Ito, K.; Nishide, Y.;
Yamada, H.; Ohtomo, K.; Ishisaka, T.;

9

Okano, K.; Ogawa, T.; et al. Novel
Platform for Regulation ofExtracellular Vesicles and Metabolites

10
11

Secretion from Cells Using a
Multi-Linkable Horizontal*

Medical Research Institute, Kanazawa Medical University, Uchinada 920-0293, Japan;s-yama@kanazawa-med.ac.jp (S.Y.); r-omura@kanazawa-med.ac.jp (R.O.);
tomosugi@kanazawa-med.ac.jp (N.T.)
Division of Translational and Clinical Oncology, Cancer Research Institute, Kanazawa University,
Kanazawa 920-0934, Japan; minamoto@staff.kanazawa-u.ac.jp
Ginrei Lab Inc., Uchinada 920-0293, Japan
Department of Urology, Jikei University School of Medicine, Tokyo 105-0003, Japan; ito.kagenori@gmail.com
Division of Cellular Signaling, National Cancer Center Research Institute, Tokyo 104-0045, Japan;
tochiya@tokyo-med.ac.jp
Shinko Chemical Co., Ltd., Kanazawa 920-0346, Japan; master@shinko-ccl.jp (Y.N.);
hyamada@shinko-ccl.jp (H.Y.); ohtomo@shinko-ccl.jp (K.O.)
Department of Head and Neck Surgery, Kanazawa Medical University, Uchinada 920-0293, Japan;
tomo-n@kanazawa-med.ac.jp (T.I.); okano-k@kanazawa-med.ac.jp (K.O.); tsujih-i@kanazawa-med.ac.jp (H.T.)
Department of Otolaryngology-Head and Neck Surgery, Gifu University Graduate School of Medicine,
Gifu 501-1194, Japan; ogawa@gifu-u.ac.jp
Department of Gastroenterological Surgery, Nagoya City University Graduate School of Medical Sciences,
Nagoya 467-8601, Japan; matsuo@med.nagoya-cu.ac.jp
it4ip S.A., 1348 Louvain-la-Neuve, Belgium; ferain@it4ip.be
Department of Molecular and Cellular Medicine, Institute of Medical Science, Tokyo Medical University,
Tokyo 160-0023, Japan
Correspondence: takeo@kanazawa-med.ac.jp; Tel.: +81-762-218-8312

Co-Culture Plate. Micromachines 2021,
12, 1431. https://doi.org/10.3390/
mi12111431
Academic Editor: Ivan Cimrák
Received: 17 September 2021
Accepted: 16 November 2021
Published: 21 November 2021

Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims inpublished maps and institutional affiliations.

Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.

Abstract: Microfluidics is applied in biotechnology research via the creation of microfluidic channels
and reaction vessels. Filters are considered to be able to simulate microfluidics. A typical example isthe cell culture insert, which comprises two vessels connected by a filter. Cell culture inserts havebeen used for years to study cell-to-cell communication. These systems generally have a bucket-inbucket structure and are hereafter referred to as a vertical-type co-culture plate (VTCP). However,
VTCPs have several disadvantages, such as the inability to simultaneously observe samples in bothcontainers and the inability of cell-to-cell communication through the filters at high cell densities.
In this study, we developed a novel horizontal-type co-culture plate (HTCP) to overcome thesedisadvantages and confirm its performance. In addition, we clarified the migration characteristics
of substances secreted from cells in horizontal co-culture vessels. It is generally assumed that lessmaterial is exchanged between the horizontal vessels. However, the extracellular vesicle (EV) transfer
was found to be twice as high when using HTCP. Other merits include control of the degree of coculture via the placement of cells. We believe that this novel HTCP container will facilitate researchon cell-to-cell communication in various fields.
Keywords: co-culture plate; multiple connection; horizontal co-culture; extracellular vesicles;
exosome; track-etched-membrane; metaboliteThis article is an open access article
distributed under the terms andconditions of the Creative Commons
Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

1. Introduction
Systems that utilize microstructures, such as microfluidic channels, are being increasingly applied in biotechnology research. Cell culture inserts, which are used for theMicromachines 2021, 12, 1431. https://doi.org/10.3390/mi12111431

https://www.mdpi.com/journal/micromachines

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co-culture of cells, have utilized microstructures for many years. The trademarked system“Transwell® ” is a well-known co-culture vessel that comprises two containers that arestacked vertically. The upper container is composed of a filter at the bottom through which
the substances in the two containers can interact. This structure is essentially the same as a
microfluidic machine because micro-materials that are secreted by the cells pass through afilter or microfluidic channel to affect distant cells. Thus, co-culture research is one of themost relevant fields in which microstructures have been applied.
Recently, several co-culture methods have emerged, and they can be broadly classified
into two types [1]: (1) direct co-culture, in which cells are mixed in a single well without
separation, and (2) indirect co-culture, in which two different cell types are cultured simultaneously and isolated from each other using a separation mechanism. Although direct
co-culture is more commonly employed, the use of this approach to analyze individual
cells is complicated. In contrast, indirect co-culture involves placing the cells in different
environments, and cell–cell interactions are mediated via humoral factors, meaning that it
is easier to observe the cells because they are not mixed. Indirect co-culture systems aremainly classified into three types: (1) separation by filters, (2) separation by a semi-solidmaterial such as a gel, with humoral factors exchanged via the gel, and (3) the formationof separate cell colonies or layers by culturing, in which complete isolation of cell types
is not possible [1]). A typical example of filter separation is a cell culture insert, which ischaracterized by a bucket-in-a-bucket structure that is constructed vertically. This type of
co-culture plate is therefore referred to as a vertical-type co-culture plate (VTCP).
The most popular type of VTCP is the Boyden chamber, which was developed by
Stephan Boyden in 1962 [2]. It was initially designed to study cell invasion by counting thenumber of cells that passed through a filter [3]. In the Boyden chamber co-culture method,
the culture fluids at the top (on the insert) and the bottom (on the bottom dish) of a system
are shared for cell–cell interaction studies. This type of VTCP is generally referred to as a“Transwell” style [4–6] or “cell culture insert” type [7].
VTCPs have long been used as the standard for co-culture, and this technique has notchanged fundamentally because of the difficulty in making novel vessels. When using thisdesign, it is expected that cells will grow on an insert, and that the liquid factors will be
exchanged with the cells in the lower wells through the filter. However, this is not always
achieved, mainly because of the fundamental problems associated with high cell densities.
There are three main issues when using VTCPs:
1.

2.

3.

The top container is invisible: Neither cell line could be simultaneously observed
under a microscope. The cells in the culture vessel are usually observed from thebottom of the vessel because the culture fluid hinders observation from the top of thevessel. The upper container cannot be observed because of the short focal length of the
microscope. Therefore, cells were only observed in the lower parts of the VTCP-type
vessels (Figure S1a).
Different materials could impact the results: The cells are not cultured on the same
type of material surface: one group of cells is cultured on the filter material and theother on a plastic material; this difference could significantly impact the experimental
results (Figure S1b).
High cell densities are not considered: A high density of cells may prevent co-culture
due to blockage of the filter. As the Boyden chamber was initially invented to evaluate
cell invasion [2,8], scenarios in which cells were present at high densities were notconsidered (Figure S1c).

To overcome these problems and facilitate studies on cell–cell communication, wedeveloped a novel horizontal-type co-culture plate (HTCP), as shown in Figure 1. This
design is based on recent advances in three-dimensional computer-aided design technology,
which offers novel possibilities for creating different culture vessels. We hypothesized thata horizontal system would solve the problems associated with VTCPs; it would enable
simultaneous observation of cells in both containers using an inverted microscope, throughdesign is based on recent advances in three-dimensional computer-aided design technology, which offers novel possibilities for creating different culture vessels. We hypotheMicromachines 2021, 12, 1431 sized that a horizontal system would solve the problems associated with VTCPs; it would 3 of 19
enable simultaneous observation of cells in both containers using an inverted microscope,
through which a variety of cellular interactions and cell morphologies during the maturation whichof eachacellline canbe observed.
varietyof cellularinteractions and cell morphologies during the maturation ofeach cell line can be observed.

Figure 1. Redesigning the vertical-type co-culture plate to produce a horizontal-type co-culture
plate (HTCP).
Figure 1. Redesigning the vertical-type co-culture plate to produce a horizontal-type co-culture
plate (HTCP).
In addition, researchers can freely choose from a variety of commercially available

filters that are 13 mm in diameter to control the sharing factors of the liquid media when
Inusing
addition,
researchers
can afreely
a varietycommercially
HTCP,
meaning thatwide choose
variety fromof filter
optionsofcanbe used withavailable
the system.
filters that are
13
mm
indiameter
tocontrolthesharingfactorsoftheliquidmedia
Intercellular communication has traditionally been considered to involvewhen
the interacusing HTCP,
meaningwide variety
of filter
options
can be used
the system.
tion of cells
withthat
theira neighbors
throughdirectinteractions
thatwith
are mediatedby soluble
Intercellular
has traditionallyconsidered
involvethe interacmolecules, communicationsuch as cell-secreted
cytokines. been
However,recentto
studieshave
revealed thattion ofExtracellularcells with theirneighbors
direct interactions
that arecommunication
mediated by soluble
vesicles(EVs)throughplay an importantrole in cell–cell[9,10]. Johnmolecules,
cell-secreted
cytokines.
However,recenthave revealed[12,13]that Exstonesuch
et al.as[11]first discovered
vesiclessecreted
bystudies
sheep reticulocytes
in 1987,
tracellular
vesicles(EVs) play annamed
importantrole inExosomescell–cell communication[9,10].
Johnwhichwere subsequently
exosomes.
are a type of EVs
that aresecretedstone et[11]cells
firstanddiscovered
vesiclessecreted
sheep reticulocytes
in 1987,
byal.
many
are present
in body
fluids,by
includingblood, urine,[12,13]
and saliva.
Althoughwhich theweresubsequently
named
exosomes.
Exosomes
are
a
type
of
EVs
that
are
secreteddetailed features of exosomes remain unknown, the discovery of microRNA and RNA
by many
cellsinandare present
in body fluids,
including blood,urine, andgenetic
saliva. transmission
Although bein EVs
2006–2007
demonstrated
the possibility
that informative
the detailed
exosomesremain unknown,
the then,discovery
microRNAhave
andturned
RNA their
tweenfeatures
cells mayofoccur
via exosomes[14,15]. Since
manyofresearchers
in EVsattention
in 2006–2007
demonstrated
thehavepossibility
that informativetransmission
to EVs,and exosomes
since become
a hot topicgenetic
of study
in various fields.
EVs
alsomay
includelarger plasma
microvesicles
apoptotic
betweencells
occurslightly
via exosomes[14,15]. membrane-derived
Since then, many researchers
haveandturnedbodies thatare released
during cell
apoptosis[16]. Although
theoflackof ainclear
definition
their attention
to EVs,and exosomes
havesince become
a hot topic
study
variousconfusion
among
researchers,
the description of an
exosome asandan EV
has beenfields. has
EVsledalsotoinclude
slightlylargerplasma membrane-derived
microvesicles
apopdriventhat
mainlyby the International
Society for[16].
Extracellulararecurrently
totic bodies
are released
during cell apoptosis
AlthoughVesicles
the lack[17].
of aEVs
clear
defithree maingroups:(1) exosomesthat areof50–150
nm inasdiameterand arenition divided
has led tointo
confusion
among
researchers,
the description
an exosome
an EV hasboundmainly
by lipidbybilayer
membranes,Society(2) microvesicles
that are
100–1000
inare
size andbeen driven
the International
for Extracellular
Vesicles[17].nmEVs
are divided
directly secreted
cells,groups:and (3) (1)apoptotic
bodiesarise from
apoptosis [16].
currently
into threebymain
exosomes
that thatare 50–150
nm cellin diameter
Since
the
precisedefinitionofexosomesremains
elusivebecauseofthelack
ofsizestandard
and are bound by lipid bilayer membranes, (2) microvesicles that are 100–1000 nm inprotocolsfor
identifying
anddistinguishing
exosomesandmicrovesicles,
exosomes
and are directly secreted by cells, and (3) apoptotic bodies that arise from cell apoptosis haverecently
begun definition
to be referred
to as EVs.
Althoughexosomesvia
exocytosis[16]. Since
the preciseof exosomes
remains
elusivebecauseareof released
the lack ofstandandmicrovesicles
arereleasedvia
plasmamembraneshedding,
EVs
are
an
appropriate
ard protocols for identifying and distinguishing exosomes and microvesicles, exosomes
term to begun
describe
becausedifferent
typesexosomesof vesiclesare
cannotbe distinguishedbyhave recently
to bothbe referredto theas EVs.
Althoughreleased
via exocyultracentrifugation.
tosis and microvesicles are released via plasma membrane shedding, EVs are an approit becameboth
clear
that extracellularplay
an essential
roledistinguishedin communication
priate termAs
to describe
becausethe differentvesicles
types ofvesiclescannot be
betweencells,
itbecamenecessary
toanalyzethemseparately.
The
EVs
can be separated
by ultracentrifugation.
from
cells
using
three
main
methods
[18]:
(1)
the
ultracentrifugation
methodthat uses
As it became clear that extracellular vesicles play an essential role in communication
weightdifferences,
(2)
using
the
properties
ofthesurfaceproteinsonextracellular
vesicles,between cells, it became necessary to analyze them separately. The EVs can be separated
and
(3)
using
the
difference
in
the
sizes
of
the
EVs.
The
size-separation
methodfrom cells using three main methods [18]: (1) the ultracentrifugation method that uses is thesuitable (2)methodwith microfluidic
devicesand requires
either a filter
weightmost
differences,usingfortheusepropertiesof the surface
proteins
on extracellularvesi- or the
fabrication of tiny pores in the device to enable size separation. The problem with thiscles, and (3) using the difference in the sizes of the EVs. The size-separation method is themethod is that extracellular vesicles are extremely small, with exosomes reaching only 50
most suitable method for use with microfluidic devices and requires either a filter or theto 150 nm in size. Track-etching technology is ideally suited for producing filters withuniform pores. Track-etched membrane filters were fabricated by irradiating a raw polymer
film with high-energy heavy ions, creating damaged linear tracks across the film, whichwere later converted into pores by selective wet chemical etching (Figure S2a). Etchingconditions can be adjusted to obtain uniform pores with a precise diameter adapted toMicromachines 2021, 12, 1431

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separate the extracellular vesicles (Figure S2b). In addition, for the efficient filter, it isnecessary to prevent substances from leaking across the pores in the filter. VTCPs are madeby molding the culture vessel and filtering together, so that the filter cannot be replaced. In
the HTCP, an O-ring is used to sandwich the filters, meaning that it is possible to replace thefilter. We are the first group to our knowledge, to commercialize containers with multiple
horizontal connections. HTCP has been reported in several studies [19,20]. However, themigration characteristics of substances secreted by HTCP cells have not yet been reported.
The novel HTCP vessel is expected to help advance cell co-culture research, including
shedding new light on the role of EVs as messengers that can deliver signals between cells.
Here, we describe the design of a HTCP system as a new type of co-culture vessel. We
also describe the results of experiments conducted to evaluate the feasibility of exchanging
liquid factors using a track-etched membrane in comparison to that of using a VTCP. The
functional ability of the track-etched membrane to separate EVs was examined to elucidate
the influence of EVs on co-cultures.
2. Materials and Methods
2.1. Device Design and Fabrication

Micromachines 2021, 12, 1431

The design of the HTCP, its assembly, size information, and how to use it are depicted in Figure 2 and Figures S3–S5. All components comprising the HTCP were designed using Rhinoceros 6. Available online: https://www.rhino3d.co.jp/ (accessed on
14 November 2021). The products were prepared via injection molding by Shinko ChemicalCo., Ltd. The design aims to achieve an easy transition from a monoculture to co-cultures,with or without an optional filter, and the option
to culture separately with the possibility
5 of 20of transitioning to the co-culture mode at any time.

Figure 2. Design and description of the horizontal-type culture plate (HTCP). The culture vessel canbe employed in two ways. (a) Initial monoculture mode. Bodies A and B are designed to allow theuse of separated cell culturing. (b) Co-culture mode by connecting the two containers (combined
state). In the connected mode, the two containers are joined from the beginning of cell culture. The
height (volume) of the culture medium is used to control the amount of liquid that traverses the twocontainers,
controllingthe extentavailableof co-culturing.
(c)Co.,
Body
This product (HTCP)
is already commerciallythrough AR Brown
Ltd. C is used for multiple connections, with a(Tokyo, Japan), Fujifilm Wako Pure Chemicals Corporation (Tokyo, Japan), Blast Inc. (Kacouplingmechanism
on
both
the
left
and
the
right
sides
wasaki, Japan), AdvantigenBioScience L.L.C. (Madison, USA), Bulldog Bio. Inc. (Ports-of the container. How to use are depicted inmouth, USA), Xceltis GmbH (Mannheim, Germany), and 1st PhileKorea Inc. (Seoul, KoFigure S4. (d) Actual pictures.

Figure 2. Design and description of the horizontal-type culture plate (HTCP). The culture vessel canbe employed in two ways. (a) Initial monoculture mode. Bodies A and B are designed to allow theuse of separated cell culturing. (b) Co-culture mode by connecting the two containers (combined
state). In the connected mode, the two containers are joined from the beginning of cell culture. The
height (volume) of the culture medium is used to control the amount of liquid that traverses the twocontainers, controlling the extent of co-culturing. (c) Body C is used for multiple connections, witha coupling mechanism on both the left and the right sides of the container. How to use are depicted
in Figure S4. (d) Actual pictures.

rea). The product is named “ICCP,” “NICO-1,””UniWells,” and “Pair-N-Share Tandem
Co-Culture Wells & Plates”, but the product itself is the same.

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This product (HTCP) is already commercially available through AR Brown Co.,
Ltd. (Tokyo, Japan), Fujifilm Wako Pure Chemicals Corporation (Tokyo, Japan), Blast
Inc. (Kawasaki, Japan), AdvantigenBioScience L.L.C. (Madison, WI, USA), Bulldog Bio.
Inc. (Portsmouth, NH, USA), Xceltis GmbH (Mannheim, Germany), and 1st PhileKorea Inc.
(Seoul, Korea). The product is named “ICCP”, “NICO-1”, “UniWells”, and “Pair-N-Share
Tandem Co-Culture Wells & Plates”, but the product itself is the same.
2.2. Cell Lines
Human pancreatic cancer PANC-1 and AsPC-1 cells were obtained from RIKEN
BRC (Tsukuba City, Japan). The cell lines were passaged for fewer than six months after
resuscitation, cultured in complete Dulbecco’s modified Eagle medium (DMEM)-highglucose with 1% L-glutamine, 1% sodium pyruvate, 10% fetal bovine serum, and 1%
penicillin-streptomycin (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), andmaintained at 37 ◦ C with 5% CO2 in DMEM. To establish stable pancreatic cancer cell lines
expressing CD63-GFP (gPANC1) and CD63-RFP (rPANC1), cells were transfected with thelentiviral vectors pCT-CD63-GFP and pCT-CD63-RFP (pCMsV, exosome/secretory, CD63
tetraspanin tag, System Biosciences, CYTO120-PA-1, CYTO120R-PA-1), respectively, asreported previously [21]. Supernatants containing lentiviral particles obtained from thegPANC1 cell line and transduced with pCT-CD63-GFP were filtered through a 0.6-µm filter
and then applied for EV isolation.
2.3. Microscopy
Microscopic images were acquired using the EVOS system (Thermo Fisher Inc.,
Waltham, MA, USA) to confirm cell conditions and obtain fluorescent images. Excitation
wavelengths of 488 nm and 561 nm were used to detect GFP fluorescence (gPANC1) and
RFP fluorescence (rPANC1), respectively.
2.4. Cell Viability
According to the manufacturer’s instructions, cell viability was analyzed using theSuperoxide Dismutase (SOD) Assay Kit-WST (FUJIFILM WAKO Pure Chemical Corporation, Osaka, Japan. S311). In brief, WST reagent (10 µL) was added to each well, and thesample was incubated for 45 min at 37 ◦ C. Absorbance was measured at 450 nm (A450)
and 620 nm (A620). The value obtained by subtracting A620 from A450 was used as theviable cell number.
2.5. Nanoparticle Tracking Analysis
Nanoparticle tracking analysis was used to visualize and measure the size of small
particles (10–1000 nm) in the suspension based on Brownian motion analysis from a video
sequence. We used the NanoSight system (NS300, Malvern Instruments Ltd., Malvern,UK) to detect EVs. The measurements were performed at least thrice; the mean value andstandard error were calculated and are represented in the graph.
2.6. EV Isolation
EVs were isolated using the MagCapture™ Exosome Isolation Kit PS 293-77601 (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) as described previously [22].
2.7. Whole-Cell Protein Extracts
Cells were lysed with radioimmunoprecipitation (RIPA) buffer (FUJIFILM Wako Pure
Chemical Corporation, Osaka, Japan) composed of 50 mM Tris-HCl (pH 7.4), 1% NP-40,
0.5% sodium deoxycholate (SDC), 0.1% sodium dodecyl sulfate (SDS), 150 mM NaCl, anda protease inhibitor cocktail (FUJIFILM WAKO Pure Chemical Corporation, Osaka, Japan).
Supernatants obtained after centrifugation at 15,000× g for 10 min at 4 ◦ C were used aswhole-cell proteins.

Micromachines 2021, 12, 1431

6 of 192.8. Measurement of Glucose, Lactate, and Ammonium Ion Concentrations
The culture supernatant was filtered at each growth phase through a 0.22 µm membrane and directly analyzed with a BioProfile FLEX2 analyzer following the manufacturer’s instructions (Nova Biomedical, Inc., Waltham, MA, USA). The measurements were
performed at least thrice; the mean value and standard error were calculated and are
represented in the graph.
2.9. Protein Assay and Western Blotting
The protein was resuspended in 5× RIPA buffer (125 mM Tris-HCl, 750 nM NaCl,
5% NP-40, 5% SDC, and 0.5% SDS), after which the suspension was sonicated for 5 min
and incubated for 15 min on ice. Protein extract concentrations were measured using
Coomassie Protein Assay Reagents (Pierce, Thermo Fisher Scientific, Waltham, MA, USA).
A 20 µg aliquot of protein was separated by SDS-polyacrylamide gel electrophoresis andanalyzed by Western blotting to detect the proteins with anti-flotillin-1 (Cell Signaling
Technology 13174), anti-CD-9 (CosmoBio Japan SHI-EXO-M01), anti-CD-81 (CosmoBio
Japan, SHI-EXO-M03), and anti-CD-63 (BD-BioScience, Franklin Lakes, NJ, USA, 556019),
followed by incubation with the secondary antibodies goat anti-mouse IgG (Thermo Fisher
Inc., #31430), and anti-rabbit IgG (Thermo Fisher Inc., #31460). Electroblotted membranes(Amersham) were blocked with 5% bovine serum albumin (BSA). The expression of β-actin
was used as a reference for the amount of protein in each cell sample. The signals were
measured using enhanced chemiluminescence.
2.10. Silver Staining
The volume of proteins that passed through the filter was determined using a silver
staining kit (FUJIFILM Wako Pure Chemical Inc., Osaka, Japan) according to the manufacturer’s instructions.
3. Results
3.1. Device Design and Description
The HTCP consists of four main parts: the top cover, O-ring, sidewall cover, andBody A or Body B, as detailed in Figure 2. Along with these, either a stand-alone structure
for separate culture (Figure 2a) or a connected vessel that allows the same medium to beshared by different cell types through the filter (Figure 2b) can be established for co-culture.
Multiple connections are also possible by using a Body C structure, which has a couplingmechanism on both the left and right sides of the container (Figure 2c), and it can allow
three, four, or multiple connections. The stand-alone mode is used for culturing under
different conditions, such as high oxygen and low oxygen, high temperature and low
temperature, or with different chemical materials. Each well is designed to hold 1.8 mLof culture medium. The outer dimensions with slide glass size adaptors are the same as
those of a standard microscope slide glass, allowing for simple time-lapse microscopic
observations. We also designed a 96-well adapter that can fit into the four parts of the HTCP
that allows multi-screening to be conducted with a microscope, enabling simultaneous
observation of both cell types under different conditions and high-resolution imaging(Figure 2d).
The two containers, Body A and Body B were tightly coupled via the insert guideto prevent leakage. The claw in Body B is structured to constrict Body A tightly past theconvex portion of the container (Figure 3a). The O-ring sandwiched between the containers
at the junction of Body A, and Body B (Figure 3b) can prevent leakage even when a filter is
not used.
The HTCP can be used in two ways (Figure S4): the separated culture method (A),where Body A and B are used independently and are only combined during the co-culture
step, and the first assembly process (B), in which the state of the co-culture is regulated bycontrolling the volume of the culture solution. The container has a structural limitation soMicromachines 2021, 12, 1431

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8 of 20that co-culturing cannot occur if less than 400 µL of culture solution is used, meaning thatthe co-culture state can be achieved at any time by increasing the volume of culture liquid.

Figure 3. The mechanism used for leakage prevention and the methods used to prevent it. The
Figure3. The mechanism
forB)leakage
prevention
used
prevent
it. The concontainers(Body A andused
Body
are tightly
coupledand
via thethe methods
insert guide
toto
prevent
leakage.
(a) Thetainers (Body A and Body B) are tightly coupled via the insert guide to prevent leakage. (a) The claw
claw in the container of Body B is structured to constrict Body A tightly past the convex portion ofin the container of Body B is structured to constrict Body A tightly past the convex portion of thethe container. (b) The O-ring at the junction of Body A and Body B prevents leakage.
container. (b) The O-ring at the junction of Body A and Body B prevents leakage.

The use of a filter allows the liquid factors to move without mixing the cells; however,
The HTCP can be used in two ways (Figure S4): the separated culture method (A),the nature of the filter may cause problems such as adsorption of the liquid factor. Therefore,
where Body A and B are used independently and are only combined during the co-culture
we conducted initial experiments without a filter to confirm the subsequent effects ofstep, and the first assembly process (B), in which the state of the co-culture is regulated byabsorption by the filter (Figure S5a). As the bottom of the HTCP is not composed of thecontrollingthe volume of the culture solution. The container has a structural limitation so
filter material, three-dimensional culturing is also possible, along with the use of gel andthat
co-culturing
occur
less
than 400
μL of culture
solutionmeaningthatcoating (Figurecannot
S5b). Oneof if
theessential
advantagesof thisdesignisisused,that because
thefiltertheisco-culture
state
can
be
achieved
at
any
time
by
increasing
thevolumeofcultureliquid.
located away from the cells, the pores in the filter are not likely to be obstructed by cells,
Theatuseof acellfilterallows the liquid factors to move without mixing the cells; however,
even
high
densities.
the nature of the filter may cause problems such as adsorption of the liquid factor. Therefore,
conducted Design
initial experiments without a filter to confirm the subsequent effects
3.2.weExperimentalof absorption
bythe(FigureS5a). As thebottom of the HTCP is not composed of the
The Experimental filter
Designfor Evaluating
HTCP
filter material,
three-dimensional
culturing
also
along withthe usebyof cells
gel and
There areno previous studies
on the israte
at possible,
which metabolites
secretedmove
coating(Figure
S5b).
One
of
the
essential
advantagesofthisdesignisthatbecausethehorizontally between vessels. The soluble substances secreted by the cells in the medium
filterlocatedtoaway
fromthe cells,
theviaporesin the filter
are not likely
to beareisthought
move
betweenvessels
diffusion.
Extracellular
vesiclesareobstructedalso thoughtby tocells,
even
at
high
cell
densities.
move between vessels via Brownian motion due to their size. However, dependingon the cellular factors, the actual situation may differ. The passing ratio of metabolites
3.2.throughExperimental
Designthe filter
was not clear. The amounts of extracellular vesicles passing through are
The
Experimental
Design for itEvaluating
HTCP they move between vessels, in theory, evenalso
unclear. Furthermore,is unclear whether
in There
the presenceof cells. studies on the rate at which metabolites secreted by cells moveare no previous
We believe
that
it will The
be usefulto substances
evaluate thesubstancetransfer
properties
using
horizontally
betweenvessels.
solublesecretedby thecells inthe medium
actual
cells
and
HTCP
for
advancingthemicromachinetechnology.
Therefore,
we
devisedare thought to move between vessels via diffusion. Extracellular vesicles are also thought
an experimental
design,in Figureoverview.
First, wemeasuredonthe
to movebetween vessels
viashown
BrownianmotionS6,
dueastoan
theirsize. However,dependingmigration
properties
ofsubstancesandextracellular
vesiclesthroughthefilter without
the cellular factors, the actual situation may differ. The passing ratio of metabolites

through the filter was not clear. The amounts of extracellular vesicles passing through areMicromachines 2021, 12, 1431

We believe that it will be useful to evaluate the substance transfer properties using
actual cells and HTCP for advancing the micromachine technology. Therefore, we devised
an experimental design, shown in Figure S6, as an overview. First, we measured the migration properties of substances and extracellular vesicles through the filter without cells(Figure 4). Second, the concentration and mass transfer produced by the cells were
8 of evalu19
ated (Figure 5). Third, the filter permeability of exosomes was investigated (Figure 6).
Fourth, we evaluated the differences between VTCPs and HTCPs in terms of extracellular
vesicle transfer (Figure 7). Finally, microscopic observation of exosomes passing throughcells (Figure 4). Second, the concentration and mass transfer produced by the cells were
the filterand (Figure
being taken
up bycells ofwas
performed.
Taking an (Figure
advantageevaluated
5). Third,
theneighboring
filter permeability
exosomes
was investigated6). ofthe characteristics
ofthethree-connectedvessel,imageswereobtainedshowing
that
Fourth, we evaluated the differences between VTCPs and HTCPs in terms of extracellular exosomes
taken
up fromcells
in boththe left andright vessels
werepassing
fused throughand processedvesicletransfer(Figure7). Finally,
microscopic
observationof exosomesthefilter and being taken up by neighboring cells was performed. Taking an advantage of thetogether.
characteristics of the three-connected vessel, images were obtained showing that exosomes
taken up from cells in both the left and right vessels were fused and processed together.

Figure 4. The results of glucose transfer without cells. Orange-colored wells with clip art of bottle
were filled with medium including glucose. The remaining wells were filled with phosphate-bufferedFigure4. Theresultstransfer
without
Orange-coloredwells
clip art wasof bottle
saline(PBS),
whichofdidglucose
not containglucose.
Whencells.
all wells
were mixed, thefinal withconcentration
wereconverted
filled withmediumThe
remainingwells werefilledwithconcentration
phosphate-buffto 100%.
The including
vertical axisglucose.
shows theconverted
concentrationin %.
Glucoseeredinsaline(PBS), which
did not containglucose.
When
all wells
mixed, the final
the medium
was measured
after 6–72
h. Theverticalaxis iswere
the concentration(%),concentrationand thewas horizontal
convertedaxis
to 100%.
The
vertical
axis
shows
the
converted
concentrationin%.
Glucoseis the time (h). The filter pore size is 0.6 µm (a–c) or 0.03 µm (d,e). (a) In caseconcenof
trationinthemediumwason
measuredafter
h. Theis the concentration(%),
2 linkages and medium
the left side.
(b)6–72
In caseof 3 verticallinkages axis
and medium
on the left side.
(c)andIn thecase of three connections and a medium in the middle. (d) In case of 2 linkages and medium on theleft side. (e) In case of 3 linkages and medium on the left side.

Micromachines 2021, 12, 1431

horizontal axis is the time (h). The filter pore size is 0.6 μm (a)–(c) or 0.03 μm (d)~(e). (a) In case of 2
linkages and medium on the left side. (b) In case of 3 linkages and medium on the left side. (c) In
9 of 19 on thecase of three connections and a medium in the middle. (d) In case of 2 linkages and medium
left side. (e) In case of 3 linkages and medium on the left side.

Figure 5. The same number of cells were placed in wells with clip art of the cell. Lactate andFigureThe same numberof cellswere
weremeasured
placed inwells withclip artof thecell. Lactate
and NH4+NH4 + 5.
concentrationin the medium
regularly
for 24–72
h. Theverticalaxis is theconcentration
thewereregularly
forresults
24–72ofh.theTheverticalis the concenconcentration,in
andthemediumhorizontal
axismeasured
is the elapsed
time. Thethreeor fouraxis
connected
tration,andthe horizontal
is the elapsedof the three results
or fouronconnected
plates areshown.
Lactic acid axis
measurement
resultstime.
on theTheleft,results
NH4 + measurement
the right. plates
4+ measurement
results equations
on the right. The
areshown.
Lacticapproximationacid measurement
oncorrelation.
the left, NH
The
polynomial
showedresults
the best
Correlation
approximation
polynomial
approximation
showedthebest
correlation.
Correlation
approximation
equationsandand correlation coefficients are shown in Supplemental Tables S1–S3. (a) In case of 3 linkages
andcorrelation
coefficients
are
shown
in
Supplemental
Table.
(a)
In
case
of
3
linkages
and
cells
on
the
cells on the left side. (b) In case of 3 linkages and cells on the middle. (c) In case of 4 linkages and
left
side.
(b)second
In casefromof 3thelinkagescells
on theleft side.and cells on the middle. (c) In case of 4 linkages and cells on thesecond from the left side.

Micromachines 2021, 12, 1431
Micromachines
2021,2021,
12, 1431
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12, 1431

11 of 20
11 of1020of 19

Figure6. Validationof theof extracellular
vesicles(EV)(EV)to pass
throughthethefilteraccordingFigure6. Validation
ofability
the abilityof extracellular
vesiclesto pass
throughfilteraccording to6. Validation
ofoftheabilityof extracellular
vesicles(EV) topass
throughcells.
the (b)filteraccordingtoFigure
pore
size.
(a)
Schemetheevaluation
methodforEVssecreted
from
PANC-1
Evaluation
pore
size.
(a)
Schemeoftheevaluation
methodforEVssecreted
from
PANC-1
cells.
(b)
Evaluation
pore
size. (a)Schemethe evaluation
methodfor TheEVs secreted
from PANC-1
cells.PANC-1(b) Evaluation
oftothepassingabilityof ofextracellular
vesicles(EVs).
same numberof EVs from
cells
of
the
passingabilityofextracellular
vesicles(EVs).
The
same
numberof
EVs
from
PANC-1cellsof thepassingof (leftextracellular
vesicles(EVs). The co-culturesame number
EVs from
wereseededon ability
one sideside) of thehorizontal-typeplateof(HTCP).
ThePANC-1
numbercellsofwere
seededon one(left side)of the horizontal-type
co-culture
plate(HTCP). The
numberofwereseededon onesideside(left
co-culture
plate(HTCP).
numberexosomes
passing
throughthe side)filter ofon thethe horizontal-type
vessel’s right side
were measured
using The
Westernblot.ofexosomes
passing
throughthefilteron
thevessel’s
right
side
were
measuredWesternblot.
The
exosomes
passing
throughthefilteron
thevessel’s
right
side
were
measuredusing
Westernblot.
The 0.03 μm filter blocked the transfer of the EVs. The absence of the exosome on the adjacent vessel
0.03 µm
μmby
thetransferofThe
absenceof ofthetheexosomeon ontheadjacent
vessel0.03
filterthetransfer
ofthe
theEVs.
EVs.
The
absenceexosometheadjacent
vesselisTheconfirmed
theblocked
Westernblotting
methodin experiments
with
pore
size 0.03μm.
MarkersofisconfirmedbytheWesternblotting
methodinexperiments
with
pore
size
0.03
μm.
Markersof ofis
confirmedbytheWesternblotting
methodinexperiments
with
pore
size
0.03
µm.
Markers
exosomes (CD9, CD63, CD81, Flotilin-1) were not detected at 72 h.
exosomes
(CD9,
CD63,
CD81,
Flotilin-1)
were
not not
detected
at 72ath.72 h.
exosomes
(CD9,
CD63,
CD81,
Flotilin-1)
weredetected

Figure 7. Validation of the ability of extracellular vesicles (EV) to pass through the filter according to
Figure7. Validation
abilityofofthe
extracellular
vesicles for(EV)
to secreted
pass throughfilter cells.
accordingco-culture
type. of(a)theSchemeevaluation methodEVsfrom the
PANC-1(b) Evalu7. Validation
the abilityof evaluation
extracellular
vesiclesfor(EV) tosecreted
pass through PANC-1
the filter according
toFigure
co-culture
type.
(a)of
Schememethodcells.
(b) ofation of thepassingabilityofofthe
extracellular
vesicles(EV)EVs
secreted fromfrom
cells. The same
numberto co-culturetype.
(a) Schemeofthe evaluation
methodforsecreted
EVs secreted
fromThe
PANC-1
cells. (b)Evaluationofthepassingabilityofextracellular
vesicles(EV)fromcells.
same
number
PANC-1ofcells
wasseeded on one
side of the horizontal-type
co-culture
plate (HTCP)
and vertical-typethepassing
extracellular
vesicles (EV)co-culture
secretedfrom
The andsame
numberofEvaluation
PANC-1 cells
wasseededability
on oneofsideof the horizontal-typeplatecells.
(HTCP)
verticalco-culture
plate(VTCP).
Theonenumberofthe
EVspassing throughthe filterplate
was (HTCP)
measuredbyverticalNanosight.
of
PANC-1cells
wasseededonsideofhorizontal-type
co-culture
andtype co-culture plate (VTCP). The number of EVs passing through the filter was measured by NaThe
numberplateof EVsper ml The
was number
then calculated.
The EV throughdensity on
thecellwassidemeasured
was set to by
100%. Thetype
co-culture(VTCP).
ofEVs
passingthefilternosight. The number of EVs per ml was then calculated. The EV density on the cell side was set Nato
HTCP
contained
times
thewasdensityEVs per cell
24 density
h, two times
atcell72 h,compared
nosight.
The
number threeof EVsper mlthenofcalculated.
TheatEVon theside
was set to the
VTCP, demonstrating the high efficiency of the HTCP for EV passage.

Micromachines 2021, 12, 1431

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Here, we have provided an in vitro experimental system using a combination of
HTCP and a track-etched membrane. Filters with pore sizes of 0.6 µm and 0.03 µm weremainly used to evaluate the mass transfer properties. Details of the filters are listed in
Supplemental Table S1.
3.3. Measurement Results
3.3.1. Differences in Mass Transfer Due to Differences in Wells Containing the Materials
We measured the change in the concentration of glucose to understand the relationships among the position, filter, and substance. Results showed that the glucose took aconsiderably longer time to pass through the 0.03 µm filter (Figure 5).
On the other hand, even the 0.03 µm filter had good permeability for NH4 + (Figure S7).
The difference in time required for the metabolites to pass through was related to theirsizes. These measurement results follow the theory of mass movement, known as the
Brownian motion. The correlation approximation equations and correlation coefficients areprovided in Supplemental Table S2. As the standard error is small and cannot be seen in
the graph, the data are provided in Supplemental Table S3 and an Excel file is available
from MDPI’s online.
3.3.2. Differences in Mass Transfer Due to Differences in Wells in Which the Cells
Were Placed
Cells produce lactate and NH4 + . Therefore, we investigated the production of lactate
and NH4 + (Figure 5) in the culture medium. The increasing curves for lactate and ammonia
both followed the polynomial approximation curve (Supplemental Table S2). In all vessels,
the correlation coefficient was almost 1. These results indicate that the lactate and NH4 +secreted by the cells are always produced at a constant rate, and the transfer between
vessels also follows the laws of physics.
3.3.3. Validation of Protein Pass Rate
The “standard protein” is a mixture of proteins of different sizes, and the sizes are
known. The standard protein was used to confirm the ability to pass through the filter.
The presence and amount of protein passage were evaluated using the SDS-PAGE method
and Coomassie protein assay. A standard protein was added to Body A (Figure S8A).
The amount of protein in Body B after 48 h was quantified by the Bradford method andconfirmed by silver staining (Figure S8B). Significant amounts of different proteins wereobserved to pass through the 0.6 µm filter, whereas barely any proteins passed through the
0.03 µm filter.
3.3.4. Difference in Exosome Transfer Due to Difference in the Pore Size of the Filter
We examined the permeability of the HTCP for EVs using filters of two sizes: 0.6 and0.03 µm. A filter with a hole diameter of 0.03 µm enables interrupting EVs from migrating
to the neighboring vessels. A filter with a hole diameter of 0.6 µm enables the exosomes (aspecific subgroup of EVs) to pass through the filter and migrate to the adjacent vessel. Thiswas confirmed by Western blot analysis of the microparticles in the right container. The
results showed no exosome surface markers (Figure 6b).
3.3.5. Comparison of the VTCP and HTCPAs the HTCP was designed to analyze cell–cell communication via EVs, we nextcompared the rate of EVs passing through filters of the same size (1.0 µm). A track-etchedmembrane, which is a thin polymer film with geometrically well-defined and accurate
pores (it4ip Inc.), was selected for this experiment. Approximately 1 × 104 cells were seededin one well, and the number of EVs in both vessels after 48 h was measured. The results
showed that the HTCP contained two times the density of EVs per cell compared to the
VTCP at 72 h, demonstrating the high efficiency of the HTCP for EV passage (Figure 7b).

seeded in one well, and the number of EVs in both vessels after 48 h was measured. The
results showed that the HTCP contained two times the density of EVs per cell comparedto the VTCP at 72 h, demonstrating the high efficiency of the HTCP for EV passage (Figure
7b).
Micromachines 2021, 12, 1431

3.3.6. Confirmation of EVs Migration and Cellular Uptake

12 of 19

One of the advantages of the proposed device is that it can be used for biological cell
culture and microscopic observation. Therefore, we used cells expressing CD63 protein
fused
fluorescent proteins
to confirm
theCellularmigrationof exosomes and their uptake by
3.3.6.toConfirmationof EVs Migration
andUptakethe cells.
CD63
is
an
exosomesurfacemarker.
Plasmidtransfection
stable panOne of the advantages of the proposed device is that it can beestablishedused for biologicalcellcreatic
cancercell
lines
expressing
CD63-GFP(gPANC1)
or
CD63-RFP(rPANC1).
culture and microscopic observation. Therefore, we used cells expressing CD63
proteingPANC1
PANC1proteins
cells wereseededthein migration
bodies A ofand
B, respectively.
wasbyobfused to andfluorescent
to confirm
exosomes
and theirGFP
uptaketheservedin cells
B after
48 h,marker.
indicatingthat exosomes
migrated
to theadjacent
cell
cells. CD63
is in
anBodyexosomesurface
Plasmidtransfection
establishedstablepancreatic
fromthecellBody
A expressing
vessel (Figure8a). Next,
to take advantageof thecharacteristicsof thecancerlines
CD63-GFP(gPANC1)
or CD63-RFP(rPANC1).
gPANC1andthreeconnected
containers,
spreadexosome-producing
cellswaslabeledwithindifferent
colPANC1
cells wereseeded inwebodiesA and
B, respectively. GFP
observed
cells in Body
orsin the48containers
at bothends (gPANC1
rPANC1).
48 h,thetheBody
centerconB afterh, indicatingthat theexosomesmigrated andto theadjacent After
cell fromA vesseltainerwasphotographed(Figure8b).
Theyellowcolor
indicates
that
the
cells
may
have
(Figure 8a). Next, to take advantage of the characteristics of the three connected containers,
processed
exogenous
exosomes atcells
the labeledsame time.
biologicalsignificanceof this experwe spread
exosome-producing
withThedifferent
colorsin the containers
at bothimentis thatit can reveal
processingof thetaken
up by cells.
as a
the ends(gPANC1
and therPANC1).
After
48 exosomesh, the centercontainer
wasHowever,photographed
limitationof theresearch,alone
cannotbe provenas significant
due
to opti(Figure 8b).
Theyellowthesecolorimages
indicatesthat
the cells
may haveprocessed
exogenous
exosomes
at issues.
the sametime.
Thesignificanceof thisexperimentthat it cancalresolution
Apart
from
thebiologicalpoint of opticalresolution,it hasnot beenispossibletorevealtheprocessingoftheexosomes
taken
up
by
cells.
However,asalimitationoftheconduct experiments to determine whether material from the left or right cell is preferenresearch,these images
alone
cannotbe provenas significant
duetheto opticalresolution
issues.
tiallyprocessed
under thesame
cultureconditions.
Changing
left andright cells
toApart fromthemakes
point ofoptical resolution,it has notthebeenpossible
conduct experiments
different
types
it possible
to check whether
centralcell toprioritizes
substances
to determine
whether material from the left or right cell is preferentially processed underfrom
either side.
the same culture conditions. Changing the left and right cells to different types makes it
possible to check whether the central cell prioritizes substances from either side.

Figure 8. Confirmation of EVs migration and cellular uptake by microscope (a) Uptake of EVs by
Figure 8. Confirmation of EVs migration and cellular uptake by microscope (a) Uptake of EVs by
CD63-GFP-expressing cells. gPANC1 cells were seeded in Body A, and a photograph of Body B was
CD63-GFP-expressing cells. gPANC1 cells were seeded in Body A, and a photograph of Body B was
taken 48 h later. (b) Uptake of extracellular vesicles from the both-side cells in the containers by threeconnected vessels. Unlabeled PANC-1 cells were seeded in Body C at the center, while gPANC1 andrPANC1 were seeded in Body A and B, respectively. Forty-eight hours later, mixed fluorescence was
detected in the cells in Body C.

4. Discussion
Originally, our aim was to investigate the interactions that occur between cancer cells
and normal cells. We believe that the results of this paper will be useful not only forrPANC1 were seeded in Body A and B, respectively. Forty-eight hours later, mixed fluorescence was
detected in the cells in Body C.

4. Discussion
Micromachines 2021, 12, 1431

13 of 19
Originally, our aim was to investigate the interactions that occur between cancer cells
and normal cells. We believe that the results of this paper will be useful not only for biological researchers who use HTCPs but also for future engineering researchers who develop
microfluidic
devices.who use HTCPs but also for future engineering researchers who
biologicalresearchers
Microfluidic
andco-culture
develop microfluidic
devices.systems have a high affinity. The co-culture method has
been classicallyusedforstudying cell–celleither witha mixturemethodof cells has
Microfluidic and co-culture
systems interactions,have a high affinity.
The co-culture[23,24]orbyaddingthesupernatant
of
one
cell
type
to
the
cultureofanothercellbeen classically used for studying cell–cell interactions, either with a mixture of cellstype[23,24][25,26].
A adding
co-culture
with upper
and
lower
is frequently
usedtotypeprevent
or by
thevessel
supernatantof onecell
typechambers
to the cultureof anothercell[25,26].
cellsAfrom
mixingvessel[27] while
examinationof the impactthat groups
havecells
co-culture
withallowing
upper andlower chambers
is frequently
used ofto cells
prevent
on eachother.
from mixing [27] while allowing examination of the impact that groups of cells have on
However,eachother. various new cell culture methods have been proposed in recent years to
overcome
the limitationsof thesetraditionalwhich
are proposedsummarized
FigureHowever,various
new cellcultureapproaches,
methods have
been
in in
recentyears
S9 (modified
andthepermitted
fromofShimasaki
et al.) [1].approaches, which are summarized in
to overcome
limitations
these traditional
Co-culture
methodsand
canpermitted
be classified
accordingto thepresenceor absence of separaFigure
S9 (modified
fromShimasaki
et al.)[1].
tion materialand structural
traditional
andconventional
methodsuse
Co-culture
methodscharacteristics.
can be classifiedTheaccordingto thepresenceor absenceof separation
a filter
and
upper
and
lower
chambers,
formingabucket-in-a-bucket
structure,
resultingmaterial and structural characteristics. The traditional and conventional methods use ain afilter
vertical(VTCP).
Evenforming
today, aVTCPs
are widely used
for co-culture.
and configuration
upper and lower
chambers,
bucket-in-a-bucket
structure,
resulting in a
However,no other effective
vesselshavebeen developed,
no comparison
verticalasconfiguration(VTCP).co-culture
Even today,
VTCPs
are widelyused for co-culture.
However,
has asbeen
madeeffective
in termsco-cultureof their performance.
no othervessels have been developed, no comparison has been madesummarized
in Figure S10 (modified from Shimasaki et al. [1] with permission),
inAs
terms
of their performance.
the majorAs
advantagesof theHTCP include
the ability
toShimasaki
confirm thevarioussummarized
in FigureS10 (modified
frometconditions
al. [1] withofpermission),major
advantages ofuse
thetheHTCP
theon
abilityconfirm
conditionsof various
cell thetypes
simultaneously,sameinclude
material
both tosides
of thethefilter,use high-dencell
types
simultaneously,
use
the
same
material
on
both
sides
of
the
filter,use
high-density
sity co-cultures with no detrimental effects, and use multiple types of filters.
co-cultures
withourno experiments
detrimental effects,
anduseHTCP
multiple
filters.
In this study,
revealed
thathas types
a highofco-culture
efficiency.
In thisour experiments
revealed
that
HTCPtohasa highefficiency.
One reason
for study,
the excellent
co-culture effectis theabilitycontroltheco-culture
volume ratioofOne reasonandfor theco-culture
effectis the ability
to control
the containers
the excellent
positional
relationshipbetweenthe vessels
and thethe volume
filter. ratio of thecontainers
and thepositional
relationshipbetweenare
thestacked
vessels means
and thethat
filter.
The structureof VTCPin whichthe “buckets”
the volume
Thestructure
of
VTCP
in
which
the
“buckets”
arestacked
means
that
the
volumeratio of the culture fluid does not become 1:1, which results in a large dilution
effect.ratiooftheculture
fluid
does
not
become1:1,
which
results
inalarge
dilution
effect.
Achieving
Achieving the same volume ratio is desirable for the effective co-culture of each cell typethe samevolumeis desirable
for theeffectiveof eachcell type(Figure(Figure9a). The
VTCPratio
systems
also allow
placing
cellsco-culture
directly onto
the filter(Figure9b), 9a).
The VTCP
systems
also allow
placing
cells
directlypreventing
onto the filter(Figure 9b),
meaningmeaningthat highcell densities
lead
to poreblockage,the exchangeof matehighthe
cellpores.
densities lead to pore blockage, preventing the exchange of material throughrial thatthroughthe pores.

Figure9. ThereasonHTCP
is moreefficientin co-culturecompared
to VTCP.
(a) TheFigure9. ThereasonwhywhyHTCP
is moreefficientin co-culturecompared
to VTCP.
(a) Themostmost
efficientconditions
are
those
in
which
the
volumeoftheculturemedium
is
the
same.
(b)
As
the cells
efficient conditions are those in which the volume of the culture medium is the same. (b) As the cells
aretopon oftoptheof filter,the filter,
the higher
the celldensity,lessermaterial
throughfilterpores.
are onthe higher
the celldensity,lessermaterial
cancanpasspass
throughthe thefilterpores.

Therefore, the decrease in the co-culture effect that can occur in cell cultures over long
periods is not visually apparent. Some researchers have argued that in VTCPs, substancesthat are secreted by the upper cells pass through the filter to affect the lower cells. However,this could not be tested because there was no container with which the efficiency could becompared. The development of the HTCP has allowed such verification to be performed.
The better performance of the HTCP is not surprising because the problems associatedwith filter blockage at high cell densities are bound to have deleterious effects on the results
obtained with VTCPs. As the substance moves to an equilibrium concentration, a 1:1
volume ratio between containers allows each substance to influence the other effectively.

Micromachines 2021, 12, 1431

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In Brownian motion, liquid factors such as EVs also pass through the filter away from thecell, meaning that it does not matter if the filter and cells are at a distance.
The track-etched membrane filter, which can be regarded as a microfluidic channel,
is an important component. Both the VTCP and HTCP connect two vessels throughtrack-etched membrane filters, which is the same mechanism as that used in microfluidic
channels. Although the vessel is not a microstructure in terms of size, the mass transfer
characteristics obtained in this study will be useful for other microstructures. We selected
a 0.03 µm filter for restricting the passage of exosomes and a 0.6 µm filter for allowing
the passage of exosomes. The experimental results showed that the size of the filter used
could control the passage of exosomes. It was also found that small molecules such as
glucose, lactic acid, and NH4 + can traverse the filer rapidly, even when pore sizes of0.03 µm were used. The ammonia in the medium was also observed to migrate rapidly
in the multi-coupled experiments, but the concentration gradient was maintained whenammonia was continuously secreted by the cells. This gradient means that the degree towhich a cell affects other cells can be controlled by considering the location of the wells
in a multiconnected experiment. In addition, as shown in the results for the 0.03 µm filter
for KLM in Figure S7, the fact that it took 72 h to equilibrate substances of extremely small
size such as NH4 + and that the results were the same in multiple experiments indicatesthat the filter sandwiching mechanism that uses the O-ring in HTCP works well and ismade with precision. This can be assumed because any leakage from a source other than
the nano-sized filter pores will allow micro materials such as NH4 + to reach equilibrium ina very short time.
As shown in Figure 10, when the cells were placed in the left container (Q), theconcentration of the substance from the cells increased in Q. When the cells were placed in
the middle container (U), the concentrations in both containers (T and V) were the same.
When the cells were placed in the second container from the left (X), W = Y did not occur
because Y was diluted by the influence of Z, resulting in W > Y > Z.
In the relationship between concentration and time, the concentration of each container
was represented by an approximate curve, with a correlation coefficient of approximately
1. These results indicate that metabolites and exosomes were not secreted randomly. They
also show that the secreted material undergoes Brownian motion and movement due
to osmotic disparity, moving precisely through the pores of the filter to the neighboringvessel. The degree of influence can change depending on the position of the cells in theconnected plates. These results may also be applicable to sensor technology via filters andmicrofluidic channels. It was very interesting that the concentration of extracellular vesicles
also changed along the approximate curve. This means that extracellular vesicles migrate
according to the theoretical concentration curve even when the vessels are separated
and filtered. In other words, the combination of horizontal co-culture vessels and thetrack-etched membrane made it possible to control the co-culture effect.
Co-culture is a powerful tool to observe the intrinsic cell–cell interaction of extracellular vesicles. To understand the reason for this, we need to know about exosome extraction
techniques and research methods.
Several techniques have been proposed for the extraction of EVs [18,28,29] and the
addition of collected EVs to other cells to confirm their effects. However, such artificial
administration of EVs cannot be performed in vivo. Moreover, these extraction methods canonly recover specific EV populations because of the nature of the approach. Techniques suchas polymer-based precipitation [30], immunoaffinity capture using surface markers [31],purification by flow cytometry, and size-exclusion chromatography [32] have all beenused to recover specific EVs. Among these methods, ultracentrifugation is the standard
approach [33] because it can recover a particular population of EVs. However, the details of
EVs have not yet been described, which means that experimental methods that can assess
the precise nature and characteristics of exosomes are required. Indeed, information on
the characteristics of exosomes remains largely unknown, although proteins and nucleic
acids appear to be involved. Exosomes include features such as lipid bilayer membranes,

Micromachines 2021, 12, 1431

Micromachines 2021, 12, 1431

15 of 19

16 of 20

tetraspanins such as CD9 and CD63, membrane transport proteins, and adhesion molecules
such as integrins.

Figure10. Material
concentration
gradient
causedby multiple
linkages(a) Whenare placed
Figure10. Material
concentration
gradient
causedby multiple
linkages(a) Whencells cells
are placed
in in
the leftmost
container
in a three-container
system,the concentration
the substances
the leftmost
container(Q) in(Q)a three-container
system,the concentrationof theofsubstances
fromfrom
the thecells increases(b) Whencellsplaced
are placed
the middle
containerof a 3-connectedcontainer,
cells increases
in Q. in(b)Q.
Whencells arein thein
middlecontainer(U) of(U)a 3-connectedcontainer,
the concentrationsof substancesfrom fromthe cells
theincontainers
at both(T andareV)thethe concentrationsof substances
the incells
the containers
at ends
both ends(TV)andaresame.
the same.
(c) If (c)theIfcells
are placedin theinsecond
vesselfromfrom
the lefta four-connectedcontainer,
W=Y
the cells
are placed
the second
vesselthe(X)
leftin(X)in a four-connectedcontainer,
W=Ywill notbetrue
becauseY
willbedilutedbytheinfluenceofZ,
resulting
in
W
>
Y
>
Z.
(d)
In
the
will not be true because Y will be diluted by the influence of Z, resulting in W > Y > Z. (d) In therelationshipbetweenconcentrationand time,
the concentrationin eachcontainer
can berepresented
relationshipbetweenconcentrationand time,
the concentrationin eachcontainer
canbe represented
by an approximate curve, with a correlation coefficient of almost 1. The approximate formulas andby an approximate curve, with a correlation coefficient of almost 1. The approximate formulas andcoefficients in each case are shown in the graphs. The data are described in Supplementary Table
coefficients in each case are shown in the graphs. The data are described in Supplementary Table S3.
S3.

Similarly, when extracting exosomes, only the effects of a particular population can
In the relationship between concentration and time, the concentration of each conbe examined. The addition of large numbers of EVs may be an artificial condition thattainer was represented by an approximate curve, with a correlation coefficient of approxwould not otherwise occur, making it difficult to reproduce the natural phenomena thatimately
1. inThese
indicatethat metabolites
andexosomes
were andnot secreted
ran- areoccur
cells.results
Althoughexperiments
in whichEVs
are extracted
administered
domly.
They
also
show
that
the
secretedmaterial
undergoes
Brownianmotionandmoveuseful, this is also a one-sided experiment. Some phenomena occur only when the cells
mentcommunicate
due to osmotic
movingthroughthe poresof the filter
withdisparity,each other.
Becauseprecisely
a living cell
has uniquemechanismsthat toaretheakin toneighboringvessel.
The
degreeofinfluencecanchangedependingonthepositionof decryptthea personal identification number, only experiments that allow such interaction cancells ainbiologicalthe connected
plates. These
results
mayregard,
also bethe
applicable
to sensor
technology
identification
number.
In thisco-culture
techniqueis usefulvia
in thatfiltersandmicrofluidic
channels.
It
was
very
interestingthat
theconcentrationof Theextrait can reproduce natural phenomena without requiring the extraction of EVs.
use ofcellularvesicles alsochanged along
the approximate
This meansthat extracellular
co-culturing
technologies
is necessary
to examinecurve.
the essential
interactionsof EVs.
vesicles migrate
to thetheoreticalconcentration
curve
when
theorvessels
To date,according
many effectsof EVsproducedby cancer cells
onevennormalcells,
vice versa,
are separated
filtered.
In otherHowever,words, thehorizontal
co-culture
ves-been
have beenand
reported[26,34,35].
thecombination
simultaneousofanalysisof these
effects has
sels andthetrack-etchedmembranemadeitpossibletocontroltheco-culture
effect.
limited owing to the lack of suitable techniques.
Co-culture is a powerful tool to observe the intrinsic cell–cell interaction of extracellular vesicles. To understand the reason for this, we need to know about exosome extraction techniques and research methods.

Micromachines 2021, 12, 1431

16 of 19

Using the HTCP experimental system, it is possible to conduct experiments withdifferent degrees of influence from other cells in other wells in the same experiment. For
example, to confirm the effect of cancer cells on normal cells, we can examine the difference
between Y and Z in the experimental system, as shown in Figure S11a. To compare thedegree of influence exerted by the two different types of cells, the experiment shownin Figure S11b is recommended. The difference in substance levels between ‘X’ and ‘Y’occurred due to the difference in the degree of influence of ‘W’ and ‘Z.’
Thus, horizontal co-culture systems offer a useful tool to unravel the characteristics
of cells. We believe that our newly designed HTCP system can help promote EV researchmethodologies and co-culture techniques. As shown in Figure 9 and Figure S11, HTCP cancontrol the effects of co-culture by selecting the location of the cells.
5. Conclusions
The combined device of a vessel and a track-etched membrane can be considered as a
microfluidic device. Vertical co-culture vessels, which have been conventionally used for along time, are not sufficient for co-culture via liquid factors. In this study, we demonstrated
a novel co-culture plate capable of controlling co-culture effects by clarifying the degree
to which materials pass through a horizontal connection. The analysis showed that an
approximate curve could represent the horizontal mass transfer rate with a high correlation
coefficient; even if the cell and the channel are located away, the secretions of the cell willpass through the fluidic channel. We hope that our results will be useful for the design ofother microfluidic devices.
For example, in the case of making a test kit using cells, the cells and fluidic channel
may be designed to be close to each other. As the extracellular vesicles are secreted after
the changes in the cell membrane, a situation where the cell membrane is in contact withthe microfluidic channel may lead to failure in the secretion of extracellular vesicles, orthe cell may block the channel itself. As our data show, the distance between the cell andthe microfluidic channel does not affect the transfer of extracellular vesicles; rather, it isnecessary to avoid blocking the microfluidic channel. We think it is important to consider
the positional relationship between the cells and the microfluidic channel.
Finally, we expect that this cell co-culture vessel will be useful for expanding research applications in various fields, especially for understanding EVs and their roles incell biology.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/
10.3390/mi12111431/s1. Figure S1. The three main problems associated with a vertical-type coculture plate (VTCP). (a) The upper container cannot be observed because of the short focal length ofthe microscope. (b) The surface material to which the cells adhere differs, meaning that the cells may
respond differently. (c) The high density of cells may prevent the exchange of materials. Figure S2.
(a) Track-etched membranes were fabricated by irradiating a raw polymer film with energetic heavyions. (b) The beamed film was then chemically etched to make uniform and precise pores. Poredensity and pore orientation were determined during the beaming step, while the etching conditions
defined the pore size. Figure S3. Size of each component. Figure S4. How to use. Methods A (Initial
monoculture mode) and B (Controlling co-culture with medium volume). (a) In a stand-alone way (b)In the connected mode. Figure S5. Schematic of various use methods: (a) filter separation, (b) without
separation, and (c) solid separation with gels (three-dimensional culture). Figure S6. Summary ofexperimental design. (a) Differences in mass transfer due to the number of containers, position of themedium, and pore size of the filter were measured. All the measurements were performed undercell-free conditions. (b) Differences in metabolite concentrations due to differences in the positions of
the containers in which the cells were placed were measured. (c) The filter passability of exosomes
was measured. (d) Comparison of the passability of exosomes between VTCP and HTCP. Figure S7.
The results of NH4+ transfer without cells. Figure S8. Migration of proteins through filters (A) The
ability of proteins to pass through filters of different sizes. (B) Proteins passing through the 0.6 µm
and 0.03 µm filters. Figure S9. Various co-culture methods. Figure S10. List of differences in features
between VTCP and HTCP. Figure S11. (a) Experimental design 1: A method to check for changes inMicromachines 2021, 12, 1431

17 of 19the cells due to different degrees of influence. (b) Experimental design 2: How to check the change inthe two types of cells due to different degrees of influence. Table S1. The density and number of poresin each filter. Table S2. The list of correlation approximation equations and correlation coefficientsshown in Figure 6. Table S3. Raw data of the results (Figures 4, 5, 7 and S7). An Excel file is available
online from the MDPI.
Author Contributions: All authors contributed to this study. Conception and design: T.S., Y.N.,
H.Y., K.O. (Kazumi Ohtomo); Acquisition of data: T.S. (Figures 1, 2 and 4–10, Supplemental
Figures S1, S3–S10), Y.N., H.Y., K.O. (Kazumi Ohtomo) (Figure 3), S.Y. (Figures 4–6), R.O. (Figure 7),
E.F. (Supplemental Figure S2), Analysis and interpretation of data: T.S., S.Y., R.O., T.I.,
K.O. (Keiichiro Okano), K.I., T.O. (Takenori Ogawa), H.T., Y.M., T.M., N.T., and T.O. (Takahiro
Ochiya); Drafting and manuscript revision: T.S., T.M. All authors have read and agreed to the
published version of the manuscript.
Funding: This research was funded by KAKENHI grant number 18K07983 and financially supportedby the Kanazawa Medical University Research Project (RP2017-09) through the Private University
Research Branding Project of the Ministry of Education, Culture, Sports, Science, and Technology(MEXT), Japan, and in part by MEXT (to TS). This work was also supported by a Grant for Promoted Research from Kanazawa Medical University (S2014-7, S2015-16, K2021-9) and an ExtramuralCollaborative Research Grant of Kanazawa University Cancer Research Institute (30-40, 31-31).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data that support the findings of this study are available from thecorresponding author upon reasonable request.
Acknowledgments: We thank Shinde (Shinko Chemical Co., Ltd.) for sales networking, Yoichi Ikeda(AR Brown Co., Ltd.) for developing NICO-1 (HTCP product name in Japan) and ICCP (HTCP’s
product name in the other lesion except for Japan), and Shimosaki for developing NICO-1 (Blast Inc.).
Conflicts of Interest: T.S. received a research grant from Kanazawa Medical University. T.S. and T.M.
received a research grant from Kanazawa University. T.S., Y.N., H.Y. and K.O. are inventors of HTCP:
patent JP Patent 11201600072S licensed to Kanazawa Medical University, and Shinko Chemical Co.,
Ltd. manufactures HTCP. Ginreilab Inc. sells HTCP. T.S. is also the CEO of Ginreilab Inc. S.Y., K.I.,
Y.N., H.Y., K.O., T.I., T.O., H.T., Y.M., T.M., N.T., T.A. and T.O. declare no conflicts of interest. E.F. isthe CEO of it4ip.

Abbreviations
DMEM
EVs
GFP
gPANC1HTCP
VTCP
rPANC1

Dulbecco’s modified Eagle’s medium
extracellular vesicles
green fluorescent protein
green fluorescence-labeled PANC-1 cellhorizontal connection type co-culture plate
vertical connection type co-culture plate
red fluorescence-labeled PANC-1 cell

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