九州大学大学院総合理工学府報告第 45 巻 第 1 号 1-6 頁 令和 5 年 9 月

Engineering Sciences Reports, Kyushu University
Vol. 45, No. 1, pp. 1-6, SEP, 2023

Cellular characterization of hiPS-CMs cultured on PMEA
analogous polymers with different bound water content
Hiroko KURITA,*1† Shingo KOBAYASHI,*2 Takahisa ANADA,*2,3
Masaru TANAKA*2,3 and Mitsugu TODO*4,5
E-mail of corresponding author: kurita.hiroko.087@s.kyushu-u.ac.jp†(Received March 22, 2023, accepted April 4, 2023)
In recent years, human iPS cell-derived cardiomyocytes (hiPS-CMs) have attracted muchattention in the arena of regenerative therapies. However, hiPS-CMs are known to be immature andhave lower contractility than the adult heart. In this study, we investigated the effect of material
properties of scaffold surfaces on hiPS-CMs with the aim of improving the contractility of hiPS-CMs.
PMEA analogs with varying surface-bound water content, which affect cell adhesion/proliferation
behavior, were selected as scaffold materials to coat PET substrates. The cell characteristics of thehiPS-CMs seeded on the scaffolds were evaluated. The initial cell adhesion area tended to be smaller
on substrates with a higher amount of bound water. With further incubation, the hiPS-CMs formed
self-pulsating aggregates on the substrate. On the substrates with high bound water content, thearea of the hiPS-CMs aggregates tended to be smaller while the pulsation behavior became greater.
The results indicate that PMEA analogues might affect the cellular properties of hiPS-CMs.
Key words: iPS cell, Cardiomyocytes, PMEA analogues, Bound water, Intermediated water

1.

Introduction

Poly(2-methoxyethyl acrylate) (PMEA) is oneof the biocompatible materials and has theproperty of inhibiting the deformation ofproteins adsorbed on its surface.
PMEA weaklyinteractswithblood
components and thus prevents the formationof blood clots.1) Due to the inertness of blood
components, PMEA has been clinically applied
as a coating material for blood-contacting
medical devices.2) Previous studies have shownthat PMEA analogues have "intermediatewater" in the layer of water molecules
adsorbed on the surface, and a correlation has
been demonstrated between the amount ofintermediate water and platelet adhesion
inhibit ability.3) Further studies applying thisproperty to the control of adhesive cell
behavior suggested a correlation between cell
*1*2*3*4*5

Department of Molecular and Material Sciences,
Graduate Student
Institute for Materials Chemistry and Engineering
Department of Applied Chemistry, Graduate School ofEngineering
Research Institute for Applied Mechanics
Department of Molecular and Material Sciences

adhesion force to PMEA analogues and theamount of bound water.4-6) In recent years, iPS
cell-derived cardiomyocytes (hiPS-CMs) haveattracted significant attention in the field of
regenerative medicine. Numerous studies
have been conducted and several problems
have emerged, including the immaturity andlow self-pulsatility of hiPS-CMs compared toadult human cardiomyocytes.7) Over the past
few years, material properties affecting thematurity, self-pulsating force and function ofhiPS-CMs have been investigated, for example,
using decellularized plant-derived scaffolds
and ECM-based scaffolds.8-10) However, few
studies have examined the effects of scaffold
surface properties on the pulsation behavior ofhiPS-CMs. This study aimed to clarify theeffect of PMEA analogues, which exhibit
different cell adhesion properties depending on
the amount of bound water, on the cellular
properties of hiPS-CMs and identify thesurface properties of materials that improve
the self-pulsation behavior of hiPS-CMs.

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Cellular characterization of hiPS-CMs cultured on PMEA analogous polymers with different bound water content

2.

Experimental

2.1 Preparation of polymer substrates
A solution of PMEA analogues wasspin-coatedonabiaxiallyorientedpoly(ethylene terephthalate) (PET) sheet(DIAFOIL®T100E,
Mitsubishi
Chemical,Japan, thickness = 125 µm). First, substratesof the same size as the bottom of the 24-wellculture plate (14 mm diameter) were cut from
PET sheet using a laser cutter. In this study,
all three PMEA analogues with different
amounts of bound water, poly(2-methoxyethylacrylate) (PMEA), poly(2-ethoxyethyl acrylate)(PEEA),
andpoly(2-methoxyethylmethacrylate) (PMEMA), were examined. The
structural formula of the PMEA analogues areshown in Fig. 1. The polymers weresynthesized using the same method as in ourprevious papers.11) The molecular weight
relative to standard polystyrene and the boundwater content of each polymer are shown in
Table 1.6) Each polymer was dissolved indehydrated toluene at a concentration of 0.5
wt/vol%, and all were filtered through a 0.20
µm pore size filter. 40 µl of PMEA analogue
solution was applied to the PET substrate andcoated using a spin coater (MS-A100, MikasaCorp., Japan) at 3000 rpm for 40 seconds.
Subsequently, the spin-coated substrates weredried under reduced pressure at 25 °C for 24hours.

Fig. 1

Chemical structures of PMEA, PEEA, andPMEMA.

Table 1 Characteristics of PMEA analogous polymers.6)

PMEMA
PEEA
PMEA

Mn
[g mol-1]

Mw / Mn

Bound water[wt%]

91,00021,00029,000

2.09
2.70
2.82

2.3
4.5
6.2

2.2 Cell culture
Each substrate was UV-sterilized for 1 hour
before seeding cells to prevent contamination.
The hiPS-CMs (Carmy-A，Myoridge Co. Ltd.,Japan), were pre-cultured for 7 days, and wereseeded on each substrate at a density of 2.5 ×105 cells/well. PET substrates coated withPMEA analogous polymer were placed on thebottom of 24-well cell ultra-nonadherent
multi-well plates (Costar® 24 Well Ultra Low
Attachment Surface Flat Bottom Lid, CorningInc.). The cells were then cultured inmaintenance medium (Myoridge Co. Ltd. ，Japan) with continuous medium changes every
2 days.
2.3 Immunostaining
The cytoskeletal protein α-actinin wasimmunostained for evaluation of single-celladhesion morphology of hiPS-CMs. The
hiPS-CMs were seeded on each substrate at a
low-density condition of 1.0 × 104 cells/well. 24hours after seeding, hiPS-CMs were fixed with
4% PFA and cell nuclei were stained with
4',6-diamidino-2-phenylindole (DAPI). Mouse
monoclonal anti-α-actinin (Sigma-Aldrich Inc.)
was added as the primary antibody, and Alexa
Fluor 488-conjugated anti-mouse IgG (H+L)
the secondary antibody (Thermo Fisher
Scientific Inc.) was added and incubated. After
staining, the α-actinin and cell nuclei wereobserved by 10x objective lens using a confocal
laser microscope (FV3000, Olympus Corp.).
The captured image data were analyzed by animage processing software, ImageJ,12) and cellarea was measured.
2.4 Cell viability assay
The cell viability of hiPS-CMs on each
substrate on the fifth day of culture wasdetermined using a cell counting kit. After thecells were washed once with PBS, WST-8
solution (Cell Counting Kit, Dojindo Co., Ltd.)
was added to each well and incubated at 37° C
for 2 hours. The absorbance of the samples wasmeasured by a plate reader (2030 ARVO X2,Perkin Elmer Co., Ltd.) at 450 nm.
2.5 Self-pulsating cell aggregate area
Video data of the self-pulsating hiPS-CMsaggregates on each substrate were captured
using an inverted microscope (CKX53,Olympus Corp.). After 5 days of incubation,
video data were captured at 40 fps for 20
seconds at 10 locations per well at ambient
temperature. The captured video data were九州大学大学院総合理工学報告

analyzed as continuous image data by digital
image correlation evaluation software, GOM
Correlate2019,13) to evaluate the displacement
distribution (subset size: 9 pixels, distance
between points: 4 pixels). The area of cellcolonies showing localized pulsating in thedisplacement distribution map was measured
using ImageJ image processing software.
2.6 Measurement of contraction behavior
The pulsation movie of the hiPS-CMs wasanalyzed for their pulsating behavior using
repeated particle image velocimetry (PIV) inImageJ.14) An image before contraction and an
image at the peak of contraction were selectedfrom the continuous image data of thepulsating movie, and the displacement anddirection associated with contraction weremeasured from the cross-correlation between
the two images. The displacement behavior
was obtained as displacement behavior in twoaxes, the X-axis and the Y-axis.
2.7 Statistical analysis
Data were calculated as the mean ± standard
deviation of the results of at least three trials.
Significant differences between means wereassessed by the multiple comparison test
Tukey HSD method using KaleidaGraph.15)
Statistical significance was set at * P ≤ 0.05
and ** P ≤ 0.001.

3.

Results and discussion

3.1

Cell adhesion area

The adhesion area and morphology of single
hiPS-CMs adhered to PMEA analogues wereevaluated. Representative cell staining imagesof hiPS-CMs adhering to the substrates for
α-actinin and cell nuclei are shown in Fig. 2.
The results of measuring the cell adhesionarea from the morphology image of α-actinin
are also shown in Fig. 3. The higher amount ofbound water content has the effect of
decreasing of protein adsorption and inhibiting
denaturation. Previous studies have shownthat bound water content affects the adhesion
morphology of cells that adhere via
proteins.16,17) The results of this study showedthat cells on PMEA and PEEA substrates withrelatively high bound water content tended tohave a spherical cell morphology compared toPET and PMEMA. Furthermore, the celladhesion area on PMEA and PEEA showedsmaller compared to other substrates. The

第 45 巻 第 1 号

3results observed in this study are consistentwith previous reports and may be attributed tosimilar effects.6)

PET

α actinin DAPI

PEEA

PMEMA

PMEA

Fig. 2 The morphology of cell adhesion after 24hours on each substrate. (scale bars : 100μm)

1.4 x 104

1.2 x 1041 x 10

4

Area (μm2)

令和 5 年度

**

**

PEEA

PMEA

8000
6000
4000
20000PET

PMEMA

Fig. 3 Average cell adhesion area of single cells.
(** P≦0.001 )4

Cellular characterization of hiPS-CMs cultured on PMEA analogous polymers with different bound water content

3.2 Evaluation of pulsating colonies ofhiPS-CMsFig. 4

Displacement distribution associated with
contraction of hiPS-CMs aggregates analyzed
by digital image correlation method. (scalebars :400 μm)

Fig. 5

Area of self- pulsating hiPS-CMs aggregates.
(** P≦0.001 ) (n=3)

0.12
NSNSNSPEEA

PMEA

0.1

Absorbance (Abs)As the hiPS-CMs on the substrate, the cells
began to show self-pulsation without any
external stimuli. Continuous image data of thepulsating hiPS-CMs were analyzed by digital
image correlation method, resulting in thedisplacement distribution shown in Fig. 4. On
the substrates, deformation due to localized
contraction of the hiPS-CMs was observed.
Furthermore, it was observed that the sites ofself-pulsation were highly consistent with theareas where cell aggregation and colonization
took place on the substrate. The results ofcomparing the area of cell colonies that
pulsating on each substrate are shown in Fig.
5. Cells on the PMEA and PEEA substrates
showed a smaller cell aggregation area thanPET and PMEMA, and showed a significant
difference compared to cells on the PET
substrate. The results of evaluating the cell
viability of hiPS-CMs on the PMEA analogues
on the fifth day of culture are shown in Fig. 6and there was no significant difference in thenumber of adherent cells on each substrate.
The results also suggested that adherent cells
tended to aggregate more on PMEA and PEEA.
This result is presumable because, on
substrates with high bound water content,
which have low cell adhesion properties,
cell-to-cellbindingforces
exceedcell-to-substrate adhesion forces, resulting inmigrating cells to be aggregated.18,19)

0.08
0.06
0.04
0.02
0Fig. 6PETPMEMA

Comparison of cell viability of hiPS-CMs
cultured on the substrate of hiPS-CMs cultured
on the substrate for 5 days. (n=3)

The self-pulsating force of the colony on thePMEA analogues was evaluated. The
distribution of the magnitude of displacement
and the direction of displacement associatedwith pulsation are shown in Fig. 7. In addition,
the displacement behavior during contraction
at each measurement point is shown in Fig. 8.
The measurement position before contraction
was defined as X = 0 µm and Y = 0 µm.
Comparing the displacement behavior on eachsubstrate, the hiPS-CMs colonies on PMEA inparticularshowedlarger
displacement
behavior than those on the other substrates.
Table 2 shows the results of a quantitative
comparison of the pulsating behavior ofhiPS-CMs on each substrate. Comparing thebehavior of displacement caused by pulsationof hiPS-CMs colonies on each substrate, thepercentage of points showing displacement of 5µm or greater increased in the order of令和 5 年度

九州大学大学院総合理工学報告第 45 巻 第 1 号

PET<PMEMA<PEEA<PMEA. Furthermore, acomparison of the average displacement
values with pulsation showed that thedisplacement of hiPS-CMs on the PMEA
substrate was the largest and differedsignificantlyfrom
othersubstrates.
Cardiomyocytes
exhibitself-contractile
capacity
andexcellent
intercellularconnectivity when adjacent cells are bound toeach other and their aggregates are
successfully developed organoids.20) Thissuggests that since substrate-cell adhesion is
low on substrates with high bound watercontent, the three-dimensional crowding ofwell-migrated cells may have resulted in cellaggregates with strong intercellular junctions
exhibiting superior pulsation behavior.21,22)

Fig. 7

Displacement maps of hiPS-CMs aggregatesanalyzed from two photographs before and afterpulsation.

Fig. 8 Displacement behavior associated withpulsation of hiPS-CMs on substrate.

5

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Cellular characterization of hiPS-CMs cultured on PMEA analogous polymers with different bound water content

Table 2Comparison of displacement behavior
accompanying contraction. (n=3)

References1)

Percentage ofdisplacement
behavior (5 μm~)
(%)

Mean
displacementwith pulsation
(μm)

0.05
0.08
0.21
0.98

0.395
0.287
0.393
0.488

PET
PMEMA
PEEA
PMEA

4.

Conclusions

In this study, hiPS-CMs were cultured on
PMEA analogues and the effect on hiPS-CMs
was investigated. Substrates were prepared bycoating biocompatible materials, PMEA
analogues, on PET. The morphology ofhiPS-CMs
singlecells
wassphericalmorphology and the adhesion area was smaller,
as the high content of binding water in thescaffold.
Theself-pulsatinghiPS-CMs
aggregates on the fifth day of culture werethen evaluated. The results showed that thearea of self-pulsating cell aggregates tended tobe smaller on substrates with higher amountsof bound water. Furthermore, the magnitudeof displacement associated with theirself-pulsating
waslarger.
Especially,hiPS-CMs cultured on PMEA showedsignificantly larger contraction behavior thanothers. This result suggests that the boundwater content of scaffolds affects cell adhesion
andweakens
cell-scaffold
interaction,
resulting in stronger intercellular binding ofcell aggregates and better self-contraction. In
previous studies, cell alignment of hiPS-CMs
with nanofiber scaffolds has been successful in
expressing many genes related to intercellularjunctions.23) There was also an example of astudy that succeeded in improving pulsation
force by geometrically aggregating thehiPS-CMs by using Faraday waves.22) This
study demonstrated that hiPS-CMs exhibit
different cell aggregation behaviors anddifferent
cellularcharacteristics
whencultured on PMEA analogues with different
amounts of bound water. In the future, it isexpected to establish a scaffold material that
can improve the pulsatility of hiPS-CMs bycontrolling the amount of bound water on thescaffold surface.

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