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ISSN: 2155-9821

Journal of Bioprocessing & Biotechniques

Nomura et al., J Bioproces Biotechniq 2011, S:3
DOI: 10.4172/2155-9821.S3-003

Research Article

Open AccessLabel-Free Shape-Based Selection of Cardiomyocytes with on-Chip
Imaging Cell Sorting System
Fumimasa Nomura, Tomoyuki Kaneko, Akihiro Hattori and Kenji Yasuda*
Department of Biomedical Information, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda, Tokyo 1010062, Japan

Abstract

We have examined the method for the label-free optical microscopic image-based selection of cardiomyocytes
from the mixture of collagenase-digested embryonic mouse heart cells using the on-chip imaging cell sorter system,
in which 1/2000 s real-time high-speed camera-based phase-contrast cell image recognition was performed. To
separate the cardiomyocytes from other cells including fibroblasts, we distinguished the roughness of the cell surface
quantitatively as the index of sorting. The selected cells with smooth surface (roughness index not more than 1.1) for
cardiomyocytes, and confirmed that the more than 80% of the collected and recultivated cells were self-beating and the
99.2% of them were identified as cardiomyocytes by immunostaining. The results indicates the potential of the labelfree cell purification using imaging cell sorting system, especially for cardiomyocytes by the index of their cell surface
roughness, which might be applicable for purification of cardiomyocytes for regenerative medicine.

Keywords: On-chip imaging cell sorting system, Cardiomyocyte, Index of roughness, Agarose microchamber
Introduction
The non-invasive purification technique of the particular target cells
from tissues, organs or cultivated cells is the essential and fundamental
for cell-based research and even for regenerative medicine. For
example, for the purification of cardiomyocytes, we need to remove
more than half of the noncardiomyocytes from whole heart cells [1].
At present, the most commonly used method for cardiomyocyte
purification is the pre-cultivation method exploiting the difference of theadhesion of cardiomyocytes and other cells including fibroblasts to thesurface of cultivation dish [2]. However, the method was probabilistic
way and is still regarded to be a rough purification. Another more
precise purification method is the fluorescence-activated cell sorting(FACS) [3] with fluorescent cardiomyocyte marker labeling, whereas
it involves the potential problems of cell damages caused by antibody
labeling. To overcome this labeling problem, we have proposed anddeveloped the on-chip imaging cell sorting system for the label-freephase-contrast imaging-based identification of cell types using theindex of cell surface shapes. Different shapes with 1/2000s real timeprocessing [4, 5]. In this paper, as a practical application of imaging cellsorting, we have examined the purification efficacy of cardiomyocytes
cells with using of the index of cell surface roughness and reported.

Materials and Methods

one is for waste drain and the other is for target cell collection. A pairof 1.5 % agarose gel electrodes containing 0.5 M NaCl (carrier of ioniccurrent) was also arranged at the connecting point of two laminar flows
for cell separation to shift target cells from the sample laminar flow
to the buffer laminar flow through their boundary by electrophoretic
force.
First for separation of target cells from the mixture of cells, thesamples were applied to the sample inlet (Figure 1b) and put the airpressure both to the sample inlet and the buffer inlet to introduce thecells into the central separation area with keeping the boundary of
two laminar flows by maintaining the same flow velocities of medium
buffers (Figure 1c). The phase-contrast real-time images of the cells
flew in the sample flow channel were directly monitored with 2000
pictures/s intervals, and were analyzed to acquire specific indexes (cellsize, cross-sectional area, cell surface roughness) of cells using the fieldprogrammable gate array (FPGA) real time image processing unit. The
system can record both of the cell image pictures and FPGA analysis
data simultaneously for the further checking and evaluation.
Then, when the target cells which satisfy the required values of
the indexes were recognized, they were sorted by shifting them fromleft laminar flow to right laminar flow by the computer-controlled
electrostatic force generation between two gel electrodes, whicharranged perpendicular to the flow direction at the separation area, by
the switching DC power supply.
Finally, only the recognized and shifted target cells were moved

Imaging cell sorter system
The on-chip phase-contrast imaging cell sorter system wasconsisted of the following five modules; [4, 5] a cell sorting chip, an
optical microscope equipped with a phase-contrast image processing
module, a 1/2000 s high-speed charge-coupled devise (CCD) camera(HAS-220; DITECT, Japan), image processing unit, and a computercontrolled switching DC power supply for cell separation (Figure1a). The cell sorting chip was made of poly dimethylsiloxane (PDMS)
attached on a 0.9 mm thin glass slide, in which two inlets and flow
channels for sample solution and buffer solution were symmetricallyarranged. The generated two laminar flow in the two channels wereconnected at the cell separation area in center of the chip, and the twolaminar flows were divided to the symmetrical two flow channels again,
J Bioproces Biotechniq*Corresponding author: Kenji Yasuda, Department of Biomedical Information,
Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University,
2-3-10 Kanda-Surugadai, Chiyoda, Tokyo 101-0062, Japan E-mail: yasuda.bmi@
tmd.ac.jp
Received September 14, 2011; Accepted December 05, 2011; Published
December 07, 2011
Citation: Nomura F, Kaneko T, Hattori A, Yasuda K (2011) Label-Free ShapeBased Selection of Cardiomyocytes with on-Chip Imaging Cell Sorting System. J
Bioprocess Biotechniq S3:003 doi:10.4172/2155-9821.S3-003
Copyright: © 2011 Nomura F, et al. This is an open-access article distributed under
the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author andsource are credited.

Cellomics and Tissueomics

ISSN:2155-9821 JBPBT, an open access journalCitation: Nomura F, Kaneko T, Hattori A, Yasuda K (2011) Label-Free Shape-Based Selection of Cardiomyocytes with on-Chip Imaging Cell Sorting
System. J Bioprocess Biotechniq S3:003 doi:10.4172/2155-9821.S3-003

Page 2 of 6
mM Na2HPO4, 1.5 mM KH2PO4, pH 7.4) containing 0.9 mM CaCl2
and 0.5 mM MgCl2 to induce heart contraction and remove corpuscles.
The hearts were then transferred to PBS without CaCl2 and MgCl2 andminced into 1-mm3 pieces with fine scissors, and incubated in PBS
containing 0.25% collagenase (Wako, Osaka, Japan) for 30 min at 37 ºC
to digest the ventricular tissue. After this procedure was repeated twice,the cell suspension was transferred to a cell culture medium (DMEM[Invitrogen Corp, Carlsbad, SC, USA] supplemented with 10% fetal
bovine serum, 100 U/ml penicillin, and 0.1 mg/ml streptomycin) at
4 ºC. The cells were filtered through a 40-μm-nylon mesh and were
centrifuged at 180 g for 5 min at room temperature. Then, the cell pellet

from the waste drain channel flow to the target sample collection
channel flow, and the target cells were acquired at the target cellreservoir arranged at the end of the target sample collection channel(Figure 1b and 1c).

Sample preparation
The cardiomyocytes were isolated from 12- to 13-day-old mouseembryos (ICR) (Saitama Experimental Animals Supply, Japan) [6]. A
mouse was anesthetized with diethyl ether and embryos were rapidly
removed. The hearts of the embryos were removed and then washedwith phosphatebuffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 8(a)DC power supply

(c)

(b)

Sampleinjection〇－＋〇

Sample inlet

Air pressure
Flow channel

Downstream
reservoir
Outlet tubes(Cultivationchamber)

Cell sorting
chip

Obj. lens

Image
Processing unit

Downstream
reservoir
Sample outlet(target cells)

Cardiomyocytes

High-speed camera

Buﬀer channel(d)Sample channel
Gel electrode
Waste drain channel(e)Target sample collection channel

Figure 1: Selection of cardiomyocytes with on-chip cell sorting system.
(a) System design of on-chip cell sorting system. (b) Chip design of on-chip cell sorting system. (c) Cross sectional view of the chip. (d) The time course imagesof cell sorting area during the collection of selected cells. Each image was obtained 33ms. Bar, 100 μm. (e) The time course images of cell sorting area during theremoving the non-selected cells. Each image was obtained 33ms. Bar, 100 μm.

Rough surface

Smooth surface
(a)

(b)

(d)

(c)

(e)

(f)

Original images

Binary images

Roughness (R)

1.04

1.04

1.05

1.20

1.22

1.23

Figure 2: Selection of cardiomyocytes with on-chip cell sorting system.
(a)-(c) Acquired images and sorting indices of smooth surface cells, and (d)-(f) rough surface cells. Bar, 10 μm.

J Bioproces Biotechniq

Cellomics and Tissueomics

ISSN:2155-9821 JBPBT, an open access journalCitation: Nomura F, Kaneko T, Hattori A, Yasuda K (2011) Label-Free Shape-Based Selection of Cardiomyocytes with on-Chip Imaging Cell Sorting
System. J Bioprocess Biotechniq S3:003 doi:10.4172/2155-9821.S3-003

Page 3 of 6
Sample(pixel)Area (pixel)

Perimeter(pixel)Radius(pixel)have defined the index of roughness of cell surface R, which compare
the real perimeter of cell surface and the estimated perimeter of cellsurface by approximation of sphere model, to distinguish the surface
roughness of cardiomyocytes and other cells quantitatively as,
l
R=
					
(1),
4π S
Where l is the perimeter of cell surface, S is the cross-sectional areaof cell (both of l and S were acquired from pixel-based digitized image
analysis of cells).

Circumference (pixel)

(a)

4232

240.6

36.7

230.6

1.04

(b)

3203

209.1

31.9

200.6

1.04

(c)

2767

195.2

29.6

186.4

1.04

(d)

2431

209.2

27.8

174.7

1.20

(e)

3787

265.0

34.7

218.1

1.21

(f)

2947

236.1

30.6

192.4

1.22

Table 1: Quantitative evaluation of cell surface roughness (Fig. 2).

was resuspended in a cell culture medium and incubated at least 20 min
in room temperature.

For

The cells were fixed with 4% paraformaldehyde and permeabilized
in 0.1% Triton X-100 and exposed for 2 hours to the mouse monoclonal
antibody to heavy chain of cardiac myosin (abcam, Japan) dissolved inblocking buffer (PBS containing 1% BSA) and incubated for 1 hourat room temperature with Alexa Fluor 546, goat anti-mouse IgG(Molecular probes, Eugene, USA). To visualize the nuclei, cells werecounterstained with Hoechst 33342 for 30 min at room temperature.
The cell images were recorded using an inverted microscope equipped
for epifluorescence (IX-70, Olympus, Japan) using cooled CCD camera(ORCA-ER, Hamamatsu photonics, Japan).

For the imaging cell sorting, the particular index of cell images isessential for their separation. Regarding separation of cardiomyocytes
from the mixture, we have already examined and found that the mostof the smooth round shaped cells should cardiomyocytes). Hence, we

7
13
19

(b)
1

3

4

5

6

8

9

10

11

12

15

14
20

21

2

17

16

3

radiusr

should

have

18

23

22

with

There are two major points for accomplishing the non-labeledseparation of cardiomyocytes from the mixture of enzyme-treatedheart extracts. One is the development of the proper quantified index
of surface roughness of cells for judging the target cells and others as
described above, and the other is the proper enzymatical treatment
for appearance of surface shape differences to be able to distinguishdifferent cells. Regarding the cardiomyocyte purification, based on our
experience, [11] the collagenase-digestion treatment was effective toappear the difference of smooth/rough surface shapes depending on
their difference of cell types. Hence, in this experiment, the sample

Index for distinguish the surface roughness2

sphereCardiomyocyte separation using the index of roughness R

Results and Discussion

1aR should be increased in proportion to the increase of the real
perimeter. Using the above index R, we can quantify the smoothness/roughness of cell surfaces. For example, as shown in Figure 2 and Table
1, the typical smooth cells have the index of roughness R less than 1.1(Figures 2a, 2b and 2c), and the typical rough cells have R larger than
1.1 (Figure 2d, 2e, and 2f). Table 1 also shows the acquired values ofeach cell calculated by the FPGA unit calculation from the captured
cell images.

Immunostaining of cardiomyocytes(a)example,R = [2π r / (4π × π r 2)1/ 2] equal to 1, and when the roughness increased,

24

4

5

6

7

8

9

10

11

12

Original
image
Binary
image
Area

560

625

694

577

517

736

645

525

713

593

516

646

Perimeter

90.67

91.50

106.57

89.50

84.67

100.91

92.33

85.84

98.33

90.67

85.26

93.98

R

1.08

1.03

1.14

1.05

1.05

1.05

13

14

15

16

17

18

1.03
19

1.06

1.04

20

21

1.05
22

1.06

1.04

23

24

Original
image
Binary
image
Area

785

419

566

656

737

797

504

591

806

485

572

610

Perimeter105.15

79.67

87.84

103.98101.40104.23

82.43

90.33

105.98

82.67

90.91

92.08

R

1.06

1.06

1.04

1.15

1.05

1.04

1.04

1.05

1.05

1.06

1.07

1.05

Figure 3: Representative images and indexes of selected smooth surface cells.
(a) Phase-contrast image of arrangement of the selected smooth surface cells settled on agarose microchamber at one by one manner using micropipette aftercell sorting purification. (b) Phase-contrast image and FPGA calculated index of cells in Figure 3a.

J Bioproces Biotechniq

Cellomics and Tissueomics

ISSN:2155-9821 JBPBT, an open access journalCitation: Nomura F, Kaneko T, Hattori A, Yasuda K (2011) Label-Free Shape-Based Selection of Cardiomyocytes with on-Chip Imaging Cell Sorting
System. J Bioprocess Biotechniq S3:003 doi:10.4172/2155-9821.S3-003

Page 4 of 6
1

(a)

(b)
Original
image

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

1

2

3

4

5

6

7

8

9

10

11

12

Binary
image
Area

535

600

570

528

416

388

396

383

565

521

489

890

Perimeter

87.50

99.15

103.74

97.98

93.64

92.23

89.15

84.57

121.54103.8891.74

181.72R
Original
image

1.07

1.14

1.23

13

14

15

1.20
16

1.30
17

1.32
18

1.26

1.22

1.44

19

20

21

1.28
22

1.17
23

1.72
24

Binary
image
Area

670

639

403

460

771

552

703

638

463

801

530

543

Perimeter105.30107.88

75.01

84.67

117.8894.57

129.68110.81

90.57

129.44106.13102.47

R

1.15

1.20

1.05

1.11

1.20

1.14

1.38

1.24

1.19

1.29

1.30

1.24

Figure 4: Representative images and indexes of removed rough surface cells.
(a) Phase-contrast image of arrangement of the selected rough surface cells settled on agarose microchamber at one by one manner using micropipette after cellsorting purification. (b) Phase-contrast image and FPGA calculated index of cells in Figure 4a.

(a)

(d)

(b)

(e)

(c)

(f)

Figure 5. Representative images of selected cells in agarose microchambers.
(a) Phase-contrast image of arrangement of the selected smooth cells on agarose microchamber at one by one manner using micropipette (same image of Figure 3a).
(b) Phase-contrast image of selected cells after 2 days cultivation. White arrowhead shows a shrinked cell. White arrow shows the microchamber in which the cell wasdisappeared during cultivation caused by apoptosis or inufficient attachment. (c) Phase-contrast image superimposed on the fluorescence image of the selected cells(2 days cultivation) after fixation with 4% Formaldehyde solution. Nucleus (Hoechst33342; blue), heavy chain cardiac myosin (red). White double arrowheads show the
no-stained cells by cardiac myosin (i.e., non cardiomyocytes). (d) Phase-contrast image of arrangement of the selected rough cells on agarose microchamber at one
by one manner using micropipette (same image of Figure 4a). (e) Phase-contrast image of selected cells after 2 days cultivation. White arrowhead shows shrinkagecell. (f) Phase-contrast image superimposed on the fluorescence image of the selected cells (2 days cultivation) after fixation with 4% Formaldehyde solution. Nucleus(Hoechst33342; blue), heavy chain cardiac myosin (red). Bars, 100 μm.

J Bioproces Biotechniq

Cellomics and Tissueomics

ISSN:2155-9821 JBPBT, an open access journalCitation: Nomura F, Kaneko T, Hattori A, Yasuda K (2011) Label-Free Shape-Based Selection of Cardiomyocytes with on-Chip Imaging Cell Sorting
System. J Bioprocess Biotechniq S3:003 doi:10.4172/2155-9821.S3-003

Page 5 of 6
0 DIV

2 DIV

staining of cardiac myosin

selected cellbeating cellno beating cell

shrinkage cell

removal cell

positive cell

cell number

242

178

19

12

33

195

negative cell
2

ratio (%)

100

73.6

7.9

5

13.6

80.6

0.8

Table 2: Quantitative evaluation of cardiomyocyte ratio

mixture of cells were acquired from collagenase-digested heart isolated
from 13-day-old mouse embryos (ICR). It should be noted for thisprocedure that the collagenase-digestion procedure and following
incubation before cell sorting is essential for appearing the differenceof smooth/rough surface of cells. Otherwise, if we use the conventional
trypsinization procedure for cell preparation, cells do not show
apparent smooth/rough surface shape differences. And the incubation
after the collagenase-treatment at least 20 min was also essential forappearing their shape differences. Without this incubation, the shapedifference was not sufficiently appeared on the surface of cells.
The functional sorting procedure of cardiomyocytes using above
on-chip imaging cell sorter system with this imaging index R was asfollows; (1) when the target smooth cells (R ≤ 1.1) flew into the sorting
area, the target cell was recognized by high-speed camera and a pulse
DC voltage was applied between a pair of agarose gel electrodes to shift
the target cells from the sample flow channel to the buffer channel tointroduce to the target sample collection channel (Figure 1d); (2) on
the other hand, when other rough cells (R > 1.1) comes into this area,
no electrostatic force was applied and the rough cells were introduced
into the waste drain channel (Figure 1e).
Each of the collected smooth surface cell in the target cell reservoir
was transferred into the 5-μm-thick 2 % (w/v) agarose (GenePure
LowMelt ISC BioExpress) microchamber [7, 8] laid on the collagencoated polystyrene cultivation dish at one by one manner [9, 10]
using micropipette (Figures 3, 4 and 5), and cultivated in a cell culture
medium (Invitrogen DMEM supplemented with 10 % fetal bovine
serum, 100 U/mL penicillin, and 100 mg/mL streptomycin) at 37 °C
with a humidified atmosphere of 95 % air and 5 % CO2.
Figure 3 shows the 24 samples of the typical target smooth cells
acquired from cell sorting system. As shown in Figure 3a, each cell
was put into the microchamber to isolate and identification of cellfor identification of their characteristics after cultivation. Figure 3b
shows the micrographs and the FPGA acquired indexes of the collectedsmooth cells. The index R of all the smooth cells except for the two
coagulated smooth cells (see no. 3 and 16) showed less than 1.08. The
samples of coagulated smooth cells (No. 3 and 16) were manuallyacquired from the rough sample cell reservoir to indicate the potential
error of the index R for appropriate smooth cells. The results indicatethat the visualized micrograph-based identification of smooth cells issuitable match to the index R < 1.1.
Figure 4 also shows the 24 samples of the typical rough surface cells
acquired from cell sorting system. Just same as Figure 3, each cell was
put into the microchamber (Figure 4a) and indicated the index R larger
than 1.1 except for the samples no. 1 and no. 15, which were manually
collected from the smooth sample reservoir to indicate the potential
error of the index R for appropriate rough cells, too. In the results, somerough surface cells were judged R< 1.1 using FPGA procedure. One ofthe possible reasons and possible improvement for overcoming thisproblem is using of higher magnitude of objective lens for acquiring
more precise perimeters.
J Bioproces Biotechniq

Evaluation of the index of surface roughness for characterizing
cardiomyocytes
After two days cultivation of the smooth surface cells (Figure 5b) andthe rough surface cells (Figure 5e) laid in the agarose microchambers,
we first categorized the cultivated cells by their ability of beating, andalso by their adhesion abilities such as shrinking and stable expansion.
For example, as shown in Figure 5b, a cell at the white arrowhead wasshrinking, and a cell at the white arrow was disappeared. Moreover,as shown in Figure 5c, after the staining of myosin heavy chain ofcells as the index of cardiomyocyte protein, a cell at the white doublearrowheads was not stained, whereas all the remaining 21 cells in the
24 microchambers were well cultivated and showed their beating. In
contrast, as shown in Figures 5d, 5e and 5f, most of rough surface cells
were not attached on the surface of cultivation dish and disappeared(white arrows). Even the attached cells did not stained by myosin heavy
chain probes (white double arrowheads) except for the rough surface
sample no. 1, having the index R = 1.07.
In the experiment, from all of the 242 smooth cells, beating cells
were 178 (73.6%), no-beating cells were 19 (7.9%), shrunk cells were 12(5.0%), unattached cells were 33 (13.6%), respectively (Table 2).
As shown in Figure 5c, cardiac myosin positive-staining cells
were 195 (99.99% of healthy cultivated cells; 80.6% of whole collectedsmooth cells), and the negative-staining cells were 2 cells (0.01% of
healthy cultivated cells; 0.8% of whole cells) (Table 2). That means,even the cells which were not beating were cardiomyocytes except for
two cells from 197 cells.
Hence, the results indicated that at least more than 80% of thecollected cells were beating cardiomyocytes, and more than 99.99%
of healthy cultivated cells were cardiomyocytes. The shrinkage andunattachment in our re-cultivation procedure might be caused by theinsufficient removal of agarose coating of cultivation microchamberarray from collagen layer. It should be noted that, although we did
not count into the myosin-positive cells, even the shrinked cells in
microchambers showed the positive-staining (see the white single
arrowhead in Figure 5c).
In conclusion, we have succeeded in separating cardiomyocyteswith label-free on-chip imaging cell sorting procedures with using of
the index of their surface roughness, and the results indicated that themore than 80% of the smooth cells were beating cardiomyocyte cells,
and also more than 99% of healthy re-cultivated cells were cardiac
myosin positive cells. The results suggested that the non-labelingsimple phase-contrast image-based cell sorting has a potential toseparate cardiomyocytes precisely from the mixture of heart cells by
the index of cell surface roughness with less than 1 % error rate.
Acknowledgements
This work was financially supported by New Energy Development Organization(NEDO).

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Cellomics and Tissueomics

ISSN:2155-9821 JBPBT, an open access journalCitation: Nomura F, Kaneko T, Hattori A, Yasuda K (2011) Label-Free Shape-Based Selection of Cardiomyocytes with on-Chip Imaging Cell Sorting
System. J Bioprocess Biotechniq S3:003 doi:10.4172/2155-9821.S3-003

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This article was originally published in a special issue, Cellomics and Tissueomics handled by Editor(s). Dr. Masamichi Kamihira, Kyushu University,
Japan.

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Cellomics and Tissueomics

ISSN:2155-9821 JBPBT, an open access journal