An automated system for high-throughput
single cell-based breeding
SUBJECT AREAS:
ASSAY SYSTEMSLAB-ON-A-CHIP

Nobuo Yoshimoto1,2, Akiko Kida1, Xu Jie3, Masaya Kurokawa4, Masumi Iijima1, Tomoaki Niimi1,
Andrés D. Maturana1, Itoshi Nikaido5, Hiroki R. Ueda5, Kenji Tatematsu2, Katsuyuki Tanizawa2,
Akihiko Kondo6, Ikuo Fujii7 & Shun’ichi Kuroda1,2

HIGH-THROUGHPUT SCREENING
BIOMATERIALS-CELLS

Received
25 October 2012
Accepted
17 January 2013
Published
1 February 2013

Correspondence andrequests for materials
should be addressed to
N.Y. (n-yosi44@agr.
nagoya-u.ac.jp) or

1

Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8601, Japan, 2The
Institute of Scientific and Industrial Research, Osaka University, Mihogaoka, Ibaraki, Osaka 567-0047, Japan, 3Furukawa ElectricCo., Ltd., Yawata-kaigandori, Ichihara, Chiba 290-8555, Japan, 4STARLITE Co., Ltd., Kamitoyama, Ritto, Shiga 520-3004, Japan,
5Functional Genomics Unit, RIKEN Center for Developmental Biology, Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047,
Japan, 6Graduate School of Science and Technology, Kobe University, Rokkodai-cho, Nada-ku, Kobe, Hyogo 657-8501, Japan,
7
Graduate School of Science, Osaka Prefecture University, Gakuen-cho, Naka-ku, Sakai, Osaka 599-8570, Japan.

When establishing the most appropriate cells from the huge numbers of a cell library for practical use of cells
in regenerative medicine and production of various biopharmaceuticals, cell heterogeneity often found in an
isogenic cell population limits the refinement of clonal cell culture. Here, we demonstrated high-throughput
screening of the most suitable cells in a cell library by an automated undisruptive single-cell analysis andisolation system, followed by expansion of isolated single cells. This system enabled establishment of themost suitable cells, such as embryonic stem cells with the highest expression of the pluripotency marker
Rex1 and hybridomas with the highest antibody secretion, which could not be achieved by conventional
high-throughput cell screening systems (e.g., a fluorescence-activated cell sorter). This single cell-based
breeding system may be a powerful tool to analyze stochastic fluctuations and delineate their molecularmechanisms.

S.K. (skuroda@agr.
nagoya-u.ac.jp)

Cell-based technology is sustained by a wide variety of cell species, such as bacteria, yeast, insect and plant,
as well as animal and human cells, for research and industrial use. In particular, cells utilized inregenerative medicine and producing various biopharmaceuticals, such as cytokines, antibodies,
enzymes, proteins, peptides and metabolites, have significantly contributed toward human welfare. For thepractical use of biopharmaceuticals, it is essential to obtain the most appropriate cells from a candidate cell
population. Thus far, conventional cell screening systems have been based on colony isolation by assuming allcells in a colony possess homogeneous characteristics1,2. However, recent single cell-based analyses have revealed
that each cell in an isogenic cell population shows diverse and heterogeneous gene expression, morphology and/or cell proliferation3–5. For example, each cell of a mouse embryonic stem (ES) cell colony shows heterogeneous
expression of the ES marker protein Rex16. Humanized immunoglobulin G (IgG)-producing Chinese hamster
ovary (CHO) cells are a mixture of clones showing stochastic fluctuations in IgG production7. Thus, a morerational approach has been considered necessary to isolate and culture the most suitable cells from a huge numberof cell candidates by single-cell isolation and expansion (i.e., single cell-based breeding). Although we haveexamined the use of a fluorescence-activated cell sorter (FACS) as a practical approach, the recovered cells
suffered from mechanical stresses probably associated with high voltage and pressure, as well as chemical stressfrom the sheath solution for cell suspension. A conventional FACS system requires cell sample consisting of ,1 3105 cell population and containing more than 0.1% positive cells8. Considering the cell sample may be limited, alarge portion of the sample is required for FACS optimization, in which cell sample is not recovered. These
current issues of FACS technology prompted us to develop a novel system capable of isolating single positive cells
from less than 1 3 105 cells under undisruptive conditions. In this study, we have developed an automated singlecell analysis and isolation system to facilitate high-throughput isolation of fluorescently labeled mammalian cells
in an undisruptive and single cell-based manner. The robot is a stand-alone machine with a microchamber arraychip (containing ,2.5 3 105 cells) and an automated micromanipulator with a glass capillary and fluorescencemicroscope system, which isolates single positive cells from ,2.5 3 105 cells under undisruptive conditions. Here,
we report single cell-based selection and expansion of mouse ES cells with a homogeneous genetic background for
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the pluripotency marker gene Rex1 and hybridomas that highly
secrete antibody, using our automated single-cell isolation system.

Results
Development of the automated single-cell analysis and isolation
system. Recently, a microchamber array chip was developed, whichallows single-cell microarray analysis of a large cell population (,2.0
3 105 cells)8. Each microchamber (10 mm in diameter) is designed toaccommodate only one cell and enables weak fluorescence detection
at a high signal to noise ratio by excluding noise signals from negative

cells. However, both the fluorescence analysis and single-cell isolation of on-chip cells have been carried out manually. A fully automated single-cell isolation system may improve screening efficiency
for the most appropriate cell from a candidate cell population.
Therefore, we constructed a robot that executed the following procedures automatically. First, acquisition of the fluorescent intensity ofeach cell on the microchamber array chip. Second, permutation ofcells by the order of their fluorescent intensities. Third, physicalretrieval of desired cells with a glass capillary attached to a micromanipulator. Fourth, movement and release of isolated cells to the

Figure 1 | Overview of the automated single-cell analysis and isolation system. (a) Automated single-cell analysis and isolation system.
(b) Microchamber array chip made of polydimethylsiloxane (PDMS, 31,360 wells, 30 mm diameters). Left, overview (10 3 16 subareas); center, subarea(14 3 14 wells); right, wells. (c) Microchamber array chip made of polystyrene (PS, 256,000 wells, 10 mm diameters). Left, overview (20 3 32 subareas);
center, subarea (20 3 20 wells); right, wells. (d) Cell numbers of hybridoma in each 30-mm PDMS microchamber. Error bars 5 SD (n 5 6).
(e) Cell numbers of yeast in each 10-mm PS microchamber. Cell numbers of hybridoma in each 30-mm PDMS microchamber. Error bars 5 SD (n 5 6).
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microchamber array was scanned with a CCD camera on the robot. Basedon the fluorescent image, microchambers containing no or more than 2
fluorescent particles were excluded from further analyses. Green circles,
excluded microchambers; red circles, selected microchambers (step (c)). A
histogram of fluorescent intensity from each cell was generated for
permutation of cells by the order of fluorescent intensities, a list ofaddresses, fluorescent intensities and images, as well as a transmission
image of each cell was generated (step (d)). Cells of interest wereautomatically retrieved with a glass capillary attached to themicromanipulator. Recovery of each cell was repeated until the
fluorescence from each microchamber was absent (step (e)). Each retrieved
cell was transferred to an assigned reservoir well (step (f)).
Scale bars 5 200 mm (b) and 30 mm (e).

reservoirs of a multi-well plate (Figure 1a). Detailed specifications of
the robot were described in ‘‘Methods section.’’ Two types of microchamber array chips were fabricated with polydimethylsiloxane(PDMS) for mammalian cells (31,360 wells on a 1.39 3 2.23 cm2
total area; 30 mm diameter, 80 mm well-to-well pitch and 30 mm
well depth) (Figure 1b), and with polystyrene (PS) for small
mammalian or yeast cells (256,000 wells on a 1.39 3 2.23 cm2 total
area; 10 mm diameter, 30 mm well-to-well pitch and 10 mm welldepth) (Figure 1c). Various numbers of hybridomas were introduced into 30-mm microchambers by brief centrifugation. Approximately 80 , 90% of microchambers were occupied by single
hybridomas (Figure 1d). Similarly, yeast cells were introduced into
10-mm microchambers at comparable efficiency (Figure 1e).
Flowchart of the automated single-cell analysis and isolation
system. Cell manipulation by the robot was carried out as follows.
Cells were introduced into microchambers by brief centrifugation(Figure 2, steps a and b) and covered with culture medium, which
could be cultured for at least 24 h. The fluorescent intensities of 9,600
microchambers on a chip were measured by the robot for 30 s
(14 min for a 256,000 microchamber array chip) (step b; Supplementary video S1 online). Microchambers containing no or morethan 2 fluorescent particles were excluded from further analyses (stepc). Finally, a histogram together with a list of correlations between theposition and fluorescent intensity of each cell was generated (step d).
Cells of interest could be virtually marked in a descending/ascending/random order of fluorescent intensity. Marked cells
were automatically collected with a glass capillary attached to themicromanipulator of the robot, which were confirmed by elimination of fluorescence in the target microchamber (step e). Upon
failure, the robot automatically repeated the collection process. Eachcell was transferred and released into the culture medium of anassigned well in 96- or 384-well plates (step f). The reciprocal movement of the glass capillary required 15 s for each cell (Supplementary
video S2 online).

Figure 2 | Flow chart of the automated single-cell analysis and isolation
system. Approximately 5.0 3 104 cells in culture medium were added tothe microchamber array chip equipped with an aluminum frame(step (a)) and then introduced into 30-mm PDMS microchambers by brief
centrifugation (50 3 g, at room temperature for 1 min) (step (b)). The
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Single cell-based breeding of mouse ES cells. Among established ES
cell lines, the expression of pluripotency markers in each cell has
often been observed in a stochastic fluctuating state3,6. When ,5.0
3 104 cells of the mouse ES cell line OLG harboring the Oct4-EGFP
gene were introduced to 30-mm PDMS microchambers in our system, the cells showed variety of expression level of Oct4 (Figure 3a,
upper panel). The mouse ES cell line clone No. 10 harboring theRex1-EGFP gene showed an even higher degree of variety of
expression level of Rex1 (Figure 3a, lower panel), indicating thateach mouse ES cell line showed a distinct distribution of stemness9.
From the cell library of clone No. 10 mouse ES cells, 24 cells with thehighest fluorescent intensity were transferred to culture medium andallowed to proliferate from 1 to ,1,000 cells over 7 d (Figure 3b).
The daughter cells formed rounder colonies with increased homogeneous Rex1-EGFP expression, compared with that of parental
cells. After 2–3 weeks, 23 clones reached ,1 3 106 cells, in which3www.nature.com/scientificreports

Figure 3 | Single cell-based breeding of mouse ES cells. (a) Oct4-EGFP and Rex1-EGFP expression in mouse ES cell lines OLG and No. 10,
respectively, were analyzed by the robot. (b) Colony formation from isolated No. 10 cells (daughter cells). Scale bar 5 50 mm. (c) Rex1-EGFP expressionof isolated No. 10 cells. Approximately 2.0 3 104 cells were analyzed by FACS. Clone numbers are indicated in the upper-left. Contents of cells with higher
fluorescent intensity (over 103) are indicated in the upper-right. A549 (an adenosquamous lung carcinoma cell line) cells were used as a negative control.

20 clones retained a higher fluorescent intensity compared with thatof the parental cell population (Figure 3c). When calculating the ratioof highest numbers of cells with higher intensity (over 103) to thosewith lower intensity (102 , 2 3 102), the daughter cells of .7.0 ratio(mean 1 3SD of parental cells, n 5 6) were judged as a single-peakgroup. Finally, we obtained 5 clones expressing higher level of Rex1,
which would be suitable for further breeding process (Figure 3c).
This result indicated that single cell-based breeding of cells isolated
from a cell library is a powerful method to expand ES cells withthe highest expression of pluripotency markers. ES and induced
pluripotent stem (iPS) cells, particularly from humans, are often
susceptible to mechanical and chemical stresses10. The automatedsingle-cell isolation system is practical for isolating suitable cells
under undisruptive conditions because of gentle manipulation ofcells in culture medium with a glass capillary.
Single cell-based breeding of hybridomas. To evaluate the secretionof anti-rabbit lactate dehydrogenase IgG1 from the hybridoma cellline HyLDH/YK-1, we treated the hybridomas with brefeldin A (aSCIENTIFIC REPORTS | 3 : 1191 | DOI: 10.1038/srep01191

protein transport inhibitor)11 to accumulate the IgG intracellularly,
followed by FACS analysis. The isogenic cells were found to secretevarious amounts of antibody and showed a dynamic fluorescentintensity range between ,102 and ,105 (Figure 4a). A traditional
limiting dilution method to isolate mammalian cells secreting highamounts of biopharmaceuticals, deposits the cell library into 96- or
384-well plates at ,1 cell per well to form colonies. After culturing
for at least 1 month, the supernatants of these cell cultures are assayedby enzyme-linked immunosorbent assay (ELISA) to detect candidate
clones, followed by establishing the selected cells, which is time
consuming with low scalability (up to ,100 candidate clones per
screen). Recently, two automatic single-colony isolation systems
became commercially available (ClonePix FL (Molecular Devices,
Sunnyvale, CA, USA) and CellCelector (AVISO, Jena, Germany)).
Both machines facilitate manipulation of a large number of cells, but
approximately 1 month is still required for colony formation andidentification of candidate cells12,13. Using a cuboid microchamberarray as the single-cell container and the solid phase of the ELISA,
candidate cells can be manually detected after culturing cells for4www.nature.com/scientificreports

Figure 4 | Single cell-based breeding of hybridomas. (a) Antibody production by mouse hybridoma HyLDH/YK-1 cells was analyzed by FACS. HEK293
cells were used as a negative control. (b) Outline of the cell-surface FIA. Cells displaying trapping molecules, goat anti-mouse Fc antibody conjugated to
DOPE-PEG2000, were allowed to capture secreted antibodies and then treated with a FITC-labeled rabbit anti-mouse F(ab9)2 antibody for
detection. (c) Alexa 488-labeled trapping molecules on the surface of HyLDH/YK-1 cells. Scale bar 5 20 mm. (d) Cytotoxicity of the trapping molecule on
HyLDH/YK-1 cells. Cell viabilities were measured by trypan blue staining; error bars, p , 0.01, n 5 12 from three independent experiments.
(e) On-chip cell-surface FIA visualized by the robot. Scale bar 5 240 mm. (f) Histogram of antibody secretion determined by the cell-surface FIA. Blackvertical lines indicate fluorescence thresholds (range 50–200). White vertical lines indicate cells with high fluorescent intensities. (g) Antibody secretion
determined by a conventional ELISA. Seven cells formed colonies at ,10 d after single-cell isolation (inset, scale bar 5 100 mm) and were subjected to
ELISA at ,20 d. Error bars, p , 0.05, n 5 6 from three independent experiments.
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several days. The selected cells are then automatically isolated by
CellCelector13. However, both machines are unable to promptly
and automatically isolate the most suitable cell from a cell library,
and retain the issue of cell heterogeneity in the isogenic cell
population. Meanwhile, an automated single cell isolation system(Cellporter system; SC World, Toyama, Japan)14 has been developed, which is likely to utilize similar picking-up system to ourstudy. But, the machine requires additional scanning system (SCScanner) for measurement of fluorescent intensity of each cell,
indicating that Cellporter system is not a completely automatedsystem for cell analysis and isolation. To identify hybridomas that
secreted higher amounts of antibodies, we developed an undisruptivecell-surface fluorescence-linked immunosorbent assay (CS-FIA) toevaluate antibody production at a single-cell level using the robot(Figure 4b). The surfaces of secreting cells were modified with a goatanti-mouse Fc antibody conjugated to dioleoyl phosphatidylethanolamine-poly-ethylene glycol 2000 (DOPE-PEG2000) as a trapping molecule15,16. Trapping molecules spontaneously assembled
around the cell surface (Figure 4c) and showed no cytotoxicity forat least 2 weeks (Figure 4d). Cells were introduced into 30-mm PDMS
microchambers to allow the capture of secreted antibodies at the cellsurface, followed by incubation with a fluorescein isothiocyanate(FITC)-labeled rabbit anti-mouse F(ab9)2 F(ab9)2 to establish the
sandwich FIA on the cell surface (Figure 4e). After analyzing

on-chip cells using the robot, 362 cells exhibited higher fluorescence among the ,5.0 3 104 cells (Figure 4f). Eight cells withthe highest level of fluorescence were isolated. Seven cells formed
colonies within 10 d and expanded to ,5 3 105 cells over 2 weeks(Figure 4g). When evaluating the amount of antibody secreted into
the culture medium by ELISA, the daughter cells were found tosecrete higher amounts of antibodies, compared with that of theparental clone. This result indicated that single cell-based analysis
and isolation allowed us to obtain hybridomas with the highest
antibody-secreting ability in only 1 day. Because the CS-FIA technique is applicable to other cell types and various secreted biomaterials,
the combination of the automated single-cell isolation system with
the CS-FIA would be more effective to improve the productivity ofbiomaterials secreted from various cells.
Single cell isolation of adhesive cells. Since ES cells and hybridomas
are less adhesive and non-adhesive cells, respectively, we herein
examined if the robot is applicable to adhesive cells, such as CHO
cells. CHO cells (,1.0 3 104 cells) were trypsinized, stained with
CytoRed (fluorescent dye for viable cells; Dojin, Kumamoto, Japan)
and then introduced into 30-mm PDMS microchambers at
comparable efficiency of ES cells and hybridomas (Figure 5a).
Within 3 h, single CHO cells could be retrieved according to theirfluorescent intensities by the robot (Figure 5b), and proliferate to ,4Figure 5 | Single cell isolation of CHO cells. (a) Cell array for CHO cells. Cells were stained with CytoRed solution. Scale bar 5 160 mm. (b) Retrieval of
single CHO cell. Scale bar 5 25 mm. (c) Colony formation from isolated CHO cells. Scale bar 5 200 mm. (d) On-chip culture of CHO cells. Cells were
cultured on microchamber array chip and stained with PKH26. Scale bar 5 100 mm. (e) Retrieval of cell cluster of CHO cells. CHO cells stained with
PKH26 were trypsinized and retrieved with the robot. Scale bar 5 25 mm. (f) Colony formation from isolated cell cluster of CHO cells. Scale bar 5
200 mm.
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3 102 cells in 6 days (Figure 5c), of which the viability was more than
80% of retrieved single cells (20/24 clones). Furthermore, single CHO
cells transferred into 50-mm ellipsoidal PS microchambers could
propagate to several cells in microchambers (Figure 5d). After
24 h, the cells were stained with PKH26 (fluorescent dye for membrane staining; Sigma-Aldrich, St Louis, MO, USA), trypsinized
briefly, and then retrieved as a cell cluster by the robot (Figure 5e).
These cell clusters were allowed to grow up to ,2 3 102 cells in 5 days
(Figure 5f), of which the viability was nearly 100% of retrieved cellclusters (10/10 cell clusters). The automated retrieval of cell clusters
might be useful for the single cell-based breeding of vulnerable
adhesive cells.

Discussion
To manufacture biopharmaceuticals, each cell used for production(e.g., hybridomas and CHO cells) should express biomaterials stably
and efficiently. In regenerative therapies, each stem cell should
express pluripotency markers homogeneously to obtain differentiated cells with less tumorigenicity. It is an important step to isolate
the most suitable cell from a candidate cell library and expand the cell
population for further optimization. However, both isolation andexpansion have been traditionally performed with a cell colony,which impairs isolation of suitable cells because of potential difficulties in excluding contaminating unsuitable cells. Furthermore, longterm culture necessary for colony formation often impairs thefavorable properties of isolated cells7. For example, the productivityof established hybridomas is sometimes decreased by long-term cell
culture17. In established ES, iPS and mesenchymal stem cells, a trace
number of cells in colonies cannot fully differentiate, which sometimes leads to severe tumorigenesis18,19. These cellular instabilities aremainly caused by stochastic fluctuation of gene expression amongindividual cells3–7, which is considered a general phenomenon invarious cell types. Although cellular instabilities have been conventionally circumvented by contingency-based cell screenings (i.e., random screening of naturally occurring suitable cells) to establish cellswith low stochastic fluctuation, the molecular mechanisms underlying stochastic fluctuation should be delineated. For example, after
purifying two types of cells with high and low stochastic fluctuation
by single-cell isolation, a comparison of gene expression profiles
might delineate the mechanisms. Taken together, cells used for producing biopharmaceuticals and regenerative therapies should beestablished by single cell-based screening to maximize favorable
properties and minimize the occurrence of stochastic fluctuation.
Here, we demonstrated that an automated single-cell isolation
system facilitates prompt and effective establishment of the mostsuitable single cells from a large number of candidate cells to avoid
the occurrence of stochastic fluctuation. Mouse ES cells with thehighest expression level of the pluripotency marker Rex1 were successfully isolated from a heterogeneous cell population. Although 3
isolated ES cells (clones D07, A03, D03) showed lower expression
level of Rex1 at 2–3 weeks after single-cell isolation, 20 cells retained
higher Rex1 expression (see Figure 3c). Particularly, 5 isolated ES
cells (clones D06, A04, D08, C04, D04) were found to show homogeneous profile of Rex1 expression. In addition, hybridomas secreting the highest amount of antibodies were isolated by the robot in
combination with a CS-FIA technique. All isolated hybridomas
showed a higher expression level of antibody than that of parental
hybridomas at ,21 days after single-cell isolation (see Figure 4g).
These results indicate that single cell-based breeding is a practical
way to obtain suitable cells with fewer effects from stochastic fluctuation. Moreover, these established cells would be useful to decipher
the molecular basis of stochastic fluctuation.
Micromanipulation under a microscope, limiting dilution and
FACS have been widely used to isolate single cells. Micromanipulation is undisruptive and reliable for manipulating single cells,
but extremely laborious. Limiting dilution is also undisruptive, but
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not reliable because of possibly excluding suitable cells during serial
dilutions. As described in the introduction, FACS is a high-throughput and reliable system for isolating cell populations, but cannot
isolate a single positive cell from less than 1 3 105 cells under undisruptive conditions. In particular, the chemical and mechanical stresses intrinsically associated with FACS are likely to alter the gene
expression profiles of isolated single cells, thus single-cell manipulation should be carried out under undisruptive conditions. The issuesof these techniques for isolating single cells have led us to develop an
automated single-cell isolation system. The robot is designed to isolate a single positive cell from less than 1 3 105 cells in about 1 ml ofculture medium. Therefore, the automated single-cell isolation system is considered suitable for high-throughput single cell-based analysis and isolation.
In high-throughput identification of hybridomas that secrete highamounts of antibodies, candidate hybridomas can be selected by
FACS, followed by colony formation and evaluation with a conventional ELISA. Cell-surface markers (e.g., CD19, CD20, CD38,
CD138)20–24 expressed in hybridomas are indirectly labeled withfluorescence, and then populations of positive cells are collected as
candidate hybridomas by FACS. This method cannot evaluate theantibody-secreting ability of each hybridoma directly, and only concentrates candidate hybridomas. Another method is treatment ofcells with brefeldin A to accumulate antibodies intracellularly. Thismethod can evaluate the antibody-secreting ability, but also severely
affects cell viability. Thus far, no reported method can evaluate theantibody-secreting ability of hybridomas directly in an undisruptive
manner and without the requirement of long-term culture for colony
formation. Here, we developed a CS-FIA that fulfills the above criteria. Combination of the CS-FIA with the robot successfully shortened the time to identify positive hybridomas from ,1 month(FACS) or ,2 months (limiting dilution) to 1 day. To further optimize the CS-FIA, the use of fluorescence-labeled antigens as detection
molecules could improve the signal to noise ratio by reducing crossreactions among displayed antibodies.
In addition to single cell-based high-throughput analysis andisolation, our robot can be used for the following applications.
Because the robot can evaluate protein-protein interactions on thecell surface, orphan ligand screenings using cells displaying receptor
libraries, and orphan receptor screenings using cells expressing cellsurface-anchored forms of ligand libraries are applicable, which maylead to drug discovery. Moreover, because of the undisruptive condition during cell manipulation, the robot would be suitable forisolation of rare cells in limited clinical samples (e.g., circulatingtumor cells25 and cancer stem cells26) to develop an innovative diagnosis system. Thus, our automated single-cell isolation system may
offer a new technology for single cell-based engineering and bringsignificant progress to cell-based research and industries.

Methods
The automated single-cell analysis and isolation system. The robot consisted of a
micromanipulator (,1 mm movement accuracy) equipped with a pencil pump
(DENSO, Aichi, Japan) and glass capillary (40.5 mm inside diameter, 58 mm outside
diameter for mammalian cell; 14.5 mm inside diameter, 21 mm outside diameter for
yeast cell), a CCD camera (Nikon, Tokyo, Japan), an objective lens (Plan Fluor 10 3/
0.3, Nikon), an excitation light source (Nikon) using a super high pressure mercury
lamp and power supply, filter units (for FITC, 465–495-nm excitation, 515–555-nm
emission, 505 nm dichroic mirror; for Cy3, 510–560-nm excitation, 572.5–647.5-nm
emission, 565 nm dichroic mirror, Nikon), a 96- or 384-well reservoir plate, amicrochamber array chip and a control PC (CPU, Pentium M 1.8 GHz; RAM
980 MB). Microfabrication on the polymethylmethacrylate (PMMA) plate was
performed by X-ray mask and lithography technology combined with synchrotron
radiation8,27. Using the patterned PMMA plate as a master plate, a nickel (Ni) moldpart was produced by an electro-forming process resulting in effective production of
the Ni mold part with replicated micro-patterns. Then, the mold part was inserted
into a mold base and injected to produce a microchamber array chip with polystyrene
or PDMS. The robot identified a minimum 1 mm single particle at ,1800 MESF fromthe fluorescence of GFP, EGFP, phycoerythrin and propidium iodide. The control PC
regulated the vertical movement of the micromanipulator, the horizontal movement7

www.nature.com/scientificreportsof the microchamber array chip, fluorescence acquisition and transmission of images
to indentify cells of interest, followed by cell recovery using the pencil pump.
Cell culture. Mouse ES cell lines, OLG and clone No. 10, were cultured in Glasgow
Minimum Essential Medium (Sigma-Aldrich, St Louis, MO, USA) supplemented
with 10% fetal bovine serum (FBS; StemCell Technologies, Vancouver, BC, Canada),
1 mM sodium pyruvate, 0.1 mM non-essential amino acids (NEAA; Invitrogen Life
Technology, Carlsbad, CA, USA), 32.7 mM sodium bicarbonate, 0.1 mM 2mercaptoethanol and 1,000 U ml21 leukemia inhibitory factor (Invitrogen). The
mouse hybridoma cell line HyLDH/YK-1 secreting anti-rabbit lactate dehydrogenase
IgG1 was cultured in RPMI-1640 (Nacalai, Kyoto, Japan) supplemented with 10%
FBS (Thermo, Waltham, MA, USA), 0.1 mM NEAA and 0.05 mM 2mercaptoethanol. Human embryonic kidney (HEK) 293 and lung adenocarcinoma
A549 cell lines were maintained in Dulbecco’s modified Eagle’s medium
supplemented with 10% FBS (PAA Laboratories, Pasching, Austria). Dihydrofolate
reductase-deficient Chinese hamster ovary (CHO) cells (CHO-DG44 cells) weremaintained in RPMI-1640 supplemented with 10% FBS (PAA Laboratories).
Flow cytometric analysis. Daughter mouse ES cells isolated by the robot were
cultured for 2–3 weeks to reach ,1 3 106 cells, and then harvested by trypsinization.
The fluorescent intensities of ,2.0 3 104 cells from each clone were measured with a
BD FACSCanto II flow cytometer (BD, Franklin Lakes, NJ, USA). Parental mouse ES
and A549 cells were used as positive and negative controls, respectively. HyLDH/YK1 cells treated with 1 mg ml21 brefeldin A (BD) for 6 h were fixed in PBS containing
4% paraformaldehyde (PFA) at room temperature for 15 min, permeabilized with
PBS containing 0.03% Triton X-100 at room temperature for 5 min and thencollected by centrifugation (200 3 g, 5 min). Cells were then incubated with
2 mg ml21 FITC-conjugated anti-mouse F(ab9)2 rabbit F(ab9)2 (Rockland,
Gilbertsville, PA, USA) in PBS at room temperature for 20 min, followed by FACS
analysis. HEK 293 cells were used as a negative control.
Preparation of trapping molecules. For the cell-surface FIA, 1.65 nM Immunopure
anti-mouse Fc goat polyclonal antibody (Thermo) was reacted with 66 mM DOPEPEG2000-N-hydroxysuccinimide (NHS; NOF, Tokyo, Japan) in 100 ml sodium
phosphate (10 mM, pH 7.6) and 250 mM NaCl at room temperature for 10 min. Free
DOPE-PEG2000-NHS molecules were removed by ultrafiltration through 50 kDa
cut-off membranes (Millipore, Billerica, MA, USA). Coupling of DOPE-PEG2000 to
the antibody was confirmed by SDS-PAGE and Coomassie Brilliant Blue R-250
staining. To observe trapping molecules on the HyLHD/YK-1 cell surface, ,2.0 3 104
cells were combined with 100 ml serum-free RPMI-1640 medium containing 16 nM
trapping molecules, incubated at 37uC for 10 min and then fixed with 4% PFA atroom temperature for 20 min. Cells were washed with PBS twice, incubated in 100 ml
PBS containing 400 ng Alexa 488-conjugated anti-goat IgG antibody (Invitrogen) atroom temperature for 20 min, washed with PBS twice and then observed under aconfocal laser-scanning microscope (FV-1000D; Olympus, Tokyo, Japan). To
evaluate the cytotoxicity of the trapping molecule (DOPE-PEG2000-antibody) in
HyLDH/YK-1 cells, ,1.25 3 105 cells in 2.5 ml culture medium were treated with thetrapping molecule at 0.16–16 nM final concentration (as a protein concentration)and then maintained at 37uC with 5% CO2 for 2 weeks. Cell viabilities were measured
by trypan blue staining.
Cell-surface FIA. Approximately 5.0 3 104 HyLDH/YK-1 cells were combined with
100 ml serum-free RPMI-1640 medium containing 16 nM trapping molecules,
incubated at 37uC for 10 min, mixed with 900 ml serum-free RPMI-1640 medium
and then placed into microchambers by brief centrifugation. After addition of 100 ml
FBS, cells on the microchamber array chip were incubated for 30 min in a CO2
incubator to allow antibody secretion and then treated with 2 mg ml21 FITC-labeledanti-mouse F(ab9)2 F(ab9)2 antibody to establish the cell-surface FIA. Afterincubation at room temperature for 10 min, cells were briefly washed twice withmedium and then subjected to the automated single-cell analysis and isolation
system.
Mouse IgG ELISA. Each well of a MaxiSorp 96-well plate (Thermo) was treated with
30 ml anti-mouse Fc antibody (10 ng ml21) at 4uC for 24 h, washed with 200 ml PBS
containing 0.1% (vol/vol) Tween 20 (PBS-T) five times and then treated with 200 ml
bovine serum albumin (5 mg ml21) in PBS at room temperature for 1 h. To measure
the amount of secreted antibody from HyLDH/YK-1 cells isolated by the robot, eachsample was added to an anti-mouse Fc-immobilized well, incubated at room
temperature for 1 h and then washed with 200 ml PBS-T five times, followed byaddition of 200 ml PBS-T containing 0.2 ml horse radish peroxidase-conjugated antimouse Ig (GE healthcare, Little Chalfont, Buckinghamshire). Plates were incubated atroom temperature for 1 h and then washed with PBS-T five times, followed byaddition of 100 ml 3,39,5,59-tetramethylbenzidine substrate (Thermo). The coloration
reaction was performed at room temperature for 30 min, stopped by 100 ml H2SO4(2 M) and then the absorbance at 450 nm was measured.
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Acknowledgments
This study was supported in part by The Japan Science Society (H22 to N.Y.), Adaptable &Seamless Technology Transfer Program through Target-driven R&D (A-STEP) by theJapan Science and Technology Agency (JST) (AS231Z04687F to N.Y., AS2311699F to S.K.),
Regional New Consortium Projects (H15 to S.K. and H17 to I.F., METI, Japan), theProgram for Promotion of Basic and Applied Researches for Innovations in Bio-oriented
Industry (H22-7, BRAIN to S.K., K. Tanizawa), the Health Labor Sciences Research Grant
from the Ministry of Health Labor and Welfare (to SK), a Grant-in-Aid for Scientific
Research (A) (21240052 to S.K.), the As One Corporation (to S.K., A. Kondo, I.F.), theSuzuken Memorial Foundation (10-013 to S.K.), the Nagase Science and Technology
Foundation (H22-1 to S.K.) and the Canon Foundation (H22-1-7 to S.K.). We thank Satoshi
Sugiyama and Ken-ichi Kimura (Furukawa) for technical support, and Masahiro
Matsushita and Kenji Uemukai (As One) for advice.

Author contributions
N.Y. and S.K. conceived the project, designed and performed experiments, processed andanalyzed data and wrote the manuscript. A. Kida, M.I., T.N., A.D.M. and K. Tatematsu8www.nature.com/scientificreports
processed and analyzed data. X.J. and M.K. developed the robot and chips. I.N. and H.R.U.
prepared mouse ES cell samples. A. Kondo, I.F., K. Tanizawa and S.K. coordinated the
project.

Competing financial interests: The authors declare no competing financial interests.

Additional information

How to cite this article: Yoshimoto, N. et al. An automated system for high-throughput
single cell-based breeding. Sci. Rep. 3, 1191; DOI:10.1038/srep01191 (2013).

Supplementary information accompanies this paper at http://www.nature.com/
scientificreports

SCIENTIFIC REPORTS | 3 : 1191 | DOI: 10.1038/srep01191

License: This work is licensed under a Creative Commons
Attribution-NonCommercial-NoDerivs 3.0 Unported License. To view a copy of thislicense, visit http://creativecommons.org/licenses/by-nc-nd/3.0/

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