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Damage-less Sorting System for Live Cells

INTRODUCTION

Research on live cells, especially on human embryonicstem (hES) cells and stem cells, is crucially important forregenerative medical treatment, clarification of oncogenesis mechanism and tailor-made medical treatment. A system, which can acquire such kinds of living cells in goodcondition, will be a very useful tool for medical researchers worldwide. Taking advantage of its proprietary technologies in fiber optics and mechatronics, Furukawa
Electric has developed and commoditized a flow cytometer (cell sorter) that features damage-less sorting for thefirst time in the world. Here we will describe the featuresand performance of our new product.

20015010050FEATURES010110210

341010FL1Figure 2

Measurement results of fluorescence sensitivity.

6Sample size

56 µm
4310 µm
23 µm
12 µm
000.025

0.05

0.075

0Transmission light (arbitrary unit)

Statistic▲

2.1 Fiber-Optic Flow Cytometer
A flow cytometer is an analytical apparatus, which isemployed to identify in a short time various kinds of cells
existing in a cell population, whereby single cells whichflow in a flow cell are illuminated by an exciting light beam
to emit the scattered and fluorescent light signals, andthese light signals are analyzed to achieve cell analysis.
We have developed an entirely new fiber optic flow cytometer that directly connects optical fibers to the flow cellwith the aim of both laser light illumination and fluorescentand scattered light reception. Figure 1 shows the structure of the optics system. Figure 2 shows the measurement results of 8 kinds of fluorescent beads with different
fluorescent intensity spectra, demonstrating that the fluorescence sensitivity of our system is below 200 MESF
(molecules of equivalent soluble flourochrome).
Moreover this optics structure allows for obtaining new
information from the transmission light (which corre-Side scatter light (arbitrary unit)

2.

sponds to the projection area of cells) instead of the forward scatter light conventionally used, taking advantageof the fact that the cells to be measured and the endfaceof the optical fiber for reception are in close proximity withFrequency

1.

1000000
100000New measurement information:transmission light
Optical ﬁber

488 nm laser
635 nm laser

Photodetector
for transmission light

No intervention of air layer,resulting in high sensitivity
SheathUse of exclusive ﬁber for each exciting light ﬂow
No need for optical axis alignment
Ease of downsizing permitting laser source addition

Figure 1

10000

MESF

Direct illumination andreception using optical ﬁber

Photomultiplier tube
for side scatter light

1000
100
10

Photomultiplier tube
for ﬂuorescence light
Sample ﬂow

Structure of flow cytometer using optical fiber.

1approximationmean110100

1000

10000

FL1Figure 3

Evaluation results of transmission light information.

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Damage-less Sorting System for Live Cells

EVALUATION EXAMPLES

Figure 5 shows the result of lymphocyte subset measurement for human peripheral blood mononuclear cells,
using the FITC-conjugated anti-CD4 and PE-conjugatedanti-CD8 antibodies. Erythrocytes and granulocytes wereremoved in advance from the sample by centrifugal separation in conjunction with a Ficoll-Paque fluid. Subsets
are identified through fluorescent signals generated byeach antibody specifically bound to its special cluster ofdifferentiation (CD).
Figure 6 shows the sorting results for megakaryocite(i.e., a kind of cell constituting blood platelet, approximately 150 μm in diameter) which is, known as one of theDensity（150305̲H.bin）

104

Under
123
4
5
6
7
8
9

FL2 Comp

103

102

101

100

Quadrant
X：19.9777
Y：9.6296

100

101

102

103

104

FL2 Comp

statistic▲

2.2 Damage-less Sorting
A flow cytometer has a function to sort target cells, besides
cell analysis function. In the conventional sorting method,cell-containing droplets are formed using a high hydraulicpressure and ultrasonic waves, and subsequently a highintensity electric charge is applied to the droplets before
sorting. Thus there is a danger that live cells may be damaged or stressed through this process, rendering in somecases this process unsuitable to be used for sorting thecells with multi differentiation potency, such as ES cells
and stem cells, as well as other tender cells.
We have introduced a new sorting method into our flow
cytometer, which has completely eliminated ultrasonicbased droplet formation and high-voltage droplet charging conventionally used. In our flow cell, multiple opticalfibers are accurately arrayed along the fluid channel,
allowing for precise measurement of the flow velocity ofeach sample. As illustrated in Figure 4, the time when atarget sample reaches the front end of the sorting nozzle
is calculated based on this flow velocity information, andin synchronization with the arrival time, the nozzle front
end is moved from the drain tank to the specified well on
the culture plate, thereby achieving sorting of the target
cell. This method has completely eliminated the use ofultrasonic waves and high-intensity electric charges that
constitute the potential factors of damage and stress tothe cells, thus realizing a damage-free sorting methodthat is boundlessly friendly to the cells. Moreover, since
this method is able to sort a single cell to an arbitrary well,
the method is expected to make a significant contribution
to the study of ES cells and stem cells in particular thatare required for regenerative medical treatment.

3.

CD8 PEeach other (i.e., several hundreds μms). Figure 3 shows
the measurement results of the beads with different sizes.
A strong correlation between the transmission light andthe beads size can be seen, demonstrating a significant
improvement in the accuracy of sample size recognition.
It is expected that this transmission light information offer
new valuable information on cell analysis in the future, sothat we are promoting development of new application
software to this end.

CD4 FITC

Figure 5

Measurement result of subset of human peripheral
blood mononuclear cells.

Immediately after sorting① Sorting② Measurement of ﬂow velocity;
calculation of sorting time

③ Damage-less sorting

After three days cultivation
Figure 6

Figure 4

Sorted megakaryocite.

Schematic of damage-less sorting system.

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200780Damage-less Sorting System for Live Cells

most fragile live cells, absolutely impossible to be treatedusing conventional cell sorters. It has been confirmed thatthe cells are alive for three days after sorting.

Figure 7 shows the result of an experiment carried out
to find neural crest stem cells. In this experiment, thegreen fluorescent protein (GFP) gene was injected into afertilized egg of mouse to obtain the mouse embryo
expressing GFP. Brain tissue was taken out 9.5 days after
fertilization, and resultant single cells were sorted into
each well on a 96-well culture plate. As shown in Figure
7, a cell colony has been formed after about 20 days ofcultivation. This result confirms that sorting has been successfully achieved with less damage even for the fragile
brain tissue.

Cultivated for 20 days by the neurosphere method

4.

Figure 8 shows the appearance of two models of ourproducts, and Table 1 main product specifications. ThePERFLOW Ana is the model for analysis only, capable ofmeasuring four fluorescence lights, as well as the scatterand side scatter lights, using its two lasers with different
wavelengths. The PERFLOW Sort is equivalent to the100
100μMmmFigure 7

Colony formed from sorted single neural crest stem cell.		

PRODUCT SPECIFICATIONPERFLOW Ana.

PERFLOW Sort.

Figure 8Appearance of PERFLOW Ana and PERFLOW Sort.

Table 1Specifications of PERFLOW Ana and PERFLOW Sort.

Model

PERFLOW Ana

PERFLOW Sort

Light source

Blue (488 nm) semiconductor laser
Red (635 nm) semiconductor laser

Measurement parameters

Transmission light (corresponding to forward scatter light),
Side scatter light, Four fluorescence lights (FITC, PI (PE), PeCy5, APC)

Detection sensitivity

FITC < 200 MESF (when Spherotech Rainbow Beads is used)

Fluorescence light detection resolution

CV value (variation coefficient): 4 % or less (when BD QC Particles is used)

Side scatter light detection sensitivity

Capable of detecting φ2-µm beads

Sample analysis speed

20,000 event/sec

Optical axis alignment

Unnecessary

Sorting method―Sample acquisition speed―

1 sample/sec

Sample acquisition function―

6~384 multiple-well plate supported

SoftwareMechanical flow switching

Exclusive control and analysis software is
installed in standard specification (FCS compatible)

Power supply

100 V AC, 50/60 Hz, 4 A

100 V AC, 50/60 Hz, 9 A

Dimensions

400 x 550 x 450 mm (WDH)

550 x 600 x 600 mm (WDH)

Mass

Approx. 40 kg

Approx. 90 kg

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2007

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Damage-less Sorting System for Live Cells

PERFLOW Ana in terms of the analysis function, and isprovided with a damage-less sorting function whichallows for sorting of a single cell to an arbitrary well. Bothmodels have achieved considerable downsizing taking
advantage of their proprietary optical fiber-based optics
system. Moreover, since the number of the optical fiber
array can be expanded to eight at the maximum, it is possible to add UV lasers and the like as options.
In an effort to achieve cost-effectiveness of this apparatus so that every laboratory can afford it, as well as tobreak down the traditional knowledge such that a flow
cytometer is an expensive apparatus, we have employed

the brand name “PERFLOW” to carry a meaning of“Personal Flow Cytometer”. It is expected that thePERFLOW featuring transmission light exploitation anddamage-less sorting will contribute to the leading-edgelife-science study involving ES cells and stem cells, aswell as to the development of regenerative medical treatment.
For more information, please contact:
Production Engineering Ctr., Production Technologyand Engineering Div., Furukawa Electric
FAX 0436-42-9334		 TEL 0436-42-1636

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