Microfluidic chip based flow cytometer

On-chip SortThe world’s first Cell Sorter using disposable microfluidic chips for damage-free and sterile sorting.

On-Chip USA

Phone: (877) 666-5426Email: usa@onchip.co.jpWeb: on-chipbio.com

7098 Miratech Drive, Suite 100San Diego, CA 92121

On-chip Biotechnologies Co., Ltd.204 Venture Port,2–24–16 Naka-cho, Koganei-city, Tokyo 184–0012, Japan

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FL4FL4FL4

LaserLaserLaser

chipchipchip

Flow pathFlow pathFlow path

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Target

Sort

PULL PUSH

Detection80 µm

Non-target

SampleSheathSheath

Waste

On-chip Innovation

Disposable chips: sterilized and cross−contamination freeMicrofluidic technology

Damage free sorting

Wide selection of sheath liquids and sample solutions

Flow system technology

Optical technology

Easy and maintenance-free operation

Fig 5. Flow and optics of microfluidic chip

Fig. 6. Principle of sortingFig . 3. The microfluidic chip enables a closed system

Fig. 2. On-chip Sort installed in biosafety cabinet

Microfluidic chip Micro flow paths

All fluids (sample, sheath and waste) stay within the chip during sorting and analysis, which prevents contamination of the instrument. The Disposable micofluidic chips are just 5.5 x 4.0 cm with 80 x 80 µm microfluidic channels. The chips are acrylic (PMMA), and are available as sterilized or unsterellized. In addition, On-chip Sort is compact enough to fit inside a bio-safety cabinet, providing the best solution to deal with biohazardous samples and strict safety regulations (Fig. 2).

Sheath and sample flow in micro channels are controlled by air pressure. Fast liquid pulses of air pressure are used to deflect target cells into the collection reservoir (Fig. 6). Non-target cells continue in a straight line to the waste reservoir, where they are fully recoverable as well.Since sorting is dependent on fluid viscosity instead of ionic strength, many different fluids can be used for the sample and sheath fluids, including culture media, serum and even oil.

Conventional cell sorters damage cells through high speed collisions (10 m/s) in the collection vessel, high pressure flow control (>45 PSI) which creates high shear stress in the cells, and strong electric fields.In contrast, On-chip Sorter causes no damage to the cells because the flow speed is less than 1 m/s and the flow control pressure is less than 0.3 PSI. Since electric fields are not used, sample and sheath fluids are unconstrained by ionic strength: users can choose buffers and media that best suit their experimental needs.Cells are detected in the laser irradiation area (detection area in Fig. 6). On-chip software determines whether a cell is a target cell while the cell approaches the sorting area.When a target cell is passing through the sorting area, push/pull pulse flow is generated for a few milliseconds to send target cells into the isolation reservoir.The electromagnetic valves and air pumps generate the pu l se f low and the technology i s the so lut ion for damage-free, closed sorting.

The chip has two sheath flows which sandwich the sample flow and narrow it to less than 10 µm in the middle of the channel. This causes cells to pass through the flow cell one by one in a line. Figure 4 is a cross section view of the chip, with the flow path located in the middle.Lasers irradiate from above the chip. Forward scatter (FSC) and fluorescence are detected through an objective lens below the chip (Fig. 5)On the other hand, s ide scatter (SSC) and FL4 are reflected by the edge slopes, and detected through optical guides (Fig. 4).

Freedom from time-consuming maintenance and cleaning: The microfluidic chips are placed in a chip holder and then loaded into the machine. The pumps and valves in On-chip provide air pressure but never contact the sample or sheath fluid, so they don’t require cleaning. After discarding the chip at the end of analysis or sorting, operation is terminated by simply turning off the power (Fig. 3).

Fig. 4. Detection of scattered light and fluorescent light

Fig. 1. Disposable microfluidic chip

Chip for both analysis and sorting

Chip Loading the sample Set in the holder Installation Run

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Analysis Sorting

2. Analysis using biohazardous materials

2. Circulating tumor cells (CTC) sorting by On-chip Sort for clinical trial.

Apoptosis assay Fungicidal capacity of Meropenem against

Analysis in a culture medium

Analysis in a culture medium

Detection of side population (SP) Live/Dead analysis of

Multicolor assay

Isolation of CTCs

CK and/or Vimentin positives CD45 negative WBC TC

Bright field

Hoechst 33342(Nuclei)

Cytokeratin

Vimentin

CD45

Gate�

Sort�

Assessment of an anti-HIV-1 drug candidate

Collaboration with Dr. OKADA Seiji, Center for AIDS Research, Kumamoto Univ.: 2012 Collaboration with Dr. WATANABE Masaru, Shizuoka Cancer Center : 2013

*Collaboration with Drs. Koh & Watanabe, Shizuoka Cancer Center, and Drs. Koizumi & Uehara, National Cancer Center Research Institute

Collaboration with Dr. SUZUKI Ikuro, Tokyo University of Technology: 2011

OCBT Dr. ISHIGE Masayuki: 2013

OCBT Dr. YAMASHITA Namiko:2011OCBT Dr. YAMASHITA Namiko:2011

OCBT Dr. TAKEDA Kazuo:2009

KATOIII cells, UV exposureAPOPCYTO Annexin V-Azami-Green Apoptosis Detection Kit

(Comparison of On-chip Sort and CellSearch™ using EpCAM expression or non-expression cell lines)

AnnexinV

Annexin V-Azami-Green

PINegative

Q4Q4

Q1

Q3

Q2

NegativePositive

Q3Negative

Positive

Live cells Apoptotic cells Dead cells

Q2Positive

Cell lineKATO-III

EpCAM expression

LowHigh

Detection rate

82.4 ± 8.5%93.8 ± 5.5%

Detection rate

On-chip Flow CellSearch®

28.8%74.7%

PC-14

A549

Null 96.0 ± 8.5% 0.0%

Sorter A: flow late = 5, Nozzle = 70 µm,cooling system = ONOn-chip Sort: pressure = 20 kPa, room temperature

Cells from hippocampus, rat E18

Sorted by Sorter A or On-chip Sort

Pl staining Culture

Neuronal cells from rat hippocampus are delicate and sensitive to mechanical stresses. Below is a comparative experiment of On-chip Sort to conventional jet-in-air sorter A.An aliquot of the sorted cells were stained by PI to check viability and the remaining cells were cultured. PI staining, the percentage of dead cells did not increase much in Sorter A nor in On-chip Sort compared to the cells without sorting.Damage for jet-in-air sorted cells appeared as early as 3 days after cultivation (data not shown).By the 7th day, neuronal cells not subjected to sorting or sorted by On-Chip Sort showed healthy, “neuron like” morphology and extended axons. In contrast, the majority of the cells sorted by sorter A died by day 3, and almost all of them were dead by day 7.We confirmed the finding is reproducible. Clearly, this damage-free sorting is strong advantage, and On-chip Sort is a pioneer in this field.

Cancer cells can be profiled as cytokeratin (CK), EpCAM, and nuclear dye (7-AAD) positive and CD45 negative. On the other hand, white blood cells are detected as CK and EpCAM negative, 7-AAD and CD45 positive. CTC were sorted by On-chip Sort for subsequent gene analysis which can be used for personalized medicine*.Furthermore, sorting trials using clinical samples have been launched which aim to detect/sort CTCs by organ/cancer specific antigens. Non-FDA approved.

Without sorting Sorter A On-chip Sort

Without sorting

Day 7

FS

PI

Day 7FL

Sorter A On-chip Sort

1. General flow cytometric analysis by On-chip Flow/Sort 1. Cell sorting by On-chip Sort : Hippocampus cells from rat fetal brain

0 hr 3 hrs

Propidium lodide(PI)

pattern 1

PE/Cy5-CD4(FL-5B)

FITC

-CD

3( F

L-2B

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Per

CP

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SS

C

PE-CD45(FL-3B)

X(nM)Uninfected Infected

6 dpi

HIV-DsRed

1 1000 100

Candidate compounds

11 dpi

APC/Cy7-CD8(FL-6R)

PE/Cy7-CD19(FL-6B)

Cytokeratin-FITC Adjacent channel

P2=CD3*CD4* T cellsP1=Lymphocyte P2=MonocytesP3=Granulocytes

Isolated CTCs are observed under microscope.Isolated cells were then subjected for sequencing to detect mutation.

P1=Lymphocytes

P3=CD3*CD8* T cells

PE-CD45(FL-3B)

P4=T cells P5=B cells

pattern 2

APOPCYTO Annexin V-Azami-Green Apoptosis Detection Kit, MBL, Code No.4690OCBT KAWASE Yoshie: 2013

Anne

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V-Az

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Pseudomonas

E. coli

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Supporting a variety of fluorescent colors/high sensitivity Specifications

Violet

Violet

Blue

Green

FL1-V

FL1

FL1

FL2-V

FL2-B

FL2

FL3-V

FL3-B

FL3

FL4-V

FL4-B

FL3-B FL4-B

FL4

FL5-V

Sample: rainbow calibration particles (8 peaks)

FITC Signal intensity (510-550 nm)FL5-B

FL5-R

FL5

FL6-V

FL6-B

FL6-R

Red FL5-R FL6-R

FL6

400 450 500 550 600 650 700 750 800

FL2 FL3 FL4 FL5 FL6Blue Green Red

Product name

On-chip Sort HS

Equipment (On-chip Sort)

On-chip Sort HSGOn-chip Sort MS5On-chip Sort MS5GOn-chip Sort MS6On-chip Sort LS5On-chip Sort LS3

Fluorescence sensitivity: FITC<200 MESF

Lasers

Blue Red VioletBlue Green VioletBlue RedBlue GreenBlue VioletBlueBlue

The number of detectors

6655653

Catalogue No.

362S3001362S3001G252S3001252S3001G262S3001152S3001132S3001

Fre

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One to three lasers can be installed in On-chip Sort, Blue (488 nm) is required for scattering, Red (637 nm), Violet (405 nm) and Green (561 nm) lasers are available as additional options. The system achieves as high as <200 MESF FITC. Up to six detectors cover a wide variety of fluorescent colors, customized laser(s) are also available such as 785 nm.

Detection of individual signals from multiple lasers is available in a single detector, and it enables smaller and inexpensive equipment.

600 MESF

Blank

Optics

Fluidics

Analysis and Sorting

Safety

Size and Weight

PC and Software

Supply

Data resolutionPulse analysis

Detection wavelength

Fluorescent sensitivitySize sensitivityMeasurement parameterLaser

Flow-cell chipMaterial of chipFlow channel sizeFlow speedSheath bufferVolume of sampleVolume of sheath

Sorting methodPurityYieldCell damageCross-contamination freeSterilizedPressureDetection speedSorting speedStart-upShutdown

Generation of aerosol

Size (W × H × W, mm)Weight (kg)

PC and SoftwareOSData format

Power requirementsPower Consumption

4 decades, 18 bit ADCHeight, Area, Width

FL1 (445/20 nm), FL2 (543/22 nm), FL3 (591.5/40 nm)FL4 (607/24 nm), FL5 (676/37 nm), FL6 (>710 nm)

< 200 MESF FITCFSC < 0.5 μm, SSC < 1.0 μm

Forward scatter (FSC), Side scatter (SSC), 6 PMTs (up to 10 parameters)Up to 3 Lasers from 405 nm, 488 nm, 561 nm, 637 nm, 785 nm

Disposable microfluidic chipAcrylic (PMMA)80 μm × 80 μm500 mm/sec

Any media can be used20-300 µL or up to 1 mL*

* You can load up to 1 mL on the chip, but sheath fluid runs out before you use up the sample. You can, however, refill the sheath fluid after taking out the chip.

4-9 mL

“Flow Shift” to generate pulse flow> 95% (depends on cell concentration)

> 80%No

Yes, because of the disposable chipYes

0.3 PSI4,000 events/sec100 targets/sec

5 minutes10 sec (no cleanup of tubing necessary)

No

620 × 330 × 39045 kg

Laptop PCWindows 7 or 8, 64 bit

Own format and FCS 3.0

AC100−240 V, 50/60 Hz< 240 VA

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Microfluidic chip based flow cytometer

On-chip SortThe world’s first Cell Sorter using disposable microfluidic chips for damage-free and sterile sorting.

On-Chip USA

Phone: (877) 666-5426Email: usa@onchip.co.jpWeb: on-chipbio.com

7098 Miratech Drive, Suite 100San Diego, CA 92121

On-chip Biotechnologies Co., Ltd.204 Venture Port,2–24–16 Naka-cho, Koganei-city, Tokyo 184–0012, Japan

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