Caught Cell

Microchannel

Cell fix for measurement/ observation

Cell Gripper

Microfluidic Channel

Acknowledgements：This work was financially supported by This work was financially supported by Grant-in-Aid forJapan Society for the Promotion of Science (JSPS) Fellows

Contact Person： Takeshi HayakawaE-mail: t-hayakawa@biorobotics.mech.nagoya-u.ac.jp Nagoya University Department of Micro-Nano Systems EngineeringE-mail: t-hayakawa@biorobotics.mech.nagoya-u.ac.jpFuro-cho, Chikusa-ku, Nagoya-City, Aichi-Pref., 464-8603, JapanTEL: 052-789-5026, FAX : 052-789-5027,

Background Single Cell Separation for Single Cell Assay Immunoassay Stem cell analysis Cancer cell analysis

Current standard: Limiting dilution method

http://www.enzolifesciences.com/

Labor intensive protocol

Dulbecco, R., & Vogt, M. (1953). Cold Spring Harbor Symp. Quant. Biol., 18, 273

Petri Dish Caught Cell

XYZ stageCell Gripper

Glass rodMicromanipulator

Petri Dish

Glass Pipette

Single Cell Pickup

Tip of Pipette

Cell Gripper

H+ + heat 

+h

PNIPAAm

Cross-linker

Bioresist

Bioresist(Patternable thermo-responsive gel)

Bioresist-i, Nissan Kagaku

Bioresist Single Cell Gripper50 μm

Microheater

Shrinking Bioresist

Trapped Cell

High Temperature (37 ̊C~) Low Temperature (~32 ̊C)

Swelling Bioresist

Microheater Caught Cell

50 μm

Reversible

To realize low initial cost and easy to handle single cell separation and dispense chip.

Purpose

Fabrication Process

D: Pattern WidthW: Hole Diameter

Max. Aspect ratio of Bioresist: ≈ 0.5

Thickness [μm]Hole Diameter [μm]

Design: 30 Design: 20

9.0±0.5 24.3±0.5 12.5±2.8

20.8±2.2 24.5±1.5 NG

We succeeded in single cell separation and dispense to culture well by one chip. Success rate of single cell separation: 50 %

single cell dispense: 75 %total: 38 %

Conclusions

Pattern Dependence of Expansion Rate

12.4 ̊C

< 32 ̊C

W: 10μm W: 30μm

50 μm

> 37 ̊C

Expansion rate can be controlled by changing pattern width.

0

10

20

30

40

50

60

70

80

90

10 20 30 40

Dis

pla

cem

en

t[μ

m]

Temperature [°C]

0

5

10

15

20

25

30

10 20 30 40

Temperature [°C]

0

5

10

15

20

25

30

10 20 30 40

Ho

le D

iam

ete

r[μ

m]

Temperature [°C]

0

10

20

30

40

50

60

70

80

90

10 20 30 40

Temperature [°C]

Displacement Hole Diameter

● W: 10 μm (Heating)× W: 10 μm (Cooling)● W: 30 μm (Heating)× W: 30 μm (Cooling)● W: 50 μm (Heating)× W: 50 μm (Cooling)● W: 70 μm (Heating)× W: 70 μm (Cooling)

Thickness: 9.0±0.5 μm Thickness: 20.8±2.2 μm Thickness: 9.0±0.5 μm Thickness: 20.8±2.2 μm

Single Cell Catch & Release

100 μm

Washed Chip100 μm

Caught Cell

Swelling Bioresist

100 μm

Trapped Cell

Shrinking Bioresist

20 μm

Single cell released in culture well

Released cells were stained by calcein-AM.

• Sample was MDCK (Madin-Darby canine kidney) cell suspension. ・Cell concentration was approximately 1.0 x 105 cell/mL.

3D Observation of Cell GripBefore Catch After Catch

50um

XZYZ

50um

XZYZ

Bright Field

Confocal Microscope

50um 50um

• MDCK cells were stained by Dojinkagaku Calcein-AM.

• Bioresist was stained by Sigma-Aldrich Rhodamine B.

Caught Cell

Microchip

Micropipette

Cell suspension

Parallel single cell separation

Microchip

Microchip

Cell Gripper Array

Frequency Response with MicroheaterClose : Under 30 °C

Channel

Bioresist

Heater

Open : Over 30 °C

50 mm-250

-200

-150

-100

-50

0

0.1 1 10

Ph

as

e (

de

g.)

Applied frequency (Hz)

0

1

2

3

4

5

Dis

pla

ce

me

nt

[mm

]

0.89V 1.03V 1.17V

K. Ito, et al, “On-Chip Gel-Valve Using Photoprocessable Thermoresponsive Gel” , Robomech Journal, Accepted

3) Development

1) Spin coating of Bioresist

Bioresist

Glass substrate

Bioresist

2) Exposure

Microfluidic channel