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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: [email protected] Nagoya University Department of Micro-Nano Systems EngineeringE-mail: [email protected], 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