Bioresist Single Cell Gripper - 名古屋大学 · Background Single Cell Separation for Single...
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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 Hayakawa E-mail: [email protected] Nagoya University Department of Micro-Nano Systems Engineering E-mail: [email protected] Furo-cho, Chikusa-ku, Nagoya-City, Aichi-Pref., 464-8603, Japan TEL: 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 stage Cell Gripper Glass rod Micromanipulator 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 Gripper 50 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 Width W: 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 Displacement [μm] 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 Hole Diameter [μm] 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 Chip 100 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 10 5 cell/mL. 3D Observation of Cell Grip Before Catch After Catch 50um XZ YZ 50um XZ YZ Bright Field Confocal Microscope 50um 50um • MDCK cells were stained by Dojin kagaku 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 Microheater Close : Under 30 °C Channel Bioresist Heater Open : Over 30 °C 50 mm -250 -200 -150 -100 -50 0 0.1 1 10 Phase (deg.) Applied frequency (Hz) 0 1 2 3 4 5 Displacement [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
Transcript of Bioresist Single Cell Gripper - 名古屋大学 · Background Single Cell Separation for Single...
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
