Micro TAS for regenerative medicineDevelopment of Bio/Nano hybrid platform technology for measuring cell functions and

regenerative medicine

Hidetoshi Kotera(Dept. of Micro engineering, Faculty of Engineering, Kyoto Univ.)

９／９／２０１０

Contents of Presentation

• Research Issue of My Lab.( Nano Metrics Lab.)

• Research for regenerative Medicine

1) How the cell communicates ?

2) Orientation action of cell for stimuli 

3) Regeneration of capillary arteriole

Research Targets of NanoMetrics Lab. 

PZT

Pt

MgO 1μm

Basic Material⇒ Piezoelectric thin‐filmBasic Material⇒ Piezoelectric thin‐film

Application⇒ Bio-MEMS & μ-TAS⇒ RF-MEMS & Optical MEMS

Application⇒ Bio-MEMS & μ-TAS⇒ RF-MEMS & Optical MEMS

Simulation⇒ Multi‐physics Simulator (MEMS‐One & Bio simulator)

Simulation⇒ Multi‐physics Simulator (MEMS‐One & Bio simulator)

Nano particles assemble

AO; Deformable mirrorCell encapsulation

Cell level simulationOrgan level simulationOrgan level simulation

POC device X-bar RF-MEMS Switch

Traveling wave micro pump Micro Mixer

Functional particles’ simulation

Single-Mask Inclined Rithography

3μm100μm

Nano Inprint

Fabrication Process⇒ Nanoimprint & Powder Filing⇒ Lithography

Fabrication Process⇒ Nanoimprint & Powder Filing⇒ Lithography

Multi target spatter

MeasurementDesignMaterial

Switch type

Meta structure and meta material

Horn-array type Millimeter wave antenna

遅波構造制御型

樹脂基板による遅波構造

Adaptive Optics system

Wavefront sensor

Wavefront generator

Reflected wavefront

Incident Wavefront

Adaptive optics is used for correction of disturbed wave front.This technology has been developed in astronomy.

Control system

Turbulence

Actuator side Mirror side

Fabrication process

SOI (Si on Insulator) Ti sputtering Pt sputtering

PZT sputteringZPN spin coatingand patterning

Cr sputtering

Cr lift off DryFilm pastingand patterning

Si dry etching

Ti Pt

PZTZPNCr

Al evaporation SiO2 wet etching

S1813 spin coating

DryFilmS1813

New！

New！

Deflection of mirror in case of single electrode is driven

① ②⑤⑥

③④

⑦

⑧

⑨⑩⑪

⑫

⑬

⑭

⑮⑯ ⑰

⑱

⑲

① ② ③

⑥⑤④

⑦ ⑧

Left; experimentRight: simulation

⑨ ⑩ ⑪

⑭⑬⑫

⑮ ⑯ ⑰

⑱ ⑲

① ②⑤⑥

③④

⑦

⑧

⑨⑩⑪

⑫

⑬

⑭

⑮⑯ ⑰

⑱

⑲

Knowledge for simulation, MEMS design and fabrication process

Knowledge DB

High end simulator

CAD

Designer system and modeling tools

Phenomena and function

Stress (Linear & non-linear）Thermal and thermal stressElectro-magnetic fieldRadio-frequency field

Tribology analysis

Wet and dry etching

Nano-inprint(Thermal and photo)

Process designer

Mask layouter

Inverse simulator for mask

Material properties

Material DB

Design

Micro fabrication

knowledge

Knowledge of professional engineer

Experimental data

Interface for commercial software

Material

Mask design

Technical Concept of MemsONE

in vitro diagnostic test system

Implantation

Point of Care

Optical Image (x50)

50μm

Orifice

Microchannel

myocyte

30µm

orifices orifices

Blood

carrier

filter

filter

valve mixer

reactorseparator

sensor

pump

outlet

circuit

signal

Piezoelectric material & Actuator

Bio-Nano Plat form/Micro Fluidic system and micro sensor for single molecule

Model and Simulator / Micro TAS for measuring cell performance

Bio Nano hybrid platform and 3D Fabrication

Micro Fluidics/ Micro TAS and SPR

Eye ballLaser

Signal

Deformable MirrorCCD Camera

Adaptive Optics

controller

Beam Spritter

Wave sensor

Education & Fabrication

ＯＣＴ ｆｏｒ retina

Contents of Presentation

• Research Issue of My Lab.( Nano Metrics Lab.)

• Research for regenerative Medicine

1) How the cell communicates ?

2) Orientation action of cell for stimuli 

3) Regeneration of capillary arteriole

Structure of ISLET（Mouse）Structure of ISLET（Mouse）

Structure of ISLET（Mouse）Structure of ISLET（Mouse）

Background_1: cell‐cell communication

stimulation reaction secretion of a stimulant reaction

stimulant

e.g. pancreas glucose Ca2+↑ insulin Ca2+↑

Q1；How to communicate the cells? And how chain reaction occur?

βcell-βcell， αcell-βcell，Other cell or tissue-βcellSeparate the cell from tissue to measuring

communicationQ2；Measuring the protein and/or small molecules which

communicate between the cells１molecule level

Q3；Regenerate the tissue from cell; Design and alignment

Q4; Direction of insulin secretion from venous vessel to arterial vessel

β Cell α Cell

ES Cell iPS Cell

encapsulation

Capillary vessel

20

Immobilize Incubate

Examine Stimulate!

Configuration control → local stimulation → Tissue analysisHighly integrated orifice array is required.

Objective 

Microfluidic devices used in the study and operating procedure 

the microfluidic chip assembling by grafting PDMS layer onto SU-8 microchip

the operatingg principle of the microfluidic chip

Material and methods_1:Microfluidic chip fabrication

The fabrication of the SU-8 chip by single-mask inclined UV photolithography for embedded network process

SiMPLE process  – inclined/rotated photolithography ‐

23

Exposure method Exposure volume

θ

ω

θr

Curved microchannel Crossed microchannel Orifice with microchannel

Single-mask Multidirectional PhotoLithography for Embedded networks

24

Fluorescent image of cell trapping 2

Suction

cell

Suction side

20μm

Cell trapping with parallel configuration

25

Fluorescent image of cell trapping 2

150μm

Suction

Suction side

20μm

Cell trapping with parallel configuration

26

Fluorescent image of cell trapping 2

150μm

Suction

Suction side

20μm

Cell trapping with parallel configuration

27

Fluorescent image of cell trapping 2

Suction

Suction side

20μm

Cell trapping with parallel configuration

28吸引20μm

Fluorescent image of cell trapping 2

150μm

Suction

Cell trapping with parallel configuration

Material and methods_2: Experimental setup for microscopic observation of single cell monitoring in micro‐fluidic device

Schematic overview of micro-fluidic device system containing pressure control, temperature control and cell and reagent load

Schematic diagram of setup for microscopic observation

β-Cell (Min 6 m9) humoral transmission

suction

cell trapping stimulation response

cellorifice

objectivelense

stimulant

culturing

1 cm 2 mm

suction holecell loading channel

stimulant loading channel

orifice-channelorifice

20 μmcell array device

Experimental Set Up

Chamber for temperature control Fluid circulation

Injection system

spriter

圧力調整スプリッター

吸引口

channel1

channel2

injector

吸引_1 吸引_2吸引_3

一穴 三穴 五穴

Ppump

Hot water

fluid system

Temperature control

Pressure controler

injector

pull pushWaste fluidpump

• Cell reaction for insulin:Ca2+ concentration（0-5min：0mM, 5-10min：5.5mM,10min-

600nMInsuline +5.5mMGlucose

Cell reaction for insulin stimulus

imobilization・cwll：MIN6-m9・flow speed：1 μl/min・orifice：φ2μm

×５速

Cell imobilization & Culturing

Experimental procedure

StimulationUptake of a stimulus (fluorescent glucose analogue: 2-NBDG)

0 min 25 min Bright Field 20 μm

Time course of the uptake of 2-NBDG

Experimental procedure

Dynamic monitoring of cytosolic Ca2+ increasing by glucose through micro‐orifices

Fluorescence images by Fluo4 of cultured MIN6-m9 cells cultured on microfluidic chip before and after glucose through micro-orifices.

To confirm the cellular uptake of stimulant through micro-orifice can evoke physiological reaction, we load 20mM glucose solution in micro-channels after MIN6-m9 cells were imobilised in micro-chamber.

immobilized cells on the micro-orifices can physiologically react to stimulation through the micro-orifices.

a

b

c

Stimulated cell

Resting cell

backgroundUpper channelsolution：bufferrate：5 μl/min

Lower channelsolution：20mM glurate：1 μl/min

10μm

conditons：5μM Fluo8-AM（Ca2+, 37℃, 1時間

20XLUCPlanFL, N.A. 0.45CCD：Orca-ER：111msec

22.2mM 22.2mM

Cell reaction ‘Ca2+ concentration’ for glucose stimulus

Fade of cell

No.3

No.2

No.1

0mM 0mM

glucose

Upper layer flowfluid：0mM gluflow：1 μl/min

Lower channelfluid：22.2mM gluflow：1 μl/min

Ca2+conceltrarion in the cell for glucose stimulus

10 μm

Contents of Presentation

• Research Issue of My Lab.( Nano Metrics Lab.)

• Research for regenerative Medicine

1) How the cell communicates ?

2) Orientation action of cell for stimuli 

3) Regeneration of capillary arteriole

Localization and control mechanism of secreting direction 1. Apply localized stimuli

Stimulate through“Pseudo-arterial capillary”

2. Measure intracellular response4D imaging (x,y,z and time)

Mimicked Tissue Micro-environment

・ Complex micro channel from Single photo mask

・ No mask alignment process

Multi-Directional UV Lithography.

Fabrication

Fabrication

Trap single cell by aspiration through micro channel.

Positioning of single cell

・Localized stimuli introduction into trapped cell・4D imaging of its uptake and intracellular responses

Setup for measuring localization and secretion

・Aperture-size/shape controlby exposure dose

Simulation

SEM image

Simulation SEM image

Micro channel and hole

Single cell is positioned at an aperture.

Cell positioning

・External substances is introduced・Glucose uptake is visualized with4D (x,y,z and time) resolution.

10 sec / frameGlucose instability and localization inside cell

Insulin-eGFP / MIN6 cell

Insulin GFP is introduce and imaged in β cell

Insulin Secretion

resident

newcomer

1μm 33 msec/frame

Insulin-eGFP / MIN6 cell

Control of transport direction – method I• In vivo: Microtubules are oriented in a 

direction for organized intracellular transport

• In vitro: Polarity of purified microtubule is random

• Microtubule orientation is necessary for engineered transport in vitro

micro.magnet.fsu.edu

Oriented MT in vivo Orientation method in vitro

Single MT orientation in nanochannelScale down to true nano!• +

MT is introduced a nanochannel by MT motility

Nanochannel

MT

MT motility Nanochannel

MT position & polarity are defined

Arrayed MTs serve as molecular transport platform

Dynein Kinesin

Dynein Kinesin

25 nm

10~100 μm

+ －Microtubule (MT)

Contents of Presentation

• Research Issue of My Lab.( Nano Metrics Lab.)

• Research for regenerative Medicine

1) How the cell communicates ?

2) Orientation action of cell for stimuli 

3) Regeneration of capillary arteriole

Vessels

100μm

Conclusions

1. All the micro-channels whose micro-orifices were occupied with cells were open in the devices with 2µm in diameter

2. MIN6-m9 cells could be cultured on the SU-8 microchip

3. the stimulant was able to exclusively reach the designated cells without leaking through the micro-orifices

4. immobilized cells on the micro-orifices of this micro-fluidic device can physiologically react to stimulation through the micro-orifices.

5. we proposed the novel device that connected vessels and microchannels. HUVECs were cultured on the micro device self organized the vessels. The vessels could be guided to the gel beads and include them.

This study is supported by CREST of JSPS:Japan Science and Technology Agency.

Acknowledgement

Regenerative medicine+ Transplantation

ＡｃｋｎｏｗｌｅｄｇｅｍｅｎｔＡｃｋｎｏｗｌｅｄｇｅｍｅｎｔ

Cell Initialization

Prof. Okitsu, Prof. IwataProf. Miura, Prof. TadaKyoto Univ.Prof. Takeuchi; Univ. of Tokyo

Dr. OkonogiProf. Tada,Prof. YokokawaProf. Suzuki, Prof. TeraoKyoto Univ. & Kagawa Univ.

Prof. Washizu, Prof. OanaProf. Murat & Dr. KiuraUniv. of Tokyo

Micro TAS for Cell communication

Prof. Fujita・Ｊ．Ｗ．Ｐａｒ

ｋUniv. of Tokyo

Molecule measurement

Staffs in the lab