Chip based microfluidic methods Chip-based

Chip-based Chip based microfluidic methods for analytical y chemistry y Andreas Manz UK U.K.

microfabrication, 1979

glass glass device, 1747

nanot tech hnol logy y 17 747

microfluidics,, 1747

many things start off with sophisticated manual methods d l into develop i t fool-proof f l f generall methods finally, crude power dominates the field

algorithm to calculate square root of a number

l logarithmic ith i tables, t bl slide lid rules l

algorithm to calculate square root of a number l logarithmic ith i tables, t bl slide lid rules l PC

information content

vision

„...ome“ complex mixture mixture 1 compoundd 1 times 1 location 1d 2d 3d space

1/min

1/s

continuously ti l

time

information content

proteomics

most analytical methods

NMR tomography space

glucose sensor time

information content

vision

time

space

“labb on a chip” “l hi ” microfabrication microfluidics μTAS miniaturized total analysis systems

“ on a chiip” “lab micrroflu uidiccs caa. 10 0,000 0 pap perss

microfluidics / scaling laws ttrick: i k every existing chemistry will work the same on small as on large g scale

scaling laws for microwell plate volume of

is a cube of

1µL

1nL

1pL

(1mm)3

(100µm)3

(10µm)3

600,000,000

600,000

600

25 / cm2

2500 / cm2

250 ,000/ cm2

17 min

10s

100ms

1.5 /min / cm2

250 /s / cm2

2,500,000 /s / cm2

# molecules (1nM solution)

# volumes In array diffusion time

# reactions (diffusion controlled)

detection in small volumes is an issue goingg nano is getting g g g worse

microfluidics / scaling laws trick works for: chemical h i l reaction ti separation dilution series etc

for more information on the topic micro TAS conference S Diego, San Di USA 2008 Cheju, Korea 2009 1,000 attendees annually impact p factor f 5.8 Reviews on Micro total analysis systems in Anal.Chem. 2002, 2004, 2006 and 2008

cited over 1,900 times

ppart 1

electrophoresis p

scaling g laws

10 fold miniaturization

100 x faster separation p 1000 x smaller volume 10 x lower reagent consumption

electrophoresis FITC labeled l b l d amino i acids id

t7 s s

u o l re c e s n c e [a r b .u n ts i ]

t7

1 22 1

r. h c n y s

3

3

4

cy e#ll# e

4

cy 5

6 7

8

1 6

0

f 0

0 4

8

0

1 0 2

DJH i D.J.Harrison, K.Flury, K Fl K.Seiler, K S il Z.Fan, ZF C.S.Effenhauser, C S Eff h A.Manz, AM Science S i 261 895-897 261, 895 897 (1993) C.S.Effenhauser, A.Manz, H.M.Widmer, Anal. Chem. 65, 2637-2642 (1993)

200

180

160

140

120

100

80

60

40

20

0

Courtesyy of Agiilent W C Waldbronn

Jul-06

Jan-06

Jul-05

Jan-05

Jul-04

Jan-04

Jul-03

Jan-03

Jul-02

Jan-02

Jul-01

Jan-01

Jul-00

Jan-00

publications per month citing 2100 bioanalyzer

220

ppart 2

serial to parallel converter SERIAL

1

2

3

CONVERTER ?

PARALEL

1 2 3 4

4

SERIAL 1 CONVERTER

PARALEL 1 2 3 4

17 SEPARATION CHANNELS

INJECTION CHANNEL

double stranded DNA separation

reaction with intercalating dye

x x x x x x x x

concentrration

double stranded DNA

DN A is slowing down at m oving front of SYBR green

x x

x x

com plex [fluorescing]

x x

x x x

SYBR green

SYBR green is slowing dow n at m oving front of D NA

fluo orescence

length of plug

length of plug

ppart 3

no scaling laws for cell biology on chip hi

size similarity of cells and microchannels cells ll hhave tto “f “feell happy” h ”

timescale 1-2 weeks

Start Position & Current Situation in Stem Cell Research Hair H i Ear

Brain

Tooth

1. Take stem cells from any origin

Blood vessels

Lung Heart Pancraes Liver Kidney

2. Induce differentiation by:

Cartilage C ge

Soluble factors

Immobilised factors

Bone

Muscle

3. Count and find model cell type to test the device

Cell Programming by Nanoscaled Devices

43

Problem 1:

The Difficulty in Comparing Results

Potenttial of differen ntiation

St Stem cell ll (SC) sources

embryonic

higher

Inner cell group

adult

In vitro ESC (no. of passage?)

Single ESC

In vitro adult SC (passage?)

lower

Quality of differentiation Q

low

high

Morphology only Specific marker (images only) Statistics Specific markers (quantitatively) Proteomecharacterisation

Functionality and cell interaction

Functionality

Complete epigenetic characterisation h t i ti Long-term stable implantation


44

Problem 2: Statistical Significance of Differentiation 1. Spontaneous differentiation depends on many factors. Not constant! 2 No 100%, 2. 100% no 0% differentiation. differentiation

90 Adult SC

Embryonic SC 80

30 20 10 0

Induce ed differentiattion

40

Spontaneous differentiation S n

50

4. Frequently many factors are changed simultaneously.

Sup ppressed diffe erentiation

60

Induced differentiation

70

3. Suppressing any differentiation in the case of ESC only. Sp pontaneous d differentiation

Amount o of differentiated ce ells in [%]

100%

5. Isotrope versus cluster differentiation.

? Cell Programming by Nanoscaled Devices

45

Problem 3: Differentiation is not in vivo Differentiation I vitro-culture In it lt off stem t cells ll

Application A li i off factors Variant 1:

SPARC

Variant 2:

X

Variant 3:

X+Y

…

Variant n:

SPARC + X +Y

… Actual single factor induction of cell differentiation! Application pp of different media! Unphysiological high concentration of factors! In addition undefined substances (e.g. FCS, Trypsin …)!


46

SC lines installed and investigated in CellPROM in vivo

Embryogenese: highest accuracy in cell location and differentiation in space and time!


47

Hundreds of Nanoscapes & Extra Equipment


48

MagnaLab: Device

Long-term Cell Cultivation & Differentiation


49

MagnaLab - Cell Cultivation on Carriers over Weeks Highly parallel

NANOSCAPES

Variable ssurface-mediated rface mediated and soluble factor application


50

Results of CellPROM P i i l off M Principle MagnaLab L b

20 Carrier

Bild MagnaLab 20 Carrier

20 Carrier

120 Microcarriers in one cultivation unit

20 Carrier

20 Carrier

20 Carrier

! Inlet and outlet tubes removed ! Cultivation for more than 20 days! Cell Programming by Nanoscaled Devices

51

R Results lt off CellPROM C llPROM

Cultivation for more than 20 days! Cell Programming by Nanoscaled Devices

52

Principle of Microcarrier-Multichannel Cultivation Nanoscape immobilised factor

Soluble factor

Spontaneous differentiation

Spontaneous differentiation

BSA

BSA

Suppressed differentiation

LIF Induced differentiation

SPARC

Suppressed differentiation

LIF Induced differentiation

SPARC


53

Beating Cardiomyocyte Clusters Colonies

X = Number of beating (synchronised) clusters/cm2

Stem Cell

Y = Number of stem cell clusters/cm2

50 up to 100 synchronized cardiomyocytes Cell Programming by Nanoscaled Devices

54

…work work in progress mm scale device handling m scale channels nm scale surface chemistry 5-15 days time scale

part 4

panic

• soft matter for microfluidics ? biocompatible self assembly potentially low cost

vesicle p production PDMS

Si

PDMS

vesicle production p

PDMS

Si 2 µm

PDMS

100 µm

vesicle production p flow direction

100 µm

side view

100 µm

side view fluorescence image

Formation of vesicles

100 µm

100 µm

100 µm 140 120

#

100 80 60 40 20 0

100 µm

2

4

6

8

10

12

d ia m e te r (µ m ) Lipid: DLPC (16:0 Phosphocholine) dye: DiI-C18

P.S.Dittrich, M.Heule, P.Renaud, A.Manz Lab Chip 6, 6 488 488-493 493 (2006)

formation of vesicles

i increase flow fl increase backside pressure

formation of vesicle tubes



Stopping the flow

P.S.Dittrich, M.Heule, P.Renaud, A.Manz Lab Chip 6, 488-493 (2006)

Formation of helices

50 µm

P.S.Dittrich, M.Heule, P.Renaud, A.Manz Lab Chip 6, 488-493 (2006)

panic i

Part 5

fun panic

hottest issues: 1 ll biology 1.cell bi l supportt 2 widening gap between academic 2.widening research and industry y needs 3.expiry of microfluidic patents in coming few years

acknowledgements g Dr.Petra Dr Petra Dittrich Dr.Jonathan West Prof.Günter Fuhr, St. Ingbert g Li Chen Lin Ch Dr.Daniel Schmidt, St.Ingbert Helke Reinhardt Prof Claude Leclerq, Prof.Claude Leclerq Paris Kaoru Tachikawa Dr.Richard Loman, Paris Claus Schumann Prof Philippe Renaud, Renaud Lausanne Prof.Philippe Dr.Joachim Franzke Dr.Martin Heule, Lausanne Prof Philip Day Prof.Philip Dr.Luc Bousse, Mountain i View i Ying Cai P f K i hi Ohno Prof.Ken-ichi Oh

the h endd