many things start off with sophisticated many things start off with sophisticated manual methods d l i t f l f l develop into fool-proof general methods methods finally, crude power dominates the field
information content vision content „...ome“ complex mixture mixture 1 d ti l mixture time 1 times 1 location 1 compound continuously 1/s 1/min 1d 2d 3d space 3d
microfluidics / scaling laws t i k trick: every existing chemistry will work the every existing chemistry will work the same on small as on large scale g
for more information on the topic for more information on the topic micro TAS conference S Di USA 2008 San Diego, USA 2008 Cheju, Korea 2009 impact factor 5.8 1,000 attendees annually p f Reviews on Micro total analysis systems in Anal.Chem. 2002, 2004, 2006 and 2008 cited over 1,900 times
electrophoresis FITC l b l d i id FITC labeled amino acids c e [ a r b . u n i t s ] 1 2 3 4 c y c l e # t 7 s s y n c h r . c e [ a r b . u n i t s ] 1 2 3 4 c y c l e # t 7 s s y n c h r . c e [ a r b . u n i t s ] 1 2 3 4 c y c l e # t 7 s s y n c h r . f l u o r e s c e n 0 4 0 8 0 1 2 0 1 6 0 5 6 7 8 f l u o r e s c e n 0 4 0 8 0 1 2 0 1 6 0 5 6 7 8 f l u o r e s c e n 0 4 0 8 0 1 2 0 1 6 0 5 6 7 8 D J H i K Fl K S il Z F C S Eff h A M S i 261 895 897 D.J.Harrison, K.Flury, K.Seiler, Z.Fan, C.S.Effenhauser, A.Manz, Science 261, 895-897 (1993) C.S.Effenhauser, A.Manz, H.M.Widmer, Anal. Chem. 65, 2637-2642 (1993)
publications per month publications per month citing 2100 bioanalyzer 200 220 C 140 160 180 Courtesy 80 100 120 y of Agi 40 60 80 ilent W 0 20 -00 -00 -01 -01 -02 -02 -03 -03 -04 -04 -05 -05 -06 -06 Waldbro Jan Jul Jan Jul Jan Jul Jan Jul Jan Jul Jan Jul Jan Jul nn
x x x x x x x x x x x x x x x x x double stranded DNA SYBR green complex [fluorescing] ration DNA is slowing down at moving front of SYBR green SYBR green is slowing down at moving front of DNA concentr length of plug orescence fluo length of plug
Start Position & Current Situation in Stem Cell Research H i Brain Blood vessels Ear Tooth 1. Take stem cells from any origin Hair Lung Heart Pancraes Liver Kidney Kidney Cartilage 2. Induce differentiation by: Soluble Immobi- lised C ge Bone factors lised factors Muscle 3. Count and find model cell type to test the device 43 Cell Programming by Nanoscaled Devices
Problem 1: The Difficulty in Comparing Results St ll (SC) Inner cell group In vitro ESC Stem cell (SC) sources tial of ntiation higher embryonic adult Single ESC In vitro ESC (no. of passage?) In vitro adult SC (passage?) Potent differen lower adult on low Morphology only Specific marker (images only) Statistics ifferentiati Specific markers (quantitatively) Proteome- charac- Quality of d Functionality Functionality and cell interaction Complete epigenetic h t i ti terisation Q high characterisation Long-term stable implantation 44 Cell Programming by Nanoscaled Devices
Problem 2: Statistical Significance of Differentiation 90 100% 1. Spontaneous differentiation depends on many factors. Not constant! 2 No 100% no 0% differentiation ] 70 80 90 Embryonic SC Adult SC 2. No 100%, no 0% differentiation. 3. Suppressing any differentiation in the case of ESC only. ells in [% 50 60 70 differentiation fferentiation 4. Frequently many factors are changed simultaneously. ntiated ce n tion 30 40 pontaneous d Induced di 5. Isotrope versus cluster differentiation. of differe differentiation ed differentiat 10 20 Sp erentiation Amount o Spontaneous Induce ? 0 ppressed diffe S ? 45 Cell Programming by Nanoscaled Devices Sup
Problem 3: Differentiation is not in vivo Differentiation I it lt f t ll In vitro-culture of stem cells A li i f Application of factors SPARC Variant 1: X Variant 2: Variant n: SPARC + X Variant 3: X + Y SPARC X SPARC + X + Y X + Y … … … Actual single factor induction of cell differentiation! Application of different media! pp Unphysiological high concentration of factors! In addition undefined substances (e.g. FCS, Trypsin …)! 46 Cell Programming by Nanoscaled Devices
SC lines installed and investigated in CellPROM in vivo Embryogenese: highest accuracy in cell highest accuracy in cell location and differentiation in space and time! 47 Cell Programming by Nanoscaled Devices
MagnaLab - Cell Cultivation on Carriers over Weeks Highly parallel NANOSCAPES Variable s rface mediated and NANOSCAPES Variable surface-mediated and soluble factor application 50 Cell Programming by Nanoscaled Devices
Results of CellPROM P i i l f M L b Principle of MagnaLab Bild MagnaLab 20 Carrier 120 Microcarriers in one cultivation unit 20 Carrier 20 Carrier unit 20 Carrier 20 Carrier 20 Carrier Cultivation for more than 20 days! ! Inlet and outlet tubes removed ! 51 Cell Programming by Nanoscaled Devices Cultivation for more than 20 days!
Beating Cardiomyocyte Clusters Stem Cell Colonies X = Number of beating (synchronised) clusters/cm2 Y = Number of stem cell clusters/cm2 50 up to 100 synchronized 54 Cell Programming by Nanoscaled Devices 50 up to 100 synchronized cardiomyocytes
work in progress …work in progress mm scale device handling m scale channels m scale channels nm scale surface chemistry nm scale surface chemistry 5-15 days time scale
hottest issues: 1 ll bi l t 1.cell biology support 2 widening gap between academic 2.widening gap between academic research and industry needs y 3.expiry of microfluidic patents in coming few years
acknowledgements g Dr Petra Dittrich Dr.Petra Dittrich Dr.Jonathan West Li Ch Prof.Günter Fuhr, St. Ingbert Lin Chen Helke Reinhardt g Dr.Daniel Schmidt, St.Ingbert Prof Claude Leclerq Paris Kaoru Tachikawa Claus Schumann Prof.Claude Leclerq, Paris Dr.Richard Loman, Paris Prof Philippe Renaud Lausanne Dr.Joachim Franzke Prof Philip Day Prof.Philippe Renaud, Lausanne Dr.Martin Heule, Lausanne i i Prof.Philip Day Ying Cai P f K i hi Oh Dr.Luc Bousse, Mountain View Prof.Ken-ichi Ohno
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