biomimetics

... talk given at Twente University, September 2018.

1. BIOMIMETICS - attempts to learn from nature Andreas Manz
2. biomimetics Leonardo da Vinci 1452-1519
3. biomimetics Leonardo da Vinci 1452-1519
4. biomimetics
5. biomimetics I guess we can learn from nature it may take time exact copies don’t make sense
6. biomimetics I guess we can learn from nature it may take time exact copies don’t make sense
7. biomimetics - microfluidics what type of microfluidics do we find in nature ? how is fluid pumping done ? what is it designed for ? lessons to the engineer
8. biomimetics - microfluidics animal vascular system plant leaf venation
9. biomimetics - microfluidics Physarum polycephalum, slime mold
10. biomimetics - microfluidics what is network designed for ? perfusion, removal equally distribute liquid from one point to large area damage tolerance mechanical stability
11. leaf venation the real leaf, fluorescein
12. leaf venation the PDMS copy, red ink Wenming Wu et al, RSC Advances 6, 22469-22475 (2016)
13. leaf venation manufacturing Wenming Wu et al, RSC Advances 6, 22469-22475 (2016)
14. leaf venation Wenming Wu et al, RSC Advances 6, 22469-22475 (2016)
15. leaf venation Wenming Wu et al, RSC Advances 6, 22469-22475 (2016)
16. filling option #1
17. filling option #1
18. filling option #2
19. filling option #2
20. filling option #3
21. filling option #3
22. What is microfluidics? “lab on chip” micro TAS? history
23. REFERENCE 1747
24. W.J.s’Gravesande, „Mathematical Elements of Natural Philosophy confirm’d by experiments, or an introduction to Sir Isaac Newton’s philosophy“, plate 120, fig.2 ,London 1747
25. micro- fluidics, 1747
26. microfabrication, 1975/79 S. Terry et al., Stanford USA
27. microfabrication, 1980s P. Bergveld et al., Twente University 1985
28. • liquid chromatography, electrochemical detection • silicon – glass chip, design 1987, fab 1987 hitachi ltd. japan • manz, miyahara, miura, watanabe, miyagi, sato, sensors & actuators B1, 249-255, 1990
29. μTAS
30. μTAS 1989/90
31. μTAS 1994
32. example micro well plate volume 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) is a cube of
33. flow injection analysis, 1989-92 Ciba-Geigy / CSEM – Sabeth Verpoorte et al. Jointly with Neuchatel University & CSEM Bart van der Schoot and Nico de Rooij
34. 1989-91 CE on chip Ciba-Geigy, Basel – Jed Harrison et al. Microfab IMT Greifensee (then part of Mettler Toledo, Ciba-Geigy)
35. • capillary electrophoresis, flow injection, electrochemical detection • glass – glass chip, design 1989, fab 1989 mettler imt switzerland • manz, fettinger, lüdi, widmer, svs bulletin 5, 4-10, 1990
36. • capillary electrophoresis, injection, electrochemical detection • glass – glass chip, design 1989, fab 1989 mettler imt switzerland • manz, harrison, fettinger, verpoorte, lüdi, widmer, proc. transducers 1991 san francisco, 939-941, 1991
37. • capillary electrophoresis, injection • glass – glass chip, design 1992, fab 1992 mettler imt switzerland • effenhauser, manz, widmer, anal.chem. 65, 2637-2642, 1993
38. flu o re s c e n c e [a rb . u n its ] tim e [s ] 0 4 0 8 0 1 2 0 1 6 0 1 2 3 4 5 6 c y c le # 7 8 t 7 s s y n c h r. flu o re s c e n c e [a rb . u n its ] tim e [s ] 0 4 0 8 0 1 2 0 1 6 0 1 2 3 4 5 6 c y c le # 7 8 t 7 s s y n c h r. flu o re s c e n c e [a rb . u n its ] tim e [s ] 0 4 0 8 0 1 2 0 1 6 0 1 2 3 4 5 6 c y c le # 7 8 t 7 s s y n c h r. electrophoresis FITC labeled amino acids 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)
39. history of “lab on chip” activities based on publications considered relevant for analytical chemistry
40. history 1989 based on publications considered relevant for analytical chemistry 1975 -
41. history 1990 based on publications considered relevant for analytical chemistry
42. history 1991 based on publications considered relevant for analytical chemistry CE on chip presented at HPLC91 Basel
43. history 1992 based on publications considered relevant for analytical chemistry
44. history 1993 based on publications considered relevant for analytical chemistry
45. history 1994 based on publications considered relevant for analytical chemistry 1st micro TAS conference
46. history 1995 based on publications considered relevant for analytical chemistry
47. history 1996 based on publications considered relevant for analytical chemistry
48. history 1997 based on publications considered relevant for analytical chemistry
49. history 1998 based on publications considered relevant for analytical chemistry
50. history 1999 based on publications considered relevant for analytical chemistry Caliper goes public
51. history 2000 based on publications considered relevant for analytical chemistry
52. history 2001 based on publications considered relevant for analytical chemistry 1st issue of „Lab Chip“ journal
53. history 2002 based on publications considered relevant for analytical chemistry
54. history 2003 based on publications considered relevant for analytical chemistry
55. history 2004 based on publications considered relevant for analytical chemistry
56. history 2005 based on publications considered relevant for analytical chemistry
57. history 2006 based on publications considered relevant for analytical chemistry
58. history 2007 based on publications considered relevant for analytical chemistry
60. • substrate materials used glass-glass pdms-pdms pdms- glass glass-silicon silicon-silicon pmma- pmma glass-glass pdms-pdms pdms-glass glass-silicon silicon-silicon pmma-pmma glass-gold-glass glass-laminate-glass glass-silicon-glass ordyl multi-layer glass pdms-copper pdms-silicon pdms-silicon-pdms quartz-quartz substrate materials
61. • integrated featurs, like metal electrodes, heaters, membranes etc integrated features metal electrodes heaters porous membrane nothing metal electrodes heaters porous membrane slit array outside liquid membrane planar waveguides x-ray source t sensor phase quides nothing
62. • topology • spider, tree, loop, network, etc topology of channels tree, spider single channel 1 loop central ch array, tree central bed, ch around binary branching structure non-binary branching well tree, spider single channel 1 loop central ch array, tree central bed, ch around binary branching structure non-binary branching well central chamber, frit, tree network central chamber, single ch
63. • interfacing type • (“chip to world interface”) interfacing type eppendorf pipets, open fused silica tubing don't know, not used plastic tubing - glue flat metal plates large holes, thick glass cover flat plastic plates large holes, thick pdms cover eppendorf pipets, open fused silica tubing don't know, not used plastic tubing - glue flat metal plates large holes, thick glass cover flat plastic plates large holes, thick pdms cover
64. • what is the chip used for? application area separation detection reaction biology basics application pumping sample prep separation detection reaction biology basics application pumping sample prep
65. • How many chips were in direct line to commercialization? commercializations commercial attempt no attempt commercial attempt no attempt
66. biomimetics - microfluidics Cell culture model (replace animal model) Perfusion allowing 3d cell cultures for longer term Liver cells, human and zebra fish Increased size of cell culture  better analysis of low abundancy metabolites
67. Increased size of cell culture  better analysis of low abundancy metabolites compounds in blood a) clinically used b) existing a) b)
68. a) b) single cell 20 x 20 x 20 um 8.10-15 m3 hormones 10-9 ... 10-12 M hormones per cell volume 8.10-21 mol/cell ... 8.10-24 mol/cell large cell culture (10 cm)3 1011 cells in culture hormones 10-9 ... 10-12 M hormones per cell volume 8.10-10 mol/culture ... 8.10-13 mol/culture larger volume cell cultures are easier for low abundancy metabolite assays, but will show average values only single cell assays can show cell variability, but only for high enough concentration metabolites
69. perfusion for cell culture (0.1 µL) differentiate HepaRG cells directly in microfluidic device CK18 / CK19 expression level (microfluidic chip by Mimetas) M.Jang et al, Biomicrofluidics 9, 034113 (2015), M.Jang et al, Biomedical Microdevices (in press 2018)
70. Porous membrane Cell Culture Medium 75µL/h Novel cell culture device for long-term toxicology studies Principle: - Cells embedded in 3D matrix (Matrigel) - Perfusion of cell culture medium through the matrix - Improved nutrient supply and removal of metabolic waste products perfusion for cell culture (1 mL)
71. filling option #3
72. perfusion for cell culture
73. perfusion for cell culture
74. perfusion for cell culture
75. perfusion for cell culture epithelial cell viability in newly developed device after 7d comparable to the non-perfused control measured by live/dead staining and flow cytometry Comparison of cell viabilities after 7d
76. perfusion for cell culture Comparison of number of cells after 7d Comparison of device and control shows significant increase in number of cells recovered from newly developed device after 7d in culture. Next steps: address reproducibility optimise perfusion apply to human liver cells zebra fish liver cells
77. perfusion for cell culture • can we cultivate large volumes of cells „on chip“? • like: Daphnia or zebra fish liver cell culture, in g quantities? • imaging of such cell culture nearly impossible • off line analysis is disruptive, flow cytometry • indirect analysis, metabolites • analysis of headspace (volatile metabolites)
78. DIFFERENTIATION OF HUMAN LIVER PROGENITOR CELL LINE (HEPARG) DIRECTLY ON A BIOCHIP
79. • HepaRG cells ? A human hepatic progenitor cell line that can differentiate into hepatocyte and biliary like cells. 9 days 2 week 5 Days Progenitor cells Hepatocyte-like cell surrounded by epithelial cells, biliary cell. Fully differentiation to hepatocyte and biliary cell 1 month 2%DMSO Introduction of HepaRG cells
80. Medium flow MG Col 14 Days Chip cultivation 5 Days Progenitor cells (PGC) Not fully differentiated cells (NFDC) 14 Days Flask cultivation Medium flow Medium flow Medium flow 2. Flow direction 3. With & Without DMSO Medium flow Medium flow 1. Matrix Flo w Flow Flo w Flow Y Z Flo w Flo w X Y • Motivation Differentiate HepaRG cells directly in microfluidic device to reduce time and DMSO treatment • Strategies - Different Matrix (Matrigel vs. Collagen I) - Flow direction ( One side vs. Both side) - DMSO treatment (with vs.without) Aim of Research
81. Medium flow MG Col PGC NFDC 1. Matrix CK19 (Biliary cells marker) Albumin (Hepatocyte marker) Matrigel vs. Collagen I  Matrigel : co-expression of albumin and CK19.  Collagen I : Albumin expression very low, only CK19 expression
82. Medium flow Medium flow Medium flow PGC NFDC  Cell cluster size PGC (One) PGC (Both) NFDC (One) NFDC (Both) 2. Flow direction One side flow vs.Both side flow  Flow influenced on generation of cell clusters.  Cells under both side of flow : spheroid is bigger
83. PGC Medium flow Medium flow Medium flow NFDC NTCP expression One side flow vs.Both side flow  Flow influenced on expression level of membrane protein. 2. Flow direction
84. PGC + DMSO NFDC - DMSO + DMSO - DMSO  Cell viability -/- +/+ -/- +/+ PGC NFDC 0 20 40 60 80 100 Cell viability % +DMSO vs. - DMSO  DMSO reduced cell viability significantly.
85. PGC  CK18/19 expression level -DMSO NFDC +DMSO PG C -D M SO PG C +D M SO N FD C -D M SO N FD C +D M SO 0 20 40 60 80 100 CK18/CK19 Ratio +DMSO vs. - DMSO  DMSO reduced both CK18,CK19 expression level.  PGC shows the highest ratio of CK18 expression level CK18(Hepatocyte)/19(Bilary cell)
86.  CYP3A4, ALBUMIN EXPRESSION PGC NFDC -DMSO +DMSO  CYP1A induction assay 3 Day 7 Day 14Day 0 10 20 30 PGC -DMSO PGC +DMSO UDC -DMSO UDC +DMSO -DMSO +DMSO -DMSO +DMSO PGC NFDC 0 1 2 3 4 5 -DMSO Fold change ng/day/cell  Albumin production +DMSO vs. - DMSO  PGC without DMSO : the best cultivation in microfluidic device
87. Distribution of cell population Z= 64 µ m Z=64µm Z=113µm XZ YZ Y X Z  CK18/ CK19 expression in PGC  30µm-80µm : stable cell spheriods  Cell distribution : CK19(Biliary cells) surrounded by CK18 (Hepatocytes)
88. Cell polarization Y X Z  NTCP (Basolateral membrane) expression in PGC Z=36µm Z=63µm Z=100µm XZ YZ  Most of Spheroid (30-80µm) express NTCP  Differentiated HepaRG cells in microfluidic device show highly polarized basolateral membrame.
89. Conclusion 1. Only matrigel induce cells to differentiate to hepatocyte but not collagen I. 2. Both side of flow helped cells to form cell cluster and express membrane protein. 3. DMSO influenced on cell viability and albumin production but not for CYP expression level. 4. Differentiated progenitor cells in microfludic device showed highly polarized membrane protein. Finally, Progenitor cells can differentiate into hepatocyte-like cells in microfluidic device for 2 weeks without DMSO.
90. ecotoxicology, chronic toxicity Metabolites of Daphnia magna (Crustacea) Keep Daphnia alive for days Take headspace (air above water) as samples Analyze contents, qualitative and quantitative  Search for biomarkers for Daphnia presence and „happyness“
91. Daphnia metabolites Human excretion, example Weight 80 kg, volume 80 L Feces per day ca 250 g, of which 60 g are solids Urine per day ca 1 L, of which 120 g are solids Total excreted metabolites ca 120 g per day Daphnia excretion ??? Weight 8 mg, volume 8 mL  Total excreted metabolites ca 12 mg per day (in 1 L of water, that is in the order of 12 ppb)
92. Daphnia metabolites head space analysis equilibrium of volatile molecules between gas and water phase depends on vapor pressure advantage: easy access easy analysis reversible gas phase water phase
93. Daphnia metabolites Method: acidification, headspace, gas chromatography, mass spectrometry Collaboration with INM, Yuliya Silina red: algae feed black: Daphnia
94. Daphnia metabolites Method: acidification, headspace, gas chromatography, mass spectrometry Collaboration with INM, Yuliya Silina red: algae feed black: Daphnia algae feed: many oxygen containing compounds, ethers, alcohols Daphnia: many fatty acid derivatives, aldehydes, ketones
95. Daphnia metabolites Next steps: Reproducibilty ? Life cycle of Daphnia ? Stressed Daphnia ? Identify biomarker molecule Can it be used for envirotox monitoring ? Develop chemical sensor / µTAS chip for it
96. Felix Löser Jun Hyung Im André-René Blaudszun Matthias Altmeyer Mi Jang Wenming Wu Leon Abelmann, prof Xiangping Li Jonathan O‘Connor Agu Vahtrik Irving Mbougue Djuemo Chang Gyun Park acknowledgements