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)
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
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
allowing 3d cell cultures for longer term Liver cells, human and zebra fish Increased size of cell culture better analysis of low abundancy metabolites
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
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)
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
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)
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
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
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
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
-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)
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
XZ YZ Y X Z CK18/ CK19 expression in PGC 30µm-80µm : stable cell spheriods Cell distribution : CK19(Biliary cells) surrounded by CK18 (Hepatocytes)
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.
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.
alive for days Take headspace (air above water) as samples Analyze contents, qualitative and quantitative Search for biomarkers for Daphnia presence and „happyness“
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)