Scaffold- free
Upgrade Your Cell-Based Assays to 3D !! GravityPLUS™ 3D Cell Culture Kits InSight™ Microtissues
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Why is 3D Cell Culture System Used? Drug-Target interactions
Drug-Cell response
Drug-Tissue response
Drug-Organism response
Increasign cost of drug development program
What is Real 3D Cell Culture?
More in-vivo like‌ Immediately after seeding
cell shape
cell-to-cell interactions
cellular organisation
One week after seeding
Sinusoidal Endothelial Cell Sinusoid
Kuppfer Cell Stellate Cell
Central vein
hepatocyte Liver
hepatic lobule
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Work smarter with better biology
3D Microtissues: to make or buy InSphero’s scaffold-free 3D Microtissues are multicellular spheroids, which are morphologically and functionally very similar to native tissue. Easily amenable to standard biochemical assays, imaging and immunostaining, they are widely used for pre-clinical toxicity and efďŹ cacy studies to improve biological relevance. Close cell-cell contacts, a gene expression proďŹ le closer to in vivo and an intact endogenous extracellular matrix make them ideal models to improve in-vitro cell-based assays.
In-house Compound Pro ďŹ ling
InSphero is the only supplier on the market that gives you the freedom of choice: You can use our assay-ready 3D InSight™ Microtissues in your lab or decide to outsource your compound proďŹ ling to our skilled team as a feefor-service project. And, if you do not ďŹ nd a suitable 3D Microtissue in our catalog, use our patented GravityPLUS™ Hanging-Drop Platform to make your own. All global top 15 pharmaceutical companies use our technology today. When do you upgrade to 3D?
Expert 3D Screening Services
Cell Biology Research
3D InSight™ Microtissues
3D InSight™ Services
GravityPLUS™ Hanging-Drop System
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Toxicity analysis of hepatoxic compounds in primary human hepatocytes in 2D and 3D-culture Simon Messner1, Paul Walker2, Heather Woodhouse2, Patricia Ragazzon2, Helen Gill2, Jan Lichtenberg1, Wolfgang Moritz1, Jens M. Kelm1 and Katya Tsaioun2 1
InSphero AG,ZĂœrich, Switzerland; 2Cyprotex, Macclesfield, UK
Shoba Shetty1, Karissa Adkins1, Marc Roy1, Irina Agarkova2, Jens M. Kelm2, and Simon Messner2 Pfizer-Drug Safety R&D, Groton, CT, 2InSphero AG, Schlieren, Switzerland
A 3D co-culture model-based assay to assess liver Kupffer-cell activation and functionality
Introduction A day 4
B
Microtissue Morphology day 4
day 10
Cell Number to Readout Correlation
day 10
Histological analysis (H&E) of 4 and 10-day old microtissues composed of DLD-1 (A) and T47-D (B).
4.
200 150
0
50
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0
LDH assay
20000 40000 cell number
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6000
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cell number
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LDH assay
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4000 cell number
2000
LDH assay
4d
4d
60000
0
0
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80 60 40 20 0
80
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40 30 20
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ATP assay
0
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20000 40000 cell number
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cell number
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ATP assay
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ATP assay
0d
0d
15 10 5 0
15 10 5 0
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70 60 50 40 30 20 10 0
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0
0
cell number
4000
Pico Green
0
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cell number
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4d
4d
Pico Green
0d
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Pico Green
20000 40000 cell number
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0d
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Size
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Size
4d
4d
A
B
taxol
IG50
compound 3
cisplatin
0 days
C
monolayer
microtissue
0
Monolayer and Microtissue Drug Screening
4
microtissue
12
37
cisplatin [μM]
80
60
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70
1
111
60
40
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0
-20
120
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0
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130
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-10
-30
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>10
1.08
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10
compound 3 [μM]
1 compound concnetration [μM]
10 compound concentration [μM]
100 compound concentration [nM]
monolayer
333
4 days of treatment wit taxol [nM]
22.1
80
120
1000
120
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100 compound concentration [nM]
100
10
10 compound concentration [μM]
taxol [nM]
19.9
1 compound concentration [μM]
70.0
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59.4
Conclusion
This indicates that a more predictive screening system may not only reduce false positive hits that lead to failure at later stage, but also increase the chance to identify novel compounds that are only active in 3D.
Within this study we could show that tumor microtissues produced by the hanging drop technology can be implemented into a screening campaign. Moreover, in contrast to the commonly known concept of multicellular resistance of cells in a 3D format, we have identified a compound which displays higher efficacy in cancer cells grown as microtissues compared to their monolayer counterparts.
Cell-based assays retain a high potential to be improved by creating a more native-like environment to reflect the biological response to substances more closely. However, the implementation into routine screening processes has been impaired by complex production processes.
8.
1
Images of DLD1 microtissues treated with indicated concentrations of taxol for 4 days (A). Dose-response curves of DLD-1 microtissue and monolayer cultures treated with taxol, cisplatin and an undisclosed compound (compound 3) (B). The resulting IG50 values are summarized in C.
7.
Marianne M. Helbling1, Maren Drewitz1, Manuela Bieri1, Carina Lotz2, Lorenza Wyder2, Francois Lehembre2, Wolfgang Moritz1, Jan Lichtenberg1, Urs Regenass2, Jens M. Kelm1 1 InSphero AG, Technoparkstrasse 1, 8005 Zurich, Switzerland 2Actelion Pharmaceuticals Ltd, Gewerbestrasse 16, 4123 Allschwil, Switzerland
Characterization and Drug Sensitivity Testing of HTS-Compatible Cancer Microtissues 1.
Material and Methods
A
50 0
0
Cell number to readout correlation for microtissue and monolayer cultures for three different assays: LDH content (CytoTox one, Promega); ATP content (CellTiter-Glo, Promega) and DNA content (PicoGreen, Invitrogen), as well as the microtissue diameter.
5.
T47-D
Dynamic Range of Assays over 4 Days of Treatment
2000
0d
0d
growth [% of control] growth [% of control]
Although the advantages of organotypic 3D microtissue models have been known for years, complex production and readout processes hampered its implementation into an industrial setting. This study presents a novel method to produce organotypic, scaffold-free cancer microtissues models (0.1-1 mm in diameter) and its implementation in an automated screening process.
formation
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DNA per well [ng]
DNA per well [ng]
2.
seeding
B
6.
rel. LDH per well
The dynamic range is demonstrated for the different assays. Data are shown for DMSO treated controls at 0 and 4 days of treatment of six biological replicas +/- sd.
1500 1000
0
500
1500 1000
0
500
growth [% of control]
growth [% of control]
growth [% of control]
ATP per well [pg] ATP per well [pg]
The hanging drop technology was adapted to standard 96- A Lid TM well format (A, GravityPLUS ) allowing for generation and processing of droplets from the top, similar to processing Rasterplate standard 96-well plates. This is enabled by a microfluidic withstrips channel connecting an Reservoir Bottomplate B inlet funnel at the top and an outlet funnel at 96-well V-bottom plate containing the bottom (B). The one microtissue per well InSphero`s GravityPLUS plate use of this technology enables manual and automated microtissue production as well as the processing of microtissues such as transfer into a 96-well receiver plate for biochemical - and cell biological analysis (C). C automated microtissue automated transfer
T47-D 250 cells
5 10 15 days after microtissue seeding
MT diameter [μm] MT diameter [μm]
Assay-ready microtissues
Microtissue Formation and Growth
0
T47-D
To assess compound effects on proliferation, the lactate dehydrogenase (LDH) content of microtissues after 4 days of treatment (4d) was measured and normalized to the initial measurement prior treatment (d0). The resulting values indicate either growth (up to 100%) or non-growth (0%). Cytotoxic effects leads to values below 0%. DLD-1 and T47-D were chosen as best suited models for the growth evaluation. GI50 values were calculated with XLfit.
3.
500 400 300 200 100 0
MT diameter [μm] MT diameter [μm]
Microtissue formation (A) and growth (B) of DLD-1 (colon cancer) and T47-D (breast cancer) over 10 days. day 4 day 8 day 0 day 6 day 2 day 10
DLD-1 500 cells
5 10 15 days after microtissue seeding
DNA per well [ng] DNA cpwe well [ng] DNA per well [ng] DNA per well [ng]
T47-D 250 cells
0
ATP perwell [pg] ATP per well [pg] ATP per well [pMol] ATP per well [pMol]
growth [% of control]
DLD-1
DLD-1 DLD-1
T47-D rel. LDH per well rel. LDH per well
DLD-1 T47-D
DLD-1 500 cells
500 400 300 200 100 0
microtissue diameter [μm]
rel. LDH per well rel. LDH per well rel. LDH per well
A
B microtissue diameter [μm]