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Upgrade Your Cell-Based Assays to 3D !! GravityPLUS™ 3D Cell Culture Kits InSight™ Microtissues

Tel : 02-3672-3188 www.hdbio.net


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


The Fastest Growing Company

ÇŚ Swiss- and US based company ÇŚ 4 patents granted, one still pending ÇŚ 12 of top 15 pharma companies among customers ÇŚ Partner Network Provides Service and Co-Marketing Opportunities

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

ÇŚ ˜˜†žǂ—Š†‰ž Čž Žˆ—”™Ž˜˜šŠ˜ Ž“ ȤȥÇ‚ œŠ‘‘ ‹”—’†™ ‹”— •—Š‰Žˆ™Ž›Š ™” Â?ŽˆŽ™ž †“‰ ÂŠÂ‹ĘŹÂˆÂ†ÂˆÂž ™Š˜™Ž“Œ ÇŚ †˜ž ™” š˜Š Ž“ ž”š— ‘†‡ š˜Ž“Œ ”š— —”‡š˜™ •—”™”ˆ”‘˜ ÇŚ ÂŽÂŒÂ?‘ž ‹š“ˆ™Ž”“†‘ †“‰ ˜™†“‰†—‰ŽÂ&#x;Š‰ ÇŚ Ž›Š—ƽ ™š’”—ƽ ˆ†—‰Ž†ˆ †“‰ ’†“ž ’”—Š ™Ž˜˜šŠ˜ †›†Ž‘†‡‘Š

ÇŚ Š™ ”š— ™Š†’ •—”‹Ž‘Š ž”š— ˆ”’•”š“‰˜ š˜Ž“Œ ”š— •†™Š“™Š‰ ™ŠˆÂ?“”‘”Œž ÇŚ ‘ŠÂ?Ž‡‘Š Â?ÂŽÂŒÂ?ǂ–š†‘Ž™ž ˜Š—›ŽˆŠ œŽ™Â? ‹†˜™ ™š—“ǂ†—”š“‰ ™Ž’Š˜ ÇŚ š˜™”’ Čž Žˆ—”™Ž˜˜šŠ †“‰ †˜˜†ž ‰Š›Š‘”•’Š“™ ‹”— ™†Ž‘”—Š‰ ˜”‘š™Ž”“˜

ÇŚ Â?Š ‘Š†‰Ž“Œ ȤȥÇ‚ †“‰ ȞȣČ&#x; ˆŠ‘‘ǂˆš‘™š—Š ˜”‘š™Ž”“ ‹”— ‰Š›Š‘”•Ž“Œ ˜ˆ†‹‹”‘‰ǂ‹—ŠŠ Čž Žˆ—”™Ž˜˜šŠ˜ ÇŚ —†›Ž™ž Č? ‘†™Š˜ †‘‘”œ ‘”“Œǂ ™Š—’ ˆš‘™š—Š †“‰ Ž’†ŒŽ“Œ ÇŚ Čž “ ÂŽÂŒÂ?™Č? Š‘‘ǂ š‘™š—Š Š‰Ž† ‹”— •Š—‹Šˆ™ —Š˜š‘™˜


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.

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LDH assay

20000 40000 cell number

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LDH assay

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Size

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A

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taxol

IG50

compound 3

cisplatin

0 days

C

monolayer

microtissue

0

Monolayer and Microtissue Drug Screening

4

microtissue

12

37

cisplatin [μM]

80

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1

111

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>10

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compound 3 [μM]

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monolayer

333

4 days of treatment wit taxol [nM]

22.1

80

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taxol [nM]

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1 compound concentration [μM]

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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

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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]


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