What is Micropatterns? CTYOO Cytophobic

coating (extra

cellular matrix , ECM, such as fibronecin, collgene, Laminin, ect)

2D + cell culture

CYTOO adhesive micropaerns

A breakthrough in quantave cell analysis

CYTOO

Cells in a standard culture dish Cells cultured on CYTOO products

micropattern micropattern

Reduce cell to cell variability

Control the locaon of cell compart-

ments and protein networks

Achieve simple and rapid image analysis

Improve assay reproducibility

Map a standard? averaged cell to be

used as a Reference Cell

Cell biology and high throughput screening

Applicaon assays:

Cell Shape and Acn Cytoskeleton

Microtubule network

Cell Polarity and Organelle Posioning

Cell Division and Mitoc Spindle Orientaon

Quantave Cell Phenotyping

Cell signaling

Toxicology

...

CYTOO

CYTOOplates

for screening

CYTOOchips

for research

The glass coverslip format with an array

of up to 20,000 micropaerns and a printed

grid for easy localizaon.

The standard glass boom microplate for-

mat presenng over 1,000 micropaerns

per well.

RPE1 V shape micropattern RICM

(credits : M Thery/ M Bornens)

http://www.youtube.com/watch?v=2ayd7H2c6fM

CYTOO micro pattern

http://www.youtube.com/watch?v=nKMDoHn8yFE

QR code QR code

CYTOOchambersTM

1 and 4 wells

micropattern micropattern

micropattern

Epithelial cells (HeLa, RPE-1, CHO, MDCK, BSC,

MCF10A)

Fibroblasts (murine NIH-3T3, BHK)

Adenocarcinoma cell lines (MDA-231, A549)

Hepac cell lines (HepG2)

Primary cells (Rat astrocytes, Rat ventricular myo-

cytes, myoblasts)

Neurons and neuron progenitors (SH-SY5Y; hip-

pocampal and corcal neurons)

Stem Cells (Human mesenchymal stem cells,

mouse embryonic stem cells/mESCs)

Check here for an updated list of cell types:

www.cytoo.com/celltypes

Efficient labeling of mitochondrial networks

in micropaerned cells for toxicity studies

Yoran Margaron, Sebasen Degot, Alexandra Fuchs, Chloe Loiraud *

Opmized protocol for mitochondrial network labeling in both live and fixed cells

Cell individualizaon and normalizaon thanks to adhesive micropaerns

StraighForward comparison between different experimental condions

seminar hp://

www.youtube.com/

watch?

v=AvwOnqSWqh4

Build a Reference Cell for powerful cell phe-

notype quanficaon

Muriel Auzan*, Violaine Chapuis*, Joanne Young*, Anne Bghin**, Pauline Mnager*, Sbasen

Degot*.

* CYTOO Cell Architects, 7 parvis Louis Nel, Grenoble,

France - www.cytoo.com

** Centre Commun de Quantimtrie, Facult de

Mdecine Rockefeller, 8 av. Rockefeller Lyon, France

*** Reference Cell

tech@biogenesis.com.tw

,,

CYTOO micropattern , ,

,

Unveiling drug-induced phenotypes on mi-

cropa$erned cells

HeLa cells were seeded in parallel on full fibronecn or on

fibronecn L micropaerns, then treated with drugs at 10M

for 1h (except for Nocodazole at 5M) or leN untreated. Nu-

cleus; Acn; Fibronecn micropaern

Robust quanficaon of drug effects with

only 50 cells

Figure 3: Reference Cell gallery depicng drug effects on the acn

distribuon in cells on L micropaerns and performed in

a 96-well CYTOOplate. Nocodazole (5M), Blebbistan (10M),

Y27632 (10M) and Cytochalasin D (10M), n=50 cells for all

condions.

JoVE

website :

JoVE 46: http://www.jove.com/

index/Details.stp?ID=2514

Reproducible internal cell organizaon in re-sponse to the geometry of the micropa$ern

Figure 2:

Top: representave images of individual cells labeled with an CD63 (leN: con-

trol, right: nocodazole) yield lile insight into the effect of this drug on the mul-

vesicular body (MVB) network.

Boom: 2D Density maps calculated for the MVB compartment show signifi-

cant differences between the control and the Nocodazole (NZ) treated cells.

Automacally calculated P value < 10-4 with only 20 cells.

Figure 1: Stable density maps that represent the or-

ganizaon of different endomembrane compart-

ments were obtained from the indicated number of

cells (n=35 to 82). 2D and 3D density maps of mul-

vesicular bodies (CD63), early endosomes (Rab5),

ER exit sites (Sec13) and the secretory compartment

(Rab6) are shown.

Lysosome compartment

Early endosomes

Endoplasmic re-

culum exit sites Trans Golgi,

secreon and retrograde

transport

Quanfying the undetectable:

Probabilisc density maps on normalized cells

centrosome Nucleus Golgi

CYTOO chips

& CYTOO plates

Molity

Cell Migraon

Cells on fibronecn-paerned lines are polarized

Model for macrophage- tumor cell pairing and streaming :

robust & physiological

Courtesy of Landes Bioscience, reproduced from IntraVital 2012;1(1):77-85. (Le$:

in vivo imaging and Right: in vitro imaging on micropa)erned 1D tracks)

Nodediameter: 70 m, Path: 100 m

Structure opmizedfor SH-SY5Y neuroblastomacellline

Protein: PDL; Cytophobigcoang: PLL-g-PEG

Formaon of neuriteconnecon between adjacent groups of cells; 6 to 10 cell

bodies per paern in average

CYTOOplate Neuro plate

Neurite outgrowth is highly bi-

direconal; no branching observed Easy single cell neurite length quanfica-

on possible But the posion of cell bodies is not con-

trolled

Bipolar neuriteoutgrowth & Migraon

Cell bodies and outgrowing processes are

clearly separated The neuronal cell bodies adhere prefer-

enally to the nodes where local adhe-

siveness is greater and are stabilized

there

Controlling neuriteoutgrowth & Cell body posion

Single neuron network with axon Guidance.

Neuron polarizaon

The curved path geometry provides a strong inhibitory

effect on axon specificaonand prevents mulple axon for-

maon. Axon rafficking studies are facilitated. Curved lines for neuriteoutgrowth are supposed to mimic

in vivo neuron path-finding in a crowded environment

Connecons are progressively created between nodes

within 72 h Quanficaon of network formaon= Number of connec-

ons per node Kinecs of neuriteoutgrowth can be measured in me

lapse

Network Formaon Assay

1

5

3 & 4

2

hp://www.youtube.com/watch?v=0rglGMAP78c

Further reading 1. Debnath J, Muthuswamy SK, Brugge JS. Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-

dimensional basement membrane cultures. Methods. 2003;30(3):256268.

2. Wendt MK, Smith JA, Schiemann WP. Transforming growth factor--induced epithelial-mesenchymal transion facilitates epi-

dermal growth factor-dependent breast cancer progression. Oncogene. 2010;29(49):64856498.

3. Kumar A, Xu J, Brady S, et al. Tissue transglutaminase promotes drug resistance and invasion by inducing mesenchymal transi-

on in mammary epithelial cells. PLoS ONE. 2010;5(10):e13390.

4. Sang L, Miller JJ, Corbit KC, et al. Mapping the NPHP-JBTS-MKS protein network reveals ciliopathy disease genes and pathways.

Cell. 2011;145(4):513528.

5. Li H, Yang W, Mendes F, Amaral MD, Sheppard DN. Impact of the cysc fibrosis mutaon F508del-CFTR on renal cyst formaon

and growth. Am. J. Physiol. Renal Physiol. 2012;303(8):F11761186.

6. Qin X-Y, Fukuda T, Yang L, et al. Effects of bisphenol A exposure on the proliferaon and senescence of normal human mammary

epithelial cells. Cancer Biol. Ther. 2012;13(5):296306.

7. Wang H, Lacoche S, Huang L, Xue B, Muthuswamy SK. Rotaonal moon during three-dimensional morphogenesis of mammary

epithelial acini relates to laminin matrix assembly. Proc. Natl. Acad. Sci. U.S.A. 2013;110(1):163168.

8. Hrm V, Virtanen J, Mkel R, et al. A comprehensive panel of three-dimensional models for studies of prostate cancer growth,

invasion and drug responses. PLoS ONE. 2010;5(5):e10431.

:

Protocol for seeding MDCK cells on CYTOOchips (MO-EXT-19)

Hints for using Matrigel (MO-EXT-20).

micropattern, ECM coating, 3D , MDCK Acini

,, Collagen-I Fibronectin Acini

CYTOO hp://www.cytoo.com/CYTOO-adhesive-

micropaerns-applicaons.php

service@biogenesis.com.tw 0800-211-667

02-26558877 037-687493 04-22602466

07-3105441 03-8463953 2013Q405

http://www.biogenesis.com.tw