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CORNING Life Sciences

Cell Culture TreatmentsCell Culture Treatments

Corning – A brief History

- A brief history

- Corning’s Brands

- Corning’s Workflows- Cell Culture Workflow

- Cell Culture surfaces- Suspension cells

- Non treated

© 2013 Corning Incorporated 2

- ULA = Ultra Low Attachment

- Adherent Cells- TC = standard treated culture

- Synthetic non specific

- Biological coating

- Syntethic defined

- Syntehtic specific

- Other products with specific cell culture surfaces- Inserts cell culture

- Assays microplates

Founded in 1851 : world leader in specialty glass and ceramics.

Corning created and make keystone components that enable high-technologysystems for consumer electronics, mobile emissions control, telecommunicationsand life sciences : bubble (first one it Edison ; touchable screens ; …

Headquarters : Corning, New York

Employees:

Approximately 25,000 worldwide

Corning – A brief History


Corning acquired Costar Corporation to become Corning Costar in 1993.

Corning acquired Axygen end of 2009

Corning acquired Gosselin end of 2010

Corning acquired Falcon DL end of 2012

Corning Life Sciences – Different Brands & Market segments

• Whatever is your application, you can now get a more comprehensive line of labresearch tools with Corning’s quality.

FALCON


cellgro® is available in Europe as Mediatech™.

Cell Culture, Drug Discovery &Liquid Handling

Molecular BiologyGenomics

Microbiology

Corning Life Science - Workflows


Corning Life Science - Workflows

Cell Culture surfaces :

- Suspension cells

- Adherent cells

Adherent Cells :

- Standard - TC

- Synthetic improved


- Synthetic improved(CellBIND ; Primaria)

- Biological(ECM – Biocoat)

- Synthetic defined(Purecoat)

- Synthetic specific(OsteoAssays / Synthemax)

StationaryMoving

Adherent culture Suspension culture

• Spinner

StationaryMoving

• Flasks• Rollerbottles

• Flasks

Corning Life Science – Cell Culture surfaces


• Spinnerflasks

• Shakerflasks

• Rollerbottles

• Bags

• Flasks• Storagebottles

• Dishes• Microplates• Transwellinserts

• Bags

bottles

• Micro-carriersin spinnerflasks

• Flasks onrockers

• Microplates

• Dishes

• Transwellinserts

• HYPERFlask

• CellSTACK

Moving

Adherent culture Suspension culture

Corning Life Science – Cell Culture surfaces


• Suspension Cell Culture :

− NT (Not Treated)

• For standard suspension cell culture

− ULA (Ultra Low Attachment)

Corning Life Science – Cell Culture surfacesSuspension Cells


• For advanced suspension cell applications (stem cells..)

• Adherent Cell Culture :

- Standard – TC

- Synthetic improved (CellBIND ; Primaria)

Corning Life Science – Cell Culture surfacesAdherent Cells

Specificity to CellsAnimal Free

YES

YES


- Biological (ECM – Biocoat)

- Synthetic defined (Purecoat)

- Synthetic specific (OsteoAssays / Synthemax)

NO

YES

YES

Cell Culture surfaces - Adherent CellsStandard Tissue Culture Surfaces

Standard Tissue Culture (TC) Treatement :

• To be used with adherent cells, the Polystyrene needs to be treated toadd charges and to get it hydrophylic.

• Polystyrene is fully hydrophobic and no natural.

• TC-surface treatment is achieved by (similar proceedure) :

− By gas plasma : placing polystyrene vessels into a vacuum chamber that


− By gas plasma : placing polystyrene vessels into a vacuum chamber thatreceives a mixture of gases with electric power.

− By corona discharge.

• The treatment will create a modification of the polystyrene altering it‘ssurface chemistry Negative charges

• Feature No specific

Cell Culture surfaces - Adherent CellsStandard Tissue Culture Surfaces

- uncharged- hydrophobic- No cell attachment

e.g. "non-treated"rounder

40 ml droplet

Conveyor belt

High energy oxygen ions

Corona Discharge Plate

Conveyor belt

High energy oxygen ions

Corona Discharge PlateCultureVessels


- charged groups- hydrophilic- cell attachment

e.g. "TC"flatter

40 ml droplet

HH H H

HH H HHH

H

H

C C C C CC C CC

HH H H

HH H HHH

H H

H

H

C C C C CC C CC

HH H H

HH H HHH

H

H

C C C C CC C CC

HH H H

HH H HHH

H H

H

H

C C C C CC C CCHydrophobic

H H

H

O HH H

O

OHH

OC C C C C C C CC

H

H

O OHHH

H H

O

OH

OH

HO

H

OC C C C C C C CCHydrophilic

H H

H

O HH H

O

OHH

OC C C C C C C CC

H

H

O OHHH

H H

O

OH

OH

HO

H


H H

H

O HH H

O

OHH

OC C C C C C C CC

H

H

O OHHH

H H

O

OH

OH

HO

H


SurfacetreatmentSurfacetreatment

Cell Culture surfaces - Adherent CellsSynthetic Non Specific – CellBIND & Primaria

Syntethic Non specific (Improved attachment) – CellBIND or Primaria :

• To be used with adherent cells, the Polystyrene needs to be treated toadd charges and to get it hydrophylic.

• Polystyrene is fully hydrophobic and no natural.

• TC-surface treatment is achieved by :

− a specific gaz mixture.


− a specific gaz mixture.

− A difference source of energy (microwaves).

• The treatment will create a modification of the polystyrene altering it‘ssurface chemistry more balanced – vs + positives

• Feature No specific ; more dedicated to difficult cells

Cell Culture surfaces - Adherent CellsSynthetic Non Specific – CellBIND & Primaria

CellBIND® TCTBD

Primaria™Not Treated

Treatment N2O O2 NH4 /proprietary

% Oxygen/

Nitrogen35% Oxygen 22% Oxygen

14.1% Oxygen

11.1% Nitrogen1.8% Oxygen

Contact


TCT

CellBIND

Contact

angle23 - 26° 56 - 63° 75° 90 - 95°

Charge Negative Negative Positive

Cell Culture surfaces - Adherent CellsECM – Biological Coating

Biological coating with ECM molecules :

• Cells are not living in 2D into a plastic box !

• They are normally „living“ in the context of other cells, surrounded and incontact with an extracellular matrix (ECM)

• The TC-treated surfaces can be coated with ECM components :


− Biological molecules from the ECM :

• Can be bought pre-coated – ready-to-use products.

• Can be bought as a chemical – to plate into the lab.

− Reconstituted ECM = Corning® Matrigel® Matrix :

• Can be bought pre-coated – ready-to-use products.

• Can be bought as a chemical – to plate into the lab.


READY-TO-USE

Biological molecules from the ECM :

Collagen I

Collagen IV

Fibronectin

Gelatin

Laminin


Laminin

Poly-Lysine

Poly-D-Lysine/Laminin

Poly-L-Ornithine/Laminin

Laminin/Fibronectin

Reconstituted ECM = Corning® Matrigel® Matrix :

Matrigel

Matrigel Growth Factors Reduced (GFR)


TO COAT INTO THE LAB :

Biological molecules from the ECM :

Collagen I, II, III, IV, V, VI

Fibronectin

Laminin

Osteopontin

Poly-Lysine


Poly-Lysine

Vitronectin

Reconstituted ECM = Corning® Matrigel® Matrix :

Matrigel

Matrigel hESC (human Embryonic Stem Cells) qualified

Matrigel HC to do tumor injection

Matrigel Phenol Red free

Matrigel GFR (Growth Factor Reduced)

Dispase & Cell Recovery Solution to recover cells from Matrigel


APPLICATIONS :• Endothelial cell migration, invasion

• Angiogenesis tube formation

• Tumor invasion

• 3-D-cell culture

• Maintenance of human embryonic stem cells

• Neurite outgrowth

• Hepatocyte culture


SEM‘s of hepatocytes cultured for two days on collagen I (1),collagen I gels (2), or BD Matrigel Matrix (3). Note the cluster ofspherical cells for hepatocytes cultured on Corning Matrigel Matrix,typical of differentiated cells.

TC Plates

After Wash,Before Wash After Wash Calcein AM Staining

APPLICATIONS :• HEK293



BD BioCoatPDL Plates

After Wash,Before Wash After Wash Calcein AM Staining

HEK-293 cells were cultured under serum-free conditions on TC-treated or BDBioCoat PDL 384-well black/clear plates. Samples were washed using aSkatron Washer (Molecular Devices).

ADVANTAGES :• More in vivo like properties

• Better way of life :

− attachment, proliferation, growth

− including difficult cells as : primary cells, neural cells, hepatocytes,endothelial cells, …

• Differentiation and functional studies possible



• Differentiation and functional studies possible

• 3-dimensional culture of cells possible, when cells are embedded in a thick gel,and not only applied to a thin coating

• Testing packs to try different molecules (mix of different surfaces)

LIMITATION :• Not Animal components free

• Limit shelf life

• An advanced cell culture surfaces, that uses a novel process tocoat the culture vessel in a very defined and reproducible way

• 2 options :− amine (+) residues

− carboxyl (-) residues

BD PureCoatTM

Cell Culture surfaces - Adherent CellsSynthetic defined - Purecoat


• Advantages :− Chemically defined

− Animal-free

− High consistency

− Low CV‘s in cell-based assays

− Stable at room temperature (3 years)

+ + + + + +

or- - - - - - -

BD PureCoatTM

Thin-film coatingCarboxyl or Amine

momomers

Cell Culture surfaces - Adherent CellsSynthetic defined - Purecoat


2 options :− Fibronectine

− Collagen

• ECM Mimetic Surface technology

Cell Culture surfaces - Adherent CellsSynthetic defined – Purecoat ECM Mimetic


• Main applications :− Human Mesenchymal Stem Cells

− Human Endothelial Progenitor Cells

− Human Keratinocytes

PuraMatrix Petpide Hydrogel :- Synthetic matrix to create a defined 3D microenvironments

- origin: synthetic, animal free

- major components: - 16 aa peptide

- average pore size 50-200 nm

- no growth factors

Cell Culture surfaces - Adherent CellsSynthetic defined – PuraMatrix


- Applications :

- Puramatrix scaffold allows creation of highly defined environment throughaddition of factors & compounds

- Does not activate RGD-dependent integrin signaling

- Highly reproducible experiments

- Compatible with most cells types in vitro & in vivo (stem cell proliferation, tumorcell migration and invasion, angiogenesis assays, tissue regeneration)

Synthemax Surface :

1 Application Stem cell

What is the Corning® Synthemax™ Surface ?

Cell Culture surfaces - Adherent CellsSynthetic Specific – Synthemax


polystyrenepolystyrene polystyrene

1. Uncoated surface 2. Acrylate coated 3. Peptide covalentlybound

Feeders ECMs Synthemax

Storage

• Need to be usedwithin a week

• Need to be stored at -70C• When working they

• No special storageconditions; can bestored at room temp

• Advantages : Time Saving & Shelf Life



need to be kept cold

Shelf Life

• Last only a week onceprepared

• Plates need to be usedas soon as possible

• 2 year shelf life

Easy-to-Use

• Harvest mouseembryos• Grow up theFibroblasts• Time intensive

• Laborious time-consuming prep

• No prep, just cutopen the package anduse

Customer Needs Research Therapeutic Screening

Traceability

10-6 Sterility

Consistent Performance

Shelf Life

No Special Storage Conditions

Ease of Use



Ease of Use

Purity Post-Harvesting

Lot Size

Multiple Formats

GMP Production

Expand & Differentiate on same Surface

Inventory Availability

Xeno-Free Substrate

Ability to visualize cells

Automation Friendly Format

Synthemax

Consistency

• Manufactured under GMP conditions• Performs as good as Matrigel• Consistent performance



Consistency • Consistent performance• 2 year shelf life

Defined, Synthetic,Xeno-Free

• Defined• Synthetic• No animal components

Easy-to-Use

• No prep, just cut open the package and use

Osteo Assay Surface :

1 Application Osteo/bone assays

Specifications :

Inorganic synthetic coating that mimics

in-vivo bone for in vitro bone cell assays

Cell Culture surfaces - Adherent CellsSynthetic Specific – Osteo Assay


in-vivo bone for in vitro bone cell assays

Surface applications :

- Consistent assay results for bone cell

- Differentiation and function

- Co-culture of bone cells / metastatic cancer cells

- Alternative to preparing dentine, bone slicesSEM image

magnified not truerepresentation

Key Benefits Critical Attributes Differentiating Attributes

Time Reduce time and cost of surfacepreparation

Precoated, sterile, ready to use

Direct Readout Detect both cell differentiation andfunctional analysis of bone cells on thesame surface

Transparent to conventional imagingtechniques

Cell Culture surfaces - Adherent CellsSynthetic Specific – Osteo Assay


same surfaceSupernatant can be sampled for cellfunction

Amenable toautomation

Standard SBS format Can be used with standard imagingtechnologies

Reliable cellresponse

Consistent assays Standard process for manufacturingUniform surface

• You can find the different cell culture surfaces on multiple vessels :

• Flasks

• Microplates from 6 to 1536 wells

• Dishes

• For specific applications :

Cell Culture surfaces – Procuts available


• Assays microplates :

• Drug discovery

• Molecules screening

• Cells Culture inserts (Transwell®) :

• Coculture

• Migration

• Invasion

• Cell Culture Selection Guide Corning

• Cell Culture Selection Guide Corning – Falcon

• Success Stories CellBIND

• Bibliography OsteoAssays

• Bibliography ULA (Ultra Low Attachment)

TOOLS TO FIND THE RIGHT SURFACE


• Different Protocols or Application notes

• European Scientific Support (2 scientists)


http://www.cultek.com/index.asp?p=soporte-productos&id_prf=84



http://www.cultek.com/index.asp?p=productos-destacados-detalles&id_pdt=473

http://www.cultek.com/index.asp?p=productos-destacados-detalles&id_pdt=473

• Glass

− High optical quality and scratch-resistant glass

− Bottom thickness 200 µm

− Intra well bottom flatness <50µm

• Copolimer

− Cyclic Olefin Copolymer(COC)

− Bottom thickness: 127 µm

− Intra-well bottom flatness 5µm

− Inter-well bottom flatness 30

Glass Bottom Microplates


− Inter-well bottom flatness 30µm

Flowell


Axygen® 5 mL Snaplock Microtubes

5mL

15mm • No necesita adaptador


• No necesita adaptador• Rotores 15 mm (15ml)

• Rotores 17 mm (15ml)

• 14.000 rpm

• 120ºC (15’ 15psi)

• Libre de ARNasas, ADNasas

Corning Life Science - AXYGEN Biosciences

Thanks a lot for your time and attention.


QUESTIONS ?

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