CORNING Life Sciences

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CORNING Life Sciences

Cell Culture Treatments

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Corning – A brief History

- A brief history - Corning’s Brands - Corning’s Workflows

- Cell Culture Workflow

- Cell Culture surfaces

- Suspension cells

- Non treated

- 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

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

Corning created and make keystone components that enable high-technology

systems for consumer electronics, mobile emissions control, telecommunications and 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

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Corning Life Sciences – Different Brands & Market segments

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

FALCON

cellgro® is available in Europe as Mediatech™.

Cell Culture, Drug Discovery & Liquid Handling

Molecular Biology Genomics

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Corning Life Science - Workflows

Cell Culture surfaces :

- Suspension cells

- Adherent cells

Adherent Cells :

- Standard - TC

- Synthetic improved

(CellBIND ; Primaria)

- Biological

(ECM – Biocoat)

- Synthetic defined

(Purecoat)

- Synthetic specific

(OsteoAssays / Synthemax)

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Stationary

Moving

Adherent culture

Suspension culture

• Spinner

Stationary

Moving

• Flasks

• Roller

bottles

• Flasks

Corning Life Science – Cell Culture surfaces

• Spinner

flasks

• Shaker

flasks

• Roller

bottles

• Bags

• Flasks

• Storage

bottles

• Dishes

• Microplates

• Transwell

inserts

• Bags

bottles

•

Micro-carriers

in spinner

flasks

• Flasks on

rockers

• Microplates

• Dishes

• Transwell

inserts

•

HYPER

Flask

• CellSTACK

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Moving

Adherent culture

Suspension culture

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•

Suspension Cell Culture :

−

NT

(Not Treated)

• For standard suspension cell culture

−

ULA

(Ultra Low Attachment)

Corning Life Science – Cell Culture surfaces

Suspension Cells

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•

Adherent Cell Culture :

- Standard – TC

- Synthetic improved

(CellBIND ; Primaria)

Corning Life Science – Cell Culture surfaces

Adherent Cells

Specificity to Cells Animal Free

YES

- Biological

(ECM – Biocoat)

- Synthetic defined

(Purecoat)

- Synthetic specific

(OsteoAssays / Synthemax)

NO

YES

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Cell Culture surfaces - Adherent Cells

Standard Tissue Culture Surfaces

Standard Tissue Culture (TC) Treatement :

•

To be used with adherent cells, the Polystyrene needs to be treated to

add 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

receives a mixture of gases with electric power.

− By corona discharge.

•

The treatment will create a modification of the polystyrene altering it‘s

surface chemistry



Negative charges

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Standard 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 Plate

Culture Vessels - charged groups - hydrophilic - cell attachment e.g. "TC" flatter 40 ml droplet H H H H H H H H H H H H C C C C C C C C C H H H H H H H H H H H H H H C C C C C C C C C H H H H H H H H H H H H C C C C C C C C C H H H H H H H H H H H H H H C C C C C C C C C Hydrophobic H H H O H H H O OH H O C C C C C C C C C H H O H OH H H H O OH OH HO H O C C C C C C C C C Hydrophilic H H H O H H H O OH H O C C C C C C C C C H H O H OH H H H O OH OH HO H O C C C C C C C C C Hydrophilic H H H O H H H O OH H O C C C C C C C C C H H O H OH H H H O OH OH HO H O C C C C C C C C C Hydrophilic Surface treatment Surface treatment

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Cell Culture surfaces - Adherent Cells

Synthetic Non Specific – CellBIND & Primaria

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

• To be used with adherent cells, the Polystyrene needs to be treated to

add charges and to get it hydrophylic.

• Polystyrene is fully hydrophobic and no natural.

• TC-surface treatment is achieved by :

− a specific gaz mixture.

− A difference source of energy (microwaves).

• The treatment will create a modification of the polystyrene altering it‘s

surface chemistry



more balanced – vs + positives

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Synthetic Non Specific – CellBIND & Primaria

CellBIND® TCT BD

Primaria™ Not Treated Treatment N2O O2 NH4/proprietary

% Oxygen/

Nitrogen 35% Oxygen 22% Oxygen

14.1% Oxygen 11.1% Nitrogen 1.8% Oxygen Contact

TCT

Contact angle 23 - 26° 56 - 63° 75° 90 - 95°

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Cell Culture surfaces - Adherent Cells

ECM – 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 in

contact 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.

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Cell Culture surfaces - Adherent Cells

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

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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

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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 of

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TC Plates

After Wash, Before Wash After Wash Calcein AM Staining

APPLICATIONS :

• HEK293

BD BioCoat PDL Plates

After Wash, Before Wash After Wash Calcein AM Staining

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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

Cell Culture surfaces - Adherent Cells

• 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 :

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•

An advanced cell culture surfaces, that uses a novel process to

coat the culture vessel in a very defined and reproducible way

•

2 options :

− amine (+) residues

− carboxyl (-) residues

BD PureCoatTM

Synthetic 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 coating Carboxyl or Amine momomers

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Cell Culture surfaces - Adherent Cells

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2 options :

− Fibronectine

− Collagen

•

ECM Mimetic Surface technology

Synthetic defined – Purecoat ECM Mimetic

•

Main applications :

− Human Mesenchymal Stem Cells

− Human Endothelial Progenitor Cells

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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 Cells

Synthetic defined – PuraMatrix

- Applications :

- Puramatrix scaffold allows creation of highly defined environment through addition 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, tumor cell migration and invasion, angiogenesis assays, tissue regeneration)

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Synthemax Surface :

1 Application  Stem cell

What is the Corning® _Synthemax™ _{Surface ?}

Synthetic Specific – Synthemax

polystyrene

polystyrene polystyrene

1. Uncoated surface 2. Acrylate coated 3. Peptide covalently bound

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Feeders ECMs Synthemax Storage • Need to be used within a week • Need to be stored at -70C

• When working they

• No special storage conditions; can be stored at room temp

•

Advantages : Time Saving & Shelf Life

need to be kept cold

Shelf Life

• Last only a week once prepared

• Plates need to be used as soon as possible

• 2 year shelf life

Easy-to-Use • Harvest mouse embryos • Grow up the Fibroblasts • Time intensive • Laborious time-consuming prep

• No prep, just cut open the package and use

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Customer Needs Research Therapeutic Screening

Traceability 

10-6 _Sterility 

Consistent Performance   

Shelf Life 

No Special Storage Conditions   

Ease of Use   

Cell Culture surfaces - Adherent Cells

Ease of Use   

Purity Post-Harvesting 

Lot Size 

Multiple Formats   

GMP Production 

Expand & Differentiate on same Surface   

Inventory Availability 

Xeno-Free Substrate 

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Synthemax

Consistency

• Manufactured under GMP conditions

• Performs as good as Matrigel

• Consistent performance

Cell Culture surfaces - Adherent Cells

Consistency _• _{Consistent performance}

• 2 year shelf life

Defined, Synthetic, Xeno-Free • Defined • Synthetic • No animal components Easy-to-Use

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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 Cells

Synthetic 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 slices

SEM image magnified not true

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Key Benefits Critical Attributes Differentiating Attributes

Time Reduce time and cost of surface

preparation

Precoated, sterile, ready to use

Direct Readout Detect both cell differentiation and functional analysis of bone cells on the same surface

Transparent to conventional imaging techniques

Cell Culture surfaces - Adherent Cells

Synthetic Specific – Osteo Assay

same surface

Supernatant can be sampled for cell function

Amenable to automation

Standard SBS format Can be used with standard imaging

technologies

Reliable cell response

Consistent assays Standard process for manufacturing

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• 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

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• 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)

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• Glass

− High optical quality and

scratch-resistant glass

− Bottom thickness

200 µm

− Intra well bottom flatness

<50

µm

• Copolimer

− Cyclic Olefin Copolymer

(COC)

− Bottom thickness:

127 µm

− Intra-well bottom flatness

5

µm

−

Inter-well bottom flatness

30

Glass Bottom Microplates

−

Inter-well bottom flatness

30

µm

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Axygen® 5 mL Snaplock Microtubes



5mL



15mm



• No necesita adaptador

• Rotores 15 mm (15ml) • Rotores 17 mm (15ml)

• 14.000 rpm

• 120ºC (15’ 15psi)

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Corning Life Science - AXYGEN Biosciences

Thanks a lot for your time and attention.