Elizabeth J. Abraham, PhDBD Biosciences

Advanced Cell Culture Surfaces: BD PureCoatsurfaces provide improved cell attachment and growth of many cell types compared to standard tissue culture vessels

Outline

Two novel advanced cell culture surfaces:BD PureCoat™ cultureware• Salient features of BD PureCoat surfaces• Cell culture parameters tested on BD PureCoat surfaces• Customer evaluation of BD PureCoat surfaces• Advantages of BD PureCoat cultureware vs. standard tissue

culture (TC) vesselsQuestions and Answers

What is BD PureCoat™ Cultureware?

Chemically defined, animal-free surfacesManufactured by a proprietary thin-film coating technology

The Next Generation of Advanced Cell Culture Surfaces

Two surface chemistries available• BD PureCoat carboxyl

Negatively charged surface• BD PureCoat amine

Positively charged surface

Enhanced cell attachment and growth in serum-reduced or serum-free culture mediaBetter attachment of cryopreserved cells post freeze-thawEnhanced attachment of transfected, primary, and fastidiouscell linesSupports neural cell attachment and differentiationCells on BD PureCoat surfaces remain functional and can be coupled for use in HTS assays such as G-Protein Coupled Receptor (GPCR), Cyclic Adenosine Monophosphate (cAMP), proteasome-inhibition, and transfections

BD PureCoat Surfaces: Application Highlights

Rat keratinocytes

Primary Non-Neural Cells

Rat brain cortical neuronsRat astrocytesRat cerebellar granule

Primary Neural Cells

EcoPack™2-293hERG-T-Rex™ 293 DAHEK-ZsGreen Proteasome SensorCHO-M4

Transfected Cell Lines

BHK-21HT-1080MRC-5HepG2PC-12HEK-293

Cell Lines

BD PureCoat carboxyl

BD PureCoat amine

Cell Types Cultured on BD PureCoat Cultureware: Surface Recommendations

Parameters Tested on BD PureCoat Cultureware

Freeze-thaw recovery and attachment of cryopreserved cellsCell attachment and proliferation• Reduced serum-supplemented medium• Serum-free medium

Attachment and differentiation of primary neural cellsCell performance in HTS-based assaysCell performance in transfection-based assays

40% increase in hERG-T-REx division arrested (DA) cell line attachment and recovery post-thaw on BD PureCoat amine

Tissue Culture Enhanced TC Competitor C

BD PureCoat amine

24h attachment assay (96-well format)

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TC Enhanced TC Competitor C BD PureCoat amine

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ativ

e A

bsor

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nits n=10 wells/surface

384-well format

Better Attachment and Freeze-Thaw Recovery of hERG-T-REx 293 DA Cells on BD PureCoat Amine

Better Freeze-Thaw Recovery of LnCAP on BD PureCoat Carboxyl

40-400% increase in LnCAP cell attachment and recovery post thawin standard culture conditions on BD PureCoat carboxyl

Tissue Culture

BD PureCoat carboxyl

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Tissue Culture BD PureCoat CarboxylTo

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seeded immediately post-freeze thaw

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Tissue Culture BD PureCoat Carboxyl

Tota

l cel

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ber

1st passage

P1

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75cm2 area flasks6 day growth assay

Parameters Tested on BD PureCoat Cultureware

Freeze-thaw recovery and attachment of cryopreserved cellsCell attachment and proliferation• Reduced serum-supplemented medium• Serum-free medium

Attachment and differentiation of primary neural cells

Tissue Culture

BD PureCoat amine

DAPI stain (24-well format)

Increased BHK-21 Cell Proliferation in Reduced Serum (+1% FBS) on BD PureCoat Amine

BD PureCoat amine supports BHK-21 cell growth in reduced serum.

3 day growth assay

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6400 cells/well 3200 cells/well 1600 cells/well

Initial seeding density

#1 #3#2 #1 #3#2 #1 #3#2

n=10 wells/surfaceTCEnhanced TC Competitor CBD PureCoat amine

(96-

wel

l for

mat

)(7

5cm

2fla

sks)

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Enhanced Cell Proliferation of MRC-5 Cells in Reduced Serum(+5% FBS) on BD PureCoat Carboxyl

10-15% increase in MRC-5 cell growth in reduced-serum containing media on BD PureCoat carboxyl

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n=16 wells/surface

3d growth assay

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TC Enhanced TCCompetitor C

BD PureCoatcarboxyl

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nits

(24-

wel

l for

mat

)

(75c

m2

flask

s)

Increased HEK-293 Cell Proliferation in Reduced Serum (1% FBS) on BD PureCoat surfacesBD PureCoat surfaces support HEK-293 cell growth in reduced-serum containing media

4d growth assay (24-well format)n=4 wells/surface

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BD-PureCoatCarboxyl

BD-PureCoatAmine

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TCEnhanced TC Competitor CBD PureCoat CarboxylBD PureCoat amine

Initial seeding density120,000 cells/well

Tissue Culture

BD PureCoat carboxyl

BD PureCoat amine

HepG2 cells seededat 8,000 cells/well (top panel) or 16,000 cells/well (lower panel) into 96-well plates and cultured for 24h were treated with drug for 3h followed by MTS assay.

Hep-G2 Cells Grown on BD PureCoat Surfaces Remain Responsive in Cytotoxicity Assays Despite Changes in Cell MorphologyPotency (EC50) of tamoxifen in cytotoxicity assays from cells grown onBD PureCoat surfaces within two-fold that of those grown on TC vessels.

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10uM 30uM 60uM

% c

ell v

iabi

lity

of c

ontro

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TC BD PureCoat Carboxyl BD PureCoat Amine

Conc: of Tamoxifen

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% c

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of c

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HT-1080 cells attach and spread uniformly on BD PureCoat carboxyl in serum-free medium.

Tissue Culture

Enhanced TC Competitor C BD PureCoat carboxyl

24h post seeding cells were fixed with methanol and stained with toluidene blue

Enhanced Attachment of HT-1080 Cells onBD PureCoat Carboxyl in Serum-free Media

Parameters tested on BD PureCoat Cultureware

Cell attachment and proliferation• Reduced serum-supplemented medium• Serum-free medium

Attachment and differentiation of primary neural cellsCell performance in HTS-based assays

>50% increase in primary rat astrocyte cell proliferation post-thaw in media containing 3% FBS & growth factors on BD PureCoat aminePrimary rat astrocytes cultured on BD PureCoat amine continue to express typical phenotypic functional markers such as Glial Fibrillary Acidic Protein (GFAP)

Fluorescent staining for α-GFAP

BD PureCoat amine

6d growth assay

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TC Enhanced TCCompetitor C

Lot 1 Lot 2

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ativ

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banc

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n=5 wells/surfaceTCEnhanced TC Competitor CBD PureCoat amine

Increased Cell Proliferation of Rat Astrocytes onBD PureCoat Amine

Tissue Culture (TC)

BD PureCoat amine

Cell Attachment of RCG Cells on BD PureCoat Amine

Rat Cerebellar Granule (RCG) cells attach and differentiate on BD PureCoatamine surface, whereas those plated on TC remain unattached and float

RCG cells immunostained with anti-tubulin IIIβ

Differentiation of RCG Cells on BD PureCoat Amine

BD PureCoat amine supports differentiation of neural cells• Neurite outgrowths from RCG cells form a network• Immunostained with markers expressed in neural cells

such as tubulin IIIβ

24h

post

-isol

atio

nA

nti-t

ubul

in II

Iβ

Ant

i-tub

ulin

IIIβ

/ DA

PI

48h post-thaw 5d post-thaw

Tissue culture

Primary Rat Brain Cortical Neuronal Cells Post-Thaw Do Not Attach on Uncoated TC VesselsPoor cell attachment and recovery on uncoated TC-treated surfaces. Cells remain unattached and float in clusters which increase in size with time.

48h post-thaw

5d post-thaw

Cell Attachment and Differentiation of Primary Rat Brain Cortical Neuronal Cells Post-Thaw on BD PureCoat AmineCells attach and differentiate on BD PureCoat Amine. Increase in neurite length observed with time.

Freeze-thaw recovery and attachment of cryopreserved cellsCell attachment and proliferation• Reduced serum-supplemented medium• Serum-free medium

Attachment and differentiation of primary neural cellsCell performance in HTS-based assaysCell performance in transfection-based assays

Parameters tested on BD PureCoat Cultureware

EcoPack2™-293 cell monolayer integrity maintained on BD PureCoatamine, whereas cells are washed off on TC-treated or Competitor C surfaces

Cells were grown in serum-free media and washed with HBSS buffer using an automated washer (Skatron EMBLA)

Before wash

After wash

384-well format

Tissue Culture Enhanced TC Competitor C

BD PureCoat amine

HTS formats—Ecopack2-293™ cells adhere to BD PureCoat Amine

EcoPack2-293 cells grown in serum-free media were washed with HBSS buffer using an automated washer (Skatron EMBLA) and stained with calcein AM dye

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lot 1 lot 1 lot 1 lot 2 lot 2 lot 2 lot 3 lot 3 lot 3

TCEnhanced TC Competitor CBD PureCoat amine

CV

1536-well format

CV

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60

lot 1 lot 1 lot 1 lot 2 lot 2 lot 2

384-well format

TC

BD PureCoat amine

HTS formats—Better Consistency of Cell-Based Assays on BD PureCoat Amine

Intra-plate coefficient of variation (CV) of multiple lots of BD PureCoat amine were < 10% for 384- and 1536-well plate formatsZ′ values were > 0.72 indicating a robust assay

Carbachol-induced calcium mobilization studies on EcoPack2-293 cells cultured in serum-free medium

Concentration of carbachol (µM)

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nits

Cells on BD PureCoat Surfaces Remain Functional and Can Be Coupled for Use in GPCR-based AssaysComparable potency (EC50) of carbachol from cells grown either on TC-treated or BD PureCoat amine

ATP-induced calcium mobilization studies on EcoPack2-293 cells cultured in serum-free medium

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TCBD PureCoat amine

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Cells on BD PureCoat Surfaces Remain Functional and Can Be Coupled for use in GPCR-based AssaysComparable responses of cells on BD PureCoat amineand TC-treated in GPCR-based assays

Utilization of BD PureCoat Surfaces for Functional Second Messenger cAMP AssaysBetter cell attachment of HEK-293 cells in serum-free medium resulted in higher cAMP responses from cells on BD PureCoat surfaces

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mea

n cA

MP

(nM

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cAMP responses of HEK-293 cells cultured in serum-free medium and treated with Isoproterenol

n=3 wells/treatment/surface

ControlIsoproterenol (20µM)

BD PureCoat Surfaces—Proteasome Inhibition Assays: Fluorometry Cells cultured on BD PureCoat surfaces remain functional in proteasome-inhibition assays

Living Colors™ HEK- 293 ZsGreen Proteasome Sensor Cells (Clontech) were treated with ALLN, a proteasome inhibitor for 20h

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ALLN conc.(µM)

n=10 wells/treatment/surface

TCBD PureCoat amineBD PureCoat carboxyl

Tissue Culture BD PureCoat carboxyl BD PureCoat amine

Untreated

10 µM ALLN

10x objective

Living Colors HEK-293 ZsGreen Proteasome Sensor Cells (Clontech) were either untreated or treated with 10 µM ALLN, a proteasome inhibitor for 12h

BD PureCoat Surfaces—Proteasome Inhibition Fluorescent Microscopy on BD Pathway™ 800 Imager

Cells cultured on TC-treated and BD PureCoat surfaces respond to ALLN

HT-1080 cells transfected with pZsGreen C1 plasmid DNA (Clontech) and images captured 24h post-transfection on the BD Pathway 800 Imager

Tissue Culture BD PureCoat carboxyl (Lot #1) BD PureCoat carboxyl (Lot #2)

Cells Cultured on BD PureCoat Surfaces Can Be Transfected with Lipofectamine 2000HT-1080 cells grown on BD PureCoat carboxyl or TC-treated surfaces can be transfected with plasmid DNA

HepG2 cells transfected with pZsGreen C1 plasmid DNA (Clontech) and images captured 24h post transfection on the BD Pathway 800 Imager

GFPFluorescence

Tissue Culture BD PureCoat amine

Cells Cultured on BD PureCoat Surfaces Can Be Transfected with Lipofectamine 2000

HepG2 cells grown on BD PureCoat Amine or TC-treated surfaces can be transfected with plasmid DNA

Featured Customer Evaluationof BD PureCoat Surfaces

Customer DataIncreased attachment of human mesenchymal stem cells (hMSC) on BD PureCoat Amine

Data generated by Academic research faculty, Australia

(TC)

Tissue Culture BD PureCoat Amine BD PureCoat Carboxyl

Culture Medium: Alpha-MEM containing 20% fetal bovine serum

hMSC morphology is normal on BD PureCoat Amine surface with increased attachment observed compared with TC and PureCoat Carboxyl.

A.

Customer DataIncreased growth of hMSCs on BD PureCoat Amine

Cell growth factor was calculated by dividing the total cells at confluence with the cell number seeded in the well at the previous passage (3 passages; total culture time of 21 days).

(TC)

47 foldAmine

3.2 foldCarboxyl

8.9 foldTissue Culture

Data generated by Academic Researcher, Australia

B. Cell growth factor calculations (6-well format)

C.

Umbilical cord MSCs isolated from Wharton’s Jelly exhibit good attachment and proliferation on BD PureCoat Amine surface (6-well format).

10X objective 20X objective

Data generated by Bio-Tech Researcher, India

Customer DataAttachment of umbilical cord-derived MSCs on BD PureCoat Amine

DMEM Knockout media (Invitrogen), supplemented with 2 mM glutamine and 10% FBS

Customer DataIncreased neuronal differentiation of Rat Bone Marrow-derived MSCs on BD PureCoat AmineRat Bone Marrow-derived MSCs on BD PureCoat amine differentiate more rapidly upon induction into neuronal lineage.

Induce with small molecule (KC-1039)

for 48h

BD PureCoat Amine

Pre-differentiation

Induce with small molecule (KC-1039)

for 48h

Tissue Culture

Pre-differentiation

Data generated by Academic Research Institute, Korea

Cells grown in DMEM containing 10% FBS (Gibco) and 1% (Penicillin/Streptomycin)

Customer DataGrowth of Rat E14d Cortex-derived Neural Stem Cells (NSCs) onBD PureCoat AmineEquivalent attachment, viability, proliferation rate and differentiation properties of rat E14d Cortex-derived NSCs on BD PureCoat Amine vs. self-coated PLO/Fibronectin TC vessels in a 4d growth assay.

Data generated by Academic Researcher, Korea

Grown in Serum-free defined Media (N2 media; Invitrogen) with 20 ng/ml bFGF.BD PureCoat amine Poly-L-Ornithine/Fibronectin

6-well format

Customer DataBetter attachment of RCG cells on BD PureCoat Amine

Self-coated PDL BD PureCoat amine

Primary neural cell: Cerebellar granule cells 8-day Rat (6-well format)

Seeding: 1.5 106 cells/mlMedium: DMEM/HAM F12 + SVF 10% + KCL 25mMCulture: 4 days

General comments:‘After 4 days, the cells seem to be better in BD PureCoat Amine compared to homemade coating in PDL’.

Data generated by Academic Researcher, France

Customer DataBetter consistency of results on BD PureCoat Carboxyl

Hamamatsu Fluorescence Assay using HEK-293 cells on BD PureCoat carboxyl.

Agonist mode Antagonist mode

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Plate #1 Plate #2 Plate #1 Plate #2

Competitor PDL BD PureCoat carboxyl

Z’va

lues

Data generated by Pharmaceutical Researcher, U.S.A

Consistently higher Z’ values obtained on BD PureCoat carboxyl vs. competitor PDL vessels in a HTS-fluorescence assay.

96-well format

Cell type: HEK-293Culture Medium: DMEM+10% FBSType of assay: HTSDetails of assay: Cell attachment and proliferation Detection reagent: MTSComparison/Format: TC 96-well format Observations: Better cell attachment, signal:noise ratio, low intra-

and inter-plate CVs on BD PureCoat amine than TC“During the assay, I have always faced a problem because HEK-293 cells are weakly adherent cells. But BD PureCoat amine plates are much better than the TC plates in terms of cell attachment.”

Customer DataBetter consistency of HTS-assay results on BD PureCoat Amine

Evaluated by Academic Researcher, U.S.A

Summary of BD PureCoat CulturewareSurface Evaluations by Customers

BD PureCoat surfaces = collagen ICompetitor collagenHus-E/26-well amine and carboxyl platesAcademic

BD PureCoat carboxyl = collagen ICompetitor collagenGP2936-well carboxyl plateAcademic

BD PureCoat amine = collagen ICollagen I coatingHEK-293T96-well amine plateAcademic

BD PureCoat surfaces > TCTCmouse primary hippocampus neuronal cell

6-well amine and carboxyl platesAcademic

BD PureCoat amine > carboxyl > TCTCPC-9100 mm amine and carboxyl dishesAcademic

BD PureCoat amine = polyethylenimineSelf coating (polyethylenimine)HEK-293T96-well amine plateAcademic

BD PureCoat carboxyl = collagen ICompetitor collagenHPAEC100 mm carboxyl dishAcademic

BD PureCoat amine > self-coated PDLSelf coating (PDL)NE4C24-well amine plateAcademic

BD PureCoat amine > TCTCST2－wt, LMACＯ24-well amine plateAcademic

BD PureCoat carboxyl > TCTCMCF76-well carboxyl plateAcademic

BD PureCoat amine > Fibronectin coated Fibronectin coatingHUVEC100 mm amine dishAcademic

Testing resultsComparisonCell TypeType evaluatedOrganization

Summary

Evaluated by Academic Researchers, Japan

BD PureCoat amine > TCTCCHOK1SV96-well amine platePharmaceutical

BD PureCoat amine = PLO/Laminin (self)Self-coat PLO/LamininEnStem-A96-well amine platePharmaceutical

BD PureCoat amine = PLO/Laminin (self)Self-coat PLO/LamininRat primary neuronal cells96-well amine platePharmaceutical

BD PureCoat amine > TCTCA70496-well amine platePharmaceutical

BD PureCoat amine = collagenCompetitor collagenHEK-29396-well amine platePharmaceutical

BD PureCoat amine > PLL coated Competitor PLL coating

Rat primary microglial cell96-well amine platePharmaceutical

Testing resultsComparisonCell TypeType evaluatedOrganization

Evaluated by Pharmaceutical Researchers, Japan

Summary

Summary: Additional Cell Types Cultured onBD PureCoat Surfaces

Human Placental Epithelial cellsHuman Epidermal Keratinocytes (neonatal)Embryonic Mouse Brain Stem CellRat E14d Cortex-Derived Neural Stem CellsPrimary Cervical Epithelial CellsPrimary Rat Pancreatic IsletMouse CardiomyocyteMesenchymal Stem Cell (Rat bone-marrow-derived)Primary FibrocytesDifferentiated Skeletal Muscle

Primary Cells

BD PureCoat carboxyl

BD PureCoat amine

N2AHeLa293TFlp-In-T-REx 293HEK-293 ZellenHEK-293U87 glioblastomaIMCD3HK2Ht22Detroit 562 cell line

Cell Lines

BD PureCoat carboxyl

BD PureCoat amine

Evaluated by Academic and Pharmaceutical Researchers, U.S.A, Europe, China and Japan

Features and Advantages – Animal-free and Chemically Defined

Increased control over cell culture environmentMore predictable biological outcomeReduced costs/time associated with rework for ECM self-coaters

Uniform and functionalized surface

What does it mean to you?Advantages

Increased confidence in results with superior consistencyPossibility to run experiment longerReduced costs associated with the use of serum

Indicated for serum-free or serum-reduced cell culture conditions

Increased productivity with improved cell yield (more cells) and shorter proliferation time Superior results with a broad range of cells that exhibit poor attachment properties

Improved cell attachment, faster proliferation and enhanced post-thaw recovery of primary, transfected and fastidious cell lines

Features and Advantages – Positive and Negative Charge Options

Broader range of cells can be cultured with BD PureCoat

Versatility

What does it mean to you?Advantages

Follows same process as with commonly used TC or biological surfaces

No special steps for cell adaptation required

SAL 10-6

No need for expensive cold rooms

Terminally sterilized

Can be stored at room temperature

High-density Black/Clear Microplates• 96-well• 384-well• 1536-well

Flasks• Coming Soon!

• Multiwell Plates and Cell Culture Dishes• 6-well• 24-well• 100 mm dishes

BD PureCoat Cultureware: Launched June 2009

Contact information:

Elizabeth Abraham, Ph.D.e-mail: elizabeth_abraham@bd.com

EcoPack and Living Colors are the property of Clontech Laboratories, Inc., a Takara Bio Company.BD, BD Logo, and all other trademarks are the property of Becton, Dickinson and Company. ©2009 BD

Questions?