Advanced Cell Culture Surfaces: BD PureCoat surfaces provide
Transcript of Advanced Cell Culture Surfaces: BD PureCoat surfaces provide
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)
0
0.02
0.04
0.06
0.08
0.1
0.12
TC Enhanced TC Competitor C BD PureCoat amine
Rel
ativ
e A
bsor
banc
e U
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
0
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
Tissue Culture BD PureCoat CarboxylTo
tal c
ell n
umbe
r
seeded immediately post-freeze thaw
0
1000000
2000000
3000000
4000000
5000000
6000000
7000000
Tissue Culture BD PureCoat Carboxyl
Tota
l cel
l num
ber
1st passage
P1
P2
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
0
0.5
1
1.5
2
2.5
3
Rel
ativ
e A
bsor
banc
e U
nits
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)
0
500000
1000000
1500000
2000000
2500000
3000000
3500000
TC BD PureCoat Amine
Tota
l cel
l yie
ld
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
0
10
20
30
40
50
60
70
80
TC BD PureCoat Carboxyl
% m
ean
conf
luen
cy
n=16 wells/surface
3d growth assay
0
500000
1000000
1500000
2000000
2500000
TC BD PureCoat Carboxyl
tota
l cel
l yie
ld
0
0.2
0.4
0.6
0.8
TC Enhanced TCCompetitor C
BD PureCoatcarboxyl
Rel
ativ
e A
bsor
banc
e U
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
0
0.1
0.2
0.3
0.4
0.5
TC Enhanced TCCompetitor C
BD-PureCoatCarboxyl
BD-PureCoatAmine
Rel
ativ
e A
bsor
banc
e Va
lues
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.
0
20
40
60
80
100
120
140
10uM 30uM 60uM
% c
ell v
iabi
lity
of c
ontro
l
TC BD PureCoat Carboxyl BD PureCoat Amine
Conc: of Tamoxifen
0
20
40
60
80
100
120
140
10uM 30uM 60uM
% c
ell v
iabi
lity
of c
ontr
ol
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
0
0.2
0.4
0.6
0.8
1
TC Enhanced TCCompetitor C
Lot 1 Lot 2
Rel
ativ
e A
bsor
banc
e U
nits
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
0
10
20
30
40
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
0
15
30
45
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)
Rel
ativ
e Fl
uore
scen
ce U
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
0
20
40
60
80
100
120
Rel
ativ
e Fl
uore
scen
ce U
nits
ATP (µM)
TCBD PureCoat amine
0 30 300
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
0
2
4
6
8
10
12
TC BD PureCoat carboxyl BD PureCoat amine
mea
n cA
MP
(nM
)
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
0
200
400
600
800
1000
1200
1400
1600
1800
0.1 1 10
Rel
ativ
e Fl
uore
scen
t Uni
ts
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
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
Competitor PDL BD PureCoat carboxyl0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
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: [email protected]
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?