Development of Novel Advanced Cell Culture Surfaces that Provide Better Cell Growth and Attachment for Cell-Based Assays

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1 Development of Novel Advanced Cell Culture Surfaces that Provide Better Cell Growth and Attachment for Cell-Based Assays Elizabeth J. Abraham, Ph.D. BD Biosciences

2 Outline Overview of surfaces and culture microenvironment Two novel advanced cell culture surfaces: BD PureCoat cultureware Salient features of BD PureCoat surfaces Cell culture parameters tested on BD PureCoat surfaces Advantages of BD PureCoat cultureware vs. standard tissue culture (TC) vessels Questions and Answers

3 Impact of Surface Treatments on Cell-Based Assays Cell-based assays provide predictive physiological model systems Cells respond to cues in their microenvironment and surface treatment influences the physiologic behavior of cells: morphology, growth, migration, differentiation, etc.

4 Surface Requirements for Cell-Based Assays Surface requirements for high throughput screening assays provides robust cell attachment superior consistency in cell performance low background cells remain functional Surface requirements for cell growth assays enhance cell proliferation cells remain functional phenotypic integrity maintained

5 Overview of TC Surface Treatment Traditional TC-surface chemistry is achieved by placing polystyrene vessels into a vacuum chamber that receives a mixture of gases or by corona discharge Under specific conditions a gas plasma is produced that reacts with the polystyrene thus altering it s surface chemistry Typically used to culture anchorage-dependent cells Surface features that influence cell culture Rigidity Hydrophilicity Charge on the surface

6 What is BD PureCoat Cultureware? The Next Generation of Advanced Cell Culture Surfaces Chemically defined, animal-free surfaces Manufactured by a proprietary thin-film coating technology

7 BD PureCoat Cultureware: Surface Chemistry Two surface chemistries currently available: BD PureCoat carboxyl Negatively charged surface BD PureCoat amine Positively charged surface

8 BD PureCoat Surfaces: Application Highlights Enhanced cell attachment and growth in serum-reduced or serum-free culture media Better freeze-thaw recovery of cryopreserved cells Enhanced attachment of transfected, primary, and fastidious cell lines Supports neural cell attachment and differentiation Cells 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

9 Cell Types Cultured on BD PureCoat Cultureware: Surface Recommendations Cell Lines BHK-21 HT-1080 MRC-5 HepG2 PC-12 BD PureCoat amine BD PureCoat carboxyl HEK-293 Transfected Cell Lines EcoPack herg-t-rex 293 DA HEK-ZsGreen Proteasome Sensor CHO-M4 Primary Neural Cells Rat brain cortical neurons Rat astrocytes Rat cerebellar granule Primary Non-Neural Cells Rat keratinocytes

10 Cell Types Cultured on BD PureCoat Cultureware: Customer Recommendations Cell Lines N2A HeLa 293T Flp-In T-REx 293 HEK-293 Zellen Primary Cells Human Placental Epithelial cells Human Epidermal Keratinocytes (neonatal) Embryonic Mouse Brain Stem Cell Rat E14d Cortex-Derived Neural Stem Cells Primary Cervical Epithelial Cells Primary Rat Pancreatic Islet Mouse Cardiomyocyte Mesenchymal Stem Cell (Rat bone-marrow-derived) BD PureCoat amine BD PureCoat carboxyl

11 Parameters Tested on BD PureCoat Cultureware Freeze-thaw recovery and attachment of cryopreserved cells Cell attachment and proliferation Reduced serum-supplemented medium Serum-free medium Attachment and differentiation of primary neural cells Cell performance in HTS-based assays Cell performance in transfection-based assays

12 Uniform LnCAP Cell Attachment Post Thaw on BD PureCoat Carboxyl Images captured 24h post seeding (100x magnification) Tissue Culture BD PureCoat carboxyl Competitor C

13 Better Freeze-Thaw Recovery of LnCAP on BD PureCoat Carboxyl 25% increase in LnCAP cell attachment and recovery post-thaw in standard culture conditions on BD PureCoat carboxyl 24h attachment assay (96-well format) 0.8 n=14 wells/surface 140 n=3 experiments Relative Absorbance Units % increase over TC TC Competitor C BD PureCoat carboxyl 0 TC Competitor C BD PureCoat carboxyl

14 Better Attachment and Freeze-Thaw Recovery of herg-t-rex 293 DA Cells on BD PureCoat Amine 384-well format 24h attachment assay (96-well format) 0.12 n=10 wells/surface Tissue Culture Relative Absorbance Units TC Competitor C BD PureCoat amine Competitor C 40% increase in herg-t-rex division arrested (DA) cell line attachment and recovery post-thaw on BD PureCoat amine BD PureCoat amine

15 Parameters Tested on BD PureCoat Cultureware Freeze-thaw recovery and attachment of cryopreserved cells Cell attachment and proliferation Reduced serum-supplemented medium Serum-free medium Attachment and differentiation of primary neural cells

16 Increased BHK-21 Cell Proliferation in Reduced Serum (+1% FBS) on BD PureCoat Amine DAPI stain 3d growth assay (96-well format) Tissue Culture Relative Absorbance Units n=10 wells/surface TC Competitor C BD PureCoat amine 0 #1 #2 #3 #1 #2 #3 #1 #2 # cells/well 3200 cells/well 1600 cells/well BD PureCoat amine Initial seeding density BD PureCoat amine supports BHK-21 growth in reduced serum

17 Increased BHK-21 Cell Proliferation in Reduced Serum (+1% FBS) on BD PureCoat Amine 60-70% increase in BHK-21 cell growth in multiple lots (lots #1-3) and formats of BD PureCoat amine 3d growth assay (24-well format) Relative Absorbance Units #1 #2 #3 n=4 wells/surface #1 #2 #3 TC Competitor C BD PureCoat amine #1 #2 #3 40,000 cells/well 20,000 cells/well 10,000 cells/well Initial seeding density

18 Enhanced Cell Proliferation of MRC-5 Cells in Reduced Serum (+5% FBS) on BD PureCoat Carboxyl 10-40% increase in MRC-5 cell growth in reduced-serum containing media on BD PureCoat carboxyl 3d growth assay (24-well format) 3d growth assay (96-well format) 0.8 n=16 wells/surface n=2 experiments 200 Relative Absorbance Units % Increase over TC TC Competitor C BD PureCoat carboxyl 0 TC Competitor C BD PureCoat carboxyl

19 Increased HEK-293 Cell Proliferation in Reduced Serum (1% FBS) on BD PureCoat Amine ~70% increase in cell growth on BD PureCoat amine supports HEK-293 growth in reduced-serum containing 60,000 cells/ well 4d growth assay (24-well format) 0.6 n=4 wells/surface TC Tissue Culture Relative Absorbance Values Competitor C BD PureCoat amine ,000 cells/well 60,000 cells/well 30,000 cells/well BD PureCoat amine Initial seeding density

20 Better Hep-G2 Cell Proliferation in Reduced Serum (+5% FBS) on BD PureCoat Carboxyl 4d growth assay (96-well format) 25-65% increase in cell proliferation in reduced serum on BD PureCoat carboxyl 25-50% increase in cell proliferation in standard culture conditions on BD PureCoat carboxyl Relative Absorbance Values Relative Absorbance Values MEM + 5% serum n=7 wells/surface TC BD PureCoat carboxyl MEM + 10% serum n=7 wells/surface 6400 cells/well 3200 cells/well Initial seeding density

21 Hep-G2 Cells Grown on BD PureCoat Surfaces Remain Responsive in Cytotoxicity Assays Despite Changes in Cell Morphology Potency (EC 50 ) of tamoxifen in cytotoxicity assays from cells grown on BD PureCoat surfaces within two-fold that of those grown on TC vessels. 140 Tissue Culture BD PureCoat carboxyl BD PureCoat amine % cell viability of control % cell viability of control uM 30uM 60uM TC BD PureCoat Carboxyl BD PureCoat Amine 10uM 30uM 60uM Concentration of Tamoxifen HepG2 cells seeded at 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.

22 Enhanced Attachment of HT-1080 Cells on BD PureCoat Carboxyl in Serum-free Media HT-1080 cells attach and spread uniformly on BD PureCoat carboxyl in serum-free medium 24h post seeding cells were fixed with methanol and stained with toluidene blue Tissue Culture Competitor C BD PureCoat carboxyl

23 Parameters tested on BD PureCoat Cultureware Cell attachment and proliferation Reduced serum-supplemented medium Serum-free medium Attachment and differentiation of primary neural cells Cell performance in HTS-based assays

24 Increased Cell Proliferation of Rat Astrocytes on BD PureCoat Amine > 50% increase in primary rat astrocyte cell proliferation post-thaw in media containing 3% FBS and growth factors on BD PureCoat amine Primary rat astrocytes cultured on BD PureCoat amine continue to express typical phenotypic functional markers such as Glial Fibrillary Acidic Protein (GFAP) 6d growth assay Fluorescent staining for α-gfap Relative Absorbance Units TC Competitor C BD PureCoat amine n=5 wells/surface TC Competitor C Lot 1 Lot 2 BD PureCoat amine

25 Cell Attachment of RCG Cells on BD PureCoat Amine Rat Cerebellar Granule (RCG) cells attach and differentiate on BD PureCoat amine, whereas those plated on TC-treated or Competitor C surfaces remain unattached and float Tissue Culture BD PureCoat amine Competitor C

26 Differentiation of RCG Cells on BD PureCoat Amine BD PureCoat amine supports differentiation of neural cells Neurite outgrowths from RCG cells form a network Increase of neurite migration with time Immunostained with markers expressed in neural cells such as tubulin IIIβ RCG cells immunostained with anti-tubulin IIIβ 24h post-isolation 48h post-isolation

27 Primary Rat Brain Cortical Neuronal Cells Post- Thaw Do Not Attach on Uncoated TC Vessels Poor 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 Tissue culture

28 Cell Attachment and Differentiation of Primary Rat Brain Cortical Neuronal Cells Post-Thaw on BD PureCoat Amine Cells attach and differentiate on BD PureCoat amine. Increase in neurite length observed with time. 5d post-thaw 48h post-thaw

29 Parameters tested on BD PureCoat Cultureware Freeze-thaw recovery and attachment of cryopreserved cells Cell attachment and proliferation Reduced serum-supplemented medium Serum-free medium Attachment and differentiation of primary neural cells Cell performance in HTS-based assays Cell performance in transfection-based assays

30 HTS formats Ecopack2-293 cells adhere to BD PureCoat Amine EcoPack2-293 cell monolayer integrity maintained on BD PureCoat amine, 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) Tissue Culture Competitor C BD PureCoat amine Before wash 384-well format After wash

31 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 formats Z values were > 0.72 indicating a robust assay 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 384-well format well format 40 TC TC 30 Competitor C BD PureCoat amine 45 BD PureCoat amine CV 20 CV lot 1 lot 1 lot 1 lot 2 lot 2 lot 2 lot 3 lot 3 lot 3 0 lot 1 lot 1 lot 1 lot 2 lot 2 lot 2

32 Cells on BD PureCoat Surfaces Remain Functional and Can Be Coupled for Use in GPCR-based Assays Comparable potency (EC 50 ) of carbachol from cells grown either on TC-treated or BD PureCoat amine Carbachol-induced calcium mobilization studies on EcoPack2-293 cells cultured in serum-free medium Relative Fluorescence Units Concentration of carbachol (µm)

33 Cells on BD PureCoat Surfaces Remain Functional and Can Be Coupled for use in GPCR-based Assays Comparable responses of cells on BD PureCoat amine and TC-treated in GPCR-based assays 120 ATP-induced calcium mobilization studies on EcoPack2-293 cells cultured in serum-free medium Relative Fluorescence Units TC BD PureCoat amine ATP (µm)

34 Utilization of BD PureCoat Surfaces for Functional Second Messenger camp Assays Better cell attachment of HEK-293 cells in serum-free medium resulted in higher camp responses from cells on BD PureCoat surfaces camp responses of HEK-293 cells cultured in serumfree medium and treated with Isoproterenol mean camp (nm) Control Isoproterenol (20µM) n=3 wells/treatment/surface 0 TC BD PureCoat carboxyl BD PureCoat amine

35 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 Relative Fluorescent Units n=10 wells/treatment/surface TC BD PureCoat amine BD PureCoat carboxyl ALLN conc.(µm)

36 BD PureCoat Surfaces Proteasome Inhibition Fluorescent Microscopy on BD Pathway 800 Imager Cells cultured on TC-treated and BD PureCoat surfaces respond to ALLN Living Colors HEK-293 ZsGreen Proteasome Sensor Cells (Clontech) were either untreated or treated with 10 µm ALLN, a proteasome inhibitor for 12h Tissue Culture BD PureCoat carboxyl BD PureCoat amine Untreated 10x objective 10 µm ALLN

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

38 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 HepG2 cells transfected with pzsgreen C1 plasmid DNA (Clontech) and images captured 24h post transfection on the BD Pathway 800 Imager GFP Fluorescence Tissue Culture BD PureCoat Amine

39 Advantages of BD PureCoat Cultureware vs. Standard TC-treated Surfaces Enhanced cell attachment and growth in serum reduced or serum-free culture media Better freeze-thaw recovery of cryopreserved cells Enhanced attachment of transfected, primary, and fastidious cell lines Supports neural cell attachment and differentiation Cells on BD PureCoat surfaces remain functional and can be coupled for use in HTS assays such as GPCR, camp, proteasome-inhibition, and transfection-based assays Chemically defined, animal-free surfaces Sterile (SAL 10-6 ) Stored at room temperature Available in a variety of formats

40 BD PureCoat Cultureware: Launched June 2009 High-density Black/Clear Microplates 96-well 384-well 1536-well Multiwell Plates and Cell Culture Dishes 6-well 24-well 100 mm dishes Flasks Coming Soon!

41 Questions? Contact information: Elizabeth Abraham, Ph.D. EcoPack and Living Colors are the property of Clontech Laboratories, Inc., a Takara Bio Company. Flp-In and T-REx are the property of Invitrogen Corporation. BD, BD Logo, and all other trademarks are the property of Becton, Dickinson and Company BD