Microporous Membrane- Based Cell Culture Insert Systems Introduction and Key Applications

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1 Microporous Membrane- Based Cell Culture Insert Systems Introduction and Key Applications Marshall Kosovsky, Ph.D. May 12, 2009

2 Topics for Discussion Overview of microporous membrane insert platform Membrane types Insert formats Insert handling Applications

3 Membrane Supports for Cell Culture Cell culture inserts provide a two-compartment culture system suitable for a variety of complex cell-based assays Insert wells contain a microporous membrane floor composed of polyethylene teraphthalate (PET) available with different pore diameters Pores allow exchange of media, nutrients, molecules, and the passage of cells (pore size dependent on cell type) Small pore diameters (0.4 and 1.0 µm) prevent cell passage; large pore diameters (3.0 and 8.0 µm) allow cell passage

4 Benefits of PET Membrane Chemical properties minimize non-specific binding of compounds and small molecules Durable material that will not break, bend, or curl when manipulated Available in transparent, translucent, and BD FluoroBlok (fluorescence blocking) Pore sizes: 0.4, 1.0, 3.0, and 8.0 µm pore diameter Low and high pore density (0.4 and 3.0 µm only)

5 Applications (Clear and BD FluoroBlok Membrane) Angiogenesis Endothelial Cell Migration/Invasion Tumor Cell Biology Tumor Cell Migration/Invasion Inflammation Monocyte, Leukocyte Chemotaxis Transendothelial Cell Migration ADME/Tox Cell Differentiation Blood Brain Barrier Models Co-culture studies (primary cells, stem cells) Drug Discovery (single parameter or multiplexing) Cell Imaging Transport and Permeability Toxicity Assays

6 Applications by Pore Size Pore Diameter 0.4, 1.0 µm 3.0 µm 8.0 µm Applications ADME/Tox (compound transport and permeability through cell barrier; toxicity assays) Co-culture; cell differentiation Cell migration/invasion Blood cell chemotaxis or transendothelial migration Rat glioma migration Cell migration/invasion Transendothelial migration Blood cell chemotaxis Cell Types Caco-2 MDCK Neuronal Endothelial Leukocytes Neuronal Epithelial Tumor-derived Leukocytes Fibroblasts

7 Clear PET Membrane Transparent membrane allows visualization of cells by light microscopy Translucent membrane exhibits high pore density, which allows maximal basolateral diffusion for studies of compound bioavailability (i.e., cell physiology/adme applications such as compound transport, permeability, or absorption)

8 BD FluoroBlok PET Membrane Treated with proprietary blue dye Unique fluorescence-blocking membrane blocks light transmission from nm Quantitative analysis using fluorescence detection Increases productivity and assay throughput No need to dismantle, wash, and manually count cells Eliminates multiple handling steps just add cells, label, and read 8 µm pores visible light

9 BD Falcon FluoroBlok Cell Culture Inserts The blue dyed membrane physically and visually separates cells above the membrane from those below the membrane Insert (individual or multiwell) Apical Chamber Cross section (not to scale) Track-Etched Pores Basal Chamber Base plate 3.0 and 8.0 µm pore diameters Individual, 24-Multiwell, and 96-Multiwell formats

10 BD Falcon and BD BioCoat Individual Cell Culture Inserts General Features Hanging design facilitates pipeting and allows for co-culture Non-TC-treated insert housing minimizes cell growth on insert walls Clear or BD FluoroBlok membrane Variety of pore sizes (0.4, 1.0, 3.0, and 8.0) and formats (6-, 12-, or 24-well) Packaged in individual blister packs 48 inserts/case Must be used with companion plates sold separately BD extracellular matrix coatings for studying cells in physiological environment BD Matrigel Matrix Fibronectin Laminin Collagens I and IV Fibrillar Collagen

11 Individual Insert Handling + Individual Inserts (w/flanges) Companion Plate (w/notches) = Insert Flanges Resting in Notch

12 Individual Insert Handling

13 Insert System Handling Bubbles should be eliminated at all steps Chemoattractant should be added to bottom chamber via access port To minimize bubbles, add to the apical chamber first and then to basal chamber bubble under insert will influence reading (cells may not migrate or stain in that area)

14 BD Falcon and BD BioCoat Multiwell Insert Systems

15 Multiwell Insert Handling Repeating Pipettor recommended for 24-Multiwell Inserts (also suitable for individual inserts) Multi-channel Pipettor required for 96-Multiwell Inserts The 96 square-well receiver plate must be kept level

16 Applications Cell imaging Compound transport and permeability

17 Confocal Analysis of Caco-2/bbe (C2) Cells using BD Falcon Cell Culture Inserts xy xz merged xz (2X) Control NH 2 Cl Cy2-tagged -subunit of Na + -K + -ATPase in C2 cells. The - subunit localizes to the apical pole in the presence of NH 2 Cl. Data kindly provided by Dr. Mark Musch, University of Chicago; Figure adapted from Musch, M.W., et al., Am. J. Physiol. Gastrointest. Liver Physiol. 290: 222 (2006).

18 BD Falcon 24- and 96-Multiwell Insert Systems Automation compatible 24- and 96-Multiwell Inserts Generous Sampling Ports 24-Multiwell format (1.0, 3.0, and 8.0 µm; clear membrane) 96-Multiwell format (1.0 µm; clear membrane)

19 P-Glycoprotein Function in Caco-2 Cells using the BD BioCoat HTS Caco-2 Assay System Transmission EM of Caco-2 cells cultured for 3 days on fibrillar collagencoated PET membrane. Differentiated cells exhibit microvilli, desmosomes, and cellular interdigitation. P-Glycoprotein function was assessed by analyzing the distribution of radiolabeled Vinblastine. Inhibition of P-Glycoprotein was examined in presence of 100 m Verapamil.

20 BD Falcon FluoroBlok 24- and 96- Multiwell Insert Systems Unique fluorescence-blocking PET membrane Increased productivity and throughput; simplified fluorescence detection Available in 3.0 and 8.0 µm pore sizes Suitable for real-time kinetic analysis Automation compatible

21 Key Applications for BD FluoroBlok Inserts BD Falcon FluoroBlok Individual or Multiwell Inserts Cell migration and invasion Tumor or endothelial cell migration using uncoated or self-coated inserts Tumor or endothelial cell invasion using self-coated inserts Cell differentiation and co-culture Variety of cell types using self-coated inserts BD BioCoat FluoroBlok Multiwell Inserts Tumor cell biology: BD BioCoat Tumor Invasion Systems Endothelial cells: BD BioCoat Angiogenesis Systems Blood cells (e.g., monocyte migration): BD BioCoat Inserts pre-coated with fibronectin

22 Cell Labeling Dyes Any fluorescent dye derived from the fluorescein, rhodamine and cyanine families can all be used with BD FluoroBlok inserts emission wavelength must be between nm your dye here Ultraviolet-inducible dyes tend to be incompatible with the BD Falcon FluoroBlok insert since they tend to emit light in the blue range For more information on spectra and alternative fluorophore choices, consult BD Biosciences Technical Bulletin #451 Spectrum image from under GNU free documentation license.

23 Cell Labeling Methods Pre-Labeling Labeling cells in vitro prior to assay Post-labeling Labeling cells on the underside of membrane following migration/invasion Transfected cells that are intrinsically-labeled Over-expression of Green Fluorescent Protein or analogs (e.g., RCFP)

24 Typical Migration or Invasion Assay using Post-Labeling 1. Rehydrate ECM coating (2h) 2. Aspirate media 3. Seed cells 24-well: 25,000 50,000 cells/well 96-well: 10,000 20,000 cells/well 4. Add chemoattractant (titration of chemoattractant recommended) 5. Incubate for hours/overnight/days (dependent on cell type) 6. Stain cells with appropriate dye, such as calcein AM (incubate 1h) 7. Read with bottom-reading fluorescence plate reader

25 Applications Angiogenesis

26 BD BioCoat Angiogenesis Systems Endothelial Cell Migration 24-or 96-Multiwell BD FluoroBlok insert (3.0 µm pore size) Coated with human fibronectin BD Human Umbilical Vein Endothelial Cells (BD HUVEC-2) Pre-qualified for VEGF responsiveness and for use with endothelial cell migration assay Endothelial Cell Invasion 24-Multiwell BD FluoroBlok insert (3.0 µm pore size) Coated with BD Matrigel Matrix Endothelial Cell Tube Formation Comprised of a 96-well black/clear plate coated with BD Matrigel Matrix (non-insert system)

27 Human Umbilical Vein Endothelial Cells Most commonly used human EC type for studies of angiogenesis Source, isolation procedure, and initial culturing conditions can influence response to pro-angiogenic factors (e.g. VEGF, bfgf) BD HUVEC-2 cells (cat. no ) Pre-qualified for responsiveness to VEGF in endothelial cell migration assay Tested for presence of von Willebrand factor (vwf), CD31, uptake of Dil-Ac- LDL, and absence of alpha actin

28 Analysis of Endothelial Cell Migration and Invasion Using BD FluoroBlok Membrane Inserts Fibronectin (migration) or BD Matrigel Matrix (invasion) Attractant Excitation (485 nm) Emission (530 nm) BD FluoroBlok PET membrane (3 m pores)

29 BD HUVEC-2 Cells Exhibit Concentration- Dependent Migration Towards VEGF Cell migration assessed using the BD BioCoat Angiogenesis System: Endothelial Cell Migration (fibronectin-coated BD FluoroBlok membrane, 96-Multiwell format).

30 Migration Activity Using Different EC Types HAEC Cells HUVEC-2 Cells Fold increase over control mean + se (n=4) Fold increase over control mean + se (n=4) VEGF (ng/ml) VEGF (ng/ml) 8-10 fold stimulation 2-3 fold stimulation

31 BD BioCoat Angiogenesis System: Endothelial Cell Invasion Effect of MMP inhibitor 1'10' Phenanthroline on HMVEC Invasion Fluorescent Units Control VEGF(4ng/ml) 0.1ug/ml 1ug/ml 10ug/ml 20ug/ml VEGF(4ng/ml)+ 1'10' Phenathroline

32 Applications Tumor Cell Biology

33 Analysis of Tumor Cell Invasion using the BD BioCoat Matrigel Invasion Chamber Individual insert format (6- and 24-well) Clear PET membrane, 8.0 µm pore size Pre-coated with BD Matrigel Matrix [standard or growth factor reduced (GFR)] HT-1080 cell emerges from pore on underside of the membrane after digestion of BD Matrigel Matrix. NIH 3T3 and HT-1080 cells were incubated for hours, stained, and analyzed for invasion activity.

34 BD BioCoat Tumor Invasion Systems Combined Benefits of BD FluoroBlok and BD Matrigel Matrix Reproducibility Optimized Protocols Available in 24- and 96-Multiwell Formats (8.0 µm pore size)

35 Analysis of Tumor Cell Invasion Using BD FluoroBlok Membrane Inserts BD Matrigel Matrix (invasion) Attractant Excitation (485 nm) Emission (530 nm) BD FluoroBlok PET membrane (8.0 m pores)

36 MDA-MB-231 Human Breast Adenocarcinoma Cell Invasion Through BD Matrigel Matrix Fluorescently labeled cells residing on the bottom of the membrane post-invasion

37 Inhibition of MDA-MB-231 Cell Invasion Through BD Matrigel Matrix by Doxycycline

38 Detection Instrument A fluorescent plate reader with bottom-reading capability, and an inverted fluorescent microscope for confirmation and troubleshooting A fluorescence imager Technical Bulletin # 436: Set Up Guidelines and Dimensional Templates for Fluorescence Plate Readers Used With BD Falcon HTS FluoroBlok Insert Systems and BD BioCoat Multiwell Insert Cell-Based Assays technical_resources/pdf/tb436_fluoroblok.pdf

39 Other Representative Applications Monocyte differentiation Seo, K.S., et al., J. Leukocyte Biology 85: (2009) Stem cell differentiation Yokoyama, Y., et al., Blood, pre-published online March 25, 2009 (2009) Organotypic slice culture Chameau, P., et al., PNAS 106: (2009) Semino, C.E., et al., Tissue Engineering 10: (2004) Neuronal motogen screening Hassoun, A.T., et al., J. Neuroscience Methods 166: (2007) Glioma invasion Beadle, C., et al., Molecular Biology of the Cell 19: (2008)

40 The Advantages of BD Falcon and BD BioCoat Cell Culture Inserts and Insert Systems Features Wide selection of individual and multiwell formats and pore sizes PET membrane Unique BD FluoroBlok Membrane Automation compatible 24- and 96- Multiwell Insert Systems BD BioCoat inserts coated with ECM proteins Benefits Increases experimental flexibility Minimizes non-specific binding of small molecules; transparent membrane ideal for imaging Increases productivity by automating fluorescence detection; allows rapid analysis with fewer handling steps; highly reproducible Increases productivity and throughput by facilitating cell-based assays; reduced risk of contamination Provides physiological environment; saves preparation time and increases consistency

41 References Cell Imaging Musch, M., et al., Roles of ZO-1, occludin, and actin in oxidant-induced barrier disruption. Am. J. Physiol. Gastrointest Liver Physiol. 290: (2006). ADME/Cell Physiology Sasabe, H., et al., Differential involvement of multidrug resistance-associated protein 1 and P-glycoprotein in tissue distribution and excretion of grepafloxacin in mice. J. Pharmacol. Exp. Ther. 310:648 (2004). Kipp, H., et al., More than apical: distribution of SGLT1 in Caco-2 cells. Am. J. Physiol. Cell Physiol. 285:C737 (2003).

42 References Angiogenesis Potapova, I.A., et al., Mesenchymal stem cells support migration, extracellular matrix invasion, proliferation, and survival of endothelial cells in vitro. Stem Cells 25: (2007). Favier, B., et al., Neurophilin-2 interacts with VEGFR-2 and VEGFR-3 and promotes human endothelial cell survival and migration. Blood 108: (2006). Davis, G.E. and Senger, D.R. Endothelial extracellular matrix: biosynthesis, remodeling, and functions during vascular morphogenesis and neovessel stabilization. Circulation Res. 97: (2005). Tumor Cell Biology Stasinopoulos, I. Silencing of cyclooxygenase-2 inhibits metastasis and delays tumor onset of poorly differentiated metastatic breast cancer cells. Molecular Cancer Research 5: (2007). Wang, Z., Down-regulation of forkhead box M1 transcription factor leads to the inhibition of invasion and angiogenesis of pancreatic cancer cells. Cancer Research 67: (2007).

43 Questions? Technical Support: In the U.S. tel: Outside the U.S. Visit bdbiosciences.com/webinars For research use only. Not intended for use in diagnostic or therapeutic procedures. BD, BD Logo, and all other trademarks are the property of Becton, Dickinson and Company BD

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