Microporous MembraneBased Cell Culture Insert
Systems
Introduction and Key Applications
Marshall Kosovsky, Ph.D.
May 12, 2009
Topics for Discussion
•
Overview of microporous membrane insert platform
– Membrane types
– Insert formats
– Insert handling
•
Applications
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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
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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)
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Applications
(Clear and BD FluoroBlok™ Membrane)
• Angiogenesis
– Endothelial Cell Migration/Invasion
• Tumor Cell Biology
– Tumor Cell Migration/Invasion
• Cell Differentiation
– Blood Brain Barrier Models
– Co-culture studies (primary
cells, stem cells)
– Monocyte, Leukocyte Chemotaxis
• Drug Discovery
(single parameter or
multiplexing)
– Transendothelial Cell Migration
• Cell Imaging
• Inflammation
• ADME/Tox
– Transport and Permeability
– Toxicity Assays
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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
• Caco-2
• MDCK
• Neuronal
• Cell migration/invasion
• Blood cell chemotaxis or
transendothelial migration
• Rat glioma migration
• Endothelial
• Leukocytes
• Neuronal
• Cell migration/invasion
• Transendothelial migration
• Blood cell chemotaxis
•
•
•
•
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Cell Types
Epithelial
Tumor-derived
Leukocytes
Fibroblasts
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)
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BD FluoroBlok PET Membrane
• Treated with proprietary blue dye
• Unique fluorescence-blocking membrane blocks light
transmission from 490–700 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
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8 µm pores – visible light
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
Track-Etched Pores
(not to scale)
Basal Chamber
Base plate
3.0 and 8.0 µm pore diameters
Individual, 24-Multiwell, and 96-Multiwell formats
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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
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Individual Insert Handling
+
Individual Inserts (w/flanges)
•
Companion Plate (w/notches)
=
Insert Flanges Resting in Notch
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Individual Insert Handling
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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)
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BD Falcon and BD BioCoat Multiwell
Insert Systems
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Multiwell Insert Handling
• Repeating Pipettor
recommended for
24-Multiwell Inserts
(also suitable for
individual inserts)
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• Multi-channel Pipettor required
for 96-Multiwell Inserts
• The 96 square-well receiver plate
must be kept level
Applications
• Cell imaging
• Compound transport and permeability
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Confocal Analysis of Caco-2/bbe (C2) Cells
using BD Falcon Cell Culture Inserts
xy
Control
--------------------
NH2Cl
--------------------
xz
merged
xz (2X)
Cy2-tagged -subunit of Na+-K+-ATPase in C2 cells. The subunit localizes to the apical pole in the presence of NH2Cl.
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).
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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)
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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.
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P-Glycoprotein function was assessed by
analyzing the distribution of radiolabeled
Vinblastine. Inhibition of P-Glycoprotein was
examined in presence of 100 m Verapamil.
BD Falcon FluoroBlok 24- and 96Multiwell 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
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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
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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 490-700 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 http://en.wikipedia.org/wiki/Image:Srgbspectrum.png under GNU free documentation license.
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Cell Labeling Methods
• Pre-Labeling
–
•
Post-labeling
–
•
Labeling cells in vitro prior to assay
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)
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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
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Applications
• Angiogenesis
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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)
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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. 354151)
– Pre-qualified for responsiveness to VEGF in endothelial cell migration assay
– Tested for presence of von Willebrand factor (vWf), CD31, uptake of Dil-AcLDL, and absence of alpha actin
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Analysis of Endothelial Cell Migration and
Invasion Using BD FluoroBlok Membrane Inserts
Fibronectin
(migration)
or
BD Matrigel™
Matrix (invasion)
Attractant
Excitation
(485 nm)
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BD FluoroBlok
PET membrane
(3 m pores)
Emission
(530 nm)
BD HUVEC-2 Cells Exhibit ConcentrationDependent Migration Towards VEGF
Cell migration assessed using the BD BioCoat Angiogenesis
System: Endothelial Cell Migration (fibronectin-coated
BD FluoroBlok membrane, 96-Multiwell format).
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Migration Activity Using Different EC Types
HAEC Cells
HUVEC-2 Cells
8
6
4
2
0
2.0
mean + se (n=4)
Fold increase over control
2.5
10
mean + se (n=4)
Fold increase over control
12
1.5
1.0
0.5
0.0
0.000
1.000
3.125
6.250
12.500
25.000
0.000
1.000
3.100
6.200
12.500
VEGF (ng/ml)
VEGF (ng/ml)
8-10 fold stimulation
2-3 fold stimulation
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25.000
BD BioCoat Angiogenesis System:
Endothelial Cell Invasion
1800
1600
1400
1200
1000
g/
m
l
l
20
u
g/
m
10
u
l
g/
m
1u
g/
m
l
0.
1u
VE
G
F(
4n
g
/m
l)
800
600
400
200
0
C
on
tr o
l
Fluorescent Units
Effe ct of M M P inhibitor 1'10' P he na nthroline on
HM V EC Inva sion
V EGF(4ng/ml)+ 1'10' Phenathroline
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Applications
• Tumor Cell Biology
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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.
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NIH 3T3 and HT-1080 cells were
incubated for 18-20 hours, stained,
and analyzed for invasion activity.
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)
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Analysis of Tumor Cell Invasion Using
BD FluoroBlok Membrane Inserts
BD Matrigel™
Matrix (invasion)
Attractant
Excitation
(485 nm)
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BD FluoroBlok™
PET membrane
(8.0 m pores)
Emission
(530 nm)
MDA-MB-231 Human Breast Adenocarcinoma
Cell Invasion Through BD Matrigel Matrix
Fluorescently labeled cells residing on the
bottom of the membrane post-invasion
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Inhibition of MDA-MB-231 Cell Invasion Through
BD Matrigel Matrix by Doxycycline
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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
– http://www.bdbiosciences.com/discovery_labware/
technical_resources/pdf/tb436_fluoroblok.pdf
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Other Representative Applications
Monocyte differentiation
• Seo, K.S., et al., J. Leukocyte Biology 85:606-616 (2009)
Stem cell differentiation
• Yokoyama, Y., et al., Blood, pre-published online March 25, 2009 (2009)
Organotypic slice culture
• Chameau, P., et al., PNAS 106:7227-7232 (2009)
• Semino, C.E., et al., Tissue Engineering 10:643-655 (2004)
Neuronal motogen screening
• Hassoun, A.T., et al., J. Neuroscience Methods 166:178-194 (2007)
Glioma invasion
• Beadle, C., et al., Molecular Biology of the Cell 19:3357-3368 (2008)
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The Advantages of BD Falcon and BD BioCoat
Cell Culture Inserts and Insert Systems
Features
Benefits
Wide selection of individual and
multiwell formats and pore sizes
Increases experimental flexibility
PET membrane
Minimizes non-specific binding of small
molecules; transparent membrane ideal for
imaging
Unique BD FluoroBlok Membrane
Increases productivity by automating
fluorescence detection; allows rapid analysis
with fewer handling steps; highly reproducible
Automation compatible 24- and 96Multiwell Insert Systems
Increases productivity and throughput by
facilitating cell-based assays; reduced risk of
contamination
BD BioCoat inserts coated with ECM
proteins
Provides physiological environment; saves
preparation time and increases consistency
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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. 290:222-231 (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).
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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 25:17611768 (2007).
• Favier, B., et al., Neurophilin-2 interacts with VEGFR-2 and VEGFR-3 and promotes
human endothelial cell survival and migration. Blood 108:1243-1250 (2006).
• Davis, G.E. and Senger, D.R. Endothelial extracellular matrix: biosynthesis,
remodeling, and functions during vascular morphogenesis and neovessel stabilization.
Circulation Res. 97:1093-1107 (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 5:435-442 (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
67:8293-8300 (2007).
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