Microporous MembraneBased Cell Culture Insert
Systems
Introduction and Key Applications
Marshall Kosovsky, Ph.D.
March 16, 2009
Topics for Discussion
•
Overview of microporous membrane insert platform
– Membrane types
– Insert formats
– Insert handling
•
Applications
2
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 terephthalate (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 mm) prevent cell passage
– Large pore diameters (3.0 and 8.0 mm) allow cell passage
3
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) membranes
• 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)
4
Applications
(Clear and BD FluoroBlok™ Membranes)
•
Angiogenesis
– Endothelial Cell Migration/Invasion
•
Tumor Cell Biology
– Tumor Cell Migration/Invasion
•
Inflammation
– Monocyte, Leukocyte Chemotaxis
– Transendothelial Cell Migration
•
ADME/Tox
– Transport and Permeability
– Toxicity Assays
•
Cell Differentiation
– Blood Brain Barrier Models
– Co-culture studies (primary cells, stem cells)
•
•
Drug Discovery (single parameter or multiplexing)
Cell Imaging
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Applications by Pore Size
Pore Diameter
0.4, 1.0 µm
Applications
Cell Types
• ADME/Tox (compound transport and permeability
through cell barrier; toxicity assays)
• Caco-2
• Co-culture; cell differentiation
3.0 µm
8.0 µm
• MDCK
• Neuronal
• Cell migration/invasion
• Endothelial
• Blood cell chemotaxis or transendothelial migration
• Leukocytes
• Rat glioma migration
• Neuronal
• Cell migration/invasion
• Epithelial
• Transendothelial migration
• Tumor-derived
• Blood cell chemotaxis
• Leukocytes
• Fibroblasts
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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
8 μm pores – visible light
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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
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 and formats (6-, 12-, or 24-well)
Packaged in individual blister packs; 48 inserts/case
For use with Notched Companion Plates – sold separately
Extracellular matrix coatings (BD BioCoat cultureware) 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 rest in notches
11
Individual Insert Handling
12
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 the
basal chamber
bubble under insert will
influence acquisition of
data (cells may not migrate
or stain in that area)
13
BD Falcon™ and BD BioCoat™ Multiwell
Inserts
14
Multiwell Insert Handling
Repeating Pipettor
recommended for
24-Multiwell Inserts
(also suitable for
individual inserts)
15
•
Multi-channel Pipettor
required for 96-Multiwell Insert
plate
•
The 96-square well receiver
plate should be level
Applications
• Cell imaging
• Compound transport and permeability
16
Confocal Analysis of Caco-2/bbe (C2) Cells
using BD Falcon Cell Culture Inserts
xz
merged
xy
Control
--------------------
NH2Cl
--------------------
xz (2X)
Cy2-tagged α-subunit of Na+-K+-ATPase in C2 cells. The αsubunit 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. (2006) Am J Physiol – Gastrointest. Liver Physiol. 290: 222.
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BD Falcon™ 24- and 96-Multiwell Insert
Systems
•
Automation compatible 24- and
96-Multiwell insert systems
•
Generous sampling ports
•
24-Multiwell format (1.0, 3.0, and 8.0 µm;
clear membrane)
•
96-Multiwell format (1.0 µm; clear
membrane)
Pic from p 1
of 96well sell
sheet
18
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
collagen-coated PET membrane.
Differentiated cells exhibit microvilli,
tight junctions (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 μm Verapamil.
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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
20
Key Applications for BD FluoroBlok Inserts
•
BD Falcon FluoroBlok Individual or Multiwell Inserts:
– Cell migration and invasion
¾ Tumor or endothelial cell migration using uncoated
or self-coated inserts
¾ Tumor or endothelial cell invasion using self-coated inserts
– Cell differentiation and co-culture
¾ 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 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
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.
22
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)
23
Typical Migration or Invasion Assay
using Post-Labeling
•
Rehydrate ECM coating for 2h (if BD Matrigel matrix)
•
Aspirate media
•
Seed cells
¾
24-well: 25,000 - 50,000 cells/well
¾
96-well: 10,000 - 20,000 cells/well
•
Add chemoattractant
(titration of chemoattractant recommended)
•
Incubate for hours/overnight/days (dependent on cell type)
•
Stain cells with appropriate dye, such as calcein AM (incubate 1h)
•
Read with bottom-reading fluorescence plate reader
24
Applications
• Angiogenesis
25
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)
- Validated protocols
26
Human Umbilical Vein Endothelial Cells
•
Most commonly used human endothelial cell 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-Ac-LDL, and absence of alpha actin
27
Analysis of Endothelial Cell Migration and
Invasion Using BD FluoroBlok Membrane
Inserts
Fibronectin
(migration)
or
BD Matrigel matrix
(invasion)
Attractant
BD FluoroBlok
PET Membrane
(3.0 μm pores)
Excitation
(485 nm)
Emission
(530 nm)
28
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).
29
Migration Activity Using Different
Endothelial Cell 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
0.000
25.000
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
20
ug
/m
l
m
1u
g/
10
ug
/m
l
l
l
m
ug
/
0.
1
VE
G
F(
4n
g/
m
l)
1800
1600
1400
1200
1000
800
600
400
200
0
C
on
tro
l
Fluorescent Units
Effect of MMP inhibitor 1'10' Phenanthroline on
HMVEC Invasion
VEGF(4ng/ml)+ 1'10' Phenathroline
31
Applications
• Tumor Cell Biology
32
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)]
NIH 3T3 and HT-1080 cells were
incubated for 18-20 hours, stained,
and analyzed for invasion activity.
HT-1080 cell digests BD Matrigel matrix
and invades through pore.
33
BD BioCoat Tumor Invasion Systems
•
Combined Benefits of
BD FluoroBlok inserts and
BD Matrigel matrix
•
Reproducibility
•
Optimized protocols
•
Available in 24- and 96Multiwell formats (8.0 μm
pore size)
34
Analysis of Tumor Cell Invasion Using
BD FluoroBlok Membrane Inserts
BD Matrigel matrix
(invasion)
Attractant
BD FluoroBlok
PET Membrane
(8.0 μm pores)
Excitation
(485 nm)
Emission
(530 nm)
35
MDA-MB-231 Human Breast Adenocarcinoma
Cell Invasion Through BD Matrigel Matrix
Fluorescently labeled cells residing on the
bottom of the membrane post-invasion
36
Inhibition of MDA-MB-231 Cell Invasion
Through BD Matrigel Matrix by Doxycycline
37
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/external_files/dl/doc/tech_bulletin/live/web_enabled/tb436.p
df
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Other Representative Applications
•
Monocyte differentiation
– Seo, K.S., et al. (2009) J. Leukocyte Biology 85:606-616
•
Stem cell differentiation
– Yokoyama, Y., et al. (2009) Blood, prepublished online March 25, 2009
•
Organotypic slice culture
– Chameau, P., et al. (2009) PNAS 106:7227-7232.
– Semino, C.E., et al. (2004) Tissue Engineering 10: 643-655.
•
Neuronal motogen screening
– Hassoun, A.T., et al. (2007) J. Neuroscience Methods 166:178-194
•
Glioma invasion
– Beadle, C., et al. (2008) Molecular Biology of the Cell 19: 3357-3368
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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 Increases experimental flexibility
formats and pore sizes
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
40
References
• Cell Imaging
– Musch, M., et al. (2006) Roles of ZO-1, occludin, and actin in
oxidant-induced barrier disruption. Am. J. Physiol. –
Gastrointest Liver Physiol. 290:222-231.
• ADME/Cell Physiology
– Sasabe, H., et al. (2004) 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.
– Kipp, H., et al. (2003) More than apical: distribution of SGLT1 in
Caco-2 cells. Am. J. Physiol. Cell. Physiol. 285:C737.
41
References
•
Angiogenesis
– Potapova, I.A., et al. (2007) Mesenchymal stem cells support migration,
extracellula matrix invasion, proliferation, and survival of endothelial cells in vitro.
Stem Cells 25:1761-1768.
– Favier, B., et al. (2006) Neurophilin-2 interacts with VEGFR-2 and VEGFR-3 and
promotes human endothelial cell survival and migration. Blood 108:1243-1250.
– Davis, G.E. and Senger, D.R. (2005) Endothelial extracellular matrix:
biosynthesis, remodeling, and functions during vascular morphogenesis and
neovessel stabilization. Circulation Res 97:1093-1107.
•
Tumor Cell Biology
– Stasinopoulos, I. (2007) Silencing of cyclooxygenase-2 inhibits metastasis and
delays tumor onset of poorly differentiated metastatic breast cancer cells.
Molecular Cancer Research 5:435-442.
– Wang, Z. (2007) 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.
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•
•
•
•
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Visit Us at Booth # 524
bdbiosciences.com/events
• April 27, 2010
• BD FluoroBlok Insert Systems Tips and Techniques
Webinar
• bdbiosciences.com/webinars
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Questions?
Technical Support:
In the U.S.
tel: 877.232.8995
e-mail: [email protected]
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