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Microporous Membrane- Based Cell Culture Insert Systems
Marshall Kosovsky, Ph.D.March 16, 2009
Introduction and Key Applications
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 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
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) 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)
5
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
6
Applications by Pore Size
Pore Diameter Applications Cell Types
0.4, 1.0 µm
• ADME/Tox (compound transport and permeability through cell barrier; toxicity assays)
• Co-culture; cell differentiation
• Caco-2
• MDCK
• Neuronal
3.0 µm
• Cell migration/invasion
• Blood cell chemotaxis or transendothelial
migration
• Rat glioma
migration
• Endothelial
• Leukocytes
• Neuronal
8.0 µm
• Cell migration/invasion
• Transendothelial
migration
• Blood cell chemotaxis
• 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 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
9
BD Falcon FluoroBlok Cell Culture Inserts
•
The blue dyed membrane physically and visually separates cells above the membrane from those below the membrane.
3.0 and 8.0 µm pore diametersIndividual, 24-Multiwell, and 96-Multiwell formats
Cross section(not to scale)
Track-Etched Pores
Basal Chamber
Base plate
Apical Chamber
Insert (individual or
multiwell)
10
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
11
Individual Insert Handling
Companion plate (w/notches)
+
=
Individual inserts (w/flanges)
Insert flanges rest in notches
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
the basal chamber
bubble under insert will influence acquisition of
data (cells may not migrate or stain in that area)
14
BD Falcon™ and BD BioCoat™ Multiwell Inserts
15
Multiwell Insert Handling
Repeating Pipettor recommended for
24-Multiwell Inserts (also suitable for individual inserts)
•
Multi-channel Pipettor required for 96-Multiwell Insert plate
•
The 96-square well receiver plate should be level
16
Applications
•
Cell imaging
•
Compound transport and permeability
17
Confocal Analysis of Caco-2/bbe (C2) Cells using BD Falcon Cell Culture Inserts
Cy2-tagged α-subunit of Na+-K+-ATPase
in C2 cells. The α-
subunit localizes to the apical pole in the presence of NH2
Cl.
--------------------
--------------------
Control
NH2 Cl
xy xz (2X)xzmerged
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.
18
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
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
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.
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 invasionTumor or endothelial cell migration using uncoated or self-coated insertsTumor or endothelial cell invasion using self-coated inserts
–
Cell differentiation and co-cultureVariety 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 be used with BD FluoroBlok Inserts► emission wavelength must be between 490-700 nm
•
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.
— your dye here —
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
•
Rehydrate ECM coating for 2h (if BD Matrigel matrix)
•
Aspirate media
•
Seed cells
24-well: 25,000 - 50,000 cells/well
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
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)
-
Validated protocols
27
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
28
BD FluoroBlok PET Membrane(3.0 μm pores)
Excitation(485 nm)
Attractant
Emission(530 nm)
Fibronectin (migration)
or
BD Matrigel matrix (invasion)
Analysis of Endothelial Cell Migration and Invasion Using BD FluoroBlok Membrane Inserts
29
Cell migration assessed using the BD BioCoat Angiogenesis System: Endothelial Cell Migration (Fibronectin-coated BD FluoroBlok membrane, 96-Multiwell format).
BD HUVEC-2 Cells Exhibit Concentration- Dependent Migration Towards VEGF
30
8-10 fold stimulation 2-3 fold stimulation
HAEC Cells HUVEC-2 Cells
0.000 1.000 3.125 6.250 12.500 25.0000
2
4
6
8
10
12
VEGF (ng/ml)
Fold
incr
ease
ove
r con
trol
mea
n+
se (n
=4)
0.000 1.000 3.100 6.200 12.500 25.0000.0
0.5
1.0
1.5
2.0
2.5
VEGF (ng/ml)Fo
ld in
crea
se o
ver c
ontr
olm
ean
+se
(n=4
)
Migration Activity Using Different Endothelial Cell Types
31
BD BioCoat Angiogenesis System: Endothelial Cell Invasion
Effect of MMP inhibitor 1'10' Phenanthroline on HMVEC Invasion
0200400600800
10001200140016001800
Contro
lVEGF(4n
g/ml)
0.1ug
/ml
1ug/m
l
10ug
/ml
20ug
/ml
VEGF(4ng/ml)+ 1'10' Phenathroline
Fluo
resc
ent U
nits
32
Applications
•
Tumor Cell Biology
33
HT-1080 cell digests BD Matrigel matrix and invades through pore.
NIH 3T3 and HT-1080 cells were incubated for 18-20 hours, stained,
and analyzed for invasion activity.
•
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)]
Analysis of Tumor Cell Invasion using the BD BioCoat Matrigel Invasion Chamber
34
BD BioCoat Tumor Invasion Systems
•
Combined Benefits of BD FluoroBlok inserts and BD Matrigel matrix
•
Reproducibility
•
Optimized protocols
•
Available in 24- and 96- Multiwell formats (8.0 μm pore size)
35
BD FluoroBlok PET Membrane(8.0 μm pores)
Excitation(485 nm)
Attractant
Emission(530 nm)
BD Matrigel matrix (invasion)
Analysis of Tumor Cell Invasion Using BD FluoroBlok Membrane Inserts
36
Fluorescently labeled cells residing on the bottom of the membrane post-invasion
MDA-MB-231 Human Breast Adenocarcinoma Cell Invasion Through BD Matrigel Matrix
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
•
•
http://www.bdbiosciences.com/external_files/dl/doc/tech_bulletin/live/web_enabled/tb436.p
df
39
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
40
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 96-
Multiwell 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
The Advantages of BD Falcon and BD BioCoat Cell Culture Inserts and Insert Systems
41
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.
42
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.
43
Upcoming Events
•
April 18–21, 2010•
American Association of Cancer Research
•
Visit Us at Booth # 524•
bdbiosciences.com/events
•
April 27, 2010•
BD FluoroBlok Insert Systems Tips and Techniques Webinar
•
bdbiosciences.com/webinars
44
Questions?
Technical Support:
In the U.S.tel: 877.232.8995e-mail:
[email protected] the U.S.e-mail: [email protected] 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. ©2010 BD