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

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•

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•

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•

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44

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