Download Topics for Discussion The Extracellular Matrix

Driving Cellular Communication with the ECM: Optimization of Cell Growth and Differentiation- Marshall Kosovsky
Driving Cellular Communication with
the Extracellular Matrix: Optimization
of Cell Growth and Differentiation
Marshall J. Kosovsky, Ph.D.
Technical Support Manager
BD Biosciences – Discovery Labware
Topics for Discussion
• Extracellular Matrix (ECM) Composition and Function
• Cell Types and Applications
Slide 1
Welcome. Today’s webinar will focus on the extracellular matrix
(ECM). The title is, Driving Cellular Communication with the
ECM: Optimization of Cell Growth and Differentiation. I’m
Marshall Kosovsky, the Technical Support Manager for BD
Biosciences - Discovery Labware. I’m happy that you could join
us today, and I want to emphasize at the outset that at the end of
the talk, I will be providing my contact information, and that of
Technical Support. You are welcome to contact us at any point.
We are very happy to discuss your questions and applications.
Slide 2
Today, I’m going to talk about the ECM, what it is, why it’s
important, and get into a number of cell types and applications to
illustrate how the ECM modulates cell functionality. We’ll talk
about epithelial cells, tumor cells, endothelial cells, and neuronal
cells.
• Epithelial
• Hepatocytes
• Mammary epithelial cells
• Tumor cells
• Endothelial
• Primary neurons
The Extracellular Matrix
• Complex mixture containing glycoproteins,
collagens, and proteoglycans
• Forms structural framework that stabilizes
tissues and provides mechanical support for
cell attachment
• Plays important role in cell proliferation,
migration, shape, orientation, and differentiation
(e.g., signal transduction, gene expression, enzymatic
activities)
Slide 3
Before I discuss the applications, I will talk about the material
itself, and give some background. The ECM is a complex
mixture, containing a number of different types of molecules –
glycoproteins, collagens, proteoglycans. It’s well known that
these molecules come together to form the structural framework
that stabilizes tissues and provides mechanical support for cell
attachment. Importantly, this material plays a very important
functional role in cell behavior, including cell proliferation,
migration, shape, orientation, and most importantly, cell
differentiation. The maintenance and induction of cell
differentiation is governed by signal transduction pathways and
the regulation of gene expression, which occurs in a tissuespecific manner and dictates how cells carry out their necessary
functions.
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Driving Cellular Communication with the ECM: Optimization of Cell Growth and Differentiation- Marshall Kosovsky
The Basal Lamina: A Thin ‘Mat’ that Underlies
Epithelial Cell Sheets and Tubes
basal lamina = basement membrane
BD Matrigel™ Matrix = reconstituted basement membrane
Figure: Molecular Biology of the Cell (3rd Edition)
Basal Lamina in Chick Embryo Cornea
epithelial cells
basal lamina
collagen fibrils
Slide 4
The ECM is present in two formats or types – the basal lamina,
or basement membrane, and the connective tissue. The basal
lamina underlies epithelial and endothelial cell sheets. And the
connective tissue is present in all tissue types, and contains a
variety of macromolecules and structures. We see in this image,
or schematic, the connective tissue contains cells such as
fibroblasts, which secrete ECM molecules such as collagen and
fibronectin. We also see a capillary, indicating that the
connective tissue is vascularized. The exception to this is
cartilage, which is avascular. When you look at these two
different types of ECM, they differ with respect to composition
and structure. At the bottom of the schematic, we see some
terminology. I will talk a number of times throughout this talk
about our most physiological product, BD Matrigel™ Matrix,
which is a reconstituted basement membrane. This material is
used with a number of key cell types and provides a very
effective model for a number of applications.
Slide 5
In this electron micrograph, we see a beautiful view of the basal
lamina, which is illustrated in the middle of the image. The basal
lamina appears as a thin mat, or rug-like material. This is the
basal lamina of the chick embryo cornea, and what we see here
are corneal epithelial cells resting on the basal lamina. And
underlying the basal lamina is the prominent display of collagen
fibrils of the connective tissue. This electron micrograph
beautifully illustrates the three-dimensionality of biological
materials and cells within tissues. And as we move through the
presentation, I will emphasize that point a number of times, and
get into a brief discussion of 3D vs. 2D cell culture, and talk
about how cells are dependent on structure as well as the
biochemical composition of the environment
Figure: Molecular Biology of the Cell (3rd Edition)
BD, BD logo, and all other trademarks are the property of Becton, Dickinson and Company. ©2008 BD
Driving Cellular Communication with the ECM: Optimization of Cell Growth and Differentiation- Marshall Kosovsky
ECM Provides a Physiological
Growth Substrate
• Although tissue culture plastic is used for many
cell types, this environment is not physiological
• ECM-based growth substrates provide a
physiological environment that supports and
promotes key cell functions
ECM Contributes to Intracellular
Signaling Pathways
• ECM molecules interact with cell surface
receptors (e.g., regulation of integrin signaling by
fibronectin:integrin interactions)
• The ECM appears to function in the storage and
presentation of growth factors
Slide 6
So why do we care about the ECM? The ECM is a physiological
growth substrate. As we know, people have been studying cells
for many years using standard tissue culture-treated plasticware
as a growth substrate. Obviously, plastic is not physiological.
When people typically culture cells on TC plastic, they do so in
the presence of media, which usually contains serum. And the
serum itself contains ECM molecules. So yes, the cells are
exposed to ECM, where you have a number of molecules such
as fibronectin and vitronectin within the serum, that can make
their way to the bottom of the plate or dish, and serve as binding
sites for cells. But this is different than an ECM-based substrate
that can provide a surface that is more physiological. In contrast
to the standard system of TC plastic and serum-containing
media, a tissue culture flask or dish that is coated with a uniform
layer of ECM provides cells with a surface containing a high
density of potential contact sites. When cells bind to such a
surface, they form functional interactions that can lead to
integrin-mediated signaling and cell functionality. This activity
may increase the sensitivity or raise the level of detection of the
biological activity under investigation. Now, whether that surface
is two-dimensional or three-dimensional will dramatically impact
how the cells function, and I will get into some examples of that
as we move ahead.
Slide 7
The ECM contributes to intercellular signaling pathways. The
cell surface molecules that principally interact with ECM are
integrin receptors. The most well-known interaction of this type
is the fibronectin/integrin interaction. When this interaction
occurs, there is a functional connection or communication
between these two molecules that leads to the activation of the
integrin, which promotes a signaling pathway leading to the
series of events that ultimately regulate gene expression and cell
functionality. The ECM also plays a very important role in the
storage and presentation of growth factors. In storing and
presenting growth factors, there are well-known cases where a
specific growth factor binds to an ECM molecule, and in doing
so, is sequestered or prevented from interacting with its cognate
receptor. Some activity within the tissue, some regulatory event
that will result in the release of that growth factor would then
allow it to interact with its receptor.
BD, BD logo, and all other trademarks are the property of Becton, Dickinson and Company. ©2008 BD
Driving Cellular Communication with the ECM: Optimization of Cell Growth and Differentiation- Marshall Kosovsky
ECM Composition
Glycoproteins
Fibronectin
Laminin
Vitronectin
Slide 8
The ECM contains a number of glycoproteins such as
fibronectin, laminin, vitronectin, thrombospondin, and tenascin.
There are a number of redundant functionalities, but these
molecules are expressed in a tissue-specific manner, and the
redundancies are therefore important. I’ll talk in more detail
about fibronectin and laminin, since these glycoproteins are
prominent in a number of our products such as vialed reagents
as well as coated plasticware and cell environments.
Thrombospondin
Tenascin
Glycoproteins
Fibronectin
• Large dimeric protein (multiple isoforms)
• Contributes to matrix organization
• Promotes cell adhesion via interaction
between FN ‘RGD motif’ and integrin receptors
Slide 9
Fibronectin is a large dimeric protein. There are multiple isoforms
that result from differential splicing of the RNAs. This ECM
contributes to the organization of the matrix, and is predominantly
found in the connective tissue. Fibronectin promotes cell
adhesion via interaction between cellular receptors (integrins) and
the fibronectin RDG motif, which is the well characterized arginineglycine-aspartate motif. And this interaction results in the
promotion of cell differentiation and functionality via integrinmediated signaling and the regulation of gene expression.
• Promotes cell differentiation and functionality
(e.g., cell migration, integrin signaling, gene expression)
BD, BD logo, and all other trademarks are the property of Becton, Dickinson and Company. ©2008 BD
Driving Cellular Communication with the ECM: Optimization of Cell Growth and Differentiation- Marshall Kosovsky
Glycoproteins
G
R
R
R
R
Fibronectin
Figure: Molecular Biology of the Cell (4th Edition)
Glycoproteins
Laminin
• Large heterotrimeric proteins (11 isoforms)
• Primarily found in basal lamina
• Major structural component of basal lamina
• Promotes cell adhesion via integrin and
non-integrin receptors
• Promotes cell differentiation and functionality
Slide 10
In this schematic, we see the basic structure of the fibronectin
dimer. The fibronectin dimer is comprised of two polypeptides
associated by disulfide bonding at the C terminus. The colored
dots represent multiple domains of the protein, which is a typical
property of many proteins, that there are a number of functional
domains. For example, the green colored dots represent collagen
binding domains. Therefore, ECM molecules interact with other
ECMs, and it is that interaction that will then contribute to the
organization of these molecules within the material or the
connective tissue. We also see yellow dots, labeled as selfassociation or fibronectin binding sites. Fibronectin polypeptides
self-associate to form a fibrillar matrix. And in addition, the red
dots represent cell-binding domains. And for example, this is
where you might have an RGD motif that will interact with the cell
via the integrin receptor. And the images on the right hand side of
the figure are 3D structures that have been derived from X-ray
crystallographic data, illustrating the structural domains within the
cell-binding motif of fibronectin. The RGD motif is sticking out from
the structure and is accessible to the cell receptor. The electron
micrographs on the left hand side simply show that it is an open
structure, and flexible, and therefore, has the potential to carry out
a number of functions at the same time – for example, interacting
with an ECM while interacting with a cell.
Slide 11
Laminin is another large ECM molecule. It is a heterotrimeric
protein. There are at least 11 or more isoforms that result from
the combination of different subunits. This molecule is
predominantly found in the basal lamina, where it is a major
structural component and promotes cell adhesion via integrin, as
well as non-integrin receptors. Again, there is a common theme,
where we have ECM interacting with a cell surface receptor that
can promote cell differentiation and functionality via integrin
signaling pathways, as well as other types of receptors. Laminin
plays a very important role in neuronal differentiation in the
developing of the brain, and plays a broad role in receptormediated signaling.
(e.g., neurite outgrowth, receptor signaling, gene expression)
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Driving Cellular Communication with the ECM: Optimization of Cell Growth and Differentiation- Marshall Kosovsky
Glycoproteins
Laminin
entactin binding
col IV binding
Heparin binding
cell binding
cell binding
Slide 12
In this schematic, we see the classic cruciform structure of
Laminin, where there are multiple functional regions such as ECM,
heparin, and cell binding domains. The different arms of the
molecule have the potential to simultaneously engage in multiple
activities. Laminin is quite large, each subunit greater than 1,500
amino acids. And you can see very nicely in the electron
micrographs on the right, the inherent flexibility of the structure,
the openness, the potential ability to interact with multiple target
molecules and cells.
col IV binding
Laminin
Figure: Molecular Biology of the Cell (4th Edition)
ECM Composition
Collagens
• Most ubiquitous ECM molecules (19 types)
• Fibrous proteins that provide structure and
resiliency to tissues
Slide 13
Collagens are the most ubiquitous ECM molecules. There are at
least 19 known types. There are over 30 genes that encode the
subunits that make up those different types. And these are fibrous
proteins that provide structure and resiliency to tissues. They are
the major component of skin and bone, and the most abundant
proteins in mammals, making up about 25% of the total protein
mass.
• Major component of skin and bone
• Most abundant protein in mammals (~ 25% of total
protein mass)
Examples of Collagen Types and
Tissue Distribution
Fibrillar
Type
Tissue Distribution
I, V
bone, skin, tendon,
cornea, internal organs
II
cartilage, notochord
IV
basal lamina
(polymerized fibrils)
Network-Forming
(sheetlike network)
Slide 14
We can see in this table that there are two main classes of
collagens – fibrillar and network-forming collagens. The fibrillar
collagens form polymerized fibrils, and I’ll show a beautiful picture
of that in a moment. A couple examples of these types of
collagens are the Type I and V, which are present in a number of
tissues – bone, skin, tendon; and Type II, which is predominantly
found in cartilage, notochord. The network-forming collagens – for
example, collagen IV, is prominent in the basal lamina. Now, this
collagen, in contrast to the fibrillar collagens, forms an entirely
different structure. It participates in a sheet-like network, or
structure within the basal lamina, and does not form polymerized
fibrils.
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Driving Cellular Communication with the ECM: Optimization of Cell Growth and Differentiation- Marshall Kosovsky
Collagen Fibrils in Connective Tissue of Skin
Slide 15
In this electron micrograph of connective tissue from skin, we see
the prominent collagen fibrils. We see two fibroblasts within this
tissue that would be actively engaged in secreting ECM into the
environment. The long strands that you see are collagen bundles.
Going back to basic biochemistry, the collagen monomer (triple
helix) forms head-to-tail polymers, which come together to form
bundles. And that’s what you’re seeing in these longitudinal
structures. These are bundles of collagen polymers, and
interestingly, the dots are collagen bundles that are perpendicular
to the longitudinal structures in this cross sectional view. I think
this is a fantastic image of the weave-like structure that collagen
forms in this context, a structure that contributes to the strength of
the tissue.
Figure: Molecular Biology of the Cell (3rd Edition)
BD Matrigel™ Matrix:
Reconstituted Basement Membrane
Composition
Laminin
~ 60%
Collagen IV
~ 30%
Entactin
~ 8%
Heparan sulfate proteoglycan (perlecan)
Growth factors (e.g., PDGF, EGF, TGF-β)
Matrix metalloproteinases
BD Matrigel™ Matrix High Concentration
Protein concentration: ~ 18-22 mg/ml
Applications
•
Human tumor xenograft in immunodeficient nude mice
- Subcutaneous injection of Matrigel mixture containing human tumor
cells (e.g., lung, prostate, breast, colon)
•
‘Matrigel Plug Assay’ for in vivo angiogenesis
- Subcutaneous injection of Matrigel mixture containing test substances
(e.g., antibodies, growth factors, synthetic peptides) and/or tumor cells
- Removal of plug and analysis of new vessel formation
Slide 16
I’ll talk briefly about Matrigel to give some background, because I
discuss this in a number of application areas. BD Matrigel™
Matrix is a reconstituted basement membrane isolated from EHS
mouse tumors. These tumors are highly vascularized, which is an
excellent source of basement membrane – that is, the basement
membrane associated with the vasculature. BD Matrigel™ Matrix
is approximately 60% laminin in composition, about 30% collagen
IV, 8% entactin – which is a bridging molecule that interacts with
the laminin and the collagen IV, contributing to the structural
organization of these ECM molecules – Heparan sulfate
proteoglycan (perlecan), and a number of growth factors – e.g.,
PDGF, EGF. There’s also some residual matrix
metalloproteinases from the tumor cells. This ‘regular’
composition has been successful in many applications for
promoting cell growth and differentiation. In a number of
application areas, the high growth factor content is not desired.
Therefore, we developed a growth factor reduced formulation of
Matrigel, which doesn’t eliminate the growth factors, but greatly
reduces a majority of the growth factors.
Slide 17
In addition to the regular Matrigel, which typically exhibits a protein
concentration in the range of 9 to 12 mg/ml, we offer a high
concentration version of the BD Matrigel™ Matrix, which exhibits a
protein concentration in the range of 18 to 22 mg/ml. This material
is especially useful for in vivo applications, such as the formation
of human tumors in immunodeficient mice, as well the Matrigel
plug assay for studying in vivo angiogenesis. Typically, when
these studies are done and Matrigel is prepared for injection into
an animal, the material is diluted and supplemented with
molecules of interest such as growth factors, peptides, and/or
cells. When using the high concentration material, it is easier to
perform the necessary dilutions and inject a firm plug into the
animal.
BD, BD logo, and all other trademarks are the property of Becton, Dickinson and Company. ©2008 BD
Driving Cellular Communication with the ECM: Optimization of Cell Growth and Differentiation- Marshall Kosovsky
High Concentration Laminin/Entactin
Complex
• Major component of basement membrane in
Engelbreth-Holm-Swarm (EHS) mouse tumors
• Protein concentration: ≥ 10 mg/ml
• Purity >90% by SDS-PAGE
• Used to form 3D gel that more closely models the
cellular microenvironment in vivo
• Supports cell differentiation (e.g., mouse submandibular
cells, endothelial cell tube formation)
• Supports maintenance of undifferentiated human
embryonic stem cells
Factors that Influence Cell
Differentiation and Functionality
•
Biological composition of the culture environment
(e.g., cell types, ECMs, growth factors)
•
Molecular interactions and cell adhesion (cell:cell, cell:ECM,
ECM:ECM, cell:growth factor, and ECM:growth factor)
•
Mechanical strength and structural properties
(degree of rigidity, 3D architecture)
•
Size scale (i.e., pore or fiber size relative to cell size;
microfibers vs. nanofibers)
Slide 18
We recently developed a high concentration laminin/entactin
complex, which is derived from EHS mouse tumors, and is a
partially purified form of BD Matrigel™ Matrix. The
laminin/entactin complex does not contain collagen IV. The
material exhibits a protein concentration of greater than or equal to
10 mg/ml, it is greater than 90% pure by SDS-PAGE, and is useful
for forming a 3D gel. The laminin/entactin complex can be used to
study cells in 3D, in contrast to our other vialed laminin products
that not suitable for creating 3D substrates. The laminin/entactin
complex supports cell differentiation using a number of key cell
types such as mouse submandibular cells, as well as endothelial
cells. It has also been shown to support the maintenance of
undifferentiated human embryonic stem cells.
Slide 19
It is important to understand the key factors that influence cell
differentiation and functionality. Clearly, the biological composition
of the environment is critical. What cell types are you using? Are
you using one cell type, are you using multiple cell types in a coculture system, and what molecules are the cells exposed to? Are
the cells interacting with the right ECMs? Are they interacting with
the right growth factors? It is critical for cells to exist in an optimal
environment that contains the most appropriate mix of ECMs and
growth factors, and further, provides optimal structure for
supporting the variety of interactions that contribute to cell
functionality (cell:cell, cell:ECM, ECM:ECM, ECM:growth factor,
cell:growth factor).
BD, BD logo, and all other trademarks are the property of Becton, Dickinson and Company. ©2008 BD
Driving Cellular Communication with the ECM: Optimization of Cell Growth and Differentiation- Marshall Kosovsky
2D vs. 3D Cell Culture
2D
3D
Growth Substrate
Rigid; inert
Mimics natural tissue
environment
Architecture
Not physiological; cells ‘Physiological’;
partially interact
promotes close
interactions between
cells, ECMs, growth
factors
Cell Encapsulation
No
Yes
Growth Factor
Diffusion
Rapid
Slow; chemical and
biological gradients
regulate signaling, cellcell communication
Products for 3D Cell Culture
•
•
•
•
Collagens (animal derived, human recombinant)
HC Laminin/Entactin Complex
BD Matrigel™ Matrix (standard and high concentration)
3D Scaffolds (Collagen, OPLA® Calcium Phosphate)
Epithelial Cells
• Line all cavities and free surfaces of the body
• Tightly bound into sheets or ‘epithelia’
• Epithelia are barriers to water, solutes, cells
• ECM that underlies epithelia: basal lamina
Slide 20
In comparing 2D versus 3D, a 2D growth substrate (plastic dish,
plate, flask) provides a rigid surface. It’s relatively inert. You can
coat ECM onto it, and add to the functionality, which in many
cases will help, and allow for an appropriate system for studying
some cell types. In a 3D environment, there is a more
physiological structure that is closer to that found in natural tissue.
The architecture of 2D is not physiological. The architecture of 3D
is physiological, and allows for close interactions between cells,
ECMs and growth factors in a dynamic way, as I was alluding to
earlier, that gives cells more information and approximates the
situation in vivo. For example, many labs using primary cells and
stem cells rely on the use of a 3D environment. Cell
encapsulation is another way to study cells in a 3D environment
by initially mixing cells into a biological material, to put them into a
3D environment. This cannot be done with a 2D substrate. A 3D
environment, such as a gel or a scaffold, supports the
establishment of chemical and biological gradients. These
gradients contribute to the regulatory activities that modulate cell
behavior
Slide 21
We have a number of products for 3D cell culture, including animal
and human recombinant collagens, laminin/entactin complex, BD
Matrigel Matrix, and a number of 3D scaffolds that exhibit distinct
compositions. The 3D scaffolds can be used for culturing cells in
vitro in a 3D environment, or can used for in vivo studies. These
scaffolds are 3mm x 5mm disks, and fit in the well of a 96 well
plate. So they’re quite small, and appropriate for small animal
model studies.
Slide 22
So now, I will discuss of a number of key applications and cell
types. I’ll start with epithelial cells. These cells line all of the
cavities and free surfaces of the body, and are tightly bound into
sheets or epithelia. The epithelia are barriers to water, solutes
and cells, and the ECM that underlies the epithelia is the basal
lamina.
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Driving Cellular Communication with the ECM: Optimization of Cell Growth and Differentiation- Marshall Kosovsky
Slide 23
Epithelial cells are found in many tissues – liver hepatocytes and
bile duct epithelial cells; skin keratinocytes; and glandular cells
Epithelial Cells
• Liver (hepatocytes, bile duct epithelial cells)
• Skin (e.g., keratinocytes)
• Gut, Lung
• Exocrine glands (e.g., mammary, sweat)
• Hormone secreting glands (e.g., pituitary)
Hepatocytes
Applications
• Drug metabolism studies, toxicity assays
• Liver regeneration, tissue engineering
• Liver-specific gene expression and signaling
• Biology of hepatitis viruses
Primary Hepatocytes Exhibit Differentiated
Morphology on 3D Growth Substrates
Col I (2D thin coat)
Col I (3D gel)
BD Matrigel™ Matrix (3D)
Slide 24
Hepatocytes serve as an excellent example of a cell type that
behaves differentially in response to ECM composition and
structure. I will focus on primary hepatocytes. These cells are
used in a number of key applications such as drug metabolism
studies, toxicity assays, liver regeneration/tissue engineering,
studies of hepatocyte progenitor cells and stem cell biology, liverspecific gene expression, and the biology of hepatitis viruses.
Slide 25
In this slide, primary hepatocytes were cultured on a number of
ECM-based substrates. If cultured on plastic (no ECM coating),
the cells would not survive very long. Looking from left to right,
the cells cultured on 2D collagen I, a 3D collagen gel, and 3D BD
Matrigel Matrix. When cultured on 2D collagen I, we see is a
flattened morphology. This is indicative of dedifferentiation. So the
cells are not happy on the 2D coating. In contrast, cells on a 3D
collagen I gel start to round up and come together into clusters,
with some flattened cells. The cells are beginning to adopt, or are
more readily coming together into a differentiated morphology. On
the 3D BD Matrigel Matrix, we see tight clusters of rounded cells
that exhibit a differentiated morphology. As you look across the
figure from left to right, it is clear that the ECM composition and
structure has a dramatic impact on the cell morphology. In
addition to morphology, we examined cell functionality by looking
at a number of different key activities within hepatocytes – the liver
enriched transcription factor C/EBP and cytochrome P450
enzymes. While the activities were low on the 2D collagen, higher
levels of activity were seen with the 3D substrates. The 3D BD
Matrigel Matrix supported optimal levels of C/EBP and cytochrome
P450 activities. So hepatocytes represent a very good example of
cells that are dramatically impacted by the structure of the
environment and its composition.
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Driving Cellular Communication with the ECM: Optimization of Cell Growth and Differentiation- Marshall Kosovsky
Mammary Epithelial Cells
Applications
•
Analysis of normal cell growth and differentiation
MCF-10A cells form acinar-like
morphology on BD Matrigel™ Matrix
The tumor cell invasion assay is widely used to study mammary
derived tumor cells.
Data provided by Dr. Joan Brugge,
Harvard Medical School
•
Slide 26
Mammary epithelial cells are another cell type that responds
dramatically to the structure and composition of the environment.
These cells are used to study normal cell growth and
differentiation. This data was provided by Dr. Joan Brugge from
Harvard Medical School. MCF-10A cells were cultured in a 3D
Matrigel overlay environment. Under these conditions, published
in the journal Methods – and I will show the reference at end of the
presentation – the cells adopt differentiated morphology, which
was not observed using a 2D culture system. We see the acinar
structure that is adopted by the cells.
Analysis of tumor cell invasion
Analysis of Tumor Cell Invasion Using
Fluorescence Blocking PET Membrane Cell
Culture Inserts
BD Matrigel™
Matrix
Attractant
Excitation
(485 nm)
BD FluoroBlok™
PET Membrane
(8 μm pores)
Emission
(530 nm)
Slide 27
To study tumor cells in a tumor invasion assay, the system shown
here can be used. This schematic shows a cell culture insertbased system for studying cell invasion or migration. In the context
of tumor cell biology, the insert is coated with BD Matrigel™ Matrix
(pores occluded for invasion assay). BD Matrigel serves as a
model for the basement membrane associated with the tumor
vasculature. The schematic shows a cross-section of an insert,
where the bottom of the insert contains a porous membrane. The
membrane separates the system into two compartments – a
compartment above the membrane, and one below. Tumor cells
that exhibit invasive activity will be able to ‘invade’ through the BD
Matrigel Matrix by enzymatically degrading through the material,
and then getting through the pores. Since the Matrigel occludes
the pores, a cell must secrete matrix metalloproteinases that
effectively degrade through the matrix to provide access to the
pore. After invading through the pore, the cells reside on the
bottom of the membrane. Cells that invaded can then quantitated
using a fluorescence plate reader. In the system shown, the
membrane is colored blue to indicate a specialized form of
membrane called BD FluoroBlok PET Membrane. The BD
FluoroBlok membrane blocks fluorescent light between 490 and
700 nanometers. For a tumor invasion or cell migration assay, one
can use a fluorescent plate reader to detect the cells on the bottom
after labeling with a fluorescent dye. Because that membrane is
blocking fluorescence, any cells that are on top of it are blocked
from detection. Therefore, it’s a rapid, effective way of conducting
a quantitative invasion or migration assay.
BD, BD logo, and all other trademarks are the property of Becton, Dickinson and Company. ©2008 BD
Driving Cellular Communication with the ECM: Optimization of Cell Growth and Differentiation- Marshall Kosovsky
MDA-MB-231 Human Breast Adenocarcinoma
Cell Invasion Through BD Matrigel™ Matrix
Slide 28
Here, we’re looking at the bottom of a membrane following an
invasion assay using MDA-MB-231 breast adenocarcinoma cells.
The BD FluoroBlok™ membrane can be used to simplify and
expedite the quantitation of this result.
Fluorescently labeled cells residing on the
bottom of the membrane post-invasion
Inhibition of MDA-MB-231 Cell Invasion
Through BD Matrigel™ Matrix by Doxycycline
Endothelial Cells
• Line closed internal body cavities
(e.g., bile ducts, blood vessels)
• Angiogenesis (e.g., neovascularization
during wound healing and tumorigenesis)
• Promote blood cell adhesion during
inflammatory response
Slide 29
This is an example of quantitative data generated using the BD
FluoroBlok™ platform for a tumor cell invasion assay. The BD
BioCoat Tumor Invasion System was used to examine the invasion
activity of MDA-MB-231 cells in the presence or absence of the
inhibitor doxycycline. In the presence of increasing concentrations
of the inhibitor, there is a decreased level of invasion with an IC50
of approximately 80 micromolar. So for the purpose of basic
studies of tumor cell invasion or migration, insert systems such as
this can be used to quantitate these activities, and are useful in the
context of drug development.
Slide 30
Endothelial cells line the closed internal body cavities – the bile
ducts, blood vessels, and are the principal cells involved in the
process of angiogenesis, which is the formation of new blood
vessels during wound healing and tumorigenesis. Angiogenesis is
critical, and required for tumor growth and survival. Therefore, the
angiogenesis pathway is a key target for anti-cancer drug
discovery. Endothelial cells also play an important role in the
promotion of blood cell adhesion during the inflammatory response.
BD, BD logo, and all other trademarks are the property of Becton, Dickinson and Company. ©2008 BD
Driving Cellular Communication with the ECM: Optimization of Cell Growth and Differentiation- Marshall Kosovsky
Angiogenesis
Endothelial Cell Assay Systems
• Migration (human fibronectin)
•
BD™ HUVEC-2 Cells, pre-qualified for
responsiveness to VEGF
• Invasion (BD Matrigel™ Matrix)
• Tube Formation (BD Matrigel Matrix)
Human Microvascular Endothelial Cells Form
Tubules on BD Matrigel™ Matrix
Collagen I
BD Matrigel Matrix
2D
3D
Analysis of Endothelial Cell Tubulogenesis
Using the BD Pathway™ Bioimager
Confocal collapsed stack
Non-confocal single plane
BD™ HUVEC-2 cells stained with calcein AM; 4X
confocal images show the entire tubule network
Slide 31
We’ve developed a number of assay systems for studying
endothelial cells for basic research and drug discovery. Migration
and invasion assays that are based on the BD FluoroBlok™ insert
platform. The migration assay is comprised of a BD FluoroBlok 24or 96-well insert coated with human fibronectin. The fibronectin
does not occlude the pores, so endothelial cell migration occurs
independent of enzymatic degradation. We also offer pre-qualified
BD HUVEC-2 cells. These cells are pre-qualified for
responsiveness to VEGF in the migration assay, and are also
suitable for use with the invasion and tube formation assays. The
invasion assay system is built on the BD FluoroBlok system (24well only), and the membrane is coated with BD Matrigel™ Matrix
(pores occluded). The tube formation system is not an insertbased system. It is built on a 96-well black clear plate coated with
BD Matrigel Matrix that is optimized for the formation of the tubular
network
Slide 32
In this slide, we’re looking at microvascular endothelial cells
cultured on 2D collagen I (on left), essentially as a housekeeping
substrate. It keeps the cells very happy, but does not promote
differentiation. When cultured on 3D BD Matrigel Matrix, the
endothelial cells differentiate and for the tubule networks.
Slide 33
The BD BioCoat™ Angiogenesis System: Endothelial Tube
Formation has been used successfully in conjunction with the BD
Pathway Bioimager to image and then quantitate tube formation
activity using a software algorithm developed for this application.
The picture on the left is derived from a collapsed stack of confocal
images. Utilizing this approach, a series of confocal images are
brought together into a ‘collapsed stack’ to effectively reflect the
occurrence of the tubule network throughout the 3D sample. So
you’re looking at tubes that have formed throughout the 3D BD
Matrigel Matrix substrate. The image on the right shows a nonconfocal image of a single plane, which offers very poor resolution
and sub-optimal data. For this experiment, BD HUVEC-2 cells
were used with this system to examine endothelial cell tube
formation.
BD, BD logo, and all other trademarks are the property of Becton, Dickinson and Company. ©2008 BD
Driving Cellular Communication with the ECM: Optimization of Cell Growth and Differentiation- Marshall Kosovsky
Different Analysis Parameters –
Similar Results
350
7000
Tube Complexity
(total # of segments)
Total Tube Length
(pixels)
8000
6000
5000
4000
3000
IC50 = 2.93E-05
2000
1000
0
1.0×10 -7
1.0×10 -6
1.0×10 -5
1.0×10 -4
300
250
200
150
100
IC50 = 2.97E-05
50
0
1.0×10 -7
1.0×10 -3
1.0×10 -6
1.0×10 -5
1.0×10 -4
Log [M] Suramin
Log [M] Suramin
1.0×10 -3
Slide 34
When this data was quantitated using the associated software
algorithm, a number of experimental parameters were considered
(tube length, total tube area, and tube complexity). Tube formation
was examined in the absence or presence of the angiogenesis
inhibitor Suramin. Regardless of the parameter that was chosen,
the IC50 values are comparable. This data illustrates how the tube
formation system can be used in the context of drug screening and
basic research focused on the characterization of endothelial cell
differentiation and functionality.
70000
Total Tube Area
(pixels)
60000
50000
40000
30000
20000
IC50 = 2.94E-05
10000
0
1.0×10 -7
1.0×10 -6
1.0×10 -5
1.0×10 -4
1.0×10 -3
Log [M] Suramin
Cells of the Nervous System
• Primary Neurons
• Central nervous system/brain
• Peripheral neurons (e.g., sensory, motor)
• Primary Glial Cells
• Astrocytes
• Oligodendrocytes
• Schwann cells
Neurons
dendrite cell body
axon
• Axons conduct and transmit signals
• Dendrites receive signals
• Neuronal communication mediated by
neurotransmitters (e.g., glutamate, dopamine,
Slide 35
I will now focus on neuronal cells. If we consider cells of the
nervous system, there are multiple types of primary neurons and
primary glial cells (astrocytes, oligodendrocytes and schwann
cells). As with other primary cell types, the study of neuronal cell
biology requires careful consideration of the structure and
composition of the growth environment.
Slide 36
This is a textbook photo of a neuron. We understand that the
axons conduct and transmit neurotransmitter-induced signals, and
the dendrites receive such signals. Neuronal communication is
mediated by neurotransmitters such as glutamate, dopamine,
GABA. Since neuronal cells are highly specialized, it is critical to
culture these cells under conditions that effectively supports the
molecular interactions and signaling events that are required for
optimal functionality. This functionality includes activities such as
cell migration, receptor activation, participation in critical signaling
pathways, and interactions with important components of the
environment (cells, ECMs, growth factors).
serotonin, GABA)
BD, BD logo, and all other trademarks are the property of Becton, Dickinson and Company. ©2008 BD
Driving Cellular Communication with the ECM: Optimization of Cell Growth and Differentiation- Marshall Kosovsky
Rat Cortical Neurons Exhibit Axonal Outgrowth
and Secondary Branching on BD BioCoat™
Laminin/Fibronectin
Slide 37
In this experiment, primary rat cortical neurons derived from
neonatal rats were cultured on a laminin/fibronectin substrate.
While this is a 2D substrate, a great deal of research has
demonstrated that such an environment is suitable for neuronal cell
attachment and differentiation. In this particular case, the cells
exhibit axonal outgrowth and secondary branching. We also see
light blue cells in this image – those are glial cells. So this is a coculture, where the glial cells and the ECM coating are supporting
the neuronal cells. So this combination matrix was found to be
quite useful, and optimal for promoting the differentiated
morphology. Laminin and fibronectin are abundant in the
developing brain. Therefore, it is not surprising that the rat
neonatal neurons are happy on this substrate. Comparable results
were obtained when these cells were cultured on laminin alone,
fibronectin alone, or even poly-lysine.
Rat Cortical Neurons Exhibit Glutamate Receptor
Activity on BD BioCoat™ Laminin/Fibronectin
Slide 38
This experiment is an extension of the work from the previous
slide. In addition to differentiated morphology, this experiment
examined the functionality of these cells under comparable
conditions. Primary rat cortical neurons were examined with
respect to the activity of the glutamate receptor. Cells were
cultured on the laminin/fibronectin substrate in the presence of a
saline control or the presence of 100 micromolar glutamate. The
cells were loaded with a calcium binding dye, and in the presence
of glutamate, the glutamate receptor is activated. This activity
results in the release of calcium and an associated elevation of
fluorescence. In contract to the saline control, there is a marked
increase in calcium release and fluorescence, indicating that these
cells are exhibiting functional activity of that receptor. Studies were
also done with other neuronal receptors such as GABA and acetylCoA receptors – using a variety of substrates such as laminin
alone, fibronectin alone, or poly-lysine. The other receptors also
functioned comparably on the different substrates. So, the
laminin/fibronectin complex provides an environment that supports
the functionality and morphology of these cells. In addition, there
are alternative substrates that we offer as pre-coated plasticware,
that have been widely used for a variety of neuronal cell types to
promote differentiation and functionality.
Saline Control
100 μM Glutamate
BD, BD logo, and all other trademarks are the property of Becton, Dickinson and Company. ©2008 BD
Driving Cellular Communication with the ECM: Optimization of Cell Growth and Differentiation- Marshall Kosovsky
Analysis of PC-12 Cell Neurite Outgrowth
Using the BD Pathway™ Bioimager
Control
200 ng/ml NGF
β-tubulin staining, 20x objective
30
25
20
15
10
5
0
Neurite Total Length
Response Level
Total Neurite Length per Cell
(Well Average)
35
160
120
A001
80
40
0
0.01
0.1
1.0
NGF Dose (uM, on log scale)
B001
Immunofluorescence Analysis of EcoPack Cells
(derived from HEK-293) Stained with Integrin αv
Uncoated
Slide 39
In this study, neuronal cell differentiation was examined in the
presence or absence of nerve growth factor using the BD Pathway
Bioimager. In the presence of 200 ng/ml, the cells exhibit marked
neurite outgrowth. This system utilizes a software algorithm that
was developed for the purpose of quantitating neurite outgrowth
activity. The bottom part of the slide shows representative
quantitative data based on total neurite length. On the bottom
right, PC12 neurite outgrowth occurs in response to increasing
concentrations of NGF.
Collagen I
PDL
Slide 40
In this final data slide, expression of integrin alpha v was examined
using EcoPack cells, a transfected cell line derived from HEK293
cells. The immunofluorescence data shows that these substrates
differentially impact the expression of this surface receptor. While
the cells attach and grow effectively on the uncoated substrate,
the integrin αν activity is very low. When cultured on collagen or
poly-lysine, the expression is markedly increased. Therefore, this
data suggests that the level of integrin activity in these cells is
dependent on the growth substrate. If you were using these cells
to study a biological process involving integrin-mediate signaling, it
is likely that the level of detection or functional activity would be
optimal on the coated substrates. When considering your own cell
type and experimental requirements (what assay is in use, what
activity is measured), it will be beneficial to consider the complete
environment and how the overall composition and structure will
impact your study. What substrate is best for your cells? Should
you use a 2D or 3D environment? Is a single cell type sufficient, or
would co-culture be more appropriate? Are the right growth factors
present?
If you’re embarking on a new field of research or would like to
investigate new opportunities for expanding your studies, we would
be very happy to talk to your application and do our best to help
you further optimize your research.
Summary
ECM provides a physiological growth substrate that
supports key cellular functions
•
•
•
Structural organization of cells and tissue
Cell attachment and proliferation
Slide 41
In summary, I hope I’ve convinced you that the ECM is a critical
component of the cell environment. It provides a physiological
growth substrate that supports key functionality – not only the
structural organization of cells and tissue, cell attachment and
proliferation, but in particular, the induction and maintenance of cell
differentiation via signal transduction pathways and the regulation
of gene expression.
Induction and maintenance of cell differentiation
- signal transduction pathways
- gene expression
BD, BD logo, and all other trademarks are the property of Becton, Dickinson and Company. ©2008 BD
Driving Cellular Communication with the ECM: Optimization of Cell Growth and Differentiation- Marshall Kosovsky
References
Epithelial
Slide 42
In the next two slides, I have provided a set of recent references
that support the information presented today.
Epithelial cells, tumor cell biology,
Kass L, et al. (2007) Mammary epithelial cell: Influence of extracellular matrix composition and
organization during development and tumorigenesis. Intl J Biochem & Cell Biol 39: 1987-1994.
Park CC, et al. (2006) β1 integrin inhibitory antibody induces apoptosis of breast cancer cells, inhibits
growth, and distinguishes malignant from normal phenotype in 3D cultures and in vivo. Cancer Res
66: 1526-1535.
Debnath J, et al. (2003) Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini
grown in three-dimensional basement membrane cultures. Methods 30:256-268.
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.
References
.
Slide 43
Endothelial cells, and neuronal cells
Endothelial
Potapova IA, et al. (2007) Mesenchymal stem cells support migration, extracellular 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 GE and Senger, DR (2005) Endothelial extracellular matrix: biosynthesis, remodeling, and
functions during vascular morphogenesis and neovessel stabilization. Circulation Res 97:1093-1107.
Neuronal
Hur E-M, Kim K-T (2007) A role of local signalling in the establishment and maintenance of the
asymmetrical architecture of a neuron. J Neurochem 101: 600-610.
Dityatev A and Schachner M (2003) Extracellular matrix molecules and synaptic plasticity. Nat Rev
Neurosci 4:456-468.
Arakawa Y, et al. (2003) Control of axon elongation via an SDF-1a/Rho/mDia pathway in cultured
cerebellar granule neurons. J Cell Biol 161:381-391.
Contact Information
Marshall J. Kosovsky, Ph.D.
BD Biosciences - Discovery Labware
296 Concord Road, Suite 280
Billerica, MA 01821
Tel: 978-901-7433
Email: [email protected]
Slide 44
I give you now my contact information, and that of Technical
Support. I encourage you to call if you have any questions, or
would like to discuss your applications. We look forward to the
opportunity to discuss and learn about your research. Thank you
so much for your attention today. Best of luck with your work, and
take care
Technical Support
Tel: 877-232-8995 (US)
978-901-7300 (outside US)
Email: [email protected]
bdbiosciences.com/webinars
BD, BD logo, and all other trademarks are the property of Becton, Dickinson and Company. ©2008 BD