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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. 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 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) 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 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. 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 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. 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 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. 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 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