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Fourth Cycle Funded Project, 2005 Project Title: Project Manager: Prof. of Molecular Pathology Director of Molecular Biology Research Unit Assiut University, Assiut, Egypt Some Applications of Cell Culture Techniques in Biology Univ. Prof. Mokhtar Taha Prof. of Molecular Pathology Director of the Molecular Biology Research Unit Assiut University, Egypt Research Studies In Vitro In Vivo Cell & Tissue Culture Exp. Animals Samples Cell line Primary culture Exam. Scope of the lecture 1- Stem cell research 2- Tissue engineering applications 3- Regenerative medicine 4- Oncology 5- Hybridoma technology 6- Cloning 7- Viral diagnosis 8- Toxicity studies Stem Cell Research Stem cells are undifferentiated cells with the capacity for unlimited or prolonged self-renewal and the ability to give rise to differentiated cells. Differentiation can be defined as qualitative changes in the cellular phenotype that are a consequence of the onset of synthesis of new gene products. Embryonic Stem Cells High malleability Potential for undesired development (teratomas) Infinite lifespan unlimited supply High ethical burden Uncertain legal status Adult Stem Cells Limited developmental potential Better behaved, easier to manage Lose their ability to proliferate/differentiate after a time in culture Less moral ambiguity Less legal controversy Procedure to develop ES cell lines Methods for Isolating Stem Cells from Adult Tissue 1. Flow cytometry 2. 3. 4. Hematopoietic stem cells--CD34+, HLA-DRNeural crest stem cells--p75+ Epidermal stem cells--β1 integrin Microdissection/colony-forming assay Retinal stem cells Hair follicle stem cells Cell adhesion or lack of adhesion --Human epidermal stem cells adhered most rapidly to Type IV collagen, fibronectin or keratinocytes ECM. --Late attachment of human breast epithelial stem cells on plastic. Contact insensitive growth on cell mat Putative human kidney epithelial stem cells. Location of Adult Stem Cells Example 1 Mouse Endbuds and Budding Structures Formed by Two Types of HBEC Example 2 In Vitro Clinical Applications of Stem Cells 1.Hematopoiesis as a model of stem and progenitor cell transplantation . ■ Fanconi Anemia —an autosomal recessive disorder characterized by progressive bone marrow failure leading to aplastic anemia. ■ Bone marrow ablative doses of chemotherapy and radiotherapy to eliminate endogenous cancer〔 leukemia (CML), lymphoma (Hodgkin’s), multiple myeloma, breast cancer〕. 2. Transplantation therapy for acute and chronic degenerative disease * Epidermal--skin graft for patients with severe burns * Neuronal--Parkinson’s disease, multiple sclerosis and other neurodegenerative disease (brain gangliosidosis, demyelinating disease). * Liver--For Liver damage by drugs, toxins or viral infection. * Islet-for insulin non --producing pancreas * Skeletal muscle satellite--muscular dystrophies or muscle loss; heart disease * Mesenchymal—tissue engineering in plastic reconstructive surgery 3. Gene therapy Tissue Engineering The design, specification and fabrication of cells,biomaterials, or biomolecules to restore or modify the biological function of tissues Tissue Engineering Applications NEEDS: * Scaffold to support tissue growth * In vitro models to study such as Matrigel, Vitrogen General Approaches to Tissue Engineering Regenerative Medicine Definition: It is a technique to replace or repair defective or disease tissue or organ by in vitro design while in vivo usage Ideal Tissue or Organ Design Stem Cells Biofactor Engineering Cell Selection Scaffolds Design & Fabrication Stem Cells Differentiation In vitro cell culture on Scaffolds Transplantation Scaffolds degradation New permanent tissue or organ formation Applications in Oncology *Although the activity of carcinogens can be assayed in intact animals, such experiments are difficult to quantitate and control. *The development of in vitro assays to detect the conversion of normal cells to tumor in culture, a process called Cell Transformation, therefore represented a major advance in cancer research. * Cell lines differ in important ways from their normal progenitors in the tissue from which they were derived. Focus assay: is based on the ability to recognize a group of transformed cells as a morphologically distinct “focus” against background of normal cells on the surface of a culture dish. Primary and immortalized cell lines could be used as a model to compare efficacy and toxicity of a given treatment and its mechanism of action. Properties of Cancer Cells in Cultures Not sensitive to densitydependent inhibition. Reduced growth factor requirements loss of contact inhibition less stringent regulation of cell- cell and cell- matrix interactions As tumors develop they enter in contact with cells from nonmalignant tissue T4-2 phenotype (tumor) 6 8 10 S1 phenotype (normal) days Contact co-culture: T4-2 cells grow toward S1 glandular structures and surround these structures. T4-2cells were added to pre-formed S1 acini and cultured for 10 days. T4-2 cells were stained with DiI (red) prior to being plated, and S1 cells are stably transfected with green fluorescent protein (GFP). Images show co-cultures of S1 and T4-2 cells at days 6, 8, and 10. At day 6: Tumor cells (red) proliferate when in contact with S1 structures (green, arrow). Day 8: T4-2 cells expand over several S1 structures (5 glandular structures can be seen in green). Day 10: A single tumor (red) is in development; at least four S1 glandular structures (green) have been engulfed. Monoclonal Antibody Production Uses for monoclonal antibodies Monoclonal antibodies are widely used as diagnostic and research reagents. Their introduction into human therapy has been much slower. In some in vivo applications, the antibody itself is sufficient. Once bound to its target, it triggers the normal effector mechanisms of the body. In other cases, the monoclonal antibody is coupled to another molecule, for example a fluorescent molecule to aid in imaging the target a strongly-radioactive atom, such as Iodine-131 to aid in killing the target. Some monoclonal antibodies that have been introduced into human medicine To suppress the immune system Muromonab-CD3 (OKT3) Used to prevent acute rejection of organ, e.g., kidney, transplants. Infliximab (Remicade®). Binds to tumor necrosis factor-alpha (TNF-α). Shows promise against some inflammatory diseases such as rheumatoid arthritis. Cloning and Tissue Culture Benefits: - Recombinant DNA technology - Gene therapy •Knockout technology inactivating certain genes and tracing their effects •Providing DNA for the Human Genome Project and other species’ genome projects Why do Plant Tissue Culture? • Allows fast commercial propagation of new cultivars • Rare and endangered plants can be cloned safely • Give a continuous supply of young plants • Provide virus free plants •Plant ‘tissue banks’ can be frozen, then regenerated through tissue culture • Easier to export Virus Cultivation, Assay and Diagnosis Hosts for Virus Cultivation 1- Laboratory animals: play an essential role in studies of viral pathogenesis. 2- Embryonated chicken eggs: Several viruses cause discrete and characteristic foci providing a method for identification, quantification or assessing pathogenicity. 3- Cell culture: most widely used and most powerful hosts for cultivation and assay of viruses. Lab Methods for Confirmation of Orthopoxvirus Diagnosis . PCR related methods for DNA identification, (e.g., real-time PCR) . Electron microscopy . Histopathology . Culture . Serology − Antigen detection (IFA, EIA ag capture) − IgM capture − Neutralization antibodies − IgG ELISA Negative Stain Electron Microscopy vaccinia Orthopoxvirus Cell Culture Isolation HEK 293 HeLa BSC-40 Uninfected, 24 hpi Variola infected, 24hpi Cell culture isolation can be an important and very sensitive method for detection of vaccinia since it also amplifies the virus for further characterization. Growth of Viruses in Embryonated Chicken Eggs Foci formation on the chorioalantoic membrane by vaccinia virus Small Pox Vaccine Infected Chorioalantoic Membrane Development of a Cytopathic Effect (time course): Mixture of Small Cells and Syncytia Focus Formation Cells pile up rather than grow as a monolayer Hemadsorption Adsorption of erythrocytes to the surface of infected cells Foamy Virus CPE ECHO Virus: Round Cells Syncytium Formation Syncytium Formation Foamy Virus Vacuoles Toxicity Studies Liver cell culture can be used in co-culture with brain cell aggregates, providing a potential model for complementary toxicological and metabolic studies. Tissue culture has been used for analysis of feed stuffs and fungal extracts for the presence of mycotoxins . Toxicity Studies In vitro methods provide a necessary and useful adjunct to the in vivo studies in testing potential biomaterials by using cell lines. Primary and immortalized cell lines could be used as a model in order to compare efficacy and toxicity of a given experimental treatment and to exploit the differences in the mechanism of action of a drug.