2007 seminar 3

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Information about 2007 seminar 3

Published on October 12, 2007

Author: Charlie

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Slide1:  COMMONWEALTH OF AUSTRALIA Copyright Regulations 1969 WARNING This material has been reproduced and communicated to you by or on behalf of the University of Sydney pursuant to Part VB of the Copyright Act 1968 (the Act). The material in this communication may be subject to copyright under the Act. Any further reproduction or communication of this material by you may be the subject of copyright protection under the Act. Do not remove this notice 15 Greatest Milestones in Health and Medicine:  15 Greatest Milestones in Health and Medicine Presented by the University of Sydney in association with the BMJ and with the support of the Medical Foundation Tissue Culture :  Tissue Culture Anthony L Cunningham Westmead Millennium Institute & The University of Sydney Some Key Milestones in the History of Tissue Culture:  Some Key Milestones in the History of Tissue Culture 1870s Sydney Ringer develops salt solutions for maintaining isolated animal hearts outside the body in culture 1885 William Roux: embryonic chicken maintained in warm saline for several days 1907* Ross Granville Harrison cultures frog neural tissue in vitro 1912 Alexis Carrel cultures chick heart cells in vitro; develops sterile techniques 1923 Thomas Strangeways and Honor Fell establish cell culture lab at the University of Cambridge to study cell biology in bone and joint disease and also radio-biology and cancer cells Slide5:  Ross Granville Harrison Examined nerve cells in hanging drop Slide6:  To grow his tissue explants, Harrison adapted the hanging drop technique that microbiologists used to study live bacteria. Slide7:  Alexis Carrel 1873-1944 French surgeon working at Rockefeller Institute. Won Nobel prize for work on surgical anastamoses Surgical skill + sterile technique  tissue culture History of Tissue/cell culture:  History of Tissue/cell culture 1949 Enders, Weller and Robbins awarded the Nobel Prize (1953) for developing cell culture for the culture of (poliomyelitis) viruses in vitro; then other viruses including varicella zoster, rubella and cytomegaloviruses. 1951 First ‘immortal’ cell lines from cervical cancer which killed Henrietta lacks (HeLa) with ‘primary’ cell cultures which have limited number of cell divisions 1955 Gey established single cell culture in suspension. Led to high volume spinner flasks and bioreactors (eg for vaccine production) 1960s Chromosomes obtained from dividing cells  cytogenetics Late 1970s T-cell growth factor (interleukin 2) discovered enabling growth of T-lymphocytes – R Gallo 1980s Donald Metcalf discovers colony stimulating factors in Melbourne which enables culture of normal and leukaemic marrow cells. Slide10:  John Franklin Enders Slide11:  McLimans' group developed the first "spinner flasks" in 1957. Slide12:  Glass spinner flasks (1980s): operate at slower speeds with less damage to sensitive cells Slide13:  First electron micrograph of a cell Importance of Tissue Culture:  Importance of Tissue Culture Enabled one third of Nobel prizes since 1953 e.g. cancer viruses (1975) monoclonal antibodies (1984) growth factors (1986) oncogenes (cancer causing genes) RNA interference (2006) Importance of tissue culture:  Importance of tissue culture Enabled the: discovery of almost all animal/human viruses (except viruses discovered recently by molecular techniques). discovery and study of bacteria associated with cells such as Chlamydia Growth and attenuation of viruses in culture as vaccines e.g. rubella, polio, chickenpox/shingles, measles, mumps rabies, yellow fever etc. studies of the cellular basis of cancer, immunology, neuroscience, embryology and in vitro fertilization. Sciences of Virology and Immunology:  Sciences of Virology and Immunology Dependent on cell culture or small animal (mouse) models Viruses completely dependent on cells for survival and growth Use molecular machinery in nucleus (usually DNA viruses) or cytoplasm (usually RNA viruses) Growth of virus requires cell culture Therefore for decades diagnosis depended solely on cell culture Development of antivirals and vaccines requires growth in cell culture Interaction between cells and viruses now the major research frontier Slide21:  T Lymphocyte infected by HIV NEJM 349:2283-2285 Slide22:  Surface of a T Lymphocyte infected by HIV NEJM 349:2283-2285 Slide24:  Results: UL37-VP5-gG Slide27:  Immunology Antibodies versus cellular immunology All require tissue culture for production and study B lymphocytes produce antibodies T lymphocytes produce cytokines and may kill virus infected or cancer cells Therapy: Antibodies Cytokines Killer T-cells – e.g. EBV-lymphoma Importance of Tissue Culture:  Importance of Tissue Culture Genomics revolution. Cell culture provided enough DNA for sequencing of the human genome. - current spin-offs include the characterisation of all genes expressed in the cell through DNA microarrays and definition of changes in protein expression in a cell (proteomics). Production of monoclonal antibodies from hybridomas basic studies of drugs against viruses and cancer. development of gene and stem cell therapy (or regenerative medicine). Slide29:  Prophase Telophase Metaphase Interphase Slide31:  8K HUMAN cDNA MICROARRAYS. EACH cDNA SPOTTED TWICE. TOTAL OF 16,000 SPOTS Monoclonal Antibodies:  Monoclonal Antibodies Formed by fusing cells (Kohler & Milstein 1975) Immortal myeloma cell line and Mouse B lymphocytes producing a specific antibody Production of only one (clone) of antibody indefinitely, exquisite specificity But mouse antibody  immunogenic in humans  ‘humanised’ Wide applications: Research Diagnostics Therapy Rheumatoid arthritis Lymphoma Macular degeneration Immunosuppression for transplantation Tissue culture: Social Issues :  Tissue culture: Social Issues Tissue culture in the early days (between WWI and WWII), invoked as much controversy as stem cell research does today. People imagined babies being born in test tubes and manipulated in the laboratories by scientists for nefarious purposes. even discussed in books such as the Brave New World (Aldous Huxley). The Future:  The Future Molecular manipulation of cells to allow growth of new or previously uncultivable viruses. -will also allow production of attenuated vaccines (including specifically mutated viruses for loss of virulence). Powerful tools of genomics and proteomics should enable full molecular definition and modification of various cell types including stem cells and: a detailed catalogue of the successive interactions between viral and host proteins during the virus replication cycle). -should facilitate development of novel drug targets. SARS Cell Culture:  SARS Cell Culture 1.Uninfected 2. Day 6 post infection Vero E6 The Future:  The Future Culture of previously uncultivable body cells, including cancer and leukaemia cells, especially leukaemia stem cells- may allow definition of critical mutations leading to malignancy Interactions between cancer causing viruses and their host cells at a molecular level should shed more light on the cancer causing steps Complex cultures of multiple cell types in three dimensions will allow a better understanding of cancer cell growth and their response to novel drugs, especially those aimed at the cell signalling pathways. Slide41:  Fibroblasts ALL cells migrated into BMF ALL cells on BMF Bone Marrow Stroma as a support for ALL Cells The Future:  The Future Advances in culturing of nerve and brain cells and examining the interactions between different cell types will facilitate novel genomic, proteomic and imaging studies which should unravel the basis of neuro-degeneration as in Alzheimer’s disease. Stem cell research, from adults or (controversially) from embryos, should advance therapies for human disease, especially diabetes, Parkinson’s disease, heart disease, Alzheimer’s and many other serious human diseases. Selection and molecular modification of cultured cells is still necessary for the growth of modified viral vectors which underlie the development of gene therapy. Such viral vectors should be simplified to include just a few essential protein molecules but still retain the properties of the current whole (but altered) viruses. Stem cells / Regenerative Medicine:  Stem cells / Regenerative Medicine Able to renew themselves through cell division Can differentiate into wide range of cell types (totipotent versus pluripotent vs multipotent) 2 main categories: Embryonic stem cells (ESC) from 4-5 day old embryo, totipotent or pluripotent Adult stem cells (ASC) Undifferentiated cells found throughout the body (e.g. bone marrow), ? Pluripotent or multipotent (cord blood) Slide47:  Derived from inner cell mass of blastocyst Culture with leukemia inhibitory factor, “feeder” cells Genetic modification by addition of DNA constructs Growth and selection under conditions promoting formation of one or many cell types ES cell derived tissues Injection into host embryo to create chimeric mice Genetically modified mice D. Loebel, CMRI Culture and use of mouse embryonic stem cells Major scientific debate:  Major scientific debate Are adult stem cells fully ‘pluripotent’ (properties can be altered in vitro) Social debate Origin of ESC (like IVF) vs limitation to ASC Concerns are that ESS will lead to reproductive cloning of humans Treatment with stem cells:  Treatment with stem cells Current: blood derived stem cell therapy for leukaemia (bone marrow  now ‘mobilised’ CD34+ blood cells) Future: (for tissues unable to self-repair) Parkinson’s disease Spinal cord injury Ischaemic heart disease diabetes Conclusions:  Conclusions Tissue culture: the rather mundane technology on which much of modern medical science is built Needed for the genomics revolution Essential for diagnostics, vaccines and drugs In the future we will be able to biochemically characterise cells completely and manipulate them easily for better vaccines and drugs Acknowledgements:  Acknowledgements Slides: Ian Alexander; gene therapy Patrick Tam: rat stem cells Linda Bendall: Leukaemia cells Dominic Dwyer: viruses in culture Monica Miranda: nerve cells Nitin Saksena: HIV Helen Briscoe: monoclonal antibodies Nick Nicola: CSFs Ultrastructural Evidence for Apoptosis:  Ultrastructural Evidence for Apoptosis

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