SUBJECT
Cell, Tissue and Organ Culture in Biomedical Research
lecture
master
2
Semesters 1-4
Autumn/Spring semester
1. Introduction. General overview of cell, tissue and organ culture systems: in vitro methods, their advantages and disadvantages, conditions and levels of their applications. The most important conditions and factors for in vitro survival, safety measures, the analyis and evaluation of the results, conclusion to in vivo processes.
2. Perfusion systems: in situ and isolated-perfused organ systems and organs, main types of arrangements (constant flow-rate and pressure, single-pass and recirculation systems), technical ground, perfusion fluids.
3. Some examples of organ perfusion systems: liver, kidney, heart, pancreas.
4. Organ cultures without perfusion. Embryonic and non-embryonic cultures, organitypic growth, solid, semisolid and liquid media, the problems of oxigenation, tissue slices in short term cultures, perifusion (superfusion systems).
5. In vitro fertilization and mammalian cultures before implantation, whole embryo cultures and their utilization in studying organogenesis.
6. Disaggregation of organs and tissue into cells and gropus of cells; the utilization of the isolated parts in short term in vitro systems. The methods of isolation: mechanical, enzymatid, perfusion, immersion and combined methods, separation of cell types by physical methods.
7. The experimental system of isolated liver cell suspensions and their use in cellular bichemistry and physiology, electroporation of isolated hepatocytes.
8. Isolated Langerhans islands, kidney glomeruli and tubules, pancreas and salivary gland acini and their experimental utilization. Electron microscopic sampling and processing of isolated cells.
9. Starting real tissue cultures from tissue explants and isolated cells. Primary cultures, selection factors (migration, cell division speed etc...), cell lines and their life history, finite and continuous cell lines, monolayers and suspension cultures. Cell strans, clones, and subclones. The rules of nomenclature in tissue cultre work.
10. The composition and the characteristics of tissue culture media. The water, balanced salt solutions, essential bulk ions, buffer systems, gas phase, pH, ionic strength, osmotic concentration. Semi synthetic media, essential (BME, MEM, DMEM etc...) and complex media (the F and the MCDB series etc...) and their composition.
11. The most important technical requirements and equipment of cell and tissue culture: sterility and sterilization, culture vessels, substrates and attachment factors, feeder layers; storage, freezing, transportation.
12. General problems and methods of cloning of animal cells; synchronization, reaggregation cultures, reimplantation.
13. The serum and serum-free media. Maintenance of cultures, medium change and subculturing.
14. Cell type selection by culturing conditions, differential attachment and detachment; cell fusion, selection of drug-resistence cell lines and their use in somatic cell hybridization, HAT selection.
Textbooks and/or other information media in use or recommended:
General Techniques of Cell Culture (Handbooks in Practical Animal Cell Biology) by Maureen A.
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General Techniques of Cell Culture (Handbooks in Practical Animal Cell Biology) by Maureen A. Harrison and Ian F. Rae, 2001, Oxford
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Culture of Animal Cells: A Manual of Basic Technique, 5th Edition by R. Ian Freshney, Wiley, 2005