Practical 4 07


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Practical 4 07

  1. 1. Practical 4 In vitro cell cultivation
  2. 2. Tissue cultures Cell cultures In vitro cell cultivation
  3. 3. MAIN TYPES OF CELL CULTURE according to the cultivation period (and survival in culture) <ul><li>primary cultures – derived from excised, normal animal tissue </li></ul><ul><li>continuous cultures – comprised of a single cell type – cell lines </li></ul><ul><ul><li>normal diploid cells – maintain some degree of differentiation, finite life (die after 30 divisions) </li></ul></ul><ul><ul><li>established cell lines – tumour cells, transformed cells – aneuploid number of chromosomes – limitless availability (unlimited life span) </li></ul></ul>
  4. 4. MAIN TYPES OF CELL CULTURE <ul><li>Animal cell culture </li></ul><ul><ul><li>Mammalian cells </li></ul></ul><ul><li>Plant tissue culture </li></ul><ul><ul><li>frequently used for production of genetically modified plants </li></ul></ul>culture of macrophages plant tissue culture - callus
  5. 5. MAIN TYPES OF CELL CULTURE According to cell morphology <ul><li>epithelial cells </li></ul><ul><ul><li>HeLa – tumour cell derived from the carcinoma of human cervix – patient He len La cks </li></ul></ul><ul><li>fibroblasts </li></ul><ul><ul><li>CHO – C hinese H amster O vary Cells </li></ul></ul><ul><ul><li>BHK – Syrian Hamster Kidney Cells </li></ul></ul><ul><li>lymphoblasts </li></ul><ul><ul><li>HL60 – H uman L eukemia – derived cells </li></ul></ul><ul><li>neuroblasts </li></ul><ul><ul><li>SH-SY5Y – from human neuroblastoma </li></ul></ul>
  6. 6. THE CELL ENVIRONMENT <ul><li>temperature 37 ºC (incubator) </li></ul><ul><li>in some cases atmosphere in the incubator contains carbon dioxide (approximately 5%) </li></ul><ul><li>sterile conditions (disinfection of the work surfaces, microbiological safety cabinets) </li></ul><ul><ul><li>(Main types of microbial contaminants – bacteria, fungi, Mycoplasma, viruses) </li></ul></ul><ul><li>culture medium </li></ul><ul><ul><li>Basic Constituents of Media </li></ul></ul><ul><ul><ul><li>Inorganic salts </li></ul></ul></ul><ul><ul><ul><li>Trace elements </li></ul></ul></ul><ul><ul><ul><li>Buffering systems </li></ul></ul></ul><ul><ul><ul><ul><li>pH range should be 7,2 – 7,4 </li></ul></ul></ul></ul><ul><ul><ul><li>Carbohydrates </li></ul></ul></ul><ul><ul><ul><li>Aminoacids </li></ul></ul></ul><ul><ul><ul><li>Vitamins </li></ul></ul></ul><ul><ul><ul><li>Proteins and peptides </li></ul></ul></ul><ul><ul><ul><li>Fatty acids and lipids </li></ul></ul></ul><ul><ul><ul><li>Serum – fetal bovine serum </li></ul></ul></ul><ul><ul><ul><li>Antibiotics </li></ul></ul></ul><ul><li>cultivation flasks and tubes (plastic ware) </li></ul>
  7. 7. The biohazard box
  8. 8. Growth curve of the cell culture log N time lag log stationary phase decrease
  9. 9. Written test <ul><li>10 minutes </li></ul><ul><li>Don't forget to put down your name, your group and the test version. </li></ul><ul><li>In multiple choice questions more than 1 statement could be correct. </li></ul><ul><li>Don't write anything on the question sheet! </li></ul>
  10. 10. Presentation Cell cultures X laboratory animals
  11. 11. Tasks
  12. 12. Task 1 INVERTED MICROSCOPE <ul><li>I nverted microscope is widely used for direct observation of cells in cultivation flasks. </li></ul><ul><li>Observe various cell cultures using this microscope. Compare their morphology and density. </li></ul>
  13. 13. Task 2 OBSERVATION OF HELA CELLS <ul><li>Observe colonies of HeLa cells in the small Petri dish. They were fixed with methanol and stained with the trypan blue solution. </li></ul><ul><li>Before the observation remove the lid of the dish and observe colonies in the optical microscope using 10x magnification. Note the morphology of cells. </li></ul>
  14. 14. Task 3 CELL QUANTIFICATION <ul><li>For the majority of manipulations using cell cultures, such as transfections, cell fusion techniques, cryopreservation and subculture routines it is necessary to quantify the number of cells prior to use. Using a consistent number of cells will maintain optimum growth and also help to standardise procedures using cell cultures. </li></ul><ul><li>Estimate cell density using the Bürker haemocytometer. </li></ul><ul><li>Both sides of the chamber are filled with the cell suspension (2 x 10 microliters). View the cells under the light microscope using 20x magnification. </li></ul><ul><li>Count the number of cells inside squares of the haemocytometer. </li></ul><ul><li>Calculate the cell density (CD) using equation </li></ul><ul><li>CD = n . 104 / s </li></ul><ul><li>CD – cell density (cells/millilitre) </li></ul><ul><li>n – number of cells </li></ul><ul><li>s – number of small squares </li></ul>enumeration square coverslip
  15. 15. Task 3: Cell quantification using hemocytometer See microscopes on the table number 6.
  16. 16. Task 4 (see photo) CHROMOSOMES OF THE CHO CELL LINE <ul><li>CHO is a continuous fibroblast cell line derived from the C hinese H amster O vary. </li></ul><ul><li>Count chromosomes of the CHO cell in the photo. </li></ul><ul><li>Describe the shape of chromosomes and explain your result. </li></ul>
  17. 17. Task 4 (see photo s ) <ul><li>Count chromosomes of the CHO cell in the photo. </li></ul><ul><li>Describe the shape of chromosomes and explain your result </li></ul>
  18. 18. Results
  19. 19. Task 2: Observation of HeLa cells Mitotic cells have circular shape.
  20. 20. Task 4: CHO cells 21 chromosomes Aneuploid number of chromosomes is a consequence of cell transformation during the long time cultivation. 7 6 4 4 5 6 3 7
  21. 21. Karyotype of CHO cells 21 chromosomes CHO cells have some features of tumor ones.
  22. 22. Karyotype of normal and tumor cell normal male cell tumor cell Rapid and uncontrolled proliferation of tumor cells leads to accumulation of numerical and structural chromosomal abnormalities.
  23. 23. APPLICATION OF CELL CULTURES <ul><li>clinical genetics </li></ul><ul><ul><li>prenatal examination (cultivation of amniocytes, chorionic villi or fetal blood lymphocytes) </li></ul></ul><ul><ul><li>postnatal examination (cultivation of peripheral blood lymphocytes) </li></ul></ul><ul><ul><li>chromosomal examination of tumors </li></ul></ul><ul><li>screening of mutagenicity (genotoxicity) and carcinogenicity of chemical compounds </li></ul><ul><li>mapping of the human genome </li></ul><ul><li>cell differentiation studies (histology, embryology) </li></ul><ul><li>cell cycle and growth control studies (important for oncology) </li></ul><ul><li>production of monoclonal antibodies </li></ul><ul><li>cloning of mammals (including man) </li></ul><ul><li>maintaining and isolation of viruses </li></ul>
  24. 24. Next practicals <ul><li>Chromosomal analysis (prenatal, postnatal), karyotyping </li></ul><ul><li>Test in previous topics (only practicals) </li></ul>
  25. 25. See you next week! CHO cells