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Tissue culture

Lab. Culture activity. Tissue culture. Aulannni’am Biochemistry Laboratory Chemistry department Brawijaya University. History of tissue culture. Disadvantages/Advantages of TC. Not at all like an animal Environment is not like in vivo Effects of serum, plasma, etc. Single cell type!

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Tissue culture

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  1. Lab. Culture activity Tissue culture Aulannni’am Biochemistry Laboratory Chemistry department Brawijaya University Aulani " GE" Presentation 10

  2. History of tissue culture Aulani " GE" Presentation 10

  3. Disadvantages/Advantages of TC • Not at all like an animal • Environment is not like in vivo • Effects of serum, plasma, etc. • Single cell type! • Controlled conditions • Superb access Aulani " GE" Presentation 10

  4. Tissue Culture The objective of tissue culture Cellular models of pathophysiology Sterility Routine Cell Culture Experiments in culture Primary cell culture Cell preservation Cell cloning Culture changes Media and Salt Solutions Vendors Aulani " GE" Presentation 10

  5. “Tissue culture can be a powerful technique if conducted properly and a great waste of time and money when done sloppily” Aulani " GE" Presentation 10

  6. Aseptic Technique • Sterile Hood - All manipulations must be carried out in a sterile cabinet • Turn the UV light off. • Open the cabinet • Wipe down with disinfectant (70% ethanol or 40% isopropyl alcohol or Amphyl-type disinfectants) • Bring materials into the hood • Light up the flame or gas • Begin your work Aulani " GE" Presentation 10

  7. Aseptic Technique • Flame all caps and lids • Tightly close all bottles and caps • Remove materials from the hood • Turn off gas • Wash the hood surface • Turn the UV light on to disinfect Aulani " GE" Presentation 10

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  17. Culture medium • Contains amino acids • contains salts, Mg, Ca, K, Na, Cl • contains trace metals, Se, Zn, Cr • contains carbon source: glucose, either high or low, low = 1%, high = 4.5 % • contains B-vitamins, cofactors • contains serum, or plasma • Antibiotic, antimycotic Aulani " GE" Presentation 10

  18. Serum • Contains release products from platelets • includes PDGF, PDECGF • serotonin, ADP, ATP • also contains Platelet Factor IV • also contains insulin, transferrin, ferritin • LDL’s, albumin, factors bound to albumin Aulani " GE" Presentation 10

  19. Plasma • Prepared by spinning out platelets • Should contain low levels of PDGF • PDGF is a growth factor for smooth muscle and fibroblasts, might not want this present for culturing cells not of these origins Aulani " GE" Presentation 10

  20. Antibiotic/antimycotic • Amphotericin B • Streptomycin • Theory - eliminates, keeps out contamination • Fact - once contaminated, that is it… • Fact - it is possible to salvage cultures... Aulani " GE" Presentation 10

  21. Heparin • Highly negatively charged proteoglycan • Binds and stabilizes growth factors, e.g. IGF, b-FGF, ECGF • inhibits growth of smooth muscle and fibroblast cells • used at 90-100 ug/ml Aulani " GE" Presentation 10

  22. Growth factors • ECGF - endothelial cell growth factor • VEGF - vascular endothelial growth factor • bFGF, aFGF - fibroblast growth factor • IGF - insulin-like growth factor • IL-2 - Lymphocyte growth factor • GMCSF, CSF - granulocytes, macs, mono • EGF - epidermal growth factor (salivaries!) • HGF - hepatocyte GF, (‘scatter factor’) Aulani " GE" Presentation 10

  23. BD - ECGF • Homogenize brains • Precipitate fat • Precipitate lipid further with streptomycin • filter • lyophilize • = crude ECGF • can be further purified on heparin columns Aulani " GE" Presentation 10

  24. Cell matrix products • Often used to promote adhesion of specific cells e.g. endothelial cells • Fibronectin, 10 ug/ml • Laminin, 10 ug/ml • Gelatin - 0.5 -2% • Vitronectin • Necessary for some cell to grow, e.g. EC’s Aulani " GE" Presentation 10

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  26. Sterilization Techniques • Metal - H, A • Glass - H, A, R • Plastics • polycarbonate A, R, G • polyethylene A, R, G • polypropylene A, R, G • polystyrene R, G • Medium - R, F • Serum - R, F • Salt solutions - A, R, F Aulani " GE" Presentation 10

  27. Filtration • Always filter through 0.2 um filter to remove bacteria • Can pre-filter through 0.45 first to remove particulates • Mycoplasma and viruses will still filter through • Filter come in many sizes, all are $$$ Aulani " GE" Presentation 10

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  38. Tissue culture flasks • T-flasks • Petri dishes • coverslips • 6 well, 12, 48 or 96 wells Aulani " GE" Presentation 10

  39. Isolation of cells • Mechanical dissociation (mincing) • Chemical dissociation • collagenase (Clostridium perfringens) • dispase (bacterial) • trypsin serves to dissociate cells within tissues, why is that important? Aulani " GE" Presentation 10

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  41. Explant culture • Involves placing a piece of tissue into the tissue culture dish and allowing cells to migrate out from the tissue • Performed in the case of cells which are protease sensitive • Smooth muscle cells, Bone cells Aulani " GE" Presentation 10

  42. Explant culture Aulani " GE" Presentation 10

  43. Cell selection • Selective medium • Growth factors • Cell type specific toxins (thimerosal – kills fibroblasts) • Complement mediated cell killing (Thy 1) • Selection by nutrients (D-valine) Aulani " GE" Presentation 10

  44. Cell morphology • Monolayer - single cell layer, epi’s, endo’s • Not contacted inhibited, fibroblasts • Streaming, fibroblasts • Islands - epithelia, T-84, MDCK • Domes - MDCK and transporting epi’s Aulani " GE" Presentation 10

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  46. Growth curves • How many cells do you have per cm2? • How do you determine that? • Hemocytometer • Coulter counter - electrical resistance • Normal range - 50,000 - 100,000 cells per cm2, transformed cells can get 10 X higher! Aulani " GE" Presentation 10

  47. Growth rate • BrDU - bromodeoxyuridine DNA • DAPI - diaminophenylindole DNA - vital • 3H-thymidine - scintillation counting • Coulter counting - vital Aulani " GE" Presentation 10

  48. Aging cell cultures • Most cell cultures ‘age’ in culture, and often lose characteristics • example: endothelial cells often lose the ability to upregulate P-selectin, or express P-selectin in culture. • Cells which are not transformed can undergo roughly 50 population doublings Aulani " GE" Presentation 10

  49. Transformed cell lines • These types of cells do not age in culture • They are ‘immortal’ • They often lose contact inhibition • They often lose many normal characteristics • They are not dependent on growth factors • They may express ‘large T-antigen’ a p53 inhibitor Aulani " GE" Presentation 10

  50. Passaging of cells I • Cells are passaged or sub-cultivated using trypsin-EDTA • EDTA is a calcium chelator • removes calcium (1.2 mM) causes cell rounding • low calcium causes cells to internalize adhesion molecules, rounding or frank detachment Aulani " GE" Presentation 10

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