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

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

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

4. Aulani " GE" Presentation 10 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

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

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

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

8. Aulani " GE" Presentation 10

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

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

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

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

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

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

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

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

25. Aulani " GE" Presentation 10

26. Sterilization Techniques Metal - H, A Metal - H, A Glass - H, A, R Glass - H, A, R Plastics Plastics polycarbonateA, R, G polycarbonateA, R, G polyethyleneA, R, G polyethyleneA, R, G polypropyleneA, R, G polypropyleneA, R, G polystyreneR, G polystyreneR, G Medium - R, F Medium - R, F Serum - R, F Serum - R, F Salt solutions - A, R, F Salt solutions - A, R, F

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

28. Aulani " GE" Presentation 10

38. Tissue culture flasks T-flasks T-flasks Petri dishes Petri dishes coverslips coverslips 6 well, 12, 48 or 96 wells 6 well, 12, 48 or 96 wells

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

40. Aulani " GE" Presentation 10

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

42. Aulani " GE" Presentation 10 Explant culture

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

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

45. Aulani " GE" Presentation 10

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

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

48. Aulani " GE" Presentation 10 Aging cell cultures Most cell cultures age in culture, and often lose characteristics 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. 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 Cells which are not transformed can undergo roughly 50 population doublings

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

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

51. Aulani " GE" Presentation 10 Trypsin - highly active, relatively non-specific, cheap protease derived from pancreas Trypsin - highly active, relatively non-specific, cheap protease derived from pancreas Cleaves proteins on the cell surface and extracellular matrix Cleaves proteins on the cell surface and extracellular matrix Detaches cells Detaches cells Over-trypsinization causes cell injury Over-trypsinization causes cell injury Passaging of cells II

52. Aulani " GE" Presentation 10 Cell Markers - ID Endothelial cells - Factor VIII, LDL-rec, P-selectin Endothelial cells - Factor VIII, LDL-rec, P-selectin Epithelial cells - Epi. Spec. Antigen Epithelial cells - Epi. Spec. Antigen Transformed cells - Large T antigen Transformed cells - Large T antigen Smooth muscle cells - smooth muscle actins Smooth muscle cells - smooth muscle actins Fibroblasts - vimentin Fibroblasts - vimentin

53. Aulani " GE" Presentation 10 Cloning Cloning simply means growing a culture that has all the properties of the cell you selected Cloning simply means growing a culture that has all the properties of the cell you selected Cloning cylinders Cloning cylinders Irradiation Irradiation Scraping Scraping Cloning by dilution - may require the use of conditioned medium Cloning by dilution - may require the use of conditioned medium

54. Aulani " GE" Presentation 10 Cloning cylinders

55. Aulani " GE" Presentation 10 Freezing cells Cells can be trypsinized and then frozen! Cells can be trypsinized and then frozen! A STATE OF SUSPENDED ANIMATION A STATE OF SUSPENDED ANIMATION Cells are suspended in 5% DMSO, 95% FCS and frozen in tubes inside of styrofoam Cells are suspended in 5% DMSO, 95% FCS and frozen in tubes inside of styrofoam Styrofoam cools at 1 degree C/min and with DMSO prevents ice crystal formation Styrofoam cools at 1 degree C/min and with DMSO prevents ice crystal formation Can be stored for years!!! Can be stored for years!!! Viability decreases over time Viability decreases over time

56. Aulani " GE" Presentation 10 Contamination Happens to even the best… Happens to even the best… Fungal -yeast Fungal -yeast Bacterial Bacterial Mycoplasma - filterable bacteria which will pass across 0.2 um filter. Big problem. It is treated with cephalosporin antibiotics. Can take over laboratories Mycoplasma - filterable bacteria which will pass across 0.2 um filter. Big problem. It is treated with cephalosporin antibiotics. Can take over laboratories

57. Aulani " GE" Presentation 10

58. Incubators Maintains 37 C, 100% humid environment Maintains 37 C, 100% humid environment Contains 5-10% carbon dioxide Contains 5-10% carbon dioxide CO2 + H2O yields H+ HCO3- buffer system, Bicarbonate is also necessary for cell nutrition CO2 + H2O yields H+ HCO3- buffer system, Bicarbonate is also necessary for cell nutrition Some use Good buffers, HEPES, MOPS but this only helps somewhat, and high concentrations are toxic, can interact with NO, oxidants, etc. Some use Good buffers, HEPES, MOPS but this only helps somewhat, and high concentrations are toxic, can interact with NO, oxidants, etc.

59. Aulani " GE" Presentation 10 Special techniques Cell matrix growth - cells can be cultured inside of collagen gels, often used for contraction and wound healing studies Cell matrix growth - cells can be cultured inside of collagen gels, often used for contraction and wound healing studies Cells can be cultured inside of blood clots, can also be used to study clot retraction Cells can be cultured inside of blood clots, can also be used to study clot retraction Cells can also be cultured on permeable supports, e.g. filters, 0.45 um - 8 um pores for permeability studies or migration studies. Cells can also be cultured on permeable supports, e.g. filters, 0.45 um - 8 um pores for permeability studies or migration studies.

60. Aulani " GE" Presentation 10 Microcarrier Culture Cells can also be cultured on beads Cells can also be cultured on beads Beads are usually inert materials, e.g. sephadex, gelatin, DEAE Beads are usually inert materials, e.g. sephadex, gelatin, DEAE Coated with matrix, gelatin, etc. Coated with matrix, gelatin, etc. Need to be stirred continuously Need to be stirred continuously Microcarrier flasks Microcarrier flasks Benefits? Production of cell derived factors Benefits? Production of cell derived factors Enormous surface area 1 ml = 328 cm2! Enormous surface area 1 ml = 328 cm2!

61. Aulani " GE" Presentation 10 Microcarrier cultures Stirs at 60 RPM Low shear Massive numbers of cells possible

62. Aulani " GE" Presentation 10 Transfection Calcium phosphate Calcium phosphate DEAE dextran DEAE dextran Virally mediated gene transfer Virally mediated gene transfer Plasmid mediated gene transfer (lipofectin, superfect) uses liposomes Plasmid mediated gene transfer (lipofectin, superfect) uses liposomes Protein transfection (transfecting in the protein of choice) uses liposomes Protein transfection (transfecting in the protein of choice) uses liposomes Gene gun - biolistic devices Gene gun - biolistic devices Electroporation Electroporation

63. Aulani " GE" Presentation 10 Tissue culture is unavoidable... As hard as we try, we cant avoid doing TC As hard as we try, we cant avoid doing TC Relatively easy Relatively easy Fast Fast Inexpensive Inexpensive Can be done Can be done Must always correlate with in vivo Must always correlate with in vivo

64. Aulani " GE" Presentation 10