Aulani " GE" Presentation 10 Tissue culture Lab. Culture activity Aulannniam Biochemistry Laboratory...

Aulani " GE" Presentation 10Aulani " GE" Presentation 10

Tissue cultureTissue cultureLab. Culture activityLab. Culture activity

Aulannni’amAulannni’amBiochemistry LaboratoryBiochemistry LaboratoryChemistry departmentChemistry departmentBrawijaya UniversityBrawijaya University

Aulani " GE" Presentation 10

History of tissue cultureHistory of tissue culture


Disadvantages/Advantages of TCDisadvantages/Advantages of TC

►Not at all like an animalNot at all like an animal►Environment is not like in vivoEnvironment is not like in vivo►Effects of serum, plasma, etc.Effects of serum, plasma, etc.

►Single cell type!Single cell type!►Controlled conditionsControlled conditions►Superb accessSuperb access


Tissue Culture

The objective of tissue cultureCellular models of pathophysiology

SterilityRoutine Cell CultureExperiments in culturePrimary cell cultureCell preservationCell cloningCulture changesMedia and Salt SolutionsVendors


“Tissue culture can be a powerful technique if conducted properly and a great waste of time and money when done sloppily”


Aseptic TechniqueAseptic Technique

► Sterile Hood - All manipulations must be Sterile Hood - All manipulations must be carried out in a sterile cabinetcarried out in a sterile cabinet

► Turn the UV light off.Turn the UV light off.► Open the cabinetOpen the cabinet► Wipe down with disinfectant (70% ethanol or Wipe down with disinfectant (70% ethanol or

40% isopropyl alcohol or Amphyl-type 40% isopropyl alcohol or Amphyl-type disinfectants)disinfectants)

► Bring materials into the hoodBring materials into the hood► Light up the flame or gasLight up the flame or gas► Begin your workBegin your work


Aseptic TechniqueAseptic Technique

► Flame all caps and lidsFlame all caps and lids► Tightly close all bottles and caps Tightly close all bottles and caps ► Remove materials from the hoodRemove materials from the hood► Turn off gasTurn off gas► Wash the hood surfaceWash the hood surface► Turn the UV light on to disinfectTurn the UV light on to disinfect


Culture mediumCulture medium

► Contains amino acidsContains amino acids► contains salts, Mg, Ca, K, Na, Clcontains salts, Mg, Ca, K, Na, Cl► contains trace metals, Se, Zn, Crcontains trace metals, Se, Zn, Cr► contains carbon source: glucose, either contains carbon source: glucose, either

high or low, low = 1%, high = 4.5 %high or low, low = 1%, high = 4.5 %► contains B-vitamins, cofactorscontains B-vitamins, cofactors► contains serum, or plasmacontains serum, or plasma► Antibiotic, antimycoticAntibiotic, antimycotic


SerumSerum

►Contains release products from plateletsContains release products from platelets► includes PDGF, PDECGFincludes PDGF, PDECGF►serotonin, ADP, ATPserotonin, ADP, ATP►also contains Platelet Factor IValso contains Platelet Factor IV►also contains insulin, transferrin, ferritinalso contains insulin, transferrin, ferritin►LDL’s, albumin, factors bound to albuminLDL’s, albumin, factors bound to albumin


PlasmaPlasma

►Prepared by spinning out plateletsPrepared by spinning out platelets►Should contain low levels of PDGFShould contain low levels of PDGF►PDGF is a growth factor for smooth PDGF is a growth factor for smooth

muscle and fibroblasts, might not want muscle and fibroblasts, might not want this present for culturing cells not of this present for culturing cells not of these originsthese origins


Antibiotic/antimycotiAntibiotic/antimycoticc

► Amphotericin BAmphotericin B► StreptomycinStreptomycin► Theory - eliminates, keeps out Theory - eliminates, keeps out

contaminationcontamination► 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...


HeparinHeparin

►Highly negatively charged Highly negatively charged proteoglycanproteoglycan

►Binds and stabilizes growth factors, Binds and stabilizes growth factors, e.g. IGF, b-FGF, ECGFe.g. IGF, b-FGF, ECGF

► inhibits growth of smooth muscle and inhibits growth of smooth muscle and fibroblast cellsfibroblast cells

►used at 90-100 ug/mlused at 90-100 ug/ml


Growth factorsGrowth factors

► ECGF - endothelial cell growth factorECGF - endothelial cell growth factor► VEGF - vascular endothelial growth factorVEGF - vascular endothelial growth factor► bFGF, aFGF - fibroblast growth factorbFGF, aFGF - fibroblast growth factor► IGF - insulin-like growth factorIGF - insulin-like growth factor► IL-2 - Lymphocyte growth factorIL-2 - Lymphocyte growth factor► GMCSF, CSF - granulocytes, macs, monoGMCSF, CSF - granulocytes, macs, mono► EGF - epidermal growth factor (salivaries!)EGF - epidermal growth factor (salivaries!)► HGF - hepatocyte GF, (‘scatter factor’)HGF - hepatocyte GF, (‘scatter factor’)


BD - ECGFBD - ECGF

► Homogenize brainsHomogenize brains► Precipitate fatPrecipitate fat► Precipitate lipid further with streptomycinPrecipitate lipid further with streptomycin► filterfilter► lyophilizelyophilize► = crude ECGF= crude ECGF► can be further purified on heparin columnscan be further purified on heparin columns


Cell matrix productsCell matrix products

►Often used to promote adhesion of Often used to promote adhesion of specific cells e.g. endothelial cellsspecific cells e.g. endothelial cells

►Fibronectin, 10 ug/mlFibronectin, 10 ug/ml►Laminin, 10 ug/mlLaminin, 10 ug/ml►Gelatin - 0.5 -2%Gelatin - 0.5 -2%►Vitronectin Vitronectin ►Necessary for some cell to grow, e.g. Necessary for some cell to grow, e.g.

EC’sEC’s


Sterilization TechniquesSterilization Techniques► Metal - H, AMetal - H, A► Glass - H, A, RGlass - H, A, R► Plastics Plastics

polycarbonatepolycarbonate A, R, GA, R, G polyethylenepolyethylene A, R, GA, R, G polypropylenepolypropyleneA, R, GA, R, G polystyrenepolystyrene R, GR, G

► Medium - R, FMedium - R, F► Serum - R, FSerum - R, F► Salt solutions - A, R, FSalt solutions - A, R, F


FiltrationFiltration

► Always filter through 0.2 um filter to remove Always filter through 0.2 um filter to remove bacteriabacteria

► Can pre-filter through 0.45 first to remove Can pre-filter through 0.45 first to remove particulatesparticulates

► Mycoplasma and viruses will still filter throughMycoplasma and viruses will still filter through► Filter come in many sizes, all are $$$Filter come in many sizes, all are $$$


Tissue culture flasksTissue culture flasks

► T-flasksT-flasks► Petri dishesPetri dishes► coverslipscoverslips► 6 well, 12, 48 or 96 wells6 well, 12, 48 or 96 wells


Isolation of cellsIsolation of cells

► Mechanical dissociation (mincing)Mechanical dissociation (mincing)► Chemical dissociationChemical dissociation

collagenase (Clostridium perfringens)collagenase (Clostridium perfringens) dispase (bacterial)dispase (bacterial) trypsintrypsin

serves to dissociate cells within tissues, why is that serves to dissociate cells within tissues, why is that important?important?


Explant cultureExplant culture

► Involves placing a piece of tissue into the Involves placing a piece of tissue into the tissue culture dish and allowing cells to tissue culture dish and allowing cells to migrate out from the tissuemigrate out from the tissue

► Performed in the case of cells which are Performed in the case of cells which are protease sensitiveprotease sensitive

► Smooth muscle cells, Bone cellsSmooth muscle cells, Bone cells


Explant culture


Cell selectionCell selection

► Selective mediumSelective medium► Growth factorsGrowth factors► Cell type specific toxins (thimerosal – kills Cell type specific toxins (thimerosal – kills

fibroblasts)fibroblasts)► Complement mediated cell killing (Thy 1)Complement mediated cell killing (Thy 1)► Selection by nutrients (D-valine)Selection by nutrients (D-valine)


Cell morphologyCell morphology

► Monolayer - single cell layer, epi’s, endo’sMonolayer - single cell layer, epi’s, endo’s► Not contacted inhibited, fibroblastsNot contacted inhibited, fibroblasts► Streaming, fibroblastsStreaming, fibroblasts► Islands - epithelia, T-84, MDCKIslands - epithelia, T-84, MDCK► Domes - MDCK and transporting epi’sDomes - MDCK and transporting epi’s


Growth curvesGrowth 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?► HemocytometerHemocytometer► Coulter counter - electrical resistanceCoulter counter - electrical resistance► Normal range - 50,000 - 100,000 cells per cm2, transformed Normal range - 50,000 - 100,000 cells per cm2, transformed

cells can get 10 X higher!cells can get 10 X higher!


Growth rateGrowth rate

► BrDU - bromodeoxyuridine DNA BrDU - bromodeoxyuridine DNA ► DAPI - diaminophenylindole DNA - vitalDAPI - diaminophenylindole DNA - vital► 3H-thymidine - scintillation counting3H-thymidine - scintillation counting► Coulter counting - vitalCoulter counting - vital


Aging cell culturesAging cell cultures

► Most cell cultures ‘age’ in culture, and Most cell cultures ‘age’ in culture, and often lose characteristicsoften lose characteristics

► example: endothelial cells often lose the example: endothelial cells often lose the ability to upregulate P-selectin, or ability to upregulate P-selectin, or express P-selectin in culture.express P-selectin in culture.

► Cells which are not transformed can Cells which are not transformed can undergo roughly 50 population undergo roughly 50 population doublingsdoublings


Transformed cell linesTransformed cell lines

► These types of cells do not age in cultureThese types of cells do not age in culture► They are ‘immortal’They are ‘immortal’► They often lose contact inhibitionThey often lose contact inhibition► They often lose many normal characteristicsThey often lose many normal characteristics► They are not dependent on growth factorsThey are not dependent on growth factors► They may express ‘large T-antigen’ a p53 inhibitorThey may express ‘large T-antigen’ a p53 inhibitor


Passaging of cells IPassaging of cells I

► Cells are passaged or sub-cultivated using Cells are passaged or sub-cultivated using trypsin-EDTAtrypsin-EDTA

► EDTA is a calcium chelatorEDTA is a calcium chelator► removes calcium (1.2 mM) causes cell removes calcium (1.2 mM) causes cell

roundingrounding► low calcium causes cells to internalize low calcium causes cells to internalize

adhesion molecules, rounding or frank adhesion molecules, rounding or frank detachmentdetachment


► Trypsin - highly active, relatively non-Trypsin - highly active, relatively non-specific, cheap protease derived from specific, cheap protease derived from pancreaspancreas

► Cleaves proteins on the cell surface and Cleaves proteins on the cell surface and extracellular matrixextracellular matrix

► Detaches cellsDetaches cells► Over-trypsinization causes cell injuryOver-trypsinization causes cell injury

Passaging of cells IIPassaging of cells II


Cell Markers - IDCell Markers - ID

► Endothelial cells - Factor VIII, LDL-rec, P-selectinEndothelial cells - Factor VIII, LDL-rec, P-selectin► Epithelial cells - Epi. Spec. AntigenEpithelial cells - Epi. Spec. Antigen► Transformed cells - Large T antigenTransformed cells - Large T antigen► Smooth muscle cells - smooth muscle actinsSmooth muscle cells - smooth muscle actins► Fibroblasts - vimentinFibroblasts - vimentin


CloningCloning

► Cloning simply means growing a culture Cloning simply means growing a culture that has all the properties of the cell that has all the properties of the cell you selectedyou selected

► Cloning cylindersCloning cylinders► IrradiationIrradiation► ScrapingScraping► Cloning by dilution - may require the Cloning by dilution - may require the

use of conditioned mediumuse of conditioned medium


Cloning cylinders


Freezing cellsFreezing cells

► Cells can be trypsinized and then frozen!Cells can be trypsinized and then frozen!► A STATE OF SUSPENDED ANIMATIONA STATE OF SUSPENDED ANIMATION► Cells are suspended in 5% DMSO, 95% Cells are suspended in 5% DMSO, 95%

FCS and frozen in tubes inside of FCS and frozen in tubes inside of styrofoamstyrofoam

► Styrofoam cools at 1 degree C/min and Styrofoam cools at 1 degree C/min and with DMSO prevents ice crystal formationwith DMSO prevents ice crystal formation

► Can be stored for years!!!Can be stored for years!!!► Viability decreases over timeViability decreases over time


ContaminationContamination

► Happens to even the best…Happens to even the best…► Fungal -yeastFungal -yeast► BacterialBacterial► Mycoplasma - filterable bacteria which will Mycoplasma - filterable bacteria which will

pass across 0.2 um filter. Big problem. It is pass across 0.2 um filter. Big problem. It is treated with cephalosporin antibiotics. Can treated with cephalosporin antibiotics. Can take over laboratoriestake over laboratories


IncubatorsIncubators

► Maintains 37 C, 100% humid environmentMaintains 37 C, 100% humid environment► Contains 5-10% carbon dioxideContains 5-10% carbon dioxide► CO2 + H2O yields H+ HCO3- buffer system, CO2 + H2O yields H+ HCO3- buffer system,

Bicarbonate is also necessary for cell nutritionBicarbonate is also necessary for cell nutrition► Some use ‘Good’ buffers, HEPES, MOPS but this Some use ‘Good’ buffers, HEPES, MOPS but this

only helps somewhat, and high concentrations are only helps somewhat, and high concentrations are

toxic, can interact with NO, oxidants, etctoxic, can interact with NO, oxidants, etc..


Special techniquesSpecial techniques

► Cell matrix growth - cells can be cultured Cell matrix growth - cells can be cultured inside of collagen gels, often used for inside of collagen gels, often used for contraction and wound healing studiescontraction and wound healing studies

► Cells can be cultured inside of blood clots, Cells can be cultured inside of blood clots, can also be used to study clot retractioncan also be used to study clot retraction

► Cells can also be cultured on permeable Cells can also be cultured on permeable supports, e.g. filters, 0.45 um - 8 um supports, e.g. filters, 0.45 um - 8 um pores for permeability studies or pores for permeability studies or migration studies.migration studies.


Microcarrier CultureMicrocarrier Culture

► Cells can also be cultured on beadsCells can also be cultured on beads► Beads are usually inert materials, e.g. sephadex, Beads are usually inert materials, e.g. sephadex,

gelatin, DEAEgelatin, DEAE► Coated with matrix, gelatin, etc.Coated with matrix, gelatin, etc.► Need to be stirred continuouslyNeed to be stirred continuously► Microcarrier flasksMicrocarrier flasks► Benefits? Production of cell derived factorsBenefits? Production of cell derived factors► Enormous surface area 1 ml = 328 cm2!Enormous surface area 1 ml = 328 cm2!

Aulani " GE" Presentation 10Aulani " GE" Presentation 10

MicrocarrierMicrocarrierculturescultures

Stirs at 60 RPMStirs at 60 RPM

Low shearLow shear

Massive numbers of cells possibleMassive numbers of cells possible


TransfectionTransfection

► Calcium phosphateCalcium phosphate► DEAE dextranDEAE dextran► Virally mediated gene transferVirally mediated gene transfer► Plasmid mediated gene transfer (lipofectin, Plasmid mediated gene transfer (lipofectin,

superfect) uses liposomessuperfect) uses liposomes► Protein transfection (transfecting in the protein Protein transfection (transfecting in the protein

of choice) uses liposomesof choice) uses liposomes► Gene ‘gun’ - biolistic devicesGene ‘gun’ - biolistic devices► ElectroporationElectroporation


Tissue culture is Tissue culture is unavoidable...unavoidable...

► As hard as we try, we can’t avoid doing TCAs hard as we try, we can’t avoid doing TC► Relatively easyRelatively easy► FastFast► InexpensiveInexpensive► Can be doneCan be done► Must always correlate with in vivoMust always correlate with in vivo

Aulani " GE" Presentation 10 Tissue culture Lab. Culture activity Aulannniam Biochemistry Laboratory...

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