CulturingandVisualizingCells

Today• Cellculture

– Celllinevs.Primarycellculture– Howtofindtheinformation?

• Cellimaging• Sortingcell– FACS

• Moleculartechniques!• Invivoimaging

CulturingandVisualizingCells

4.1GrowingandStudyingCellsinCulture4.2LightMicroscopy:ExploringCellStructureandVisualizingProteinsWithinCells

Typesofculture

Animalcell

culture

Bacteriacell

culture

Suspendingcellculture Agarplatecellculture

Flaskculture

Cellculture

Plate

Nutrient

Cells

Charged?

Whyred?

Whattypes?

Cellculture

• Celllineculture

• PrimarycellculturePrimarycellshaveafinitelifespan;transformedtumorcellsandcelllinecellscangrowindefinitely.

Infinitelifespan,tumor-likeproperty

Cellculture

• Isolatedanimalcellsareculturedinanutrient-richliquid,calledculturemedium,inspeciallycoatedplasticdishesorflasksinincubatorsundercontrolledtemperature,atmosphere,andhumidityconditions.

• Culturemediumsuppliesessentialaminoacids(notsynthesizedbycells);vitamins;salts(minerals);fattyacids;glucose(energysource);andbloodserum,whichcontainsproteinfactorssuchashormonesandgrowthfactors.

• Mostanimalcellsonlythrivewhenattachedtoextracellularmatrixmoleculesattachedtoasolidsubstrate.

Stagesintheestablishmentofacellculture.

(a) Someisolatedcellsdie.Othersgrowforalimitednumberofgenerations(~50)ofharvesting,diluting,andreplating beforeundergoingsenescenceanddying.

(b)Veryrarecellscanaccumulateoncogenicmutationsthatallowthemtosurviveandcontinuedividinguntiltheirprogenyovergrowtheculture,constitutinganimmortalcellline,whichgrowsindefinitelyifitisappropriatelydilutedandfedwithnutrients.ThehumanHeLacelllinewasestablishedin1952fromamalignantuterinecervixcarcinomatumorandisstillusedtoday.

HeLacellline

Organism Homo sapiens, human

Tissue cervix

Cell Type epithelial

Product Format frozen

Morphology epithelial

Culture Properties adherent

Biosafety Level 2 [Cells contain human

papilloma virus]Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.

Disease adenocarcinoma

Age 31 years adult

Gender female

Ethnicity Black

Applications These cells are a suitable transfection host.This cell line can be used to screen for Escherichia coli strains with invasive potential.

Storage Conditions liquid nitrogen vapor phase

www.atcc.org

Liveimaging

Lightmicroscope

Developmentofthelightmicroscope

Cellculture&Primarycellculture

Culturedhippocampalneuron

Hippocampalcellculture(primarycellculture)

https://www.youtube.com/watch?v=eHDapIC6QvY

3Dbioprinting

Printedorgans

Threechallenges. https://www.youtube.com/watch?v=9RMx31GnNXY

Printedorganshttps://www.youtube.com/watch?v=9RMx31GnNXY

Future?

DramaWestWorld.https://www.youtube.com/watch?v=elkHuRROPfk

Understandingthecells…

Markingthecells

• Fluorescentdye• Fluorescentprotein

Differencesofcellscanbefoundeveninonecelllineculture….

FluorescenceFluorescence istheemissionof light byasubstancethathasabsorbedlightorother electromagneticradiation.

Bio-compatibleandusedasamarker!

FindingGreenFluorescentProtein(GFP)!

Osamu ShimomuraIn1962,theirworkculminatedinthediscoveryoftheproteins aequorin and greenfluorescentprotein (GFP)inthesmall,mouse-sizedumbrella-shaped bioluminescent jellyfish Aequoreavictoria; forthiswork,hewasawardedathirdoftheNobelPrizeinChemistryin2008.

GFPandNobelprizeRogerChen

Osamu Shimomura first isolated GFP from the jellyfish Aequorea victoria, which drifts with the currents off the west coast of North America. He discovered that this protein glowed bright green under ultraviolet light.

Martin Chalfie demonstrated the value of GFP as a luminous genetic tag for various biological phenomena. In one of his first experiments, he coloured six individual cells in the transparent roundworm Caenorhabditis elegans with the aid of GFP.

Roger Y. Tsien contributed to our general understanding of how GFP fluoresces. He also extended the colour palette beyond green allowing researchers to give various proteins and cells different colours. This enables scientists to follow several different biological processes at the same time.

Manydifferentcolors offluorescentproteinsarenowavailable.

Manydifferentcolorsoffluorescentproteinsarenowavailable.

Fluorescentdye

• Rhodamine• FITC• Alexseries• Cyseries

Ex>FITC– excitationandemissionwavelengths

Nowyoucanmarkthecells!

EGFPgeneexpressioninlivecell RFPgeneexpressioninlivecell

Howtosortthecells?

Oneexampleusingthefluorescentdye…

Afluorescence-activatedcellsorter(FACS)separatescellshavingdifferentlevelsoffluorescence.

Step1:Labeledcellsmixedwithsheathfluidbuffersolutionpasssinglefilethroughalaserlightbeam.Step2:Bothfluorescentlightemittedandlightscatteredfromeachcellaremeasuredandusedtodeterminecellshapeandsize.Step3:Forcingthecellsuspensionthroughanozzleformstinydropletscontainingatmostasinglecell;eachdropletobtainsanegativeelectricchargeproportionaltofluorescenceofthatcell.Step4:Passagethroughanelectricfielddifferentiallydeflectsthedropletsintodifferentbins;asmanyas10millioncellsperhourcanpassthroughthemachine.

FACS

https://www.youtube.com/watch?v=B2zreF2dnWk

Tcellsboundtofluorescence-taggedantibodiestotwocell-surfaceproteinsareseparatedfromotherwhitebloodcellsbyFACS.

Calciumimaging

InamovingleukocytecontainingtheCa2+fluorochrome fura-2,aCa2+gradientisestablishedwithhighestCa2+ concentrations(green)attherearofthecell,

Calciumimaginginneurons

ApplicationFRET

Protein-proteininteractionscanbevisualizedbyFRET.

• Excitation(433nm)ofthecyanfluorescentprotein(CFP)fusedtoproteinXemits475nmlight,whichcanexcitethefluorescentprotein(YFP)fusedtoproteinYtoemit530nmlight(insteadof475nmlight),iftheproteininteractionbringstheCFPandYFPcloseenoughtogether.

Förster resonanceenergytransfer(FRET)

FRETdata

b) FRETrevealsinteraction(yellow-orange)betweenanactiveregulatoryprotein(Rac)anditsbindingpartneratthefrontofamigratingmousefibroblastcell.

FRETbiosensorscandetectlocalbiochemicalenvironments.

FRET- YC3.60FRET reporter for Ca2+ inside primary cilia

Fluorescentmicroscope

Usualmicroscopevs.confocalWhat’stheadvantage?

a) Theconfocalmicroscopicimageissharp,particularlyinthecenterofthemitoticspindle,becausefluorescenceisdetectedonlyfrommoleculesinthefocalplane,generatingaverythinopticalsection.

Confocalmicroscopyproducesanin-focusopticalsectionthroughthickcells.

Stillblurred?

Super-resolutionmicroscopycangeneratelight-microscopeimageswithup-to-nanometerresolution.

Invivoimaging

Canyouseethedeepregions?

Speedismatter!

Howcanyouseedup?

Lightsheetmicroscopy

Lightsheet

bringsthetwopartsofGFPintocloseproximitysothattheGFPfluorescenceisrestored.

GCAMP6inaction!

https://www.youtube.com/watch?v=XEy_WtgrF7A

Light-sheetmicroscopycanimagerapideventsinlivingtissue.TransientincreasesinCa2+arerevealed(false-coloredred)inlivingzebrafishbraincells.

Studythefixedcell

Livecellimaging…What’stheadvantageanddisadvantage?

FixationInthefieldsof histology, pathology,and cellbiology, fixation isacriticalstepinthepreparationofhistologicalsectionsbywhich biologicaltissues arepreservedfromdecay,therebypreventing autolysis or putrefaction.

The broad objective of tissue fixation is to preserve cells and tissue components and to do this in such a way as to allow for the preparation of thin, stained sections.

Theeasywayistocrosslinkresiduesofmoleculesincell.

Thenwhatcanyoudo?

• Storethesampleforlongtime• Immunocytochemistry• Immunohistochemistry• Insituhybridization

Insteadofmembrane…

• Youcanusetheculturedcelllayer…

Specificantibody

Immunocytochemistry!

Cutintothinsections.

Immunohistochemistry

Antibody

Locationoflysosomesandmitochondriainaculturedlivingbovinepulmonaryarteryendothelialcell.

Thecellwasstainedwithagreen-fluorescingdyethatisspecificallyboundtomitochondria andared-fluorescingdyethatisspecificallyincorporatedintolysosomes.

fluorescein-stainedtubulin(left)andRhodamine-stainedactin(right),

Discussionwithfriends

1. Describewaysoffixationandtheirmechanisms.Whenyouwanttoassaythetrypsinactivityincells,canyouusethefixation?Explainthereasonofyouranswer.

2. Pleasedescribefluorescencequenching.Howcanyouapplythequenchingtostudythecellbiology?Findthepaper,‘NuclearTranslocationofCAM-AssociatedProteinActivatesTranscriptionforLong-TermFacilitationin Aplysia’andseetheSupplementaryfigure3(FRAPassay).

3. Pleasefindwhatisthe‘CLARITY’.Whichtypeofmicroscopeisthebestforitandwhy?

4. ThinkanddescribethedisadvantageofSTEDmicroscopecomparedtousualconfocalmicroscope.

참고 (etc)

Fluorescencerecoveryafterphotobleaching (FRAP)revealsthedynamicsofmolecules.

Super-resolutionmicroscopycangeneratelight-microscopeimageswithup-to-nanometerresolution.

Super-resolutionmicroscopycangeneratelight-microscopeimageswithup-to-nanometerresolution.

Super-resolutionmicroscopycangeneratelight-microscopeimageswithup-to-nanometerresolution.