growing plhc1 cells in culture

8/18/2019 Growing PLHC1 Cells in Culture

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Growing Cells in Culture

Part 1: Terminology


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Cell CultureCell Culture

• Pros

• Use of animals reduced

• Cells from one cell line are homogenous andhave same growth requirements, optimizinggrowing patterns.

• In vitro models allow for control of the

extracellular environment• Able to monitor various elements and

secretions without interference from otherbiological molecules that occurs in vivo

The maintenance of cells outside of the living animal in

vitro! for easier experimental manipulation and regulationof controls.


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• Cons• "emoval of cells from their in vivo environment

means removing the cells, hormones, supportstructures and various other chemicals that thecells interact with in vivo.

• #t is nearl$ impossible to recreate the in vivo environment. The artificial conditions couldcause cells to de%differentiate which will causethem to behave differentl$ and produceproteins other than it would in vivo. & Genotype: the genetic ma'e%up of the cell

& Phenotype: the appearance and behavior of a cellas a result of their genot$pe. (ost often, scientistsare loo'ing at phenot$pic changes in their anal$sisof cells in culture


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Classification of Cell

Cultures• Primary Culture

& Cells ta'en directl$ from a tissue to adish

• Secondary Culture

& Cells ta'en from a primar$ culture andpassed or divided in vitro.

& These cells have a limited number ofdivisions or passages. After the limit,the$ will undergo apoptosis.• Apoptosis is programmed cell death


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)rimar$ culture from Poeciliopsis

lucida the desert topminnow!


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(a'ing a )rimar$ Culture


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• Cell Line

& Cells that have undergone a mutation andwon*t undergo apoptosis after a limitednumber of passages. The$ will growindefinitel$.

• Transformed cell line & A cell line that has been transformed b$ a

tumor inducing virus or chemical. Can causetumors if in+ected into animal.

• Hybrid cell line (hybridoma)

& Two cell t$pes fused together withcharacteristics of each

Cell Lines


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ur Cell -ine

• )-C%/ )oeciliopsis lucida topminnow!

• epatocellular carcinoma

• riginiall$ from the liver so the$ are

0hepatoc$tes1

• 2pithelial cells

• ATCC C"-%3456

• http788www.atcc.org8

• -awrence 2. ightower*s lab, in culture since

/9:;.

http://www.atcc.org/http://www.atcc.org/


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• An immortal cell line, but not tumorogenic,

will reach contact inhibited state

• riginall$ used to stud$ heat shoc' response

• These cells maintain a number of

differentiated cell functions of hepatoc$tes.

The cells possess inducible and stablec$tochrome )4;5 Cot 'nown to harbor an agent 'nown to

cause disease in humans

ur Cell Line


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Part !: "nderstanding Cell

#eha$ior


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Confluency

• ow 0covered1 the growingsurface appears

• This is usuall$ a guess

• ptimal confluenc$ for

moving cells to a new dish is

?5%:5@

& too low, cells will be in lag

phase and won*t proliferate

& Too high and cells ma$

undergo unfavorable changes

and will be difficult to remove

from plate.


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Contact %nhibition

• hen cells contact

each other, the$ cease

their growth.

• Cells arrest in B5 phaseof the cell c$cle

• Transformed cells will

continue to proliferate

and pile upon each

other


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&nchorage 'eendence

• Cells that attach to surfaces in vivo require

a surface to attach to in vitro.

& ther cells or speciall$ treated plastic or other

biologicall$ active coatings

• lood cells are primar$ exception.

• Transformed cells ma$ not require

attachment.


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Passage number

• The number of times the cells have been

removed or 0split1! from the plate and re%

plated.• Alwa$s write this on $our plate or flas' as

)D


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Part : Solutions used in cell

culture


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Phoshate #uffered Saline * Ca!+

,g!+ -ree (P#S)

• Used to wash8remove excess serum

that inhibits the function of Tr$psin%

2ETA.

• Calcium will also inhibit the function of

T"2E.

• (ust be warmed in the water bathbefore use so cells are not shoc'ed b$

cold liquid.


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Trysin .'T&• An enz$me used to detach the cells from a

culture dish.

• Tr$psin cleaves peptide bonds -


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Tryan #lue

• An exclusion d$e

• -iving cells cannot ta'e up the d$e and will

appear bright and refractile.

• Eead cells with bro'en membranes will

absorb the d$e and appear blue.

• Usuall$ add 355 µl of tr$pan blue to 355 µlof cell suspension in eppendorf tube


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#leach

• Used to destro$ an$ remaining cells in

dishes and tubes before the$ are

tossed in the trash can.

• Add enough to change media to clear,

& wait ; minutes,

& rinse solution down sin'

& throw awa$ the dish8flas'8plate in the trash

can.


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Growing Cells in CultureGrowing Cells in Culture

Part / : .0uiment


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C! incubator

• maintains C3

level ;%/5@!,

humidit$ and

temperature G?o

C! to simulate in

vivo conditions.


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ater bath

• To warm media, T"2Eand )F before placing

on cells

• Can harbor fungi and

bacteria, spra$ all items

with ?5@ ethanol before

placing in the hood.

• Usuall$ ta'es /5 %/;minutes for media to

warm, ;%/5 for T"2E to

thaw


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2acuum um

• =or permanent

aspiration of liquids

media, )F and

T"2E!.

• Use unplugged

glass pasteur

pipets, throw intosharps box when

done.


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%n$erted Phase ,icroscoe

• A phase contrast

microscope with

ob+ectives below the

specimen.• A phase plate with an

annulus will aid in

exploiting differences in

refractive indices indifferent areas of the cells

and surrounding areas,

creating contrast

,echanics of hase,echanics of hase


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,echanics of hase,echanics of hase

microscoymicroscoy

Shifting of phase by ½ a wavelength

Add and subtract amplitudes to create

more contrast


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A comparison

Phase contrast microscopy Light microscopy

Can be used on living cells requires stain, thus killing cells


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#asic cell culture

instructions


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&setic Techni0ue

• =or best results in tissue culture, we want to wor' to'eep microbial bacteria, $east and molds!contamination to a minimum. To do this, there are certain

things $ou must be aware of and guidelines to follow.• or' in a culture hood set%aside for tissue culturepurposes. (ost have filtered air that blows across thesurface to 'eep microbes from settling in the hood. Turnoff the UH8antimicrobial light and turn on the hood G5minutes prior to entering the hood.

• ear short sleeves or roll $our sleeves up. Turn $ourbaseball caps bac' if $ou (UFT wear them, tie long hairbac' and remove rings and watches.


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• ash hands with soap and water beforebeginning the procedure and rewash if $ou touchan$thing that is not sterile or within the hood.

• Fpra$ down $our hands, wor' surface, andan$thing that will go into the hood with ?5@ethanol. "ewipe at intervals if $ou are wor'ingfor a long time in the hood. This will reduce thenumbers of bacteria and mold considerabl$.

• Eo not breathe directl$ into $our cultures, bottlesof media, etc. This also means to 'eep tal'ing toa minimum. >o singing or chewing gum.


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• or' as quic'l$ as $ou can within limits of $ourcoordination. Also, 'eep bottles and flas's closed when$ou are not wor'ing with them. Avoid passing $our arm orhand over an open bottle.

• Use onl$ sterilized pipets, plates, flas's and bottles in thehood for procedures.

• Ta'e special precautions with the sterile pipets. "emovethem from the pac'age +ust before use. (a'e certain toset up the numbers on the pipet so that the$ face $ou.

>ever mouth%pipet, use the pipetting aid. Change pipetsfor each manipulation. #f the tip of the pipet touchessomething outside of the flas' or bottle, replace with anew one. >ever use a pipet twice.


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#asic Cell Culture Procedure for

&nchorage 'eendent Cells

• Hiew cells using inverted phase microscope• Asepticall$ aspirate media• "inse media with )F

• Add Tr$psin%2ETA to cells• Aspirate Tr$psin%2ETA• #ncubate cells with la$er of Tr$psin%2ETA at G?I

C

• "esuspend cells with fresh media• Ta'e sample and count cells• Calculate how man$ cells are needed to add to

new plate or flas'


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"emember

• Some $olumes don3t need to be e4actin cell culture

• "insing volume of )F as long as it fits in the dishand is sufficient to rinse the serum!.

• Holume of tr$psin 2ETA as long a bottom of plate orflas' can be covered.

• Holume of media used to resuspend $our cells. Thesame number of cells will be there despite the volumeof media used.

& Too little resuspension media will result in ver$ high cell count andwould require more dilution and higher dilution factor!. The volumeneeded to seed $our next plate would then be ver$ small, ma$be toosmall to wor' with.

& Too much media would result in low cell count8ml and $ou ma$ need alarge volume to add to $our new plate.


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• Holume of cells removed for cell counting.

&


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Troubleshooting -ow

emac$tometer Counts


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Tr$psinization not complete

• Tr$psin is ineffective

& too cold, be sure to warm sufficientl$

& self digested or expired chec' date, donJt

warm too long

& too much serum left on plate rinse

plate thoroughl$ with )F


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Tr$psinization technique

• Tr$psin doesnJt coat plate, completel$ add full 3mls, la$ flas' down, count to /5, then remove

• tr$psin left on plate too long and thenaspirated...cells removed along with tr$psin

• not left long enough in incubator depends on cellline GTG%-/ can go /%; minutes

• flas' ma$ need to be tapped or slapped tofacilitate cell removalthis varies b$ cell line, but o' for GTGs!


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"esuspension technique

& too much media added more media results in lowcell8ml, but overall cells on plate should remain thesame

& cells not spra$ed off surface properl$

& media and cells not pipetted gentl$! up and down G%4times to brea' up clumps

& too long of time before retrieving sample from flas'

cells ma$ settle!. After mixing with tr$pan, donJt waittoo long before loading hemac$tometer. Bethemac$tometer read$ while tr$psinizing cells inincubator


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Ftubborn cells

• cells left on plate a long time K4 da$s! will

be more difficult to remove

• ver$ confluent plate will require more

aggressive tr$psinization because tr$psin

cannot recach plate surface effectivel$


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5eeing a good lab

noteboo6


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• -ab noteboo's provide a convenient place for $ou to'eep all of $our procedures, data and observations inone place.

• #f written well, a lab noteboo' should contain ever$thing

$ou need to 'now to allow $ou or someone else torepeat an$ experiment $ou have ever performed.

• #t can be useful in finding the source of errors andunexpected results when problems arise.

• Fhould $our wor' ever be disputed, a lab noteboo' willprovide testimon$ to $our research.

• $ following the simple guidelines below, $ou will learnhow to 'eep a good lab noteboo'.


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• The noteboo' should be bound no spiral noteboo's,please!.

• The pages should be numbered either b$ hand orpreprinted before using the boo'.

• Use onl$ permanent in'.• rite $our name, contact information, and dates the

noteboo' covers on the first page.

• F'ip the next 3%G pages for a Table of Contents. =ill in

the experiment name and page numbers as the$ arecompleted.

• rite the date, experiment title, and partner*s name atthe top of each page.

f


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The first time $ou use a

procedure• rite the whole procedure in $our own words

into the noteboo' " tape in the t$ped version• #nclude a reference to the lab manual page or

the published procedure.

• >ote an$ changes made to the originalprocedure.

• Eo not +ust cop$ the lab manual or procedureword for wordL restate each step simpl$ and

clearl$.• #f $ou repeat this procedure later, reference the

page where it was first performed and writedown an$ changes made.


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• All data and observations should be written in$our noteboo' at the time $ou too' themeasurement. Eo not write on scratch paper tobe copied later into $our noteboo' & little piecesof paper ma$ be lost and data forever lost.

• "emember $our lab noteboo' isextemporaneous writing. Meep it neat but do notwaste too much time ma'ing it perfect. 2rrorsshould be crossed out with a single lineexample!. Eo not scribble out mista'es.


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• rite down all calculations, no matter how simple, in$our noteboo'. =or example, ever$ time $ou perform acell count, cell viabilit$ must be calculated and recorded.

• )ermanentl$ attach glue or tape! images, computerprint outs, and other data in $our noteboo'. Eate andinitial over the corner of the attachment. e sure to labelthe image with an$ pertinent information. N=or example, if$ou place a estern lot image into $our noteboo', labelthe lanes with what was in each, and the gelcomposition. #f the l$sates were prepared on a date

different from the date the gel was run ma'e a referenceto the page that contains information on how the l$sateswere made.O )artners ma$ photocop$ original data forinclusion in the lab noteboo'.


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• #ncluding complete chemical equations, statisticalequations, sample calculations, and s'etches or bloc'diagrams of an$ apparatus used is also good practice.

• "ecord start and stop times.

• #nclude conclusions from this data. hat does it meanand did it wor' as expectedP #f unexpected results occur,explain wh$. #nclude expected values with reference!where appropriate.

• Eo not s'ip pages. Use ever$ page of the noteboo'. #f$ou need to rewrite a page, draw a large Q through thepage, date, initial, and start over on the next page. Thesame applies if $ou don*t fill an entire page draw a linethrough the remaining space, date, and initial.


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Fix 2ssential Calculations


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Hemacytometer

• Fpecialized chamberwith etched grid

used to count the

number of cells in asample.

• use of tr$pan blue

allows differentiationbetween living and

dead cells


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"sing the Hemacytometer

• "emove the hemac$tometerand coverslip carefull$! from

2t and dr$ thoroughl$ with a

'imwipe.

• Center coverslip on

hemac$tometer

• arel$ fill the grid under the

coverslip via the divet with $our

cell suspension.

• Count cells in ten squares ; on

each side! b$ following diagram

at station.


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Loo6ing atthe grid

under the

hase

contrast

microscoe


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ow the cells will appear • right refractile 0spheres1 are

living cells,

• lue cells about the same size

as the other cells are dead.

• Meep a differential count ofblue vs. clear for viabilit$

determination.

• Fometimes there will be serum

debris, and this will loo' red orblue and string$ or glopp$%%

don*t count itR

These are blood cells,

You will not have this many


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Count 17 s0uares

&ny 17 will do but we

will follow con$ention

atch for stringy8 reddish

material9those aren3t cellsserum


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To grou

Count cells that

touch to and

left lines

' ;TCount cells thattouch bottom and

right lines


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#ottom Grou

C l l t ll < l


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Calculate your cells


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'etermine your ercent $iability

• Hiabilit$ is a measure how man$ of $our

cells survived $our cell culture technique.

D of viable living! cells x /55

total number of cells counted

ur example ;4863 x /55 S:?.59@

C l l t t t l = f ll i


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Calculate total = of cells in

original susension

>umber of cells per ml x total mls of original suspension

Let3s assume 17ml original susension

1>!/ 417? 4 17 @1>!/ 4 17A cell total

Total = of $iable cells a$ailable in original susension

Total number of cells in original suspension x @ viabilit$/.34x/56 x :?@ S/.5:x /56 viable cells in the original suspension

' t i th b f ll


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'etermine the number of cells

you need to add to your flas6

•


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&n .4ercise&n .4ercise•


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Hessel GTG%-/ final count

/: hour doubling rate

G.;cm or 6 well plate /x/56

6cm dish or T3; flas' 3 x/56

/5cm dish ; x /56

'etermine how many mls of cell


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y

susension much to add to your

flas6

D of cells needed

cells8ml

'etermine total = mls fresh media


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you will need to add to dish or

flas6• Use table in H#FTA to see how man$ mls

will fit in $our flas' or we will tell $ou!.

Holume flas' will hold & mls suspension to

$ou plan to add


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Part ?: The rotocol

bser$ing cells in culture


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bser$ing cells in culture

• Chec' color of media

& ealth$ growth usuall$ leaves media

slightl$ orange

& Too $ellow means bacterial growth

& Too purple means low carbon dioxide, cells

dead

• bserve cells under phase microscope

& Fpread out or roundedP

& ow confluentP


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hat to do with growing cells

• #f the$ are at least ?5%:5@ confluent

• Fubculture them

& Also called passing or

splitting

• "emove media, remove

cells, resuspend and

transfer some to a newplate

• #f the$ are not ver$confluent

• -ift and replace onto same

plate

& Culture more than 4 da$s old

for our cells

& "emove old media, lift cells

from plate and resuspend in

fresh media on same plate• 0=eed1 them

& Culture less than 4 da$s old

& "emove old media and replace

with fresh, warm media


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#rief subculturing re$iew

• "emove media, lift cells from plate

• "esuspend cells in fresh media

• Count cells and determine viabilit$

• Feed new plates with appropriate D of

cells and volume of media

Fome volumes that do not need to beFome volumes that do not need to be


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exactbut follow ourexactbut follow our

recommendations until $ou arerecommendations until $ou arecomfortablecomfortable

• "insing volume of )F

• Holume of tr$psin 2ETA

• Holume of media to resuspend cells

& Record how much

• Holume of cells removed for counting

• 2xact D of cells to be plated

ill d t t t


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ou will need to return to

ta6e care of your cells

• Thursda$ or =rida$ is an in between

point before next wee'.

• =irst time through ma$ require up to anhour

• #f one member cannot ma'e the return

time, that person should wor' in hoodtonight.

• Choose times that will be consistent

growing plhc1 cells in culture

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