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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Growing Cells in Culture
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 $our plate or flas' as
)D
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Growing Cells in Culture
Part : Solutions used in cell
culture
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Phoshate #uffered Saline * Ca!+
,g!+ -ree (P#S)
• Used to wash8remove excess serum
that inhibits the function of Tr$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
$ou plan to add
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Growing Cells in Culture
Part ?: The rotocol
bser$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
top related