REF: P10453-IM

CARDIAC INNOPROFILETMIMMORTALIZED HUMAN CARDIAC FIBROBLASTS
Product Type:

Immortalized Human Cardiac Fibroblasts

Catalog Number: P10453-IM
Immortalization:

SV40 Large T Antigen

Number of cells:> 1 x106 cells in Cryopreserved vials

Storage:

Liquid NitrogenP10453ǦIM have been obtained immortalizing

RecommendedMedium

HumanPrimaryCardiacFibroblastswithLentiǦ

FibroblastMediumǦ2(Ref:P60166)

SV40 Lentivirus. Immortalized cells wereoRef:P60166

controlled passaging side by side with theprimarycells.Primarycellsgointosenescenceafter the 3rd passage while the SV40Ǧ



tranducedcellsgobeyond30passages.

The cardiac fibroblasts (CF) are the principal

celltypeoftheheart.Theyprovidestructuralsupport for cardiac myocytes and are

responsible for extracellular matrix synthesisinThe cells test negative for HIVǦ1, HBV, HCV,

theheart

during

growthand

pathophysiological conditions. CF are an

mycoplasma,bacteria,yeastandfungiimportant cellular component of myocardial

responsestoinjuryandthesourceofparacrinegrowth factors. Their proliferation andsynthesis of matrix is essential for scar
formation at sites of myocardial infarction,
cardiac fibrosis, and is often complicated by
cardiac hypertrophy . CF culture has been
widely used as a model to study the cardiac
matrix remodeled by physiological (exercise)
andpathological(hypertension)stressors.

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ProductUseSaleofthisitemissubjectedtothecompletion
ofaMaterialTransferAgreement(MTA)bythepurchasing individual/institution for eachorder.Ifyouhaveanyquestionsregardingthis,
pleasecontactusatinnoprot@innoprot.com










INSTRUCTIONSFORCULTURINGCELLS
IMPORTANT:Cryopreservedcellsareverydelicate.Thawthevialina37ºCwaterbathandreturnthemtocultureasquicklyaspossiblewithminimalhandling!


Setupcultureafterreceivingtheorder:
1. Prepare a polyǦLǦlysine coated flask (2Ɋg/cm2, TǦ75 flask is recommended).
Add 10 ml of sterile water to a TǦ75
flask and then add 15 Ɋl of polyǦLǦ
lysine stock solution. Leave the flaskinincubatorovernight(minimumone
hourat37ºCincubator).

2.
Preparecomplete
medium:decontaminate the external surfaces
ofmediumandmediumsupplements
with70%ethanolandtransferthemto
sterile field. Aseptically open eachsupplementtubeandaddthemtothebasal medium with a pipette. Rinse
eachtubewithmediumtorecovertheentirevolume.

3.RinsethepolyǦLǦlysinecoatedflaskwith
sterile water twice and add 20 ml ofcompletemediumtotheflask.Leavetheflaskinthehoodandgotothaw
thecells.

4.Placethevialina37ºCwaterbath,holdand rotate the vial gently until thecontents are completely thawed.
Remove the vial from the waterbath
immediately,wipeitdry,rinsethevial
with 70% ethanol and transfer it to asterile field. Remove the cap, being
careful not to touch the interiorthreads with fingers. Using 1 ml
eppendorf pipette gently resuspendthecontentsofthevial.
5. Dispense the contents of the vial intothe equilibrated, polyǦLǦlysine coated
culture vessels. A seeding density of
5,000cells/cm2isrecommended.

INNOVATIVE TECHNOLOGIES IN BIOLOGICAL SYSTEMS, S.L.

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Tel.: +34 944005355 | Fax: +34 946579925
INNOPROT

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
Note: Dilution and centrifugation of cells
after thawing are not recommended
sincetheseactionsaremoreharmfulto the cells than the effect of DMSO
residue in the culture. It is also
important that fibroblasts are platedinpolyǦLǦlysinecoatedculturevessels
thatpromotecellattachment.

6. Replace the cap or cover, and gently
rockthevesseltodistributethecells
evenly. Loosen cap if necessary to
permitgasexchange.

7. Return the culture vessels to theincubator.

8. For best result, do not disturb thecultureforatleast16hoursaftertheculturehasbeeninitiated.Changethegrowth medium the next day to
remove the residual DMSO and
unattachedcells,theneveryotherday
thereafter.


MaintenanceofCulture:
1. Change the medium to fresh
supplemented medium the nextmorning after establishing a culturefromcryopreservedcells..

2. Change the medium every three days
thereafter, until the culture is
approximately70%confluent.

3. Once the culture reaches 70%confluence, change medium every
other day until the culture is
approximately90%confluent.


Subculture:
1.Subculturethecellswhentheyareover
90%confluent.

2. Prepare polyǦLǦlysine coated flasks (2Ɋg/cm2)onedaybeforesubculture.

3. Warm medium, trypsin/EDTA solution
(T/E, cat. no. 0103), trypsin
neutralization solution (TNS, cat. no.0113),andDPBStoroomtemperature.
We do not recommend warming thereagents and medium at 37ºC
waterbathpriortouse.

4.RinsethecellswithDPBS.
5.Add8mlofDPBSfirstandthen2mlof
trypsin/EDTAsolutionintoflask(inthecase of TǦ75 flask); gently rock theflasktomakesurecellsarecoveredby
trypsin/EDTA solution; incubate theflask at 37ºC incubator for 1 to 3
minutes or until cells are completelyroundedup(monitoredwithinverted
microscope). During incubation,
prepare a 50 ml conical centrifuge
tubewith5mloffetalbovineserum;
transfer trypsin/EDTA solution fromtheflasktothe50mlcentrifugetube(afewpercentofcellsmaydetached);
continueincubatetheflaskat37ºCfor
1 minutes (no solution in the flask at
this moment); at the end of
trypsinisation,onehandholdonesideofflaskandtheotherhandgentlytap
the other side of the flask to detachcellsfromattachment;checktheflask
under inverted microscope to make
sureallcellsaredetached,add5mlof
trypsin neutralization solution to theflask and transfer detached cells to
the 50 ml centrifuge tube; add
another 5 ml of TNS to harvest theresiduecellsandtransferittothe50
mlcentrifugetube.Examinetheflask
under inverted microscope to make
sure the cell harvesting is successful
bylookingatthenumberofcellsleft
behind.Thereshouldbelessthan5%.






Note: DPBS, trypsin/EDTA solution &trypsin neutralization solution are
includedinthe“PrimaryCellsDetach
Kit provided by Innoprot (Cat. Nº
P60305).

6. Centrifuge the 50 ml centrifuge tube
(harvested cell suspension) at 1000
rpm (Beckman Coulter Allegra 6R
centrifuge or similar) for 5 min; reǦsuspendcellsingrowthmedium.

7.Countcellsandplatecellsinanew,polyǦ
LǦlysinecoatedflaskwithcelldensityasrecommended.

Caution: Handling human derived products is
potentially bioharzadous. Although each cell
straintestesnegativeforHIV,HBVandHCVDNA,
diagnostic tests are not necessarily 100%
accurate, therefore, proper precautions mushbetakentoavoidinadvertentexposure.
Always wear gloves and safety glasses when
workingthesematerials.Nevermouthpipette.
We recommend following the universal
procedures for handling products of human
origin as the minimum precaution against
contamination[1].

[1]. Grizzle, W. E., and Polt, S. S. (1988)
Guidelinestoavoidpersonalcontaminationby
infective agents in research laboratories thatuse human tissues. J Tissue Culture Methods.
11(4).



大日本住友製薬グループ〒564-0053 大阪府吹田市江の木町33番94号

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