REF: P10652-IM

HEPATIC INNOPROFILETMIMMORTALIZED HUMAN HEPATIC SINUSOIDAL ENDOTHELIAL CELLS
Product Type:

Immortalized Hepatic Endothelial Cells

Catalog Number: P10652-IM
Immortalization:

SV40 Large T Antigen

Number of cells:> 1 x106 cells in Cryopreserved vials

Storage:

Liquid NitrogenP10652ǦIM have been obtained immortalizingProductCharacterization

HumanPrimaryHepaticSinusoidalEndothelial

Positivefor:

CellswithLentiǦSV40Lentivirus.Immortalized

ovWF/FactorVIIIoCD31(PǦCAM)

oVECadherincells were controlled passaging side by sidewith the primary cells. Primary cells go into
senescence after the 3rd passage while theNegativefor:

SV40Ǧtranducedcellsgobeyond30passages.

xCytokeratin18

x

Cytokeratin19

SECaremicrovascularendothelialcellswitha
unique phenotype reminiscent of dendritic
cells and a unique function as antigenǦ
presenting cells for CD4+ T cells. Thus, SEC

representanewtypeoforganǦresident"nonǦ

professional" antigenǦpresenting cell that

The cells test negative for HIVǦ1, HBV, HCV,

appearstobeinvolvedinthelocalcontrolof

mycoplasma,bacteria,yeastandfungi

the immune response and the induction of

immune tolerance in the liver. Primary SEC

ProductUse

express wellǦcharacterized surface receptors
and differ morphologically and metabolically

Saleofthisitemissubjectedtothecompletion

from largeǦvessel endothelia. It has reportedofaMaterialTransferAgreement(MTA)bythethat SEC are dynamic regulators of porosity

purchasing individual/institution for each

that

to

order.Ifyouhaveanyquestionsregardingthis,

environmental zonal stimuli during liver

pleasecontactusatinnoprot@innoprot.com

regeneration.

respond

rapidly

and

locally
INNOVATIVE TECHNOLOGIES IN BIOLOGICAL SYSTEMS, S.L.

Parque Tecnológico Bizkaia, Edf. 502, 1ª Planta | 48160 | Derio | Bizkaia
Tel.: +34 944005355 | Fax: +34 946579925
innoprot@innoprot.com | www.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 fibronectin coated flask (2Ɋg/cm2, TǦ75 flask is recommended).
Add 10 ml of sterile DPBS (Ca++ and
Mg++free)toaTǦ75flaskandthenadd
150Ɋloffibronectinstocksolution(1
mg/ml,Ref:P8248).Leavetheflaskin
incubatorovernight.
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.Aspiratefibronectinsolutionandadd20
ml of complete medium to the flask.
Leavetheflaskinthehoodandgoto
thaw the cells. The fibronectin
solutioncanbeusedtwice.
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 a 1 ml
eppendorf pipette gently reǦsuspendthecontentsofthevial.
5. Dispense the contents of the vial intothe equilibrated, fibronectin coated
culture vessels. A seeding density of
5,000 cells/cm2 is recommended.
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.

INNOVATIVE TECHNOLOGIES IN BIOLOGICAL SYSTEMS, S.L.

Parque Tecnológico Bizkaia, Edf. 502, 1ª Planta | 48160 | Derio | Bizkaia
Tel.: +34 944005355 | Fax: +34 946579925
innoprot@innoprot.com | www.innoprot.com

It is also important that endothelial cellsareplatedinfibronectincoatedflask
that promotes cell attachment and
growth.
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. A healthy culture willdisplaycobblestoneorspindleshaped
morphology, nonǦgranular cytoplasm
and the cell number will be double
aftertwotothreedaysinculture.

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 fibronectin coated flasks (2Ɋg/cm2)onedaybeforesubculture.
3. Warm medium, trypsin/EDTA solution,
trypsin neutralization solution, 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10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 2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 minute (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%.

6. Centrifuge the 50 ml centrifuge tube
(harvested cell suspension) at 1000
rpm (Beckman Coulter Allegra 6R
centrifuge or similar) for 5 min; reǦ
suspendcellsingrowthmedium.

7. Count cells and plate cells in a new,
fibronectin 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

INNOVATIVE TECHNOLOGIES IN BIOLOGICAL SYSTEMS, S.L.

Parque Tecnológico Bizkaia, Edf. 502, 1ª Planta | 48160 | Derio | Bizkaia
Tel.: +34 944005355 | Fax: +34 946579925
innoprot@innoprot.com | www.innoprot.com（お問い合わせ窓口）
受注・発送に関するお問い合わせ
TEL 06-6990-8051 FAX 06-6325-6058
テクニカルサポート
TEL 06-6337-5896 FAX 06-6337-6020
URL : http://www.dspbio.co.jp
Eメール : dspb-ls@bio.ds-pharma.co.jp