REF: P10361-IM

NEUROSCIENCE INNOPROFILETMIMMORTALIZED HUMAN BRAIN MICROVASCULAR ENDOTHELIAL CELLS
Product Type:

Immortalized Brain Endothelial Cells

Catalog Number: P10361-IM
Immortalization:

SV40 Large T Antigen

Number of cells:> 1 x106 cells in Cryopreserved vials

Storage:

Liquid NitrogenP10361ǦIM have been obtained immortalizing
Human

PrimaryBrain

MicrovascularProductCharacterization
Positivefor:

Endothelial Cells with LentiǦSV40 Lentivirus.
Immortalized cells were controlled passagingsidebysidewiththeprimarycells.Primarycellsgointosenescenceafterthe3rdpassagewhile

ovWF/FactorVIIIoCD31(PǦCAM)

oVECadherin

Negativefor:

the SV40Ǧtranduced cells go beyond 30

x

Cytokeratin18

passages.

xCytokeratin19The Brain MEC are the major element of thebloodǦbrain barrier and comprise the primarylimitation to passage of substances, both

soluble and cellular, from the blood into thebrain.BrainMECutilizesuniquefeaturesthat

The cells test negative for HIVǦ1, HBV, HCV,distinguish themselves from those of

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

peripheral endothelial cells. Like peripheral

endothelialcells,however,brainMECexpress,ProductUse

or can be induced to express, cell adhesionmolecules on their surface that regulate theSaleofthisitemissubjectedtothecompletion

extravasation of leukocytes into the brain. In

ofaMaterialTransferAgreement(MTA)bytheviewoftheorganspecificityofMEC,BrainMEC

purchasing individual/institution for eachhas been widely used for studying theorder.Ifyouhaveanyquestionsregardingthis,molecular and cellular basis of bloodǦbrainpleasecontactusatinnoprot@innoprot.com

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

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




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:

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 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. Prepare a fibronectin coated flask (2Ɋg/cm2, TǦ75 flask is recommended).
Add 10 ml of sterile Dulbecco’s
phosphate buffered saline (DPBS,
Ca++ and Mg++ free) to a TǦ75 flask
and then add 150 Ɋl of fibronectin
stocksolution(1mg/ml,Innoprotcat.
no. 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 resuspendthecontentsofthevial.
5. Dispense the contents of the vial intothe equilibrated, fibronectin coated
culture vessels. A seeding density of
10,000cells/cm2isrecommended.

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

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 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; 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
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