REF: P10880-IM

OCULAR CELL SYSTEM INNOPROFILETMIMMORTALIZED HUMAN RETINAL ENDOTHELIAL CELLS

Cell Type:

Cryo-preserved Endothelial Cells

Catalog Number: P10880-IM

Ref:

P10880‐IM

Immortalization:

SV40 Large T Antigen

Source:Human Retinal Tissue

Number of Cells:

5 x 105 Cells / vial (1ml)

Storage:

Liquid Nitrogen

have

been

obtained

immortalizing Human Retinal Endothelial Cells

Recommended Mediumwith Lenti‐SV40 Lentivirus. Immortalized cells

Endothelial Cell Medium(Reference: P60104)

were controlled passaging side by side withthe primary cells. Primary cells go into
senescence after the 4th passage while theSV40‐tranducedcells

gobeyond30passages.t.

The eye contains highly vascularised andcompletelyavasculartissues

incloseapposition. This specialized anatomy requires
tight regulation of the balance between
vascular quiescence and vascular growth.
Retinal vascularization is a coordinatedinteraction

ofvascularcells,

includingendothelial cells, pericytes, and astrocytes,

Product Characterization
Immunofluorescent method
oCD31 (P‐CAM

Functional Assay: Tubule Formation Assay
The cells test negative for HIV‐1, HBV, HCV,
mycoplasma, bacteria, yeast and fungi

and a balanced production of positive andnegative regulatory factors. In eye diseases
associated with angiogenesis, this delicate
balance is disturbed. Angiogenesis plays acrucial role in disorders responsible for mostblindness.

Product Use
THESE PRODUCTS ARE FOR RESEARCH USE
ONLY. Not approved for human or veterinaryuse, for application to humans or animals, orfor use in vitro diagnostic or clinicalprocedures

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 CELLSIMPORTANT: Cryopreserved cells are very delicate. Thaw the vial in a 37 ºC waterbathand return them to culture as quickly as possible with minimal handling!
Set up culture after receiving the order:
1. Prepare a fibronectin coated flask (2μg/cm2, T‐75 flask is recommended).
Add 10 ml of sterile Dulbecco’s
phosphate buffered saline (DPBS) toa T‐75 flask and then add 150 μl offibronectin stock solution (1 mg/ml,Innoprot cat. no. P8248). Leave theflask in incubator overnight.
2.

Preparecompletemedium:
decontaminate the external surfacesof medium and medium supplementswith 70% ethanol and transfer themto sterile field. Aseptically open eachsupplement tube and add them tothe basal medium with a pipette.
Rinse each tube with medium torecover the entire volume.

3. Aspirate fibronectin solution and add
20 ml of complete medium to theflask. Leave the flask in the hood andgo to thaw the cells. The fibronectinsolution can be used twice.
4. Place the vial in a 37 ºC waterbath, holdand rotate the vial gently until thecontents are completely thawed.
Remove the vial from the waterbath
immediately, wipe it dry, rinse the vialwith 70% ethanol and transfer it to asterile field. Remove the cap, being
careful not to touch the interior
threads with fingers. Using a 1 ml
eppendorf pipette gently re‐suspend
the contents of the vial.
5. Dispense the contents of the vial into
the equilibrated, fibronectin coatedculture vessels. A seeding density of
6,000 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

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 areplated in fibronectin coated flask that
promotes cell attachment andgrowth.
6. Replace the cap or cover, and gently
rock the vessel to distribute the cells
evenly. Loosen cap if necessary topermit gas exchange.
7. Return the culture vessels to theincubator.
8. For best result, do not disturb theculture for at least 16 hours after theculture has been initiated. Change
the growth medium the next day toremove the residual DMSO andunattached cells, then every otherday thereafter. A healthy culture will
display cobblestone or spindle
shaped morphology, non‐granularcytoplasm and the cell number will
be double after two to three days inculture.

Maintenance of Culture:
1.

Change the medium to fresh
supplemented medium the nextmorning after establishing a culture
from cryopreserved cells.

2. Change the medium every three days
thereafter, until the culture isapproximately 70% confluent.
3.

Once the culture reaches 70%
confluence, change medium every
other day until the culture isapproximately 90% confluent.

Subculture:
1. Subculture the cells when they are over
90% confluent.
2. Prepare fibronectin coated flasks (2μg/cm2) one day before subculture.
3. Warm medium, trypsin/EDTA solution(T/E, Solution) trypsin neutralization
solution (TNS), and DPBS to room
temperature. We do not recommend
warming the reagents and medium at37ºC waterbath prior to use.

4. Rinse the cells with DPBS.
5. Add 10 ml of DPBS first and then 2 ml oftrypsin/EDTA solution into flask (inthe case of T‐75 flask); gently rockthe flask to make sure cells arecovered by trypsin/EDTA solution;
incubate the flask at 37oC incubatorfor 1 to 2 minutes or until cells arecompletely rounded up (monitoredwith inverted microscope). During
incubation, prepare a 50 ml conicalcentrifuge tube with 5 ml of fetal
bovine serum (FBS); 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 oftrypsinisation, one hand hold one
side of flask and the other handgently tap the other side of the flask
to detach cells from attachment;
check the flask under inverted
microscope to make sure all cells aredetached, add 5 ml of trypsin
neutralization solution to the flaskand transfer detached cells to the 50ml centrifuge tube; add another 5 mlof TNS to harvest the residue cells
and transfer it to the 50 ml centrifuge
tube. Examine the flask under
inverted microscope to make surethe cell harvesting is successful bylooking at the number of cells left
behind. There should be less than 5%.

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

Note: DPBS, trypsin/EDTA solution &trypsin neutralization solution are
included in the “Primary Cells Detach
Kit provided by Innoprot (Cat. NºP60305).
6. Centrifuge the 50 ml centrifuge tube
(harvested cell suspension) at 1000rpm (Beckman Coulter Allegra 6R
centrifuge or similar) for 5 min; re‐suspend cells in growth medium.
7. Count cells and plate cells in a new,fibronectin coated flask with celldensity as recommended

Caution: Handling human derived products is
potentially bioharzadous. Although each cellstrain testes negative for HIV, HBV and HCVDNA, diagnostic tests are not necessarily 100%
accurate, therefore, proper precautions mushbe taken to avoid inadvertent exposure. Always
wear gloves and safety glasses when working
these materials. Never mouth pipette. We
recommend following the universal proceduresfor handling products of human origin as theminimum precaution against contamination [1].
[1]. Grizzle, W. E., and Polt, S. S. (1988)
Guidelines
toavoidpersonal
contamination by infective agents inresearch laboratories that use humantissues. J Tissue Culture Methods.
11(4).