REF: P10872-IM

OCULAR CELL SYSTEM INNOPROFILETMIMMORTALIZED HUMAN KERATOCYTES

Product Type:

Immortalized Keratocytes

Catalog Number:

P10872-IM

Immortalization:

HPV16 E6/E7 gene

Number of Cells:

1 x 106 Cells / vial (1ml)

Storage:

Liquid Nitrogen

P10872‐IM have been obtained immortalizing

Recommended Medium

Human Primary Keratocytes with HPV16 E6/E7

gene. Immortalized cells were controlled

Fibroblast Medium(Reference: P60108)

passaging side by side with the primary cells.
Primary cells go into senescence after the 6thpassagewhiletheHPV16 E6/E7 gene‐tranduced cells go beyond 50 passages.
The keratocyte, or corneal fibroblast, is ahighly specialized cell that is sandwiched
between orthogonally arranged layers ofcollagen lamellae in the corneal stroma. In theevent of a corneal injury or trauma, theProduct Characterization
Immunofluorescent method

active,oFibronectin

synthesizing cells and rapidly replace damaged

oCollagen I

keratocytes

differentiate

intostromal matrix. Cultured human keratocytes

The cells test negative for HIV‐1, HBV, HCV,

express functional IL‐4Rs and IL‐17R on cell

mycoplasma, bacteria, yeast and fungi

surface, suggesting that these cells may
contribute to the role of IL‐4 and IL‐17 as
mediators of allergic reactions in the cornea.

Product Use

Changes in gene expression were reproducibly

THESE PRODUCTS ARE FOR RESEARCH USE

observed on keratocytes after interleukin‐1ONLY. Not approved for human or veterinary

treatment which provides important insight inuse, for application to humans or animals, orgenefor use in vitro diagnostic or clinical procedures

expression

andsuggests

novel

therapeutic targets for the control of corneal
inflammation.

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 poly‐L‐lysine coated flask (2μg/cm2, T‐75 flask is recommended)
and leave the flask in incubator
overnight (minimum one hour at 37oCincubator).
2.

Preparecompletemedium:decontaminate the external surfacesof medium and medium supplementswith 70% ethanol and transfer them tosterile field. Aseptically open eachsupplement tube and add them to thebasal medium with a pipette. Rinse
each tube with medium to recover theentire volume.

3. Rinse the poly‐L‐lysine coated flask withsterile water twice and add 20 ml ofcomplete medium to the flask. Leave
the flask in the hood and go to thaw
the cells.
4. Place the vial in a 37ºC 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 1 ml
eppendorf pipette gently resuspend
the contents of the vial.
5. Dispense the contents of the vial into
the equilibrated, poly‐L‐lysine coatedculture vessels. A seeding density of
5,000 cells/cm2 is recommended.
Note: Dilution and centrifugation ofcells after thawing are notrecommended since these actions are
more harmful to the cells than theeffect of DMSO residue in the culture.

INNOVATIVE TECHNOLOGIES IN BIOLOGICAL SYSTEMS, S.L.
INNOPROT
Parque Tecnológico
Bizkaia,Edf.
502, |1ªBizkaia
Planta | 48160 | Derio | Bizkaia
Zitek-Mintegia- U.P.V.
| 48940| Leioa
946579925
Tel.: +34 944005355 | Fax: +34 946013455
innoprot@innoprot.com | www.innoprot.com

It is also important that cells are plated inpoly‐L‐lysine coated culture vesselsthat promote fibroblast attachment.

6. Replace the cap or cover, and gently
rock the vessel to distribute the cells
evenly. Loosen caps 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 thegrowth medium the next day toremove the residual DMSO andunattached cells, then every other day
thereafter. A healthy culture will
display stellate or spindle‐shaped cellmorphology, nongranular cytoplasm,
and the cell number will be doubled
after two to three days in culture.

Maintenance of Culture:
1.

Change the medium to fresh
supplemented medium the nextmorning after establishing a culture
from cryopreserved cells. For
subsequent subcultures, change
medium 48 hours after establishing
the subculture.

2. Change the medium every other day
thereafter, until the culture isapproximately 50% confluent.
3.

Once the culture reaches 50%
confluence, change medium every
day until the culture is approximately
80% confluent.

Subculture:
1. Subculture the cells when they are over
90% confluent.
2. Prepare poly‐L‐lysine coated cell cultureflasks.
3. Warm medium, trypsin/EDTA solution,trypsin neutralization solution, andDPBS to room temperature. We do
not recommend warming thereagents and medium at 37ºC
waterbath prior to use.
4. Rinse the cells with DPBS.
5. Incubate cells with 10 ml of trypsin/EDTAsolution (in the case of T‐75 flask) until
80% of cells are rounded up(monitored with microscope). Add 10
ml of trypsin neutralization solution tothe digestion immediately and gently
rock the culture vessel.
6. Harvest and transfer released cells into
a 50 ml centrifuge tube. Rinse theflask with another 10 ml of growth
medium to collect the residue cells.
Examine the flask under microscope
to make sure the harvesting is
successful by looking at the numberof cells left behind. There should beless than 5%.
7.

Centrifuge the harvested cellsuspension at 1000 rpm for 5 min andresuspend cells in growth medium.

8. Count cells and plate them in a new,poly‐L‐lysine coated flask with celldensity as 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

Caution: Handling human derived products is
potentially bioharzadous. Although each cellstrain testes negative for HIV, HBV and HCV DNA,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).