Conditionally Immortalized Human Retinal Pigment Epithelial Cells

Name of Cells: Conditionally Immortalized Human Retinal Pigment Epithelial Cells

Catalogue Number: (HRPEs) UBP-0063

Product Format: Frozen Vial

Cell Number: > 5 x 10⁵cells/vial

General Information: HRPEs (UBP-0037) are initiated by dissecting Retinal Pigment tissue and digestion with collagenase. CI-HRPEc (UBP-0063) are selected from Puromycin resistant HRPEs after infected with lentiviruses expressing SV40 LT under the control of TetON system. CI-HRPEs are maintained with proliferative capacity in the presence of Doxycycline^NOTE1 in HRPEs growth medium (HRPEs growth medium, contains 10% serum and growth supplements, UBP-33).

 Characterization of the cells :HRPEs are positive for REPs-specific markers CRALBP and RPE-65 HRPEs are negative for HIV-1, HBV, HCV, and mycoplasma.

Product Use: HRPEs are for research use only ^NOTE3. Shipping: Frozen Vials in Dry Ice packages.

Handling of Arriving Cells :When you receive the cells in a frozen vial, you can transfer the vial of cells into a -80ºC freezer for short-period storage or a liquid nitrogen tank for long-term storage.  Thaw the cells in a 37°C water bath, and then quickly transfer the cells into a T75 flask with 15 ml MSCGM in the presence of 1ug/ml of Doxycycline and incubated overnight in a 37ºC^NOTE2 CO2 incubator and change the medium next day (15 ml complete MSCGM) and every other thereafter.

Subculture Protocol: 

 Rinse the cells in T75 flask with 15ml HBSS (Room Temperature, RT) twice.

1. Add 4ml of Universal Detachment Solution (RT) (UBP-23) into one T75 flask (make sure the whole surface of the T75 flask is covered with Universal Detachment Solution), and gently dispose the excessive Universal Detachment Solution within 30 seconds with aspiration.
2. Leave the T75 flask with the cells at RT for 1 minute (the cells usually will detach from the surface within 1-2 minutes). You can monitor the cells under microscope and when most of cells become rounded up, hit the flask against the bench surface, and the cells will move on the surface of the flask when monitoring under microscope.
3. Add 10ml Trypsin Neutralization Buffer and spin the cells down with 800g for 5 minutes.
4. Re-suspend the cell pellet with 30-45 ml of MSCGM in the presence of 1ug/ml Doxycycline and the cell suspension is transferred directly into 2 or 4 pre-coated T75 flasks (15ml each, and the cells are sub-cultured at 1:2 or 1:3 ratios)
5. Change medium every 2-3 days and cells usually become confluent within 7 days (when split at a 1:3 ratio).

NOTE 1:  To minimize the effect of SV40 LT antigen for your studies, cells could be cultured in the absence Doxycycline for 3-5 days before your final experiments.

NOTE 2: Although wild type SV40 LT antigen is used for cell immortalization, we noticed that the immortalized cells are growing better at 33-34ºC, we encourage the end users to switch to 33-34ºC if cells are growing slower at 37ºC.

NOTE 3: The purchase of this product conveys to the buyer the nontransferable right to use the purchased amount of the product and all replicates and derivatives for research purposes conducted by the buyer in his laboratory only (whether the buyer is an academic or for-profit entity). The buyer cannot sell or otherwise transfer (a) this product (b) its components or (c) materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes.