KC-1416

CT26-GPC3-Cell-Line

22097

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Background of CT26-GPC3-Cell-Line

GPC3 is a family of heparan sulfate proteoglycans that are composed of a membrane-associated protein core substituted with a variable number of heparan sulfate chains and attached to the cell surface by a glycosylphosphatidylinositol (GPI) anchor. GPC3 play a role in the control of cell division and growth regulation. GPC3 is also found to be overexpressed in differentiating hepatocellular carcinoma (HCC) and dysplastic changes in cirrhotic livers, which might have the utility for diagnosis or treatment in cancer patient.

Specifications

Catalog Number	KC-1416
Cell Line Name	CT26-GPC3-Cell-Line
Host Cell Line	Mouse CT26 cell line
Description	CT26.WT cell line stable expressing exogenous human GPC3 gene
Quantity	Two vials of frozen cells (≥2-10⁶/vial)
Stability	Stable in culture over a minimum of 10 passages
Application	Drug screening and biological assays
Freezing Medium	70% DMEM+20% FBS+10% DMSO
Propagation Medium	DMEM+10%FBS+10µg/mL Puromycin
Selection Marker	Puromycin
Morphology	Fibroblast
Subculture	Split saturated culture 1:4-1:10 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 24 hours
Mycoplasma Status	Negative
In Vivo Validation	Yes

Cell Line Generation

CT26.WT human GPC3 cell line was generated using lentiviral vector expressing human GPC3 sequence.

Characterization

Cell Resuscitation

Prewarm culture medium (DMEM + 10% FBS + 10µg/mL Puromycin)in a 37°C water bath.
Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
Incubate the flask at 37°C, 5% CO₂ incubator.
Split saturated culture 1:4-1:10 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% DMEM + 20% FBS + 10% DMSO) fresh immediately before use.
Keep the freezing medium on ice and label cryovials.
Transfer cells to a sterile, conical centrifuge tube, and count the cells.
Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
Resuspend the cells at a density of at least 3×10⁶ cells/mL in chilled freezing medium.
Aliquot 1 mL of the cell suspension into each cryovial.
Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
Transfer vials to liquid nitrogen for long-term storage

References

Ishiguro, Takahiro, Yuji Sano, Shun-Ichiro Komatsu, Mika Kamata-Sakurai, Akihisa Kaneko, Yasuko Kinoshita, Hirotake Shiraiwa, et al. 2017. ¡ùAn Anti-Glypican 3/CD3 Bispecific T Cell-Redirecting Antibody for Treatment of Solid Tumors.¡ì Science Translational Medicine 9 (410). American Association for the Advancement of Science: eaal4291. doi:10.1126/scitranslmed.aal4291.