KC-5265

293T-cyno-EPCAM Cell Line

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Background of 293T-cyno-EPCAM Cell Line

EPCAM (Epithelial Cell Adhesion Molecule), also known as CD326, is a transmembrane glycoprotein that mediates Ca²⁺-independent homotypic adhesion in epithelial tissues. It is highly expressed on the basolateral surface of most normal epithelial cells and is markedly overexpressed in a wide range of carcinomas (e.g., colorectal, breast, and ovarian cancers). Beyond its role in adhesion and signaling, EPCAM serves as a definitive marker for isolating circulating tumor cells (CTCs), enabling liquid biopsy-based cancer diagnostics and monitoring. Therapeutically, it is a validated target for bispecific antibodies (e.g., catumaxomab) and CAR-T cell therapies, leveraging its dense surface expression on tumors to direct potent anti-tumor immune responses.

Specifications

Catalog Number	KC-5265
Cell Line Name	293T-cyno-EPCAM Cell Line
Clone Number	5-4#
Host Cell Line	293T
Description	Stable 293T cell line expressing exogenous cyno-epcam 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 +1μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:8 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 30 hours
Mycoplasma Status	Negative

Cell Line Generation

293T-cyno-EPCAM cell line was generated using lentivirus expressing cyno-EPCAM sequence.

Characterization

Figure 1. Characterization of cyno-EPCAM over-expression in the 293T-cyno-EPCAM stable clone using FACS.

Figure 2. Characterization of cyno-EPCAM over-expression in the 293T-cyno-EPCAM stable clone using PCR.

Cell Resuscitation

Prewarm culture medium (DMEM supplemented with 10% FBS, 1μg/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 1:4-1:8 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

Yu X, Ge N, Guo X, Shen S, Liang J, Huang X, Wan S, Xing J, Huang Q, Yang Y. Genetic variants in the EPCAM gene is associated with the prognosis of transarterial chemoembolization treated hepatocellular carcinoma with portal vein tumor thrombus. PLoS One. 2014 Apr 9;9(4):e93416. doi: 10.1371/journal.pone.0093416. PMID: 24718422; PMCID: PMC3981717.
Beta M, Khetan V, Chatterjee N, Suganeswari G, Rishi P, Biswas J, Krishnakumar S. EpCAM knockdown alters microRNA expression in retinoblastoma--functional implication of EpCAM regulated miRNA in tumor progression. PLoS One. 2014 Dec 12;9(12):e114800. doi: 10.1371/journal.pone.0114800. Erratum in: PLoS One. 2015 Apr 10;10(4):e0119745. doi: 10.1371/journal.pone.0119745. PMID: 25502397; PMCID: PMC4264963.