KC-1523

293T-PVRL4 Cell Line

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Background of 293T-PVRL4 Cell Line

PVRL4, also named nectin4, is a type I transmembrane glycoprotein belonging to the nectin family of Ig superfamily. PVRL4 is found upregulation in many human ductal breast, non-small cell lung and other carcinomas. In 2019, the PVRL4-directed antibody drug conjugate, enfortumab vedotin-ejfv was approved by the FDA to treat metastatic urothelial cancer.

Specifications

Catalog Number	KC-1523
Cell Line Name	293T-PVRL4 Cell Line
NCBI/UniProt Accession Number	NM_030916.3
Host Cell Line	293T
Description	293T cell line stably expressing exogenous PVRL4 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 + 1 μg/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
In Vivo Validation	Yes

Cell Line Generation

293T-PVRL4 cell line was generated using a lentiviral vector expressing the PVRL4 sequence.

Characterization

Figure1: Characterization of PVRL4 overexpressing in 293T stable clones using FACS.

Figure2: Validation of in vivo tumorigenicity of 293T cells stably expressing PVRL4 via subcutaneous implantation in BALB/c nude, with tumor growth monitored by measuring volume (V=0.5×length×width²) and body weight.

Cell Resuscitation

Prewarm culture medium (DMEM + 10% FBS + 1 μg/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

Challita-Eid, P. M. et al. Enfortumab vedotin antibody-drug conjugate targeting nectin-4 is a highly potent therapeutic agent in multiple preclinical cancer models. Cancer Res. 76, 3003ÿ3013 (2016).