KC-2992

293T-CLDN18.2-PDL1 Cell Line

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Background of 293T-CLDN18.2-PDL1 Cell Line

Claudins, a family of at least 27 transmembrane proteins, are important components and functional structures of tight cell junctions. Claudin-18 is a major component of tight junctions located on the cell membrane surface; it plays an important role in the maintenance of cell polarity and barrier function and promotes acid resistance. The human CLDN18 gene locus on chromosome 3q22 has a molecular weight of approximately 35 kb and contains 6 exons and 5 introns. The first exon of CLDN18 can be alternatively spliced, forming two different splice mutants (CLDN18.1 and CLDN18.2) that have highly homologous amino acid sequences. Both the C-terminus and the N-terminus of CLDN 18 are located in the cytoplasm. Two CLDN18 protein isoforms are expressed in a tissue-specific manner—CLDN18.1 and CLDN18.2 are specifically expressed in normal stomach and lung tissues, respectively. CLDN18 is also expressed in cancer tissues and has altered functions that are linked to tumour formation, proliferation, invasion and migration. The interaction of programmed cell death 1 ligand 1 (PDL1) with its receptor programmed cell death 1 (PD1) inhibits T cell responses, and blockade of this interaction has proven to be an effective immunotherapy for several different cancers. PDL1 can be expressed on the surface of tumour cells, immune cells and other cells in the tumour microenvironment but is also found in extracellular forms. In patients with melanoma, exosomal PDL1 is also a marker of immune activation early after initiation of therapy with PD1-blocking antibodies and predicts a clinical response to PD1 blockade.

Specifications

Catalog Number	KC-2992
Cell Line Name	293T-CLDN18.2-PDL1 Cell Line
Host Cell Line	293T
Description	Stable HEK293T clone expressing exogenous human CLDN18.2 and PDL1 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+150μg/mL hygromycin B
Selection Marker	Puromycin, Hygromycin B
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	NA

Cell Line Generation

293T-CLDN18.2-PDL1 cell line was generated using a lentiviral vector expressing the human CLDN18.2 and PDL1 sequence.

Characterization

Figure 1: Characterization of human CLDN18.2 overexpression in the 293T-CLDN18.2-PDL1 stable clone using FACS.

Figure 2: Characterization of human PDL1 overexpression in 293T-CLDN18.2-PDL1 cells using FACS.

Cell Resuscitation

Prewarm culture medium (DMEM supplemented with 10% FBS, 1μg/mL puromycin and 150μg/mL hygromycin B)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

Chen J, Xu Z, Hu C, Zhang S, Zi M, Yuan L, Cheng X. Targeting CLDN18.2 in cancers of the gastrointestinal tract: New drugs and new indications. Front Oncol. 2023 Mar 10;13:1132319. doi: 10.3389/fonc.2023.1132319. PMID: 36969060; PMCID: PMC10036590.
Daassi D, Mahoney KM, Freeman GJ. The importance of exosomal PDL1 in tumour immune evasion. Nat Rev Immunol. 2020 Apr;20(4):209-215. doi: 10.1038/s41577-019-0264-y. Epub 2020 Jan 21. PMID: 31965064.