KC-2991

MKN45-CLDN18.2-PDL1 Cell Line

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Home » MKN45-CLDN18.2-PDL1 Cell Line

Background of MKN45-CLDN18.2-PDL1 Cell Line

The claudin18.2 (CLDN18.2) protein, an isoform of claudin18, a member of the tight junction protein family, is a highly selective biomarker with limited expression in normal tissues and often abnormal expression during the occurrence and development of various primary malignant tumors, such as gastric cancer/gastroesophageal junction (GC/GEJ) cancer, breast cancer, colon cancer, liver cancer, head and neck cancer, bronchial cancer and non-small-cell lung cancer. CLDN18.2 participates in the proliferation, differentiation and migration of tumor cells. Recent studies have identified CLDN18.2 expression as a potential specific marker for the diagnosis and treatment of these tumors. Zolbetuximab (claudiximab, IMAB362), a monoclonal antibody (mAb) against CLDN18.2, have been developed. The engagement of programmed cell death protein 1 (PD-1; encoded by the PDCD1 gene) receptor expressed on activated T cells and its ligand programmed death-ligand 1 (PD-L1; encoded by the CD274 gene) is a major co-inhibitory checkpoint signaling that controls T-cell activities. Various types of cancers express high levels of PD-L1 and exploit the PD-L1/PD-1 signaling to evade T-cell immunity. Blocking the PD-L1/PD-1 pathway has consistently shown remarkable anti-tumor effects in patients with advanced cancers and is recognized as the gold standard for developing new immune checkpoint blockade (ICB) and combination therapies.

Specifications

Catalog NumberKC-2991
Cell Line NameMKN45-CLDN18.2-PDL1 Cell Line
Clone Number6#
Host Cell LineMKN45
DescriptionStable MKN45 cell line expressing exogenous human CLDN18.2 and PDL1 gene
QuantityOne vial of frozen cells (≥2-106/vial)
StabilityStable in culture over a minimum of 10 passages
ApplicationDrug screening and biological assays
Freezing Medium70% RPMI1640 + 20% FBS + 10% DMSO
Propagation MediumRPMI1640 + 10% FBS + 1μg/mL Puromycin+300μg/mL Hygromycin B
Selection MarkerPuromycin | Hygromycin B
MorphologyFibroblastoid cells growing as a monolayer
SubcultureSplit saturated culture 1:3-1:4 every 2-3 days; seed out at about 1-3 × 105 cells/mL
Incubation37 °C with 5% CO2
StorageLiquid nitrogen immediately upon receiving
Doubling TimeApproximately 30 hours
Mycoplasma StatusNegative
In Vivo ValidationNA

Cell Line Generation

MKN45-Human-CLDN18.2-PDL1 cell line was generated using a lentiviral vector expressing the human CLDN18.2 and PDL1 sequence.

Characterization

Figure 1: Characterization of CLND18.2 overexpression in MKN45 stable clones using FACS.

Figure 2: Characterization of human PDL1 overexpression in MKN45-Human-CLDN18.2-PDL1 stable clones using FACS.

Figure 3: Characterization of human CLDN18.2 and PDL1 overexpression in MKN45-Human-CLDN18.2-PDL1 stable clones using PCR Sequencing.

Cell Resuscitation

1. Prewarm culture medium (RPMI1640 + 10% FBS + 1μg/mL Puromycin+300μg/mL Hygromycin B)in a 37°C water bath. 2. Thaw the frozen vial in a 37°C water bath for 1-2 minutes. 3. Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol. 4. Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium. 5. Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet. 6. Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask. 7. Incubate the flask at 37°C, 5% CO2 incubator. 8. Split saturated culture 1:3-1:4 every 2-3 days; seed out at about 1-3 × 105 cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% RPMI1640 + 20% FBS + 10% DMSO) fresh immediately before use. 2. Keep the freezing medium on ice and label cryovials. 3. Transfer cells to a sterile, conical centrifuge tube, and count the cells. 4. Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium. 5. Resuspend the cells at a density of at least 3×106 cells/mL in chilled freezing medium. 6. Aliquot 1 mL of the cell suspension into each cryovial. 7. Freeze cells in the CoolCell freezing container overnight in a -80°C freezer. 8. Transfer vials to liquid nitrogen for long-term storage.

References

1. Cao W, Xing H, Li Y, Tian W, Song Y, Jiang Z, Yu J. Claudin18.2 is a novel molecular biomarker for tumor-targeted immunotherapy. Biomark Res. 2022 May 31;10(1):38. doi: 10.1186/s40364-022-00385-1. PMID: 35642043; PMCID: PMC9153115. 2. Cha JH, Chan LC, Li CW, Hsu JL, Hung MC. Mechanisms Controlling PD-L1 Expression in Cancer. Mol Cell. 2019 Nov 7;76(3):359-370. doi: 10.1016/j.molcel.2019.09.030. Epub 2019 Oct 24. PMID: 31668929; PMCID: PMC6981282.

Use License Agreement

Research Use Only.
Not for use in diagnostic procedures or therapeutic applications.
Redistribution of the cell line or its derivatives is prohibited without prior written permission from Kyinno Biotechnology.
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