KC-2576

CT26 human MSLN Cell Line

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Background of CT26 human MSLN Cell Line

Mesothelin (MSLN) is a cell surface glycoprotein normally expressed on serosal tissues such as pleura, pericardium, and peritoneum, but is not in the parenchyma of any vital organs. It is commonly expressed on a number of solid tumors, such as mesothelioma, pancreatic adenocarcinoma, ovarian cancer and others. It has no known physiologic function, but may play a role in tumorigenesis and malignancy. Preclinical and clinical studies showed that aberrant MSLN expression on tumor cells plays an important role in promoting proliferation and invasion. MSLN has also been identified as a receptor of CA125 that mediates cell adhesion. The interaction of CA125 and MSLN plays an important role in ovarian cancer cell peritoneal implantation and increases the motility and invasion of pancreatic carcinoma cells. The overexpression of MSLN could activate the NFκB, MAPK, and PI3K pathways and subsequently induce resistance to apoptosis or promote cell proliferation, migration, and metastasis by inducing the activation and expression of MMP7 and MMP9. Because of its strong differential expression, it has become a popular target for a directed anti-neoplastic therapies, including monoclonal antibodies, antibody-drug conjugates (ADCs), radioimmunotherapy (RIT), CAR-T cells, and vaccines.

Specifications

Catalog Number	KC-2576
Cell Line Name	CT26 human MSLN Cell Line
Host Cell Line	CT26
Description	CT26 stable cell line expressing exogenous human MSLN 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 30 hours
Mycoplasma Status	Negative
In Vivo Validation	Yes

Cell Line Generation

CT26 human MSLN cell line was generated using a lentiviral vector expressing the human MSLN sequence.

Characterization

Figure1: Characterization of human MSLN overexpression in the CT26 stable clone using FACS.

Figure2: Characterization of human MSLN overexpression in CT26 cells using FACS.

Cell Resuscitation

Prewarm culture medium (DMEM supplemented with 10% FBS and 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

Lv J, Li P. Mesothelin as a biomarker for targeted therapy. Biomark Res. 2019 Aug 23;7:18. doi: 10.1186/s40364-019-0169-8. PMID: 31463062; PMCID: PMC6708176.
Hagerty BL, Pegna GJ, Xu J, Tai CH, Alewine C. Mesothelin-Targeted Recombinant Immunotoxins for Solid Tumors. Biomolecules. 2020 Jun 28;10(7):973. doi: 10.3390/biom10070973. PMID: 32605175; PMCID: PMC7408136.