KC-1705

H22-mPDL1-KO-2C3 Cell Line

22653

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Background of H22-mPDL1-KO-2C3 Cell Line

Programmed death protein 1 (PD1) is a common immunosuppressive member on the surface of T cells and plays an imperative part in downregulating the immune system and advancing self-tolerance. Its ligand programmed cell death ligand 1 (PDL1) is overexpressed on the surface of malignant tumor cells, where it binds to PD1, inhibits the proliferation of PD1-positive cells, and participates in the immune evasion of tumors leading to treatment failure. PD-L1 is expressed on the surface of tumor cells and it is able to bind to PD-1 on the surface of activated T cells and B cells. The binding of PD-L1 to PD-1 leads to an immunosuppressive effect and allows the tumor to evade immune destruction.

Specifications

Catalog Number	KC-1705
Cell Line Name	H22-mPDL1-KO-2C3 Cell Line
Host Cell Line	H22
Description	Stable H22 clone with human mPDL1 gene knockout, No.2C3
Quantity	One vial 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% RPMI1640 + 20% FBS + 10% DMSO
Propagation Medium	RPMI1640+10%FBS
Selection Marker	NA
Morphology	Mostly single, round cells in suspension
Subculture	Split saturated culture 1:4-1:5 every 2-3 days; seed out at about 1 × 10⁵ cells/mL
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 48 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

H22-mPDL1-KO cell line was generated using CRISPR method.

Characterization

Figure 1: Characterization of H22-mPDL1-KO-2C3 cell line stable clone using PCR sequencing.

Figure 2: Characterization of H22-mPDL1-KO-2C3 Cell Line stable clone using FACS.

Cell Resuscitation

Prewarm culture medium (RPMI1640 + 10% FBS) 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 1:4-1:5 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% RPMI1640 + 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

Liu J, Chen Z, Li Y, Zhao W, Wu J, Zhang Z. PD - 1/PD - L1 checkpoint inhibitors in tumor immunotherapy. Front Pharmacol. 2021;12:731798. doi: 10.3389/fphar.2021.731798. Epub 2021 Sep 1. PMID: 34539412.

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.