KC-1704

H22-mPDL1-KO-2B1 Cell Line

22651

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Background of H22-mPDL1-KO-2B1 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-1704
Cell Line Name	H22-mPDL1-KO-2B1 Cell Line
Host Cell Line	H22
Description	Stable H22 clone with human mPDL1 gene knockout, No.2B1
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% 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 30 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-2B1 cell line stable clone using PCR sequencing.

Figure 2: Characterization of H22-mPDL1-KO-2B1 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.