KC-2204-DW

NCI-H929-GPRC5D-KO Cell Line

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Background of NCI-H929-GPRC5D-KO Cell Line

GPRC5D (G Protein-Coupled Receptor Class C Group 5 Member D) is an orphan receptor highly expressed on malignant plasma cells in multiple myeloma, with restricted expression in normal tissues limited primarily to hair follicles and nail beds. Its specific expression on tumor cells, independent of BCMA, makes it a promising therapeutic target for T-cell redirecting therapies, particularly in relapsed/refractory multiple myeloma. Clinical studies with GPRC5D-targeting bispecific antibodies (e.g., talquetamab) and CAR-T cells have demonstrated significant efficacy. The main challenges include managing unique adverse events related to on-target, off-tumor effects in keratinized tissues (nail changes, skin rash, taste disturbances) and overcoming potential antigen escape-mediated resistance.

Specifications

Catalog Number	KC-2204-DW
Cell Line Name	NCI-H929-GPRC5D-KO Cell Line
NCBI/UniProt Accession Number	55507/Q9NZD1
Clone Number	2B4
Host Cell Line	NCI-H929
Description	Stable NCI-H929 clone with human GPRC5D gene knockout, No.2B4
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	RPMI1640+20% FBS+10% DMSO
Propagation Medium	RPMI1640+10% FBS+0.05mM 2-mercaptoethanol
Selection Marker	NA
Morphology	lymphoblast
Subculture	Split saturated culture 1:3-1:5 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 50 hours
Mycoplasma Status	Negative

Cell Line Generation

NCI-H929-GPRC5D-KO cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of NCI-H929-GPRC5D-KO cell line stable clone using PCR sequencing.

Figure 2: Characterization of NCI-H929-GPRC5D-KO cell line stable clone using FACS.

Cell Resuscitation

Prewarm culture medium (RPMI1640+10% FBS+0.05mM 2-mercaptoethanol) 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 1:3-1:5 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

Mailankody S, Devlin SM, Landa J, et al. GPRC5D-Targeted CAR T Cells for Myeloma. N Engl J Med. 2022; 387(13): 1196-1206. doi: 10.1056/NEJMoa2209900. PMID: 36170501; PMCID: PMC10309537.