KC-2203

NCI-H929-GPRC5D-KO-1A1-Cell-Line

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Background of NCI-H929-GPRC5D-KO-1A1-Cell-Line

GPRC5D is a membrane of the G protein-coupled receptor family, and is predominately expressed on malignant bone marrow plasma cells, whereas its normal tissue expression is limited. Several therapeutical antibodies, and CART against GPCR5D have been developed rapidly to treat the multiple myeloma.

Specifications

Catalog Number	KC-2203
Cell Line Name	NCI-H929-GPRC5D-KO-1A1-Cell-Line
Host Cell Line	Human NCI-H929 cell line
Description	NCI-H929 cell line with GPRC5D gene knockout
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+0.05mMβ-ME
Morphology	Lymphoblast
Subculture	Split saturated culture 1:2-1:5 every 2-3 days; seed out at about 1-2 × 10⁵ cells/mL
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 50 hours
Mycoplasma Status	Negative
In Vivo Validation	Yes

Cell Line Generation

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

Characterization

Cell Resuscitation

Prewarm culture medium (RPMI1640 + 10% FBS + 0.05mMβ-ME) 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 1:2-1:5 every 2-3 days; seed out at about 1-2 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% RPMI-1640 + 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

Br?uner-Osborne, H. et al. Cloning and characterization of a human orphan family C G-protein coupled receptor GPRC5D. Biochim. Biophys. Acta 1518, 237ÿ48 (2001).
Pillarisetti, K. et al. A T-cell-redirecting bispecific G-protein-coupled receptor class 5 member D x CD3 antibody to treat multiple myeloma. Blood 135, 1232ÿ1243 (2020).