KC-1723

293T-GPRC5D Cell Line

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Background of 293T-GPRC5D 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-1723
Cell Line Name	293T-GPRC5D Cell Line
Clone Number	3#
Host Cell Line	293T
Description	Stable 293T cell line expressing exogenous human GPRC5D 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 + 1ug/mL Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:5 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 30 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

293T GPRC5D cell line was generated using a lentiviral vector expressing the human GPRC5D sequence.

Characterization

Figure 1: Characterization of human GPRC5D overexpression in the 293T GPRC5D stable clone using FACS.

Cell Resuscitation

1. Prewarm culture medium (DMEM + 10% FBS + 1ug/ml Puromycin)in a 37°C water bath.
2. Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
3. Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
4. Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
5. Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
6. Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
7. Incubate the flask at 37°C, 5% CO₂ incubator.
8. Split saturated culture 1:4-1:5 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% DMEM + 20% FBS + 10% DMSO) fresh immediately before use.
2. Keep the freezing medium on ice and label cryovials.
3. Transfer cells to a sterile, conical centrifuge tube, and count the cells.
4. Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
5. Resuspend the cells at a density of at least 3×10⁶ cells/mL in chilled freezing medium.
6. Aliquot 1 mL of the cell suspension into each cryovial.
7. Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
8. Transfer vials to liquid nitrogen for long-term storage.

References

1.Bräuner-Osborne, H. et al. Cloning and characterization of a human orphan family C G-protein coupled receptor GPRC5D. Biochim. Biophys. Acta 1518, 237–48 (2001).
2. 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, 1232–1243 (2020).