KC-4850

K562-BCMA-CD19-GFP-Luc2 Cell Line

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Background of K562-BCMA-CD19-GFP-Luc2 Cell Line

CD19 is a B-lineage-specific transmembrane glycoprotein, the expression of which is maintained on more than 95% B-cell malignancies. This strict lineage restriction makes CD19 an ideal target for immune therapies using chimeric antigen receptors (CARs).CD19 is nearly ubiquitously expressed on B-lymphocytes and in B-cell malignancies. The anti-CD19 monoclonal antibody tafasitamab, paired with the immunomodulator lenalidomide, mediates antibody-dependent cellular toxicity and phagocytosis; the antibody-drug conjugate loncastuximab tesirine delivers the DNA cross-linking agent tesirine via CD19 binding and internalization; and CD19-directed chimeric antigen receptor T-cell therapy (CAR-T) products are engineered from autologous T cells.
B cell maturation protein (BCMA), also named CD269, is a membrane of the TNF receptor super family (TNFRSF17) and mainly expressed in mature B-lymphocytes, and play an important role in B cell development and autoimmune response.

Specifications

Catalog Number	KC-4850
Cell Line Name	K562-BCMA-CD19-GFP-Luc2 Cell Line
Clone Number	12-12#
Host Cell Line	K562-GFP-Luc2
Description	Stable K562-GFP-Luc2 cell line expressing exogenous CD19 and BCMA 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% RPMI1640 + 20% FBS + 10% DMSO
Propagation Medium	RPMI1640 + 10% FBS + 1ug/ml Puromycin + 300ug/ml HygromycinB
Selection Marker	Puromycin, Hygromycin B
Morphology	lymphoblast
Subculture	Split saturated culture 1:2-1:4 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 30~48 hours
Mycoplasma Status	Negative

Cell Line Generation

K562-BCMA-CD19-GFP-Luc2 Cell Line was generated using a lentiviral vector expressing the human CD19 and BCMA sequence.

Characterization

Figure1: Characterization of human CD19 and BCMA overexpression in K562-BCMA-CD19-GFP-Luc2 stable clones using FACS.

Figure2: Characterization of the K562-BCMA-CD19-GFP-Luc2 cell line stable clone using Bright-Lite Luciferase Assay System in the conditions of different cell number.

Figure3: Characterization of human BCMA and CD19 overexpression in K562-BCMA-CD19-GFP-Luc2 stable clones using PCR.

Cell Resuscitation

1. Prewarm culture medium (RPMI1640 supplemented with 10% FBS and 1ug/ml puromycin + 300ug/ml HygromycinB)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 1:2-1:4 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% RPMI1640 + 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. Sermer D, Elavalakanar P, Abramson JS, Palomba ML, Salles G, Arnason J. Targeting CD19 for diffuse large B cell lymphoma in the era of CARs: Other modes of transportation. Blood Rev. 2023 Jan;57:101002.
2. Hatzoglou, A. et al. TNF Receptor Family Member BCMA (B Cell Maturation) Associates with TNF ReceptorAssociated Factor (TRAF) 1, TRAF2, and TRAF3 and Activates NF- B, Elk-1, c-Jun N-Terminal Kinase, and p38 Mitogen-Activated Protein Kinase. The Journal of Immunology 165, 1322ÿ1330 (2000).