KC-3335

293T cyno-CD19-High Cell Line

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Background of 293T cyno-CD19-High 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.

Specifications

Catalog Number	KC-3335
Cell Line Name	293T cyno-CD19-High Cell Line
Host Cell Line	293T
Description	Stable 293T clone expressing exogenous cyno CD19 gene in high level
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 + 1μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:8 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 cyno-CD19-high cell line was generated using a lentiviral vector expressing the cyno-CD19 sequence.

Characterization

Figure1: Characterization of 293T-cyno-CD19-High Cell Line stable clone using PCR sequencing.

Figure2: Characterization of cyno CD19 expression in 293T cells using FCAS.

Figure3: Characterization of cyno CD19 overexpression in the 293T-cyno-CD19-High stable clone using FCAS.

Cell Resuscitation

1. Prewarm culture medium (DMEM supplemented with 10% FBS and 1μg/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 1:4-1:8 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. Ramos CA, Savoldo B, Dotti G. CD19-CAR trials. Cancer J. 2014 Mar-Apr.
2. 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.
3. Lownik J, Boiarsky J, Birhiray R, Merchant A, Mead M. Sequencing of Anti-CD19 Therapies in the Management of Diffuse Large B-Cell Lymphoma. Clin Cancer Res. 2024 Jul 15;30(14):2895-2904.