KC-6337

293T-EGFR-B7H3-KO-B7H3 Cell Line

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Background of 293T-EGFR-B7H3-KO-B7H3 Cell Line

B7H3, also known as CD276, is a type I transmembrane protein which belongs to the B7 superfamily of immune checkpoint molecules. B7-H3 is an immune checkpoint molecule that inhibits tumor antigen-specific immune responses. B7-H3 is present at low levels in most normal tissues but is overexpressed in a wide variety of cancers, including bladder, breast, cervical, colorectal, esophageal, glioma, kidney, liver, lung, ovarian, pancreatic, prostate, intrahepatic cholangiocarcinoma, liver, oral squamous cell carcinoma, endometrial cancer, and squamous cell carcinoma and gastric cancer, glioma, and melanoma. B7-H3 also possesses non-immunological pro-tumorigenic functions such as promoting migration, invasion, angiogenesis, chemoresistance, epithelial-to-mesenchymal transition, and affecting tumor cell metabolism.

Specifications

Catalog Number	KC-6337
Cell Line Name	293T-EGFR-B7H3-KO-B7H3 Cell Line
NCBI/UniProt Accession Number	NM_001024736.2
Clone Number	5#
Host Cell Line	293T-EGFR-B7H3-KO
Description	Stable 293T-EGFR-B7H3-KO cell line expressing exogenous B7H3 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% basal medium+20% FBS+10% DMSO
Propagation Medium	DMEM+10% FBS +1μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Epithelial-like
Subculture	Split saturated culture 1:4-1:8 every 2-3 days
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 30 hours
Mycoplasma Status	Negative

Cell Line Generation

293T-EGFR-B7H3-KO-B7H3 cell line was generated using a lentiviral vector expressing the B7H3 sequence.

Characterization

Figure 1: Characterization of B7H3 overexpression in the 293T-EGFR-B7H3-KO-B7H3 stable clone using FACS.

Figure 2: Characterization of B7H3 in the 293T-EGFR-B7H3-KO-B7H3 stable clone using PCR sequencing.

Cell Resuscitation

Pre-warm complete culture medium (basal medium and 10% FBS) in a 37°C water bath.
Rapidly thaw the cryovial in a 37°C water bath for 1-2 minutes with gentle agitation.
Transfer the vial to a biosafety cabinet, and disinfect the exterior with 70% ethanol.
Aseptically transfer the cell suspension dropwise into a sterile centrifuge tube containing 9.0 mL of pre-warmed complete medium.
Centrifuge at approximately 125 × g for 5–7 minutes at room temperature, carefully aspirate the supernatant without disturbing the cell pellet.
Gently resuspend the pellet in an appropriate volume of complete medium and transfer the suspension into a T25 flask.
Incubate the flask in a 37°C in a humidified 5% CO₂ incubator.
Assess cell viability and morphology after 24 hours. If cells appear healthy, replace the medium with fresh medium supplemented with the appropriate selective antibiotic.
Subculture the cells at a ratio of 1:4-1:8 every 2-3 days upon reaching 80%–90% confluency.

Cell Freezing

Prepare the freezing medium (70% basal medium, 20% FBS and 10% DMSO) freshly before use.
Pre-chill the freezing medium on ice and label the cryovials accordingly.
Transfer the cell suspension to a sterile conical tube and perform a cell count to determine total viability and density.
Centrifuge the cells at 250×g for 5 minutes at room temperature; carefully aspirate the supernatant.
Gently resuspend the cell pellet in chilled freezing medium, ensuring a minimum cell density of 3×10⁶ cells/mL.
Aliquot 1 mL of the cell suspension into each pre-labeled cryovial.
Place the cryovials into a CoolCell® container and store at -80°C overnight for controlled-rate cooling.
Transfer the cryovials to the liquid nitrogen for long-term storage the following day.

References

1.Mortezaee K. B7-H3 immunoregulatory roles in cancer. Biomed Pharmacother. 2023 Jul;163:114890.
2. Harland N, Maurer FB, Abruzzese T, Bock C, Montes-Mojarro IA, Fend F, Aicher WK, Stenzl A, Amend B. Elevated Expression of the Immune Checkpoint Ligand CD276 (B7-H3) in Urothelial Carcinoma Cell Lines Correlates Negatively with the Cell Proliferation. Int J Mol Sci. 2022 Apr 29;23(9):4969.
3. Guo C, Figueiredo I, Gurel B, Neeb A, Seed G, Crespo M, Carreira S, Rekowski J, Buroni L, Welti J, Bogdan D, Gallagher L, Sharp A, Fenor de la Maza MD, Rescigno P, Westaby D, Chandran K, Riisnaes R, Ferreira A, Miranda S, Calì B, Alimonti A, Bressan S, Nguyen AHT, Shen MM, Hawley JE, Obradovic A, Drake CG, Bertan C, Baker C, Tunariu N, Yuan W, de Bono JS. B7-H3 as a Therapeutic Target in Advanced Prostate Cancer. Eur Urol. 2023 Mar;83(3):224-238.