KC-1315

CHOK1-OS8-B7H7 (HHLA2) Cell Line

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Background of CHOK1-OS8-B7H7 (HHLA2) Cell Line

HHLA2, also named as B7H7, is a member of the B7 family of immune regulatory proteins, B7H7 is expressed monocytes and is up-regulated by LPS and IFN-gamma stimulation.
CHOK1-OS8 stable cell was used as an artificial antigen presenting cells (APCs), OS8 could function as a membrane anchored T cell engager that directly activates TCR in T cell-based assay.

Specifications

Catalog Number	KC-1315
Cell Line Name	CHOK1-OS8-B7H7 (HHLA2) Cell Line
Clone Number	NA
Host Cell Line	CHOK1
Description	CHOK1 cell line stable expressing membrane OKT3 scFV and human B7H7 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+10μg/ml puromycin+750µg/ml Hygromycin B
Selection Marker	Puromycin, Hygromycin B
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 24 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

CHO-K1 OS8-B7H7 (HHLA2) Cell Line was generated using lentiviral vector expressing membrane OKT3 scFV and human B7H7 gene.

Characterization

Figure 1: Characterization of B7H7 overexpressing in CHOK1-OS8 table clones using FACS.

Cell Resuscitation

1. Prewarm culture medium (RPMI1640+10% FBS+10μg/ml puromycin+750µg/ml Hygromycin B)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% 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. Zhao, Ruihua, Jordan M Chinai, Susan Buhl, Lisa Scandiuzzi, Anjana Ray, Hyungjun Jeon, Kim C Ohaegbulam, et al. 2013. HHLA2 Is a Member of the B7 Family and Inhibits Human CD4 and CD8 T-Cell Function. Proceedings of the National Academy of Sciences of the United States of America 110 (24). National Academy of Sciences: 9879–84. doi:10.1073/pnas.1303524110.
2. Koirala, Pratistha, Michael E Roth, Jonathan Gill, Jordan M Chinai, Michelle R Ewart, Sajida Piperdi, David S Geller, et al. 2016. HHLA2, a Member of the B7 Family, Is Expressed in Human Osteosarcoma and Is Associated with Metastases and Worse Survival. Scientific Reports, August. Nature Publishing Group, 1–9. doi:10.1038/srep31154.