KC-2173

A375-OS8 Cell Line

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Background of A375-OS8 Cell Line

A375-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-2173
Cell Line Name	A375-OS8 Cell Line
Host Cell Line	A375
Description	A375 cell line stable expressing membrane OKT3 scFV sequence
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 + 100μg/mL Hygromycin
Selection Marker	Hygromycin B
Morphology	Epithelial
Subculture	Split the saturated culture at a ratio of 1:4~1:5 every 2~3 days; seed out at about 1-3 x 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

A375 OS8 cell line was generated using lentiviral vector expressing a ScFV sequence of anti-human CD3 mAb OKT3 and a C-terminal domain of mouse CD8a which consist of transmembrane and cytoplasmic domains.

Characterization

Figure: Characterization of OS8 overexpressing in A375 stable clone using FACS.

Cell Resuscitation

Prewarm culture medium (DMEM supplemented with 10% FBS and 100μg/mL Hygromycin)in a 37°C water bath.
Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
Incubate the flask at 37°C, 5% CO₂ incubator.
Split saturated culture 1:4-1:5 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

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

References

atouche, Jean-Baptiste; Sadelain, Michel (2000). "Induction of human cytotoxic T lymphocytes by artificial antigen-presenting cells". Nature Biotechnology. 18 (4): 405–409.
Perica, Karlo; Kosmides, Alyssa K; Schneck, Jonathan P (2015). "Linking form to function: Biophysical aspects of artificial antigen presenting cell design". Biochimica et Biophysica Acta (BBA) - Molecular Cell Research. 1853 (4): 781–790