KC-1148

293T-OS8-hPDL1-Cell-Line

21611

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Background of 293T-OS8-hPDL1-Cell-Line

PD-L1, also called human programed cell death ligand 1, is a transmembrane protein that play a major role in suppressing the immune system during particular events such as pregnancy, tissue allografts, autoimmune disease, virus infection and cancer. PD-L1 binds to its receptor PD-1 on activated T cells, B cells, and myeloid cells, to modulate activation or inhibition. Upregulation of PD-L1 can allow cancer cell to evade the host immune system.

293T-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-1148
Cell Line Name	293T-OS8-hPDL1-Cell-Line
Host Cell Line	293T
Description	HEK293T cell line stable expressing exogenous membrane OKT3 scFV and human PDL1 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+1µg/mL Puromycin+140µg/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 30 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

293T OS8 cell line was generated uisng lentiviral vector expessing a ScFV sequence of anti-human CD3 mAb OKT3 and a C-terminal domain of mouse CD8a which consisit of transmembrane and cytoplasmcic domains, and another lentiviral vector expessing human PDL1 molecules.

Characterization

Figure: Characterization of OS8 and PDL1 overexpressing in 293T stable clones using FACS.

Cell Resuscitation

1. Prewarm culture medium (DMEM supplemented with 10% FBS, 1µg/mL Puromycin and 140µg/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% 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. Latouche, Jean-Baptiste; Sadelain, Michel (2000). Induction of human cytotoxic T lymphocytes by artificial antigen-presenting cells. Nature Biotechnology. 18 (4): 405–409.
2. 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
3. Sunshine, J. & Taube, J. M. PD-1/PD-L1 inhibitors. Current Opinion in Pharmacology 23, 32–38 (2015).
4. Boussiotis, V. A. Molecular and Biochemical Aspects of the PD-1 Checkpoint Pathway. N Engl J Med 375, 1767– 1778 (2016).
5. Topalian, S. L. et al. Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med 366, 2443–2454 (2012).