KC-2385

Hepa1-6-mGPC3-KO-human-GPC3 Cell Line

23952

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Background of Hepa1-6-mGPC3-KO-human-GPC3 Cell Line

Glypican-3 (GPC3) is Glypican ™ A member of the family. Glypicans are proteoglycans that attach to the cell surface through glycosylphosphatidylinositol anchoring. They regulate the signal transduction activity of various growth factors, including Wnts. This regulation is based on the ability of glypicans to stimulate or inhibit the interaction between these growth factors and their respective signaling receptors. Glypican-3 (GPC3) plays an important role in regulating cell growth, differentiation, and migration. Expressed in 75% of hepatocellular carcinoma (HCC), but not in healthy liver tissue or other organs. Targeting GPC3 may provide a new tool for treating HCC.

Specifications

Catalog Number	KC-2385
Cell Line Name	Hepa1-6-mGPC3-KO-human-GPC3 Cell Line
Clone Number	1A3
Host Cell Line	Hepa1-6-mGPC3-KO
Description	Stable Hepa1-6-mGPC3-KO cell line expressing exogenous human GPC3 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% DMEM + 20% FBS + 10% DMSO
Propagation Medium	DMEM + 10% FBS + 2ug/ml Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:2-1:3 every 2 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

Hepa1-6-mGPC3-KO-human-GPC3 cell line was generated using lentiviral vector expressing human GPC3 sequence.

Characterization

Figure 1: Characterization of human GPC3 overexpression in the Hepa1-6-mGPC3-KO-human-GPC3 stable clone using FACS.

Cell Resuscitation

1. Prewarm culture medium (DMEM + 10% FBS + 2ug/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 1:2-1:3 every 2 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.Dargel C, Bassani-Sternberg M, Hasreiter J, Zani F, Bockmann JH, Thiele F, Bohne F, Wisskirchen K, Wilde S, Sprinzl MF, Schendel DJ, Krackhardt AM, Uckert W, Wohlleber D, Schiemann M, Stemmer K, Heikenwälder M, Busch DH, Richter G, Mann M, Protzer U. T Cells Engineered to Express a T-Cell Receptor Specific for Glypican-3 to Recognize and Kill Hepatoma Cells In Vitro and in Mice. Gastroenterology. 2015 Oct;149(4):1042-52.
2. Mounajjed T, Zhang L, Wu TT. Glypican-3 expression in gastrointestinal and pancreatic epithelial neoplasms. Hum Pathol. 2013 Apr;44(4):542-50.