HepG2 in Cell Culture

HepG2 (liver hepatocellular carcinoma): cell culture and transfection ...

HepG2 (liver hepatocellular carcinoma): cell culture and transfection protocol

HepG2 in Cell Culture

HepG2 Cell Line Characteristics

HepG2 is an immortalized cell line consisting of human liver carcinoma cells, derived from the liver tissue of a 15-year-old Caucasian male who had a well-differentiated hepatocellular carcinoma. Hepatocellular carcinoma is the fifth most-common cancer worldwide. The morphology of HepG2 cells is epithelial and contains 55 chromosome pairs. HepG2 cells can be grown successfully at a large scale, and secrete many plasma proteins, such as transferrin, fibrinogen, plasminogen and albumin. They can be stimulated with human growth hormone. HepG2 cells are adherent, epithelial-like cells growing as monolayers and in small aggregates.

Copyright picture from Altogen.com. Reproduced with permission from Altogen Biosystems.

HepG2 Cytogenetics

HepG2 cells are hyperdiploid karyotype – 52(47-54)<2n>XY, +2, +14, +17, +20, +2mar, t(1;21) (p22.2;p11-12), i(17q)/der(17)t(17;17)(p11;q11)

HepG2 Cell Culturing Protocol

HepG2 complete medium

Eagle’s Minimum Essential Medium (EMEM) supplemented with 10% FBS; DMEM and RPMI1640 are also alternatives that work well. Aspirate and add fresh culture medium every 2-3 days.  HepG2 cell doubling time is 48 hours.

1. To passage cells, rinse cell monolayer with 1x PBS twice and add pre-warmed (37°C) 0.05% Trypsin-EDTA solution to cover the bottom of the flask; incubate for 5 – 7 minutes
2. As cells detach, neutralize the Trypsin by adding 4x volume of complete growth medium with 10% FBS and gently resuspend the cells by pipetting
3. To avoid clumping do not agitate the cells by shaking the flask while waiting for detachment
4. Split cells 1:4 every 3 days or 1:8 every 6 days
5. Cultures should be incubated at 37°C in a humidified atmosphere with 5% CO2

Subculture Troubleshooting Procedures

Low cell viability after passaging:

• Dissociation agent left on cells too long; only expose cells to dissociation agent long enough for cell detachment
• Pipette gently during passaging procedures; cells are fragile when exposed to dissociation agents

Cells are difficult to detach:

• Cell-to-cell junctions are tight due to cell growth being 100% confluent and dissociation agent cannot reach cell interface; subculture cells before confluent
• Use higher concentration of dissociation agent; incubate flask at 37°C to increase enzymatic activity
• Wash flask twice with sterile 1x PBS prior to addition of the dissociation agent

Clumps form after detachment:

• Cells were centrifuged too fast; do not spin cells faster than 100 x g to pellet
• Place the flask or vial on ice to decrease aggregation before use

HepG2 Cell Line Derived Xenograft

HepG2 cells are inoculated in immunocompromised mice to create the HepG2 Cell Line Derived Xenograft (CDX) mouse model. The HepG2 xenograft of human hepatocellularcarcinoma (HCC) enables studies targeting antiangiogenesis (i.e. rapamycin, bevacizumab) or tumor growth inhibition (e.g. sorafenib).

Stable Cell Line Generation

HepG2 cells have been demonstrated to be Neomycin G418 resistant (400 µg/mL). Development of HepG2 stable cell line services are provided by Altogen Labs CRO

*NOTE: All IP rights to the HepG2 cell line belongs solely to the Wistar Institute.

HepG2 Resources

HepG2 Transfection Kit

Xenograft Models

siRNA Delivery – In Vivo Transfection Kits

CRO Pre-clinical Research Services: Xenograft animal models

HepG2 Cell Banking services

Generation of Stably Expressing Cell Lines in 28 Days

Encapsulation of Protein, RNA, mRNA, and DNA Molecules into Liposomes

HepG2 cells Forum: Research methods & Laboratory techniques: Link