Introduction to Hep G2 Cell Line

The Hep G2 cell line is derived from the liver tumor of a 15-year-old male with hepatocellular carcinoma. Widely used in cell biology, genetic toxicology, virology, and cancer research, Hep G2 cells are known for their infinite lifespan, stable phenotype, and ease of handling.

Hep G2 cells play a pivotal role in studying liver cancer development, drug metabolism, and toxicity, making them an essential tool for research into liver-related diseases and therapies.

Key Features of Hep G2 Cells

Cell Names: Hep G2 Cell Line Human (HepG2, HEPG2) (ATCC HB-8065); a.k.a. Human hepatoma-derived cell line, hepatocellular carcinoma, hepatoblastoma cell line

Cell Line Origin: 15-year-old Caucasian male patient with liver cancer (hepatocellular carcinoma)

Growth Characteristics: Adherent growth, epithelial-like morphology, often containing vacuoles  

Expression marker: Insulin-like growth factor II (IGF II)

Culture Conditions: DMEM + 10% FBS  

Culture Environment: 95% air + 5% CO₂; 37°C  

Applications of Hep G2 Cells

Hep G2 cells play a critical role in various research fields:

1. Liver Cancer Research: Hep G2 is widely used to study the mechanisms of liver cancer development, drug resistance, and cancer therapy.

2. Drug Metabolism and Hepatotoxicity Studies: Given its similarity in morphology and function to human liver tissue, Hep G2 is used to assess drug metabolism and liver toxicity, aiding in the evaluation of new drug safety and efficacy.

3. Virology Research: Hep G2 is valuable for studying the replication and pathogenic mechanisms of hepatitis viruses such as hepatitis B virus (HBV) and hepatitis C virus (HCV).

4. Gene Editing and Functional Studies: Researchers use gene editing technologies on Hep G2 to investigate specific gene functions and their roles in disease progression.

5. Cytotoxicity Assays: Hep G2 cells are also used to assess the potential liver toxicity of chemicals and drugs.

Best Practices for Hep G2 Cell Culture

Maintaining high-quality cell culture conditions is essential for the success of Hep G2 cell experiments. The following points should be noted during cell culture to ensure optimal cell growth:

1. Serum Quality: Use high-quality, low-endotoxin fetal bovine serum (FBS)—which should not be heat-inactivated—to enhance cell adhesion and monolayer formation.

2. Vacuolation: Hep G2 cells typically form vacuoles as they reach confluence. This is normal and does not affect cell growth.

3. Cell Density: Control the passaging ratio to prevent overcrowding or sparse growth, as this can affect cell viability.

4. Cell Digestion: Ensure that cell clumps are properly dispersed during digestion.

5. Medium Observation: Regularly monitor the culture medium color and change it timely to avoid impacting cell adhesion.

FAQ: Growth and Morphology of Hep G2 Cell Cultures

1. How do you culture Hep G2 cells?

Hep G2 cells are cultured in Dulbecco′s Modified Eagle′s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), which should not be heat-inactivated, under 95% air and 5% CO₂ at 37°C. High-quality fetal bovine serum (FBS) helps cells adhere and form a monolayer. They exhibit adherent growth with an epithelial-like morphology, initially attaching in small patches while many clusters remain in suspension. Over time, growth expands outward from the adherent cell colonies, and it is common for Hep G2 cells to form a multilayered appearance. During cultivation, vacuoles easily form within the cells, which is a normal characteristic and does not affect cell growth.

2. Is it normal to see floating HepG2 cells after cryopreservation?

While Hep G2 is an adherent cell line, attachment can sometimes be slow in certain cultures. During the first week post-cryopreservation, floating cells are normal for Hep G2. If this occurs, it’s recommended to leave the cultures undisturbed for the first three days.

3. What should I do if floating cells remain after thawing Hep G2 cells?

If a significant number of floating cells persist, assess their viability using trypan blue. Viable cells can be gently centrifuged (125 xg for 5-10 minutes) and returned to the adherent population when refreshing the medium every 2-3 days. Discarding viable floating cells can result in a dilute culture, slowing or halting growth.

4. How can I improve the attachment of Hep G2 cells?

If attachment is slow, increasing the serum concentration to 15-20% or using high-quality, low-endotoxin FBS can improve cell adhesion and promote faster growth.

Even minor changes in pH or serum quality may influence the degree of clumping. For optimal results, supplement the growth medium with high-quality,

5. What factors influence the clumping of Hep G2 cells?

While HepG2 cells tend to clump, the most common cause of clumping is the presence of free DNA and cell debris in the culture medium. Minor changes in pH or serum quality can also affect the degree of clumping. Ensuring the growth medium is supplemented with high-quality, non-heat-inactivated FBS can help reduce clumping and enhance cell attachment.

Gene Editing Hep G2 Cells with Smart-CRISPR™

Cyagen offers Smart-CRISPR™ gene editing services for Hep G2 cells. These services include gene knockout, point mutation, knock-in (KI), overexpression, and interference, delivering monoclonal Hep G2 cell lines with enhanced accuracy and reliability.

Key Gene Editing Projects for Hep G2

Cyagen has optimized the monoclonal preparation method for Hep G2 to deliver gene-edited monoclonal cells, significantly enhancing the accuracy and reliability of experiments and providing a more solid foundation for liver-related disease research.

  • Gene Knockout (KO): Precise deletion of target genes in Hep G2 aid functional research related to liver diseases and cancer.
  • Point Mutation & Knock-In (KI): Smart-CRISPR™ enables high-efficiency homologous recombination in Hep G2 cells to introduce precise mutations or gene insertions.
  • Overexpression & RNA Interference: Cyagen offers stable Hep G2 cell line generation with gene overexpression or RNA interference (suppression) elements, critical for protein function studies.

Best Practices for Gene Editing in Hep G2 Cells:

  • Ensure the cells are in good condition prior to transfection, with approximately 70% confluency.
  • During digestion, disperse the cells into single-cell suspensions for better transfection results.
  • Use the clone-picking method to prepare monoclonal cells. Use Cyagen's custom culture medium conditions for monoclonal cell preparation and perform clonal selection using the limiting dilution method, inoculating the cells into a 10 cm dish. After a period of growth, Transfer clones to 96-well plates for continued culturing.

Hep G2 Subcutaneous Tumor Xenograft (CDX) Model

Cyagen has validated over 200 tumor cell lines for developing in vivo CDX (Cell-Derived Xenograft) models, including Hep G2. These CDX models are critical for:

  • Gene function research
  • Drug target screening
  • Pharmacodynamics studies


Building on our stable cell line model validation data, our Hep G2 subcutaneous tumor models have been successfully used for antitumor efficacy studies. We possess extensive experience with multiple IND evaluation projects, offering a wide variety, large quantity, and superior quality of developed anti-tumor efficacy models and preclinical R&D services.

Hep G2 CDX Mouse Model Protocol

Figure 1. Growth Curves of Hep G2 Liver Cancer Subcutaneous Xenograft Tumor and Body Weight (n=8).

Hep G2 Cells were subcutaneously injected into 7-week-old NKG and 8-week-old NOD-Scid immunodeficient mice, and tumor volume was measured at various time points. The injection dose was 9×10⁶ and 1×10⁷ cells per mouse, with data presented as Mean ± SEM. The results showed that Hep G2 forms tumors in both NKG and NOD-Scid immunodeficient mice. Tumor volumes typically reach 100-200 mm³ by day 22 post-injection and 2000 mm³ by day 45, marking the experimental endpoint. The drug administration window was estimated to be around 23 days.

Hep G2 Cells: Frequently Asked Questions (FAQs)

1. What is the origin of Hep G2 cells?

Hep G2 cells originate from the liver tumor of a 15-year-old Caucasian male diagnosed with hepatocellular carcinoma. They are widely used in liver cancer research due to their stable phenotype and ease of handling.

2. What are the applications of Hep G2 cells?

Hep G2 cells are primarily used in liver cancer research, drug metabolism studies, hepatotoxicity testing, virology research, and gene editing projects.

3. Can Hep G2 cells be used for xenograft models?

Yes, Hep G2 cells can be used to develop subcutaneous xenograft (CDX) tumor models, providing a valuable platform for studying tumor growth and drug efficacy in vivo.

4. How is gene editing performed in Hep G2 cells?

Gene editing in Hep G2 cells can be performed using Smart-CRISPR™ technology, allowing for gene knockout, point mutation, knock-in, and overexpression, with high precision and reliability.

Conclusion

Hep G2 cells offer immense value in liver cancer research, drug metabolism studies, and gene editing. With the availability of advanced tools like Smart-CRISPR™ and validated cell-derived xenograft (CDX) mouse models, researchers can explore disease mechanisms and therapeutic interventions more effectively. Contact Cyagen today to learn more about our Hep G2 gene editing and CDX model services.

Contact Cyagen today for expert support in gene editing, cell culture, and tumor model development for your liver cancer research projects.

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