Hep 3B / Hep 3B-luc Orthotopic Mouse Model Service for Liver Cancer

The Hep 3B / Hep 3B-luc orthotopic mouse model for liver cancer establishes a hepatitis B virus-associated, p53-deficient human hepatocellular carcinoma as a primary tumor in the livers of nude mice, combining clinically meaningful molecular features with quantitative imaging readouts. Working exclusively in the preclinical space, Alfa Cytology provides this xenograft platform as an integrated service, from authenticated Hep 3B / Hep 3B-luc cell supply through intrahepatic implantation, scheduled in vivo bioluminescence imaging, and endpoint histopathological verification.

Overview of Hep 3B / Hep 3B-luc Orthotopic Mouse Model for Liver Cancer

Hep 3B is a human hepatocellular carcinoma cell line established in 1972 by Aden and colleagues at the University of California, San Francisco, from the tumor of an 8-year-old male patient, making it one of the oldest HCC lines in continuous use. The line carries several features that mirror the human disease: integrated hepatitis B virus DNA, from which HBx sequences and surface antigen expression are detectable, and a homozygous deletion of TP53 that leaves the cells without p53 protein—a profile that has made Hep 3B the reference p53-null HCC system for restoration-of-function and synthetic-lethality studies. The cells grow adherently with an epithelial morphology, express α-fetoprotein (AFP), and are tumorigenic in immunocompromised mice, unlike some other historic liver lines. ATCC lists Hep 3B as HB-8064 with a defined STR profile for authentication.

Fig 1. Longitudinal bioluminescence imaging of Hep3B-Luc orthotopic hepatocellular carcinoma following direct intrahepatic implantation, demonstrating progressive liver tumor growth and suppression after modulation of the miR-423-5p/MALAT1 axis. (Bocchetti, Marco, et al., 2025)

In orthotopic xenograft work, luciferase-tagged Hep 3B cells are injected directly into the hepatic parenchyma—commonly the left lobe—at inocula ranging from about 0.5 × 10⁶ to 3 × 10⁶ cells, with or without Matrigel as a carrier; reported tumor take rates reach 95–99%, and bioluminescence after D-luciferin administration, usually 150 mg/kg intraperitoneally with signal capture at 5–15 minutes, quantifies tumor burden from the first week onward, with in vitro calibration curves confirming a linear relationship between cell number and luminescent output. The resulting intrahepatic tumors support treatment windows of two to four weeks and have been used to evaluate GPC3-targeted CAR-T cells, mRNA-based p53 restoration combined with mTOR inhibition, epigenomic MYC repressors, sorafenib combinations, and photothermal nanoplatforms. Figure 1 summarizes how the model is constructed and followed longitudinally.

Cell Line Information: Hep 3B / Hep 3B-luc

Hep 3B is among the most deeply characterized liver cancer lines, and its luciferase-tagged derivatives are the workhorses of modern orthotopic HCC imaging. The key attributes are compiled below.

Our Services

A Hep 3B / Hep 3B-luc program at Alfa Cytology is run as a single accountable pipeline: authenticated and luciferase-calibrated cell banks, meticulous left-lobe intrahepatic surgery, imaging sessions on a pre-agreed cadence, and a terminal package that pairs photon-flux curves with AFP assay, ex vivo organ imaging, and H&E/IHC-confirmed pathology.

Workflow of Hep 3B / Hep 3B-luc Orthotopic Mouse Model Construction

Establishing a Hep 3B or Hep 3B-luc orthotopic liver tumor is a surgical xenograft workflow in which cell authentication, luciferase calibration, precise parenchymal injection, and serial imaging each determine data quality. The sequence below reflects standard execution of this platform.

1. Cell Bank Authentication and Expansion: Hep 3B or Hep 3B-luc stocks are verified by STR profiling and mycoplasma testing, then expanded in EMEM- or DMEM-based medium and harvested in logarithmic phase.
2. Luciferase Calibration (Hep 3B-luc Only): A standard curve relating in vitro cell number to bioluminescent output is generated, confirming that in vivo photon flux can be read as relative tumor burden.
3. Mouse Preparation and Randomization: Female BALB/c nude or NSG mice, typically 4–6 weeks old, are acclimatized and randomized by body weight, with anesthesia and perioperative analgesia per approved protocols.
4. Laparotomy and Left-Lobe Exposure: Under aseptic conditions the peritoneal cavity is opened through a small midline or transverse incision, and the left hepatic lobe is gently exteriorized for direct injection.
5. Intrahepatic Inoculation: Approximately 0.5–3 × 10⁶ cells in 10–50 µl of PBS or a 1:1 Matrigel mixture are injected slowly into the parenchyma; the needle track is compressed briefly on withdrawal to limit reflux, and any spilled cells are absorbed.
6. Closure and Postoperative Recovery: The abdominal wall and skin are sutured in layers, and animals recover under warming with daily health checks until fully ambulatory.
7. Longitudinal Bioluminescence Monitoring: At scheduled intervals (commonly weekly), mice receive intraperitoneal D-luciferin (150 mg/kg) and are imaged under isoflurane 5–15 minutes later, with region-of-interest analysis quantifying intrahepatic tumor growth and flagging extrahepatic signal.
8. Endpoint Analysis: At humane endpoint (e.g., >20% body-weight loss or a pre-defined photon threshold), the liver is resected for ex vivo imaging and weighing, blood is collected for AFP assay, abdominal organs are surveyed for metastatic foci, and tissues are processed for H&E and immunohistochemistry (Ki67, cleaved caspase-3).

Fig 2. Hep 3B / Hep 3B-luc orthotopic mouse model construction workflow.

Case Study-Hep 3B / Hep 3B-luc Orthotopic Mouse Model Development

Alfa Cytology has executed preclinical programs on the Hep 3B / Hep 3B-luc orthotopic platform for clients pursuing p53-pathway restoration, HBV-associated HCC biology, GPC3-directed cell therapy, and nanoparticle-based treatment strategies, producing calibrated bioluminescence growth curves, ex vivo liver quantification, AFP measurements, and histopathology-confirmed endpoint packages. As each engagement is built around the client’s specific therapeutic concept, study outlines and representative datasets are available upon request.

Why Choose Alfa Cytology?

Hep 3B / Hep 3B-luc orthotopic studies live and die by execution quality: uncalibrated imaging, imprecise injections, or unverified cell banks can quietly undermine weeks of work. Alfa Cytology’s layered quality controls protect the integrity of every dataset that leaves the facility.

• STR- and mycoplasma-verified Hep 3B / Hep 3B-luc banks, with luciferase output calibrated before in vivo use.
• Refined left-lobe intrahepatic injection technique with disciplined reflux control and postoperative care.
• In-house IVIS bioluminescence imaging on pre-agreed schedules for quantitative tumor tracking in vivo.
• Molecularly relevant endpoints including AFP assay and p53-pathway IHC alongside standard histology.
• Immunocompromised hosts (nude, SCID, NSG) matched to the client’s study design and effector requirements.
• One dedicated team owning the study from feasibility through the final consolidated data package.

Contact Us

From p53-restoration strategies to CAR-T and nanomedicine programs, Alfa Cytology can configure a Hep 3B / Hep 3B-luc orthotopic study that speaks directly to your therapeutic hypothesis. Contact us today to align on study design, imaging cadence, and endpoints, or reach out to our scientific team for a customized quotation.

For research use only. Not intended for any clinical use.