H22 Xenograft Model

H22 xenograft model

The H22 cell line (mouse liver) is used to create the CDX (Cell Line Derived Xenograft) H22 xenograft mouse model.  The H22 xenograft model enables the study of potential therapeutics for HCC, including immunotherapies in the orthotopic model.  The orthotopic H22 model has been shown to increase the immune cell population of regulatory T cells and myeloid-derived suppressor cells in the bone marrow, spleen and tumor tissues.

Basic study design

1. All flasks are maintained at a phase of exponential growth prior to collection.
2. The H22 cells are collected for injection by trypsinization.  Cell count and viability is determined by a trypan blue exclusion test (min 98% cell viability).  Cell suspensions are adjusted to the required density for inoculation.
3. One million cells (in a volume of 100 uL) of the matrigel/H22 cell suspension is injected subcutaneously into the hind leg of each mouse (NOD/SCID or athymic BALB/C, 10-12 weeks).
4. The injection sites are monitored until tumors are established.  Digital calipers are used to measure tumors until they reach an sizes of 50-150 mm3.
5. Post-sorting into the treatment cohorts (randomization), the compound of interest is administered according to the treatment schedule.
6. Daily tumor measurements are taken and mouse weights are recorded up to 3 times weekly.
7. As the predetermined tumor size limit is reached (or 2,000 mm2), animals are euthanized.
8. As defined for the termination of the experiment, a necropsy and tissue collection is performed.
9. All tumors are removed and weighed, then documented via digital imaging.
10. Tissues are collected for downstream analysis by performing standard gross necropsies.
11. All tumors and tissues can be snap frozen, prepared for histology, stabilized in RNAlater or nucleic acid isolated for genetic analysis.

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H22 Xenograft Model

Xenograft animal models are used to assess the effectiveness of drugs against specific types of cancer. New medicines are tested on staged tumor growths that have been engrafted via subcutaneous or orthotopic inoculation in an immunocompromised mouse or rat model. All clinically approved anti-cancer agents have been evaluated with conventional preclinical in vivo models. Xenograft studies can be highly complex, starting with the selection of the appropriate animal model, choice of tumorigenic cell line, administration method, dosing, analysis of tumor growth rates and tumor analysis (histology, mRNA and protein expression levels).

Altogen Labs provides an array of laboratory services using over 30 standard Cell Line Derived Xenograft (CDX) models and over 20 PDX models. Researchers investigating the role of specific proteins or gene products in regulating tumor growth can benefit from development of protein overexpression (genetically engineered to ectopically express proteins, tumor suppressors, or oncogenes) and RNAi cell lines with long term gene silencing. Altogen Labs provides quantitative gene expression analysis of mRNA expression (RT-PCR) and protein expression analysis using the WES system (ProteinSimple).

The dosing of the experimental compound of interest is initiated, for a staged study, when the mean tumor size reaches a specified volume (typically 50-100 mm3). In an unstaged study, the dosing of the compound of interest is initiated immediately after xenografting. Mice are dosed once or twice a day for 28 days (or other desired study duration) via the chosen route of administration. Tumor volume (mm3) is calculated via the “(W x W x L) / 2” formula, where W is tumor width and L is tumor length.

Animal handling and maintenance at the Altogen Labs facility is IACUC-regulated and GLP-compliant. Following acclimatization to the vivarium environment, mice are sorted according to body mass. The animals are examined daily for tumor appearance and clinical signs. We provide detailed experimental procedures, health reports and data (all-inclusive report is provided to the client that includes methods, results, discussion and raw data along with statistical analysis). Additional services available include collection of tissue, histology, isolation of total protein or RNA and analysis of gene expression. Our animal facilities have the flexibility to use specialized food or water systems for inducible gene expression systems.

Following options are available for the H22 xenograft model:

  • H22 Tumor Growth Delay (TGD; latency)
  • H22 Tumor Growth Inhibition (TGI)
  • Dosing frequency and duration of dose administration
  • Dosing route (intravenous, intratracheal, continuous infusion, intraperitoneal, intratumoral, oral gavage, topical, intramuscular, subcutaneous, intranasal, using cutting-edge micro-injection techniques and pump-controlled IV injection)
  • H22 tumor immunohistochemistry
  • Alternative cell engraftment sites (orthotopic transplantation, tail vein injection and left ventricular injection for metastasis studies, injection into the mammary fat pad, intraperitoneal injection)
  • Blood chemistry analysis
  • Toxicity and survival (optional: performing a broad health observation program)
  • Gross necropsies and histopathology
  • Positive control group employing cyclophosphamide, at a dosage of 50 mg/kg administered by intramuscular injection to the control group daily for the study duration
  • Lipid distribution and metabolic assays
  • Imaging studies: Fluorescence-based whole body imaging, MRI

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H22 Xenograft Model