Procedure
1. Cell Harvesting
Collect cells in the logarithmic growth phase with 80–90% confluence into a sterile centrifuge tube.
Note: Cells must be harvested during exponential growth. Cell status greatly influences tumor formation efficiency. If the tumorigenicity of a cell line is low, Matrigel may be added to the injection mixture to provide a supportive extracellular matrix.
2. Cell Preparation
Centrifuge cells at 1500 rpm, 4°C for 5 minutes. Remove supernatant and resuspend in 1 mL PBS. Wash cells 2–3 times.
Digest cells with trypsin, and then neutralize with PBS or serum-free medium. Centrifuge again 1–2 times to remove trypsin and resuspend in PBS or medium to a final concentration of 1–5 × 10⁸ cells/mL. Keep cell suspension on ice.
Note: Ensure homogeneous mixing of the cell suspension for accurate cell counting. After trypsinization, the cells should be injected subcutaneously into nude mice as soon as possible, preferably within 30 minutes. If a large number of injections are required, divide the process into multiple batches to ensure timely implantation.
3. Cell-Matrigel Mixture Preparation
Mix the cell suspension and Matrigel at a 1:1 ratio on ice to obtain a final concentration of 1–5 × 10⁷ cells/mL. Store on ice until injection.
Immediately before injection, pipette thoroughly to disperse cell clumps and ensure even distribution.
Note: Keep the mixture cold to reduce cell metabolism and prevent premature gelling.
4. Subcutaneous Injection
Inject 100–200 µL of the cell-Matrigel suspension subcutaneously in areas with good blood supply, such as the posterior axilla or inguinal region.
Use your thumb and forefinger to gently pinch the skin over the shoulders and upper back to restrain the mouse and prevent it from kicking the syringe. Hold the animal in an upright position, and disinfect the dorsal skin at the injection site 2–3 times using a cotton swab soaked in 75% ethanol.
5. Injection Technique
Insert the needle gently into the subcutaneous space, avoiding muscle. Slowly inject the cell suspension. Wait a few seconds before withdrawing the needle to minimize leakage.
Note: Ensure all air is removed from the syringe before injection. Insert the needle ~1 cm under the skin, moving slightly before injection to confirm it is not in the dermis or muscle. If the needle moves freely, it is in the subcutaneous layer.
6. Post-Injection Monitoring
Return mice to their cages and monitor daily for survival, tumor growth, body weight, appetite, and overall health.
Tumor formation typically occurs within 1 week to 1 month. According to experimental design, euthanize mice before the tumor exceeds 1000 mm³ and take photographs.
Note: Avoid tumor burden >1500 mm³ for animal welfare reasons.
7. Tumor Harvesting
After euthanasia, freeze part of the tumor in liquid nitrogen for future RNA/protein extraction.
Fix the remaining tissue in formalin for immunohistochemistry or immunofluorescence analyses.
Precautions and Notes
I. Cells
1. Cell Line Characteristics
Tumorigenicity varies by cell line. Perform a pilot study to evaluate tumor formation and review relevant literature beforehand.
2. Cell Condition
Maintain sterile technique to avoid contamination. Mycoplasma infection is common and alters cell metabolism, proliferation, and morphology, affecting tumor reproducibility.
Confirm mycoplasma-negative status before use. Only use cells in the log phase. Check cell viability before injection and optionally after injection as well.
Keep cells on ice to preserve viability. Avoid excessive in vitro passaging (limit to 3–5 passages before implantation).
II. Animal Models
Common immunodeficient mouse strains include Nude, NOD-Scid, and NCG. Tumor formation rates vary depending on the degree of immune deficiency and the age of the mice. It is recommended to select an appropriate animal model based on pilot studies and published literature. Avoid using older mice, as immune function becomes more active with age. For example, in nude mice, NK cell activity begins to increase after 8 weeks. Mice aged 4–6 weeks are generally preferred, as their immature immune systems result in higher tumor take rates.
III. Injection Sites
There are four commonly used subcutaneous injection sites (as illustrated below). It is recommended to select the injection site based on the tumorigenicity of the cell line and the specific goals of the experiment. The subaxillary region (site #3) is generally considered optimal due to its rich blood supply and ease of access on the right side. For fast-growing tumor types, alternative sites such as the lower limbs or dorsal region—with relatively less vascularization—can be used to slow tumor progression. Alternatively, reducing the number of injected cells may also help control tumor growth rate.
IV. Cell Dosage
The optimal cell dose varies depending on the cell line, mouse strain, and injection site. It is recommended to conduct pilot studies with different dose gradients to determine the most suitable cell concentration for tumor formation.
In addition, auxiliary factors may be required depending on the cell type. For most solid tumor cell lines, mixing the cells with Matrigel at a 1:1 ratio is sufficient. In ovarian cancer models, estrogen supplementation is commonly required — typically administered via a separate injection after cell implantation.
V. Animal Housing
Mice can experience stress responses during transportation, leading to a range of pathophysiological changes that may introduce variability or errors in experimental results. Therefore, animals should not be used for experiments immediately upon arrival. A 1–2 week acclimation period is strongly recommended before beginning procedures.
Maintain a highly controlled housing environment. Immunodeficient mice must be housed in SPF (specific pathogen-free) barrier facilities, and must be kept in separate rooms from immunocompetent mice to prevent cross-contamination and infection.
Nude mice, when chronically exposed to weak antigens in the environment, may gradually develop increased activity of NK cells and other residual immune cells. This can lead to enhanced nonspecific immune responses against tumor cells, reducing tumor take rates over time.
NCG mice and related strains, which are severely immunodeficient, are highly susceptible to pathogens. Therefore, their housing environment must be kept clean and rigorously disinfected to minimize infection risks.
FAQs:
Why use nude mice instead of regular mice?
Nude mice lack a thymus and have severely compromised immune systems. This makes them tolerant to xenografts and ideal for tumor engraftment. In contrast, regular mice have intact immune systems that may trigger immune rejection against transplanted tumor cells, interfering with tumor formation.
Why did no tumors form despite following the protocol correctly?
Possible reasons include:
- Poor cell viability or low tumorigenicity of the cell line.
- Cell line may require the addition of Matrigel or increased inoculation volume to improve engraftment.
- Host mice may be immunologically mature or insufficiently immunodeficient, resulting in immune rejection. Consider using mice with more severe immunodeficiency.
- Tumor development takes time; visible nodules may appear after 1 week, while solid tumors may require 1–2 months. Continuous observation is needed.
Why did the tumor initially grow but then shrink or disappear?
This may result from an inflammatory response or immune clearance of tumor cells by the host. If the tumor regresses and does not resume growth, consider using mice with higher degrees of immunodeficiency.
Why is there size variability among tumors in the same group?
Tumor growth may be influenced by:
- Biological variation among mice (e.g., age, weight, immune status).
- Differences in cell suspension volume, concentration, or injection technique.
- Inconsistencies in site selection or Matrigel use.
What is the appropriate tumor size for endpoint analysis?
According to animal welfare regulations, tumor burden should not exceed 10% of the mouse's body weight or 15 mm in any dimension.
For biomarker discovery or short-term studies: target tumor volume of 150–250 mm³.
For long-term drug efficacy studies: 50–150 mm³ is recommended.
How to accurately measure irregularly shaped tumors?
Monitor animals daily for tumor progression and overall health.
Subcutaneous tumors can be measured using calipers.
Internal tumors can be assessed via in vivo fluorescence imaging or post-mortem dissection.
Use the formula: Tumor Volume = (Length × Width²) / 2 for volume estimation.
Can multiple tumors be induced on the same mouse to increase success rate?
No. Only one injection site per mouse is allowed. Multiple tumors in the same host can interfere with each other and complicate interpretation.
What if the mouse develops necrosis or ulceration during tumor growth?
This may indicate infection or excessive tumor burden.
Initiate appropriate treatment (e.g., antibiotics).
If ulceration/necrosis exceeds 1.5 cm or persists for 48–72 hours without healing, humane euthanasia should be considered.
How should the mice be handled after the tumor study is completed?
Follow institutional guidelines for animal welfare. Euthanasia should be performed if animals exhibit:
- Persistent weight loss
- Severe diarrhea or abnormal excretions
- Tumor ulceration, necrosis, or infection
- Rapid tumor growth compromising the animal’s wellbeing
How to evaluate tumor metastasis?
- Gross necropsy: Visible metastatic lesions in liver, kidneys, or other organs.
- In vivo imaging: If tumor cells are labeled (e.g., luciferase or fluorescent proteins), metastasis can be monitored using optical imaging systems.
- Small-animal MRI: Allows precise detection of metastases in organs like the liver or lungs.