Matrix Gel is widely used in xenograft and tumorigenesis models to facilitate tumor cell implantation and growth. When mixed with tumor cells before subcutaneous or orthotopic injection, Matrix Gel provides an extracellular matrix–like environment that promotes cell survival, attachment, and angiogenesis, leading to more consistent and reproducible tumor formation.

I. Protocol for In Vivo Tumor Formation

Using a HepG2 cell xenograft tumor model in nude mice as an example, Ceturegel™ Matrix Gel is mixed with cell suspension at a 1:1 ratio and subcutaneously injected into 4–5 week-old female BALB/c-nu mice. The experimental workflow is as follows:

1. Prepare HepG2 cells in logarithmic growth phase with 80–90% confluence. Change to fresh medium the night before cell collection.

2. Digest cells with trypsin. When cells begin to round up but have not detached, remove trypsin and resuspend in serum-free medium. Centrifuge once, then resuspend to a final concentration of 1 × 10⁸ cells/mL.

3. At 4 °C, mix cell suspension and Ceturegel™ High Concentration Matrix Gel at a 1:1 ratio to achieve a final concentration of 5 × 10⁷ cells/mL.

4. Hold the nude mouse with the left hand and inject subcutaneously at the right shoulder. Insert the needle deeper (~1 cm) under the skin to minimize leakage of the cell suspension after injection. Inject 200 μL. (Complete the process within 30 min; keep the cell suspension on ice during the procedure to reduce apoptosis and prevent premature gelation.)

5. Return the mouse to the cage for continued housing. Tumors typically appear within 1–4 weeks. Euthanize the mouse according to experimental design when tumor volume does not exceed 1000 mm³, and take photos.

[Note]: The control group consists of a mixture of medium and cell suspension at the same final cell density as the Matrix Gel group.

II. Experimental Results

Figure 1. Angiogenesis results and immunofluorescence staining images

III.Troubleshooting

Q1: What is the recommended ratio of Matrix Gel to cell suspension?

A1: Typically, a 1:1 (v/v) mixture is used to achieve optimal viscosity and tumor take rate.

Q2: Does Matrix Gel interfere with drug treatment studies?

A2: No, it does not interfere chemically but ensures more physiologically relevant tumor growth for therapeutic evaluation.

Q3: How should Matrix Gel be handled before injection?

A3: Keep on ice until immediately before injection to prevent gelation in the syringe.

Q4: Can normal mice be used for this experiment?

A4: Nude mice lack a thymus and have compromised immunity, making them unable to reject tumor cells, thus facilitating tumor formation.

Q5: Why is the tumor not growing?

A5: ① Tumor growth may take 1–2 months.

② Possible reasons include insufficient cell number, poor cell viability, or the cell line’s low tumorigenicity, which may require supportive factors such as Matrix Gel.

[Npte]: It is recommended to use immunodeficient mice aged 4–6 weeks.

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