Subcutaneous graft tumor model construction

Summary

A certain amount of tumor cells were injected subcutaneously into nude mice with congenital immunodeficiency to form tumors, thus observing the development of the tumors and the interference of drugs, etc.

Operation method

Modeling of tumor cell subcutaneous graft invasion

Materials and Instruments

1. Animal selection: Generally, nude mice around 6 weeks of age and weighing 18~22 g are selected and kept in an isolated barrier environment after quarantine.
2. Tumor cell preparation: Pay attention to the calculation of the amount of cells, the amount of cells is generally 5 × 10
6
The amount of cells is generally 5 × 10 6 /200 μl (the inoculum amount may be different for different cell lines).
3、Preparation of consumables: PBS × 1, 75% alcohol × 1, filter paper × 3, gloves, instrument box (mainly curved scissors, forceps, 1 ml syringe and hemostatic clamp), cotton balls, etc.

Move

1. Tumor cell expansion:

(a) Take tumor cells for recovery and expansion, digest with 0.25% trypsin, centrifuge at 1 000 rpm.

(b) Remove the supernatant, add 1×PBS to wash the cells once, then add 1×PBS to resuspend them, count them, and dilute the cell suspension proportionally, adjusting it to about 5 × ¹⁰⁶ cells/200 μl of cell suspension.

(c) Blow and centrifuge the cells twice with serum-free medium, then resuspend and mix them with serum-free medium, place them in 0.5 ml EP tubes, and set them aside on ice (in consideration of the cell activity, they should be injected into the subcutaneous area of nude mice as soon as possible).

2. Tumor formation process:

(a) Group the mice according to the experimental requirements, with at least 5 mice in each group.

(b) Grasp the whole back and head of the nude mice with the thumb and forefinger, and press the calf and tail with the ring finger and little finger. The injection site (back or axilla) is wiped with a cotton ball dipped in alcohol. Use a 1 ml syringe (a conventional 1 ml syringe is sufficient) to draw up 200 μl of cell suspension and inject it into the subepidermis of the nude mice.

3. After tumor formation:

(a) The tumor cells are usually observed visually 3 days after injection, and the weight of the nude mice is recorded at each observation.

(b) After tumor formation, it is necessary to record the changes of the long and short axes of the tumor with a ruler at regular intervals and record the statistics.

(c) According to the experimental requirements, the nude mice were executed by cervical dislocation after reaching the time, and the tumors were removed and photographed. When taking photos, pay attention to the light and focus clearly.

(d) The formula for calculating the tumor volume is: long axis × wide axis 2/2.

Caveat

(a) The concentration of serum and antibiotics can be increased appropriately in order to ensure the activity of the cells when they are expanded in large numbers.(b) Criteria for successful subcutaneous injection: subcutaneous injection forms a visible protrusion that can be pushed (refer to chickenpox).(c) Stay for a few seconds after subcutaneous injection of tumor cell suspension before removing the needle to prevent the suspension from leaking with the needle.(d) Mice are prone to die after injection of tumorigenic mice, pay attention to the experimental cycle.(e) Pre-experimentation can be conducted in groups of 3 mice each. If tumor formation is not possible in nude mice, other immunodeficient mice such as NOG and SCID mice can be selected.(f) Tumor formation in nude mice may not be possible due to individual differences, so try to have as many as possible in a group, or randomize the group after tumor formation.(g) Increasing the concentration of cells can increase the rate of tumor formation, but the range is not fixed and difficult to grasp.

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