The process of tumor drug development requires the evaluation of the biological activity, pharmacological effects, and therapeutic value of substances used at biochemical and cellular levels. The entire screening process needs to be applied to pharmacological experimental models, with animal experimental models being a common pharmacological toxicity experimental model. On September 29th, in the Eastern Time Zone of the United States, the United States Senate passed the FDA Modernization Act 2.0, which repealed the provision in the 1938 Food, Drug, and Cosmetic Act (FDCA) that required all drugs to be tested on animals to eliminate toxicity. It is worth noting that the law does not prohibit the use of animal testing but allows drug developers to use alternative methods where feasible. Other methods such as organoids and AI (Artificial Intelligence) may also serve as experimental approaches.

Figure: Comparison of Characteristics of Different Model Systems [1]

 

PDO Model

Patient-Derived Organoids (PDOs) are entities obtained by culturing tumor tissues obtained from patients through biopsy, puncture, or surgical resection in vitro for a certain period. While maintaining the heterogeneity of the source tumor and between patients, tumor organoids also exhibit uniform morphology and scale among individuals, providing a more convenient solution for drug screening. Matrix gel is used as an important material in the preparation process of PDOs, which accelerates the formation of PDOs.

The main components of matrix gel include laminin, type IV collagen, heparan sulfate proteoglycans (HSPG), nidogen, and other growth factors such as TGF-beta, EGF, IGF, FGF, tissue plasminogen activator, and other growth factors present in EHS tumors. It provides support, tensile strength, and scaffold support for tissues and cells. Arcegel^TM Matrix Gel developed and produced by Arcegen Biology is free from LDEV (lactate dehydrogenase-elevating virus), has an ultra-low endotoxin content, and has been thoroughly tested for mycoplasma to ensure no contamination, including different types of matrix gels such as basic concentration, high concentration, and low growth factor concentration. 

 

 Application Directions

 

Product Features

High Safety: Free from LDEV (lactate dehydrogenase-elevating virus)

Diverse Concentrations: Concentration range between 8 to 20 mg/mL

Good Batch Stability: Strict production quality inspection processes ensure stable performance between batches

Low Endotoxin Levels: Endotoxin content < 8 EU/mL

Contaminant Detection: Tested free of mycoplasma, bacteria, and fungal residues

High Yield: Production exceeds 30L/month

Excellent Compatibility: Compatible with any type of cell culture medium

 

In Vivo Tumorigenesis Experiment

Experimental Procedure

Taking the example of subcutaneous tumor formation experiment using HepG2 cells in nude mice, Arcegel high-concentration matrix gel and cell suspension are diluted at a 1:1 ratio and subcutaneously injected into 4-5 week-old female BALB/c-nu mice. The experimental procedure is as follows:

1. Prepare logarithmic-phase, cells with a density of approximately 80-90% before cell collection, and replace the medium with fresh culture medium the night before cell collection.

2. Digest the cells with trypsin, remove the trypsin when the cells become round but have not detached from the culture dish, add serum-free medium to make cell suspension, centrifuge and wash once, and resuspend to a final concentration of 5×10⁷ cells/mL.

3. Dilute the cell suspension and Arcegel high-concentration matrix gel at a 1:1 ratio at 4°C to prepare a final concentration of 5×10⁷ cells/mL.

4. Hold the nude mouse with the left hand and subcutaneously inject the mixture into the right shoulder of the mouse. When inoculating, insert the needle slightly deeper subcutaneously, about 1cm deep, to reduce the overflow of cell suspension from the needle hole after injection. The injection volume is 200μL. (This process should be completed within half an hour, and the cell suspension should be placed on ice to slow down cell apoptosis and prevent gelation).

5. Return the nude mouse to the cage for continued breeding. Tumors can be observed approximately 1 week to 1 month later. According to the experimental design, euthanize the nude mouse when the tumor volume reaches the required size and take photographs.

Note: The control group consists of a suspension of medium and cells, with a final density identical to the matrix gel experimental group.

 

Experiment Results

Figure: Tumor Formation in BALB/c-nu Mice with HepG2 Cells After 1 Month

 

FAQ

01. What is the reason for the color change (from pale yellow to deep red) observed in the obtained matrix?

For matrix gel containing phenol red, the main reason for the color change is the interaction between phenol red and bicarbonate with CO₂. However, the color difference will decrease after balancing with 5% CO2. After thawing, gently shaking the reagent bottle to evenly disperse the matrix gel.

02. What precautions should be taken when handling the matrix gel?

All operations should be conducted in a sterile environment, and pre-chilled pipettes should be used to ensure that the matrix gel is evenly dispersed.

03. How should the matrix gel be aliquoted and stored for later use?

After thawing, the Arcegel matrix gel can be aliquoted into multiple tubes, all of which should be pre-chilled storage tubes. Rapidly freeze and store to avoid repeated freeze-thaw cycles. All items involved in use should be pre-chilled, and matrix gel handling should be done using pre-chilled pipettes, tips, and tubes.

04. Can regular mice be used for the experiment?

The use of nude mice is advantageous due to their lack of a thymus, resulting in lower immunity and no immune response to tumor cells, making them more susceptible to tumor formation.

05. What could be the reasons for the tumor not growing?

Possible reasons include injecting too few cells, cells with poor viability, or cells that are not tumorigenic and require a longer breeding period. It is recommended to use immunodeficient mice of moderate age (4-6 weeks).

 

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