**3. Air pouch methodology**

Air pouch inflammation model.

In general, a common process to induce air pouches is performed as follows:


**Note:** Inflammatory agents commonly used to induce the inflammation air pouch model are ultraviolet radiation, 12-o-tetracanoilphorbol-13-acetate, oxazolone in acetone, turpentine, carrageenan, brewer's yeast, formaldehyde, dextran, egg albumin, kaolin, aerosil, implantation of pellets of compressed cotton, collagen, incomplete Freund's adjuvant, and papaya latex [60].

**Figure 3.** Air pouch formation. The rodent back is shaved (A); the animal is held, and air is injected subcutaneously into

the back (B); and air pouch inflation is observed (C and D). The air pouch completely formed (E).

**Figure 2.** Step-by-step sterile air filtration in laminar flow hood. A sterile syringe that can contain 5 mL of air and a 0.2 μm filter (A) packages is opened inside a laminar flow hood (B), the syringe is carefully removed (C), the needle is

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detached (D), the syringe is attached to the filter (E), and 5 mL of air is loaded into the syringe (F).

**4.** On day 8, the pouches of the experimental groups are filled with necessary doses of the compound to test, according to our requirements.

**Figure 2.** Step-by-step sterile air filtration in laminar flow hood. A sterile syringe that can contain 5 mL of air and a 0.2 μm filter (A) packages is opened inside a laminar flow hood (B), the syringe is carefully removed (C), the needle is detached (D), the syringe is attached to the filter (E), and 5 mL of air is loaded into the syringe (F).

**3. Air pouch methodology**

180 Cell Culture

Air pouch inflammation model.

of each mouse (**Figure 3**).

In general, a common process to induce air pouches is performed as follows:

(0.22 μm) directly into a 10 mL syringe (**Figure 2**).

compound to test, according to our requirements.

**1.** Sterile air is obtained in a laminar flow station by filtration through a Millipore filter

**Table 1.** Comparison between the *in vitro* and the *in vivo* air pouch model cell line culturing and drug discovery.

**2.** Five milliliters of sterile air is injected subcutaneously into the shaved skin site on the back

**3.** The pouches are allowed to settle for 3 days to permit the healing of the wound. The pouch is then reinflated with 5 mL of sterile air and left for 3 more days before treatments.

**4.** On day 8, the pouches of the experimental groups are filled with necessary doses of the

**Figure 3.** Air pouch formation. The rodent back is shaved (A); the animal is held, and air is injected subcutaneously into the back (B); and air pouch inflation is observed (C and D). The air pouch completely formed (E).

**Note:** Inflammatory agents commonly used to induce the inflammation air pouch model are ultraviolet radiation, 12-o-tetracanoilphorbol-13-acetate, oxazolone in acetone, turpentine, carrageenan, brewer's yeast, formaldehyde, dextran, egg albumin, kaolin, aerosil, implantation of pellets of compressed cotton, collagen, incomplete Freund's adjuvant, and papaya latex [60].

Air pouch modified for tumor induction


Protocol 1: Air pouch model for tumor implantation


The appropriate age range of the rodents depends on the experiment and relevant age for human disease. For example, 3- to 6-month C57BL6/J mouse is comparable with 20–30 human years, 10–14 months compares to 38–47 human years, and 18–24 months compares to 56–69

pouch (A). The mouse was sacrificed 12 days after for tumor evaluation. The skin from the air pouch (B) and tumor (B)

were detached (the arrow points to the tumor). Tumor sections were stained with hematoxylin and eosin (C).

viable 4 T1 cells in 100 μL of PBS were inoculated into an air

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**Animal sex:** This can also affect the therapy outcome. This is particularly true for targeted therapies, and it has become a requirement for any applications for the National Institutes of Health (NIH) to report the male and female ratio in preclinical studies [62]. Genes encoded by the Y chromosome include inflammatory pathway genes, while X chromosome encodes genes for Toll-like receptors, cytokine receptors, and transcriptional and translational regulator genes. Although X chromosome is present in both sexes, female X polymorphism allows for mosaicism and randomly silenced alleles. It has been observed that male sex leads to data alteration in pharmacology and neuroscience, and female sex affects immunology results [62, 63]. The National Institutes of Health (NIH) announced in May 2014 that they "require applicants to report their plans for the balance of male and female cells and animals in preclinical studies

**Therapy administration:** Carcinogenesis is described as a three-stage process, initiation, promotion, and progression. Compounds that prevent or delay any stage of carcinogenesis are called chemopreventive agents. Chemoprevention is defined as the use of compounds to reduce the risk or delay the development of cancer or to avoid its recurrence. Bio-compounds such as food-derived polyphenols, usually inhibit initiation and promotion, of induced cancer, and therefore are considered chemopreventive agents [64]. When testing antitumor activity of natural or synthetic compounds, highly variable results can be obtained based on the type of tumor, the administration dose and frequency, and the evaluation parameters: tumor incidence, growth, or metastasis. Therefore, the purpose and parameters of the preclinical

human years [61].

**Figure 4.** Tumor cell inoculation into the air pouch. 1 × 106

in all future applications" [62].

testing must be clearly defined [65].

**3.** For therapy evaluation, mice are sacrificed after 9 days of treatment, and tumor is resected (**Figure 4B**) and can be stained with hematoxylin and eosin (**Figure 4C**).

Protocol 2: Air pouch model for tumor progression


**Note:** For each experiment design, it is important to take into consideration variables that could alter the results, such as animal age and sex, and treatment administration.

**Animal age:** A study performed by Jackson et al. revealed that there is a discrepancy on the age of rodents used for neuroscience, immunology, cancer, genetics, physiology, and toxicology research. The age can vary from 2 to 160 weeks, and 8 to 12 weeks old are the most used.

Air pouch modified for tumor induction

(**Figure 3**).

182 Cell Culture

line.

line.

controls, respectively.

filter (to avoid any contaminant particles) (**Figure 2**).

Protocol 1: Air pouch model for tumor implantation

Protocol 2: Air pouch model for tumor progression

growth controls, respectively.

inoculated 48 hours after the air pouch induction (**Figure 4A**).

**1.** Inside the laminar flow hood, 5 mL of air is charged into a sterile syringe through a 0.2 μm

**2.** The animal back is shaved and sprayed with alcohol (70%). The animal is hold by the scruff and tail (as shown in **Figure 2B**), and the sterile air is injected subcutaneously using a 21G × 1 ¼ caliber needle. The formation of the air pouch is immediately observed

**3.** After 72 hours the procedure is repeated once again to avoid disinflation. Tumor cells are

**1.** Viable tumor cells in 100 μL of phosphate-buffered saline are injected subcutaneously into the air pouch using a 30G × 1/2 caliber needle. Cell number varies depending on the cell

**2.** Treatment administration begins 24 hours later. Treatment administration scheme depends on the tested drug. We recommend adding a group treated with placebo (injectable solution) and with the reported treatment (e.g., chemotherapy), as positive and negative tumor

**3.** For therapy evaluation, mice are sacrificed after 9 days of treatment, and tumor is resected

**1.** Viable tumor cells in 100 μL of phosphate-buffered saline are injected subcutaneously into the air pouch using a 30G × 1/2 caliber needle. Cell number varies depending on the cell

**2.** Treatment administration begins 7 days after tumor inoculation (days can vary depending on tumor growth characteristics). Treatment administration scheme depends on the tested drug. We recommend adding a group treated with placebo (injectable solution) and with the reported treatment group (e.g., chemotherapy), as positive and negative tumor growth

**Note:** For each experiment design, it is important to take into consideration variables that

**Animal age:** A study performed by Jackson et al. revealed that there is a discrepancy on the age of rodents used for neuroscience, immunology, cancer, genetics, physiology, and toxicology research. The age can vary from 2 to 160 weeks, and 8 to 12 weeks old are the most used.

(**Figure 4B**) and can be stained with hematoxylin and eosin (**Figure 4C**).

**3.** For therapy evaluation, mice are sacrificed after 21 days of treatment.

could alter the results, such as animal age and sex, and treatment administration.

**Figure 4.** Tumor cell inoculation into the air pouch. 1 × 106 viable 4 T1 cells in 100 μL of PBS were inoculated into an air pouch (A). The mouse was sacrificed 12 days after for tumor evaluation. The skin from the air pouch (B) and tumor (B) were detached (the arrow points to the tumor). Tumor sections were stained with hematoxylin and eosin (C).

The appropriate age range of the rodents depends on the experiment and relevant age for human disease. For example, 3- to 6-month C57BL6/J mouse is comparable with 20–30 human years, 10–14 months compares to 38–47 human years, and 18–24 months compares to 56–69 human years [61].

**Animal sex:** This can also affect the therapy outcome. This is particularly true for targeted therapies, and it has become a requirement for any applications for the National Institutes of Health (NIH) to report the male and female ratio in preclinical studies [62]. Genes encoded by the Y chromosome include inflammatory pathway genes, while X chromosome encodes genes for Toll-like receptors, cytokine receptors, and transcriptional and translational regulator genes. Although X chromosome is present in both sexes, female X polymorphism allows for mosaicism and randomly silenced alleles. It has been observed that male sex leads to data alteration in pharmacology and neuroscience, and female sex affects immunology results [62, 63]. The National Institutes of Health (NIH) announced in May 2014 that they "require applicants to report their plans for the balance of male and female cells and animals in preclinical studies in all future applications" [62].

**Therapy administration:** Carcinogenesis is described as a three-stage process, initiation, promotion, and progression. Compounds that prevent or delay any stage of carcinogenesis are called chemopreventive agents. Chemoprevention is defined as the use of compounds to reduce the risk or delay the development of cancer or to avoid its recurrence. Bio-compounds such as food-derived polyphenols, usually inhibit initiation and promotion, of induced cancer, and therefore are considered chemopreventive agents [64]. When testing antitumor activity of natural or synthetic compounds, highly variable results can be obtained based on the type of tumor, the administration dose and frequency, and the evaluation parameters: tumor incidence, growth, or metastasis. Therefore, the purpose and parameters of the preclinical testing must be clearly defined [65].
