The current work is supported by grants from the (1) National Nature Science Foundation (81973877 and 82174408), (2) National Key R&#38;D Program of China (2020YFE0201600), (3) Shanghai Top Priority Research Center construction project (2022ZZ01009), (4) Shanghai Collaborative Innovation Center of Industrial Transformation of Hospital TCM Preparation, and (5) Research Projects within Budget of Shanghai University of Traditional Chinese Medicine (2021LK047).

This protocol describes the detailed gavage technique in osteosarcoma model mice by using the Yiqi Jiedu decoction as a sample. Four-week-old, male athymic mice (BALB/c nude) were used in this experiment and kept in the SPF-grade breeding room of Laboratory Animal Center in Shanghai University of Traditional Chinese Medicine. All animal experiments were approved by the Animal Management and Committee of Shanghai University of Traditional Chinese Medicine, complied with the Code of Ethics for Laboratory Animals (Animal Ethics Approval Number: PZSHUTCM2304060007), and conducted in strict accordance with the international requirements related to laboratory animals.
1. Materials
<ol>
	<li>Obtain the following materials: Chinese herbs (Ginseng 42 g, Hedyotis diffusa 52.5 g, Barbed skullcap herb 31.5 g), 1 mL syringe, and 12G/55 mm elbow gavage needle (Figure 1).</li>
</ol>
2. Dosage determination
<ol>
	<li>Calculate the daily dosage as follows: the daily dosage for adults with a standard body weight of 60 kg is Ginseng 12 g, Hedyotis diffusa 15 g, and Barbed skullcap herb 9 g, with a total of 36 g decoction-ready medicine, that is, 0.6 g decoction-ready medicines/kg body weight. Referring to the 12 times for equivalent dose, 6 times for low dose, and 24 times for high dose between the adults with standard body weight of 60 kg and the mice with standard body weight of 25 g according to the body surface area11, as well as our preliminary data, use a 20 times dose ratio of Yiqi Jiedu decoction for the mice in the current study, therefore, the dosage for mice was 12 g decoction-ready medicines/kg body weight.</li>
	<li>Calculate the dosage for each mouse by using the equation: 
	dosage for mouse (stand body weight) = dosage for adult (stand body weight) x 20 (designated equivalent dose ratio). 
	The daily dosage of each mouse is 0.3 g [(12 g decoction-ready medicines/1000 g body weight) x 25 g body weight] in 200 &#181;L with a final concentration of 1.5 g/mL.</li>
	<li>Calculate the total decoction-ready medicines for mice (n=7) in the Yiqi Jiedu decoction group with gavage administration for 28 days to be 58.8 g. Concentrate this to 39.2 mL to reach a final concentration of 1.5 g/mL.</li>
	<li>Considering possible drug loss during the decoction and the gavage stages, and for the convenience of calculation, prepare a total of 126 g (Ginseng 42 g, Hedyotis diffusa 52.5 g, Barbed skullcap herb 31.5 g) decoction-ready medicine, concentrate into a final volume of 84 mL.</li>
</ol>
3. Cell source and culture
<ol>
	<li>Purchase human-derived 143B osteosarcoma cells from ATCC cell bank.</li>
	<li>Seed 1 x 106 143B cells in 10 mL of MEM medium containing 10% FBS, 100 U/mL penicillin, and 100 &#181;g/mL streptomycin in a 10 cm cell culture dish.</li>
	<li>Culture 143B cells at 37 &#176;C in a 5% CO2 incubator until the cells are 80%-90% confluent.</li>
</ol>
4. Modeling methods
<ol>
	<li>Collect the 80% confluent cultured 143B cells, resuspend into 6 mL of 10% serum-containing MEM medium and mix with basement membrane matrix gel at a volume ratio of 1:1 to make a cell suspension with a final concentration of 1 x 107 cells/mL.</li>
	<li>Inject 10 &#181;L of the cell suspension into the left tibia of each athymic mouse to establish an in situ xenograft osteosarcoma mouse model, as being described in our previous publication12. The steps in brief are as follows.
	<ol>
		<li>Wash the cells 2x with PBS. Trypsinize the cells with 1.5 mL of 0.25% trypsin for 3 min. Add 6 mL of 10% serum-containing MEM media. Collect the cells in a 15 mL centrifuge tube.</li>
		<li>Aspirate 20 &#181;L of cell suspension into the chamber of the cell counting plate and calculate cell concentration using an automatic cell counter. Centrifuge the cells at 800 x g for 5 min at room temperature.</li>
		<li>Discard the supernatant with a pipette, resuspend the cell pellet in 8.5 mg/mL basement membrane matrix, and place them on ice.</li>
		<li>Anesthetize the mice by exposing them to 2% isoflurane and 98% oxygen (oxygen flow rate, 2 L/min). Apply a small amount of vet ointment on the eyes to prevent dryness while under anesthesia.</li>
		<li>Perform the entire procedure in a well-ventilated area. Before osteosarcoma cell injection, ensure that each mouse is under deep anesthesia by gently touching the foot; if the mouse still has responses, such as twitch or jerk.&#160;Wait for a longer time if the mouse still has responses, such as twitch or jerk, until these responses disappear.</li>
		<li>Place the mouse in a supine position. Hold the ankle of the mouse using the thumb and index finger and disinfect the injection site of the tibia with three alternating rounds of betadine and 70% ethanol swab.</li>
		<li>Attach the needle to a 1 mL syringe and point the needle tip toward the injection site paralleling to the axis of the tibia. Percutaneously insert the needle to drill a hole through the tibia platform toward the distal end of the tibia.</li>
		<li>Load the osteosarcoma cell suspension previously being placed on ice into a microvolume syringe and replace the 1 mL syringe in the tibia with the osteosarcoma cell-loaded microvolume syringe. Slowly inject approximately 10 &#181;L of (1 x 107 cells/mL) osteosarcoma cell suspension into each athymic mouse&#39;s tibia.</li>
		<li>Remove the microvolume syringe from the injection site, and press the injection site with the cotton swab for about 30 s. Place the mouse back into a clean cage and observe its recovery for 10 min.</li>
	</ol>
	</li>
</ol>
5. Preparation of traditional Chinese medicine decoction formula for gavage (&#160;Figure 2)
<ol>
	<li>Calculate the dosage as per step 2.</li>
	<li>Soak all the medicinal herbs for decoction, except for the expensive herb Ginseng, in drinking water with a level 3 cm higher than the medicine surface, for 30 min, and then perform the decoction 2x as described below.</li>
	<li>Perform the first decoction by heating to boiling over a strong fire, and then heating for 15-25 min over a mild fire. Mild fire refers to a low-power heat with temperatures typically ranging from 80 &#176;C to 100 &#176;C, while strong fire refers to a high-power heat typically ranging from 200 &#176;C to 300 &#176;C.</li>
	<li>Pour the decoction liquid into one container.</li>
	<li>Perform the second decoction by heating the medicinal herbs in drinking water, with a volume just enough to submerge the medicine surface, to boiling over a strong fire, and then heating for 15-25 min over a mild fire.</li>
	<li>Pour the decoction liquid into the same container as in step 5.4.</li>
	<li>Soak and decoct 42 g of Ginseng separately, with drinking water 3 cm higher than the medicine surface, since it is expensive.</li>
	<li>Perform the first decoction of soaked Ginseng by heating it over a strong fire for 60 min. 
	Pour the decoction liquid into the same container as in step 5.4.</li>
	<li>Perform the second decoction of Ginseng in drinking water with a volume just enough to submerge the medicine surface, by heating over a mild fire for 40 min. Pour the decoction liquid into the same container as in step 5.4. 
	NOTE: The containers used for decoction should be chemically stable, such as casseroles (the most commonly used in clinical practice), ceramics, glass, and stainless steel to avoid possible chemical reactions with the medicinal substances. All decoction liquid obtained from different steps must be put in the same container.</li>
	<li>Mix and filter the decoction liquid using three layers of gauze fixed at the mouth of a measuring cup with rubber bands.</li>
	<li>Boil the filtered decoction on a strong fire in a pot, then change to mild fire and keep the mild fire until reaching the target concentration (a final volume of 84 mL with a final concentration of 1.5 g/mL).</li>
	<li>Prepare 21 mL aliquots of the concentrated decoction according to the dose required for intragastric administration for 1 week, and store the aliquots in a refrigerator at -20 &#176;C.</li>
</ol>
6. Gavage administration in mice by using a gavage device
<ol>
	<li>Place the mouse on the surface of the mouse cage or other rougher surface.</li>
	<li>Grasp the mouse&#39;s tail with the thumb and index finger of the right hand. Then, grasp the mouse&#39;s neck with the thumb, index finger, and middle finger of the left hand.</li>
	<li>Hold the mouse tail with the other two fingers of the left hand to make the head, neck, and body of the mouse in a straight line. 
	NOTE: Make sure to keep the mouse relaxed during the entire grasping process. If the mouse keeps struggling to move, the grasping process should be performed until the mouse is relaxed.</li>
	<li>Hold the gavage device in the right hand and load 200 &#181;L of decoction formula for a mouse with 20 g body weight (100 &#181;L decoction formula/10 g body weight of mouse).</li>
	<li>Insert the gavage needle into the mouth from the corner and push the tongue against the upper jaw of mouse.</li>
	<li>Keep the mouse body in the same direction and gently push the gavage needle toward the stomach along the esophagus until about 10 mm of the gavage needle is left outside. 
	NOTE: There is a hollow feeling when the gavage needle enters into the esophagus.The length of the gavage needle inserted into the mouse should be measured to the top of the sternum. The total length of the gavage needle is 60 mm (Figure 3), therefore, about 50 mm of the gavage needle should be inserted into the body of mouse before the decoction formula is injected into the stomach of mouse.</li>
	<li>Inject the decoction formula into the stomach of mouse. Meeting resistance or liquid coming out of the mouth corner means misplacement of the gavage needle, which must be avoided. In addition, the injection speed must be slowed down if the decoction formula is thick.</li>
	<li>Release the mouse and observe them for 10 min. The gavage is successful if no breathing abnormality is observed and return the mouse into the cage (Figure 4).</li>
</ol>
7. Course of treatment
<ol>
	<li>To explore the therapeutic efficacy of Yiqi Jiedu decoction, set up a control group and a Yiqi Jiedu decoction group. For mice in the control group, administer physiological saline, once a day for 28 days. For mice in the Yiqi Jiedu decoction group, administer the Yiqi Jiedu decoction once a day for 28 days.</li>
</ol>
8. Adverse reactions
<ol>
	<li>Closely observe the status of the mice after being placed back into the cage, including weight loss and decreased mental status due to the inability of food to enter into the stomach.</li>
	<li>Mice will experience difficulty in breathing and shortness of breath due to food-caused tracheal obstruction and will cough in an attempt to clear the trachea. If any of these symptoms is observed during gavage, immediately stop the gavage and&#160;euthanize the mice.</li>
</ol>

Herb Decoction Formulation and Gavage Standardization in Osteosarcoma Mice

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	<li>Gao, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discussion+on+advantages+and+improvement+of+traditional+Chinese+medicine+decoction.">Discussion on advantages and improvement of traditional Chinese medicine decoction.</a> Chinese Community Physicians (Comprehensive Edition). (21), 12 (2005).</li><li>Chinese Pharmacopoeia Commission. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Pharmacopoeia of the People's Republic of China. , (2015).</li><li>An, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interpretation+on+specification+of+traditional+Chinese+medicine+decoction.">Interpretation on specification of traditional Chinese medicine decoction.</a> Herald Med. 42 (11), 1648-1652 (2023).</li><li>Chen, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Research+strategies+in+standard+decoction+of+medicinal+slices.">Research strategies in standard decoction of medicinal slices.</a> China J Chinese Materia Medica. 41 (8), 1367-1375 (2016).</li><li>Luo, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+soaking+traditional+Chinese+medicine+before+decoction+on+active+components.">Effect of soaking traditional Chinese medicine before decoction on active components.</a> Shanghai Yiyao. 40 (9), 63-64 (2019).</li><li>Liu, F., Chen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+standardization+of+decocting+methods+of+modern+decoction.">On the standardization of decocting methods of modern decoction.</a> Global Trad Chinese Med. 7 (5), 377-378 (2014).</li><li>Li, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+decocting+times+and+instrument+on+the+quality+of+decoction.">Influence of decocting times and instrument on the quality of decoction.</a> J Trad Chinese Med. 50 (6), 550-552 (2009).</li><li>Li, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herbal+decoctosome+is+a+novel+form+of+medicine.">Herbal decoctosome is a novel form of medicine.</a> Sci China. Life Sci. 62 (3), 333-348 (2019).</li><li>Lan, Y., Hu, Y., Huang, W., Tang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reflections+on+PK+-+PD+model+based+on+animal+experiments+for+study+of+Chinese+medicine.">Reflections on PK - PD model based on animal experiments for study of Chinese medicine.</a> Lishizen Med Materia Medica Res. 34 (2), 395-397 (2023).</li><li>Li, Y., Bai, M., Song, Y., Guo, H., Miao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Research+and+reflection+on+standard+decoction+of+Chinese+materia+medica.">Research and reflection on standard decoction of Chinese materia medica.</a> Chinese Trad Herbal Drugs. 49 (17), 3977-3980 (2018).</li><li>. Estimating the maximum safe starting dose in initial clinical trials for therapeutics in adult healthy volunteers Available from: <a target="_blank" href="https://www.fda.gov/regulatory-information/search-fda-guidance-documents/estimating-maximum-safe-starting-dose-initial-clinical-trials-therapeutics-adult-healthy-volunteers">https://www.fda.gov/regulatory-information/search-fda-guidance-documents/estimating-maximum-safe-starting-dose-initial-clinical-trials-therapeutics-adult-healthy-volunteers</a> (2005)</li><li>Chang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intratibial+osteosarcoma+cell+injection+to+generate+orthotopic+osteosarcoma+and+lung+metastasis+mouse+models.">Intratibial osteosarcoma cell injection to generate orthotopic osteosarcoma and lung metastasis mouse models.</a> J Vis Exp. (176), 63072 (2021).</li><li>He, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+animal+science.">Laboratory animal science.</a> , (2006).</li><li>Patten, A. R., Fontaine, C. J., Christie, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+the+different+animal+models+of+fetal+alcohol+spectrum+disorders+and+their+use+in+studying+complex+behaviors.">A comparison of the different animal models of fetal alcohol spectrum disorders and their use in studying complex behaviors.</a> Fronti Pedia. 2, 93 (2014).</li><li>Jones, C. P., Boyd, K. L., Wallace, J. 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The authors declare that they have nothing to disclose.

The clinical efficacy of the prescribed herbs in traditional Chinese medicine is largely associated with the administered dose and dosage form; therefore, the establishment of a standardized decoction strategy and a skilled administration process are necessary in clinical practice, as well as in improving the reliability of experimental results.
In this study, we first provided a detailed strategy for the standardized decoction of Chinese herbs used in clinical practice of traditional Chinese ...

Decoction formula is the most commonly used dosage form of the traditional Chinese medicine and the liquid medicinal drugs1,2. The decoction formula of traditional Chinese medicine is processed by decocting the herbs in water after being soaked, followed by filtering to discard the dregs.
Because the decoction formula can be individualized based on clinical necessities, especially in line with the characteristics of evidence-based treatment in traditional Chinese medicine, this dosage form via trans-oral administration has been applied in clinical practice for thousands of years with specific and irreplaceable advantages3,4. The quality of the decoction formula is directly related to the clinical efficacy.&#160;Therefore, the decoction formula must be prepared based on the principles for the standardized decoction methods to ensure the active ingredients are maximally extracted and protected3. Meanwhile, decoction-ready medicines are the main form of raw Chinese herbs used in clinical practice; the quality standards and production methods of prescribed decoction-ready medicines must comply with the provisions of the Chinese Pharmacopoeia2. Moreover, the prescribed decoction-ready medicines must be soaked in cold drinking water for at least 30 min before being decocted in a special pharmaceutical pot by heating5. Studies have shown that most of the active ingredients can be extracted by decocting for two times, and the&#160;Code of Practice for the Administration of Decoction Rooms of Traditional Chinese Medicines in Medical Institutions also prescribes decoction to be done twice4,6,7. If special Chinese herbs, such as one with hard texture or tonics, are prescribed, the decoction time is recommended to be extended. The decoction utensils and storage containers that directly come in contact with the decoction formula, starting from the container in which the prescribed decoction-ready medicines are soaked, should be chemically stable and have lids, such as casseroles, ceramics, glass and stainless steel. However, aluminum and ordinary plastic products should not be used, while iron and other corrosive utensils should be prohibited to avoid possible chemical reactions, which will reduce therapeutic efficacy and even produce harmful effects4,6,7.
Due to the limitations of performing clinical experiments, small animals bearing the human diseases, such as mice, are often used in medical research to explore the therapeutic efficacy and comprehensive mechanisms of different interventions including the decoction formula of traditional Chinese medicine8,9; however, unlike humans, the decoction formulas should be prepared every day, mixed together, and freshly administered 2x a day in clinical practice. Animals are unable to voluntarily take their medications on time with the required dosage, and most of the animal experiments related to traditional Chinese medicine are gavage-based oral administration of the decoction formulas at present. The decoction formulas used for in vivo animal experiments should be prepared in advance, aliquoted, stored at -20 &#176;C and thawed just before use3,4,10. Moreover, substandard animal immobilization and gavage needle manipulation increase the risk of unnecessary injuries and negative emotions. Therefore, as an important method of trans-oral drug delivery, the skilled gavage technique is particularly important to avoid possible esophageal injury and drug spillage, thus ensuring that each model animal receives an equal amount of prescribed herbal medicine to achieve accurate experimental results. Here, we provide a gavage protocol for the decoction formula of traditional Chinese medicine.

<table><tbody><tr><td>143B cell line</td><td>ATCC</td><td>CRL-8303</td><td>osteosarcoma cell line</td></tr><tr><td>Anesthesia machine</td><td>Shenzhen RWD Life Technology Co.,Ltd</td><td>R500IP</td><td>The Equipment of Anesthesia mice</td></tr><tr><td>Automatic cell counter</td><td>Shanghai Simo Biological Technology Co., Ltd</td><td>IC1000</td><td>Counting cells</td></tr><tr><td>BALB/c athymic mice</td><td>Shanghai SLAC Laboratory Animal Co, Ltd.</td><td>Male</td><td>Animal</td></tr><tr><td>Barbed skullcap herb</td><td>Shanghai Yanghetang Traditional Chinese Medicine Tablet Co., Ltd.</td><td>20230606</td><td>Decoction-ready medicines&amp;nbsp;</td></tr><tr><td>Basement Membrane Matrix</td><td>Shanghai Uning BioscienceTechnology Co., Ltd</td><td>356234, BD, Matrigel</td><td>re-suspende cells</td></tr><tr><td>Centrifuge tube (15 mL)</td><td>Shanghai YueNian Biotechnology Co., Ltd</td><td>430790, Corning</td><td>Centrifuge the cells</td></tr><tr><td>Elbow gavage needle (12-gauge/ 55 mm)</td><td>RWD Life Science Co., Ltd.</td><td>C21014-12</td><td>Component of gastric perfusion device</td></tr><tr><td>Gauze</td><td>Haishi Hainuo Group Co., Ltd.</td><td>LC45</td><td>Decoction filter</td></tr><tr><td>Ginseng</td><td>Shanghai Wanshicheng Pharmaceutical Co., Ltd.</td><td>20220704-2</td><td>Decoction-ready medicines&amp;nbsp;</td></tr><tr><td>Hedyotis diffusa</td><td>Shanghai Yanghetang Traditional Chinese Medicine Tablet Co., Ltd.</td><td>2023071107</td><td>Decoction-ready medicines&amp;nbsp;</td></tr><tr><td>isoflurane</td><td>Shenzhen RWD Life Technology Co., Ltd</td><td>VETEASY</td><td>Anesthesia mice</td></tr><tr><td>Micro-volume syringe</td><td>Shanghai high pigeon industry and trade Co., Ltd</td><td>0-50 &amp;mu;L</td><td>Inject precise cells into the tibia</td></tr><tr><td>Phosphate-buffered saline</td><td>Beyotime Biotechnology</td><td>ST447</td><td>wash the human osteosarcoma cells</td></tr><tr><td>Rubber bands</td><td>Shanghai Hengfei Biotechnology Co., Ltd.</td><td>XPJ</td><td>Fixing gauze on the measuring cup</td></tr><tr><td>Syringe (1 mL )</td><td>Shanghai Mishawa Medical Industry Co., Ltd.</td><td>SBM0040</td><td>Component of gastric perfusion device</td></tr><tr><td>Trypsin (0.25%)</td><td>Shanghai YueNian Biotechnology Co., Ltd</td><td>25200056, Gibco</td><td>trypsin treatment of cells</td></tr></tbody></table>

gavage strategy for decoction formula of traditional chinese medicine in osteosarcoma model mice

Decoction formula is the most commonly used dosage form in traditional Chinese medicine and applied in clinical practice for thousands of years by trans-oral administration, which is characterized by quick effect, easy absorption, and individualized treatment based on the specific syndromes of patients. The quality of the decoction formula is directly responsible for the clinical efficacy of traditional Chinese medicine; therefore, the standardization process of the decoction formula is important to avoid differences in decoction quality caused by subjective factors. Meanwhile, due to the limitations of performing clinical experiments, small animals bearing human diseases, such as mice, are often used in medical research to explore the therapeutic efficacy and comprehensive mechanisms of different interventions, including the decoction formula for traditional Chinese medicine.&#160;Consequently, as an important trans-oral administration method, the skilled gavage technique is particularly important to avoid potential esophagus damage and drug spillage, which will ensure an equal amount of medicine being administered to each model animal, leading to accurate experimental results. Furthermore, the standardized method of decoction formula preparation and skilled gavage strategy are necessary to protect animal welfare and minimize the number of animals used. Here, we reported a detailed standardization process of the decoction formula and gavage technique with Yiqi Jiedu decoction in osteosarcoma mouse model as an example. The efficacy was evaluated by the tumor volume. This protocol will maximize animal protection and improve the reliability of research data, therefore providing effective strategies for future investigating therapeutic efficacy and molecular mechanisms of decoction formula for traditional Chinese medicine in vivo.

The inhibitory effect of Yiqi Jiedu decoction on the growth of osteosarcoma in vivo was determined in an intra-tibial xenograft osteosarcoma mouse model, which was prepared by injecting the 143B osteosarcoma cells into the tibia of athymic mice. The Yiqi Jiedu decoction was administered to the mice of the Yiqi Jiedu decoction group by gavage for 28 days starting from the second day of the 143B injection. Physiological saline was given to the mice in the control group. All mice were euthanized after 28 days. The ...

Author Spotlight: Standardized Herbal Decoction Protocol for Enhanced Animal Studies

The protocol provides a detailed standardization process of decoction formula and gavage technique with Yiqi Jiedu decoction in the osteosarcoma mouse model as an example. It describes animal protection and improves reliability of research data, providing effective strategies for investigating therapeutic efficacy and molecular mechanisms of decoction formulas in vivo.

Gavage Strategy for Decoction Formula of Traditional Chinese Medicine in Osteosarcoma Model Mice

Decoction formula is the most commonly used dosage form in traditional Chinese medicine and applied in clinical practice for thousands of years by ...

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Research

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Medicine

514 Views.  Shanghai University of Traditional Chinese Medicine. The protocol provides a detailed standardization process of decoction formula and gavage technique with Yiqi Jiedu decoction in the osteosarcoma mouse model as an example. It describes animal protection and improves reliability of research data, providing effective strategies for investigating therapeutic efficacy and molecular mechanisms of decoction formulas in vivo.

Video: Author Spotlight: Standardized Herbal Decoction Protocol for Enhanced Animal Studies

Practical Considerations in Studying Metastatic Lung Colonization in Osteosarcoma Using the Pulmonary Metastasis Assay

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung colonization in osteosarcoma (OS) by fluorescence microscopy. This article provides a detailed description of the protocol, and discusses examples of obtaining image data on metastatic growth using widefield or confocal fluorescence microscopy platforms. The flexibility of the PuMA model permits researchers to study not only the growth of OS cells in the lung microenvironment, but also to assess the effects of anti-metastatic therapeutics over time. Confocal microscopy allows for unprecedented, high-resolution imaging of OS cell interactions with the lung parenchyma. Moreover, when the PuMA model is combined with fluorescent dyes or fluorescent protein genetic reporters, researchers can study the lung microenvironment, cellular and subcellular structures, gene function, and promoter activity in metastatic OS cells. The PuMA model provides a new tool for osteosarcoma researchers to discover new metastasis biology and assess the activity of novel anti-metastatic, targeted therapies.

The goal of this article is to provide a detailed description of the protocol for the pulmonary metastasis assay (PuMA). This model permits researchers to study metastatic osteosarcoma (OS) cell growth in lung tissue using a widefield fluorescence or confocal laser-scanning microscope.

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung ...

Cancer Research

A Preclinical Mouse Model of Osteosarcoma to Define the Extracellular Vesicle-mediated Communication Between Tumor and Mesenchymal Stem Cells

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under the influence of specific environmental signals, these adult stem cells can release paracrine mediators leading to accelerated tumor growth and metastasis. Defining the crosstalk between tumor and MSCs is of primary importance to understand the mechanisms underlying cancer progression and identify novel targets for therapeutic intervention. 
Cancer cells produce high amounts of extracellular vesicles (EVs), which can profoundly affect the behavior of target cells in the tumor microenvironment or at distant sites. Tumor EVs enclose functional biomolecules, including inflammatory RNAs and (onco)proteins, that can educate stromal cells to enhance the metastatic behavior of cancer cells or to participate in the pre-metastatic niche formation. In this article, we describe the development of a preclinical cancer mouse model that enables specific evaluation of the EV-mediated crosstalk between tumor and mesenchymal stem cells. First, we describe the purification and characterization of tumor-secreted EVs and the assessment of the EV internalization by MSCs. We then make use of a multiplex bead-based immunoassay to evaluate the alteration of the MSC cytokine expression profile induced by cancer EVs. Finally, we illustrate the generation of a bioluminescent orthotopic xenograft mouse model of osteosarcoma that recapitulates the tumor-MSC interaction, and show the contribution of EV-educated MSCs to tumor growth and metastasis formation. 
Our model provides the opportunity to define how cancer EVs shape a tumor-supporting environment, and to evaluate whether blockade of the EV-mediated communication between tumor and MSCs prevents cancer progression.

Direct injection of cancer-derived extracellular vesicles (EVs) leads to reprogramming of bone marrow supporting tumor progression; however, which cells mediate this effect is unclear. Herein, we describe a step-by-step protocol to investigate EV-mediated tumor-mesenchymal stem cell (MSC) interactions in vivo, revealing a crucial role for EV-educated MSCs in metastasis.

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under ...

Modeling Osteosarcoma Using Li-Fraumeni Syndrome Patient-derived Induced Pluripotent Stem Cells

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including osteosarcoma--one of the most frequent primary non-hematologic malignancies in the childhood and adolescence. Therefore, LFS provides an ideal model to study this malignancy. Taking advantage of iPSC methodologies, LFS-associated osteosarcoma can be successfully modeled by differentiating LFS patient iPSCs to mesenchymal stem cells (MSCs), and then to osteoblasts--the cells of origin for osteosarcomas. These LFS osteoblasts recapitulate oncogenic properties of osteosarcoma, providing an attractive model system for delineating the pathogenesis of osteosarcoma. This manuscript demonstrates a protocol for the generation of iPSCs from LFS patient fibroblasts, differentiation of iPSCs to MSCs, differentiation of MSCs to osteoblasts, and in vivo tumorigenesis using LFS osteoblasts. This iPSC disease model can be extended to identify potential biomarkers or therapeutic targets for LFS-associated osteosarcoma.

Here, we present a protocol for the generation of induced pluripotent Stem Cells (iPSCs) from Li-Fraumeni Syndrome (LFS) patient derived fibroblasts, differentiation of iPSCs via mesenchymal stem cells (MSCs) to osteoblasts, and modeling in vivo tumorigenesis using LFS patient-derived osteoblasts.

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including ...

Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with diagnostic or prognostic difficulties. OS comprises genotypically and phenotypically heterogeneous cancer cells. Bone microenvironment elements are proved to account for tumor heterogeneity and disease progression. Bone extracellular matrix (BEM) retains the microstructural matrices and biochemical components of native extracellular matrix. This tissue-specific niche provides a favorable and long-term scaffold for OS cell seeding and proliferation. This article provides a protocol for the preparation of BEM model and its further experimental application. OS cells can grow and differentiate into multiple phenotypes consistent with the histopathological complexity of OS clinical specimens. The model also allows visualization of diverse morphologies and their association with genetic alterations and underlying regulatory mechanisms. As homologous to human OS, this BEM-OS model can be developed and applied to the pathology and clinical research of OS.

The bone extracellular matrix (BEM) model for osteosarcoma (OS) is well established and shown here. It can be used as a suitable scaffold for mimicking primary tumor growth in vitro and providing an ideal model for studying the histologic and cytogenic heterogeneity of OS.

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with ...

A Syngeneic Orthotopic Osteosarcoma Sprague Dawley Rat Model with Amputation to Control Metastasis Rate

The most recent advance in the treatment of osteosarcoma (OS) occurred in the 1980s when multi-agent chemotherapy was shown to improve overall survival compared to surgery alone. To address this problem, the aim of the study is to refine a lesser-known model of OS in rats with a comprehensive histologic, imaging, biologic, implantation, and amputation surgical approach that prolongs survival. We used an immunocompetent, outbred Sprague-Dawley (SD), syngeneic rat model with implanted UMR106 OS cell line (originating from a SD rat) with orthotopic tibial tumor implants into 3-week-old male and female rats to model pediatric OS. We found that rats develop reproducible primary and metastatic pulmonary tumors, and that limb amputations at 3 weeks post implantation significantly reduce the incidence of pulmonary metastasis and prevent unexpected deaths. Histologically, the primary and metastatic OSs in rats were very similar to human OS. Using immunohistochemistry methods, the study shows that rat OS are infiltrated with macrophages and T cells. A protein expression survey of OS cells reveals that these tumors express ErbB family kinases. Since these kinases are also highly expressed in most human OSs, this rat model could be used to test ErbB pathway inhibitors for therapy.

Here, a syngeneic orthotopic implantation followed by an amputation procedure of the osteosarcoma with spontaneous pulmonary metastasis that can be used for preclinical investigation of metastasis biology and development of novel therapeutics is described.

The most recent advance in the treatment of osteosarcoma (OS) occurred in the 1980s when multi-agent chemotherapy was shown to improve overall survival ...

Intratibial Osteosarcoma Cell Injection to Generate Orthotopic Osteosarcoma and Lung Metastasis Mouse Models

Osteosarcoma is the most common primary bone cancer in children and adolescents, with lungs as the most common metastatic site. The five-year survival rate of osteosarcoma patients with pulmonary metastasis is less than 30%.Therefore, the utilization of mouse models mimicking the osteosarcoma development in humans is of great significance for understanding the fundamental mechanism of osteosarcoma carcinogenesis and pulmonary metastasis to develop novel therapeutics. Here, detailed procedures are reported to generate the primary osteosarcoma and pulmonary metastasis mouse models via&#160;intratibia injection of osteosarcoma cells. Combined with the bioluminescence or X-ray live imaging system, these living mouse models are utilized to monitor and quantify osteosarcoma growth and metastasis. To establish this model, a basement membrane matrix containing osteosarcoma cells was loaded in a micro-volume syringe and injected into one tibia of each athymic mouse after being anesthetized. The mice were sacrificed when the primary osteosarcoma reached the size limitation in the IACUC-approved protocol. The legs bearing osteosarcoma and the lungs with metastasis lesions were separated. These models are characterized by a short incubation period, rapid growth, severe lesions, and sensitivity in monitoring the development of primary and pulmonary metastatic lesions. Therefore, these are ideal models for exploring the functions and mechanisms of specific factors in osteosarcoma carcinogenesis and pulmonary metastasis, the tumor microenvironment, and evaluating the therapeutic efficacy in vivo.

The present protocol describes intratibia osteosarcoma cell injection to generate mouse models bearing orthotopic osteosarcoma and pulmonary metastasis lesions.

Osteosarcoma is the most common primary bone cancer in children and adolescents, with lungs as the most common metastatic site. The five-year survival ...

Protocol for Osteosarcoma PDX Mouse Models: Studying Growth and Drug Resistance

Establishment of Patient-Derived Xenograft Mouse Model with Human Osteosarcoma Tissues

Osteosarcoma is the most common primary malignant bone tumor in children and adolescents. Despite the development of new treatment plans in recent years, the prognosis for osteosarcoma patients has not significantly improved. Therefore, it is crucial to establish a robust preclinical model with high fidelity. The patient-derived xenograft (PDX) model faithfully preserves the genetic, epigenetic, and heterogeneous characteristics of human malignancies for each patient. Consequently, PDX models are considered authentic in vivo models for studying various cancers in transformation studies. This article presents a comprehensive protocol for creating and maintaining a PDX mouse model that accurately mirrors the morphological features of human osteosarcoma. This involves the immediate transplantation of freshly resected human osteosarcoma tissue into immunocompromised mice, followed by successive passaging. The described model serves as a platform for studying the growth, drug resistance, relapse, and metastasis of osteosarcoma. Additionally, it aids in screening the target therapeutics and establishing personalized treatment schemes.

Author Spotlight: Replicating Human Osteosarcoma Progression in Immunodeficient Mice for Cancer Study

The present protocol describes the method for establishing a patient-derived xenograft (PDX) mouse model using human osteosarcoma tissue.

Osteosarcoma is the most common primary malignant bone tumor in children and adolescents. Despite the development of new treatment plans in recent years, ...

Preparation Of Gushukang (GSK) Granules for In Vivo and In Vitro Experiments

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang (GSK) granules, a marketed prescription in China, have bone-protective effects in treating POP. Before administration to the body, one standard preparation procedure is commonly required, which aims to promote the release of active constituents from raw herbs and enhance the pharmacological effects as well as therapeutic outcomes. This study proposes a detailed protocol for using GSK granules in in vivo and in vitro experimental assays. The authors first provide a detailed protocol to calculate the animal-appropriate dosages of granules for in vivo investigation: weighing, dissolving, storage, and administration. Second, this article describes protocols for micro-CT scanning and the measurement of bone parameters. Sample preparation, protocols for running the micro-CT machine and quantification of bone parameters were evaluated. Third, serum-containing GSK granules are prepared, and drug-containing serum is extracted for in vitro osteoclastogenesis and osteoblastogenesis. GSK granules were intragastrically administered twice per day to rats for three consecutive days. Blood was then collected, centrifuged, inactivated, and filtered. Finally, serum was diluted and used for performing osteoclastogenesis and osteoblastogenesis. The protocol described here can be considered a reference for pharmacological investigations of herbal prescription medicines, such as granules.

Preparation Of Gushukang GSK Granules for In Vivo and In Vitro Experiments

This article provides a detailed protocol for preparing a working solution of Gushukang granules for animal studies and GSK granule containing serum for in vitro experiments. This protocol can be applied to pharmacological investigations of herbal medicines as well as prescriptions for both in vivo and in vitro experiments.

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang ...

HE Staining and Micro-CT to Evaluate Murine Trabecular Bone Microarchitecture

Trabecular Bone Microarchitecture Evaluation in an Osteoporosis Mouse Model

Bone microstructure refers to the arrangement and quality of bone tissue at the microscopic level. Understanding the bone microstructure of the skeleton is crucial for gaining insight into the pathophysiology of osteoporosis and improving its treatment. However, handling bone samples can be complex due to their hard and dense properties. Secondly, specialized software makes image processing and analysis difficult. In this protocol, we present a cost-effective and easy-to-use solution for trabecular bone microstructure analysis. Detailed steps and precautions are provided. Micro-CT is a non-destructive three-dimensional (3D) imaging technique that provides high-resolution images of trabecular bone structure. It allows for the objective and quantitative evaluation of bone quality, which is why it is widely regarded as the gold standard method for bone quality assessment. However, histomorphometry remains indispensable as it offers crucial cellular-level parameters, bridging the gap between two-dimensional (2D) and 3D assessments of bone specimens. As for the histologic techniques, we chose to decalcify the bone tissue and then perform traditional paraffin embedding. In summary, combining these two methods can provide more comprehensive and accurate information on bone microstructure.

Author Spotlight: An Economic and Efficient Method for Quantitative Evaluation of Bone Microarchitecture in a Murine Osteoporosis Model 

This protocol presents an economical and efficient method for quantitative evaluation of bone microarchitecture in a mouse model of osteoporosis by combining Hematoxylin-Eosin (HE) staining and micro-computed tomography (Micro-CT) techniques.

Bone microstructure refers to the arrangement and quality of bone tissue at the microscopic level. Understanding the bone microstructure of the skeleton ...

Impact of Sanleng Jiashen Formula on Orthotopic Hepatocellular Carcinoma in Mice

Establishment of Hepatocarcinoma in BALB/c-nu Mice and Investigation of the Therapeutic Effect of the Sanleng Jiashen Formula

The lethality of liver cancer and the resistance to chemical drugs have forced the search for effective prescriptions of traditional herbs for liver cancer. Animal models that are repeatable, easy to manipulate, and highly mimic the pathophysiological processes of liver cancer are the prerequisite for the successful screening of effective drug candidates. Meanwhile, reliable drug efficacy evaluation indicators and means are also the guarantee of anti-liver cancer drug research and development.
Sanleng Jiashen formula, a representative prescription of traditional Chinese medicine containing Sparganium stoloni erum, Buch. -Ham. (Sanleng), Panax ginseng C. A. Mey. (ginseng), Rheum officinale Baill. (rhubarb), and Ligusticum chuanxiong Hort. (Chuanxiong), is prescribed to nourish the liver and clear heat, remove toxins, and promote blood circulation to treat liver cancer. This experimental protocol describes the preparation of lyophilized Sanleng Jiashen formula and the establishment process of in-situ liver cancer in BALB/c-nu mice. Histopathological staining, immunohistochemical detection of cancer markers, in vivo imaging of mice, and chick embryo chorioallantoic membrane test were used to explore the inhibition and anti-angiogenesis effect of Sanleng Jiashen formula on malignant proliferation of liver cancer tissue. The data show that the Sanleng Jiashen formula can effectively resist the malignant proliferation of liver cancer tissue, which is manifested by reduced tumor mass volume, improved pathological damage, and lower levels of the cancer marker ki67.
The superior inhibition of angiogenesis also suggests that the Sanleng Jiashen formula may have the potential to treat and prevent the progression and deterioration of liver cancer. The whole experimental scheme shows a comprehensive process of traditional Chinese medicine components in the treatment of mouse liver cancer, which provides a reference for the establishment and optimization of a liver cancer model, as well as the research and development of drugs to prevent and treat liver cancer.

Here, we present the step-by-step protocol to investigate the therapeutic and vascular inhibitory effects of the Sanleng Jiashen formula on BALB/c-nu mice with orthotopic hepatocellular carcinoma.

The lethality of liver cancer and the resistance to chemical drugs have forced the search for effective prescriptions of traditional herbs for liver ...