The authors would like to thank the staff of the Experimental Surgery and Animal Laboratory at the College of Medicine, King Saud University, for their cooperation and support-especially Hussain Almukhayzim and Hisham Aloudah. The authors would also like to acknowledge the media team at King Saud bin Abdul-Aziz University for preparing the visual material-especially Muath bin Ghannam and Abdulwahab Alsulami.

The protocol follows the animal care guidelines of King Saud University&#39;s ethical committee. Surgical procedures, anesthetics, and other medications used on animals are approved by King Saud University&#39;s ethical committee. All experimental work is performed by appropriately trained personnel.
1. Cell Sheet Construction
<ol>
	<li>Coat the culture dishes (3.5-cm temperature-responsive culture dishes) with undiluted fetal bovine serum (FBS) and ensure to cover all the surface of the dish. 
	NOTE: This step is important for the detachment of the cell sheet since it supports the growth of the cells in a monolayer and prevents cell aggregation.</li>
	<li>Incubate the dishes for 24 h at 37 &#176;C in a humidified atmosphere containing 5% CO2 and 95% air.</li>
	<li>Remove the excess FBS from the coated dishes and allow the dishes to dry at room temperature (RT) for at least 1 h.</li>
	<li>Prepare the three cell suspensions: 1 x 106 HepG2 cells, 1 x 106 HepG2 cells with BMMSCs, and 1 x 106 HepG2 cells with UCMSCs (in a ratio of 4:1 for both BMMSCs and UCMSCs).</li>
	<li>Seed the appropriate cell suspension to each labeled dish.</li>
	<li>Suspend all cells in DMEM culture medium supplemented with 10% FBS and 1% penicillin/streptomycin.</li>
	<li>Incubate the cells at 37 &#176;C in a humidified atmosphere containing 5% CO2 and 95% air.</li>
</ol>
2. Cell Sheet Detachment
<ol>
	<li>At 100% cell confluence, take the dishes out of the 37 &#176;C incubator.</li>
	<li>Incubate the HepG2 cell sheet dish for 1 h at RT, while incubating the HepG2/BMMSC and HepG2/UCMSC cell sheet dishes for 30 - 40 min at 20 &#176;C in a humidified atmosphere containing 5% CO2 and 95% air. 
	NOTE: The cell sheet made from only HepG2 is fragile; reducing the temperature to 20 &#176;C causes damages to the structure of the sheet.</li>
</ol>
3. Performance of the Surgical Procedure
Note: During the incubation time for the cell sheet detachment, start the animal procedure.
<ol>
	<li>Instructions for the preparation of the surgery area
	<ol>
		<li>Conduct all surgical procedures in a sterile separate area within the laboratory, such as a hood, laminar flow cabinet, or an aseptic surgery room. Ensure that all surfaces of the surgery area are non-porous, sealed, durable, and sanitizable.</li>
		<li>Wear proper protective gear, including scrubs, gloves, facemasks, and head and shoe covers.</li>
		<li>Use surgical tools that are clean and sterilized (via autoclaving; saturated stem under high pressure) at the beginning of the surgery.</li>
		<li>Change gloves between rats.</li>
	</ol>
	</li>
	<li>Preparation of the animal for surgery
	<ol>
		<li>Use 8- to 12-week-old nude rats.</li>
		<li>Create injectable anesthesia for rats.
		<ol>
			<li>To prepare the anesthesia solution, ketamine-xylazine, mix 2 mL of ketamine, 1 mL of xylazine (at a concentration of 20 mg/mL), and 1 mL of sterile physiologic saline (e.g., 0.9% sodium chloride) in a sterile 10-mL serum collection vial and shake well before use.</li>
			<li>To apply anesthesia, inject 0.2 mL of the ketamine-xylazine solution per 100 g of body weight of the rat intraperitoneally. 
			NOTE: The total dose of the solution is 100 mg/kg of ketamine and 10 mg/kg of xylazine.</li>
		</ol>
		</li>
		<li>Remove visible dirt/debris from the surgical site.</li>
		<li>Check the depth of anesthesia by testing the pedal withdrawal reflex (foot pad pinch on both hind feet). 
		NOTE: If the foot pad pinch causes a response, repeat at a dose of 0.05 mL/100 g (approximately every 30 min), then recheck the anesthetic depth.</li>
		<li>Disinfect the surgery area with a two-stage scrub by povidone-iodine/isopropanol.</li>
		<li>Cover the rat with a sterile drape to avoid contamination of the incision.</li>
		<li>Inject the rats subcutaneously with buprenorphine (0.01 - 0.05 mg/kg)8 prior to making the incision.</li>
		<li>Using a sterile scalpel, make a 7- to 10-cm cut between the skins and underlying muscles to expose the liver.
		<ol>
			<li>Separate the abdominal muscle from the skin with the flat back edge of the scalpel.</li>
			<li>Carefully stab the linea alba using a scalpel and extend the cut with sharp scissors.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
4. Cell Sheet Transplantation
Note: Cell sheet transplantation is performed on the liver .
<ol>
	<li>Collect the cell sheet from the culture dish.
	<ol>
		<li>Gently tap the culture dish over the bench to detach the sheet from its surface.</li>
		<li>Separate the cell sheet from the dish&#39;s surface by scraping the edge of the sheet in a half circle using a sterile pipette tip.</li>
		<li>Gently remove the entire medium from the culture dish.</li>
		<li>Ensure the sheet is intact without any damage; otherwise, it cannot be used (Figure 1). 
		NOTE: The resulting sheets can be detected directly by the eye.</li>
	</ol>
	</li>
	<li>Prepare a sterile membrane (filter paper) to harvest the cell sheet from the dish.
	<ol>
		<li>First, soak the membrane with normal saline. Then, remove the excess saline with a sterile cotton pad.</li>
		<li>Place the wet membrane over the cell sheet, while avoiding creases and air bubbles between the membrane and the cell sheet.</li>
		<li>Add few drops of normal saline over the membrane and the cell sheet to collect them easier. 
		Note:&#160;To avoid breaking the cell sheet, do not use cold saline and lift the membrane and the cell sheet with forceps.</li>
		<li>Leave the membrane for a few seconds to ensure that the cell sheet is properly attached to the membrane, and then collect it.</li>
	</ol>
	</li>
	<li>Prepare the liver for cell sheet transplantation.
	<ol>
		<li>Make sure the liver surface is dry by gently tapping it with a sterile cotton pad.</li>
		<li>Using sterile forceps, place the membrane with the cell sheet over a single lobe of the rat&#39;s liver.</li>
		<li>Add a few drops of sterile saline and apply a gentle pressure to the membrane to detach the cell sheet from it.</li>
		<li>Wait for 5 min before carefully removing the membrane. 
		NOTE: This is done to ensure the cell sheet is properly attached to the liver.</li>
		<li>Finally, close the underlying muscles and skin incisions with 5-0 nylon sutures.</li>
		<li>Disinfect the surgery area with povidone-iodine. 
		Note: Figure 2 shows a diagram of the cell sheet transplantation procedure on the liver of a nude rat.</li>
	</ol>
	</li>
</ol>
5. Post-surgery Care
<ol>
	<li>Immediately after the surgery, house the rat individually to avoid cannibalism or suffocation, and monitor it regularly (at least every 15 min) until it is conscious and fully ambulant.</li>
	<li>Then, assess the rats daily for 5 - 14 d, at least 5 d post-operatively, to ensure that there are no complications. During the daily assessment, make sure of the following.
	<ol>
		<li>The wound is closed, and the sutures are intact, without being excessively tight.</li>
		<li>There are no signs of infection in the incision site, such as heat, excessive swelling, or purulent discharge.</li>
		<li>There are no signs associated with pain, such as decreased food and water consumption, weight loss, dehydration, skin tenting, or rapid and open mouth breathing. To control moderate to severe post-surgery pain, if any, inject the rats subcutaneously with buprenorphine (0.01 - 0.05 mg/kg) every 6 - 8 h9 for a minimum of 48 - 72 h postoperatively.</li>
	</ol>
	</li>
</ol>
6. Analysis of the Transplanted Area
<ol>
	<li>One month after the transplantation, scarifice the rat and collect the transplanted area for histological and immunohistochemical analysis.</li>
</ol>

<ol><li>Marra, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+targets+and+oxidative+stress+biomarkers+in+hepatocellular+carcinoma%3A+an+overview%5BTitle%5D)+AND+%22Journal+of+Translational+Medicine%22%5BJournal%5D)">Molecular targets and oxidative stress biomarkers in hepatocellular carcinoma: an overview.</a> Journal of Translational Medicine. 9, 171(2011).</li>
<li>Yamada, K. M., Cukierman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modeling+tissue+morphogenesis+and+cancer+in+3D%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Modeling tissue morphogenesis and cancer in 3D.</a> Cell. 130 (4), 601-610 (2007).</li>
<li>Kushida, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Decrease+in+culture+temperature+releases+monolayer+endothelial+cell+sheets+together+with+deposited+fibronectin+matrix+from+temperature-responsive+culture+surfaces%5BTitle%5D)+AND+%22Journal+of+Biomedical+Materials+Research%22%5BJournal%5D)">Decrease in culture temperature releases monolayer endothelial cell sheets together with deposited fibronectin matrix from temperature-responsive culture surfaces.</a> Journal of Biomedical Materials Research. 45 (4), 355-362 (1999).</li>
<li>Okano, T., Yamada, N., Sakai, H., Sakurai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+recovery+system+for+cultured+cells+using+plasma-treated+polystyrene+dishes+grafted+with+poly+%28N-isopropylacrylamide%29%5BTitle%5D)+AND+%22Journal+of+Biomedical+Materials+Research+Part+A%22%5BJournal%5D)">A novel recovery system for cultured cells using plasma-treated polystyrene dishes grafted with poly (N-isopropylacrylamide).</a> Journal of Biomedical Materials Research Part A. 27 (10), 1243-1251 (1993).</li>
<li>Chamberlain, G., Fox, J., Ashton, B., Middleton, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Concise+review%3A+mesenchymal+stem+cells%3A+their+phenotype%2C+differentiation+capacity%2C+immunological+features%2C+and+potential+for+homing%5BTitle%5D)+AND+%22Stem+Cells%22%5BJournal%5D)">Concise review: mesenchymal stem cells: their phenotype, differentiation capacity, immunological features, and potential for homing.</a> Stem Cells. 25 (11), 2739-2749 (2007).</li>
<li>Waterman, R. S., Tomchuck, S. L., Henkle, S. L., Betancourt, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+new+mesenchymal+stem+cell+%28MSC%29+paradigm%3A+polarization+into+a+pro-inflammatory+MSC1+or+an+Immunosuppressive+MSC2+phenotype%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">A new mesenchymal stem cell (MSC) paradigm: polarization into a pro-inflammatory MSC1 or an Immunosuppressive MSC2 phenotype.</a> PLoS One. 5 (4), e10088(2010).</li>
<li>Waterman, R. S., Henkle, S. L., Betancourt, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mesenchymal+stem+cell+1+%28MSC1%29-based+therapy+attenuates+tumor+growth+whereas+MSC2-treatment+promotes+tumor+growth+and+metastasis%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Mesenchymal stem cell 1 (MSC1)-based therapy attenuates tumor growth whereas MSC2-treatment promotes tumor growth and metastasis.</a> PLoS One. 7 (9), e45590(2012).</li>
<li>Curtin, L. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+buprenorphine+in+a+postoperative+pain+model+in+rats%5BTitle%5D)+AND+%22Comparative+Medicine%22%5BJournal%5D)">Evaluation of buprenorphine in a postoperative pain model in rats.</a> Comparative Medicine. 59 (1), 60-71 (2009).</li>
<li>Nowland, M. H. Guidelines on Anesthesia and Analgesia in Rats - ULAM Guidelines and SOPs - Michigan Medicine Confluence. , 
 <a target="_blank" href="https://wiki.med.umich.edu/display/ULAMGSOP/Guidelines+on+Anesthesia+and+Analgesia+in+Rats">https://wiki.med.umich.edu/display/ULAMGSOP/Guidelines+on+Anesthesia+and+Analgesia+in+Rats</a> (2017).</li>
<li>Alshareeda, A. T., Sakaguchi, K., Abumaree, M., Mohd Zin, N. K., Shimizu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+potential+of+cell+sheet+technique+on+the+development+of+hepatocellular+carcinoma+in+rat+models%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">The potential of cell sheet technique on the development of hepatocellular carcinoma in rat models.</a> PLoS One. 12 (8), e0184004(2017).</li>
<li>Russo, J., Russo, I. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Atlas+and+histologic+classification+of+tumors+of+the+rat+mammary+gland%5BTitle%5D)+AND+%22Journal+of+Mammary+Gland+Biology+and+Neoplasia%22%5BJournal%5D)">Atlas and histologic classification of tumors of the rat mammary gland.</a> Journal of Mammary Gland Biology and Neoplasia. 5 (2), 187-200 (2000).</li>
<li>Lin, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Applications+and+limitations+of+genetically+modified+mouse+models+in+drug+discovery+and+development%5BTitle%5D)+AND+%22Current+Drug+Metabolism%22%5BJournal%5D)">Applications and limitations of genetically modified mouse models in drug discovery and development.</a> Current Drug Metabolism. 9 (5), 419-438 (2008).</li>
<li>Driscoll, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+preclinical+new+drug+research+program+of+the+National+Cancer+Institute%5BTitle%5D)+AND+%22Cancer+Treatment+Reports%22%5BJournal%5D)">The preclinical new drug research program of the National Cancer Institute.</a> Cancer Treatment Reports. 68 (1), 63-76 (1984).</li>
<li>Suzuki, R., Aruga, A., Kobayashi, H., Yamato, M., Yamamoto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+a+novel+in+vivo+cancer+model+using+cell+sheet+engineering%5BTitle%5D)+AND+%22Anticancer+Research%22%5BJournal%5D)">Development of a novel in vivo cancer model using cell sheet engineering.</a> Anticancer Research. 34 (9), 4747-4754 (2014).</li>
<li>Chen, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Application+of+the+cell+sheet+technique+in+tissue+engineering%5BTitle%5D)+AND+%22Biomedical+Reports%22%5BJournal%5D)">Application of the cell sheet technique in tissue engineering.</a> Biomedical Reports. 3 (6), 749-757 (2015).</li>
<li>Matsuura, K., Haraguchi, Y., Shimizu, T., Okano, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+sheet+transplantation+for+heart+tissue+repair%5BTitle%5D)+AND+%22Journal+of+Controlled+Release%22%5BJournal%5D)">Cell sheet transplantation for heart tissue repair.</a> Journal of Controlled Release. 169 (3), 336-340 (2013).</li>
<li>Matsuura, K., Shimizu, T., Okano, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Toward+the+development+of+bioengineered+human+three-dimensional+vascularized+cardiac+tissue+using+cell+sheet+technology%5BTitle%5D)+AND+%22International+Heart+Journal%22%5BJournal%5D)">Toward the development of bioengineered human three-dimensional vascularized cardiac tissue using cell sheet technology.</a> International Heart Journal. 55 (1), 1-7 (2014).</li>
<li>Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((What+is+the+evidence+that+tumors+are+angiogenesis+dependent%3F%5BTitle%5D)+AND+%22Journal+of+the+National+Cancer+Institute%22%5BJournal%5D)">What is the evidence that tumors are angiogenesis dependent?</a> Journal of the National Cancer Institute. 82 (1), 4-6 (1990).</li>
</ol>

The authors have nothing to disclose.

An extensive amount of research is dedicated to developing an adequate in vivo preclinical animal model that resembles human cancers. Currently, the major approaches used to create cancer animal models involve genetic engineering and cell transplantation11. Genetically modified animal models are good tools for the identification and validation of target genes, as well as understanding the molecular mechanisms underlying drug-induced toxicity. However, this model is associated with some li...

HCC is a primary cancer of the liver that is significantly associated with a poor prognosis. Annually, almost a half million new patients are diagnosed with HCC, representing 85% of liver cancer patients worldwide1.&#160;Hepatocarcinogenesis is not a single-form disease; rather,it is a collection of diseases that have different histopathological features and genetic and genomic variability, in addition to varied prognostic outcomes1. Therefore, the main challenges in the development of an effective therapeutic strategy for HCC are the limited knowledge of HCC biology and&#160;the lack of a suitable experimental animal model that can help understand this complex disease. An in vivo animal model that mimics human cancer is necessary for selecting candidate genes and identifying prognostic/predictive markers implicated in cancer induction, as well as for investigating different factors that may affect cancer responses to therapeutic agents.
In vitro cancer studies are still associated with major limitations. This is due to the fact that cancer cells lose many of their in vivo features when maintained in culture. The changes that occur to cells in vitro result from the absence of the whole tissue physiology in an ex vivo setting. Cancer cell-to-cell interaction (stromal, immune, vasculature, epithelial, etc.) within the tumor microenvironment reflects greatly on cancer cell characteristics2. The tumor microenvironment could alter cancer cell gene/protein expression and phenotypic characteristics, in addition to angiogenetic and metastatic potentials. The two-dimensional (2-D) in vitro culture system also lacks a suitable tissue matrix, which is necessary to regulate tumor progression. Thus, due to these limitations, in vivo models should always be utilized to support the preliminary findings of in vitro models. In this study, we use cell sheet technology to develop an in vivo animal model that elucidates the complete biological process underlying HCC.
More than a decade ago, Okano&#39;s laboratory established a novel method of tissue engineering based on cell sheet technology3. This technique utilizes a thermo-responsive culture plastic to allow reversible cell adhesion/detachment by controlling the surface hydrophobicity. This method allows a gentle harvesting of cultured cells in an intact three-dimensional (3-D) format (i.e.,cell sheet), with a well-kept extracellular matrix (ECM) and cell-to-cell interactions. The cell sheet technique requires the precoating of culture dishes with a temperature-responsive polymer poly(N-isopropylacrylamide) (PIPA Am), which is commercially available and ready for use. At a temperature below 20 &#176;C, PIPA Am polymers become hydrated and dissolve in aqueous solutions, whereas, at a higher temperature (37 &#176;C), the polymers become dehydrated and transform into a turbid precipitate. The polymer contains hydrophilic amide chains and hydrophobic side chains (isopropyl groups). At high temperatures, the Brownian movement of the water molecules is intensified, whereas, at a low temperature, the molecules of the water surrounding the isopropyl groups of the hydrated structure breakdown and the groups of hydrophobic isopropyl aggregate because of hydrophobic interactions. Therefore, the entire chain of the polymer aggregates and precipitates4.
In the presented study, this technique is employed to develop an HCC animal model using three different cell sheets. The first sheet employed consists of HCC cell line cells only, whereas the other two sheets consist of a combination of HCC cell line cells and MSCs from two different sources: bone marrow MSCs (BMMSCs) and umbilical cord MSCs (UCMSCs). MSCs are non-hematopoietic stromal cells that are capable of differentiating intocell derivatives of the mesenchymal lineage, including adipocytes, osteocytes, chondrocytes, and myocytes5. The reason we employ these cells when creating the cancer cell sheet is the inconsistent reporting on the effect of MSCs on cancers. It has been suggested that MSCs can have two distinct phenotypes: &#34;MSC1&#34;, a proinflammatory phenotype, and &#34;MSC2&#34;, an immunosuppressive phenotype6. MSCs express toll-like receptors (TLRs). TLR4 priming of MSCs increases their secretion of proinflammatory factors, whereas TLR3 priming increases their secretion of immunosuppressive factors6. An in vitro study of these two phenotypes reported that a co-culture of MSC1 with cancer cell lines attenuated cancer cell growth, while MSC2 co-culture had the opposite effect7. This implicates that MSCs can be either pro-cancer or anticancer, depending on their phenotype. Thus, in addition to developing the HCC animal model using cell sheet technology, we want to explore the effect of MSC transplants on tumor development, and whether using these cells will enhance or reduce the development of this model.

<table><tbody><tr><td>&lt;strong&gt;Reagents&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>FBS&amp;nbsp;</td><td>Gibco/Invitrogen</td><td>10270106</td><td></td></tr><tr><td>DMEM high glucose&amp;nbsp;</td><td>Sigma</td><td>D5671-500ML</td><td></td></tr><tr><td>Penicillin/streptomycin&amp;nbsp;</td><td>Life Technology</td><td>15070063</td><td></td></tr><tr><td>Sterile physiologic saline</td><td>Sigma&amp;nbsp;</td><td>S0817-1GA</td><td></td></tr><tr><td>Human HepG2 cell line</td><td>ATCC, USA</td><td>HB-8065</td><td></td></tr><tr><td>Human bone marrow MSCs cell line</td><td>PromoCell, USA</td><td>C-12974</td><td></td></tr><tr><td>human umbilical cord tissue MSCs</td><td>PromoCell, USA&amp;nbsp;</td><td>C-12971</td><td></td></tr><tr><td>Ketamine 50%</td><td>Rompun, Bayer</td><td></td><td></td></tr><tr><td>Xylazine 2%</td><td>Rompun</td><td>23076-35-9</td><td></td></tr><tr><td>Alphadine&amp;reg;&amp;nbsp;solution.</td><td>Riyadh Pharma</td><td>LBL0816</td><td></td></tr><tr><td>&lt;strong&gt;Disposables:&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>15mL Polypropylene High Clarity PP Centrifuge Tube</td><td>Falcon&amp;nbsp;</td><td>352097</td><td></td></tr><tr><td>3.5 cm sterile UpCell culture dishes with the filter paper (membrane)</td><td>Sigma</td><td>174904-1CS</td><td></td></tr><tr><td>100-1000 &amp;micro;l &amp;nbsp;Pipette Tips&amp;nbsp;</td><td>Sigma</td><td>CLS4868-1000EA&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Basic Procedure Drape</td><td>Thermofisher</td><td>PMD5293.0</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Plus pipette, variable volume</td><td>Eppendorf&amp;reg;&amp;nbsp;Research&amp;reg;</td><td>Z683779-1EA</td><td></td></tr><tr><td>Tissue culture incubator 37 &amp;deg;C, 5% CO&lt;sub&gt;2&lt;/sub&gt;</td><td>Any brand</td><td></td><td></td></tr><tr><td>Biological safety cabinet</td><td>Any brand</td><td></td><td></td></tr><tr><td>Tissue culture incubator 20 &amp;deg;C, 5% CO&lt;sub&gt;2&lt;/sub&gt;</td><td>Any brand</td><td></td><td></td></tr><tr><td>Sterile surgical tools and nude rats:&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Forceps</td><td></td><td></td><td></td></tr><tr><td>Scissors</td><td></td><td></td><td></td></tr><tr><td>scalpel&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;Nylon Suture&amp;nbsp; 5-0</td><td>Accutome</td><td>AB-3854S</td><td>Monofilament, Lancet</td></tr><tr><td>1 ml Tuberculin Syringes</td><td>Fisher Scientific</td><td>14-826-88</td><td></td></tr><tr><td>Nude rats&amp;nbsp;</td><td>Charles river</td><td></td><td></td></tr></tbody></table>

exploring the potential of mesenchymal stem cell sheet on the development of hepatocellular carcinoma in vivo

An in vivo animal model that mimics human cancer could have various applications that deliver significant clinical information. The currently used techniques for the development of in vivo cancer models have considerable limitations. Therefore, in this study, we aim to implement cell sheet technology to develop an in vivo cancer model. Hepatocellular carcinoma (HCC) is successfully developed in nude rats using cell sheets created from HCC cell line cells. The cancer cell sheets are generated through intracellular adhesion and the formation of a stratified structure, controlled by the extracellular matrix. This allows for the HCC sheet transplantation into the liver and the creation of a tumor-bearing animal model within a month. In addition, the role of mesenchymal stem cells (MSC) in the development of this cancer model is investigated. In addition to the HCC cell line sheet, another two cell sheets are created: a sheet of HCC cells and bone marrow MSCs (BMMSCs) and a sheet of HCC cells and umbilical cord MSCs (UCMSCs). Sheets that have a combination of both HCC cells and MSCs are also capable of producing a tumor-bearing animal. However, the addition of MSCs reduces the size of the formed tumor, and this adverse effect on tumor development varies depending on the used MSCs' source. This indicates that a cell sheet made of certain MSC subtypes could be utilized in tumor management and control.

Tumorigenicity of Transplanted Cell Sheets in Rats:
One month after the transplantation, all transplanted cell sheets in rats&#39; livers have developed tumors (Figure 3). The average size of the developed tumors from HepG2, HepG2/BMMSC, and HepG2/UCMSC cell sheets were 4.5 cm, 4 cm, and 2.5 cm, respectively10.
<p class="jove_content" fo:keep-together.wit...

Watch this Scientific Journal Video about Exploring the Potential of Mesenchymal Stem Cell Sheet on The Development of Hepatocellular Carcinoma In Vivo at JoVE.com

Exploring the Potential of Mesenchymal Stem Cell Sheet on The Development of Hepatocellular Carcinoma In Vivo

Here, we present a protocol to develop an in vivo cancer model using cell sheet technology. Such a model could be very useful for the evaluation of anticancer therapeutics.

Exploring the Potential of Mesenchymal Stem Cell Sheet on The Development of Hepatocellular Carcinoma In Vivo

An in vivo animal model that mimics human cancer could have various applications that deliver significant clinical information. The currently used ...

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Bioengineering

5.8K Views.  Ministry of National Guard Health Affairs. Hier presenteren we een protocol voor de ontwikkeling van een in vivo kanker model met behulp van blad celtechnologie. Een dergelijk model zou zeer nuttig zijn voor de evaluatie van antikanker therapeutics.

Video: Exploring the Potential of Mesenchymal Stem Cell Sheet on The Development of Hepatocellular Carcinoma In Vivo

An Oncogenic Hepatocyte-Induced Orthotopic Mouse Model of Hepatocellular Cancer Arising in the Setting of Hepatic Inflammation and Fibrosis

The absence of a clinically relevant animal model addressing the typical immune characteristics of hepatocellular cancer (HCC) has significantly impeded elucidation of the underlying mechanisms and development of innovative immunotherapeutic strategies. To develop an ideal animal model recapitulating human HCC, immunocompetent male C57BL/6J mice first receive a carbon tetrachloride (CCl4) injection to induce liver fibrosis, then receive histologically-normal oncogenic hepatocytes from young male SV40 T antigen (TAg)-transgenic mice (MTD2) by intra-splenic (ISPL) inoculation. Androgen generated in recipient male mice at puberty initiates TAg expression under control of a liver-specific promoter. As a result, the transferred hepatocytes become cancer cells and form tumor masses in the setting of liver fibrosis/cirrhosis. This novel model mimics human HCC initiation and progression in the context of liver fibrosis/cirrhosis and reflects the most typical features of human HCC including immune dysfunction.

Here, we describe the development of a clinically relevant murine model of liver cancer recapitulating the typical immune features of hepatocellular cancer (HCC).

The absence of a clinically relevant animal model addressing the typical immune characteristics of hepatocellular cancer (HCC) has significantly impeded ...

Cancer Research

A Biomimetic Model for Liver Cancer to Study Tumor-Stroma Interactions in a 3D Environment with Tunable Bio-Physical Properties

Hepatocellular carcinoma (HCC) is a primary liver tumor developing in the wake of chronic liver disease. Chronic liver disease and inflammation leads to a fibrotic environment actively supporting and driving hepatocarcinogenesis. Insight into hepatocarcinogenesis in terms of the interplay between the tumor stroma micro-environment and tumor cells is thus of considerable importance. Three-dimensional (3D) cell culture models are proposed as the missing link between current in vitro 2D cell culture models and in vivo animal models. Our aim was to design a novel 3D biomimetic HCC model with accompanying fibrotic stromal compartment and vasculature. Physiologically relevant hydrogels such as collagen and fibrinogen were incorporated to mimic the bio-physical properties of the tumor ECM. In this model LX2 and HepG2 cells embedded in a hydrogel matrix were seeded onto the inverted transmembrane insert. HUVEC cells were then seeded onto the opposite side of the membrane. Three formulations consisting of ECM-hydrogels embedded with cells were prepared and the bio-physical properties were determined by rheology. Cell viability was determined by a cell viability assay over 21 days. The effect of the chemotherapeutic drug doxorubicin was evaluated in both 2D co-culture and our 3D model for a period of 72h. Rheology results show that bio-physical properties of a fibrotic, cirrhotic and HCC liver can be successfully mimicked. Overall, results indicate that this 3D model is more representative of the&#160;in vivo&#160;situation compared to traditional 2D cultures.&#160;Our 3D tumor model showed a decreased response to chemotherapeutics, mimicking drug resistance typically seen in HCC patients.&#160;

This protocol presents a 3D biomimetic model with accompanying fibrotic stromal compartment. Prepared with physiologically relevant hydrogels in ratios mimicking the bio-physical properties of the stromal extracellular matrix, an active mediator of cellular interactions, tumor growth and metastasis.

Hepatocellular carcinoma (HCC) is a primary liver tumor developing in the wake of chronic liver disease. Chronic liver disease and inflammation leads to a ...

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.

Human tumor xenografts in immunodeficient mice are valuable tools to study cancer biology. Specific protocols to generate subcutaneous and intrahepatic xenografts from human hepatocellular carcinoma cells or tumor fragments are described. Liver regeneration induced by partial hepatectomy in recipient mice is presented as a strategy to facilitate intrahepatic engraftment.

In  vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate  the human disease provide a valuable platform for research into disease  ...

Medicine

The In Ovo Chick Chorioallantoic Membrane (CAM) Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

The chick chorioallantoic membrane (CAM) begins to develop by day 7 after fertilization and matures by day 12. The CAM is naturally immunodeficient and highly vascularized, making it an ideal system for tumor implantation. Furthermore, the CAM contains extracellular matrix proteins such as fibronectin, laminin, collagen, integrin alpha(v)beta3, and MMP-2, making it an attractive model to study tumor invasion and metastasis. Scientists have long taken advantage of the physiology of the CAM by using it as a model of angiogenesis. More recently, the CAM assay has been modified to work as an in vivo xenograft model system for various cancers that bridges the gap between basic in vitro work and more complex animal cancer models. The CAM assay allows for the study of tumor growth, anti-tumor therapies, and pro-tumor molecular pathways in a biologically relevant system that is both cost- and time-effective. Here, we describe the development of CAM xenograft model of hepatocellular carcinoma (HCC) with embryonic survival rates of up to 93% and reliable tumor take leading to growth of three-dimensional, vascularized tumors.

The In Ovo Chick Chorioallantoic Membrane CAM Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

The chick chorioallantoic membrane (CAM) is immunodeficient and highly vascularized, making it a natural in vivo model of tumor growth and angiogenesis. In this protocol, we describe a reliable method of growing three-dimensional, vascularized hepatocellular carcinoma (HCC) tumors using the CAM assay.

The chick chorioallantoic membrane (CAM) begins to develop by day 7 after fertilization and matures by day 12. The CAM is naturally immunodeficient and ...

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

The metastatic spread of cancer is the major cause of cancer-related deaths. We provide an in-depth description of our survival surgery methodology for establishing a &#8220;patient-like&#8221; orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...

Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low cell retention and poor survival within the target zone. However, during the in vitro construction process, a solution for maintaining stem cell bioactivity and increasing the cell amount within the cell sheet is urgently needed. Here, this protocol presents a method for constructing a multilayered cell sheet with favorable stem cell bioactivity and optimal operability. Decellularized porcine pericardium (DPP) is prepared by phospholipase A2 (PLA2) decellularization method as the cell sheet scaffold, and rat bone marrow mesenchymal stem cells (BMSCs) are isolated and expanded as the seeded cells. The temporary multilayered cell sheet structure is constructed by using RAD16-I peptide hydrogel. Finally, the cell sheet is cultured with a dynamic perfusion system to stabilize the three-dimensional (3D) structure, and the cell sheet could be obtained following a 48-hour culture in vitro. This protocol provides an efficient and feasible method for constructing a multilayered stem cell sheet, and the cell sheet could be developed as a favorable stem cell therapy product in the future.

This article provides an efficient and feasible method for constructing multilayered stem cell sheets with favorable stem cell property.

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low ...

Isolation of CD133+ Liver Stem Cells for Clonal Expansion

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In addition, liver stem cells are hypothesized to be the precursors for a subset of liver cancer, Hepatocellular carcinoma. One of the primary challenges to stem cell work in any solid organ like the liver is the isolation of a rare population of cells for detailed analysis. For example, the vast majority of cells in the liver are hepatocytes (parenchymal fraction), which are significantly larger than non-parenchymal cells. By enriching the specific cellular compartments of the liver (i.e. parenchymal and non-parenchymal fractions), and selecting for CD45 negative cells, we are able to enrich the starting population of stem cells by over 600-fold.The proceduresdetailed in this report allow for a relatively rare population of cells from a solid organ to be sorted efficiently. This process can be utilized to isolateliver stem cells from normal murine liver as well as chronic liver injury models, which demonstrate increased liver stem cell proliferation. This method has clear advantages over standard immunohistochemistry of frozen or formalin fixed liver as functional studies using live cells can be performed after initial co-localization experiments. To accomplish the procedure outlined in this report, a working relationship with a research based flow-cytometry core is strongly encouraged as the details of FACS isolation are highly dependent on specialized instrumentation and a strong working knowledge of basic flow-cytometry procedures. The specific goal of this process is to isolate a population of liver stem cells that can be clonally expanded in vitro.

Here we describe the isolation of CD133 expressing liver stem cells and cancer stem cells from whole murine liver, a process that requires tissue digestion, cell enrichment, and flow cytometry isolation. We include methods for advanced single cell isolation and clonal expansion.

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In ...

Biology

Transplantation of Adipose Tissue-Derived Stem Cell Sheet to Reduce Leakage After Partial Colectomy in A Rat Model

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of clinical efficacy, they are until now imperfect solutions. Stem cell therapy using ASC sheets could provide a solution to this problem. ASCs are considered as promising candidates for promoting tissue healing because of their trophic and immunomodulatory properties. Here, we provide methods to produce high-density ASC sheets, that are transplanted onto a colorectal anastomosis in a rat model to reduce the leakage. ASCs formed cell sheets in thermo-responsive culture dishes that could be easily detached. On the day of the transplantation, a partial colectomy with a 5-suture colorectal anastomosis was performed. Animals were immediately transplanted with 1 ASC sheet per rat. ASC sheets adhered spontaneously to the anastomosis without any glue, suture, or any biomaterial. Animal groups were sacrificed 3 and 7 days postoperatively. Compared to transplanted animals, the incidence of anastomotic abscesses and leakage was higher in control animals. In our model, the transplantation of ASC sheets after colorectal anastomosis was successful and associated with a lower leakage rate.

The preparation and transplantation of adipose tissue derived stem cell (ASC) sheets onto insufficiently sutured colorectal anastomoses in a rat model is presented. This novel application shows that ASC sheets can reduce colorectal anastomosis leakage.

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of ...

An Efficient Method for Directed Hepatocyte-Like Cell Induction from Human Embryonic Stem Cells

The potential functions of hepatocyte-like cells (HLCs) derived from human embryonic stem cells (hESCs) hold great promise for disease modeling and drug screening applications. Provided here is an efficient and reproducible method for differentiation of hESCs into functional HLCs. The establishment of an endoderm lineage is a key step in the differentiation to HLCs. By our method, we regulate the key signaling pathways by continuously supplementing Activin A and CHIR99021 during hESC differentiation into definitive endoderm (DE), followed by generation of hepatic progenitor cells, and finally HLCs with typical hepatocyte morphology in a stagewise method with completely defined reagents. The hESC-derived HLCs produced by this method express stage-specific markers (including albumin, HNF4&#945; nuclear receptor, and sodium taurocholate cotransporting polypeptide (NTCP)), and show special characteristics related to mature and functional hepatocytes (including indocyanine green staining, glycogen storage, hematoxylin-eosin staining and CYP3 activity), and can provide a platform for the development of HLC-based applications in the study of liver diseases.

This manuscript describes a detailed protocol for differentiation of human embryonic stem cells (hESCs) into functional hepatocyte-like cells (HLCs) by continuously supplementing Activin A and CHIR99021 during hESC differentiation into definitive endoderm (DE).

The potential functions of hepatocyte-like cells (HLCs) derived from human embryonic stem cells (hESCs) hold great promise for disease modeling and drug ...

Immunology and Infection

Assembloid Technology: Pioneering Liver Development Research

In Vitro Cultivation Techniques for Modeling Liver Organogenesis, Building Assembloids, and Designing Synthetic Tissues using Human Cell Lines

Chronic liver disease has reached epidemic proportions, affecting over 800 million people globally. The current treatment, orthotopic liver transplantation, has several limitations. Promising solutions have emerged in the field of liver regenerative medicine, with liver organogenesis holding significant potential. Early liver organogenesis, occurring between E8.5 and 11.5, involves the formation of epithelial-mesenchymal interactions leading to morphogenesis, hepatic cord formation, and collective migration. However, there is a lack of methods for in vitro modeling of this process. In this study, a detailed series of methods is presented, enabling the modeling of various stages and aspects of liver organogenesis with human cell lines. In one method series, assembloid technology with hepatic (HEP) and mesenchymal (MES) spheroids are utilized, replicating early structures found in liver organogenesis, modeling early morphogenesis, and demonstrating interstitial cell migration as seen in vivo. These innovative assembloid systems help identify factors influencing assembloid formation and migration. HEP spheroid cultivation systems were also employed to model collective migration and branching morphogenesis. Mesenchymal-conditioned media (M-CM) plays a significant role in initiating dose-dependent branching migration. All presented methods were shown to be highly reproducible with a high success rate. By linking these events together, our work will enable recreating sequential, morphogenetic events to reverse engineer organogenesis in vitro and in vivo.

Organoids revolutionize personalized tissue modeling for organ development, drug discovery, and disease research. Organoid engineering advances into creating intricate synthetic tissues. The aim is to integrate morphogenesis, assembloid technology, and biomatrices to advance tissue engineering. This protocol aids in modeling liver organogenesis and establishing guidelines for synthetic tissue construction.

Exploring the Potential of Mesenchymal Stem Cell Sheet on The Development of Hepatocellular Carcinoma In Vivo

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