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 dis...

<ol>
	<li>Marra, M., 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=Molecular+targets+and+oxidative+stress+biomarkers+in+hepatocellular+carcinoma:+an+overview.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+tissue+morphogenesis+and+cancer+in+3D.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decrease+in+culture+temperature+releases+monolayer+endothelial+cell+sheets+together+with+deposited+fibronectin+matrix+from+temperature-responsive+culture+surfaces.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+recovery+system+for+cultured+cells+using+plasma-treated+polystyrene+dishes+grafted+with+poly+(N-isopropylacrylamide).">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concise+review:+mesenchymal+stem+cells:+their+phenotype,+differentiation+capacity,+immunological+features,+and+potential+for+homing.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+mesenchymal+stem+cell+(MSC)+paradigm:+polarization+into+a+pro-inflammatory+MSC1+or+an+Immunosuppressive+MSC2+phenotype.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cell+1+(MSC1)-based+therapy+attenuates+tumor+growth+whereas+MSC2-treatment+promotes+tumor+growth+and+metastasis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+buprenorphine+in+a+postoperative+pain+model+in+rats.">Evaluation of buprenorphine in a postoperative pain model in rats.</a> Comparative Medicine. 59 (1), 60-71 (2009).</li><li>. Guidelines on Anesthesia and Analgesia in Rats - ULAM Guidelines and SOPs - Michigan Medicine Confluence Available from: <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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+of+cell+sheet+technique+on+the+development+of+hepatocellular+carcinoma+in+rat+models.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atlas+and+histologic+classification+of+tumors+of+the+rat+mammary+gland.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+and+limitations+of+genetically+modified+mouse+models+in+drug+discovery+and+development.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+preclinical+new+drug+research+program+of+the+National+Cancer+Institute.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+novel+in+vivo+cancer+model+using+cell+sheet+engineering.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+the+cell+sheet+technique+in+tissue+engineering.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+sheet+transplantation+for+heart+tissue+repair.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+the+development+of+bioengineered+human+three-dimensional+vascularized+cardiac+tissue+using+cell+sheet+technology.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+is+the+evidence+that+tumors+are+angiogenesis+dependent?.">What is the evidence that tumors are angiogenesis dependent?.</a> Journal of the National Cancer Institute. 82 (1), 4-6 (1990).</li></ol>

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 border="0" cellpadding="0" cellspacing="0" style="width:787px;" width="787">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:501px;">Reagents&#160;</td>
			<td></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:309px;">FBS&#160;</td>
			<td style="width:192px;">Gibco/Invitrogen</td>
			<td style="width:119px;">10270106</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:309px;">DMEM high glucose&#160;</td>
			<td style="width:192px;">Sigma</td>
			<td style="width:119px;">D5671-500ML</td>
			<td></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:309px;">Penicillin/streptomycin&#160;</td>
			<td style="width:192px;">Life Technology</td>
			<td style="width:119px;">15070063</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:309px;">Sterile physiologic saline</td>
			<td style="width:192px;">Sigma&#160;</td>
			<td style="width:119px;">S0817-1GA</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:309px;">Human HepG2 cell line</td>
			<td style="width:192px;">ATCC, USA</td>
			<td style="width:119px;">HB-8065</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Human bone marrow MSCs cell line</td>
			<td style="width:192px;">PromoCell, USA</td>
			<td>C-12974</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:309px;">human umbilical cord tissue MSCs</td>
			<td style="width:192px;">PromoCell, USA&#160;</td>
			<td style="width:119px;">C-12971</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:309px;">Ketamine 50%</td>
			<td style="width:192px;">Rompun, Bayer</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:309px;">Xylazine 2%</td>
			<td style="width:192px;">Rompun</td>
			<td style="width:119px;">23076-35-9</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Alphadine&#174;&#160;solution.</td>
			<td style="width:192px;">Riyadh Pharma</td>
			<td style="width:119px;">LBL0816</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:501px;">Disposables:&#160;</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:309px;">15mL Polypropylene High Clarity PP Centrifuge Tube</td>
			<td style="width:192px;">Falcon&#160;</td>
			<td style="width:119px;">352097</td>
			<td></td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:309px;">3.5 cm sterile UpCell culture dishes with the filter paper (membrane)</td>
			<td style="width:192px;">Sigma</td>
			<td style="width:119px;">174904-1CS</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:309px;">100-1000 &#181;l &#160;Pipette Tips&#160;</td>
			<td style="width:192px;">Sigma</td>
			<td style="width:119px;">CLS4868-1000EA&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Basic Procedure Drape</td>
			<td style="width:192px;">Thermofisher</td>
			<td style="width:119px;">PMD5293.0</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:501px;">Equipment&#160;</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:309px;">Plus pipette, variable volume</td>
			<td style="width:192px;">Eppendorf&#174;&#160;Research&#174;</td>
			<td style="width:119px;">Z683779-1EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Tissue culture incubator 37 &#176;C, 5% CO2</td>
			<td style="width:192px;">Any brand</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Biological safety cabinet</td>
			<td style="width:192px;">Any brand</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:309px;">Tissue culture incubator 20 &#176;C, 5% CO2</td>
			<td style="width:192px;">Any brand</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td colspan="2" height="22" style="height:22px;width:501px;">Sterile surgical tools and nude rats:&#160;</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td colspan="2" height="22" style="height:22px;width:501px;">Forceps</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td colspan="2" height="22" style="height:22px;width:501px;">Scissors</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td colspan="2" height="22" style="height:22px;width:501px;">scalpel&#160;</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:309px;">&#160;Nylon Suture&#160; 5-0</td>
			<td style="width:192px;">Accutome</td>
			<td style="width:119px;">AB-3854S</td>
			<td>Monofilament, Lancet</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:309px;">1 ml Tuberculin Syringes</td>
			<td style="width:192px;">Fisher Scientific</td>
			<td style="width:119px;">14-826-88</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:309px;">Nude rats&#160;</td>
			<td style="width:192px;">Charles river</td>
			<td style="width:119px;"></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 ...

Hello, I'm Dr.Alaa Alshareeda from King Abdullah International Medical Research Center at the Department of Stem Cells and Regenerative Medicine. Today we will be demonstrating the effect of mesenchymal stem cells on the development of hepatocellular carcinoma on rat model by using cell sheet technology. Hello, I am Abdullah Almubarak.I'm working in Experimental Surgery and Animal Lab King Saud University. We use the nude rat to avoid any rejections after transplanting the cell sheet. This diagram shows cell sheet transplantation procedure on nude rats.Eight to 12 weeks old nude rats will be use in this study. First, nick down the rats and make a seven to ten centimeter cut between the skin and the underlying muscles by using scalpel in order to expose the liver. To transfer the cell sheet, use a sterile membrane.Place the membrane over cell sheet to collect it. Then, place the cell sheet with the membrane over one lobe of the liver. Cell sheet construction.Before seeding the cells, coat 3.5 centimeter temperature-responsive UpCell culture dishes with undiluted FBS. Coating dishes with FBS supports the growth of the cells in the membrane layer and prevents the degradation of cells. Ensure to cover all the surface of the dishes.Incubate the dishes for 24 hours at 37 Celsius in a humidified atmosphere containing 5%carbon dioxide and 95%air. On the next day remove the excess FBS and allow the dishes to dry in room temperature for at least one hour. Seed at least one million of HepG2 cancer cells alone or co-cultured with mesenchymal stem cells into DMEM culture medium.The medium supplemented with 10%FBS and 1%penicillin-streptomycin. The ratio used of cancer cells to mesenchymal stem cells in this study is 4:1. After adding the cells gently shake the dishes.Incubate the cells at 37 Celsius in a humidified atmosphere containing 5%carbon dioxide and 95%air. Detachment of the cell sheet. At 100%cell confluence take the dishes out of the 37 Celsius incubator.Now, incubate the HepG2 cancer cell sheet for one hour at room temperature, while incubate the HepG2 co-cultured with mesenchymal stem cell for 30 to 40 minute at 20 Celsius in a humidified atmosphere containing 5%carbon dioxide at 95%air. During the incubation time for the cell sheet detachment start the animal procedure. All studies on animal were approved by King Saud University ethical committee.Performing the surgical procedure. Check the depth of anesthesia by testing the pedal withdrawal reflex. Disinfect the surgery area with iodine.Now, apply a second scrub with iodine. Cover the animal with a sterilized drape to avoid contamination of the incision. Using a sterilized scalpel make a cut between the skin and underlying muscles, about seven to ten centimeter in size to expose the liver.Separate the abdominal muscle from the skin with the flat back edge of the scalpel. Carefully stab the linea alba using a scalpel and extend the cut with a sharp scissor. Cell sheet transplantation on rat's liver.Gently tap the culture dish over the bench to detach the sheet from its surface. Separate the cell sheet from the dish's surface by scraping the sheet edges in a half circle. Now, gently remove all the culture medium from the dish.Ensure the sheet is intact without any damage, otherwise, it cannot be used. Prepare a sterile membrane to harvest and transfer the cell sheet. So first, soak the membrane with normal saline, then remove the excess saline with a sterile cotton bud.Place the wet membrane over the cell sheet, while avoiding any air bubbles between the membrane and the cell sheet. Add few drops of normal saline over the membrane and the cell sheet to collect the material. Leave the membrane for a few seconds to ensure that the cell sheet is properly attached on the membrane, then collect it.Now, prepare the liver for transplantation. Make sure the liver surface is dry. Place the membrane with the cell sheet over a single lobe of the rat's liver.Add few drops of sterilized saline to detach the cell sheet from the membrane. Wait for five minutes before carefully removing the membrane. The cell sheet can be seen attached on the liver in this video.Finally, close the underlying muscles and skin incisions with five nylon suture. After closing the incision, disinfect the surgery area with betadine. The time from anesthetic off to head up is variable, but is usually somewhere between five and 20 minutes Results.This figure shows the developed tumor on rat's liver after one month of HepG2 cell sheet transplantation. Here, the tumor was developed after the transplantation of cell sheet fabricated HepG2 cancer cells and bone marrow mesenchymal stem cell. Whereas this one shows, the smallest developed tumor on rat's liver after transplantation of cell sheet fabricated from HepG2 cancer cells and umbilical chord mesenchymal stem cell, This figure shows H and E staining of rat's liver after one month of cell sheet transplantation.Where A represent normal rat liver cells, and B shows cancer rat liver cells.Conclusion. So after watching this video you should be able to develop hepatocellular carcinoma model on nude rat by using cell sheet technology. Thank you for watching.

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JoVE Journal

Bioengineering

5.7K vistas.  Ministry of National Guard Health Affairs. Hello, I'm Dr.Alaa Alshareeda from King Abdullah International Medical Research Center at the Department of Stem Cells and Regenerative Medicine. Today we will be demonstrating the effect of mesenchymal stem cells on the development of hepatocellular carcinoma on rat model by using cell sheet technology. Hello, I am Abdullah Almubarak.I'm working in Experimental Surgery and Animal Lab King Saud University. We use the nude rat to avoid any rejections after transplanting the cell sheet. ...