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 Views.  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. ...

Video: 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...

development and optimization of a human hepatocellular carcinoma patient-derived organoid model for potential target identification and drug discovery

Author Spotlight: Investigating Liver Cancer Pathogenesis Using Patient-Derived Organoids 

We provide a comprehensive overview and refinement of existing protocols for hepatocellular carcinoma (HCC) organoid formation, encompassing all stages of organoid cultivation. This system serves as a valuable model for the identification of potential therapeutic targets and the assessment of drug candidate...

Development and Optimization of A Human Hepatocellular Carcinoma Patient-Derived Organoid Model for Potential Target Identification and Drug Discovery

study of viral vectors in a three-dimensional liver model repopulated with the human hepatocellular carcinoma cell line hepg2

Study of Viral Vectors in a Three-dimensional Liver Model Repopulated with the Human Hepatocellular Carcinoma Cell Line HepG2

The recellularized extracellular matrix of a decellularized rat liver can be used as a humanized, three-dimensional ex vivo model to study the distribution and transgene expression of a virus or viral...

construction of a multilayered mesenchymal stem cell sheet with a 3d dynamic culture system

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

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

a three-dimensional spheroid model to investigate the tumor-stromal interaction in hepatocellular carcinoma

A Three-Dimensional Spheroid Model to Investigate the Tumor-Stromal Interaction in Hepatocellular Carcinoma

Comprehensive in vitro models that faithfully recapitulate the relevant human disease are lacking. The current study presents three-dimensional (3D) tumor spheroid creation and culture, a reliable in vitro tool to study the tumor-stromal interaction in human hepatocellular...

the in ovo chick chorioallantoic membrane (cam) assay as an efficient xenograft model of hepatocellular carcinoma

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...

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

generation of subcutaneous and intrahepatic human hepatocellular carcinoma xenografts in immunodeficient mice

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

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...

isolation of human mesenchymal stem cells and their cultivation on the porous bone matrix

Isolation of Human Mesenchymal Stem Cells and their Cultivation on the Porous Bone Matrix

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs the bovine bone matrix Nukbone (NKB) as a...

mast cells in the microenvironment of hepatocellular carcinoma confer favorable prognosis: a retrospective study using qupath image analysis software

Author Spotlight: Investigating Immune Cell Dynamics in the Tumor Microenvironment &amp;#8212; Challenges and Innovations in Cancer Prognosis

The presence of mast cells in the inner margin and peritumor areas of hepatocellular carcinoma after resection confers a favorable prognosis. This study endorses QuPath image analysis software as a promising platform that could meet the need for reproducibility, consistency, and accuracy in digital pathology.

Mast Cells in the Microenvironment of Hepatocellular Carcinoma Confer Favorable Prognosis: A Retrospective Study using QuPath Image Analysis Software

a "patient-like" orthotopic syngeneic mouse model of hepatocellular carcinoma metastasis

A &quot;Patient-Like&quot; Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma 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...

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

development of compendium for esophageal squamous cell carcinoma

Author Spotlight: Integrative Bioinformatics Approach for the Discovery of Differentially Expressed Molecules in Esophageal Squamous Cell Carcinoma

This protocol describes a useful tool for identifying significant molecular changes in cancer and leads to the development of new diagnostic and therapeutic approaches for esophageal squamous cell...

Development of Compendium for Esophageal Squamous Cell Carcinoma

non-invasive pet/mr imaging in an orthotopic mouse model of hepatocellular carcinoma

Non-Invasive PET/MR Imaging in an Orthotopic Mouse Model of Hepatocellular Carcinoma

Here, we present a protocol to create orthotopic hepatocellular carcinoma xenografts with and without hepatic artery ligation and perform non-invasive positron emission tomography (PET) imaging of tumor hypoxia using [18F]Fluoromisonidazole ([18F]FMISO) and [18F]Fluorodeoxyglucose...

transradial access chemoembolization for hepatocellular carcinoma patients

Transradial Access Chemoembolization for Hepatocellular Carcinoma Patients

Transarterial chemoembolization (TACE) is the standard therapy for patients in the intermediate stage of hepatocellular carcinoma and is typically performed through femoral artery access. Compared with transfemoral access, transradial access (TRA) can decrease the rate of bleeding complications and improve patient tolerance. A method is presented here to perform transarterial chemoembolization via radial artery...

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single-cell sorting of immunophenotyped mesenchymal stem cells from human exfoliated deciduous teeth

Author Spotlight: Importance of Single Cell Sorting in Isolating Purified Populations of Mesenchymal Stem Cells 

This protocol describes the use of fluorescence-activated cell sorting of human mesenchymal stem cells using the single-cell sorting method. Specifically, the use of single-cell sorting can achieve 99% purity of the immunophenotyped cells from a heterogeneous population when combined with a multiparametric flow cytometry-based...

Fluorescence-Activated Single-Cell Sorting of Human Mesenchymal Stem Cells

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assessment of the immunomodulatory properties of human mesenchymal stem cells (mscs)

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells MSCs

The immunomodulatory properties of human mesenchymal stem cells (MSC) appear increasingly relevant for clinical application. Using a co-culture system of MSCs and peripheral blood leukocytes pre-stained with the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE), we describe the in vitro assessment of MSC immunomodulation on effector leukocyte proliferation and specific...

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells (MSCs)

hepatocellular carcinoma tissue dissociation for organoid culture

Hepatocellular Carcinoma Tissue Dissociation for Organoid Culture  

the aortic ring co-culture assay: a convenient tool to assess the angiogenic potential of mesenchymal stromal cells in vitro

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Here, we present a novel application of the aortic ring assay where prelabelled mesenchymal cells are co-cultured with rat aorta-derived endothelial networks. This novel method allows visualization of Mesenchymal Stromal Cells (MSCs) homing and integration with endothelial networks, quantification of network properties, and evaluation of MSC immunophenotypes and gene...

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

y-90 radioembolization and pd-1 inhibitor as neoadjuvant treatment in hepatocellular carcinoma

Author Spotlight: Treatments to Mitigate Postoperative Recurrence in Hepatocellular Carcinoma

This study illustrates the methodological potential of combining Yttrium-90 Trans-Arterial Radioembolization (Y-90 TARE) with an anti-PD-1 monoclonal antibody as an effective neoadjuvant strategy leading to hepatectomy in hepatocellular carcinoma (HCC) patients with a high initial recurrence risk. It emphasizes the safety, feasibility, and step-by-step procedural guidance of this...

Y-90 Radioembolization and PD-1 Inhibitor as Neoadjuvant Treatment in Hepatocellular Carcinoma

generating rabbit liver carcinoma model: a surgical procedure to implant tumor tissue in left lobe of liver for induction of hepatocellular carcinoma

In this video, we demonstrate the surgical implantation of tumor tissue into a rabbit liver to induce hepatocellular carcinoma. The procedure helps establish a reliable animal model to study tumor behavior and development of locoregional treatments targeting cancers of the...

Generating Rabbit Liver Carcinoma Model: A Surgical Procedure to Implant Tumor Tissue in Left Lobe of Liver for Induction of Hepatocellular Carcinoma

evaluation of stem cell properties in human ovarian carcinoma cells using multi and single cell-based spheres assays

Evaluation of Stem Cell Properties in Human Ovarian Carcinoma Cells Using Multi and Single Cell-based Spheres Assays

In vitro spheres assays are commonly used to identify cancer stem cells. Here we compare single with multi cell-based spheres assays. The more laborious single cell-based assays or methylcellulose supplementation give more accurate results while multi cell-based assays performed in liquid medium can be highly influenced by cell...

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to high levels of NK cells after 4 weeks culture and can undergo further 2-log expansion with artificial antigen presenting cells. hESC- and iPSC-derived NK cells developed in this system have a mature phenotype and function. The production of large numbers of genetically modifiable NK cells is applicable for both basic mechanistic as well as anti-tumor studies. Expression of firefly luciferase in hESC-derived NK cells allows a non-invasive approach to follow NK cell engraftment, distribution, and function. We also describe a dual-imaging scheme that allows separate monitoring of two different cell populations to more distinctly characterize their interactions in vivo. This method of derivation, expansion, and dual in vivo imaging provides a reliable approach for producing NK cells and their evaluation which is necessary to improve current NK cell adoptive therapies.

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and iPSCs.

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to maintain cellular homeostasis. Puncture injury with glass needles provides a direct method to trigger a rapid epidermal wound response that activates wound transcriptional reporters, which can be visualized by a localized reporter signal in living embryos or larvae. Puncture or laser injury also provides signals that promote the recruitment of hemocytes to the wound site. Surprisingly, severe (through and through) puncture injury in late stage embryos only rarely disrupts normal embryonic development, as greater than 90% of such wounded embryos survive to adulthood when embryos are injected in an oil medium that minimizes immediate leakage of hemolymph from puncture sites. The wound procedure does require micromanipulation of the Drosophila embryos, including manual alignment of the embryos on agar plates and transfer of the aligned embryos to microscope slides. The Drosophila epidermal wound response assay provides a quick system to test the genetic requirements of a variety of biological functions that promote wound healing, as well as a way to screen for potential chemical compounds that promote wound healing. The short life cycle and easy culturing routine make Drosophila a powerful model organism. Drosophila clean wound healing appears to coordinate the epidermal regenerative response, with the innate immune response, in ways that are still under investigation, which provides an excellent system to find conserved regulatory mechanisms common to Drosophila and mammalian epidermal wounding.

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

The embryonic epidermis of very late stage Drosophila embryos provides an in vivo system for rapid puncture wound response analysis and can be combined with genetic manipulations or chemical microinjection treatments to advance studies in wound healing for translation into mammalian models.

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to ...

Procedure for the Development of Multi-depth Circular Cross-sectional Endothelialized Microchannels-on-a-chip

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, and observation and quantification are very challenging. However, conventional in vitro microvessel assays have limitations when representing in vivo microvessels with respect to three-dimensional (3D) geometry and providing continuous fluid flow. Using a combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics, we have developed a multi-depth circular cross-sectional endothelialized microchannels-on-a-chip, which mimics the 3D geometry of in vivo microvessels and runs under controlled continuous perfusion flow. A positive reflowable photoresist was used to fabricate a master mold with a semicircular cross-sectional microchannel network. By the alignment and bonding of the two polydimethylsiloxane (PDMS) microchannels replicated from the master mold, a cylindrical microchannel network was created. The diameters of the microchannels can be well controlled. In addition, primary human umbilical vein endothelial cells (HUVECs) seeded inside the chip showed that the cells lined the inner surface of the microchannels under controlled perfusion lasting for a time period between 4 days to 2 weeks.

A microchannels-on-a-chip platform was developed by the combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics. The endothelialized microchannels platform mimics the three-dimensional (3D) geometry of in vivo microvessels, runs under controlled continuous perfusion flow, allows for high-quality and real-time imaging&#160;and can be applied for microvascular research.

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, ...

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&#949;-caprolactone) Electrospun Yarns

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell number. While researchers commonly reference their seeding density this does not necessarily indicate the actual number of cells that have adhered to the material in question. This is particularly the case for materials, or scaffolds, that do not cover the base of standard cell culture well plates. This study investigates the initial attachment of human mesenchymal stem cells seeded at a known number onto electrospun poly(&#949;-caprolactone) yarn after 4 hr in culture. Electrospun yarns were held within several different set-ups, including bioreactor vessels rotating at 9 rpm, cell culture inserts positioned in low binding well plates and polytetrafluoroethylene (PTFE) troughs placed within petri dishes. The latter two were subjected to either static conditions or positioned on a shaker plate (30 rpm). After 4 hr incubation at 37&#160;oC, 5% CO2, the location of seeded cells was determined by cell DNA assay. Scaffolds were removed from their containers and placed in lysis buffer. The media fraction was similarly removed and centrifuged &#8211; the supernatant discarded and pellet broken up with lysis buffer. Lysis buffer was added to each receptacle, or well, and scraped to free any cells that may be present. The cell DNA assay determined the percentage of cells present within the scaffold, media and well fractions. Cell attachment was low for all experimental set-ups, with greatest attachment (30%) for yarns held within cell culture inserts and subjected to shaking motion. This study raises awareness to the actual number of cells attaching to scaffolds irrespective of the stated cell seeding density.

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&amp;#949;-caprolactone) Electrospun Yarns

This article describes a range of set-ups for seeding human mesenchymal stem cells onto materials, in this case electrospun yarns, that do not cover the base of standard culture well plates in order to maximize and quantify the number of cells that initially attach compared to the known seeding density.

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell ...

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that replicate the biofidelity of three-dimensional native human myocardium, while also enabling a controlled level of biological complexity, and allowing non-destructive longitudinal monitoring of tissue contractile function. Initially, human engineered cardiac tissues (hECT) were created using the entire cell population obtained from directed differentiation of human pluripotent stem cells, which typically yielded less than 50% cardiomyocytes. However, to create reliable predictive models of human myocardium, and to elucidate mechanisms of heterocellular interaction, it is essential to accurately control the biological composition in engineered tissues. 
To address this limitation, we utilize live cell sorting for the cardiac surface marker SIRP&#945; and the fibroblast marker CD90 to create tissues containing a 3:1 ratio of these cell types, respectively, that are then mixed together and added to a collagen-based matrix solution. Resulting hECTs are, thus, completely defined in both their cellular and extracellular matrix composition.
Here we describe the construction of defined hECTs as a model system to understand mechanisms of cell-cell interactions in cell therapies, using an example of human bone marrow-derived mesenchymal stem cells (hMSC) that are currently being used in human clinical trials. The defined tissue composition is imperative to understand how the hMSCs may be interacting with the endogenous cardiac cell types to enhance tissue function. A bioreactor system is also described that simultaneously cultures six hECTs in parallel, permitting more efficient use of the cells after sorting.

This manuscript describes the creation of defined engineered cardiac tissues using surface marker expression and cell sorting. The defined tissues can then be used in a multi-tissue bioreactor to investigate mechanisms of cardiac cell therapy in order to provide a functional, yet controlled, model system of the human heart.

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that ...

Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths from the myocardium. Genetic program defects in the Notch signaling cascade are involved in ventricular defects such as Left Ventricular Non-Compaction Cardiomyopathy or Hypoplastic Left Heart Syndrome. Using this protocol, it can be determined that shear stress driven trabeculation and Notch signaling are related to one another. Using Light-sheet Fluorescence Microscopy, visualization of the developing zebrafish heart was possible. In this manuscript, it was assessed whether hemodynamic forces modulate the initiation of trabeculation via Notch signaling and thus, influence contractile function occurs. For qualitative and quantitative shear stress analysis, 4-D (3-D+time) images were acquired during zebrafish cardiac morphogenesis, and integrated light-sheet fluorescence microscopy with 4-D synchronization captured the ventricular motion. Blood viscosity was reduced via gata1a-morpholino oligonucleotides (MO) micro-injection to decrease shear stress, thereby, down-regulating Notch signaling and attenuating trabeculation. Co-injection of Nrg1 mRNA with gata1a MO rescued Notch-related genes to restore trabeculation. To confirm shear stress driven Notch signaling influences trabeculation, cardiomyocyte contraction was further arrested via tnnt2a-MO to reduce hemodynamic forces, thereby, down-regulating Notch target genes to develop a non-trabeculated myocardium. Finally, corroboration of the expression patterns of shear stress-responsive Notch genes was conducted by subjecting endothelial cells to pulsatile flow. Thus, the 4-D light-sheet microscopy uncovered hemodynamic forces underlying Notch signaling and trabeculation with clinical relevance to non-compaction cardiomyopathy.

Here, we present a protocol to visualize developing hearts in zebrafish in 4-Dimensions (4-D). 4-D imaging, via light-sheet fluorescence microscopy (LSFM), takes 3-Dimensional (3-D) images over time, to reconstruct developing hearts. We show qualitatively and quantitatively that shear stress activates endocardial Notch signaling during chamber development, which promotes cardiac trabeculation.

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths ...

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 ...

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development.

Extracellular matrix ligands can be patterned onto polyacrylamide hydrogels to enable the culture of human embryonic stem cells in confined colonies on compliant substrates. This method can be combined with traction force microscopy and biochemical assays to examine the interplay between tissue geometry, cell-generated forces, and fate specification.

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that ...

Isolation, Expansion, and Differentiation of Mesenchymal Stem Cells from the Infrapatellar Fat Pad of the Goat Stifle Joint

The IFP, present in the knee joint, serves as a promising source of MSCs. The IFP is an easily accessible tissue as it is routinely resected and discarded during arthroscopic procedures and knee replacement surgeries. Additionally, its removal is associated with minimal donor site morbidity. Recent studies have demonstrated that IFP-MSCs do not lose their proliferation capacity during in vitro expansion and have age-independent osteogenic differentiation potential. IFP-MSCs possess superior chondrogenic differentiation potential compared to bone marrow-derived MSCs (BMSCs) and adipose-derived stem cells (ADSCs). Although these cells are easily obtainable from aged and diseased patients, their effectiveness is limited. Hence, using IFP-MSCs from healthy donors is important to determine their efficacy in biomedical applications. As access to a healthy human donor is challenging, animal models could be a better alternative to enable fundamental understanding. Large animals such as dogs, horses, sheep, and goats play a crucial role in translational research. Amongst these, the goat could be a preferred model since the stifle joint of the goat has the closest anatomy to the human knee joint. Moreover, goat-IFP can fulfill the higher MSC numbers needed for tissue regeneration applications. Furthermore, low cost, availability, and compliance with the 3R principles for animal research make them an attractive model. This study demonstrates a simple protocol for isolating IFP-MSCs from the stifle joint of goats and in vitro culture conditions for their expansion and differentiation. The aseptically isolated IFP from the goat was washed, minced, and digested enzymatically. After filtration and centrifugation, the collected cells were cultured. These cells were adherent, had MSCs-like morphology, and demonstrated remarkable clonogenic ability. Further, they differentiated into adipogenic, chondrogenic, and osteogenic lineages, demonstrating their multipotency. In conclusion, the study demonstrates the isolation and expansion of MSCs, which show potential in tissue engineering and regenerative medicine applications.

Infrapatellar fat pad mesenchymal&#160;stem cells (IFP-MSCs) can be isolated easily from the infrapatellar fat pad of the knee joint. They proliferate well in vitro, form CFU-F colonies, and differentiate into adipogenic, chondrogenic, and osteogenic lineages. Herein, the methodology for the isolation, expansion, and differentiation of IFP-MSCs from goat stifle joint is provided.

The IFP, present in the knee joint, serves as a promising source of MSCs. The IFP is an easily accessible tissue as it is routinely resected and discarded ...

Prospective, Randomized, and Controlled Study of a Human Umbilical Cord Mesenchymal Stem Cell Injection for Treating Diabetic Foot Ulcers

With the development of society and the economy, the incidence of diabetic foot ulcers continues to increase. Currently, conventional debridement with dressing changes, hyperbaric oxygen, and vacuum sealing drainage are the main conservative treatments in clinical practice, and large wounds often require skin grafts or skin flap grafts. However, the treatment effects are not ideal, and many complications exist. Due to its complex pathogenesis, long treatment time, significant associated difficulties, and high disability rate, diabetic foot ulcers cause a heavy burden to patients, society, and medical care. According to our previous study, the pharmacological effects of human umbilical cord blood stem cells include nonspecific immune regulation; increased secretion of growth factors, vasoactive factors, and anti-inflammatory factors; enhanced anti-infectious ability of the human body; elimination of inflammation; and promotion of angiogenesis and ulcer healing. These effects suggest stem cells may be useful as an autologous or allogeneic treatment for refractory wounds. Therefore, we are conducting a clinical trial to treat refractory diabetic wounds with human umbilical cord stem cells in our clinic for diabetic foot ulcer patients who meet the inclusion criteria.

The present protocol describes a prospective, randomized, controlled clinical study that evaluates a human umbilical cord mesenchymal stem cell injection for the treatment of chronic diabetic foot ulcers.

With the development of society and the economy, the incidence of diabetic foot ulcers continues to increase. Currently, conventional debridement with ...