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<article dtd-version="3.0" article-type="Invited Methods Article - Author Produced Video">
  <front>
    <journal-meta>
      <journal-id journal-id-type="publisher">jove</journal-id>
      <journal-id journal-id-type="email">editorial-office@jove.com</journal-id>
      <journal-title-group>
        <journal-title>Journal of Visualized Experiments</journal-title>
      </journal-title-group>
      <issn pub-type="ppub">1940-087X</issn>
      <issn pub-type="epub"></issn>
    </journal-meta>
    <article-meta>
      <article-id pub-id-type="publisher-id">JoVE58624</article-id>
      <article-id pub-id-type="manuscript">JoVE58624</article-id>
      <article-categories>
        <subj-group subj-group-type="Article Type">
          <subject>Invited Methods Article - Author Produced Video</subject>
        </subj-group>
        <subj-group subj-group-type="Category">
          <subject>JoVE Bioengineering</subject>
        </subj-group>
        <subj-group subj-group-type="Classification">
          <subject>bioengineering (general)</subject>
          <subject>Life sciences</subject>
          <subject>Life Sciences (General)</subject>
        </subj-group>
      </article-categories>
      <title-group>
        <article-title>Construction of multilayered mesenchymal stem cell sheet with a 3D dynamic culture system</article-title>
      </title-group>
      <contrib-group>
        <contrib contrib-type="author">
          <role content-type="1" />
          <name>
            <surname>Wang</surname>
            <given-names>Yingwei</given-names>
          </name>
          <email>vinmi_wong@126.com</email>
          <xref ref-type="aff" rid="aff1" />
        </contrib>
        <contrib contrib-type="author">
          <role content-type="2" />
          <name>
            <surname>Lu</surname>
            <given-names>Cheng</given-names>
          </name>
          <email>lc471015366@126.com</email>
          <xref ref-type="aff" rid="aff2" />
        </contrib>
        <contrib contrib-type="author">
          <role content-type="3" />
          <name>
            <surname>He</surname>
            <given-names>Chengzhi</given-names>
          </name>
          <email>1732513111@qq.com</email>
          <xref ref-type="aff" rid="aff3" />
        </contrib>
        <contrib contrib-type="author">
          <role content-type="4" />
          <name>
            <surname>Chen</surname>
            <given-names>Baoxin</given-names>
          </name>
          <email>baoxin_chen@126.com</email>
          <xref ref-type="aff" rid="aff4" />
        </contrib>
        <contrib contrib-type="author">
          <role content-type="5" />
          <name>
            <surname>Zheng</surname>
            <given-names>Youling</given-names>
          </name>
          <email>youling_z@126.com</email>
          <xref ref-type="aff" rid="aff5" />
        </contrib>
        <contrib contrib-type="author">
          <role content-type="6" />
          <name>
            <surname>Zheng</surname>
            <given-names>Junming</given-names>
          </name>
          <email>646880087@qq.com</email>
          <xref ref-type="aff" rid="aff6" />
        </contrib>
        <contrib contrib-type="author">
          <role content-type="7" />
          <name>
            <surname>Zhang</surname>
            <given-names>Jianhua</given-names>
          </name>
          <email>zhangjh@jnu.edu.cn</email>
          <xref ref-type="aff" rid="aff7" />
        </contrib>
        <contrib contrib-type="author" corresp="yes">
          <role content-type="8" />
          <name>
            <surname>Wu</surname>
            <given-names>Zheng</given-names>
            <prefix>Dr.</prefix>
          </name>
          <email>wuzheng@jnu.edu.cn</email>
          <xref ref-type="aff" rid="aff8" />
        </contrib>
        <contrib contrib-type="editor">
          <name>
            <surname>DSouza</surname>
            <given-names>Alisha</given-names>
          </name>
          <degrees>Ph.D.</degrees>
          <email>alisha.dsouza@jove.com</email>
          <xref ref-type="aff" rid="aff9" />
        </contrib>
        <aff id="aff1">
          <institution>Jinan University</institution>
          <institution content-type="dept">Department of Developmental and Regenerative Biology</institution>
          <addr-line content-type="addrline1">#601, Huangpudadaoxi, Tianhe District</addr-line>
          <country>CHINA</country>
          <addr-line content-type="city">Guangzhou</addr-line>
          <addr-line content-type="state">Guangdong</addr-line>
          <addr-line content-type="zipcode">510632</addr-line>
        </aff>
        <aff id="aff2">
          <institution>Jinan University</institution>
          <institution content-type="dept">Key laboratory for Regenerative Medicine, Ministry of Education</institution>
          <addr-line content-type="addrline1">#601, Huangpudadaoxi, Tianhe District</addr-line>
          <country>CHINA</country>
          <addr-line content-type="city">Guangzhou</addr-line>
          <addr-line content-type="zipcode">510632</addr-line>
        </aff>
        <aff id="aff3">
          <institution>Nansha College Preparatory Academy</institution>
          <addr-line content-type="addrline1">#180, Gangqiandadaonan, Nansha District</addr-line>
          <country>CHINA</country>
          <addr-line content-type="city">Guangzhou</addr-line>
          <addr-line content-type="zipcode">511458</addr-line>
        </aff>
        <aff id="aff4">
          <institution>Jinan University</institution>
          <institution content-type="dept">Key laboratory for Regenerative Medicine, Ministry of Education</institution>
          <addr-line content-type="addrline1">#601, Huangpudadaoxi, Tianhe District</addr-line>
          <country>CHINA</country>
          <addr-line content-type="city">Guangzhou</addr-line>
          <addr-line content-type="zipcode">510632</addr-line>
        </aff>
        <aff id="aff5">
          <institution>Jinan University</institution>
          <institution content-type="dept">Key laboratory for Regenerative Medicine, Ministry of Education</institution>
          <addr-line content-type="addrline1">#601, Huangpudadaoxi, Tianhe District</addr-line>
          <country>CHINA</country>
          <addr-line content-type="city">Guangzhou</addr-line>
          <addr-line content-type="zipcode">510632</addr-line>
        </aff>
        <aff id="aff6">
          <institution>Jinan University</institution>
          <institution content-type="dept">Key laboratory for Regenerative Medicine, Ministry of Education</institution>
          <addr-line content-type="addrline1">#601, Huangpudadaoxi, Tianhe District</addr-line>
          <country>CHINA</country>
          <addr-line content-type="city">Guangzhou</addr-line>
          <addr-line content-type="zipcode">510632</addr-line>
        </aff>
        <aff id="aff7">
          <institution>First affilitated Hospital of Jinan University</institution>
          <institution content-type="dept">Department of Cardiology</institution>
          <addr-line content-type="addrline1">#613, Huangpudadaoxi, Tianhe District</addr-line>
          <country>CHINA</country>
          <addr-line content-type="city">Guangzhou</addr-line>
          <addr-line content-type="state">Guangdong</addr-line>
          <addr-line content-type="zipcode">510632</addr-line>
        </aff>
        <aff id="aff8">
          <institution>Key Laboratory for Regenerative Medicine, Ministry of Education</institution>
          <institution content-type="position">Researcher</institution>
          <institution content-type="dept">Department of Developmental and Regenerative Biology</institution>
          <addr-line content-type="addrline1">#601, Huangpudadaoxi, Tianhe District</addr-line>
          <country>CHINA</country>
          <addr-line content-type="city">Guangzhou</addr-line>
          <addr-line content-type="state">Guangdong</addr-line>
          <addr-line content-type="zipcode">510632</addr-line>
          <phone content-type="primary">+8620 85222711</phone>
        </aff>
        <aff id="aff9">
          <institution>JoVE</institution>
          <addr-line>1 Alewife Center</addr-line>
          <addr-line>Cambridge, MA  02140</addr-line>
          <country>UNITED STATES</country>
        </aff>
      </contrib-group>
      <history>
        <date date-type="received">
          <day>14</day>
          <month>06</month>
          <year>2018</year>
        </date>
        <date date-type="rev-recd">
          <day>20</day>
          <month>08</month>
          <year>2018</year>
        </date>
        <date date-type="accepted">
          <day>29</day>
          <month>08</month>
          <year>2018</year>
        </date>
      </history>
      <abstract>
        <p>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 cell amount within the cell sheet is urgently needed. Here, this protocol presents a method for constructing multilayeredcell sheet with favorable stem cell bioactivity and optimal operability. Decellularized porcine pericardium (DPP) is prepared by phospholipase A2 (PLA2) decellularized 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 was constructed by using RAD16-I peptide hydrogel. Finally, the cell sheet was cultured with a dynamic perfusion system to stabilize the 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 multilayered stem cell sheet, and the cell sheet could be developed as a favorable stem cell therapy product in the future.</p>
      </abstract>
      <kwd-group>
        <kwd>Bioengineering, tissue engineering, cell sheet, stem cell bioactivity, 3D cell culture, tissue regeneration</kwd>
      </kwd-group>
      <funding-group />
      <counts>
        <fig-count count="0" />
      </counts>
      <custom-meta-group>
        <custom-meta>
          <meta-name>Please indicate whether this article will be Standard Access or Open Access. </meta-name>
          <meta-value>Standard Access (US$1200);</meta-value>
        </custom-meta>
        <custom-meta>
          <meta-name>Black and White Image Count</meta-name>
          <meta-value>0</meta-value>
        </custom-meta>
        <custom-meta>
          <meta-name>Color Image Count</meta-name>
          <meta-value>0</meta-value>
        </custom-meta>
        <custom-meta>
          <meta-name>Acquisitions Editor</meta-name>
          <meta-value>Lyndsay Troyer</meta-value>
        </custom-meta>
        <custom-meta>
          <meta-name>Assistant who entered lead</meta-name>
          <meta-value>Kathleen Wang</meta-value>
        </custom-meta>
        <custom-meta>
          <meta-name>ALA Access Type</meta-name>
          <meta-value>ASV Standard Access ($1200)</meta-value>
        </custom-meta>
      </custom-meta-group>
    </article-meta>
  </front>
  <body>
    <fig>
      <label>Figure 1</label>
      <graphic xlink:href="Figure 1.tif" xmlns:xlink="http://www.w3.org/1999/xlink" />
    </fig>
    <fig>
      <label>Figure 2</label>
      <graphic xlink:href="Figure 2.psd" xmlns:xlink="http://www.w3.org/1999/xlink" />
    </fig>
    <fig>
      <label>Figure 3</label>
      <graphic xlink:href="Figure 3.psd" xmlns:xlink="http://www.w3.org/1999/xlink" />
    </fig>
    <fig>
      <label>Figure 4</label>
      <graphic xlink:href="Figure 4.psd" xmlns:xlink="http://www.w3.org/1999/xlink" />
    </fig>
    <table-wrap>
      <label>Table of Materials</label>
      <graphic xlink:href="JoVE Materials.xls" xmlns:xlink="http://www.w3.org/1999/xlink" />
    </table-wrap>
    <fig>
      <label>Figure 1</label>
      <graphic xlink:href="Figure 1-JoVE58624.tif" xmlns:xlink="http://www.w3.org/1999/xlink" />
    </fig>
    <fig>
      <label>Figure 3</label>
      <graphic xlink:href="Figure 3-JoVE58624.tif" xmlns:xlink="http://www.w3.org/1999/xlink" />
    </fig>
    <fig>
      <label>Figure 4</label>
      <graphic xlink:href="Figure 4-JoVE58624.tif" xmlns:xlink="http://www.w3.org/1999/xlink" />
    </fig>
    <fig>
      <label>Figure 5</label>
      <graphic xlink:href="Figure 5-JoVE58624.tif" xmlns:xlink="http://www.w3.org/1999/xlink" />
    </fig>
    <fig>
      <label>Figure 2</label>
      <graphic xlink:href="Figure 2-JoVE58624.tif" xmlns:xlink="http://www.w3.org/1999/xlink" />
    </fig>
  </body>
</article>