An Improved Harvest and in Vitro Expansion Protocol for Murine Bone Marrow-Derived Mesenchymal Stem Cells
Compared to bone marrow (BM) derived mesenchymal stem cells (MSCs) from human origin or from other species, the in vitro expansion and purification of murine MSCs (mMSCs) is much more difficult because of the low MSC yield and the unwanted growth of non-MSCs in the in vitro expansion cultures. We de...
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| Veröffentlicht in: | BioMed research international 2010-01, Vol.2010 (2010), p.1-10 |
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| creator | Vanderkerken, Karin De Becker, Ann Van Camp, Ben Van Riet, Ivan Xu, Song |
| description | Compared to bone marrow (BM) derived mesenchymal stem cells (MSCs) from human origin or from other species, the in vitro expansion and purification of murine MSCs (mMSCs) is much more difficult because of the low MSC yield and the unwanted growth of non-MSCs in the in vitro expansion cultures. We describe a modified protocol to isolate and expand murine BM derived MSCs based on the combination of mechanical crushing and collagenase digestion at the moment of harvest, followed by an immunodepletion step using microbeads coated with CD11b, CD45 and CD34 antibodies. The number of isolated mMSCs as estimated by colony forming unit-fibroblast (CFU-F) assay showed that this modified isolation method could yield 70.0% more primary colonies. After immunodepletion, a homogenous mMSC population could already be obtained after two passages. Immunodepleted mMSCs (ID-mMSCs) are uniformly positive for stem cell antigen-1 (Sca-1), CD90, CD105 and CD73 cell surface markers, but negative for the hematopoietic surface markers CD14, CD34 and CD45. Moreover the immunodepleted cell population exhibits more differentiation potential into adipogenic, osteogenic and chondrogenic lineages. Our data illustrate the development of an efficient and reliable expansion protocol increasing the yield and purity of mMSCs and reducing the overall expansion time. |
| doi_str_mv | 10.1155/2010/105940 |
| format | Article |
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We describe a modified protocol to isolate and expand murine BM derived MSCs based on the combination of mechanical crushing and collagenase digestion at the moment of harvest, followed by an immunodepletion step using microbeads coated with CD11b, CD45 and CD34 antibodies. The number of isolated mMSCs as estimated by colony forming unit-fibroblast (CFU-F) assay showed that this modified isolation method could yield 70.0% more primary colonies. After immunodepletion, a homogenous mMSC population could already be obtained after two passages. Immunodepleted mMSCs (ID-mMSCs) are uniformly positive for stem cell antigen-1 (Sca-1), CD90, CD105 and CD73 cell surface markers, but negative for the hematopoietic surface markers CD14, CD34 and CD45. Moreover the immunodepleted cell population exhibits more differentiation potential into adipogenic, osteogenic and chondrogenic lineages. Our data illustrate the development of an efficient and reliable expansion protocol increasing the yield and purity of mMSCs and reducing the overall expansion time.</description><identifier>ISSN: 1110-7243</identifier><identifier>ISSN: 2314-6133</identifier><identifier>EISSN: 1110-7251</identifier><identifier>EISSN: 2314-6141</identifier><identifier>DOI: 10.1155/2010/105940</identifier><identifier>PMID: 21197440</identifier><language>eng</language><publisher>Cairo, Egypt: Hindawi Puplishing Corporation</publisher><subject>Animals ; Biological and medical sciences ; Bone marrow ; Bone Marrow Cells - cytology ; Cell Culture Techniques - methods ; Cell Differentiation - physiology ; Cell Separation - methods ; Colony-Forming Units Assay ; Disease ; Flow Cytometry ; Hematopoietic Stem Cell Mobilization ; Histocytochemistry ; Medical sciences ; Mesenchymal Stromal Cells - cytology ; Methodology Report ; Mice ; Mice, Inbred C57BL ; Pharmacology. Drug treatments ; Statistics, Nonparametric ; Stem cells</subject><ispartof>BioMed research international, 2010-01, Vol.2010 (2010), p.1-10</ispartof><rights>Copyright © 2010 Song Xu et al.</rights><rights>2015 INIST-CNRS</rights><rights>Copyright © 2010 Song Xu et al. Song Xu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</rights><rights>Copyright © 2010 Song Xu et al. 2010 Copyright © 2010 Song Xu et al.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c457t-cc13f379dff0c2eb323d2a16536f0a5854627fbc78f5a3ad1884ea4a96994c233</citedby><cites>FETCH-LOGICAL-c457t-cc13f379dff0c2eb323d2a16536f0a5854627fbc78f5a3ad1884ea4a96994c233</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3010643/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3010643/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,885,27924,27925,53791,53793</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=25883535$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/21197440$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Byrne, Barry J.</contributor><creatorcontrib>Vanderkerken, Karin</creatorcontrib><creatorcontrib>De Becker, Ann</creatorcontrib><creatorcontrib>Van Camp, Ben</creatorcontrib><creatorcontrib>Van Riet, Ivan</creatorcontrib><creatorcontrib>Xu, Song</creatorcontrib><title>An Improved Harvest and in Vitro Expansion Protocol for Murine Bone Marrow-Derived Mesenchymal Stem Cells</title><title>BioMed research international</title><addtitle>J Biomed Biotechnol</addtitle><description>Compared to bone marrow (BM) derived mesenchymal stem cells (MSCs) from human origin or from other species, the in vitro expansion and purification of murine MSCs (mMSCs) is much more difficult because of the low MSC yield and the unwanted growth of non-MSCs in the in vitro expansion cultures. We describe a modified protocol to isolate and expand murine BM derived MSCs based on the combination of mechanical crushing and collagenase digestion at the moment of harvest, followed by an immunodepletion step using microbeads coated with CD11b, CD45 and CD34 antibodies. The number of isolated mMSCs as estimated by colony forming unit-fibroblast (CFU-F) assay showed that this modified isolation method could yield 70.0% more primary colonies. After immunodepletion, a homogenous mMSC population could already be obtained after two passages. Immunodepleted mMSCs (ID-mMSCs) are uniformly positive for stem cell antigen-1 (Sca-1), CD90, CD105 and CD73 cell surface markers, but negative for the hematopoietic surface markers CD14, CD34 and CD45. Moreover the immunodepleted cell population exhibits more differentiation potential into adipogenic, osteogenic and chondrogenic lineages. Our data illustrate the development of an efficient and reliable expansion protocol increasing the yield and purity of mMSCs and reducing the overall expansion time.</description><subject>Animals</subject><subject>Biological and medical sciences</subject><subject>Bone marrow</subject><subject>Bone Marrow Cells - cytology</subject><subject>Cell Culture Techniques - methods</subject><subject>Cell Differentiation - physiology</subject><subject>Cell Separation - methods</subject><subject>Colony-Forming Units Assay</subject><subject>Disease</subject><subject>Flow Cytometry</subject><subject>Hematopoietic Stem Cell Mobilization</subject><subject>Histocytochemistry</subject><subject>Medical sciences</subject><subject>Mesenchymal Stromal Cells - cytology</subject><subject>Methodology Report</subject><subject>Mice</subject><subject>Mice, Inbred C57BL</subject><subject>Pharmacology. 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Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>BioMed research international</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Vanderkerken, Karin</au><au>De Becker, Ann</au><au>Van Camp, Ben</au><au>Van Riet, Ivan</au><au>Xu, Song</au><au>Byrne, Barry J.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>An Improved Harvest and in Vitro Expansion Protocol for Murine Bone Marrow-Derived Mesenchymal Stem Cells</atitle><jtitle>BioMed research international</jtitle><addtitle>J Biomed Biotechnol</addtitle><date>2010-01-01</date><risdate>2010</risdate><volume>2010</volume><issue>2010</issue><spage>1</spage><epage>10</epage><pages>1-10</pages><issn>1110-7243</issn><issn>2314-6133</issn><eissn>1110-7251</eissn><eissn>2314-6141</eissn><abstract>Compared to bone marrow (BM) derived mesenchymal stem cells (MSCs) from human origin or from other species, the in vitro expansion and purification of murine MSCs (mMSCs) is much more difficult because of the low MSC yield and the unwanted growth of non-MSCs in the in vitro expansion cultures. We describe a modified protocol to isolate and expand murine BM derived MSCs based on the combination of mechanical crushing and collagenase digestion at the moment of harvest, followed by an immunodepletion step using microbeads coated with CD11b, CD45 and CD34 antibodies. The number of isolated mMSCs as estimated by colony forming unit-fibroblast (CFU-F) assay showed that this modified isolation method could yield 70.0% more primary colonies. After immunodepletion, a homogenous mMSC population could already be obtained after two passages. Immunodepleted mMSCs (ID-mMSCs) are uniformly positive for stem cell antigen-1 (Sca-1), CD90, CD105 and CD73 cell surface markers, but negative for the hematopoietic surface markers CD14, CD34 and CD45. Moreover the immunodepleted cell population exhibits more differentiation potential into adipogenic, osteogenic and chondrogenic lineages. Our data illustrate the development of an efficient and reliable expansion protocol increasing the yield and purity of mMSCs and reducing the overall expansion time.</abstract><cop>Cairo, Egypt</cop><pub>Hindawi Puplishing Corporation</pub><pmid>21197440</pmid><doi>10.1155/2010/105940</doi><tpages>10</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Animals Biological and medical sciences Bone marrow Bone Marrow Cells - cytology Cell Culture Techniques - methods Cell Differentiation - physiology Cell Separation - methods Colony-Forming Units Assay Disease Flow Cytometry Hematopoietic Stem Cell Mobilization Histocytochemistry Medical sciences Mesenchymal Stromal Cells - cytology Methodology Report Mice Mice, Inbred C57BL Pharmacology. Drug treatments Statistics, Nonparametric Stem cells |
| title | An Improved Harvest and in Vitro Expansion Protocol for Murine Bone Marrow-Derived Mesenchymal Stem Cells |
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