Xeno-free extraction, culture, and cryopreservation of human adipose-derived mesenchymal stem cells

Molecules of animal or bacterial origin, which pose a risk for zoonoses or immune rejection, are com-monly used for extraction, culture, and cryopreservation of mesenchymal stem cells. There is no se-quentialandorderlyprotocolforproducinghumanadipose-derivedstemcells(hASCs)underxeno-freeconditions....

Full description

Autores:
Escobar, Carlos Hugo
Chaparro, Orlando
Tipo de recurso:
Article of journal
Fecha de publicación:
2016
Institución:
Fundación Universitaria de Ciencias de la Salud - FUCS
Repositorio:
Repositorio Digital Institucional ReDi
Idioma:
eng
OAI Identifier:
oai:repositorio.fucsalud.edu.co:001/2699
Acceso en línea:
https://repositorio.fucsalud.edu.co/handle/001/2699
Palabra clave:
Adipose
Adult stem cells
Xeno-free production
Human platelet lysate
Explant culture
Cryopreservation
Criopreservación
Tejido
Antígenos de plaqueta humana
Adipocitos
Células madre adultas
Anticuerpos heterófilos
Rights
openAccess
License
Atribución-NoComercial-SinDerivadas 4.0 Internacional (CC BY-NC-ND 4.0)
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dc.title.eng.fl_str_mv Xeno-free extraction, culture, and cryopreservation of human adipose-derived mesenchymal stem cells
title Xeno-free extraction, culture, and cryopreservation of human adipose-derived mesenchymal stem cells
spellingShingle Xeno-free extraction, culture, and cryopreservation of human adipose-derived mesenchymal stem cells
Adipose
Adult stem cells
Xeno-free production
Human platelet lysate
Explant culture
Cryopreservation
Criopreservación
Tejido
Antígenos de plaqueta humana
Adipocitos
Células madre adultas
Anticuerpos heterófilos
title_short Xeno-free extraction, culture, and cryopreservation of human adipose-derived mesenchymal stem cells
title_full Xeno-free extraction, culture, and cryopreservation of human adipose-derived mesenchymal stem cells
title_fullStr Xeno-free extraction, culture, and cryopreservation of human adipose-derived mesenchymal stem cells
title_full_unstemmed Xeno-free extraction, culture, and cryopreservation of human adipose-derived mesenchymal stem cells
title_sort Xeno-free extraction, culture, and cryopreservation of human adipose-derived mesenchymal stem cells
dc.creator.fl_str_mv Escobar, Carlos Hugo
Chaparro, Orlando
dc.contributor.author.none.fl_str_mv Escobar, Carlos Hugo
Chaparro, Orlando
dc.subject.proposal.eng.fl_str_mv Adipose
Adult stem cells
Xeno-free production
Human platelet lysate
Explant culture
Cryopreservation
topic Adipose
Adult stem cells
Xeno-free production
Human platelet lysate
Explant culture
Cryopreservation
Criopreservación
Tejido
Antígenos de plaqueta humana
Adipocitos
Células madre adultas
Anticuerpos heterófilos
dc.subject.decs.none.fl_str_mv Criopreservación
Tejido
Antígenos de plaqueta humana
Adipocitos
Células madre adultas
Anticuerpos heterófilos
description Molecules of animal or bacterial origin, which pose a risk for zoonoses or immune rejection, are com-monly used for extraction, culture, and cryopreservation of mesenchymal stem cells. There is no se-quentialandorderlyprotocolforproducinghumanadipose-derivedstemcells(hASCs)underxeno-freeconditions. Afterstandardizinga humanplateletlysate (hPL) productionprotocol,four humanadiposetissue samples were processed through explants with fetal bovine serum (FBS)-supplemented or hPL-supplemented media for extracting the adipose-derived stem cells. The cells were cultivated in cellculture medium + hPL (5%) or FBS (10%). The cellular replication rate, immunophenotype, and differ-entiation potential were evaluated at fourth passage. Cellular viability was evaluated before and aftercryopreservation of the cells, with an hPL-based solution compared with an FBS-based solution. Theexplants cultured in hPL-supplemented media showed earlier and faster hASC proliferation than didthosesupplementedwithFBS.Likewise,cellsgrowninhPL-supplementedmediashowedagreaterpro-liferation rate, without losing the immunophenotype. Osteogenic differentiation of xeno-free hASCwas higher than the hASC produced in standard conditions. However, adipogenic differentiationwas reduced in xeno-free hASC. Finally, the cells cryopreserved in an hPL-based solution showed ahigher cellular viability thanthecells cryopreserved inanFBS-based.In conclusion, we have developeda complete xeno-free protocol for extracting, culturing, and cryopreserving hASCs that can be safelyimplemented in clinical studies.
publishDate 2016
dc.date.issued.none.fl_str_mv 2016-02
dc.date.accessioned.none.fl_str_mv 2022-04-20T13:41:58Z
dc.date.available.none.fl_str_mv 2022-04-20T13:41:58Z
dc.type.spa.fl_str_mv Artículo de revista
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dc.language.iso.spa.fl_str_mv eng
language eng
dc.relation.ispartof.none.fl_str_mv Stem cells translational medicine
dc.relation.citationendpage.spa.fl_str_mv 365
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dc.relation.citationstartpage.spa.fl_str_mv 358
dc.relation.citationvolume.spa.fl_str_mv 5
dc.relation.ispartofjournal.spa.fl_str_mv Stem cells translational medicine
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dc.rights.creativecommons.spa.fl_str_mv Atribución-NoComercial-SinDerivadas 4.0 Internacional (CC BY-NC-ND 4.0)
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dc.format.extent.spa.fl_str_mv 8 p.
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dc.publisher.spa.fl_str_mv AlphaMed Press
dc.publisher.place.spa.fl_str_mv Reino Unido
dc.source.spa.fl_str_mv https://stemcellsjournals.onlinelibrary.wiley.com/doi/10.5966/sctm.2015-0094
institution Fundación Universitaria de Ciencias de la Salud - FUCS
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spelling Escobar, Carlos Hugof8678d9e7ce2d62715756f53062e57a0Chaparro, Orlando16708af86b656654f767e3137e1e4ca42022-04-20T13:41:58Z2016-022022-04-20T13:41:58ZAlphaMed PressReino UnidoMolecules of animal or bacterial origin, which pose a risk for zoonoses or immune rejection, are com-monly used for extraction, culture, and cryopreservation of mesenchymal stem cells. There is no se-quentialandorderlyprotocolforproducinghumanadipose-derivedstemcells(hASCs)underxeno-freeconditions. Afterstandardizinga humanplateletlysate (hPL) productionprotocol,four humanadiposetissue samples were processed through explants with fetal bovine serum (FBS)-supplemented or hPL-supplemented media for extracting the adipose-derived stem cells. The cells were cultivated in cellculture medium + hPL (5%) or FBS (10%). The cellular replication rate, immunophenotype, and differ-entiation potential were evaluated at fourth passage. Cellular viability was evaluated before and aftercryopreservation of the cells, with an hPL-based solution compared with an FBS-based solution. Theexplants cultured in hPL-supplemented media showed earlier and faster hASC proliferation than didthosesupplementedwithFBS.Likewise,cellsgrowninhPL-supplementedmediashowedagreaterpro-liferation rate, without losing the immunophenotype. Osteogenic differentiation of xeno-free hASCwas higher than the hASC produced in standard conditions. However, adipogenic differentiationwas reduced in xeno-free hASC. Finally, the cells cryopreserved in an hPL-based solution showed ahigher cellular viability thanthecells cryopreserved inanFBS-based.In conclusion, we have developeda complete xeno-free protocol for extracting, culturing, and cryopreserving hASCs that can be safelyimplemented in clinical studies.https://stemcellsjournals.onlinelibrary.wiley.com/doi/10.5966/sctm.2015-00948 p.application/pdfXeno-free extraction, culture, and cryopreservation of human adipose-derived mesenchymal stem cellsArtículo de revistahttp://purl.org/coar/resource_type/c_6501http://purl.org/coar/resource_type/c_2df8fbb1info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionTexthttps://purl.org/redcol/resource_type/WPhttp://purl.org/coar/version/c_970fb48d4fbd8a852157-6564https://repositorio.fucsalud.edu.co/handle/001/26992157-6580engStem cells translational medicine36533585Stem cells translational medicineinfo:eu-repo/semantics/openAccessAtribución-NoComercial-SinDerivadas 4.0 Internacional (CC BY-NC-ND 4.0)https://creativecommons.org/licenses/by-nc-nd/4.0/http://purl.org/coar/access_right/c_abf2AdiposeAdult stem cellsXeno-free productionHuman platelet lysateExplant cultureCryopreservationCriopreservaciónTejidoAntígenos de plaqueta humanaAdipocitosCélulas madre adultasAnticuerpos heterófilosPublicationORIGINALXeno-Free Extraction, Culture, and Cryopreservation of Human Adipose-Derived Mesenchymal Stem Cells.pdfXeno-Free Extraction, Culture, and Cryopreservation of Human Adipose-Derived Mesenchymal Stem Cells.pdfArtículoapplication/pdf1918323https://repositorio.fucsalud.edu.co/bitstreams/54d22c6b-4acb-42e8-b7f6-e93bdb71eb90/download76cff6c034307b64b42afb7180c54f8cMD51LICENSElicense.txtlicense.txttext/plain; charset=utf-815424https://repositorio.fucsalud.edu.co/bitstreams/c5d51018-ff90-4cbe-b4a8-3939ddec9568/downloadf6cfdd0a0be8a7a8b575d6cd88a94402MD52TEXTXeno-Free Extraction, Culture, and Cryopreservation of Human Adipose-Derived Mesenchymal Stem Cells.pdf.txtXeno-Free Extraction, Culture, and Cryopreservation of Human Adipose-Derived Mesenchymal Stem Cells.pdf.txtExtracted texttext/plain1https://repositorio.fucsalud.edu.co/bitstreams/58955985-6661-4ce9-a776-6980c86a6ff9/download68b329da9893e34099c7d8ad5cb9c940MD53THUMBNAILXeno-Free Extraction, Culture, and Cryopreservation of Human Adipose-Derived Mesenchymal Stem Cells.pdf.jpgXeno-Free Extraction, Culture, and Cryopreservation of Human Adipose-Derived Mesenchymal Stem Cells.pdf.jpgGenerated Thumbnailimage/jpeg17248https://repositorio.fucsalud.edu.co/bitstreams/7b07430d-9aaa-41ce-94e1-6e694a206789/download8b43583e0f50cfb216a7b081bde61266MD54001/2699oai:repositorio.fucsalud.edu.co:001/26992024-02-02 13:08:01.113https://creativecommons.org/licenses/by-nc-nd/4.0/open.accesshttps://repositorio.fucsalud.edu.coRepositorio Digital de la Fundación Universitaria de Ciencias de la 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