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A Reliable Protocol for the Isolation of Viable, Chondrogenically Differentiated Human Mesenchymal Stem Cells from High-Density Pellet Cultures

Administration of chondrogenically differentiated mesenchymal stem cells (MSC) is discussed as a promising approach for the regenerative treatment of injured or diseased cartilage. The high-density pellet culture is the standard culture for chondrogenic differentiation, but cells in pellets secrete...

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Autores principales: Ullah, Mujib, Hamouda, Houda, Stich, Stefan, Sittinger, Michael, Ringe, Jochen
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Mary Ann Liebert, Inc. 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3559221/
https://www.ncbi.nlm.nih.gov/pubmed/23514965
http://dx.doi.org/10.1089/biores.2012.0279
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author Ullah, Mujib
Hamouda, Houda
Stich, Stefan
Sittinger, Michael
Ringe, Jochen
author_facet Ullah, Mujib
Hamouda, Houda
Stich, Stefan
Sittinger, Michael
Ringe, Jochen
author_sort Ullah, Mujib
collection PubMed
description Administration of chondrogenically differentiated mesenchymal stem cells (MSC) is discussed as a promising approach for the regenerative treatment of injured or diseased cartilage. The high-density pellet culture is the standard culture for chondrogenic differentiation, but cells in pellets secrete extracellular matrix (ECM) that they become entrapped in. Protocols for cell isolation from pellets often result in cell damage and dedifferentiation towards less differentiated MSC. Therefore, our aim was to develop a reliable protocol for the isolation of viable, chondrogenically differentiated MSC from high-density pellet cultures. Human bone marrow MSC were chondrogenically stimulated with transforming growth factor-β3, and the cartilaginous structure of the pellets was verified by alcian blue staining of cartilage proteoglycans, antibody staining of cartilage collagen type II, and quantitative real-time reverse-transcription polymerase chain reaction of the marker genes COL2A1 and SOX9. Trypsin and collagenases II and P were tested alone or in combination, and for different concentrations and times, to find a protocol for optimized pellet digestion. Whereas trypsin was not able to release viable cells, 90-min digestion with 300 U of collagenase II, 20 U of collagenase P, and 2 mM CaCl(2) worked quite well and resulted in about 2.5×10(5) cells/pellet. The protocol was further optimized for the separation of released cells and ECM from each other. Cells were alcian blue and collagen type II positive and expressed COL2A1 and SOX9, verifying a chondrogenic character. However, they had different morphological shapes. The ECM was also uniformly alcian blue and collagen type II positive but showed different organizational and structural forms. To conclude, our protocol allows the reliable isolation of a defined number of viable, chondrogenically differentiated MSC from high-density pellet cultures. Such cells, as well as the ECM components, are of interest as research tools and for cartilage tissue engineering.
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spelling pubmed-35592212013-03-20 A Reliable Protocol for the Isolation of Viable, Chondrogenically Differentiated Human Mesenchymal Stem Cells from High-Density Pellet Cultures Ullah, Mujib Hamouda, Houda Stich, Stefan Sittinger, Michael Ringe, Jochen Biores Open Access Original Research Articles Administration of chondrogenically differentiated mesenchymal stem cells (MSC) is discussed as a promising approach for the regenerative treatment of injured or diseased cartilage. The high-density pellet culture is the standard culture for chondrogenic differentiation, but cells in pellets secrete extracellular matrix (ECM) that they become entrapped in. Protocols for cell isolation from pellets often result in cell damage and dedifferentiation towards less differentiated MSC. Therefore, our aim was to develop a reliable protocol for the isolation of viable, chondrogenically differentiated MSC from high-density pellet cultures. Human bone marrow MSC were chondrogenically stimulated with transforming growth factor-β3, and the cartilaginous structure of the pellets was verified by alcian blue staining of cartilage proteoglycans, antibody staining of cartilage collagen type II, and quantitative real-time reverse-transcription polymerase chain reaction of the marker genes COL2A1 and SOX9. Trypsin and collagenases II and P were tested alone or in combination, and for different concentrations and times, to find a protocol for optimized pellet digestion. Whereas trypsin was not able to release viable cells, 90-min digestion with 300 U of collagenase II, 20 U of collagenase P, and 2 mM CaCl(2) worked quite well and resulted in about 2.5×10(5) cells/pellet. The protocol was further optimized for the separation of released cells and ECM from each other. Cells were alcian blue and collagen type II positive and expressed COL2A1 and SOX9, verifying a chondrogenic character. However, they had different morphological shapes. The ECM was also uniformly alcian blue and collagen type II positive but showed different organizational and structural forms. To conclude, our protocol allows the reliable isolation of a defined number of viable, chondrogenically differentiated MSC from high-density pellet cultures. Such cells, as well as the ECM components, are of interest as research tools and for cartilage tissue engineering. Mary Ann Liebert, Inc. 2012-12 /pmc/articles/PMC3559221/ /pubmed/23514965 http://dx.doi.org/10.1089/biores.2012.0279 Text en Copyright 2012, Mary Ann Liebert, Inc.
spellingShingle Original Research Articles
Ullah, Mujib
Hamouda, Houda
Stich, Stefan
Sittinger, Michael
Ringe, Jochen
A Reliable Protocol for the Isolation of Viable, Chondrogenically Differentiated Human Mesenchymal Stem Cells from High-Density Pellet Cultures
title A Reliable Protocol for the Isolation of Viable, Chondrogenically Differentiated Human Mesenchymal Stem Cells from High-Density Pellet Cultures
title_full A Reliable Protocol for the Isolation of Viable, Chondrogenically Differentiated Human Mesenchymal Stem Cells from High-Density Pellet Cultures
title_fullStr A Reliable Protocol for the Isolation of Viable, Chondrogenically Differentiated Human Mesenchymal Stem Cells from High-Density Pellet Cultures
title_full_unstemmed A Reliable Protocol for the Isolation of Viable, Chondrogenically Differentiated Human Mesenchymal Stem Cells from High-Density Pellet Cultures
title_short A Reliable Protocol for the Isolation of Viable, Chondrogenically Differentiated Human Mesenchymal Stem Cells from High-Density Pellet Cultures
title_sort reliable protocol for the isolation of viable, chondrogenically differentiated human mesenchymal stem cells from high-density pellet cultures
topic Original Research Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3559221/
https://www.ncbi.nlm.nih.gov/pubmed/23514965
http://dx.doi.org/10.1089/biores.2012.0279
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