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Evaluation of different protocols for culturing mesenchymal stem cells derived from murine bone marrow

INTRODUCTION: Culturing bone marrow mesenchymal stem cells (BM-MSCs) is a key point in different fields of research, including tissue engineering and regenerative medicine and studies of the bone marrow microenvironment. However, isolating and expanding murine BM-MSCs in vitro has challenged researc...

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Autores principales: Pissarra, Mariana Ferreira, Torello, Cristiane Okuda, Saad, Sara Teresinha Olalla, Lazarini, Mariana
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Sociedade Brasileira de Hematologia e Hemoterapia 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9605884/
https://www.ncbi.nlm.nih.gov/pubmed/34034994
http://dx.doi.org/10.1016/j.htct.2021.02.005
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author Pissarra, Mariana Ferreira
Torello, Cristiane Okuda
Saad, Sara Teresinha Olalla
Lazarini, Mariana
author_facet Pissarra, Mariana Ferreira
Torello, Cristiane Okuda
Saad, Sara Teresinha Olalla
Lazarini, Mariana
author_sort Pissarra, Mariana Ferreira
collection PubMed
description INTRODUCTION: Culturing bone marrow mesenchymal stem cells (BM-MSCs) is a key point in different fields of research, including tissue engineering and regenerative medicine and studies of the bone marrow microenvironment. However, isolating and expanding murine BM-MSCs in vitro has challenged researchers due to the low purity and yield of obtained cells. In this study, we aimed to evaluate five different protocols to culture murine BM-MSCs in vitro. METHODS: All protocols were based on the adhesion capacity of BM-MSCs to the tissue culture plastic surface and varied in the types of plate, culture media, serum, additional supplementation and initial cell density. Flow cytometry analysis was used to investigate lineage purity after expansion. RESULTS: The expression of CD45 and CD11b was detected in the cultures generated according to all protocols, indicating low purity with the presence of hematopoietic cells and macrophages. The cellular growth rate and morphology varied between the cultures performed according to each protocol. Cells cultured according to protocol 5 (8 × 10(7)cells/plate, Roswell Park Memorial Institute (RPMI) culture medium during first passage and then Iscove's Modified Delbecco's Medium (IMDM) culture medium, both supplemented with 9% fetal bovine serum, 9% horse serum, 12µM L-glutamine) presented the best performance, with a satisfactory growth rate and spindle-shape morphology. CONCLUSION: Our results point out that the purity and satisfactory growth rate of murine BM-MSC cultures are not easily achieved and additional approaches must be tested for a proper cell expansion.
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spelling pubmed-96058842022-10-28 Evaluation of different protocols for culturing mesenchymal stem cells derived from murine bone marrow Pissarra, Mariana Ferreira Torello, Cristiane Okuda Saad, Sara Teresinha Olalla Lazarini, Mariana Hematol Transfus Cell Ther Original Article INTRODUCTION: Culturing bone marrow mesenchymal stem cells (BM-MSCs) is a key point in different fields of research, including tissue engineering and regenerative medicine and studies of the bone marrow microenvironment. However, isolating and expanding murine BM-MSCs in vitro has challenged researchers due to the low purity and yield of obtained cells. In this study, we aimed to evaluate five different protocols to culture murine BM-MSCs in vitro. METHODS: All protocols were based on the adhesion capacity of BM-MSCs to the tissue culture plastic surface and varied in the types of plate, culture media, serum, additional supplementation and initial cell density. Flow cytometry analysis was used to investigate lineage purity after expansion. RESULTS: The expression of CD45 and CD11b was detected in the cultures generated according to all protocols, indicating low purity with the presence of hematopoietic cells and macrophages. The cellular growth rate and morphology varied between the cultures performed according to each protocol. Cells cultured according to protocol 5 (8 × 10(7)cells/plate, Roswell Park Memorial Institute (RPMI) culture medium during first passage and then Iscove's Modified Delbecco's Medium (IMDM) culture medium, both supplemented with 9% fetal bovine serum, 9% horse serum, 12µM L-glutamine) presented the best performance, with a satisfactory growth rate and spindle-shape morphology. CONCLUSION: Our results point out that the purity and satisfactory growth rate of murine BM-MSC cultures are not easily achieved and additional approaches must be tested for a proper cell expansion. Sociedade Brasileira de Hematologia e Hemoterapia 2022 2021-05-08 /pmc/articles/PMC9605884/ /pubmed/34034994 http://dx.doi.org/10.1016/j.htct.2021.02.005 Text en © 2021 Associação Brasileira de Hematologia, Hemoterapia e Terapia Celular. Published by Elsevier España, S.L.U. https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Original Article
Pissarra, Mariana Ferreira
Torello, Cristiane Okuda
Saad, Sara Teresinha Olalla
Lazarini, Mariana
Evaluation of different protocols for culturing mesenchymal stem cells derived from murine bone marrow
title Evaluation of different protocols for culturing mesenchymal stem cells derived from murine bone marrow
title_full Evaluation of different protocols for culturing mesenchymal stem cells derived from murine bone marrow
title_fullStr Evaluation of different protocols for culturing mesenchymal stem cells derived from murine bone marrow
title_full_unstemmed Evaluation of different protocols for culturing mesenchymal stem cells derived from murine bone marrow
title_short Evaluation of different protocols for culturing mesenchymal stem cells derived from murine bone marrow
title_sort evaluation of different protocols for culturing mesenchymal stem cells derived from murine bone marrow
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9605884/
https://www.ncbi.nlm.nih.gov/pubmed/34034994
http://dx.doi.org/10.1016/j.htct.2021.02.005
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