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Automated—Mechanical Procedure Compared to Gentle Enzymatic Tissue Dissociation in Cell Function Studies
The first step to obtain a cellular suspension from tissues is the disaggregation procedure. The cell suspension method has to provide a representative sample of the different cellular subpopulations and to maximize the number of viable functional cells. Here, we analyzed specific cell functions in...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9138555/ https://www.ncbi.nlm.nih.gov/pubmed/35625628 http://dx.doi.org/10.3390/biom12050701 |
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author | Montanari, Mariele Burattini, Sabrina Ciacci, Caterina Ambrogini, Patrizia Carloni, Silvia Balduini, Walter Lopez, Daniele Panza, Giovanna Papa, Stefano Canonico, Barbara |
author_facet | Montanari, Mariele Burattini, Sabrina Ciacci, Caterina Ambrogini, Patrizia Carloni, Silvia Balduini, Walter Lopez, Daniele Panza, Giovanna Papa, Stefano Canonico, Barbara |
author_sort | Montanari, Mariele |
collection | PubMed |
description | The first step to obtain a cellular suspension from tissues is the disaggregation procedure. The cell suspension method has to provide a representative sample of the different cellular subpopulations and to maximize the number of viable functional cells. Here, we analyzed specific cell functions in cell suspensions from several rat tissues obtained by two different methods, automated–mechanical and enzymatic disaggregation. Flow cytometric, confocal, and ultrastructural (TEM) analyses were applied to the spleen, testis, liver and other tissues. Samples were treated by an enzymatic trypsin solution or processed by the Medimachine II (MMII). The automated–mechanical and enzymatic disaggregation procedures have shown to work similarly in some tissues, which displayed comparable amounts of apoptotic/necrotic cells. However, cells obtained by the enzyme-free Medimachine II protocols show a better preservation lysosome and mitochondria labeling, whereas the enzymatic gentle dissociation appears to constantly induce a lower amount of intracellular ROS; nevertheless, lightly increased ROS can be recognized as a complimentary signal to promote cell survival. Therefore, MMII represents a simple, fast, and standardized method for tissue processing, which allows to minimize bias arising from the operator’s ability. Our study points out technical issues to be adopted for specific organs and tissues to obtain functional cells. |
format | Online Article Text |
id | pubmed-9138555 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-91385552022-05-28 Automated—Mechanical Procedure Compared to Gentle Enzymatic Tissue Dissociation in Cell Function Studies Montanari, Mariele Burattini, Sabrina Ciacci, Caterina Ambrogini, Patrizia Carloni, Silvia Balduini, Walter Lopez, Daniele Panza, Giovanna Papa, Stefano Canonico, Barbara Biomolecules Article The first step to obtain a cellular suspension from tissues is the disaggregation procedure. The cell suspension method has to provide a representative sample of the different cellular subpopulations and to maximize the number of viable functional cells. Here, we analyzed specific cell functions in cell suspensions from several rat tissues obtained by two different methods, automated–mechanical and enzymatic disaggregation. Flow cytometric, confocal, and ultrastructural (TEM) analyses were applied to the spleen, testis, liver and other tissues. Samples were treated by an enzymatic trypsin solution or processed by the Medimachine II (MMII). The automated–mechanical and enzymatic disaggregation procedures have shown to work similarly in some tissues, which displayed comparable amounts of apoptotic/necrotic cells. However, cells obtained by the enzyme-free Medimachine II protocols show a better preservation lysosome and mitochondria labeling, whereas the enzymatic gentle dissociation appears to constantly induce a lower amount of intracellular ROS; nevertheless, lightly increased ROS can be recognized as a complimentary signal to promote cell survival. Therefore, MMII represents a simple, fast, and standardized method for tissue processing, which allows to minimize bias arising from the operator’s ability. Our study points out technical issues to be adopted for specific organs and tissues to obtain functional cells. MDPI 2022-05-14 /pmc/articles/PMC9138555/ /pubmed/35625628 http://dx.doi.org/10.3390/biom12050701 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Montanari, Mariele Burattini, Sabrina Ciacci, Caterina Ambrogini, Patrizia Carloni, Silvia Balduini, Walter Lopez, Daniele Panza, Giovanna Papa, Stefano Canonico, Barbara Automated—Mechanical Procedure Compared to Gentle Enzymatic Tissue Dissociation in Cell Function Studies |
title | Automated—Mechanical Procedure Compared to Gentle Enzymatic Tissue Dissociation in Cell Function Studies |
title_full | Automated—Mechanical Procedure Compared to Gentle Enzymatic Tissue Dissociation in Cell Function Studies |
title_fullStr | Automated—Mechanical Procedure Compared to Gentle Enzymatic Tissue Dissociation in Cell Function Studies |
title_full_unstemmed | Automated—Mechanical Procedure Compared to Gentle Enzymatic Tissue Dissociation in Cell Function Studies |
title_short | Automated—Mechanical Procedure Compared to Gentle Enzymatic Tissue Dissociation in Cell Function Studies |
title_sort | automated—mechanical procedure compared to gentle enzymatic tissue dissociation in cell function studies |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9138555/ https://www.ncbi.nlm.nih.gov/pubmed/35625628 http://dx.doi.org/10.3390/biom12050701 |
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