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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...

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Autores principales: Montanari, Mariele, Burattini, Sabrina, Ciacci, Caterina, Ambrogini, Patrizia, Carloni, Silvia, Balduini, Walter, Lopez, Daniele, Panza, Giovanna, Papa, Stefano, Canonico, Barbara
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
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.
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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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