Ute Grässer1, Monika Bubel2, Daniela Sossong3, Martin Oberringer4, Tim Pohlemann5, Wolfgang Metzger6. 1. Department of Trauma, Hand and Reconstructive Surgery, Saarland University, Building 57, 66421 Homburg, Germany. Electronic address: u.graesser@web.de. 2. Department of Trauma, Hand and Reconstructive Surgery, Saarland University, Building 57, 66421 Homburg, Germany. Electronic address: monika.bubel@uks.eu. 3. Department of Trauma, Hand and Reconstructive Surgery, Saarland University, Building 57, 66421 Homburg, Germany. Electronic address: daniela.sossong@uks.eu. 4. Department of Trauma, Hand and Reconstructive Surgery, Saarland University, Building 57, 66421 Homburg, Germany. Electronic address: martin.oberringer@uks.eu. 5. Department of Trauma, Hand and Reconstructive Surgery, Saarland University, Building 57, 66421 Homburg, Germany. Electronic address: tim.pohlemann@uks.eu. 6. Department of Trauma, Hand and Reconstructive Surgery, Saarland University, Building 57, 66421 Homburg, Germany. Electronic address: johann-wolfgang.metzger@uks.eu.
Abstract
BACKGROUND: Spheroids are considered to reflect the natural organization of cells better than 2D cell cultures, but their analysis by flow cytometry requires dissociation into single cells. METHODS: We established protocols for dissociation of mono- and co-culture spheroids consisting of human fibroblasts and human endothelial cells. Cell recovery rate and viability after dissociation were evaluated with hemocytometer and by flow cytometry. The diameter of cells and the amount of cell aggregates were quantified by Casy®-technology and the cellular composition was analyzed by flow cytometry. RESULTS: Optimal dissociation conditions with low cell aggregation were determined by size, cultivation time and cellular composition of the spheroids. Smaller spheroids (10,000 cells) could be dissociated with Accutase®, whereas larger spheroids (50,000 cells) required more stringent dissociation conditions. The size of the cells decreased with increasing cultivation time. Cell recovery rate was dependent upon cellular composition and spheroid size. The highest cell recovery rate was found for co-culture spheroids. The highest cell viability was detected for dissociated fibroblast spheroids. A quantitative analysis of the cellular composition of dissociated co-culture spheroids was possible. DISCUSSION: Spheroids can be successfully dissociated into singular cells for subsequent flow cytometric analysis. Dissociation conditions as well as cell recovery rate and cell viability depend on size, cultivation time and cellular composition of the spheroids. The observed decrease in cell size in spheroids over time might be responsible for the well-known time-dependent decrease in spheroid size.
BACKGROUND: Spheroids are considered to reflect the natural organization of cells better than 2D cell cultures, but their analysis by flow cytometry requires dissociation into single cells. METHODS: We established protocols for dissociation of mono- and co-culture spheroids consisting of human fibroblasts and human endothelial cells. Cell recovery rate and viability after dissociation were evaluated with hemocytometer and by flow cytometry. The diameter of cells and the amount of cell aggregates were quantified by Casy®-technology and the cellular composition was analyzed by flow cytometry. RESULTS: Optimal dissociation conditions with low cell aggregation were determined by size, cultivation time and cellular composition of the spheroids. Smaller spheroids (10,000 cells) could be dissociated with Accutase®, whereas larger spheroids (50,000 cells) required more stringent dissociation conditions. The size of the cells decreased with increasing cultivation time. Cell recovery rate was dependent upon cellular composition and spheroid size. The highest cell recovery rate was found for co-culture spheroids. The highest cell viability was detected for dissociated fibroblast spheroids. A quantitative analysis of the cellular composition of dissociated co-culture spheroids was possible. DISCUSSION: Spheroids can be successfully dissociated into singular cells for subsequent flow cytometric analysis. Dissociation conditions as well as cell recovery rate and cell viability depend on size, cultivation time and cellular composition of the spheroids. The observed decrease in cell size in spheroids over time might be responsible for the well-known time-dependent decrease in spheroid size.
Authors: Sara Darakhshan; Ali Bidmeshki Pour; Reza Kowsari-Esfahan; Massoud Vosough; Leila Montazeri; Mohammad Hossein Ghanian; Hossein Baharvand; Abbas Piryaei Journal: Tissue Eng Regen Med Date: 2020-07-14 Impact factor: 4.169
Authors: Carolina Orlando Vaso; Níura Madalena Bila; Fabiana Pandolfi; Daniela De Vita; Martina Bortolami; Jean Lucas Carvalho Bonatti; Rosângela Aparecida De Moraes Silva; Larissa Naiara Carvalho Gonçalves; Valeria Tudino; Roberta Costi; Roberto Di Santo; Maria José Soares Mendes-Giannini; Caroline Barcelos Costa-Orlandi; Luigi Scipione; Ana Marisa Fusco-Almeida Journal: Pharmaceutics Date: 2022-05-12 Impact factor: 6.525