Spheroid-Based Approach to Assess the Tissue Relevance of Analysis of Dispersed-Settled Tissue Cells by Cytometry of the Reaction Rate Constant.
Anal Chem
; 92(13): 9348-9355, 2020 07 07.
Article
en En
| MEDLINE
| ID: mdl-32522000
ABSTRACT
Cytometry of Reaction Rate Constant (CRRC) uses time-lapse fluorescence microscopy to measure a rate constant of a catalytic reaction in individual cells and, thus, facilitate accurate size determination for cell subpopulations with distinct efficiencies of this reaction. Reliable CRRC requires uniform exposure of cells to the reaction substrate followed by their uniform imaging, which in turn, requires that a tissue sample be disintegrated into a suspension of dispersed cells, and these cells settle on the support surface before being analyzed by CRRC. We call such cells "dispersed-settled" to distinguish them from cells cultured as a monolayer. Studies of the dispersed-settled cells can be tissue-relevant only if the cells maintain their 3D tissue state during the multi-hour CRRC procedure. Here, we propose an approach for assessing tissue relevance of the CRRC-based analysis of the dispersed-settled cells. Our approach utilizes cultured multicellular spheroids as a 3D cell model and cultured cell monolayers as a 2D cell model. The CRRC results of the dispersed-settled cells derived from spheroids are compared to those of spheroids and monolayers in order to find if the dispersed-settled cells are representative of the spheroids. To demonstrate its practical use, we applied this approach to a cellular reaction of multidrug resistance (MDR) transport, which was followed by extrusion of a fluorescent substrate from the cells. The approach proved to be reliable and revealed long-term maintenance of MDR transport in the dispersed-settled cells obtained from cultured ovarian cancer spheroids. Accordingly, CRRC can be used to determine accurately the size of a cell subpopulation with an elevated level of MDR transport in tumor samples, which makes CRRC a suitable method for the development of MDR-based predictors of chemoresistance. The proposed spheroid-based approach for validation of CRRC is applicable to other types of cellular reactions and, thus, will be an indispensable tool for transforming CRRC from an experimental technique into a practical analytical tool.
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Esferoides Celulares
/
Microscopía Fluorescente
Tipo de estudio:
Prognostic_studies
Límite:
Humans
Idioma:
En
Revista:
Anal Chem
Año:
2020
Tipo del documento:
Article
País de afiliación:
Canadá