Automated—Mechanical Procedure Compared to Gentle Enzymatic Tissue Dissociation in Cell Function Studies

Abstract

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.

Flow cytometric analysis of hepatopancreatic cells from Armadillidium vulgare highlights terrestrial Isopods as efficient environmental bioindicators in ex vivo settings

“…In the starting phase of the study, to collect hepatopancreatic cells, we applied two different mechanical disaggregations: manual and automated (Medimachine II) tissue disaggregation (Montanari et al, 2022).…”

“…Several endogenous uorophores are known to cause auto uorescence in many tissues, for these reasons we evaluated auto uorescence in each instrument channel, with particular attention to FL1 (520 nm), corresponding to the wavelength of emission of several probes employed in the study (CFDA, SYBRGreen, Leadmium, Fly). Indeed for hepatopancreatic cells, overall auto uorescence emission contains considerable information because it is the sum of auto uorescence contributions from uorochromes involved in metabolism, (i.e., NAD(P)H, avins, lipofuscins, retinoids, porphyrins, bilirubin, and lipids) (Montanari et al, 2022). Accordingly, samples from C sites show higher auto-uorescence values compared to MP and SP samples (Fig.…”

“…In this study we investigated cell functions and viability of the hepatopancreatic cells (S and B cells) of Isopods from sites of different degree of ecological disturbance, in order to detect possible differences in stress parameters and to verify if these parameters correspond to the environmental stresses. We successfully applied mechanical-automated procedures previously optimised on murine and rat tissues (Montanari et al, 2022).…”

Nuclei on the Rise: When Nuclei-Based Methods Meet Next-Generation Sequencing

“…Enzymatic digestion is one of the most commonly used methods for cellular dissociation. Enzymes such as trypsin, collagenase, papain and dispase can be used to break down cell–cell and cell–matrix interactions, releasing single cells [ 11 , 12 , 13 , 14 ] ( Figure 1 A). The strength of the enzyme needed for dissociation can vary depending on the type of tissue being dissociated.…”

Nuclei on the Rise: When Nuclei-Based Methods Meet Next-Generation Sequencing

“…Enzymatic digestion is one of the most commonly used methods for cellular dissociation. Enzymes such as trypsin, collagenase, papain and dispase can be used to break down cellcell and cell-matrix interactions, releasing single cells [11][12][13][14] (Figure 1A). The strength of the enzyme needed for dissociation can vary depending on the type of tissue being dissociated.…”