Impact of buffer composition on biochemical, morphological and mechanical parameters: A tare before dielectrophoretic cell separation and isolation

Paolo G Bonacci; Giuseppe Caruso; Grazia Scandura; Clarissa Pandino; Alessandra Romano; Giorgio I Russo; Ronald Pethig; Massimo Camarda; Nicolò Musso

doi:10.1016/j.tranon.2022.101599

Impact of buffer composition on biochemical, morphological and mechanical parameters: A tare before dielectrophoretic cell separation and isolation

Transl Oncol. 2023 Feb:28:101599. doi: 10.1016/j.tranon.2022.101599. Epub 2022 Dec 12.

Authors

Paolo G Bonacci¹, Giuseppe Caruso², Grazia Scandura³, Clarissa Pandino³, Alessandra Romano³, Giorgio I Russo⁴, Ronald Pethig⁵, Massimo Camarda⁶, Nicolò Musso¹

Affiliations

¹ Department of Biomedical and Biotechnological Sciences, University of Catania, Catania 95123, Italy.
² Department of Drug and Health Sciences, University of Catania, Catania 95125, Italy; Oasi Research Institute-IRCCS, Troina 94018, Italy. Electronic address: giuseppe.caruso2@unict.it.
³ Haematological section, University of Catania, Catania 95125, Italy.
⁴ Urology section, University of Catania, Catania 95125, Italy.
⁵ Institute for Integrated Micro and Nano Systems, Joint Research Institute for Integrated Systems, School of Engineering, The University of Edinburgh, Edinburgh EH9 3JF, UK.
⁶ STLab srl, Catania 95126, Italy.

Abstract

Dielectrophoresis (DEP) represents an electrokinetic approach for discriminating and separating suspended cells based on their intrinsic dielectric characteristics without the need for labeling procedure. A good practice, beyond the physical and engineering components, is the selection of a buffer that does not hinder cellular and biochemical parameters as well as cell recovery. In the present work the impact of four buffers on biochemical, morphological, and mechanical parameters was evaluated in two different cancer cell lines (Caco-2 and K562). Specifically, MTT ([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]) assay along with flow cytometry analysis were used to evaluate the occurring changes in terms of cell viability, morphology, and granulocyte stress formation, all factors directly influencing DEP sorting capability. Quantitative real-time PCR (qRT-PCR) was instead employed to evaluate the gene expression levels of interleukin-6 (IL-6) and inducible nitric oxide synthase (iNOS), two well-known markers of inflammation and oxidative stress, respectively. An additional marker representing an index of cellular metabolic status, i.e. the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, was also evaluated. Among the four buffers considered, two resulted satisfactory in terms of cell viability and growth recovery (24 h), with no significant changes in cell morphology for up to 1 h in suspension. Of note, gene expression analysis showed that in both cell lines the apparently non-cytotoxic buffers significantly modulated IL-6, iNOS, and GAPDH markers, underlining the importance to deeply investigate the molecular and biochemical changes occurring during the analysis, even at apparently non-toxic conditions. The selection of a useful buffer for the separation and analysis of cells without labeling procedures, preserving cell status, represents a key factor for DEP analysis, giving the opportunity to further use cells for additional analysis.

Keywords: Biochemical parameters; Cell isolation and separation; Cell status; Circulating tumor cells; Dielectrophoresis; Microfluidics.