Efficacy of Local Anesthetics in Detachment of Normal 3T3 Mouse Fibroblasts and Prostate Cancer AT-2 Cells from Substrata, in Maintenance of Viable Cells in a Non-Adherent State, and in Preservation of Cell Surface Markers Detected with FlowSight Image Cytometry

Abstract

Cells from substrata, in maintenance of viable cells in a non-adherent state, and in preservation of cell surface markers detected with FlowSight image cytometry. Folia Biologica (Kraków) 63: 249-255. The local anesthetics procaine, lidocaine and tetracaine permit the reversible detachment of viable cells and their passaging or preservation in a non-adherent state in the absence of proteolytic enzymes. The effects of these anesthetics, dissolved in various media, on cell viability, cell detachment from substrata and preservation of cells in a non-adherent state, were compared using the AT-2 line of rat prostate carcinoma cells of moderate malignancy and the 3T3 mouse fibroblast cell line. It was found that all three local anesthetics can induce cell rounding followed by detachment of over 95% of viable cells in both lines in Ca 2+ /Mg 2+-free PBS. Tetracaine in 1 mM concentration was the most effective in induction of fast cell detachment. However, procaine and lidocaine in 16 mM concentrations were found to be optimal for preservation of cells in a non-adherent state and for the maintenance of cell viability for at least 2 h. The tested anesthetics also cause cell rounding and detachment when present in various cell culture media but these processes occurred much more slowly and less efficiently than in Ca 2+ /Mg 2+-free PBS. Normal 3T3 mouse fibroblasts after detachment and passaging undertake growth reaching the same saturation density in cultures after detachment with procaine or lidocaine as after passaging using trypsin solution. The results suggest that the application of local anesthetics can be a very simple and effective technique for cell passaging in tissue cultures. This technique might decrease side-effects and cell injury caused by trypsinization or cell scraping. The preservation of cells in suspension in a non-adherent state may facilitate analysis of cell surface properties and fractionation of cell mixtures. Avoiding the use of trypsin allows for the preservation of cell surface proteins ICAM, CXCR4, and HCAM analyzed with FlowSight® image flow cytometry.

Explant-Like Passaging of Cells Growing on Portable Substrata Permits the Avoidance of Enzyme Application and Facilitates the Passage Procedure

“…Routine cell passaging procedures based on the use of proteolytic enzymes require the centrifugation of cell suspensions. In addition, the detachment of cells from substrats is associated with cell rounding and the disassembly of stress fibers followed by great changes in cell cytoskeleton architecture due to the release of cell isotonic contractions (KOROHODA & WOHLFARTH-BOTTERMANN 1976;ANGHILERI & DERMIETZEL 1976;FURCHT & WENDELSCHAFER-CRABB 1978;KOROHODA & KAJSTURA 1982;KOROHODA et al 2015). It has been shown that, proteolytic enzymes should not be used in analytical or immunological studies requiring the intact composition of the cell surface.…”

“…Trypsin, the most commonly used enzyme, modifies the epitopes of cell surface markers causing changes in a cell membrane's physical properties and shows mitogenic activity (BURGER 1970(BURGER , 1971SEFTON & RUBIN 1970RUSSELL & PASTAN 1973;MOLINARI & PLATT 1975;ANGHILERI & DERMIETZEL 1976;FURCHT & WENDELSCHAFER-CRABB 1978;GRAY et al, 2001;HUANG et al 2010). Due to the negative side-effects caused by cell trypsinization and the scraping of cells with a silicone rubber, attempts have been taken to develop alternate methods of viable cell detachment (RABINOVITCH & DESTEFANO 1975;KOROHODA et al 2015).…”

Imipramine Treatment Alters Sphingomyelin, Cholesterol, and Glycerophospholipid Metabolism in Isolated Macrophage Lysosomes

“…To aid in the detachment from the flask bottom, the mexAMs were incubated with a local anesthetic lidocaine (20 mM, cat # J63035.14, Thermo Fisher Scientific) for 4 min at 37 • C and then gently scraped from the flask bottom. The usage of lidocaine for the detachment of mexAMs and other cell types has been used previously by our laboratory and others and has been shown to preserve the cell surface proteins that are otherwise lost through trypsinization or scraping alone [21][22][23]. For the drug treatments, the mexAMs were incubated at a cell density of 10 6 cells/mL in a complete media without growth factors, with either imipramine (25 µM, cat # 113-52-0, Cayman Chemical, Ann Arbor, MI, USA) or U18666A (1 µg/mL, cat # 3039-71-2, Cayman Chemical) for 24 h prior to the lysosome isolations.…”

Bupivacaine inhibits the malignant biological behavior of oral squamous cell carcinoma cells by inhibiting the activation of ERK1/2 and STAT3

“…Local anesthesia is part of the patient's preoperative analgesic regimen. It has been reported that using local anesthetics clinically could decrease recurrence of breast cancer (7)(8)(9), thyroid cancer (10), prostate cancer (11), and OSCC (12). Bupivacaine (Bupi) is a widely used long-acting local anesthetic.…”