Closed Chamber System for Delivery of Ethanol to Cell Cultures

Abstract

The accuracy and consistency of the delivery of ethanol to cultured cells is important to determine effects on morphologic, biochemical and physiologic alterations. Open and closed chamber systems were evaluated to determine cytotoxic vs sublethal, potentially teratogenic effects on neonatal rat cardiac myocytes. The open system employed a variety of cell culture vessels. Cardiac cells were exposed directly to ethanol in the growth media at concentrations of 5-50 mM in Petri dishes, multiwell slides and multiwell chambers. Ethanol concentrations in the media in these open vessels decreased over 60% in a 24 hr incubation period. A closed system consisted of tightly sealed plastic containers in which the same vessels were used. The vessels were placed on a platform over a bath of ethanol-water. Cells were acclimated for 24 hr with ethanol in the bath at 200% of the final desired media concentration. Ethanol gradually diffused into the media to reach peak levels of 5, 10, 25 or 50 mM at 24 hr. After the 24 hr period, ethanol was added to both the media and bath at the desired concentration. Cells exposed gradually to ethanol in the closed chambers remained viable, but showed slower division and growth. A period of gradual acclimation is required to induce sublethal cellular effects rather than lethal effects. The diversity of cell systems and manipulations of cultures to study the potential teratogenic effects of ethanol are improved using such a closed chamber system.

Ethanol Affects Transforming Growth Factor β1-Initiated Signals: Cross-Talking Pathways in the Developing Rat Cerebral Wall

“…All cultures were maintained in sealed containers to stabilize the ethanol concentration in the medium (Adickes et al, 1988). Culture dishes were suspended over a bath containing ethanol in the same concentrations as in the medium, i.e., 0, 200, 400, or 800 mg/dl.…”

Ethanol Suppresses PGC-1α Expression by Interfering with the cAMP-CREB Pathway in Neuronal Cells

“…We used two plasmids for the promoter activity assay; plasmid PGC-1-alpha-promoter-2 kb-luciferase [26], where the wild type PGC-1α promoter sequences (∼2 kb) was cloned upstream of reporter luciferase; and plasmid PGC-1-alpha-promoter-luciferase-delta CRE, wherein the CREB-binding CRE sequences were deleted [32]. The delta-CRE-PGC-1α promoter plasmid and PGC-1α-promoter-2 kb-luciferase control plasmid, was transfected into alcohol-treated and untreated SH-SY5Y cells and luciferase activity was measured.…”

“…To maintain and stabilize the ethanol concentration in the medium, all cultures were maintained in sealed containers [26].…”

Ethanol inhibition of aspartyl-asparaginyl-β-hydroxylase in fetal alcohol spectrum disorder: Potential link to the impairments in central nervous system neuronal migration

“…For ethanol treatment, cultures were placed in sealed humidified chambers in which 50 mM ethanol was supplied to both the culture medium and reservoir tray (Adickes et al, 1988; Karl and Fisher, 1993). Control cultures were identically treated but with only water added to the reservoir tray.…”