Unexpected fluctuations of trace element levels in cell culture medium in vitro: caveat emptor

Abstract

Copper is an essential trace element micronutrient in human and animal nutrition. Trace amounts present even in ultrapure water, serum and other cell culture medium components are sufficient to support the health requirements of most cell types in culture. Analysis of a variety of different types of basal media from a number of different suppliers revealed large fluctuations in the levels of copper, and also of other micronutrients including zinc, iron, selenium and cobalt. Investigations on proliferating Caco-2 cells revealed reductions in growth with increasing copper concentrations within the range seen in the commercial media and changes in expression of apoptosis- and autophagy-related proteins were noted. Even at concentrations of 1 μM CuSO where there was no significant change in cell growth, there was a significant decrease in procaspase-3 expression. These results stress the importance of batch testing of basal media when undertaking trace metal research since the baseline levels may vary. Batch variation of serum is well established but our results suggest that batch variation of the media may also be important.

PLEKHA5, PLEKHA6, and PLEKHA7 bind to PDZD11 to target the Menkes ATPase ATP7A to the cell periphery and regulate copper homeostasis

“…For immunofluorescence microscopy of copper-dependent ATP7A trafficking, MDCK or mCCD culture medium containing 315 µM CuCl 2 (Sigma-Aldrich, C3279), diluted from aqueous 1500x stock solution, was added to the cells (Greenough et al, 2004), overnight before fixation in the case of cysts, 4 or 5 hours before fixation for MDCK or mCCD cells grown on Transwells, respectively. Basal copper conditions correspond to regular MDCK or mCCD culture medium, whose main source of copper is fetal bovine serum (10-20%) and copper concentration is less than 1 µM (Arredondo et al, 2000;Bauerly et al, 2004;Keenan et al, 2018). For copper washout, cells were treated with CuCl 2 as described, washed once with culture medium, and then incubated for 4h at 37°C in culture medium containing 200 µM of the copper chelating agent bathocuproinedisulfonic acid (BCS) (Santa Cruz Biotechnology, Sc-217698) and 50 µg/ml cycloheximide (protein synthesis inhibitor), with replacement with fresh medium every 80 min (Holloway et al, 2013).…”

Ionophore Ability of Carnosine and Its Trehalose Conjugate Assists Copper Signal in Triggering Brain-Derived Neurotrophic Factor and Vascular Endothelial Growth Factor Activation In Vitro

“…Our findings show that carnosine and its conjugate not only permeate the cellular membrane increasing the intracellular peptide level (Figure 2) but also enhance the copper content inside the cell (Figure 3A); BCS counteracts this last effect blocking the metal extracellular pool Consistently, the changes in Ctr1 and Sp1 expression levels testify the ability of the metallostasis network to appreciate the low variation of copper level as the one caused by the partial influx of micromolar metal amounts present in the culture medium (Figure 3) [143]. This ionophore effect of the studied molecules that show a moderate copper affinity [106,117] recalls what was found for MPAC ligand, a term coined to indicate "organic small molecule that, without the high affinities of chelators, can ligate adventitial metal ions related to proteinopathies and redistribute them to a safer compartment" [144].…”

Extracellular Cu2+ pools and their detection: From current knowledge to next-generation probes

“…[127,128] Keenan et al provided evidence of fluctuation between different batches of Minimal Essential Medium (MEM) and Dulbecco's Modified Eagle Media (DMEM) from the same suppliers and between different suppliers, with concentrations varying from undetectable to 0.55 µM and 0.64 µM, respectively. [129] With the addition of supplements, Cu content tends to increase even further. Common supplements in cell culture media include foetal bovine serum (FBS), penicillin-streptomycin (PS), horse serum (HS), which all contain traces of Cu at 0.160 mg/L (2.51 µM), 0.330 mg/L (5.20 µM), and 0.120 mg/L (1.89 µM), respectively.…”