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Evaluation of impedance-based label-free technology as a tool for pharmacology and toxicology investigations.

Atienzar FA, Gerets H, Tilmant K, Toussaint G, Dhalluin S - Biosensors (Basel) (2013)

Bottom Line: In addition, specific RTCA profiles (signatures) were generated when HepG2 and HepaRG cells were exposed to calcium modulators, antimitotics, DNA damaging and nuclear receptor agents, with a percentage of prediction close to 80% for both cellular models.In a subsequent experiment, HepG2 cells were exposed to 81 proprietary UCB compounds known to be genotoxic or not.Based on the DNA damaging signatures, the RTCA technology allowed the detection of ca. 50% of the genotoxic compounds (n = 29) and nearly 100% of the non-genotoxic compounds (n = 52).

View Article: PubMed Central - PubMed

Affiliation: UCB Pharma SA, Non Clinical Development, Chemin du Foriest, 1420 Braine-l'Alleud, Belgium; E-Mails: helga.gerets@ucb.com (H.G.); karen.tilmant@ucb.com (K.T.); gaelle.toussaint@ucb.com (G.T.); stephane.dhalluin@ucb.com (S.D.).

ABSTRACT
The use of label-free technologies based on electrical impedance is becoming more and more popular in drug discovery. Indeed, such a methodology allows the continuous monitoring of diverse cellular processes, including proliferation, migration, cytotoxicity and receptor-mediated signaling. The objective of the present study was to further assess the usefulness of the real-time cell analyzer (RTCA) and, in particular, the xCELLigence platform, in the context of early drug development for pharmacology and toxicology investigations. In the present manuscript, four cellular models were exposed to 50 compounds to compare the cell index generated by RTCA and cell viability measured with a traditional viability assay. The data revealed an acceptable correlation (ca. 80%) for both cell lines (i.e., HepG2 and HepaRG), but a lack of correlation (ca. 55%) for the primary human and rat hepatocytes. In addition, specific RTCA profiles (signatures) were generated when HepG2 and HepaRG cells were exposed to calcium modulators, antimitotics, DNA damaging and nuclear receptor agents, with a percentage of prediction close to 80% for both cellular models. In a subsequent experiment, HepG2 cells were exposed to 81 proprietary UCB compounds known to be genotoxic or not. Based on the DNA damaging signatures, the RTCA technology allowed the detection of ca. 50% of the genotoxic compounds (n = 29) and nearly 100% of the non-genotoxic compounds (n = 52). Overall, despite some limitations, the xCELLigence platform is a powerful and reliable tool that can be used in drug discovery for toxicity and pharmacology studies.

No MeSH data available.


Related in: MedlinePlus

Effect of methylprednisolone (nuclear receptor modulator) on cell index curves (RTCA) in HepG2, fresh HepaRG and A549 cells. HepG2 (A) and HepaRG (B) cells were exposed for at least 72 h to 0 (0.5% DMSO, red curve), 0.1 (green curve), 1 (dark blue curve), 10 (purple curve) and 100 (light blue curve) µM and A549 cells to 0 (control DMSO, red curve) and 20 µM (green curve) of methylprednisolone (C from Abassi et al., [9]). Cell indexes were normalized with the last time point before compound addition. Panels A and B: each data point was calculated from triplicate values (except for control cells n = 6). Data represent the average ± standard deviation (except for panel C). The normalized time point is indicated by the vertical line. For more details, please refer to the Materials and Methods section.
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biosensors-03-00132-f006: Effect of methylprednisolone (nuclear receptor modulator) on cell index curves (RTCA) in HepG2, fresh HepaRG and A549 cells. HepG2 (A) and HepaRG (B) cells were exposed for at least 72 h to 0 (0.5% DMSO, red curve), 0.1 (green curve), 1 (dark blue curve), 10 (purple curve) and 100 (light blue curve) µM and A549 cells to 0 (control DMSO, red curve) and 20 µM (green curve) of methylprednisolone (C from Abassi et al., [9]). Cell indexes were normalized with the last time point before compound addition. Panels A and B: each data point was calculated from triplicate values (except for control cells n = 6). Data represent the average ± standard deviation (except for panel C). The normalized time point is indicated by the vertical line. For more details, please refer to the Materials and Methods section.

Mentions: The specific RTCA patterns obtained in HepG2, fresh HepaRG and A549 cells exposed to one of the compounds belonging to each of the four mechanisms of action (i.e., tamoxifen, nocodazole, strophanthidin and methylprednisolone) are presented in Figure 3, Figure 4, Figure 5, Figure 6, respectively. It is noteworthy that for the fresh HepaRG cells, the background reading was done on the E-plates seeded with the cells. The background reading is normally done in absence of the cells, but since the E-plates were sent to Biopredic, to avoid any contamination problems, we decided not to perform the background reading in absence of cells. Consequently, as the reference index value is zero (in presence of the cells), negative values can be obtained in case of cytotoxic effects or specific signatures, such as calcium modulators. For the HepG2 cells, the lowest cell index value was zero (in case of 100% cell mortality), as the plates were prepared in house and the background measurement was performed with 100 µL medium in absence of cells. Typically, compounds that control calcium efflux induce a decrease in cell index immediately after compound addition. This effect was observed in A549 cells exposed to 20 µM tamoxifen (Figure 3(C)), as well as in HepG2 (Figure 3(A)) and HepaRG (Figure 3(B)) cells at 100 µM. It takes generally a few hours (e.g., 5–10 h) for the cell index to reach its minimum value, which can last for a few days (Figure 3). Antimitotic compounds produce a flat and/or wavy cell index kinetic, due to the inhibition of the cell division. In other words, cells stay alive, but do not proliferate. This signature was detected in HepG2 cells exposed to all concentrations of nocodazole (0.1–100 µM; Figure 4(A)), in HepaRG cells at 10 and 100 µM (Figure 4(B)) and in A549 cells at 20 µM (Figure 4(C)). Compounds that damage DNA can produce an increase in cell index (more or less pronounced) compared to control, followed by a decrease in cell index. This specific pattern was observed in HepaRG cells exposed to 10 µM strophanthidin (Figure 5(B)), in A549 cells at 20 µM (Figure 5(C)), but not in HepG2 cells in the range 0.1–100 µM (Figure 5(A)). Finally, the compounds that target nuclear receptors induce generally an increase in cell index in comparison to control that can last for a few days. This signature was observed in HepG2 cells exposed to 1–100 µM methylprednisolone (Figure 6(A)), in HepaRG cells at all concentrations tested (0.1–100 µM) (Figure 6(B)) and in A549 cells at 20 µM (Figure 6(C)).


Evaluation of impedance-based label-free technology as a tool for pharmacology and toxicology investigations.

Atienzar FA, Gerets H, Tilmant K, Toussaint G, Dhalluin S - Biosensors (Basel) (2013)

Effect of methylprednisolone (nuclear receptor modulator) on cell index curves (RTCA) in HepG2, fresh HepaRG and A549 cells. HepG2 (A) and HepaRG (B) cells were exposed for at least 72 h to 0 (0.5% DMSO, red curve), 0.1 (green curve), 1 (dark blue curve), 10 (purple curve) and 100 (light blue curve) µM and A549 cells to 0 (control DMSO, red curve) and 20 µM (green curve) of methylprednisolone (C from Abassi et al., [9]). Cell indexes were normalized with the last time point before compound addition. Panels A and B: each data point was calculated from triplicate values (except for control cells n = 6). Data represent the average ± standard deviation (except for panel C). The normalized time point is indicated by the vertical line. For more details, please refer to the Materials and Methods section.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263586&req=5

biosensors-03-00132-f006: Effect of methylprednisolone (nuclear receptor modulator) on cell index curves (RTCA) in HepG2, fresh HepaRG and A549 cells. HepG2 (A) and HepaRG (B) cells were exposed for at least 72 h to 0 (0.5% DMSO, red curve), 0.1 (green curve), 1 (dark blue curve), 10 (purple curve) and 100 (light blue curve) µM and A549 cells to 0 (control DMSO, red curve) and 20 µM (green curve) of methylprednisolone (C from Abassi et al., [9]). Cell indexes were normalized with the last time point before compound addition. Panels A and B: each data point was calculated from triplicate values (except for control cells n = 6). Data represent the average ± standard deviation (except for panel C). The normalized time point is indicated by the vertical line. For more details, please refer to the Materials and Methods section.
Mentions: The specific RTCA patterns obtained in HepG2, fresh HepaRG and A549 cells exposed to one of the compounds belonging to each of the four mechanisms of action (i.e., tamoxifen, nocodazole, strophanthidin and methylprednisolone) are presented in Figure 3, Figure 4, Figure 5, Figure 6, respectively. It is noteworthy that for the fresh HepaRG cells, the background reading was done on the E-plates seeded with the cells. The background reading is normally done in absence of the cells, but since the E-plates were sent to Biopredic, to avoid any contamination problems, we decided not to perform the background reading in absence of cells. Consequently, as the reference index value is zero (in presence of the cells), negative values can be obtained in case of cytotoxic effects or specific signatures, such as calcium modulators. For the HepG2 cells, the lowest cell index value was zero (in case of 100% cell mortality), as the plates were prepared in house and the background measurement was performed with 100 µL medium in absence of cells. Typically, compounds that control calcium efflux induce a decrease in cell index immediately after compound addition. This effect was observed in A549 cells exposed to 20 µM tamoxifen (Figure 3(C)), as well as in HepG2 (Figure 3(A)) and HepaRG (Figure 3(B)) cells at 100 µM. It takes generally a few hours (e.g., 5–10 h) for the cell index to reach its minimum value, which can last for a few days (Figure 3). Antimitotic compounds produce a flat and/or wavy cell index kinetic, due to the inhibition of the cell division. In other words, cells stay alive, but do not proliferate. This signature was detected in HepG2 cells exposed to all concentrations of nocodazole (0.1–100 µM; Figure 4(A)), in HepaRG cells at 10 and 100 µM (Figure 4(B)) and in A549 cells at 20 µM (Figure 4(C)). Compounds that damage DNA can produce an increase in cell index (more or less pronounced) compared to control, followed by a decrease in cell index. This specific pattern was observed in HepaRG cells exposed to 10 µM strophanthidin (Figure 5(B)), in A549 cells at 20 µM (Figure 5(C)), but not in HepG2 cells in the range 0.1–100 µM (Figure 5(A)). Finally, the compounds that target nuclear receptors induce generally an increase in cell index in comparison to control that can last for a few days. This signature was observed in HepG2 cells exposed to 1–100 µM methylprednisolone (Figure 6(A)), in HepaRG cells at all concentrations tested (0.1–100 µM) (Figure 6(B)) and in A549 cells at 20 µM (Figure 6(C)).

Bottom Line: In addition, specific RTCA profiles (signatures) were generated when HepG2 and HepaRG cells were exposed to calcium modulators, antimitotics, DNA damaging and nuclear receptor agents, with a percentage of prediction close to 80% for both cellular models.In a subsequent experiment, HepG2 cells were exposed to 81 proprietary UCB compounds known to be genotoxic or not.Based on the DNA damaging signatures, the RTCA technology allowed the detection of ca. 50% of the genotoxic compounds (n = 29) and nearly 100% of the non-genotoxic compounds (n = 52).

View Article: PubMed Central - PubMed

Affiliation: UCB Pharma SA, Non Clinical Development, Chemin du Foriest, 1420 Braine-l'Alleud, Belgium; E-Mails: helga.gerets@ucb.com (H.G.); karen.tilmant@ucb.com (K.T.); gaelle.toussaint@ucb.com (G.T.); stephane.dhalluin@ucb.com (S.D.).

ABSTRACT
The use of label-free technologies based on electrical impedance is becoming more and more popular in drug discovery. Indeed, such a methodology allows the continuous monitoring of diverse cellular processes, including proliferation, migration, cytotoxicity and receptor-mediated signaling. The objective of the present study was to further assess the usefulness of the real-time cell analyzer (RTCA) and, in particular, the xCELLigence platform, in the context of early drug development for pharmacology and toxicology investigations. In the present manuscript, four cellular models were exposed to 50 compounds to compare the cell index generated by RTCA and cell viability measured with a traditional viability assay. The data revealed an acceptable correlation (ca. 80%) for both cell lines (i.e., HepG2 and HepaRG), but a lack of correlation (ca. 55%) for the primary human and rat hepatocytes. In addition, specific RTCA profiles (signatures) were generated when HepG2 and HepaRG cells were exposed to calcium modulators, antimitotics, DNA damaging and nuclear receptor agents, with a percentage of prediction close to 80% for both cellular models. In a subsequent experiment, HepG2 cells were exposed to 81 proprietary UCB compounds known to be genotoxic or not. Based on the DNA damaging signatures, the RTCA technology allowed the detection of ca. 50% of the genotoxic compounds (n = 29) and nearly 100% of the non-genotoxic compounds (n = 52). Overall, despite some limitations, the xCELLigence platform is a powerful and reliable tool that can be used in drug discovery for toxicity and pharmacology studies.

No MeSH data available.


Related in: MedlinePlus