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Microfluidic Impedimetric Cell Regeneration Assay to Monitor the Enhanced Cytotoxic Effect of Nanomaterial Perfusion.

Rothbauer M, Praisler I, Docter D, Stauber RH, Ertl P - Biosensors (Basel) (2015)

Bottom Line: In the last decade, the application of nanomaterials (NMs) in technical products and biomedicine has become a rapidly increasing market trend.To bridge this technological gap, we here present a microfluidic cell culture system containing embedded impedance microsensors to continuously and non-invasively monitor the effects of NMs on adherent cells under varying flow conditions.As a model, the impact of silica NMs on the vitality and regenerative capacity of human lung cells after acute and chronic exposure scenarios was studied over an 18-h period following a four-hour NM treatment.

View Article: PubMed Central - PubMed

Affiliation: BioSensor Technologies, AIT Austrian Institute of Technology GmbH, 1190 Vienna, Austria. mario.rothbauer@gmail.com.

ABSTRACT
In the last decade, the application of nanomaterials (NMs) in technical products and biomedicine has become a rapidly increasing market trend. As the safety and efficacy of NMs are of utmost importance, new methods are needed to study the dynamic interactions of NMs at the nano-biointerface. However, evaluation of NMs based on standard and static cell culture end-point detection methods does not provide information on the dynamics of living biological systems, which is crucial for the understanding of physiological responses. To bridge this technological gap, we here present a microfluidic cell culture system containing embedded impedance microsensors to continuously and non-invasively monitor the effects of NMs on adherent cells under varying flow conditions. As a model, the impact of silica NMs on the vitality and regenerative capacity of human lung cells after acute and chronic exposure scenarios was studied over an 18-h period following a four-hour NM treatment. Results of the study demonstrated that the developed system is applicable to reliably analyze the consequences of dynamic NM exposure to physiological cell barriers in both nanotoxicology and nanomedicine.

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(a) Fluorescence and bright-field images of H441 cells stained with tetramethylrhodamine ethyl ester perchlorate (TMRE) mitochondrial membrane potential dye (live cells: red; top panel) and the live/dead cytotoxicity assay kit (live cells: green; dead cells: red; bottom panel) post-nanoparticle administration and regeneration. (b) Cytotoxicity of silica nanoparticles (AmSil30) towards 50% and 100% confluent H441 epithelial cell layers. The data presented are derived from the metabolic MTT assay (n = 3) and expressed as % mean value ± % standard deviation.
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biosensors-05-00736-f003: (a) Fluorescence and bright-field images of H441 cells stained with tetramethylrhodamine ethyl ester perchlorate (TMRE) mitochondrial membrane potential dye (live cells: red; top panel) and the live/dead cytotoxicity assay kit (live cells: green; dead cells: red; bottom panel) post-nanoparticle administration and regeneration. (b) Cytotoxicity of silica nanoparticles (AmSil30) towards 50% and 100% confluent H441 epithelial cell layers. The data presented are derived from the metabolic MTT assay (n = 3) and expressed as % mean value ± % standard deviation.

Mentions: Before conducting on-chip microfluidic regeneration assays, the cell viability and metabolic activity of epithelial H441 cells were investigated using standard static cell cultures to identify sub-toxic, as well as EC50 values for AmSil30 silica nanoparticles. Results of this nanotoxicological study using TMRE mitochondrial membrane potential dye to indicate cell viability are shown in Figure 3a and revealed that AmSil30 nanoparticle concentrations between 600 µg/mL and 6 mg/mL are highly toxic for H441 cells, while 60 µg/mL showed reduced and 6 µg/mL almost no cytotoxicity effects. Similar results were obtained using a live/dead cytotoxicity assay kit, as shown in Figure 3a (bottom panel); however, for 60 µg/mL of silica nanoparticles, which was previously reported to have no significant effect on H441 cell viability [40], the two viability assays distinctly differed and showed that cytotoxic potential is clearly dependent on cell surface coverage. Therefore, to further investigate the effects of increasing silica nanoparticle concentrations on H441 epithelial cell cultures at different cell surface coverages, quantitative analysis of AmSil30 cytotoxicity was performed using MTT assays in subsequent experiments. Results of the viability study are shown in Figure 3b demonstrating a distinct dose-time response in the presence of different cell culture densities. While an EC50 value of 120 µg/mL was calculated for 50% confluence, a significant EC50 shift to 240 µg/mL was observed using a fully-confluent (100%) cell layer. These results indicate that islands of proliferating cells, as well as non-confluent cell layers react more sensitively to cytotoxic nanomaterials than intact epithelial cell monolayers that resemble an epithelial lung barrier.


Microfluidic Impedimetric Cell Regeneration Assay to Monitor the Enhanced Cytotoxic Effect of Nanomaterial Perfusion.

Rothbauer M, Praisler I, Docter D, Stauber RH, Ertl P - Biosensors (Basel) (2015)

(a) Fluorescence and bright-field images of H441 cells stained with tetramethylrhodamine ethyl ester perchlorate (TMRE) mitochondrial membrane potential dye (live cells: red; top panel) and the live/dead cytotoxicity assay kit (live cells: green; dead cells: red; bottom panel) post-nanoparticle administration and regeneration. (b) Cytotoxicity of silica nanoparticles (AmSil30) towards 50% and 100% confluent H441 epithelial cell layers. The data presented are derived from the metabolic MTT assay (n = 3) and expressed as % mean value ± % standard deviation.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4697142&req=5

biosensors-05-00736-f003: (a) Fluorescence and bright-field images of H441 cells stained with tetramethylrhodamine ethyl ester perchlorate (TMRE) mitochondrial membrane potential dye (live cells: red; top panel) and the live/dead cytotoxicity assay kit (live cells: green; dead cells: red; bottom panel) post-nanoparticle administration and regeneration. (b) Cytotoxicity of silica nanoparticles (AmSil30) towards 50% and 100% confluent H441 epithelial cell layers. The data presented are derived from the metabolic MTT assay (n = 3) and expressed as % mean value ± % standard deviation.
Mentions: Before conducting on-chip microfluidic regeneration assays, the cell viability and metabolic activity of epithelial H441 cells were investigated using standard static cell cultures to identify sub-toxic, as well as EC50 values for AmSil30 silica nanoparticles. Results of this nanotoxicological study using TMRE mitochondrial membrane potential dye to indicate cell viability are shown in Figure 3a and revealed that AmSil30 nanoparticle concentrations between 600 µg/mL and 6 mg/mL are highly toxic for H441 cells, while 60 µg/mL showed reduced and 6 µg/mL almost no cytotoxicity effects. Similar results were obtained using a live/dead cytotoxicity assay kit, as shown in Figure 3a (bottom panel); however, for 60 µg/mL of silica nanoparticles, which was previously reported to have no significant effect on H441 cell viability [40], the two viability assays distinctly differed and showed that cytotoxic potential is clearly dependent on cell surface coverage. Therefore, to further investigate the effects of increasing silica nanoparticle concentrations on H441 epithelial cell cultures at different cell surface coverages, quantitative analysis of AmSil30 cytotoxicity was performed using MTT assays in subsequent experiments. Results of the viability study are shown in Figure 3b demonstrating a distinct dose-time response in the presence of different cell culture densities. While an EC50 value of 120 µg/mL was calculated for 50% confluence, a significant EC50 shift to 240 µg/mL was observed using a fully-confluent (100%) cell layer. These results indicate that islands of proliferating cells, as well as non-confluent cell layers react more sensitively to cytotoxic nanomaterials than intact epithelial cell monolayers that resemble an epithelial lung barrier.

Bottom Line: In the last decade, the application of nanomaterials (NMs) in technical products and biomedicine has become a rapidly increasing market trend.To bridge this technological gap, we here present a microfluidic cell culture system containing embedded impedance microsensors to continuously and non-invasively monitor the effects of NMs on adherent cells under varying flow conditions.As a model, the impact of silica NMs on the vitality and regenerative capacity of human lung cells after acute and chronic exposure scenarios was studied over an 18-h period following a four-hour NM treatment.

View Article: PubMed Central - PubMed

Affiliation: BioSensor Technologies, AIT Austrian Institute of Technology GmbH, 1190 Vienna, Austria. mario.rothbauer@gmail.com.

ABSTRACT
In the last decade, the application of nanomaterials (NMs) in technical products and biomedicine has become a rapidly increasing market trend. As the safety and efficacy of NMs are of utmost importance, new methods are needed to study the dynamic interactions of NMs at the nano-biointerface. However, evaluation of NMs based on standard and static cell culture end-point detection methods does not provide information on the dynamics of living biological systems, which is crucial for the understanding of physiological responses. To bridge this technological gap, we here present a microfluidic cell culture system containing embedded impedance microsensors to continuously and non-invasively monitor the effects of NMs on adherent cells under varying flow conditions. As a model, the impact of silica NMs on the vitality and regenerative capacity of human lung cells after acute and chronic exposure scenarios was studied over an 18-h period following a four-hour NM treatment. Results of the study demonstrated that the developed system is applicable to reliably analyze the consequences of dynamic NM exposure to physiological cell barriers in both nanotoxicology and nanomedicine.

Show MeSH
Related in: MedlinePlus