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Controlled electromechanical cell stimulation on-a-chip.

Pavesi A, Adriani G, Rasponi M, Zervantonakis IK, Fiore GB, Kamm RD - Sci Rep (2015)

Bottom Line: The platform was validated in experiments using human bone marrow mesenchymal stem cells.These experiments demonstrated the ability for inducing changes in cell morphology, cytoskeletal fiber orientation and changes in gene expression under physiological stimuli.This novel bioengineering approach can be readily applied to various studies, especially in the fields of stem cell biology and regenerative medicine.

View Article: PubMed Central - PubMed

Affiliation: Biosym IRG, Singapore-MIT Alliance for Research and Technology, Singapore.

ABSTRACT
Stem cell research has yielded promising advances in regenerative medicine, but standard assays generally lack the ability to combine different cell stimulations with rapid sample processing and precise fluid control. In this work, we describe the design and fabrication of a micro-scale cell stimulator capable of simultaneously providing mechanical, electrical, and biochemical stimulation, and subsequently extracting detailed morphological and gene-expression analysis on the cellular response. This micro-device offers the opportunity to overcome previous limitations and recreate critical elements of the in vivo microenvironment in order to investigate cellular responses to three different stimulations. The platform was validated in experiments using human bone marrow mesenchymal stem cells. These experiments demonstrated the ability for inducing changes in cell morphology, cytoskeletal fiber orientation and changes in gene expression under physiological stimuli. This novel bioengineering approach can be readily applied to various studies, especially in the fields of stem cell biology and regenerative medicine.

No MeSH data available.


Related in: MedlinePlus

Gene expression analysis.A) Assessment of RNA qualities and concentrations using the Agilent Bioanalyzer RNA 6000 Pico Kit. RNA concentrations showed in the electropherograms varied from ~800 pg/μl to ~2 ng/μl, with an integrity number of 8.9 ± 0.1 representing an high quality RNA. (B) Agilent Bioanalyzer gel image of total RNA. (C) Histogram showing fold-changes in the expression of the selected genes following mechanical stimulation (7% strain), electrical stimulations (5 V/cm) and electromechanical stimulation (strain 7%, electrical signal 5 V/cm) compared to a control configuration without stimulation. GAPDH was chosen as housekeeping gene. Data are expressed as means ± SD.
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f6: Gene expression analysis.A) Assessment of RNA qualities and concentrations using the Agilent Bioanalyzer RNA 6000 Pico Kit. RNA concentrations showed in the electropherograms varied from ~800 pg/μl to ~2 ng/μl, with an integrity number of 8.9 ± 0.1 representing an high quality RNA. (B) Agilent Bioanalyzer gel image of total RNA. (C) Histogram showing fold-changes in the expression of the selected genes following mechanical stimulation (7% strain), electrical stimulations (5 V/cm) and electromechanical stimulation (strain 7%, electrical signal 5 V/cm) compared to a control configuration without stimulation. GAPDH was chosen as housekeeping gene. Data are expressed as means ± SD.

Mentions: As a proof-of-principle, relative RNA expression analysis was performed using cells harvested from a single microfluidic device. RNA was extracted from five devices per configuration (control, mechanical and electrical), and prior to performing quantitative real time polymerase chain reactions (qRT-PCR), the quality and concentration of extracted RNA samples were evaluated with an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara CA, USA) (Fig. 6A,B). To demonstrate the ability to measure changes in gene expression, we identified primers (see Supplementary Table 1) for the specific cardiac markers (GATA4, MEF2C, MYH7, NKX2.5, TUBB, CX43, TNNT2, and OCT4) using Primer Express 3.0 (Life Technologies), Carlsbad, CA) and optimized their measurement according to a previously reported protocol38. GAPDH was chosen as the housekeeping gene. Assays were performed in triplicate for each sample, and the data were analyzed and statistically evaluated using the delta-delta CT method, as previously described39.


Controlled electromechanical cell stimulation on-a-chip.

Pavesi A, Adriani G, Rasponi M, Zervantonakis IK, Fiore GB, Kamm RD - Sci Rep (2015)

Gene expression analysis.A) Assessment of RNA qualities and concentrations using the Agilent Bioanalyzer RNA 6000 Pico Kit. RNA concentrations showed in the electropherograms varied from ~800 pg/μl to ~2 ng/μl, with an integrity number of 8.9 ± 0.1 representing an high quality RNA. (B) Agilent Bioanalyzer gel image of total RNA. (C) Histogram showing fold-changes in the expression of the selected genes following mechanical stimulation (7% strain), electrical stimulations (5 V/cm) and electromechanical stimulation (strain 7%, electrical signal 5 V/cm) compared to a control configuration without stimulation. GAPDH was chosen as housekeeping gene. Data are expressed as means ± SD.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4488866&req=5

f6: Gene expression analysis.A) Assessment of RNA qualities and concentrations using the Agilent Bioanalyzer RNA 6000 Pico Kit. RNA concentrations showed in the electropherograms varied from ~800 pg/μl to ~2 ng/μl, with an integrity number of 8.9 ± 0.1 representing an high quality RNA. (B) Agilent Bioanalyzer gel image of total RNA. (C) Histogram showing fold-changes in the expression of the selected genes following mechanical stimulation (7% strain), electrical stimulations (5 V/cm) and electromechanical stimulation (strain 7%, electrical signal 5 V/cm) compared to a control configuration without stimulation. GAPDH was chosen as housekeeping gene. Data are expressed as means ± SD.
Mentions: As a proof-of-principle, relative RNA expression analysis was performed using cells harvested from a single microfluidic device. RNA was extracted from five devices per configuration (control, mechanical and electrical), and prior to performing quantitative real time polymerase chain reactions (qRT-PCR), the quality and concentration of extracted RNA samples were evaluated with an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara CA, USA) (Fig. 6A,B). To demonstrate the ability to measure changes in gene expression, we identified primers (see Supplementary Table 1) for the specific cardiac markers (GATA4, MEF2C, MYH7, NKX2.5, TUBB, CX43, TNNT2, and OCT4) using Primer Express 3.0 (Life Technologies), Carlsbad, CA) and optimized their measurement according to a previously reported protocol38. GAPDH was chosen as the housekeeping gene. Assays were performed in triplicate for each sample, and the data were analyzed and statistically evaluated using the delta-delta CT method, as previously described39.

Bottom Line: The platform was validated in experiments using human bone marrow mesenchymal stem cells.These experiments demonstrated the ability for inducing changes in cell morphology, cytoskeletal fiber orientation and changes in gene expression under physiological stimuli.This novel bioengineering approach can be readily applied to various studies, especially in the fields of stem cell biology and regenerative medicine.

View Article: PubMed Central - PubMed

Affiliation: Biosym IRG, Singapore-MIT Alliance for Research and Technology, Singapore.

ABSTRACT
Stem cell research has yielded promising advances in regenerative medicine, but standard assays generally lack the ability to combine different cell stimulations with rapid sample processing and precise fluid control. In this work, we describe the design and fabrication of a micro-scale cell stimulator capable of simultaneously providing mechanical, electrical, and biochemical stimulation, and subsequently extracting detailed morphological and gene-expression analysis on the cellular response. This micro-device offers the opportunity to overcome previous limitations and recreate critical elements of the in vivo microenvironment in order to investigate cellular responses to three different stimulations. The platform was validated in experiments using human bone marrow mesenchymal stem cells. These experiments demonstrated the ability for inducing changes in cell morphology, cytoskeletal fiber orientation and changes in gene expression under physiological stimuli. This novel bioengineering approach can be readily applied to various studies, especially in the fields of stem cell biology and regenerative medicine.

No MeSH data available.


Related in: MedlinePlus