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Complete cell killing by applying high hydrostatic pressure for acellular vascular graft preparation.

Mahara A, Morimoto N, Sakuma T, Fujisato T, Yamaoka T - Biomed Res Int (2014)

Bottom Line: On the other hand, when the cells were treated with the pressure of more than 200 MPa, the cells did not adhere, and the dehydrogenase activity was completely suppressed.That is, membrane permeability was disturbed with the pressure treatment of above 500 MPa.These results indicated that the pressure of 200 MPa for 10 min was enough to induce cell killing through inactivation of mitochondria activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, National Cerebral and Cardiovascular Center Research Institute, Fujishiro-dai, Suita, Osaka 565-8565, Japan.

ABSTRACT
Pressure treatment has been developed in tissue engineering application. Although the tissue scaffold prepared by a ultrahydrostatic pressure treatment has been reported, an excessive pressure has a potential to disrupt a structure of extracellular matrix through protein denaturation. It is important to understand the suitable low-pressure condition and mechanisms for cell killing. In this study, cellular morphology, mitochondria activity, and membrane permeability of mammalian cells with various pressure treatments were investigated with in vitro models. When the cells were treated with a pressure of 100 MPa for 10 min, cell morphology and adherence were the same as an untreated cells. Dehydrogenase activity in mitochondria was almost the same as untreated cells. On the other hand, when the cells were treated with the pressure of more than 200 MPa, the cells did not adhere, and the dehydrogenase activity was completely suppressed. However, green fluorescence was observed in the live/dead staining images, and the cells were completely stained as red after above 500 MPa. That is, membrane permeability was disturbed with the pressure treatment of above 500 MPa. These results indicated that the pressure of 200 MPa for 10 min was enough to induce cell killing through inactivation of mitochondria activity.

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Evaluation of cell permeability by fluorescence images of UHP treated cells under live/dead staining after pressure treatment at 3 hours. (a–e) NIH/3T3, (f–j) smooth muscle, and (k–o) endothelial cells are shown as phase-contrast images. The cells were treated with the pressure of 0 (a, f, and k), 100 (b, g, and l), 200 (c, h, and m), 500 (d, i, and n), and 1000 (e, j, and o) for 10 min. The membrane permeability was disturbed with the pressure treatment of above 500 MPa.
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fig5: Evaluation of cell permeability by fluorescence images of UHP treated cells under live/dead staining after pressure treatment at 3 hours. (a–e) NIH/3T3, (f–j) smooth muscle, and (k–o) endothelial cells are shown as phase-contrast images. The cells were treated with the pressure of 0 (a, f, and k), 100 (b, g, and l), 200 (c, h, and m), 500 (d, i, and n), and 1000 (e, j, and o) for 10 min. The membrane permeability was disturbed with the pressure treatment of above 500 MPa.

Mentions: Cell permeability and esterase activity of pressure treated cells were evaluated by CLSM images under the live/dead staining (Figure 5). The assay would mainly give the results of cell membrane damages by the pressure treatment. The green fluorescence derived from enzyme-activated calcein-AM was observed in 0, 100, and 200 MPa treated cells. After the treatment above 500 MPa, red fluorescence derived from ethidium homodimer III was mainly observed. This tendency was the same as that in each cell line. Cellular membrane was completely broken under above 500 MPa. In the condition of 200 MPa treatment, the esterase activity remained and provided the green fluorescence in the cells.


Complete cell killing by applying high hydrostatic pressure for acellular vascular graft preparation.

Mahara A, Morimoto N, Sakuma T, Fujisato T, Yamaoka T - Biomed Res Int (2014)

Evaluation of cell permeability by fluorescence images of UHP treated cells under live/dead staining after pressure treatment at 3 hours. (a–e) NIH/3T3, (f–j) smooth muscle, and (k–o) endothelial cells are shown as phase-contrast images. The cells were treated with the pressure of 0 (a, f, and k), 100 (b, g, and l), 200 (c, h, and m), 500 (d, i, and n), and 1000 (e, j, and o) for 10 min. The membrane permeability was disturbed with the pressure treatment of above 500 MPa.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4022071&req=5

fig5: Evaluation of cell permeability by fluorescence images of UHP treated cells under live/dead staining after pressure treatment at 3 hours. (a–e) NIH/3T3, (f–j) smooth muscle, and (k–o) endothelial cells are shown as phase-contrast images. The cells were treated with the pressure of 0 (a, f, and k), 100 (b, g, and l), 200 (c, h, and m), 500 (d, i, and n), and 1000 (e, j, and o) for 10 min. The membrane permeability was disturbed with the pressure treatment of above 500 MPa.
Mentions: Cell permeability and esterase activity of pressure treated cells were evaluated by CLSM images under the live/dead staining (Figure 5). The assay would mainly give the results of cell membrane damages by the pressure treatment. The green fluorescence derived from enzyme-activated calcein-AM was observed in 0, 100, and 200 MPa treated cells. After the treatment above 500 MPa, red fluorescence derived from ethidium homodimer III was mainly observed. This tendency was the same as that in each cell line. Cellular membrane was completely broken under above 500 MPa. In the condition of 200 MPa treatment, the esterase activity remained and provided the green fluorescence in the cells.

Bottom Line: On the other hand, when the cells were treated with the pressure of more than 200 MPa, the cells did not adhere, and the dehydrogenase activity was completely suppressed.That is, membrane permeability was disturbed with the pressure treatment of above 500 MPa.These results indicated that the pressure of 200 MPa for 10 min was enough to induce cell killing through inactivation of mitochondria activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, National Cerebral and Cardiovascular Center Research Institute, Fujishiro-dai, Suita, Osaka 565-8565, Japan.

ABSTRACT
Pressure treatment has been developed in tissue engineering application. Although the tissue scaffold prepared by a ultrahydrostatic pressure treatment has been reported, an excessive pressure has a potential to disrupt a structure of extracellular matrix through protein denaturation. It is important to understand the suitable low-pressure condition and mechanisms for cell killing. In this study, cellular morphology, mitochondria activity, and membrane permeability of mammalian cells with various pressure treatments were investigated with in vitro models. When the cells were treated with a pressure of 100 MPa for 10 min, cell morphology and adherence were the same as an untreated cells. Dehydrogenase activity in mitochondria was almost the same as untreated cells. On the other hand, when the cells were treated with the pressure of more than 200 MPa, the cells did not adhere, and the dehydrogenase activity was completely suppressed. However, green fluorescence was observed in the live/dead staining images, and the cells were completely stained as red after above 500 MPa. That is, membrane permeability was disturbed with the pressure treatment of above 500 MPa. These results indicated that the pressure of 200 MPa for 10 min was enough to induce cell killing through inactivation of mitochondria activity.

Show MeSH