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Altered Functions of Human Blood-Derived Vascular Endothelial Cells by Simulated Microgravity

Vidhya Ramaswamy

Vidhya Ramaswamy

Department of Materials Science and Engineering, University of FloridaGainesville,
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Ramaswamy, Vidhya
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Allison Goins

Allison Goins

  • Department of Materials Science and Engineering, University of FloridaGainesville,
  • Institute of Cell and Tissue Science and Engineering, University of FloridaGainesville,
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Goins, Allison
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Josephine B. Allen

Josephine B. Allen

  • Department of Materials Science and Engineering, University of FloridaGainesville,
  • Institute of Cell and Tissue Science and Engineering, University of FloridaGainesville,
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Allen, Josephine B.
  
Jul 17, 2020
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Article Category: Research Article
Published Online: Jul 17, 2020
Page range: 2 - 16
DOI: https://doi.org/10.2478/gsr-2016-0001
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© 2016 Vidhya Ramaswamy et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Figure 1

Bioreactor set-up and operation: (A) Synthecon RWV bioreactor set-up (B) RWV bioreactor detachable chamber (C) Representative 20X phase contrast image of HE-like cells cultured on cytodex-1 microcarrier beads. Scale bar is 100 μm. In panel C, the arrows indicate cell-covered beads.
Bioreactor set-up and operation: (A) Synthecon RWV bioreactor set-up (B) RWV bioreactor detachable chamber (C) Representative 20X phase contrast image of HE-like cells cultured on cytodex-1 microcarrier beads. Scale bar is 100 μm. In panel C, the arrows indicate cell-covered beads.

Figure 2

Isolation of PBMNCs and differentiation into ECs: (A) Representative phase contrast image of PBMNC-derived colony formation of HE-like cells; Scale bar 200 μm (B) Representative phase contrast image of characteristic endothelial cobblestone morphology of HE-like cells in subsequent culture (20X). Representative 20X fluorescent images of positive immunocytochemical staining of HE-like cells for (C) CD31 and (D) von Willebrand factor, both shown in green; nuclei were counterstained with Hoechst 33342, shown in blue. Panel B - Panel D scale bar is 50 μm.
Isolation of PBMNCs and differentiation into ECs: (A) Representative phase contrast image of PBMNC-derived colony formation of HE-like cells; Scale bar 200 μm (B) Representative phase contrast image of characteristic endothelial cobblestone morphology of HE-like cells in subsequent culture (20X). Representative 20X fluorescent images of positive immunocytochemical staining of HE-like cells for (C) CD31 and (D) von Willebrand factor, both shown in green; nuclei were counterstained with Hoechst 33342, shown in blue. Panel B - Panel D scale bar is 50 μm.

Figure 3

Proliferation kinetics: Representative proliferation kinetics curve HE-like cells cultured under microgravity (μg) vs. cells maintained in normal gravity (ng), as measured by DNA concentration in cell lysates. Data shown are Mean +/− SD; n=3.
Proliferation kinetics: Representative proliferation kinetics curve HE-like cells cultured under microgravity (μg) vs. cells maintained in normal gravity (ng), as measured by DNA concentration in cell lysates. Data shown are Mean +/− SD; n=3.

Figure 4

Gene expression: Representative relative gene expression of HSPA4 by microgravity (μg) cultured HE-like cells vs. cells maintained in normal gravity (ng) over 2 and 4 days of culture. Data shown are Mean +/− SD; n=3. N.S. indicates no statistically significant difference (p>0.05) and * indicates a statistically significant increase (p<0.05).
Gene expression: Representative relative gene expression of HSPA4 by microgravity (μg) cultured HE-like cells vs. cells maintained in normal gravity (ng) over 2 and 4 days of culture. Data shown are Mean +/− SD; n=3. N.S. indicates no statistically significant difference (p>0.05) and * indicates a statistically significant increase (p<0.05).

Figure 5

Secretory functions of cells: Representative results of quantification of release of (A) Nitric oxide and (B) Interleukin-6 by microgravity (μg) cultured HE-like cells vs. cells maintained in normal gravity (ng) over 2 and 4 days of culture. Data shown are Mean +/− SD; n=3. N.S. indicates no statistically significant difference (p>0.05) and * indicates a statistically significant increase (p<0.05).
Secretory functions of cells: Representative results of quantification of release of (A) Nitric oxide and (B) Interleukin-6 by microgravity (μg) cultured HE-like cells vs. cells maintained in normal gravity (ng) over 2 and 4 days of culture. Data shown are Mean +/− SD; n=3. N.S. indicates no statistically significant difference (p>0.05) and * indicates a statistically significant increase (p<0.05).

Figure 6

Plasma clotting kinetics: Recalcified plasma clotting kinetics for human plasma incubated with HE-like cells cultured in normal vs. simulated microgravity for (A) 2 days and (B) 4 days. Data show absorbance plotted vs. time. Arrows indicate the rightward shift in the normal gravity conditions, indicative of delayed clotting. SD error bars have been omitted for visual clarity. Data shown are Means; n=3. (C) Representative result showing quantification of the time taken for recalcified plasma to clot (in minutes) when incubated with HE-like cells cultured for 2 and 4 days in normal vs. simulated microgravity. Data shown are Mean +/− SD; n=6. * indicates a statistically significant increase (p<0.05).
Plasma clotting kinetics: Recalcified plasma clotting kinetics for human plasma incubated with HE-like cells cultured in normal vs. simulated microgravity for (A) 2 days and (B) 4 days. Data show absorbance plotted vs. time. Arrows indicate the rightward shift in the normal gravity conditions, indicative of delayed clotting. SD error bars have been omitted for visual clarity. Data shown are Means; n=3. (C) Representative result showing quantification of the time taken for recalcified plasma to clot (in minutes) when incubated with HE-like cells cultured for 2 and 4 days in normal vs. simulated microgravity. Data shown are Mean +/− SD; n=6. * indicates a statistically significant increase (p<0.05).
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