Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Jun 14;7(1):3538.
doi: 10.1038/s41598-017-03788-5.

Microfluidic co-cultures of retinal pigment epithelial cells and vascular endothelial cells to investigate choroidal angiogenesis

Li-Jiun Chen  1 , Shuntaro Ito  1 , Hiroyuki Kai  1 , Kuniaki Nagamine  1 , Nobuhiro Nagai  2 , Matsuhiko Nishizawa  1 , Toshiaki Abe  2 , Hirokazu Kaji  3
Affiliations

Microfluidic co-cultures of retinal pigment epithelial cells and vascular endothelial cells to investigate choroidal angiogenesis

Li-Jiun Chen et al. Sci Rep. .

Abstract

Angiogenesis plays a critical role in many diseases, including macular degeneration. At present, the pathological mechanisms remain unclear while appropriate models dissecting regulation of angiogenic processes are lacking. We propose an in vitro angiogenesis process and test it by examining the co-culture of human retinal pigmental epithelial cells (ARPE-19) and human umbilical vein endothelial cells (HUVEC) inside a microfluidic device. From characterisation of the APRE-19 monoculture, the tight junction protein (ZO-1) was found on the cells cultured in the microfluidic device but changes in the medium conditions did not affect the integrity of monolayers found in the permeability tests. Vascular endothelial growth factor (VEGF) secretion was elevated under low glucose and hypoxia conditions compared to the control. After confirming the angiogenic ability of HUVEC, the cell-cell interactions were analyzed under lowered glucose medium and chemical hypoxia by exposing ARPE-19 cells to cobalt (II) chloride (CoCl2). Heterotypic interactions between ARPE-19 and HUVEC were observed, but proliferation of HUVEC was hindered once the monolayer of ARPE-19 started breaking down. The above characterisations showed that alterations in glucose concentration and/or oxygen level as induced by chemical hypoxia causes elevations in VEGF produced in ARPE-19 which in turn affected directional growth of HUVEC.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Microfluidic device configuration. (A) Exploded view and schematic. (B) Cross-section (left) and top (right) view of the device. (C) SEM image of the porous membrane. (D) Photograph of a device with dyes in fluidic channels.
Figure 2
Figure 2
Characterisations of ARPE-19 cells. (A–C) Qualitative assessment of the monolayer inside a microfluidic device. (A) Optical microscopic image, (B) 3D confocal microscopic image (cell tracker green), and (C) fluorescent image (blue: nucleus; red: ZO-1). (D,E) Plots of permeability to FITC-Dextran and concentrations of VEGF secretion under different conditions imposed by altering glucose concentration and/or adding CoCl2 to the medium. Data sets were analyzed using 2-way ANOVA tests (showing a significant interaction effect for (E), p = 0.04); the asterisks * indicate significant findings from Tukey’s post-hoc tests. (D) mean FITC intensity (%) from lower channels is significantly different than those from upper channels (*p = 0.0005); pairwise comparisons between conditions are insignificant. (E) Pairwise comparisons between experimental conditions are insignificant while pairwise comparisons among the condition (red and blue bars for low glucose and low glucose & hypoxia) are significant (**p = 0.0022 and *p = 0.0162, respectively). N = 9 for (D) and (E).
Figure 3
Figure 3
(A) Snapshots of HUVEC migrating through the porous membrane taken at different time points. (B) Migration (%) of HUVEC at VEGF-free and with VEGF added to the upper channel. Data sets were analysed using Student’s t-test with the asterisk * indicated a significant difference found between the groups (*p = 0.035, n = 6).
Figure 4
Figure 4
Analysis of the co-culture model. (A) Anticipated behaviour of cells within the microfluidic device where ARPE-19 detached due to the invasion of HUVEC; (B) Overlaid images of ARPE-19 and HUVEC at different time points; top row: control, middle row: low glucose, bottom row: low glucose with the addition of CoCl2; ARPE-19 is depicted in red and HUVEC in green in all cases. (C,D) Quantification of the ARPE-19 and HUVEC growth area under different medium conditions and monoculture of ARPE in DME High glucose medium. Data were analyzed using 2-way ANOVA tests (showing a significant interaction effect, p < 0.0001 for (C) and (D)). Significant difference between the ARPE monolayer (purple line) and the co-culture groups (blue, red and green lines) is observed from t = 7 and onwards (p ≤ 0.0001). Pairwise comparisons between co-culture control and low glucose & hypoxia at t = 14 h (p = 0.023), and between all co-culture groups at t = 21 and t = 28 h (p < 0.0001) are significant in (C). In (D), pairwise comparisons for low glucose and low glucose & hypoxia groups (red and green lines) at t = 14 compared to t = 0 are significant (p ≤ 0.001). N = 7.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Rezzola S, et al. In vitro and ex vivo retina angiogenesis assays. Angiogenesis. 2013;17:429–442. doi: 10.1007/s10456-013-9398-x. - DOI - PubMed
    1. Stratton, R. D., Hauswirth, W. W. & Gardner, T. W. Studies on Retinal and Choroidal Disorders. (Springer Science & Business Media, 2012).
    1. Potential Public Health Impact of Age-Related Eye Disease Study Results. Arch Ophthalmol. 2003;121:1621–1624. doi: 10.1001/archopht.121.11.1621. - DOI - PMC - PubMed
    1. Karunadharma PP, Nordgaard CL, Olsen TW, Ferrington DA. Mitochondrial DNA Damage as a Potential Mechanism for Age-Related Macular Degeneration. Invest Ophthalmol Vis Sci. 2010;51:5470–5479. doi: 10.1167/iovs.10-5429. - DOI - PMC - PubMed
    1. Chang J-H, et al. Corneal Neovascularization: An Anti-VEGF Therapy Review. Surv Ophthalmol. 2012;57:415–429. doi: 10.1016/j.survophthal.2012.01.007. - DOI - PMC - PubMed

Publication types