July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
An in vitro co-culture model of the human retinal vasculature for use in development of regenerative medicine strategies for diabetic retinopathy.
Author Affiliations & Notes
  • Jessica Jane Eyre
    Department of Eye and Vision Science, University of Liverpool, Liverpool, Merseyside, United Kingdom
  • Rachel Williams
    Department of Eye and Vision Science, University of Liverpool, Liverpool, Merseyside, United Kingdom
  • Hannah J Levis
    Department of Eye and Vision Science, University of Liverpool, Liverpool, Merseyside, United Kingdom
  • Footnotes
    Commercial Relationships   Jessica Eyre, None; Rachel Williams, None; Hannah Levis, None
  • Footnotes
    Support  Crossley Barnes Bequest Studentship- University of Liverpool
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 3546. doi:
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      Jessica Jane Eyre, Rachel Williams, Hannah J Levis; An in vitro co-culture model of the human retinal vasculature for use in development of regenerative medicine strategies for diabetic retinopathy.. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3546.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : To investigate cellular changes occurring in an in vitro co-culture model of the human retinal microvasculature in healthy versus diabetic conditions.

Methods : Human retinal microvascular endothelial (hREC) and human retinal pericyte (hRP) cells were grown as monocultures in combinations of physiological glucose (5.5mM), high glucose (33mM), 20%, 5% and 2% oxygen. Immunofluorescence (IF) imaging enabled investigation of change in expression of angiogenic and oxidative stress proteins. To develop an in vitro co-culture model, cells were seeded as mono- or co-cultures on either side of polyethylene terephthalate (PET) transwell membranes in healthy versus diabetic conditions for up to 21 days. Cells on PET membranes were imaged using confocal microscopy to explore changes in junctional proteins, oxidative stress and angiogenic responses. Culture medium was collected at 7 time points and analysed using multiplex technology to measure 9 angiogenic-related proteins (Quansys). For multiplex analysis, results are reported using 2-way ANOVA with Sidak's multiple comparisons test, standard deviation and n=3.

Results : IF microscopy revealed changes in cell morphology, junctional protein expression (CD31), oxidative stress response (SOD-1) and angiogenic response (ANG-2) in both hRP and hREC in diabetic conditions. Multiplex analysis showed significant increase in ANG-2 in diabetic hREC, with the most significant change at day 7 (p<0.0001). IL-8 is significantly decreased in diabetic conditions by day 17 (p<0.05), but this change is not seen in monocultures. By day 7, TIMP-2 is significantly decreased in the diabetic conditions for both monoculture and co-cultures. HGF is produced by the hRP by day 7, but is significantly lower in diabetes (p<0.001), a change not seen in the co-culture model.

Conclusions : Multiplex analysis confirms the in vitro human diabetic model conditions have caused significant changes in proteins involved in angiogenesis. Differences between mono- and co-cultures highlight the importance of using co-culture models and primary cells to better understand the cell-cell signalling environment in the retinal microvasculature. With prolonged onset of DR, the longer timescale used enables monitoring of changes over time. This model will aid investigation of the intricate cell signalling involved in the retinal vasculature during diabetes.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

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