June 2020
Volume 61, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2020
Dynamic Full-Field OCT to image RPE cell cultures
Author Affiliations & Notes
  • Kassandra Groux
    Langevin Institute, CNRS, ESPCI Paris, PSL Research University, Paris, France
  • Jules Scholler
    Langevin Institute, CNRS, ESPCI Paris, PSL Research University, Paris, France
  • Anna Verschueren
    Quinze Vingts National Ophthalmology Hospital, Paris, France
    Vision Institute, Sorbonne Université, INSERM CNRS, Paris, France
  • Marie Darche
    Quinze Vingts National Ophthalmology Hospital, Paris, France
    Vision Institute, Sorbonne Université, INSERM CNRS, Paris, France
  • Olivier Goureau
    Vision Institute, Sorbonne Université, INSERM CNRS, Paris, France
  • Jose Alain Sahel
    Quinze Vingts National Ophthalmology Hospital, Paris, France
    Vision Institute, Sorbonne Université, INSERM CNRS, Paris, France
  • Mathias Fink
    Langevin Institute, CNRS, ESPCI Paris, PSL Research University, Paris, France
  • Michel Paques
    Quinze Vingts National Ophthalmology Hospital, Paris, France
    Vision Institute, Sorbonne Université, INSERM CNRS, Paris, France
  • Claude Boccara
    Langevin Institute, CNRS, ESPCI Paris, PSL Research University, Paris, France
  • Olivier Thouvenin
    Langevin Institute, CNRS, ESPCI Paris, PSL Research University, Paris, France
  • Sacha Reichman
    Vision Institute, Sorbonne Université, INSERM CNRS, Paris, France
  • Kate Grieve
    Quinze Vingts National Ophthalmology Hospital, Paris, France
    Vision Institute, Sorbonne Université, INSERM CNRS, Paris, France
  • Footnotes
    Commercial Relationships   Kassandra Groux, None; Jules Scholler, None; Anna Verschueren, None; Marie Darche, None; Olivier Goureau, None; Jose Sahel, None; Mathias Fink, None; Michel Paques, None; Claude Boccara, None; Olivier Thouvenin, None; Sacha Reichman, None; Kate Grieve, None
  • Footnotes
    Support  HELMHOLTZ ERC Grant Agreement #610110, ANR "OREO", IHU FORESIGHT, MACUSTAR Project
Investigative Ophthalmology & Visual Science June 2020, Vol.61, 1867. doi:
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      Kassandra Groux, Jules Scholler, Anna Verschueren, Marie Darche, Olivier Goureau, Jose Alain Sahel, Mathias Fink, Michel Paques, Claude Boccara, Olivier Thouvenin, Sacha Reichman, Kate Grieve; Dynamic Full-Field OCT to image RPE cell cultures. Invest. Ophthalmol. Vis. Sci. 2020;61(7):1867.

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

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Abstract

Purpose : Dynamic Full-Field OCT (D-FFOCT) is able to discriminate cells with different metabolic activity without contrast agents. Retinal pigment epithelium (RPE) is involved in many retinal diseases, including Age-related Macular Degeneration (AMD). Here, we used RPE cell cultures as a model to study behavior in the basal state and in response to acute damage by live imaging with D-FFOCT.

Methods : D-FFOCT is a non-invasive micrometric live imaging technique whose contrast is created by intrinsic subcellular organelle movement. Human induced pluripotent stem cell (hiPSC)-derived RPE and primary porcine RPE cell cultures, grown on polycarbonate membranes, were analyzed. The dynamic intracellular signal was recorded with 0.5µm transverse x 1.5µm axial resolution over periods of several hours. Immunochemistry was performed in order to validate results and decipher the subcellular structures generating the D-FFOCT signal. We performed scratch assays with a scalpel blade on RPE cultures and observed and quantified the dynamic response behavior of cells.

Results : The porcine RPE closed with a speed of ~20 µm/h, while for hiPSC derived RPE, we measured ~5 µm/h. The difference in speeds between porcine and human samples may be related to their different origin (hiPSC cells versus primary culture). Interestingly, if the membrane on which the RPE is grown was cut, or if the wound was too large (>40µm), after failing to close the wound through migration, both parts retracted, increasing the wound size. D-FFOCT allowed us to quantify the increase in intracellular activity around the damaged area.

Conclusions : D-FFOCT seems a convenient tool to image live RPE cell cultures, to measure both metabolic cell activity and speed of wound closing following acute damage. D-FFOCT allowed us to document behavior of healing RPE cells, showing that large wounds tend to retract rather than close, hence mimicking atrophic AMD expansion.

This is a 2020 ARVO Annual Meeting abstract.

 

Timepoints before (top), just after (middle) and 1-2 hours after (bottom) scratch assays on porcine (left) and hiPSC-derived (right) RPE cell cultures. The wound on porcine RPE closed in almost one hour, at ~20 µm/h, whereas in human RPE, closing was slower (~5 µm/h). Colorbar indicates metabolic activity level (warm/bright = active).

Timepoints before (top), just after (middle) and 1-2 hours after (bottom) scratch assays on porcine (left) and hiPSC-derived (right) RPE cell cultures. The wound on porcine RPE closed in almost one hour, at ~20 µm/h, whereas in human RPE, closing was slower (~5 µm/h). Colorbar indicates metabolic activity level (warm/bright = active).

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