June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Development of a Schlemm’s Canal “Inner Wall on a Chip” for High Content Screening
Author Affiliations & Notes
  • Seyed Mohammad Siadat
    Biomedical Engineering, Georgia Institute of Technology, Atlanta, Georgia, United States
  • Jacques A Bertrand
    Bioengineering, Imperial College London, London, United Kingdom
  • Darryl R Overby
    Bioengineering, Imperial College London, London, United Kingdom
  • W Daniel Stamer
    Ophthalmology, Duke University, Durham, North Carolina, United States
    Biomedical Engineering, Duke University, Durham, North Carolina, United States
  • C Ross Ethier
    Biomedical Engineering, Georgia Institute of Technology, Atlanta, Georgia, United States
  • Footnotes
    Commercial Relationships   Seyed Mohammad Siadat None; Jacques Bertrand None; Darryl Overby None; W Daniel Stamer None; C Ross Ethier None
  • Footnotes
    Support  BrightFocus Foundation CG2020001; NIH 5R21EY033142-02; Georgia Research Alliance (CRE)
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 50. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Seyed Mohammad Siadat, Jacques A Bertrand, Darryl R Overby, W Daniel Stamer, C Ross Ethier; Development of a Schlemm’s Canal “Inner Wall on a Chip” for High Content Screening. Invest. Ophthalmol. Vis. Sci. 2023;64(8):50.

      Download citation file:


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

      ×
  • Supplements
Abstract

Purpose : Pores in Schlemm’s canal (SC) endothelial cells are thought to be an important modulator of IOP and are triggered by mechanical stretch. We aimed to develop an in vitro assay to rapidly assess intracellular pore (I-pore) formation in SC cells exposed to local cellular stretch.

Methods : Glass substrates were coated with biotinylated gelatin cross-linked with transglutaminase. Carboxyl ferromagnetic particles (dia. ∼5 µm, Spherotech) were added on the substrate and then primary glaucomatous human SC cells were seeded (gSC57, passage 5). Pores were detected using fluorescently labeled streptavidin tracers (Braakman+, IOVS, 2014). After 5-7 h of incubation, a first tracer was added to the media for 5 min. The first tracer was then washed away and a second tracer was added for 20 min, during which time particles were exposed to a magnetic force, thereby creating local cellular stretch in the basal to apical direction. Cells were fixed, imaged, and analyzed by counting punctate tracer signals that colocalized with particles under cells.

Results : The tracers reached and bound to the substrate at locations not covered by the cells, including at I-pore sites (Figure). The I-pores were categorized as Type I (first tracer, i.e. present without magnetic force/stretching) and Type II (second tracer, i.e. present with local cell stretching). Type I and II pores were associated with 9.1 ± 3.2% (mean ± std dev) and 19.0 ± 5.1% of particles under cells, respectively (n = 6 wells, 200 analyzed cells/well, 73 ± 24 particles/well). There were more Type II pores than Type I (2-tailed t-test, p = 0.006).

Conclusions : Our approach allows assessment of I-pores in SC cells. Some pores form spontaneously (without magnets, Type I) through mechanisms that are unclear; however, the greater number of Type II pores supports the association between pore formation and local mechanical stretch. Morphological studies of I-pores formed in this assay are ongoing.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

 

Representative micrographs showing I-pores detected by the fluorescent assay colocalizing with magnetic particles under SC cells. (A) Cell nuclei (blue) and particles (dark spots). Particles are outlined by white circles in panels B-D. (B) Actin network (red). (C) The first tracer (green). White arrow: Type I pore. (D) The second tracer (magenta). Yellow arrows: Type II pores. Note the disruption of the actin network in panel B at the position of Type II pores. Scale bars = 20 µm.

Representative micrographs showing I-pores detected by the fluorescent assay colocalizing with magnetic particles under SC cells. (A) Cell nuclei (blue) and particles (dark spots). Particles are outlined by white circles in panels B-D. (B) Actin network (red). (C) The first tracer (green). White arrow: Type I pore. (D) The second tracer (magenta). Yellow arrows: Type II pores. Note the disruption of the actin network in panel B at the position of Type II pores. Scale bars = 20 µm.

×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×