July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
Optimal magnitude of shear stress for human corneal epithelial cell proliferation in microfluidic culture system
Author Affiliations & Notes
  • SeongKwang Cha
    Physiology, Chungbuk National University, Cheongju, Korea (the Democratic People's Republic of)
  • Jae-hyung Kim
    Seoul Daabom Eye Center, Cheongju, Korea (the Republic of)
  • Yongsook Goo
    Physiology, Chungbuk National University, Cheongju, Korea (the Democratic People's Republic of)
  • Footnotes
    Commercial Relationships   SeongKwang Cha, None; Jae-hyung Kim, None; Yongsook Goo, None
  • Footnotes
    Support  This work was supported by the National Research Foundation (NRF) of Korea grant funded by the Korea government (NRF-2015R1D1A1A01056903 and NRF-2017M3A9E2056460).
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 928. doi:
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      SeongKwang Cha, Jae-hyung Kim, Yongsook Goo; Optimal magnitude of shear stress for human corneal epithelial cell proliferation in microfluidic culture system. Invest. Ophthalmol. Vis. Sci. 2019;60(9):928.

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

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Abstract

Purpose : Human corneal epithelial cells (HCEC), the outermost layer of cornea, are exposed to shear stress caused by eyelids during blinking and eye rubbing. They play an important role in corneal wound healing process against injury by external trauma or eye surgery. Previous in vitro studies with CECs and other cells suggest that shear stress may affect cell morphology and growth. However, the shear stress magnitudes were highly variable. Therefore, here, we investigated the effect of shear stress on HCEC proliferation by using in-vitro microfluidic culture system to find optimal magnitude of shear stress for in vitro study.

Methods : We used cell line of HCEC, telomerase-immortalized human corneal epithelial cells (hTCEpi) and microfluidic chamber system (Ibidi GmbH, Martinsried, Germany). After seeding 1 × 105 cells/per μ-slide (surface area: 2.5 cm2, volume: 200 μl), four different magnitudes of shear stress (0, 0.22, 0.4 and 0.8 dyn/cm2) were applied for 24 hours. After 24 hours shear stress, bromodeoxyuridine (BrdU) was applied to HCEC to measure cell proliferation. Then fixation with 3.7% paraformaldehyde, permeabilization with 0.1% triton X-100, treatment of anti-BrdU antibody and treatment of Cy3-conjugated secondary antibody were performed for BrdU staining. And then Cy3 signal was detected with confocal microscopy.

Results : The increment of HCEC cell density in μ-slides after shear stress was observed across all conditions; 0, 0.22, 0.4 and 0.8 dyn/cm2 group, 161.60 ± 27.40 cells/mm2, 448.05 ± 32.48 cells/mm2, 270.80 ± 32.48 cells/mm2 and 247.14 ± 36.77 cells/mm2, respectively (Mean ± SEM, p<0.001). As magnitude of shear stress increased, Cy3 intensity observed in nucleus of HCEC increased up to 0.4 dyn/cm2, but the difference between 0.22 and 0.4 dyn/cm2 was not significant (p>0.05). In 0.8 dyn/cm2 group, Cy3 intensity decreased compared with 0.4 dyn/cm2 group (p<0.001).

Conclusions : We investigated the effect of fluid shear stress on HCEC proliferation with microfluidic culture system. We suggest that the optimal shear stress for HCEC culture system is in between 0.22 to 0.4 dyn/cm2. In future study, we would like to investigate the molecular mechanism of shear stress-dependent cell proliferation.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

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