July 2019
Volume 60, Issue 9
Free
ARVO Annual Meeting Abstract  |   July 2019
Promoting Growth of Keratocyte from Femtosecond Laser lntrastromal Lenticules Using PDMS Substrate and Cocktail Medium
Author Affiliations & Notes
  • shenyang li
    Aier School of Ophthalmology, Central South University, ChangSha, China
  • Zekai Cui
    Aier School of Ophthalmology, Central South University, ChangSha, China
  • Jianing Gu
    Aier Eye Institute, China
  • Yini Wang
    Aier School of Ophthalmology, Central South University, ChangSha, China
  • Shibo Tang
    Aier School of Ophthalmology, Central South University, ChangSha, China
    Aier Eye Institute, China
  • Jiansu Chen
    Aier School of Ophthalmology, Central South University, ChangSha, China
    Aier Eye Institute, China
  • Footnotes
    Commercial Relationships   shenyang li, None; Zekai Cui, None; Jianing Gu, None; Yini Wang, None; Shibo Tang, None; Jiansu Chen, None
  • Footnotes
    Support  Special Funds for Major Science and Technology Projects of Guangdong Province (2015B010125007); National Natural Scientific Fund of China (81871495)
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 4653. doi:
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      shenyang li, Zekai Cui, Jianing Gu, Yini Wang, Shibo Tang, Jiansu Chen; Promoting Growth of Keratocyte from Femtosecond Laser lntrastromal Lenticules Using PDMS Substrate and Cocktail Medium. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4653.

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

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Abstract

Purpose : In vitro expansion of human keratocytes with specific property is useful and promising for cell therapy. Here, we investigate a novel procedure to propagate human keratocytes through biomimetic environments of PDMS substrate and cocktail medium.

Methods : Femtosecond laser intrastromal lenticules (FLI-lenticules) from myopic patients were digested by collagenase I. Cornea stromal cells (CSCs) were cultured in cocktail medium, which was DMEM/F12 containing RITS (ROCK inhibitor Y27632, insulin, transferrin, selenous acid), L-ascorbate 2-phosphate, fibroblast growth factor 2 (FGF2), 0.5% FBS and antibiotics (also called cocktail medium ). CSCs at passage 3 (P3) were subcultured onto PDMS substrate in 6-well plate for forming spheroid adherent growth. The characteristic expressions of CSCs were analyzed.

Results : Primary CSCs cultured on FNC coated PDMS substrate in DMEM/F12 and 10% FBS displayed positive expressions of keratocan by immunofluorescence staining when on 7d. Actin staining showed dendritic and spindle morphology(Fig. 1A,1B). However, CSCs cultured on tissue-culture plastic (TCP) in DMEM/F12 and 10% FBS showed long spindle shape when stained with lumican and vimentin (Fig. 1C,1D). CSCs enable to form spheroid adherent growth when cultured on PDMS substrate in cocktail medium and shake culture for 2 weeks. The spheroid cells in cocktail medium display higher gene expression of lumincan and ALDH3A1 while lower of vimentin and α-SMA by q-PCR assay (Fig. 2).

Conclusions : Keratocyte morphology is promoted when cultured on PDMS substrate. And CSC spheroids cultured on PDMS substrate in cocktail medium have tendency to form more keratocyte phenotypes. The study is beneficial for expansion in vitro of human keratocytes with specific property and future CSC tissue engineering research and application.

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

 

Fig.1 (A,B)Immunofluorescence of keratocan and actin expression in CSCs on FNC coated PDMS. (C,D) Immunofluorescence of lumincan and vimentin expression in cells on TCP. Nuclei were stained with DAPI.

Fig.1 (A,B)Immunofluorescence of keratocan and actin expression in CSCs on FNC coated PDMS. (C,D) Immunofluorescence of lumincan and vimentin expression in cells on TCP. Nuclei were stained with DAPI.

 

Fig.2 (A) CSCs formed spheroids on PDMS substrate in cocktail medium. (B) Quantitative PCR results showed high mRNA expression levels of ALDH3A1 and lumincan in CSC spheroid cells on PDMS, while low expression of vimentin and α-SMA.

Fig.2 (A) CSCs formed spheroids on PDMS substrate in cocktail medium. (B) Quantitative PCR results showed high mRNA expression levels of ALDH3A1 and lumincan in CSC spheroid cells on PDMS, while low expression of vimentin and α-SMA.

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