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Peiyuan Wang, Yuting Han, Yonglong Guo, Quan Yu, Jun Zhang, Jiansu Chen; The stimulatory effect of ROCK inhibitor on rabbit corneal limbal epithelial spheroids and bioprinting. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4099.
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© ARVO (1962-2015); The Authors (2016-present)
The transplantation of biomimetically engineered corneal limbal epithelial cells (LECs) layer is a promising technique for the treatment of limbal stem cell deficiency. The aim of this study is to investigate the effect of spherical cultured with ROCK inhibitor on rabbit LECs and to explore the 3D bio-printing high viability LEC spheroids on curved collagen membrane (CM) to construct LEC layer.
We used primary rabbit LECs to generate cell spheroids by multiwell agarose micromolds. The cell phenotype changes and stemness of LEC spheroids were assayed. Curved CM was produced with a spherically curved mould (Fig.1A) and then coated with collagen IV. LEC spheroids were bio-printed on CM by a self-designed bio-printer under the direction of microscopy imaging system (Fig.1B).
The adherent area percentages of LEC spheroids in Y-27632 group were obviously larger than those in control group at 8, 16 and 24 hours(n=3,*P<0.05), with immunofluorescence positive expression of CK3 and p63 (Fig.1C, D). The Live/Dead assay and EdU labeling assay showed that spherical cultured with Y-27632 had lower dead cell percentage and higher proliferative cell percentage than the control group (Fig.2A) (n=3,*P<0.05). qPCR showed expression of ABCG2, ΔNp63α and Nestin increased in the group of spherical cultured (Fig.2B) (n=3,*P<0.05). LEC spheroids cultured on curved CM migrated peripherally and formed cell sheet on Day 7 (Fig.2C). SEM exhibited LECs secreted extracellular matrix and formed tight junction on CM (Fig.2D).
LEC spheroids cultured with ROCK inhibitor improve cell proliferation and viability, as well as maintain cell phenotype and stemness, which could produce a high viability bio-ink for 3D bio-printing. LEC bio-printed spheroids can construct a transplantable cell sheet with ROCK inhibitor on curved CM, which help construct curved tissue engineering LEC layer.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
(A) A spherically curved mould and curved CM. (B) The main structure of bio-printer and microscopy imaging system. (C) The adherent area percentages of LEC spheroids. (D) Positive expressed CK3 and p63.
(A) Live/Dead and EdU labeling assay, observed by confocal microscopy. (B) qPCR showed expression of ABCG2, ΔNp63α and Nestin. (C) LEC spheroids cultured on curved CM on Day 7. (D) SEM observation.
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