Establishment of a bi-layer tissue engineered conjunctiva construct using a three-dimensional (3D) printed scaffold

Abstract

Purpose : Conjunctiva is an essential component of the ocular surface. To better address the conjunctiva diseases and other ocular surface diseases which may result in local lesions of conjunctiva, it is ideal to create a 3D model instead of conventional 2D model for an in vitro study. In this study, we established a bi-layer tissue engineered conjunctiva construct using a 3D printed poly(ε-caprolactone) (PCL) scaffold to mimic the biological structure and microenvironment of conjunctiva tissue for in vitro study.

Methods : Human conjunctival tissue was digested with dispase to isolate the conjunctival epithelial cells (CjECs), followed by collagenase A digestion to obtain conjunctival stromal cells (CjSCs). A 2-step co-culture method was employed to fabricate a bi-layer tissue engineered conjunctiva construct. The CjSCs and CjECs were characterized by qPCR and immunofluorescence staining. The construct was assessed under a confocal microscope and a scanning electronic microscope (SEM).

Results : A bi-layer tissue engineered conjunctiva construct was successfully established using a 3D printed scaffold. As revealed in Figure A, proliferating CjSCs and the extracellular matrix they produced gradually filled the 3D printed scaffold to form a stromal layer during step 1, while CjECs formed a confluent epithelial layer on a suitable surface provided by the pre-seeded scaffold during the second co-culture step. The intact epithelial layer was also illustrated by CK13 expression and cell junctions indicated in Figure B.

Conclusions : Compared with other conjunctiva substitute, this 3D printed scaffold presented as a platform which allowed cells to behave following their intrinsic biological properties and better mimicked the local microenvironment and cell interaction in vivo. The tissue engineered conjunctiva construct with a stromal layer and an intact epithelial layer was well established, and could be an option for conjunctival reconstruction as well as an in vitro model for the study of conjunctival diseases.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

 

(A) Confocal microscopic images of bi-layer conjunctiva construct. Red: CjSCs stained with Vimentin. Green: CjECs stained with CK13. Blue: nuclei stained with DAPI. (B) SEM images of bi-layer conjunctiva construct. Top: epithelial layer. Bottom: stromal layer. Black arrow: scaffold fibers. White arrow: cell junction.