In vitro biomimetic platforms featuring a perfusion system and 3D spheroid culture promote the construction of tissue-engineered corneal endothelial layers

Abstract

Corneal endothelial cells (CECs) are very important for the maintenance of corneal transparency. However, in vitro, CECs display limited proliferation and loss of phenotype via endothelial to mesenchymal transformation (EMT) and cellular senescence. In this study, we demonstrate that continuous supplementary nutrition using a perfusion culture bioreactor and three-dimensional (3D) spheroid culture can be used to improve CEC expansion in culture and to construct a tissue-engineered CEC layer. Compared with static culture, perfusion-derived CECs exhibited an increased proliferative ability as well as formed close cell-cell contact junctions and numerous surface microvilli. We also demonstrated that the CEC spheroid culture significantly down-regulated gene expression of the proliferation marker Ki67 and EMT-related markers Vimentin and α-SMA, whereas the gene expression level of the CEC marker ATP1A1 was significantly up-regulated. Furthermore, use of the perfusion system in conjunction with a spheroid culture on decellularized corneal scaffolds and collagen sheets promoted the generation of CEC monolayers as well as neo-synthesized ECM formation. This study also confirmed that a CEC spheroid culture on a curved collagen sheet with controlled physiological intraocular pressure could generate a CEC monolayer. Thus, our results show that the use of a perfusion system and 3D spheroid culture can promote CEC expansion and the construction of tissue-engineered corneal endothelial layers in vitro.

Template Curvature Influences Cell Alignment to Create Improved Human Corneal Tissue Equivalents

“…Strategies to recreate cornea‐shaped (geometrically curved) stromal tissue equivalents are still rare and mostly based on the production of collagen‐based curved gels, to which cells are subsequently added . Alternatively, mechanical strain has been used to induce spherical curvature on planar collagen hydrogels containing corneal stromal cells .…”

Corneal endothelium tissue engineering: An evolution of signaling molecules, cells, and scaffolds toward 3D bioprinting and cell sheets

“…Perfusion systems provide a higher concentration of dissolved oxygen than static cultures. Therefore, they promote cell proliferation and significantly reduce EnMT (S. Li et al, 2017). Furthermore, biomimetic substrates can mimic the structure of ECM proteins the same as DM, which is believed to have a significant influence on the proliferation and prevention of EnMT in HCECs.…”

“…Perfusion systems provide a higher concentration of dissolved oxygen than static cultures. Therefore, they promote cell proliferation and significantly reduce EnMT (S. Li et al, 2017). C. Zhu & Joyce, 2004), chondroitin sulfate and laminin (Madden et al, 2011), fibronectin (San Choi et al, 2010), and collagen I (San Choi et al, 2013), which increase the proliferation of HCECs.…”

The Ultrafast and Accurate Mapping Algorithm FANSe3: Mapping a Human Whole-Genome Sequencing Dataset Within 30 Minutes

“…This high accuracy made the FANSe algorithms optimal for the analysis of translating mRNA sequencing (RNC-seq) data, which is one of the key resource pillars in the Human Proteome Project (Zhong 2014 ) and it thus facilitates sensitive and accurate identification of proteins from shotgun mass spectrometry data (Chang 2014 ; Zhang et al 2014 ; Xu 2015 ; Zhao 2017 ). Moreover, differentially expressed genes identified from FANSe2-mapped data were perfectly validated by RT-qPCR (Li 2017 ). The FANSe2splice algorithm, which is designed to map spliced reads to a genome based on the FANSe principle, in terms of experimental verifiability outperformed other mainstream spliced mappers, such as TopHat2 (Kim 2013 ), MapSplice2 (Wang 2010 ), HISAT2 (Kim et al 2015 ), and STAR (Dobin 2013 ), and it can detect splice junctions from low-throughput semi-single-cell sequencing datasets (Mai 2017 ).…”