Multi-layered silk film coculture system for human corneal epithelial and stromal stem cells

Abstract

With insufficient options to meet the clinical demand for cornea transplants, one emerging area of emphasis is on cornea tissue engineering. In the present study, the goal was to combine the corneal stroma and epithelium into one coculture system, to monitor both human corneal stromal stem cell (hCSSC) and human corneal epithelial cell (hCE) growth and differentiation into keratocytes and differentiated epithelium in these three-dimensional tissue systems in vitro.Coculture conditions were first optimized, including the medium, air-liquid interface culture, and surface topography and chemistry of biomaterial scaffold films based on silk protein. The silk was used as scaffolding for both stromal and epithelial tissue layers because it is cell compatible, can be surface patterned, and is optically clear. Next, the effects of proliferating and differentiating hCEs and hCSSCs were studied in this in vitro system, including the effects on cell proliferation, matrix formation by immunochemistry, and gene expression by quantitative reverse transcription-polymerase chain reaction. The incorporation of both cell types into the coculture system demonstrated more complete differentiation and growth for both cell types compared to the corneal stromal cells and corneal epithelial cells alone. Silk films for corneal epithelial culture were optimized to combine a 4.0-μm-scale surface pattern with bulk-loaded collagen type IV. Differentiation of each cell type was in evidence based on increased expression of corneal stroma and epithelial proteins and transcript levels after 6 weeks in coculture on the optimized silk scaffolds.

Advances in Hydrogels in Organoids and Organs‐on‐a‐Chip

“…In one study, silk fibroin was used as scaffolds to support the growth and differentiation of hiPSC‐derived kidney organoids . Silk has been widely used to support growth of cornea, gastrointestinal tissues, and neural tissue due to its properties of biocompatibility, safety and adjustable degradability and its ability to be modified by incorporation of macromolecules. Recently, avidin was incorporated into the silk material, thus biotinylated proteins (e.g., growth factors) can be conjugated to increase their local concentration, which could possibly provide instructive signals to guide tissue morphogenesis in the scaffold .…”

In Vitro Cultivation of Limbal Epithelial Stem Cells on Surface-Modified Crosslinked Collagen Scaffolds

“…Even though our results did not indicate a short-term benefit for in vitro cultivation of LESC, surface patterning might potentially result in an ideal microenvironment for long-term LESC proliferation and preservation. In addition to F- μ CP and 3D fabrication, other groups have suggested surface tethering and bulk incorporation of laminin, collagen type III, Coll-IV, IKVAV, YIGSR, RGD, and vitronectin [46, 59, 78, 79]. All of these possibilities support the promise of collagen hydrogels in tissue engineering, not only in ophthalmology but also in other disciplines such orthopedics [80], dermatology [81], and cardiology [82].…”

Corneal pain and experimental model development

“…Recently, a corneal tissue model was generated to include the stroma, epithelium, and innervation (Wang et al, 2017). A multi-layered construct based on thin silk protein film served as the scaffolding to support the corneal epithelium and stromal layers (Ghezzi et al, 2017;Gosselin et al, 2017), while a surrounding silk porous sponge has been applied to culture cortical neurons in a 3D environment (Tang-Schomer et al, 2014). The inclusion of three cell types in co-culture at an air-liquid interface provides an important advance for the field of in vitro corneal tissue engineering, in that it allows for the study of innervation and corneal tissue development, corneal disease, and tissue responses to environmental factors (Wang et al, 2017) (Fig.…”