Human stem cell based corneal tissue mimicking structures using laser-assisted 3D bioprinting and functional bioinks

Abstract

There is a high demand for developing methods to produce more native-like 3D corneal structures. In the present study, we produced 3D cornea-mimicking tissues using human stem cells and laser-assisted bioprinting (LaBP). Human embryonic stem cell derived limbal epithelial stem cells (hESC-LESC) were used as a cell source for printing epithelium-mimicking structures, whereas human adipose tissue derived stem cells (hASCs) were used for constructing layered stroma-mimicking structures. The development and optimization of functional bioinks was a crucial step towards successful bioprinting of 3D corneal structures. Recombinant human laminin and human sourced collagen I served as the bases for the functional bioinks. We used two previously established LaBP setups based on laser induced forward transfer, with different laser wavelengths and appropriate absorption layers. We bioprinted three types of corneal structures: stratified corneal epithelium using hESC-LESCs, lamellar corneal stroma using alternating acellular layers of bioink and layers with hASCs, and finally structures with both a stromal and epithelial part. The printed constructs were evaluated for their microstructure, cell viability and proliferation, and key protein expression (Ki67, p63α, p40, CK3, CK15, collagen type I, VWF). The 3D printed stromal constructs were also implanted into porcine corneal organ cultures. Both cell types maintained good viability after printing. Laser-printed hESC-LESCs showed epithelial cell morphology, expression of Ki67 proliferation marker and co-expression of corneal progenitor markers p63α and p40. Importantly, the printed hESC-LESCs formed a stratified epithelium with apical expression of CK3 and basal expression of the progenitor markers. The structure of the 3D bioprinted stroma demonstrated that the hASCs had organized horizontally as in the native corneal stroma and showed positive labeling for collagen I. After 7 days in porcine organ cultures, the 3D bioprinted stromal structures attached to the host tissue with signs of hASCs migration from the printed structure. This is the first study to demonstrate the feasibility of 3D LaBP for corneal applications using human stem cells and successful fabrication of layered 3D bioprinted tissues mimicking the structure of the native corneal tissue.

Corneal bioprinting utilizing collagen‐based bioinks and primary human keratocytes

“…Besides the studies presented by Sorkio et al () and Isaacson et al (), only a few other studies utilized fabrication techniques to develop corneal implants. Ortega et al () presented a novel approach to corneal stem cell delivery by microfabrication of a corneal limbal implant.…”

“…In recent years, biofabrication and 3D bioprinting approaches for corneal tissue engineering have been investigated (Isaacson, Swioklo, & Connon, ; Ludwig, Huff, & Zuniga, ; Ortega, Deshpande, Gill, MacNeil, & Claeyssens, ; Sorkio et al, ). Bioprinting is an emerging technology that can be used to create a native structure of human corneal stroma, by printing collagen‐based constructs layer‐by‐layer to achieve a 3D configuration.…”

“…In recent years, biofabrication and 3D bioprinting approaches for corneal tissue engineering have been investigated (Isaacson, Swioklo, & Connon, 2018;Ludwig, Huff, & Zuniga, 2018;Ortega, Deshpande, Gill, MacNeil, & Claeyssens, 2013;Sorkio et al, 2018).…”

“…Although past studies have paved the way for future work on corneal tissue bioprinting, they lack the balanced biofunctionality, optical and geometrical characteristics similar to that of native human tissues. The main disadvantages of past studies employing bioprinting strategies to create corneal stromal substitutes were poor optical properties of the printed constructs (Isaacson et al, 2018;Sorkio et al, 2018), inaccurate geometries of the fabricated lenses (Ortega et al, 2013), and poor cell viability and phenotype after the bioprinting or biofabrication process (Isaacson et al, 2018;Sorkio et al, 2018). Previous biofabrication studies used laser-based (Sorkio et al, 2018), micro-extrusion-based (Isaacson et al, 2018), or micro-stereolithography-based processing technologies.…”

Bio‐Orthogonally Crosslinked, In Situ Forming Corneal Stromal Tissue Substitute

“…Corneal structure has been mimicked using laser-assisted 3D bioprinting, where collagen type I was used for the base of the bio-ink. [27] Griffith and collaborators synthesized an artificial cornea using collagen-chondroitin sulfate and glutaraldehyde as a crosslinking agent. [28] More recently, the same group tested recombinant human collagen (RHC) type III scaffolds, crosslinked by EDC/N-hydroxysuccinimide (NHS) that were implanted as pre-formed corneal buttons.…”