Limbal organoids and bioengineering sheets based on niche microphysiological systems and bioprinting for corneal injury repair in rabbits

Limbal stem cells (LSCs) reside in the limbal niche and have the potential to regenerate the cornea. Injuries to the limbus lead to limbal stem cell deficiency (LSCD), which can progress to corneal blindness. So, tissue-engineered sheets with LSCs and limbal stem cell niche (LSCN) in vitro are essential for the repair of LSCD. Herein, after sequential culture in Matrigel, U-shaped plates, and mesh microwells, limbal cells derived from primary rabbit cells formed homogeneous limbal organoids (LOs) that exhibited a corneal epithelial phenotype and stem cell characteristics. The cultured sheets growing from LOs exhibited an upregulation of genes and proteins associated with tight junctions, cellular stemness, and proliferation. Moreover, using microstereolithography, we fabricated annular structures from polyethylene glycol diacrylate (PEGDA). Then, we developed a collagen vitrigel (CV) membrane with an LSCN annulus by lithography and imprinting technology. Subsequently, LOs were auto-bioprinted on the CV membrane with LSCN annulus. Notably, P63 expression was markedly elevated in cells located on the niche annulus compared to those on the smooth central part of the CV membrane. Finally, such LO-sheets were transplanted onto an animal model of complete excision of the anterior superficial cornea in New Zealand rabbits, showing markedly accelerated corneal epithelial healing, reduced expression levels of the fibrosis marker α-SMA, and the inflammatory response marker CD45 compared to untreated groups. The in vitro construction of corneal sheets with an LSCN annulus after bioprinting LOs, followed by in vivo transplantation for corneal repair, lays the groundwork for clinical application to treat corneal damage. STATEMENT OF SIGNIFICANCE: The bioengineering sheets containing limbal stem cells (LSCs) and the limbal stem cell niche (LSCN) are essential for repairing limbal stem cell deficiency (LSCD). Herein, after sequential culture, limbal cells became limbal organoids (LOs). Then, we developed a scaffold with an LSCN annulus by lithography and collagen imprinting technology. Subsequently, LOs were auto-bioprinted on such a scaffold to construct bioengineering LO-sheets. Finally, the LO-sheets with LSCN annulus were transplanted onto a rabbit LSCD model of complete excision of the anterior superficial cornea, demonstrating markedly accelerated corneal epithelial healing, reduced expression levels of fibrosis, and a reduced inflammatory response compared to untreated groups. This study lays the groundwork for a successful clinical application aimed at treating corneal limbal damage.