Jeremy Mathan, Salim Ismail, Trevor Sherwin

Purpose:
We have previously developed a cell-culture approach to form stem cell enriched spheresfrom the human peripheral cornea. We propose touse each sphere as a transplantable element with aunique microenvironment for limbal stem cellrepopulation and limbus reconstruction for improvedLSCD treatment.

Method:
A microsurgical technique for the implanta-tion of spheres into human corneoscleral rims wasexplored and developed. Fluorescence based cellimaging approaches were trialled for implanted spherevisualization. Immunohistochemistry was used tocharacterize spheres pre and post-implantation. Stereoand fluorescence microscopy was used to assess sphereimplantation, survival and behaviour in tissue.

Results:
The Calcein AM cell-permeant dye effec-tively facilitated visualisation of viable cells that per-sisted up to 241 hours post implantation (T241h). Asloping, wedge shaped incision enabled all implantedspheres to remain in place for the duration of eachexperiment (up to T241h post implantation). Evi-dence of three dimensional, polarized, radial cellmigration out of the sphere into the foreign limbalenvironment was observed within T25h and theextent of cell migration increased over time. Spheresimplanted in close proximity showed a cell migrationpattern from each sphere in the direction of eachother. In an inverted culture setting, cells migratedfrom spheres centripetally from peripheral to central cornea.

Conclusion: A specific approach for delivering stemcell enriched spheres for repopulating the limbus hasbeen developed in vitro and this system will facilitatecorneal repair studies for eventual improved LSCDtreatment.