Protocol for isolating human BCAM-positive corneal progenitor cells by flow cytometry and cell sorting.

Area of Science:

• Ophthalmology
• Cell Biology
• Regenerative Medicine

Background:

• Basal limbal epithelial cells are vital for corneal surface maintenance and regeneration.
• BCAM-positive cells represent an early transit-amplifying cell population (TAC) with holoclone-forming capacity.
• Understanding these cells is key to developing strategies for corneal repair.

Purpose of the Study:

• To present a detailed protocol for isolating BCAM-positive cells from human donor corneas.
• To enable further research into the factors regulating corneal regeneration.
• To facilitate the study of corneal epithelial differentiation and holoclone formation.

Main Methods:

• Human donor corneas were dissected and dissociated.
• Cells were stained with specific antibodies for flow cytometry.
• BCAM-positive and BCAM-negative cells were purified using cell sorting.
• Purified cells were cultured for holoclone and cell sheet formation assays.

Main Results:

• A reliable protocol for isolating BCAM-positive basal limbal epithelial cells was established.
• The protocol allows for the separation of BCAM-positive and BCAM-negative cell populations.
• The isolated cells are suitable for functional assays related to corneal regeneration.

Conclusions:

• The presented protocol effectively isolates BCAM-positive cells, a key population for corneal regeneration.
• This method supports the investigation of cellular mechanisms underlying corneal epithelial differentiation.
• The findings contribute to advancing research in ophthalmology and regenerative medicine.