Isolation, culture, and characterization of endothelial cells from Schlemm's canal.
OBJECTIVE
An important goal in glaucoma research has been to understand the functional contribution of trabecular meshwork (TM) and Schlemm's canal (SC) endothelia to aqueous humor outflow resistance. To date, TM cells are routinely cultured and used as a model by several laboratories. However, there has been only limited success in isolating SC cells. The current objective was to develop a technique for selective isolation and culture of endothelial cells from human SC.


METHODS
The anterior chamber of human cadaveric eyes was cut into eight equal and radially symmetric wedge-shaped pieces. Using a dissecting microscope, a gelatin-coated suture (6-0 sterile nylon monofilament) was gently inserted into the lumen of SC and advanced into the canal. The cannulated pieces of tissue were placed in culture medium and maintained for 3 weeks. Sutures were removed from SC and cells seeded onto 3-cm culture plates. Morphology, growth characteristics, and expression of endothelial surface antigens and other cellular markers were evaluated.


RESULTS
Of the 20 pairs of eyes that were cannulated, primary cells were obtained from 13. All SC cell isolates had a fusiform morphology; formed nonoverlapping, linearly arranged monolayers; and were contact inhibited. Schlemm's canal cell isolates reacted with antibodies specific for CD44 (hyaluron receptor), CD54 (intercellular adhesion molecule-1, ICAM-1), tissue-type plasminogen activator, and TM-inducible glucocorticoid-responsive protein-myocilin (TIGR-MYOC). Unlike TM cells, however, TIGR-MYOC protein was not induced in SC cells after long-term dexamethasone treatment. Schlemm's canal cells endocytosed low-density lipoprotein and acetylated low-density lipoprotein, and in the presence of Matrigel organized into multicellular tubelike structures.


CONCLUSIONS
Cannulation of SC with gelatin-coated suture material is an effective method for the isolation of human SC cells and provides a cellular model to study the potential role of SC cells in aqueous humor outflow function.
Stamer
W D
WD
Department of Ophthalmology, Duke University Medical Center and Veterans Affairs Medical Center, Durham, North Carolina 27710-3802, USA.
Roberts
B C
BC
Howell
D N
DN
Epstein
D L
DL
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D015703:Antigens, CD / Q000378:metabolism; D015415:Biomarkers; D018929:Cell Culture Techniques / Q000379:methods; D002469:Cell Separation / Q000379:methods*; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004727:Endothelium / Q000166:cytology* / Q000378:metabolism; D005136:Eye Proteins / Q000378:metabolism; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D006023:Glycoproteins / Q000378:metabolism; D006801:Humans; D007365:Intercellular Junctions; D008875:Middle Aged; D012590:Sclera / Q000166:cytology* / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000378:metabolism; D014129:Trabecular Meshwork / Q000166:cytology* / Q000378:metabolism; D000099179:Myocilin
D015703:Antigens, CD; D015415:Biomarkers; D003598:Cytoskeletal Proteins; D005136:Eye Proteins; D006023:Glycoproteins; D010959:Tissue Plasminogen Activator; D000099179:Myocilin