Human anterior lens capsule as carrier matrix for cultivated human corneal endothelial cells.
OBJECTIVE
To evaluate the potential of human anterior lens capsule (HALC) as a carrier matrix for cultivating and transplanting human corneal endothelial cells (HCECs).


METHODS
HALCs obtained from 12 patients during cataract surgery were exposed to enzyme digestion to dissolve the lens epithelium and were plated with the epithelial side up in 6-well plates. HCECs were harvested from human donor eyes and seeded on HALCs. Cell morphology was assessed with a phase-contrast microscope after 6 hours of incubation and at days 1, 3, 5, and 7. The number of HCECs counted at each capsule was pooled, and the density was expressed as cells per square millimeter after 7 days in culture. Live/Dead viability/cytotoxicity assay was also performed and analyzed by fluorescence microscopy after 7 days of incubation during confluence. Expression of zonula occludens-1, connexin-43, Na/K-adenosine triphosphatase, and cytokeratin-3 (AE5) were investigated by immunohistochemistry.


RESULTS
Seven days after seeding on HALCs, the HCECs were grown to confluence and formed a continuous viable monolayer with a mean cell density of 3012 +/- 109 cells per square millimeter, which mimics native corneal endothelial cells. Immunohistochemistry analysis demonstrated strongly positive staining for AE5, zonula occludens-1, connexin-43, and Na/K-adenosine triphosphatase.


CONCLUSIONS
Cell density and morphology of HCECs on HALCs were similar to those of healthy corneas. Phenotypical properties of HCECs on HALCs imply that the HCEC sheets are capable of maintaining intact barrier and ionic pump functions in vitro. HALCs might, therefore, be recommended as a potential scaffold for ex vivo expansion of HCECs, possibly providing an autologous biologic substrate for therapy of isolated corneal endothelial diseases.
Yoeruek
Efdal
E
University Eye Hospital, Centre of Ophthalmology, Eberhard-Karls University of Tuebinge, Tuebingen, Germany. efdal.yoeruek@med.uni-tuebingen.de
Saygili
Oguzhan
O
Spitzer
Martin S
MS
Tatar
Olcay
O
Bartz-Schmidt
Karl U
KU
Szurman
Peter
P
D000869:Anterior Eye Segment; D002452:Cell Count; D018929:Cell Culture Techniques; D002470:Cell Survival; D017690:Cell Transplantation; D018031:Connexin 43 / Q000378:metabolism; D016039:Corneal Transplantation; D004728:Endothelium, Corneal / Q000166:cytology* / Q000378:metabolism / Q000637:transplantation; D006801:Humans; D007124:Immunoenzyme Techniques; D053558:Keratin-3 / Q000378:metabolism; D007903:Lens Capsule, Crystalline; D008565:Membrane Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008858:Microscopy, Phase-Contrast; D008875:Middle Aged; D010750:Phosphoproteins / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D054457:Tissue Scaffolds; D062826:Zonula Occludens-1 Protein
D018031:Connexin 43; D053558:Keratin-3; D008565:Membrane Proteins; D010750:Phosphoproteins; C569042:TJP1 protein, human; D062826:Zonula Occludens-1 Protein; D000254:Sodium-Potassium-Exchanging ATPase