title: Factors affecting successful isolation of human corneal endothelial cells for clinical use.
abstract: Corneal transplantation is a common transplant procedure used to improve visual acuity by replacing the opaque or distorted host tissue with clear healthy donor tissue. However, its clinical utility is limited due to a lack of donor supply of high-quality corneas. Bioengineered neocorneas, created using an expandable population of human donor-derived corneal endothelial cells (HCECs), could address this shortage. Thus, the objective of this study was to evaluate HCEC sourcing with various isolation methods, including enzymatic digestion, culture medium components, and adhesive proteins. HCECs were obtained from corneas obtained from various aged donors after endothelial keratoplasty. Under a dissection microscope, the Descemet's membrane, including the attached corneal endothelium, was stripped from the stroma, and the cells were isolated and expanded by explant culture or by enzymatic digestion with enzymes such as collagenase II, dispase, or trypsin. In order to improve the initial cell attachment, tissue culture plates were coated with collagen IV, fibronectin, or fibronectin-collagen combination coating mix (FNC) before cell plating. We were able to successfully obtain HCECs from 32% (86/269) of donor corneas. Donor age and isolation method influenced the characteristics of the resulting in vitro HCEC culture. Under all conditions tested, FNC-coated plates showed higher quality cultures than the other coatings tested. These results suggest that donor age and HCEC isolation methodology are the two factors that most directly affect the quality of the resulting HCEC culture in vitro. These factors should guide the methodological development for the clinical expansion of HCECs for the generation of bioengineered neocorneas.
authors: Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC, USA.
authors: Jin San
authors: JS
authors: Choi
authors: Eun Young
authors: EY
authors: Kim
authors: Min Jeong
authors: MJ
authors: Kim
authors: Faraaz A
authors: FA
authors: Khan
authors: Matthew
authors: M
authors: Giegengack
authors: Ralph
authors: R
authors: D'Agostino
authors: Tracy
authors: T
authors: Criswell
authors: Gilson
authors: G
authors: Khang
authors: Shay
authors: S
authors: Soker
mesh_terms: D000328:Adult; D000367:Age Factors; D000368:Aged; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D024141:Collagen Type IV / Q000737:chemistry / Q000494:pharmacology; D017364:Collagenases / Q000378:metabolism; D003470:Culture Media / Q000494:pharmacology; D042783:Endothelial Cells / Q000166:cytology* / Q000378:metabolism; D004728:Endothelium, Corneal / Q000166:cytology*; D005260:Female; D005353:Fibronectins / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D008297:Male; D008875:Middle Aged; D016017:Odds Ratio; D012737:Sex Factors
chemical_list: D024141:Collagen Type IV; D003470:Culture Media; D005353:Fibronectins; D017364:Collagenases