Cornea lenticule viability and structural integrity after refractive lenticule extraction (ReLEx) and cryopreservation.
OBJECTIVE
To assess and compare keratocyte viability and collagen structure in cornea stroma lenticules collected immediately after refractive lenticule extraction (ReLEx) and one month after cryopreservation.


METHODS
The fresh and cryopreserved human stroma lenticules procured after ReLEx were processed for ultrastructural analysis of keratocytes and collagen fibrils with transmission electron microscopy (TEM), apoptotic cell detection with deoxynucleotidyl transferase-mediated nick end labeling assay (TUNEL) assay, and cultured for keratocyte-specific gene expression analysis using reverse transcriptase polymerase chain reaction (RT-PCR).


RESULTS
The periphery of the lenticule had greater TUNEL-positive cells compared to the center of the lenticule in both fresh and cryopreserved groups. There was an increase in TUNEL-positive cells after cryopreservation, which was significantly higher in the center of the lenticule, but not in the periphery. TEM showed apoptotic, necrotic and viable quiescent keratocytes in fresh and cryopreserved lenticules. Collagen analysis with TEM showed a well preserved and well aligned structure in fresh and cryopreserved lenticules; without significant change in the total number of collagen fibrils but with an increased collagen fibril density (CFD) after cryopreservation. In vitro, isolated keratocytes derived from fresh and cryopreserved lenticules exhibited a typical fibroblastic phenotype. RT-PCR showed a positive gene expression for keratocan (KERA) and aldehyde dehydrogenase 3A1 (ALDH3A1) in cells isolated from fresh and cryopreserved lenticules.


CONCLUSIONS
The stromal lenticules extracted from ReLEx surgery remain viable after cryopreservation. Although they showed a decrease in CFD, the collagen architecture was preserved and there was good cellular viability.
Tissue Engineering and Stem Cell Group, Singapore Eye Research Institute, Singapore.
Karim
K
Mohamed-Noriega
Kah-Peng
KP
Toh
Rebekah
R
Poh
Deepashree
D
Balehosur
Andri
A
Riau
Hla M
HM
Htoon
Gary S L
GS
Peh
Shyam S
SS
Chaurasia
Donald T
DT
Tan
Jodhbir S
JS
Mehta
D000368:Aged; D000444:Aldehyde Dehydrogenase / Q000378:metabolism; D017209:Apoptosis; D015415:Biomarkers / Q000378:metabolism; D003094:Collagen / Q000378:metabolism / Q000648:ultrastructure; D060527:Corneal Keratocytes; D003319:Corneal Stroma; D048988:Corneal Surgery, Laser; D015925:Cryopreservation; D003451:Cryoprotective Agents; D005109:Extracellular Matrix / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D006801:Humans; D020287:In Situ Nick-End Labeling; D046529:Microscopy, Electron, Transmission; D008875:Middle Aged; D009216:Myopia / Q000473:pathology / Q000601:surgery*; D011509:Proteoglycans / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014022:Tissue Survival
D015415:Biomarkers; D003451:Cryoprotective Agents; C490948:KERA protein, human; D011509:Proteoglycans; D003094:Collagen; C480021:ALDH3A1 protein, human; D000444:Aldehyde Dehydrogenase