Long-term preservation of donor corneas in glycerol for keratoplasty: exploring new protocols.
OBJECTIVE
To evaluate the role of temperature and adjunctive dehydration in better long-term preservation of human corneas when preserved and stored in glycerol.


METHODS
Different preservation temperatures and effects of adding silica gel in glycerol-preserved corneal tissues were evaluated. Human corneal tissues not suitable for optical keratoplasty initially preserved in McCarey-Kaufman medium were transferred to glycerol and stored at four different temperatures for 3 months as follows: tissues in anhydrous glycerol with and without silica gel at -80°C, -20°C, 4°C and at room temperature (RT). Parameters evaluated included microbial sterility, thickness (Digimatic micrometer), transparency (slit lamp examination, UV-Vis spectrophotometer), mechanical strength (Instron 5848 Microtester), tissue integrity (H&E staining), antigenicity (immunohistochemistry) and ultrastructure of collagen (transmission electron microscopy, TEM).


RESULTS
Microbial test after 3 months of glycerol preservation confirmed sterility of the tissues. The thickness increased in corneas preserved at RT with and without silica gel (p<0.001). RT corneas had the lowest transparency and tensile strength. Tissues in anhydrous glycerol stored with and without silica gel at -80°C were the most transparent (p<0.001) and had the highest tensile strength (p<0.001). Tissue integrity was maintained and expression of Human Leukocyte Antigen D related (HLA-DR) was less in glycerol-preserved corneas at -80°C. TEM studies indicated that parallel alignment of stromal collagen was disrupted at RT-preserved corneas.


CONCLUSIONS
Corneal tissue preserved at -80°C was the best method for preservation as it maintained the sterility, thickness, optical transparency, mechanical strength and ultrastructural features.
Department of Ophthalmology, Dr Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India.
Himi
H
Tripathi
Department of Ophthalmology, Dr Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India.
Manzoor Ul
MU
Mehdi
SMITA Research Labs, Department of Textile Technology, Indian Institute of Technology, New Delhi, India.
Deepika
D
Gupta
Department of Ocular Pathology, Dr Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India.
Seema
S
Sen
Department of Ocular Pathology, Dr Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India.
Seema
S
Kashyap
Department of Anatomy, All India Institute of Medical Sciences, New Delhi, India.
Tapas Chandra
TC
Nag
Department of Pathology, All India Institute of Medical Sciences, New Delhi, India.
Manisha
M
Purwar
SMITA Research Labs, Department of Textile Technology, Indian Institute of Technology, New Delhi, India.
Manjeet
M
Jassal
SMITA Research Labs, Department of Textile Technology, Indian Institute of Technology, New Delhi, India.
Ashwini K
AK
Agrawal
Stem Cell Facility, All India Institute of Medical Sciences, New Delhi, India.
Sujata
S
Mohanty
Department of Ophthalmology, Dr Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India.
Radhika
R
Tandon
D003315:Cornea; D015925:Cryopreservation / Q000379:methods*; D055119:Elastic Modulus; D004548:Elasticity; D054459:Elasticity Imaging Techniques; D005990:Glycerol; D006684:HLA-DR Antigens / Q000378:metabolism; D006801:Humans; D015948:Keratoplasty, Penetrating; D046529:Microscopy, Electron, Transmission; D019188:Organ Preservation Solutions; D013242:Sterilization / Q000592:standards; D013696:Temperature; D014021:Tissue Preservation / Q000379:methods*
D006684:HLA-DR Antigens; D019188:Organ Preservation Solutions; D005990:Glycerol
Cornea; Eye (Tissue) Banking