97th DOG Annual Meeting 1999
P59
CRYOPRESERVATION OF RABBIT CORNEAS WITH TREHALOSE
M. Halberstadt, M. Böhnke, M. Hagenah
Purpose: To evaluate optimal conditions for corneal cryopreservation with trehalose, we previously used tissue from pigs. Since the tissue is not feasible for in-vivo experiments we investigated whether corneal cryopreservation with this method may also be performed with corneas from rabbits.
Methods: We used eye balls from 7 to 9 months old rabbits that had been slaughtered for other purposes. Corneoscleral discs were cryopreserved in MEM-medium containing 4% trehalose in combination with either 15% hydroxyethylstarch (HES) or 2.5% chondroitinsulfate (ChS) and 20% fetal calf serum (FCS) at -196º Celsius and thawed at 37º Celsius in a waterbath. Then the tissue was stored in organ culture in MEM-medium with 10% FCS for 24 hours at 37º Celsius. Endothelial cell density was determined before cryopreservation and after organ culture.
Results: The use of vials especially designed for rabbit corneas resulted in an average cell loss of 38.57 (±9.44)% using 15% HES and 38.58 (±6.95)% using a combination of 2.5% ChS and 20% FCS. Use of the 27mm tall vials resulted in a slightly higher cell loss for corneas preserved in 2.5% ChS and 20% FCS (41.99 ± 10.73%, p=0.896) and in a significantly higher cell loss for corneas preserved with 15% HES (50.33 ±9.07%, p= 0.004). All corneas showed a confluent monolayer without necrotic areas.
Conclusion: The results show that rabbit corneas may be successfully cryopreserved with extracellular cryoprotectants if the experimental environment is adapted to the specific needs of the tissue. Observation of endothelial wound healing demonstrated viability. Thus, rabbit tissue may be used as a model for the experimental transplantation of cryopreserved corneas.
This study was supported by a grant from Deutsche Forschungsgemeinschaft (Ha 1526/3-2)
Dept. Ophthalmology, University of Kiel
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