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Optimal cryopreservation conditions for limbal stem cells

Poster Details

First Author: A.Ghareeb UK

Co Author(s):    C. Osei-Bempong   M. Lako   W. Armitage   F. Figueiredo           

Abstract Details


Cryopreservation has the potential to make autologous and ex-vivo expanded limbal epithelial tissue readily available for transplant. An extensive investigation of optimal cryopreservation conditions is essential to improve low-temperature banking of limbal tissue. Our aims were twofold: to identify optimal cryopreservation conditions from a group of candidate cryoprotectants employed at different concentrations and to look for the presence of Side Population (SP) cells in cryopreserved limbal epithelial cultures.


Centre for Life, Institute of Genetic Medicine, Newcastle University


Suspension cultures obtained from human corneoscleral rims and co-cultured with 3T3 fibroblasts were cooled to 4°C and Dimethyl Sulphoxide (DMSO), Propylene Glycol (PG) or Ethylene Glycol (EG) was added as a cryoprotectant at concentrations of 5%, 10% or 15%. Cells were incubated with Trypan blue to measure membrane integrity. Reduction of Alamarblue® was used as a marker of metabolic activity. Colony-forming efficiency was measured for cells cooled under each condition. Suspension cultures were cooled with the best performing cryoprotectant at its optimal concentration and frozen for 1 week at -100°C. Cells with SP phenotype were identified by FACS.


We found that increasing cryoprotectant concentration greater than 5% reduces membrane integrity, metabolic activity and colony-forming efficiency (p<0.0001, p=0.002, P<0.0001), while changing cryoprotectant was not a significant determinant of cell tolerance (p=0.9, p=0.18, p=0.25). DMSO performed significantly worse than PG and EG in terms of Alamarblue® reduction (p=0.008, p=0.02). A small population of cells (0.1%) exhibiting SP phenotypes was identified from suspension cultures cryopreserved in 5% PG.


Increasing cryoprotectant concentrations above 5% is detrimental to cell tolerance, while different cryoprotectants performed similarly. Despite measures to reduce the effect of osmotic stress, this is likely to play a role. Thus, reducing the effect of osmotic stress will be vital to future improvements in the cryopreservation of limbal cultures. We have shown that cells exhibiting stem cell behaviours are preserved after cryopreservation with 5% PG, and therefore this cryoprotectant has potential future use in low-temperature eye banking.

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