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Stephen C. Kaufman, Jillian Ewel, Ching Yuan; Corneal Limbal Stem Cell Cryopreservation. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5123.
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To characterizes cryopreserved limbal stem cell tissue and examine the feasibility of ex-vivo limbal stem cell expansion after thawing.
Corneolimbal explants were obtained from human donor corneas, which were then placed in tissue culture using an amniotic membrane (AM) bed. The expanded limbal stem cell cultures were cryopreserved, then revived and stained with propidium iodide (PI). Additional corneolimbal tissue samples were stored in cryoprotectants and kept in liquid nitrogen for 1 month before being revived and expanded in tissue culture in the same manner. Indirect immunofluorescence stainings were performed for stem cell markers (p63-deltaN, ABCG2 and vimentin) and a corneal stem cell differentiation marker (Cytokeratin K3). The samples were further evaluated using Western blots and reverse transcription-PCR (RT-PCR).
Direct cryopreservation of the corneolimbal tissue retained extensive proliferation potential. The explant cultures of the cryopreserved corneolimbal tissue also exhibited strong staining for p63, ABCG2 and vimentin, but weak staining for K3. Western blots and RT-PCR corroborated the immunostaining results. The cryopreserved limbal stem cell expanded cultures on AM resulted in an 88% cell viability after 1 month of cryopreservation, as tested by PI staining.
Our study demonstrates that direct cryopreservation of limbal stem cell explants and expanded limbal stem cell cultures can be revived with good cellular survival and maintenance of the corneal stem cell markers. Thus, a single cornea could provide stem cell tissue that could be stored and used for multiple transplant efforts.
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