April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
Viability of human fetal retina/RPE sheets after 4 days cold storage
Author Affiliations & Notes
  • Magdalene J Seiler
    Anatomy & Neurobiology, Univ of California, Irvine, Irvine, CA
  • Gabriel Nistor
    Anatomy & Neurobiology, Univ of California, Irvine, Irvine, CA
    California Stem Cell Inc., Irvine, CA
  • Norman D Radtke
    Retina Vitreous Resource Center, Louisville, KY
  • Robert B Aramant
    Anatomy & Neurobiology, Univ of California, Irvine, Irvine, CA
  • Footnotes
    Commercial Relationships Magdalene Seiler, Ocular Transplantation LLC (C), Ocular Transplantation LLC, patent # 5,941,250; # 8,057,483 (P); Gabriel Nistor, None; Norman Radtke, None; Robert Aramant, Ocular Transplantation LLC (E), Ocular Transplantation LLC patent # 5,941,250; # 8,057,483 (P)
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 695. doi:
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    • Get Citation

      Magdalene J Seiler, Gabriel Nistor, Norman D Radtke, Robert B Aramant; Viability of human fetal retina/RPE sheets after 4 days cold storage. Invest. Ophthalmol. Vis. Sci. 2014;55(13):695.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: For development of clinical retinal transplantation trials, it would be important to maintain viability of the donor tissue for several days after dissection, and to ship the tissue in temperature controlled containers.

Methods: Permission to use fetal tissue for research was obtained from the Western Institution Review Board, Norton Healthcare Research office, and the hSCRO committee of UC Irvine. Retina together with its RPE was dissected from 6 fetal eyes (age 10.5 - 12.5 weeks post-conception, 4 donors) that were received at 1 day (d) after abortion. All tissues had been placed into cold custom-made CO2-independent hibernation medium with B27 supplements immediately after harvest. Eyecups were treated with dispase for 15-20 minutes to separate choroidal vessels from RPE. After dissection, retinas with RPE were cut into 3-5 tissue pieces (size 2-6 mm2). Of each eye, pieces were either fixed immediately, or placed inside plastic nozzles in hibernation medium in shipping tubes (18 pieces). The tubes were placed into shipping containers, and their temperature monitored (between 8 and 2.5 C). After shipping for 1, 2, or 3 d, tissues were fixed (= 4 d after harvest). Cell viability in the retinas was tested by TUNEL staining (counting TUNEL-stained(+) cells per mm2 and % TUNEL-stained cells of total cells in 10 μm cryostat cross-sections). This analysis was performed for all cells, and separately for the inner retinal layers (containing differentiating neurons) and outer neuroblastic layers (that would develop into photoreceptors and other retinal cells).

Results: Of the 18 pieces placed into shipping containers, 14 pieces retained retina-RPE contact. - A low percentage of cells (0.3%) were TUNEL-positive after dissection. Although the number of TUNEL+ cells increased with time, the percentage of TUNEL+ cells remained very low (up to 0.9% on d 2 of shipping). In the inner retinal layers, there was a higher percentage of TUNEL+ cells (0.5-1.9%) than the 0.3-0.6% TUNEL+ cells in the outer neuroblastic layers. The number of TUNEL + cells increased significantly in the inner layers after 1 d shipping, whereas the number of TUNEL+ cells in the outer neuroblastic layers did not increase significantly (from 0.3 to 0.6%) until 3 d of shipping (= 4 d after harvest).

Conclusions: These results demonstrate that intact fetal-derived sheets of retina with RPE remain viable for several days with careful dissection and handling.

Keywords: 426 apoptosis/cell death • 449 cell survival • 688 retina  
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