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K. P. Kennelly, D. M. Wallace, T. M. Holmes, D. J. Keegan; Optimizing Graft Preparation for Subretinal Cell Transplantation. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5240.
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© ARVO (1962-2015); The Authors (2016-present)
Graft failure remains an obstacle to subretinal cell transplantation. A common transplantation technique used is to culture cells and prepare a concentrated cell suspension for subretinal injection. This study aimed to optimize graft cell preparation by examining if the total number of cells cultured influenced the levels of apoptosis and cell death in the graft.
DH 01, an extended-life mouse retinal pigment epithelium cell line, is currently being used in subretinal graft studies and is cultured for 7 days before transplantation. DH 01 cells were seeded in T75 flasks at increasing cell numbers (5x105 to 2x107) and cultured for 7 days in full media. Then the degree of cell confluence was ascertained by phase contrast microscopy. Cells prepared for transplantation were resuspended in serum-free media and left on ice for 4 hours, stained for annexin V (AnV) and propidium iodide (PI) and analysed by flow cytometry. Control cell suspensions were stained immediately following resuspension. Levels of apoptosis in control and transplant cell preparations were also assessed by DNA fragmentation analysis. Subretinal DH 01 grafts at post-operative Days 1 and 3 were examined for apoptosis by TUNEL-labelling.
Control DH 01 cells seeded at 5x105 were sub-confluent at Day 7 and had low levels of cell death (PI) and apoptosis (AnV) combined (5.62% +/- 1.05). Subjecting these cells to transplant conditions did not increase apoptosis and cell death (5.10% +/- 0.50). Higher seeding levels lead to post-confluence by Day 7 and increasing levels of cell death and apoptosis following transplant conditions: 5 x 106 cells seeded (9.04% +/- 0.79); 107 cells seeded (15.23% +/- 2.40). Apoptosis measured by DNA fragmentation showed no increase under transplant conditions compared to controls in sub-confluent cell cultures. TUNEL-labelling of subretinal grafts showed low levels of apoptosis.
Subjecting DH 01 cells harvested before confluence to transplant conditions does not lead to increased levels of cell death and apoptosis. However, culturing beyond confluence leads to progressively increased levels of cell death and apoptosis. When preparing cells for subretinal transplantation, sub-confluent cells should be used.
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