Minimum number of adult human retinal pigment epithelial cells
required to establish a confluent monolayer in vitro
- Kwang-Soo Kim; Tongalp H. Tezel; Lucian V. Del Priore
- Dept. of Ophthalmology (안과학)
- Issue Date
- Current Eye Research, Vol.17(10) : 962-969, 1998
Purpose. To determine the minimum number of cells required
to establish a confluent monolayer of retinal pigment epithelium
(RPE) with an epitheloid morphology in vitro.
Methods. Primary or passaged human RPE were harvested by
trypsinization from 6 donors and plated onto bovine corneal
endothelium extracellular matrix-coated tissue culture plastic
in 96-well plates. Plating densities ranged from 1 to 66,000
viable cells/well (0.03–2062 viable cells/mm2) for primary
cells or 1 to 100,000 viable cells/well (0.03–3112 viable cells/
mm2) for passaged cells. The time required to reach confluence
was determined by monitoring the cultures daily until they
reached confluence. Mean cell area and circularity index at
confluence was calculated to determine the effect of different
plating densities on final RPE morphology.
Results. Primary RPE plated at densities above 10 viable cells/
mm2 (320 cells/well) and passaged RPE plated above 2 viable
cells/mm2 (64 cells/well) reached confluence on every occasion.
There was a negative correlation between the plating density
and time required to reach confluence. Plating densities
above 3 viable cells/mm2 (96 cells/well) and 50 viable cells/
mm2 (1600 cells/well) yielded smaller, rounder cells at confluence
for primary and passaged RPE, respectively.
Conclusions. As few as 96 primary RPE cells and 1600 passaged
RPE are required to obtain a confluent, 6mm (4-disc
diameter) patch of RPE in vitro. This suggests that autologous
RPE grafts can be prepared with high efficiency for subsequent
transplantation into the subretinal space in vivo. Curr. Eye Res.
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