P 5
Mechanical isolation and culture of adult retinal pigment epithelium from human donor eyesK. H. Eibl, A. Neubauer, U. Welge-Lüßen, A. Kampik
Purpose: To isolate adult retinal pigment epithelium (RPE) mechanically from human donor eyes to keep its physiology unchanged by the use of proteolytic enzymes for isolation.
Methods: 16 human donor eyes were enucleated after post-mortem times of about 24 hours average duration and transfered to growth medium. After corneal trepanation iris and lens were incised circumferentially and removed together with the vitreous. The retina was cut at the optic nerve head and discarded. With a silicone-tipped gauge 20-cannula or Sautter cannula RPE-patches were detached gently from the choroid. The RPE-patches were transfered to 2 ml DMEM, 20% FCS and cultured in a 35 mm cell culture dish. Growth was observed under standard cell culture conditions, photo-documented and compared between the two instruments used for isolation.
Results: After four days in culture all RPE-patches adhered to the bottom of the cell culture dish. After one week, they showed centrifugal growth starting from the edges of an RPE-patch which was associated with a fibroblastic dedifferentiation of RPE-cells. The original RPE-patch maintained its grade of differentiation. The primary RPE-cell cultures reached confluence after four weeks and can be used for further investigation.
Discussion: A mechanical method for isolation of adult human RPE-cells is a prerequisite for studies on extracellular matrix and retinoid metabolism of these cells. Their integrity and function can be easily disrupted by the use of proteolytic enzymes like trypsin for isolation (von Recum et al., Exp Eye Res 1999). A mechanical method for isolation makes proteolytic enzymes dispensable (Valtink et al., Graefes Arch Clin Exp Ophthalmol 1999).
Conclusion: Adult retinal pigment epithelial cell patches can be isolated mechanically from human donor eyes after corneal trepanation.
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