Abstract
Melanin pigment has a significant role in ocular pharmacokinetics, because many drugs bind at high extent to melanin in the retinal pigment epithelial cells. Most retinal pigment epithelial cell lines lack pigmentation and, therefore, we re-pigmented human ARPE-19 cells to generate a pigmented cell model. Melanosomes from porcine retinal pigment epithelium were isolated and co-incubated with ARPE-19 cells that spontaneously phagocytosed the melanosomes. Internalized melanosomes were functionally integrated to the cellular system as evidenced by correct translocation of cellular Rab27a protein to the melanosomal membranes. The pigmentation was retained during cell cultivation and the level of pigmentation can be controlled by altering the amount of administered melanosomes. We used these cells to study melanosomal uptake of six drugs. The uptake was negligible with low melanin-binders (methotrexate, diclofenac) whereas most of the high melanin-binders (propranolol, chloroquine) were extensively taken up by the melanosomes. This cell line can be used to model pigmentation of the retinal pigment epithelium, while maintaining the beneficial cell line characteristics, such as fast generation of cultures, low cost, long-term maintenance and good reproducibility. The model enables studies at normal and decreased levels of pigmentation to model different retinal conditions.
Highlights
Retinal pigment epithelium (RPE) is a heavily pigmented cell monolayer between the neural retina and highly vascularized choroid
In this study, isolated melanosomes from porcine RPE were administered onto human ARPE-19 cell culture to re-pigment the cells
The ARPE-19 cells were able to internalize melanosomes at levels corresponding up to 1180 pg melanin/cell, an amount that is close to the melanin levels in the isolated porcine RPE cells (1110 pg melanin/cell, Fig. 2a)
Summary
Retinal pigment epithelium (RPE) is a heavily pigmented cell monolayer between the neural retina and highly vascularized choroid. Some disorders cause alterations in the levels of melanin in skin and eye (e.g. forms of oculocutaneous albinism)[8], whereas other disorders (e.g. ocular albinism, retinitis pigmentosa, age-related macular degeneration) reduce the pigmentation in the RPE and other ocular tissues[9]. The RPE is damaged in many ocular diseases (e.g. geographic atrophy) that cause impaired vision[10] Vision threatening diseases, such as age-related macular degeneration, become more common in the aging populations and new drug treatments are needed. It is important to understand pigment related pharmacokinetics at cellular level to find optimal compounds with prolonged drug action in the retina. Human RPE cell line ARPE-1923 is widely used to study retinal cell biology, pathological conditions and pharmacology, but ARPE19 cells lack melanin pigment. Intact melanosomal membrane is expected to extend the retention of pigment bound drug depot in the cells[16]
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