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A. Maminishkis, A. Bansal, M. Crawford, S. S. Miller; Primary Human Fetal Retinal Pigment Epithelial (hfRPE) Culture Growth Optimization Using Substrates Obtained from Choroidal Cells Subcellular Fractions and Cell-Conditioned Media. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3062.
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To investigate the effects of choroidal cell conditioned media and different transwell-coating substrates on hfRPE growth in cell culture.
hfRPE cells were obtained as previously described (Maminishkis at al, IOVS, 2006). Adherent retinal cells and choroidal fibroblasts obtained from the same fetal eye were cultured using hfRPE media. Sub-cellular fractions of choroidal cells were obtained using the Qproteome kit. Primary hfRPE were grown on transwells coated with human placenta extracellular matrix (hpECM) or with different cell fractions extracts. For each condition, total tissue resistance (RT) and transepithelial potential (TEP) were measured; EM photographs were taken at two different time points.
In preliminary experiments we compared RT and TEP of hfRPE grown on transwells in 5 different conditions. Cytosolic protein fraction (CPF) obtained from choroidal cells, choroidal cell membranes fraction (CMF), and hpECM were used to coat inserts prior to seeding of hfRPE. Two other conditions were used to produce an enriched media bathing the basal side of the hfRPE. These included hfRPE grown on hpECM coated inserts and with retinal adherent or choroidal cells present in the basal compartment of the insert. Even after 3 weeks in culture, EM images revealed significant differences in cell shape, pigmentation, and appearance of apical processes in hpECM controls compared to transwells coated with CMF and CPF fractions. After 3 weeks in culture, hpECM produced a RT of 119 ± 17 Ω·cm2 and TEP of 2.0 ± 1mV (mean ± SEM, n=8); CMF produced RT of 178 ± 9 Ω·cm2 and TEP of 0.7 ± 0.6 mV (n=3). The largest increases in RT were produced by CPF; RT was 260 ± 28 Ω·cm2 (n=3) and TEP, 1.2 ± 0.4 mV (n=3). After 3 weeks of culture, the presence of choroidal or retinal cells on the basal side of the hfRPE did not significantly alter cell growth. Even after 10 weeks of culture, and starting at 4.5 wks, the presence of choroidal cells in the basal bath produced a RT of 209 ± 45 Ω·cm2 and a TEP of 4.5 ± 0.8mV. Control hfRPE cultures had a significantly larger RT of 615 ± 38 Ω·cm2 and a TEP of 2.0 ± 0.5mV (n=3, p<0.005).
Compared with our standard hpECM coating, cytosolic protein fraction (CPF) obtained from choroidal cells can be used as a substrate on inserts to significantly shorten the time required for hfRPE to reach RT >100 Ω·cm2. This resistance is sufficient to maintain the chemical composition of the apical and basal baths. Co-culture of hfRPE on inserts with choroidal cells in the basal bath stabilized RT at ~200 Ω·cm2 , comparable to RT obtained using native fetal RPE.
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