The apparent permeability coefficients (Papp) are displayed as a range, and the lines represent the average values (= 3). Since LEPI cells seemed to gain differentiated phenotype quickly in culture, we compared their barrier properties after one week in culture to ARPE19 cells that had been cultured on filters for 30 days (Figure 5b). pindolol, metoprolol, betaxolol) ranged from 0.4 10?6 cm/sec to 2.3 10?6 cm/sec depending on the drug lipophilicity. This rapidly differentiating cell collection will be an asset in ocular studies since it is usually very easily managed, it develops and differentiates quickly and does not require specialized culture conditions for differentiation. Thus, this cell collection is suitable for both small level assays and high throughput screening in drug discovery and development. = 12, *** < 0.0001, determined with unpaired = 7) form a barrier against paracellular permeation much like ARPE19 cells Ciprofloxacin hydrochloride hydrate (= 5). The apparent permeability coefficient (Papp) of mannitol was comparable in LEPI cells after culture of 7 days and in ARPE19 cells that were cultured for 30 days. The bars display average values and error bar standard deviation (SD). (c) The permeation of beta-blockers across the tight LEPI monolayers (cultured for 30 days) is related to compound lipophilicity. The apparent permeability coefficients (Papp) are displayed as a range, and the lines represent the average values (= 3). Since LEPI cells seemed to gain differentiated phenotype quickly in culture, we compared their barrier properties after one week in culture to ARPE19 cells Ciprofloxacin hydrochloride hydrate that had been cultured on Ciprofloxacin hydrochloride hydrate filters for 30 days (Physique 5b). LEPI cells form a barrier against paracellular diffusion in one week, as the apparent permeation coefficients (Papp) of mannitol were similar in these two cases (Physique 5b). However, we recorded even lower mannitol permeation values after 4-week LEPI-cultures (<0.9 10?6 cm/s, data not shown) and thus, decided to perform the further barrier evaluation with cells cultured for 4 weeks. The beta-blockers showed different permeation rates across the LEPI cell layer (Physique 5c). The drug permeation in the LEPI cultures showed the comparable rank-order as their lipophilicities (logD7.4 values, Determine 5c, and Table 2) and the Papp values in isolated bovine RPE-choroid (Table 2). The Papp values of beta-blockers are 1.9- to 10.8-fold lower in LEPI cells than in the isolated RPE-choroid (Table 2). Table 2 Permeability of drugs across LEPI cells and bovine RPE-choroid. < 0.05, decided with one-way ANOVA and the HolmCSidak method. The LEPI Papp data exceeded the normality and equivalent variance criteria decided with ShapiroCWilk and BrownCForsythe methods, respectively). 4. Discussion In this study, we present an RPE cell collection, LEPI, that differentiates within 1 to 4 weeks after sub-culture into a phenotype much like main RPE. Unlike the ARPE-19 cells, the LEPI cells rapidly form cobblestone morphology, obvious microvilli, and tight barrier in simple culture conditions. As expected the LEPI and ARPE19 cells show an exact authentication match with the original ARPE19 cell collection (Supplementary Materials), and as expected, the hfRPE cells did not have any matches in the ATCCs STR database. Cobblestone morphology can be achieved in ARPE19 cultures, but this requires months in culture and specialized culture conditions [5,10]. 4.1. Morphology and RPE-Specific Il16 Protein Expression of LEPI Cells Reveal the Differentiated Phenotype LEPI cells display a cobblestone phenotype quickly after sub-culture (Physique 1e), and they even form domes (Physique 4) that have been associated with differentiated phenotype with proper polarity and tight junctions in hfRPE cultures [8,17]. We.