Confluent monolayers of cultured human fetal retinal pigment epithelium exhibit morphology and physiology of native tissue

Abstract

Purpose: Provide a reproducible method for culturing confluent monolayers of hfRPE cells that exhibit morphology, physiology, polarity, and protein expression patterns similar to native tissue.

Methods: Human fetal eyes were dissected on arrival, and RPE cell sheets were mechanically separated from the choroid and cultured in a specifically designed medium comprised entirely of commercially available components. Physiology experiments were performed with previously described techniques. Standard techniques were used for immunohistochemistry, electron microscopy, and cytokine measurement by ELISA.

Results: Confluent monolayers of RPE cell cultures exhibited epithelial morphology and heavy pigmentation, and electron microscopy showed extensive apical membrane microvilli. The junctional complexes were identified with immunofluorescence labeling of various tight junction proteins. The mean transepithelial potential (TEP) was 2.6 +/- 0.8 mV, apical positive, and the mean transepithelial resistance (R(T)) was 501 +/- 138 Omega . cm(2) (mean +/- SD; n = 35). Addition of 100 microM adenosine triphosphate (ATP) to the apical bath increased net fluid absorption from 13.6 +/- 2.6 to 18.8 +/- 4.6 microL . cm(-2) per hour (mean +/- SD; n = 4). In other experiments, VEGF was mainly secreted into the basal bath (n = 10), whereas PEDF was mainly secreted into the apical bath (n = 10).

Conclusions: A new cell culture procedure has been developed that produces confluent primary hfRPE cultures with morphological and physiological characteristics of the native tissue. Epithelial polarity and function of these easily reproducible primary cultures closely resemble previously studied native human fetal and bovine RPE-choroid explants.

Figures

FIGURE 1

The main steps in the preparation of hfRPE cell cultures.

FIGURE 2

Microphotograph of cultured RPE cells grown on inserts for 4 weeks, taken with an inverted microscope. As in native tissues, these cells formed a single monolayer, were well pigmented, and were arranged in a regular hexagonal mosaic. Magnification, x20.

FIGURE 3

Light microscope images of RPE cultured in a flask at various times: (A) 1, (B) 5, (C) 12, and (D) 14 days.

FIGURE 4

(A) Electron micrograph of RPE cells on cell culture inserts. Each cell exhibited large apical processes, pigmentation was primarily located on the apical side, and nuclei were located close to the basal membrane. Cells appear to be growing in a single monolayer with well-pronounced tight-junctional complexes. (B, C) Basal side of RPE cell image. (C) Many RPE cells contain laminar-deposit–like structures apparently secreted through the RPE basolateral membrane (arrow). (D) Tight-junctional complex. Magnification: (A) x5,000; (B–D) x50,000.

FIGURE 5

Representative immunohistochemistry staining of hfRPE cultures grown on cell culture inserts. Bottom of each panel is an en face view of a cell culture monolayer shown in a maximum-intensity projection through the z-axis. Top of each panel is a cross section through the z-plane of multiple optical slices. Blue: DAPI-stained nuclei; red: ZO1-stained nuclei. Green: occludin (A), claudin-19 (B), claudin-10 (C), CRALBP (D), RPE65 (E), cytokeratin (F), bestrophin (G), NaK-ATPase (H), and ezrin (I).

FIGURE 6

Western blot identifying the six proteins in native hfRPE cultured cells. Cells were cultured in Primaria flasks.

FIGURE 7

The effect of changing apical bath potassium on TEP, RT, VA, VB, and RA/RB. Reduction of apical [K]o from 5 to 2 mM (solid bar) increased TEP (top) by ≈1.8 mV and RT by ≈18 Ω· cm2 (solid line and open diamonds, respectively). Concomitantly, both membranes rapidly hyperpolarized (bottom, solid lines), and RA/RB increased by threefold to 0.3 (bottom, open triangles). Representative of nine similar recordings.

FIGURE 8

The effect of changing apical [K]o from 5 to 2 mM (solid bar) on TEP, VA, VB, and RA/RB in the presence of 1 nM barium in the apical bath (hatched bar). Addition of apical Ba2+ depolarized both membranes (bottom and top traces denoted as in Fig. 7), decreased TEP and increased RT. RA/RB increased twofold, from 0.2 to 0.4. Apical Ba2+ blocked all the [K]o-induced electrical changes. Representative of four similar recordings.

FIGURE 9

The effect of changing basal bath [K]o. Bottom and top traces defined as in Figure 7. Increasing basal [K]o from 5 to 50 mM (solid bar) depolarized VA and VB, increased TEP (≈3 mV). RT transiently decreased and then increased (≈19 Ω· cm2). RA/RB increased twofold, from 0.2 to 0.9. Representative of three similar recordings.

FIGURE 10

Apical epinephrine-induced electrical responses. Bottom and top traces defined as in Figure 7. Epinephrine (10 nM) was perfused into the apical bath (solid bar) and depolarized VA and VB and increased TEP. RA/RB almost doubled from 0.2 to 0.35 and RT decreased by ≈ 10 Ω· cm2. This is representative of four similar recordings.

FIGURE 11

Apical ATP-induced changes in hfRPE membrane voltage and resistance. Bottom and top traces defined as in Figure 7. Addition of ATP (100 μM) to the apical bath (solid bar) caused a rapid depolarization of both membranes and TEP initially decreased. RT decreased and RA/RB increased twofold to 0.4. Representative of 10 similar recordings.

FIGURE 12

ATP-induced changes in TEP, RT and fluid transport. Top: JV is plotted as a function of time and net fluid absorption indicated by positive values. No JV measurements were made during the solution composition changes (period marked by “probe out”). Bottom: TEP and RT plotted as a function of time in control Ringer and in the presence of 100 μM ATP. ATP increased JV by 6 μL · cm−2 per hour, decreased RT by ≈30 Ω· cm2, and transiently decreased TEP by ≈3 mV.

FIGURE 13

Polarized secretion of VEGF and PEDF in cultured human fetal RPE. Media from apical and basal baths were collected separately at 8 weeks after the cells were seeded onto cell culture inserts. ELISA for VEGF and PEDF were performed in triplicate for each of the 12 wells (24 samples). VEGF protein in the basal bath was 1.7-fold higher than in the apical bath (n = 9; P < 0.005), whereas PEDF protein in the apical bath was 2.3-fold higher than in the basal bath (n = 9; P < 0.05).