Molecular Vision 2001; 7:14-19 <http://www.molvis.org/molvis/v7/a3/> Received 17 August 2000 | Accepted 1 February 2001 | Published 7 February 2001

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Technical Brief

A cell culture medium that supports the differentiation of human retinal pigment epithelium into functionally polarized monolayers

Jane Hu,¹ Dean Bok^1,2,3
 
 

¹Jules Stein Eye Institute, ²Department of Neurobiology, and ³Brain Research Institute, School of Medicine, University of California, Los Angeles, CA

Correspondence to: Dean Bok, PhD, Jules Stein Eye Institute, 100 Stein Plaza, Room B-182, UCLA School of Medicine, Los Angeles, CA, 90095; Phone: (310) 825-6737; FAX: (310) 794-2144; email: Bok@jsei.ucla.edu

Abstract

Purpose: The retinal pigment epithelium (RPE) in vivo is known to have polarized membrane domains that are essential for its normal function. Unless the proper cell culture conditions are used, these polarized features are often lost. In the past, the use of Chee's Essential Medium (CEM) in our RPE cultures has produced functional polarity of the cell monolayers. Unfortunately, except by custom formulation, which is costly, this product is no longer commercially available. We therefore sought to develop a replacement culture medium that would support morphological and functional polarity of RPE membrane domains when the cells are removed from the in vivo milieu.

Methods: To test the performance of this CEM replacement medium in comparison with three other culture media, we grew fetal human RPE to confluence on Millipore Millicell^TM culture wells. We then used Na,K ATPase as a membrane domain marker by displaying it with polyclonal antibodies. This marker was chosen because it is not always properly polarized in culture. Immunofluorescence was imaged by laser confocal microscopy of whole mounted intact monolayers on their Millicell supports. We also used transepithelial resistance (TER) as a measurement of functional polarity as well as bestrophin protein expression as an index of cell differentiation. The expression of Na,K ATPase and bestrophin was confirmed by Western blot analysis of whole RPE cell extracts.

Results: Immunofluorescence labeling of cultured RPE Na,K ATPase was observed exclusively on the apical membrane when the CEM replacement or DMEM with high glucose was used. However Na,K ATPase was not completely polarized in DMEM/F12 medium and the cells did not express detectable Na,K ATPase in DMEM with low glucose. Western blots showed that Na,K ATPase was expressed at similar levels in CEM replacement, DMEM with high glucose and DMEM/F12 as indicated by the intensity of an approximately 100 kDa band representing the a subunit. The CEM replacement gave superior TERs as well, ranging from about 2 to 5.6 fold higher than the other media. Bestrophin protein was readily detectable by Western blot in CEM replacement medium whereas it was barely detectable in DMEM/F12 and undetectable in DMEM with high and low glucose.

Conclusions: We have provided immunocytochemical evidence that the CEM replacement medium supports the appropriate membrane domain expression of Na,K ATPase when the cells are grown on Millicell chambers. Excellent TERs and robust expression of bestrophin are also observed. This combination of features was not observed when other, standard culture media were used. The results suggest that, under these conditions, cultured human RPE develops a highly differentiated and functional polarity appropriate for the in vitro modeling of RPE in vivo function.

Introduction

Cultured retinal pigment epithelium (RPE) has the potential to provide a useful tool for a variety of studies on the cell biology of this important cell layer. However, with respect to ion and water transport for example, one must be certain that the relevant integral membrane transporters are appropriately polarized so that vectorial transport can occur. Na,K ATPase, an essential component in this process, is polarized to the apical membrane domain in vivo where it plays an important role in ion transport as shown in a number of studies on fresh, explanted preparations including human RPE [1-4]. This early, seminal work was based on electrophysiological and pharmacological approaches. Evidence for apical localization of Na,K ATPase was subsequently supported by an autoradiographic study of ³H-ouabain binding in frog [5], as well as biochemical [6] and immunocytochemical studies in rat [7,8].

Chee's Essential Medium (CEM) supported the polarized expression of several transport proteins on the apical membrane domain in our earlier RPE culture studies. Using electrophysiological methods, we showed that the ouabain-sensitive Na,K ATPase [9] and the bumetanide sensitive Na/K/2Cl co-transporter [10] were functionally polarized to the apical membrane of cultured RPE. Additionally, it was demonstrated that the apical and basolateral membrane potassium channels were polarized with respect to their sensitivity to the potassium channel blocker, barium [11], as is the case in explanted RPE. Previous studies have also shown that RPE cells grown in CEM exhibit vectorial release of newly synthesized retinoids [12,13], and vectorial secretion of retinol binding protein and transthyretin [14] from the apical membrane. Thus, in culture, these transport activities reflect their localization in vivo. Unfortunately, CEM is no longer available except by special order and its precise formulation is proprietary. Since the ingredients of CEM are known however, we used concentrations for each component that are congruent with common use in other culture medium formulations in order to arrive at the CEM replacement described here. In the evaluation of this medium, we used immunocytochemistry and Western blotting to determine the comparative expression and localization of Na,K ATPase in cultured fetal human RPE grown in replacement CEM and in some of the standard media used in RPE culture. We also used Western blots for the detection of bestrophin, a protein that is reported to be poorly expressed in two human RPE cell lines [15]. Finally, we measured transepithelial resistances in the cell cultures as an additional index of differentiation.

Methods

RPE culture

The culture methods for human RPE cells were similar to those reported previously [9,16] and are briefly summarized as follows. The tenets of the Declaration of Helsinki were followed, and the patients (or their guardians) gave consent for donation of the tissue. Institutional Human Experimentation Committee approval was obtained for the use of human eyes. Human RPE was collected from eyes of human abortuses of 21 weeks gestation. Small sheets of RPE were dissected from the choroid in calcium- and magnesium-free balanced salt solution (CMF-BSS). The dissociated RPE cells were re-suspended and plated in 100 mm culture dishes in low-calcium culture medium, which contained calcium-free MEM (Eagle) with Earle's salts (Sigma, St. Louis, MO; catalog number M-8028) and the ingredients listed below. The calcium level was then raised to 0.05 mM Ca²⁺ by addition of calcium chloride (7.92 mg/L CaCl₂·2H₂O). The pH was adjusted to 7.35 and the osmolarity was measured to be 300 mmol/kg. The RPE cells were then placed in a 37 °C incubator which contained 5% CO₂ and 95% air. The RPE cultures reached confluence and released RPE cells into the medium in about 10 days. Batches of non-attached cells were successively collected and cryopreserved for future use. This process of amplification of the primary culture was carried out until signs of reduced viability began to appear.

Aliquots of cryopreserved cells were thawed and cultured in normal Ca²⁺ medium (see below) with 1% heat-inactivated calf serum (JRH Bioscience, Lenexa, KS) on Millicell-HA or Millicell-PCF culture wells (Millipore, Bedford, MA) coated with mouse laminin (Collaborative Research, Bedford, MA). The following culture medium was used as a replacement for CEM. All of the reagents were obtained from Sigma. The basal medium is Eagle's Minimum Essential Medium (MEM) with Earle's salts (Sigma M-7647), to which the ingredients listed in Table 1 are added.

To compare the effectiveness of this new medium with other media, we grew human RPE cells from the same donor on Millicell-HA and Millicell-PCF culture wells. We cultured the cells in replacement CEM and several other commercially available media, namely DMEM/F12 (Gibco; 11330-032), DMEM/High Glucose (Gibco, Grand Island, NY) and DME/Low Glucose (Irvine Scientific, Anaheim, CA) with 1% heat-inactivated calf serum. The pH of all media was adjusted to 7.35. Osmolarity of the media was measured with a vapor pressure osmometer (Wescor Inc, Logan, Utah) and is presented in Table 2.

Following at least 1 month of culture, transepithelial resistances were measured with an epithelial voltohmmeter (World Precision Instruments, New Haven, CT). Some of the cells from these cultures were fixed along with their filters in 4% formaldehyde (Ted Pella, Inc., Redding, CA) in 0.1 M phosphate buffer and examined en face by light microscopy.

Immunofluorescence

RPE cells were grown on filter supports for two months before fixation and immunocytochemistry. The filters with their attached cells were excised from their wells, washed three times with PBS (120 mM NaCl, 2.7 mM KCl, and 10 mM NaPO₄, pH 7.4; Sigma), and fixed for 30 min with PBS-buffered 4% formaldehyde (Ted Pella Inc.). The cells were washed twice with PBS and permeabilized with 0.1% Triton-X100/PBS (Boehringer Mannheim, Mannheim, Germany) for 2 min. They were then blocked for 1 h with 1% BSA/PBS (Intergen, Purchase, NY) and 45 ml/ml goat serum (Sigma) prior to a 2 h incubation with gentle shaking at 37 °C with a rabbit polyclonal antibody directed against the Na,K ATPase a subunit (1:100 dilution; Accurate Chemical Co., Westbury, NY) in 0.1% blocking solution/PBS. After the cells were washed 3 times for 10 min each on a shaker, they were incubated for 1 h with FITC conjugated goat anti-rabbit IgG (Molecular Probes, Eugene, OR) at a concentration of 1:200 in 0.1% blocking solution/PBS and washed thoroughly as described above. All incubations and washes were conducted at room temperature unless otherwise indicated. The treated cells on their filters were placed on glass slides, covered with mounting medium (5% n-propyl gallate in 100% glycerol) and coverslipped for viewing.

The whole mount monolayers were examined with a Laser confocal microscope (Carl Zeiss, Inc., Thornwood, NY). The 488 nm line was used to excite the FITC-label. Confocal optical sections of 1-2 mm were taken in the Z-axis. Cross-sections were obtained from the Z-axis sections using the Phi-Z mode.

Western blots

RPE cells were grown on filters as described above and were placed in Laemmli sample buffer to extract the cellular proteins. SDS-acrylamide gels (8% for Na,K ATPase and 12% for bestrophin) were run at a constant voltage of about 180 V in a Tris-glycine buffer (25 mM Trizma base, 192 mM Glycine, 3.5 mM SDS, pH 8.3), using a Bio-Rad Mini-protean II system (Bio-Rad Laboratories, Richmond, CA). The proteins were then transferred to a nitrocellulose membrane overnight at a constant current of 50 A in transblot buffer (25 mM Tris, 192 mM glycine and 20% methanol, pH 8.3; Hoefer Scientific Instruments, San Francisco, CA). The nitrocellulose membrane was blocked with 10% dry milk and 3% BSA in Tris-buffered saline with 0.1% Tween-20 for 1 h and then exposed to the primary antibody for 90 min. The antibody to the Na,K ATPase a subunit was used at a dilution of 1/500 and the P-125 antibody to bestrophin [15] was a 1/2000 dilution of a 2.5 mg/ml stock. Secondary antibodies conjugated to horseradish peroxidase were used at 1/5000 and detected with an ECL Western blotting analysis system (Amersham Corp., Arlington Heights, IL).

Results

Transepithelial resistance

Culture conditions under which the RPE cells are grown play a very important role in the development and in the maintenance of their polarity. The CEM replacement culture medium described in this report supports the development of a high transepithelial resistance. The average resistance was 834±31 ohm/cm² after two months in culture on Millicell HA filters. Values for all of the cultures are presented in Table 3.

It is important to note that all media with the exception of DMEM with low glucose showed a similiar epithelial phenotype when viewed en face by light microscopy (data not shown), although RPE cells grown in the CEM replacement formed a more compact hexagonal monolayer when compared with the others. In spite of their similar general appearance, RPE maintained in the CEM replacement had a superior transepithelial resistance, suggesting the formation of an excellent tight junctional seal.

Immunofluorescence

RPE Na,K ATPase membrane polarity differed among the culture media. Figure 1 shows immunofluorescence of cultured RPE cells grown on polycarbonate filters for two months. The upper portion of each image shows the cross-sectioned RPE monolayer in the Phi-Z mode of the confocal microscope. The lower half of each image shows a single optical section in the Z axis. The upper margin of the lower half of each image in Figure 1 shows the position at which the optical sections were taken in the Z-axis.

For RPE cells grown in CEM replacement medium (Figure 1A), the immunofluorescence decreased as the optical sections proceeded from the apical to the basal surface of the monolayer within the Z-axis. Immunofluorescence was at the level of detection on the apical side only (Figure 1A). Similarly, RPE grown in DMEM/high glucose also exhibited apical labeling (Figure 1C). However, for RPE cells grown in DMEM/F12, the immunoflurescence increased as the optical sectioning proceeded from the apical to basal surface. A single optical section in the Z-axis near the basal membrane of the monolayer reveals that Na,K ATPase labeling is localized mostly on the basolateral surface (Figure 1B). The Phi-Z section in the upper portion of Figure 1B reveals robust labeling of the lateral epithelial plasma membrane.

Western blots of whole RPE cell extracts labeled with an antibody to the a subunit of Na,K ATPase showed a single, specific band at a mass of about 100 kDa (Figure 2). This is consistent with the previously determined mass of the Na,K ATPse a subunit from the RPE [17].

Bestrophin expression

Bestrophin is an RPE-specific protein that has been implicated in the etiology of Best Vitelliform Macular Dystrophy [18]. It has recently been reported that this protein is not expressed in two human RPE cell lines [15]. In these two cases, either DMEM or DMEM/F12 culture medium was used for cell culture. Figure 3 shows a Western blot of cultured human RPE grown for two months in the various media discussed above. Lane 1 represents DMEM with low glucose, lane 2 DMEM with high glucose, lane 3 DMEM/F12 and lane 4, replacement CEM. Human RPE grown in CEM replacement (lane 4) is the only experimental sample with significant expression of bestrophin. The major cross-reacting doublet at about 42 kDa is clearly not bestrophin-related because its intensity is about the same in all lanes.

Discussion

We have presented immunocytochemical and Western blot evidence that the culture medium described here supports the appropriate membrane domain expression of Na,K ATPase in cultured human RPE. Our results are consistent with our previous electrophysiological studies showing apically-polarized Na,K ATPase in similarly cultured human RPE, as well as other studies on non-cultured RPE from bullfrog, bovine, and human RPE-choroid preparations [1-8]. Our results further suggest that the culture conditions reported here are sufficient to support a level of cell polarization and differentiation that approaches that of in vivo RPE. These cultured cells also express other proteins that have been problematic when other culture conditions are used, namely bestrophin and RPE-65 (data not shown). Another study just published provides evidence that hyaluranon, a major glycosaminoglycan in the interphotoreceptor matrix is apically-secreted by human RPE cells grown under these conditions [19]. Finally, the TERs elicited by replacement CEM are superior to the other culture media used by a factor ranging from 2 to 5.6 fold.

Clearly, the data presented here support our belief that proper culture conditions are essential for RPE cell differentiation, for tight junction formation, and for polarized distribution of membrane proteins such as Na,K ATPase. We do not yet know the critical components responsible for these effects, but this can be worked out by systematic elimination of individual components in future studies.

Acknowledgements

This work was supported by USPHS grants EY00444 and EY00331 and a Foundation Fighting Blindness Center Grant to DB. DB is the Dolly Green Professor of Ophthalmology at UCLA. The authors gratefully acknowledge the gift of the Pab-125 anti-bestrophin antibody from Dr. Alan Marmorstein.

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