Polarized, Cobblestone, Human Retinal Pigment Epithelial Cell

Mar 20, 2017 - Then, we covalently grafted these optimal synthetic polymers onto 3D substrates (nanofibers) to identify the best surface for RPE cell ...
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Polarized, Cobblestone, Human Retinal Pigment Epithelial Cell Maturation on a Synthetic PEG Matrix Yangzi Tian, Michael R Zonca, Joseph Imbrogno, Andrea M. Unser, Lauren Sfakis, Sally Temple, Georges Belfort, and Yubing Xie ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00757 • Publication Date (Web): 20 Mar 2017 Downloaded from http://pubs.acs.org on March 21, 2017

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ACS Biomaterials Science & Engineering

Polarized, Cobblestone, Human Retinal Pigment Epithelial Cell Maturation on a Synthetic PEG Matrix

Yangzi Tian,1,* Michael R. Zonca Jr.,1,* Joseph Imbrogno,2 Andrea M. Unser,1 Lauren Sfakis,1 Sally Temple,3 Georges Belfort,2,** Yubing Xie1,**

___________________________________________ Yangzi Tian, Michael R. Zonca Jr., Andrea M. Unser, Lauren Sfakis and Yubing Xie 1 Colleges of Nanoscale Science and Engineering, SUNY Polytechnic Institute, 257 Fuller Road, Albany, New York, 12203, USA Email: [email protected] (Y.Xie) Joseph Imbrogno and Georges Belfort 2 Howard P. Isermann Department of Chemical and Biological Engineering and Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute (RPI), Troy, New York, 12180, USA Email: [email protected] (G. Belfort) Sally Temple 3 Neural Stem Cell Institute, One Discovery Drive, Rensselaer, NY 12144, USA Email: [email protected] (S. Temple) _______________________________________________ * Contributed equally Corresponding Authors Footnote: ** Yubing Xie, Ph.D., Colleges of Nanoscale Science and Engineering, SUNY Polytechnic Institute, 257 Fuller Road, Albany, NY 12203 Phone: +1-518 956 7381, Fax: +1-518 437 8687, Email: [email protected] (Y. Xie) ** Georges Belfort, Ph.D., Howard P. Isermann Department of Chemical and Biological Engineering and Center for Biotechnology and Interdisciplinary Studies, RPI, Troy, NY 12180 Phone: +1-518 276 6948, Fax: +1-518 276 4030, Email: [email protected] (G. Belfort)

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Abstract Cell attachment is essential for the growth and polarization of retinal pigment epithelial (RPE) cells. Currently, surface coatings derived from biological proteins are used as the gold standard for cell culture. However, downstream processing and purification of these biological products can be cumbersome and expensive. In this study, we constructed a library of chemically-modified nanofibers to mimic the Bruch’s membrane of the retinal pigment epithelium. Using atmospheric-pressure plasma-induced graft polymerization with a high throughput screening platform to modify the nanofibers, we identified three polyethylene glycol (PEG)-grafted nanofiber surfaces (PEG methyl ether methacrylate, n= 4, 8 and 45) from a library of 62 different surfaces as favorable for RPE cell attachment, proliferation and maturation in vitro with cobblestone morphology. Compared with the biologically-derived culture matrices such as vitronectin-based peptide Synthemax, our newly discovered synthetic PEG surfaces exhibit similar growth and polarization of retinal pigment epithelial (RPE) cells. However, they are chemically defined, easy to synthesize on a large scale, cost-effective, stable with long-term storage capability, and provide a more physiologically accurate environment for RPE cell culture.

To our knowledge, no one has reported that PEG-derivatives directly support

attachment and growth of RPE cells with cobblestone morphology. This study offers a unique PEG-modified 3D cell culture system that supports RPE proliferation, differentiation, and maturation with cobblestone morphology, providing a new avenue for RPE cell culture, disease modeling and cell replacement therapy. Keywords: high throughput screening, PEG, surface chemistry, RPE, stem cells, nanofibrous matrix

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Introduction Age-related macular degeneration (AMD) is the leading cause of vision impairment for those over age 60 and accounts for 6.67% of global blindness.1 It is predicted to affect 196 million people worldwide by 2020 and continue rising to 288 million by 2040.2 According to AMD Alliance International, the global cost of visual impairment due to AMD was estimated to be $343 billion in 2010, including $255 billion in direct healthcare costs.3 Due to the increase of the aging population, the incidence and burden of AMD is expected to increase alarmingly in the coming years. Hence, there is a great need for better understanding of AMD physiopathology and its management and treatment.4 AMD is a late-onset neurodegenerative disease of the retina, stemming from the progressive degeneration of the retinal pigment epithelium (RPE) in the central retina.5 The hallmark of AMD is the buildup of drusen, yellow lipoproteinaceous extracellular deposits, in the Bruch’s membrane (BM) beneath the RPE layer, which usually results in the loss of RPE cells and alteration of extracellular matrix (ECM) deposition in the BM.6-8 Major drusen components are lipids (e.g., esterified cholesterol, phosphatidylcholine) and proteins (e.g., tissue metalloprotein inhibitor 3, clusterin, vitronectin, serum albumin, complement component 9, apolipoprotein E, ATP synthase subunit β, scavenger receptor B2, retinol dehydrogenase).9-10 The increase in size and number of drusen could potentially cause structural disruption in the ECM and degeneration of macular RPE,11 which ultimately results in the death of photoreceptors and loss of central, high acuity vision. Since dysfunction and irreversible damage of the RPE layer is the fundamental factor in the progression of AMD, in vitro culture of RPE cells offers an important platform for understanding RPE biology and AMD physiopathology, as well as for developing cell replacement therapy.

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RPE cells, retinal progenitor cells, or stem cell-derived RPE cells have been cultured with natural or synthetic substrates, including Matrigel,12 collagen,13-15 or other ECM proteins, lens capsules,16 inner limiting membrane from human donors,17 amniotic membranes,18-20 magnetite nanoparticle-enabled

cell

isopropylacrylamide,22-23

sheet,21

hydrogels

(e.g.,

thermo-responsive

poly(methyl methacrylate) (PMMA),16,

24

poly-N-

polyacrylamide25), thin

porous membranes (e.g., polyester PET,26-27 silk fibroin,28-30 silk fibroin/poly(ɛ-caprolactone) (PCL)/gelatin,31 parylene-C,32-34 polyimide,35 expanded polytetrafluoroethylene (ePTFE)36), synthetic thin films (e.g., poly-l-lactic (PLLA), poly-dl-lactic-co-glycolic acid (PLGA),37-40 polyurethanes41-42), PDMS discs,36 PCL nanowires,43 micro- and nanopatterned polyesters (e.g., PCL,44 PLGA,45 poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV)/poly(L-lactideco-D,L-lactide) (P(L/DL)LA, poly(glycerol sebacate) (PGS))46-48) or microcontact printed surfaces,49-52 electrospun nanofibrous matrices (e.g., polyamide,53 PCL,54 poly(L-lactide-co-εcaprolactone) (PLCL),55-56 poly(DL-lactide) (PLA),57 collagen type I and poly(lactic-co-glycolic acid) (PLGA)58 nanofibers), and RGD-containing elastin-like recombinamers (ELR-RGDs),59 in addition to encapsulation in alginate microbeads.60-61 In vivo transplantation of RPE cells on substrates, such as PCL43-44, 56, 62 and PLA57 nanofibers, silk fibroin/ PCL/gelatin31 and collagen membranes,15 polyurethanes films,42 carbon tube bucky membrane paper,63 micro-machined PMMA,24 micropatterned PGS,48 and temperature-responsive culture surfaces64-65 has shown ocular and/or subretinal biocompatibility of these substrates, demonstrating the feasibility of implanting RPE cell-scaffold constructs for cell therapy. Attachment of RPE cells to a biocompatible substrate is essential for preventing cell apoptosis and permitting cell survival.66 Chemical modification of substrates has been used to facilitate RPE cell adhesion and proliferation on conventional cell culture surfaces, such as

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coating with Synthemax,67 Matrigel,68-69 poly-D-lysine-laminin-fibronectin, gelatin, collagen, laminin, and other ECM components.16, 24, 70-73 Additionally, plasma modification methods, such as atmospheric-pressure plasma processing,55 ammonia gas plasma treatment,36,

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plasma

polymers (e.g., acrylic acid (AC), allyl amine (AM) and allyl alcohol (AL)75), and plasma copolymer of allylamine and octadiene in the presence of

bound glycosaminoglycans

(GAGs)76), has attracted some attention for its ability to tailor surface chemistry without alteration of the bulk properties of the substrate. Although there are a variety of synthetic substrates available for RPE cell culture, the challenge is to identify the optimal surface chemistry for functional, cobblestone RPE monolayer formation. In this study, we present a simple and novel high-throughput screening method that first seeks to identify the optimal synthetic surfaces for human retinal pigment epithelial stem cell (RPESC)-derived RPE cell attachment, by screening a comprehensive library of 62 surface chemistries using 2D substrates (flat film). Then, we covalently-grafted these optimal synthetic polymers onto 3D substrates (nanofibers) to identify the best surface for RPE cell proliferation, cobblestone formation and maturation. In addition to human embryonic stem cell-derived RPE14, 77-80

and induced pluripotent stem cell-derived RPE,73, 81-83 human RPESCs represent one of the

promising stem cell sources to derive RPE for disease study and cell therapy.27,

84

We have

discovered that PEG-grafted nanofibers surprisingly demonstrated the best support for human RPESC-derived RPE cell attachment and cobblestone monolayer formation. Although the biocompatibility of PEG for subretinal implantation has been previous reported,85 no research has shown that PEG directly supports attachment and growth of RPE cells. Additionally, previous studies demonstrated that amines permitted the highest level of pluripotent stem cell attachment and PEG methyl ether methacrylates showed the least pluripotent stem cell

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attachment.86-87 This study offers a unique PEG-modified 3D cell culture system that supports RPE proliferation, differentiation, and maturation, not only providing a new platform for RPE cell culture, disease modeling and cell replacement therapy, but also opening a new avenue for PEG-based cell culture.88

2. Experimental Section 2.1. Materials Poly(ether sulfone) (PES) 96-membrane plates (Seahorse Labware, Chicopee, MA) were used as base membranes for creating the library of surface chemistry via plasma-induced graft polymerization for cell attachment experiments. Sixty-two water- or ethanol-soluble vinyl monomers (Sigma-Aldrich, St. Louis, MO) were used as received without further purification. The polymer powder, poly(ether sulfone) (PES), was purchased from BASF (Florham Park, NJ) and used as received. The solvent 1,1,1,3,3,3-hexafluoro-2-isopropanol (HFIP) (Sigma–Aldrich, St. Louis, USA) was purchased as HPLC grade. All culture media components and Synthemax were purchased from Sigma–Aldrich (St. Louis, USA) unless otherwise specified. 2.2. High-Throughput Surface Activation and Grafting of 2D Sheet PES Membranes via Atmospheric-Pressure Plasma (HTP-APP) The modification process used has been described previously, but is reviewed here in brief.89-93 The 96-well membrane filter plates were pre-soaked in Milli-Q water overnight prior to modification. A 100 kDa molecular weight cut-off (MWCO) PES membrane is located and sealed at the base of each well. The 96 membranes were then pre-filtered with 200 µL of Milli-Q water for 2 minutes with a transmembrane pressure of 68 kPa (−20 in. Hg) at room temperature. Membranes located at the base of each well in the 96-well filter plate were exposed to an

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atmospheric-pressure plasma source (model ATOMFLO, Surfx Technologies LLC, Culver City, CA) for 8 minutes at a helium flow rate of 30.0 L min−1, an oxygen flow rate of 0.5 L min−1, and a source-to-membrane distance of 20 mm. The plasma source was operated at 150 W and driven by a radiofrequency power at 27.12 MHz. An XYZ Robot (Surfx Technologies LLC) was used to control the plasma source over the plate with a scan speed of 6 mm s−1. Following exposure to the plasma and subsequent formation of radicals at the membrane surface, 200 µL of each monomer solution at a concentration of 0.2 M was added to each well of the filter plate. Graft polymerization was immediately initiated at 60 ± 1°C for 2 hr. The reaction was terminated by adding Milli-Q water. The 96-well membrane filter plate was then rinsed and soaked with pure ethanol for 24 hr. to remove homopolymer and unreacted monomer residue from the membrane surfaces. Finally, the plates were filtered 2-3 times, before testing, with 200 µL of Milli-Q water for 2 min. through a vacuum manifold (Pall, Port Washington, NY) using a TMP of 68 kPa (−20 in. Hg). 2.3. Fabrication of Poly(ether sulfone) 3D Nanofibers The PES nanofibrous substrates were fabricated by electrospinning. Briefly, a 7.5% (wt/v) solution of PES was prepared by dissolving PES in HFIP. The electrospinning apparatus employed in this study contained a syringe pump (The New Era Pump Systems, Farmingdale, NY), a high voltage supply (Gamma High Voltage Research, Ormond Beach, FL), and a collector plate covered with aluminum foil. Dozens of 5 mm glass coverslips were placed onto the aluminum foil to collect the nanofibers. A voltage of 15 kV was applied to a syringe tip, while the collector plate was grounded. The polymer solution was loaded into a 5 mL disposable syringe with a blunt-end needle, which was controlled by a syringe pump at a feeding rate of 10 µL/min. The distance between the needle tip and collector was adjusted to 14 cm. A charged jet

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of the solution was formed and ejected towards the collector, during which time the solvent evaporated and the fibers were deposited on the surface of the glass coverslips. All electrospinning experiments were performed at a constant room temperature of 25°C and a relative humidity of 25 ± 5%. Finally, the nanofibrous substrates were placed in a fume hood to allow complete solvents evaporation. 2.4. High-Throughput Surface Activation and Grafting of PES Nanofiber via AtmosphericPressure Plasma (HTP-APP). A high-throughput platform approach, that utilizes atmospheric-pressure plasma-induced graft polymerization (HTP-APP), was applied to modify the electrospun PES nanofibers on glass coverslips. All PES nanofibers were deposited onto 5 mm glass coverslips, which were then placed into a 96-well plate using a pair of forceps before soaking. The fibers were pre-soaked in Milli-Q water for 2 hr. The Milli-Q water was then removed and 200 µL of each monomer solution at 0.2 M was added to each well and allowed to soak for 30 min. The monomer solutions were then removed and the plate was exposed to an APP source at a helium flow rate of 30 L min-1 and an oxygen flow rate of 0.5 L min-1 for 8 min with a source to fiber distance of 15 mm, forming reactive radical sites. The plasma source was operated at 150 W and driven by a radio frequency power of 27.12 MHz. An XYZ Robot was used to control the plasma source over the fibers with a scan speed of 6 mm s-1. This covalently grafted each monomer via free radical polymerization. After plasma modification, 200 µL of Milli-Q water was added to each well and left at room temperature for 2 hr. to stop the reaction and remove any unreacted monomer residue. This water was replaced with fresh Milli-Q water after 1 hr. The water was then removed and the modified fibers were stored dry until use. 2.5. Human and Bovine RPE Cell Culture

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Human RPESCs were isolated from cadaver globes obtained from qualified eye banks (such as NDRI) and cultured following a previously described protocol.84 IRB review was conducted and this work was deemed exempt, as the tissue was previously collected and deidentified. The eye banks used here are allowed to release tissue for research purposes with appropriate donor consent. We used human RPESCs isolated from two donors. Isolated human RPESCs were plated onto tissue culture plates coated with Synthemax (Sigma-Aldrich) and cultured

in

RPE

medium:

MEM-α

modified

medium,

2

mM

L-glutamine,

penicillin/streptomycin (1:100), 1% Na-Pyruvate, 10% FBS (fetal bovine serum), supplemented with THT (Taurine, Hydrocortisone, Triiodo-thyronin), and N1 (Sigma-Aldrich). Cells were incubated in a 37 °C, 5% CO2 humidified incubator and the medium was replaced every 3 days. An epithelial monolayer was observed after 1 month of culture and sub-cultured for experiments. Bovine RPE cells were also isolated according to the protocol and cultured in RPE medium as described above. 2.6. Cell Attachment Analysis To quantify RPE cell attachment on various substrates, cell attachment analysis was performed as described previously.86 Briefly, RPE cell pellets were first stained with a Cell Tracker Green CMFDA (5-chloromethylfluorescein diacetate, Molecular Probes, Life Science Technologies) working solution (5 µM) in DMEM and incubated at 37ºC for 30 min. The stained cells were then seeded at 1 × 104 cells/cm on sterile modified PES 96-well membrane plates for initial screening or nanofibrous substrates for validation and incubated at 37ºC, in 5% CO2 for 24 h. Each sample was washed with PBS three times to remove non-attached and loosely attached cells. The fluorescence intensity of Cell Tracker Green labeled cells, which remained attached to the substrate, was measured using a Tecan Infinite M200 plate reader (Tecan US, Research

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Triangle Park, NC) under the excitation wavelength of 494 nm and emission wavelength of 528 nm. The integration time was 20 µs and a total of 25 flashes were taken. The Cell Attachment Index (CAI) was defined and calculated by dividing the fluorescence intensity of cells in each monomer-grafted substrate to the average fluorescence intensity of cells on corresponding control membranes. The experiment was repeated in duplicate. 2.7. Immunocytochemistry After 8 weeks of culture, cells were fixed with 4% paraformaldehyde for analysis of mature RPE marker expression. Briefly, cells were permeabilized with 0.2% Triton X-100, and blocked with normal goat serum (5%) in 1% BSA in PBS for 1 hour, then incubated with the following primary antibodies: rabbit polyclonal anti-OTX2 (ab114138, 1:200, Abcam), rabbit polyclonal anti-MITF (ab20663, 1:200, Abcam) and Goat polyclonal anti-ZO1 (ab99462, 1:100, Abcam) overnight at 4°C. Cells were then incubated with the corresponding Alexa Fluor conjugated secondary antibodies at room temperature for 45 min. These samples were further counter-stained with Alexa Fluor 488 Phalloidin (1:100; Molecular Probes) to label F-actin cytoskeleton filaments, as well as 4’, 6-diamidino-2-phenylindole (DAPI) for cell nuclei. Samples were mounted in antifade reagent and sealed with clear nail polish. 2.8. Confocal Microscopy and Image Analysis Laser scanning confocal microscopy was performed using a Leica SP5 confocal microscope (Leica Microsystems, Mannheim, Germany). All confocal images were captured with a 63X oil-immersion lens, at 1250 x 1250 pixel resolution, using a fixed laser intensity and gain setting. To observe the localization of protein markers, z-stacks with step size of 0.5 µm were captured for all samples. To quantify protein expression and localization, a customdesigned LabVIEW program, BioLIME (Bio-LabView Image Matrix Evaluation), was used to

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analyze the confocal z-stack images. Briefly, the program was designed to quantify cellular morphology and protein localization of confluent cell monolayers using fluorescence from confocal z-stack images.94 Bio-LIME for this study was used to quantify the localization of tight junction protein, ZO-1, as well as counting the expression of OTX-2 and MITF localized within the cell nuclei. 2.9. Statistics Analysis Data are expressed as mean ± standard deviation. For cell attachment analysis, quadruplicates per PES-modified scaffolds were run and this experiment was repeated twice using RPESCs from one donor. For immunocytochemistry analysis, duplicates for each marker expression per PES-modified scaffolds were obtained and this experiment was repeated twice using RPESCs from a second donor. All data were analyzed using two-way ANOVA (GraphPad Prism 6.02; GraphPad Software, Inc., La Jolla, CA). P < 0.05 is considered significant.

3. Results 3.1. High-throughput Screening of 2D Surface Chemistry for RPE Cell Attachment To identify the ideal surface modifications for RPE cell adhesion, 62 monomers were screened, representing nine different chemical functional groups – acids (#s A1-A5, A7, A9 and A10), basic and zwitterionic (#s B1-B5), heterocyclic (#s C1-C5), hydroxyls (#s H1-H4, H6 and H7), methacrylates (#s M1-M11), amines (#s N1-N6), PEGs (#s P1-P9 and P11), aromatics (#s R1-R4) and others (#s O1-O7) from Table S1. These monomers were first successfully grafted onto light sensitive PES sheet membranes through atmospheric-pressure plasma-induced graft polymerization.

Bovine RPE cells and human RPESCs labeled with Cell Tracker Green

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CMFDA were cultured on the chemically modified PES substrates and cell adhesion results were plotted with decreasing CAI values versus different grafted surface chemistries. For bovine RPE cells, the surface chemistries exhibiting the highest CAI values were PEGs (#s P11, P7, P9, P5 and P8), acid (# A1), and aromatics (# R4 and R3) (Fig. 1a). In order to compare the performance of different functional groups with respect to cell attachment, a histogram of surface chemistries was generated based on the categories and values of CAI, e.g.,1.0 (Fig. 1b). Most PEG- and aromatic-modified surfaces were favorable for cell adhesion, mainly occupying the top category (CAI > 1). While acid (# A1) supported cell adhesion (CAI > 1), most of other acid-modified surfaces fell to the lower categories. PES substrates modified with heterocyclic compounds, hydroxyls, methacrylates, amines, basic and zwitterionic functional groups also did not support adhesion of bovine RPE cells, with certain heterocyclic compounds (# C3), amines (#s N1-N6), and in particular, methacrylates (#s M1, M3-5 and M7-10) exhibiting the lowest CAI. This is quite surprising since amines are known to bind cells, while PEGs effectively repel proteins

95-97

that facilitate cell

binding 86. Human RPESCs, which have great potential for cell therapy to treat AMD, were further used to identify the optimal surface chemistries that facilitate RPE attachment using the same high throughput platform.

Amongst chemically modified substrates with the highest CAI,

hydroxyls (#s H2, H1, H4, and H6), acids (#s A1 and A2), heterocyclic (# C5) and PEGs (#s P5, P8 and P6) occupied this category (CAI > 1) (Fig. 2a). All amines (#s N1-N6) exhibited low cell adhesion (CAI < 1) for RPESCs. While some methacrylates (# M11) also allowed significant cell adhesion, all other substrates modified with this type of functional group yielded low attachment (CAI < 1). Other substrates that resulted in little cell attachment were low molecular

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weight PEG monomers, heterocyclic compounds, basic and zwitterions and others (Fig. 2b). The trend of preferred functional groups for cell adhesion of human RPESC-derived RPE was similar to bovine RPE cells in that most PEGs (n ≥ 2) supported RPE cell adhesion while some amines exhibited less cell adhesion than expected.

3.2. Validation of Surface Chemistry Using Chemically Grafted 3D Nanofibrous Matrices To further validate the effect of surface chemistry on RPE cell adhesion, we chose representative chemistries of PEGs and amines along with others and populated them in the same 96-well plates, including acids (#A1 and A3), hydroxyl (# H2), methacrylates (# M11), amines (#s N1, N2, N4 and N5), PEGs (#s P5, P7-P9 and P11), aromatics (# R4) and others (# O3). In addition to cell adhesion to substrates, cell proliferation to form cobblestone RPE monolayer is one of the important characteristics of RPE cells. In order to monitor cell growth on chemically modified substrates, we replaced PES membrane plates (non-transparent) with electrospun PES nanofibers (transparent) with a fiber diameter of 193.8 ± 62.5 nm. The feasibility of modifying PES nanofibers with HTP-APP was demonstrated previously to support 3D growth of stem cells 98

. Therefore, to confirm cell attachment results, we rescreened these selected candidates

with the high CAI in both bovine RPE and human RPESC culture and grafted them to electrospun PES nanofibrous matrices via HTP-APP. Human RPESCs were seeded onto these chemically modified substrates and their corresponding CAI values are presented in Fig. 3. The high CAI (>1.0) in all tested substrates except # P5 and # N5 confirmed the previous results. Although hydroxyl (# H2) showed the highest average CAI value and amine (# N5) the least among this group of surface chemistries, all differences were not statistically significant.

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Proliferation of human RPESCs on these substrates was further examined using optical microscopy. Cells seeded at 1 × 104 cells/cm2 adhered to all chemically modified nanofibers initially. However, as culture time increased, some cultures seemed to proliferate significantly faster than others. Human RPESCs seeded on acids (#s A1 and A3), methacrylate (# M11), amine (# N1) and hydroxyl (# H2) did not proliferate and detached from the substrate after several media changes (Fig. 4a). While cells cultured on amine (# N2), other (# O3) and aromatics (# R4)-modified nanofibers reached similar degree of confluency, the culture produced cells with elongated, spindle-shaped morphology (Fig. 4b). Human RPESCs grown on amines (#s N4 and N5) exhibited limited growth. Notably, RPESC grown on PEG-modified surfaces (#s P7, P8, P9 and P11) exhibited cobblestone morphology at week 2 (data not shown), which resembles mature RPE in vivo. By week 4, only cultures grown on PEG-modified substrates became fully confluent and formed cobblestone RPE monolayers (Fig. 4c). 3.3. Long-term Maturation of RPESC-derived RPE on PEG-grafted Nanofibrous Matrices Among the PEG-modified substrates, #s P7, P8, P9 and P11 resulted in the most uniform cobblestone morphology of human RPESC-derived RPE cells throughout the culture. By week 4, PEG surfaces significantly outnumbered the Synthemax control in proliferation rate and number of mature human RPESC-derived RPE cells (Fig. 4c). This trend continued until week 8, with mature RPE cells occupying the entire #s P8 and P11 modified surface (Fig. 4d). Expression of characteristic RPE markers, OTX-2 and MITF, were examined to determine whether PEG-modified nanofibers could support healthy growth of mature RPE cells derived from human RPESCs. The immunocytochemistry data confirmed that all tested PEGmodified nanofibers (#s P7, P8, P9 and P11) expressed OTX-2 and MITF (Fig. 5a and b). To better compare the differential expression level between samples, we used the Bio-LIME

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program to quantify the confocal z-stack images. By measuring the fluorescence intensity of each protein channel, the LabView program was able to count the number of OTX-2 or MITF expressing cells and calculate the percent expression by dividing that number by the total number of DAPI-stained nuclei (Fig. 5c). At least 3 confocal z-stacks are used for protein expression analysis per sample. Further quantification of the confocal images using Bio-LIME showed that while RPESCs grown on Synthemax coated substrate had the highest OTX-2 expression, the percent expression by cells grown on P8 (PEG, n = 4), P9 (PEG, n = 8) and P11 (PEG, n = 45) were very similar (no statistical significance). On the other hand, cells cultured on P9 and P11 expressed the most MITF. The difference in percent expression is significant when compared to P7, but not to P8 and Synthemax-coated substrate (Fig. 5d). Tight junction protein, ZO-1, and cytoskeleton protein, F-actin, were also examined to determine whether PEG-modified nanofibers could promote apical polarization of junction proteins. Many critical RPE functions depend on the proper formation and polarization of tight junction and adherens junction proteins.99-100 By localizing at the apical side, these junction complexes help to regulate protein trafficking from the subretinal space (apical) to the choroid (basolateral).101 Since the apical surface of RPE is in close contact with the photoreceptor, the correct polarization is also crucial for outer segment phagocytosis. Immunocytochemistry data showed continuous cell-cell junctions throughout the RPE monolayer in all PEG-modified substrates, as well as the Synthemax control (Fig. 5a and b). The cross-section view of the confocal z-stack shows the position of fluorescence with respect to the nucleus (Fig. 6a). Bio-LIME was used again to quantify and compare the percent apical and basolateral polarization of the junction proteins, ZO-1, between different sample substrates. The

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program accomplished this by first measuring the average cell nucleus heights and then the total fluorescence found in the ZO-1 channel. The percent apical and basolateral localization of the ZO-1 fluorescence was determined by establishing a pixel offset using the height of the nucleus. Fluorescence expression in the top 25% of the average nucleus height was considered apically localized, while everything below was localized basolaterally. The quantification showed that the percentage of apical localization in RPESC-derived RPE cells grown on the PEG-modified substrates was very similar to Synthemax (Fig. 6b). In fact, P11 even has a slight higher expression of apically localized ZO-1 than Synthemax, though the difference is not statistically significant. These results indicate that PEG-modified surfaces are comparable to Synthemax, providing an alternative, synthetic surface for RPE monolayer growth. Altogether, PEG (n = 4, 8 and 45) supported human RPESC-derived RPE cell proliferation and maturation, leading to characteristic RPE monolayer formation and marker expression.

4. Discussion This high throughput screening study was conducted to discover the best synthetic alternative for growing human RPE cells, such as human RPESC-derived RPE cells. Cell attachment plays a vital role in maintaining cell morphology and function, as well as controlling stem cell pluripotency.86,

102

Ensuring high cell attachment onto surfaces on which they grow

enables a better control over the nature and frequency of cell division. Various synthetic polymeric membranes, thin films and nanofibrous matrices have been used for RPE cell culture. The capacity to use a 2D high-throughput chemical synthesis and screening platform allows the identification of the optimal surface chemistry for RPE cells. The requirements for the best

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performing surfaces for RPE were: high cell attachment index, formation of polarized “cobblestone” cell morphology that is a characteristic indicative of RPE cell growth, polarization, and expression of mature RPE markers. RPE plays a critical role in the function of the retina and vision in vivo. The major functions of RPE include constitution of the outer blood-retinal barrier, transport of nutrients, ions and water, visual cycle, phagocytosis of photoreceptor outer segment membranes, secretion of factors and signaling molecules, light absorption, protection against photooxidation, and maintenance of immune privilege of the eye.103-104 The establishment of a functional monolayer of RPE cells relies on the attachment of RPE cells to Bruch’s membrane,105 which is the basal lamina beneath the RPE and serves as the vessel wall of the choroid.106 In addition to this structural function, Bruch’s membrane plays an important role in transport as well. In order to facilitate nutrient transport, perforated substrates are beneficial for RPE cell culture and subretinal implantation.107 Therefore, porous substrates with biomolecular coatings have been synthesized to mimic Bruch’s membrane for RPE cell adhesion and growth.

It has been

demonstrated that porous scaffolds (e.g., PCL) promoted human fetal RPE maturity and function when compared with nonporous PCL substrates and Transwell porous membranes, displaying improved pigmentation, increased cell density, superior barrier function, up-regulation of RPEspecific genes, and polarized growth factor secretion.44 Additionally, nanofibers could further mimic the topography of Bruch’s membrane while retaining the porous feature and permeability. Nanofibers demonstrated better in vitro RPE growth compared with smooth counterparts53 and in vivo sub-retinal compatibility56-57. In this study, we used electrospun PES nanofibers as the substrate. Using the atmospheric-pressure plasma-induced graft polymerization method as our high-throughput screening platform in 2D, we were able to identify the PEG-modified surfaces

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as the most favorable modification for RPESC attachment and maturation. Unlike embryonic stem cell adhesion to chemically modified substrates through integrin β1,86 it has been demonstrated that improved RPE cell adhesion to Bruch's membrane is associated with α6β4integrin, a receptor for laminin 5.108 One reason that PEG-modified porous nanofibers facilitate RPESC attachment might be through the interaction of grafted PEGs with integrin α6 and β4. The performance of these PEG-grafted surfaces was compared with Synthemax, which is the current gold standard for stem cell-derived RPE cell culture.67 Our 8-week cell morphology study revealed that 3D PEG-modified surfaces accelerated RPESC proliferation and formation of cobblestone clusters. Through immunocytochemistry analysis, we also discovered that PEG surfaces exhibited similar level of expression of mature RPE markers, OTX2 and MITF, to the Synthemax control. Similar to other biomacromoelcular coatings for RPE cell culture (e.g., Matrigel, collagen, fibronectin, laminin, vitronectin, ECM extracts16, 24, 68-72), Synthemax is a peptide-based surface coating. It is derived from the biological protein vitronectin, which has production limitations associated with biological products, such as downstream processing and purification, lot-to-lot batch variation and limited shelf-life. Compared with this natural material-derived culture matrix, our PEG-modified surfaces are chemically defined, easy to synthesize on a large scale, highly reproducible, cost-effective, stable with long-term storage capability, easily available off-the-shelf and non-immunogenicity. In particular, this study demonstrates for the first time that PEG methyl ether methacrylates (n= 4, 8 and 45) are potential surface chemistries for RPE cell attachment, proliferation and maturation. It provides a new avenue for PEGs as the surface coating for cell culture, in particular, 3D RPE monolayer culture. Traditionally, PEGs have been grafted to other

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polymers to repel proteins, avoiding unwanted adhesion or aggregation during drug and gene delivery.109-114 Due to its limited protein fouling, cytocompatible polymerization for cell encapsulation and tunable biomechanical and biochemical properties (e.g., stiffness, cell adhesive ligand conjugation), PEG has been used for cell encapsulation or 3D culture of aggregates or organoids,115 including liver cells,116 cardiomyocytes,117 pancreatic β-cells,118 neurons,119 chondrocytes120 and 3D tumors121-122. As shown in Table S2, mono-thiolated PEG (molecular weight 5000) has been used to synthesis PEG/PLA copolymer to inhibit RPE cell adhesion and proliferation on micro-contact printed substrates.52 Nanofibrous matrices derived from methyl methacrylate and PEG methacrylate have been used as a substrate for RPE cell attachment, but only after chemical modification of the PEG chain terminus with peptide chains, making the peptides the interacting species instead of PEG.123 Hydrogels of acrylate derivative of PEG and hyaluronic acid have been used for RPE attachment and proliferation for the purpose of repairing aged Bruch’s membrane, but need to be enriched with RGD for cell adhesion.124 The ocular biocompatibility of PEGs has been investigated, indicating that the format (e.g., free in suspension, coating on substrate), molecular weight (e.g., low, medium, high), and dosage (e.g., low, high) of PEG have influence on its ocular biocompatibility.85, 125-128 For in vivo cell replacement therapy, in order to avoid potential ocular toxicity as demonstrated with PEG of molecular weight less than 400 in solution,125-127 we would choose PEG with relatively larger molecular weight such as PEG #P11 (PEG, n = 11, molecular weight = 2080) to graft 3D PES nanofiber matrices for human RPESC attachment, proliferation and differentiation into mature RPE cells. For in vitro RPE modeling, we would choose PEG #P8 (n = 4, molecular weight = 300) to grow RPESC-derived RPE cells since PEG-400 was shown to induce retina

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degeneration,126 mimicking the normal and AMD RPE structure and function, leading to mechanistic understanding, drug screening and testing for AMD.

Conclusion In this study, we have identified PEG methyl ether methacrylates (n= 4, 8 and 45) as the best surface chemistry from a library of different chemical coatings for human RPESC-derived RPE cell attachment, by using a high-throughput platform to screen a library of 62 monomers that have been grafted to 2D PES membranes via atmospheric-pressure plasma. We have further translated the PEG-based surface activation and grafting from 2D sheet membranes to 3D nanofiber matrices. In particular, we have demonstrated that PEG-modified nanofibers support human RPESC-derived RPE attachment, proliferation, polarization and maturation. To our knowledge, no data show that PEG alone without the addition of cell adhesion molecules promote cell adhesion or support monolayer cell growth. This approach provides a new avenue to PEG-modified substrates for RPE cell culture as well as other mammalian cells.

Supporting Information Available The Supporting Information is available free of charge on the ACS Publications website. List of the library of 62 surface chemistries; Comparison of PEGs reported in the literature and discovered here

Acknowledgements This material is based upon work supported by the NYSTEM under Grant No. C024352 (Temple, Stern, Xie). The U.S. Department of Energy, Basic Energy Sciences Division, Grant

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No. DE-FG02-09ER16005, funded all of the PES modification work (Belfort). We thank Dr. Timothy A. Blenkinsop for helpful discussion, and Mrs. Carol Charniga, Mrs. Sue Borden, Dr. Richard Davis and Dr. Jeff Stern for providing human RPESCs. We also thank Dr. Alexander Khmaladze and Dr. James Castracane for the BioLIME LabVIEW software.

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cells. Investigative Ophthalmology & Visual Science 2011, 52 (7), 4085-4090. DOI: 10.1167/iovs.106336.

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Figure Captions Figure 1. High-throughput screening of 2D surface chemistries for bovine retinal pigment epithelial (RPE) cell attachment. (a) Cell attachment index (CAI) values for bovine RPE cells grown on atmospheric-pressure plasma-grafted PES membranes with a library of 62 monomers. (b) Histogram of surface chemistry for bovine RPE cell adhesion. Figure 2. High-throughput screening of 2D surface chemistries for human retinal pigment epithelial stem cell (RPESC)-derived RPE cell attachment. (a) Cell attachment index (CAI) values for human RPE cells grown on atmospheric-pressure plasma-grafted PES membranes with a library of 62 monomers. (b) Histogram of surface chemistry for human RPE cell adhesion. Figure 3. Rescreening of selected representative surface chemistries grafted on 3D PES nanofibrous matrices for cell attachment of human RPESC-derived RPE cells. Figure 4. Optical micrographs of human retinal pigment epithelial stem cell (RPESC)derived RPE cells grown on representative surface chemistry-grafted nanofibrous matrices. (a) Surface chemistries that do not support human RPESC-derived RPE cell proliferation after 1 and 4 weeks, including acids (#s A1 and A3), methacrylate (# M11), amine (# N1) and hydroxyl (# H2), although these surfaces support initial cell adhesion. (b) Surface chemistries that show little sign of human RPE cell growth after 4 weeks, including amines (#s N2, N4 and N5), other (# O3) and aromatics (# R4), although these surfaces allow RPESCs to grow, but fail to form cobblestone RPE monolayer which is characteristic RPE cell growth. (c) PEG-modified nanofibers support human RPE cobblestone formation for 4 weeks, including PEGs (#s P7, P8, P9 and P11), which is comparable to RPE cells grown on Synthemax. (d) Cobblestone formation of human RPESC-derived RPE cells grown on PEG-grafted nanofibers for 8 weeks. Scale bar = 100 µm. Figure 5. Immunocytochemistry of characteristic RPE markers expression in RPESCderived RPE cells grown on PEG-modified nanofibers. (A) Confocal images of OTX2 expression (red) co-stained with ZO-1 (green) and DAPI (blue). (B) Confocal images of MITF expression (red) co-stained with phalloidin to reveal F-actin cytoskeleton filaments (green) and DAPI to reveal cell nuclei (blue). (C) Screenshot of Bio-LIME’s ability to capture the difference in intensity between the DAPI and OTX2 channels. Asterisks represent lack of OTX2 expression in cells. (D) Comparison of percent OTX2 and MITF expression in RPESC-derived RPE cells. Scale bar = 5 µm. **p