Technical Reference #1607

1607.

Engineering retinal progenitor cell and scrollable poly(glycerol-sebacate) composites for expansion and subretinal transplantation Stephen Redenti a; William L. Neeley b; Santiago Rompani c; Sunita Saigal a; Jing Yang d; Henry Klassen d; Robert Langer b; Michael J. Young, Harvard Medical School, Biomaterials, 30(1607), (2009)
Link To Paper

Abstract:
Retinal degenerations cause permanent visual loss and affect millions world-wide. Presently a noveltreatment highlights the potential of using biodegradable polymer scaffolds to induce differentiation anddeliver retinal progenitor cells for cell repl

Keywords:
biodegradation; cell adhesion; elastomer; stem cell; nerve tissue engineering; ophthalmology

Materials & Methods:
2.1. Mouse progenitor cell isolation and culture All experiments were performed according to the Schepens Eye Research Institute Animal Care and Use Committee and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Isolation of mRPCs was performed as previously described [4]. Retinas were isolated from post-natal days 0–3 enhanced green fluorescent protein positive (GFPþ) transgenic mice (C57BL/6 background). Pooled retinas were dissociated by mincing and digested with 0.1% type 1 collagenase (Sigma–Aldrich; St. Louis MO) for 20 min. The liberated mRPCs were passed through a 100 mm mesh filter centrifuged at 850 rpm for 3 min re-suspended in culture medium Neurobasal (NB; Invitrogen-Gibco Rockville MD) containing 2 mM L-glutamine 100 mg/ml penicillin–streptomycin 20 ng/ml epidermal growth factor (EGF; Promega Madison WI) and neural supplement (B27; Invitrogen-Gibco) and plated into culture wells (Multiwell Becton Dickinson Labware Franklin Lakes NJ). Cells were provided 2 ml of fresh culture medium on alternating days for 3 weeks until mRPCs were visible as expanding non-adherent spheres. mRPCs were passaged 1:3 every 7 days. 2.2. Polymer fabrication 2.2.1. Microfabrication of polydimethylsiloxane (PDMS) All fabrication procedures were carried out in a class 10000 clean room. An 80 mm thick layer of SU8-2050 was spin-coated on a silicon wafer (4 inch diameter) following the manufacturer’s instructions. The photoresist was patterned using a transparency mask (PageWorks Cambridge MA) with the ink-side down and developed using washes of propylene glycol monomethyl ether acetate and isopropanol. The patterned silicon wafer was prepared for PDMS replica molding by treating with a low surface energy release agent (tridecafluoro-1122-tetrahydrooctyl) trichlorosilane. Briefly two drops of (tridecafluoro-1122-tetrahydrooctyl) trichlorosilane were applied to a glass slide which was placed on the floor of a vacuum chamber containing the patterned silicon wafer. A vacuum was applied and the (tridecafluoro-1122-tetrahydrooctyl) trichlorosilane vapor allowed to react with the wafer for at least 20 min. The PDMS negative mold was prepared from the patterned silicon wafer as described [9]. 2.2.2. Fabrication of PGS scaffolds Fabrication of the PGS scaffoldswas also carried out in a class 10000 clean room. The PDMS negative mold was oxidized by plasma treatment for 1 min to create a hydrophilic surface [8–10]. A 61.5% aqueous sucrose solution (0.2 mM filtered) was spin-coated at 3000 rpm for 30 s on the oxidized PDMS mold within 5 min of plasma treatment. The sucrose-coated PDMS was immediately baked at 135 C in an oven for 10 min and then transferred to a 120 C hotplate. Approximately 6.5 g of molten PGS (150 C) were spin-coated at 3000 rpm for 30 s on the sucrose-coated PDMS molds. The PGS on the PDMS mold was cured at 120 C under a vacuum of 15 mTorr for 48 h. Subsequently the mold was submerged in ddH2O for 16 days to loosen the PGS from the PDMS mold. The PGSwas precut into pieces using a razorblade and the pieces were gently peeled off the PDMS mold while submerged in ddH2O using forceps. To examine the PGS scaffold by scanning electron microscope (SEM) the scaffold was coated with Au/Pd using a Hummer Sputter Coater according to the manufacturer’s instructions using an evaporator (Anatech Inc. Hayward CA) [17]. 2.3. Polymer preparation cell seeding and culture PGS scaffolds were cut with a sterile scalpel to 1 1 mm for proliferation immunocytochemistry and explants/transplants and to 2 2 mm for transplant simulation protocols. In our control analyses of mRPC proliferation we seeded known numbers of cells into culture wells with a piece of PGS covering the well floor. The proliferation rates of mRPCs in culture wells alone or on PGS in identical wells showed no significant differences. Also 1 1 mm sections of glass were cut from cover glass (VWR) for differentiation (Imaris) and calcium imaging controls and prepared in an identical manner to PGS for cell seeding. PGS and glass squares were incubated in 70% ethanol for 24 h and rinsed 3 times with Phosphate Buffered Saline (PBS) Solution. PGS scaffolds were placed into single wells of 12 well culture plates and incubated in 100 mg/ml mouse laminin (Sigma) in PBS for 1 h. Polymers were then rinsed 3 times with PBS and transferred to 0.4 mm pore culture well inserts (Falcon) in 12 well plates. Scaffolds were then submerged in 1 ml of NB and incubated for 1 h at 37 C. Cultured GFPþ mRPCs were dissociated into single cell suspensions and seeded onto PGS membrane and glass. The total volume of NB in each well was brought to 2 ml with NB media and mRPCs were allowed to proliferate on the polymer for 7 days at 37 C. 2.4. Scanning electron microscopy Prior to imaging the cells were fixed and dehydrated. Each sample was rinsed twice in PBS and then soaked in a primary fixative of 3% glutaraldehyde 0.1 M sodium cacodylate and 0.1 M sucrose for 72 h. The surfaces were subjected to two 5 min washes with a buffer containing 0.1 M sodium cacodylate and 0.1 M sucrose. The cells were then dehydrated by replacing the buffer with increasing concentrations of ethanol for 10 min each. The cells were dried by replacing ethanol with hexamethyldisilazane (HMDS) (Polysciences) for 10 min and subsequently air-dried for 30 min. As discussed previously following mounting samples were sputtercoated with a 15 nm layer of Au/Pd at a current of 20 mA and a pressure of 0.05 mbar for 45 s. SEM imaging was conducted on a FEI XL30 Sirion Scanning Electron Microscope at 5 kV. 2.5. Cell proliferation on PGS Expansion of GFPþ mRPCs was analyzed on PGS. To establish a standard mRPC population curve total mRPC GFPþ signals were detected from known cell number curves (n¼ 5) from 1 103 to 1.5 105 cells in a 96 well plate using a Tecan Genios microplate reader. 1 1 mm PGS seeded with 2.5 105 mRPCs were cultured and imaged for 7 days. Total GFPþ emissions from mRPCs on each polymer were taken at days 1 3 and 7 under identical conditions. The signals from mRPCs on PGS and standard population curve signals were then correlated to establish cell density on each day. After the initial seeding of cells a Spot ISA-CE camera (Diagnostic Instruments Sterling Heights CA) attached to a Nikon Eclipse TE800 microscope was used to visualize cell proliferation across the surface and within the pores of PGS. The composites were also imaged at 10 magnification on days 1 3 and 7. 2.6. Immunofluorescence After culturing mRPCs for 7 days mRPC–PGS and mRPC–glass composites were rinsed 3 times with PBS (warmed to 37 C) and fixed in 4% paraformaldehyde for 1 h. mRPCs to be analyzed for polymer-influenced differentiation were then processed for immunocytochemistry as described below. Explanted and transplanted tissue was then cryoprotected first in 10% sucrose for 12 h and then in 30% sucrose for 12 h. Cryoprotected composites were frozen in Optimal Cutting Temperature Compound (Sakura Finetek Torrence CA) at 20 C and cut into 20 mm sections using a Minotome Plus (Triangle Biomedical Sciences Durham NC). All samples were then rinsed 3 times for 10 min each in PBS blocked and permeabilized in PBS containing 10% goat serum 1% BSA and 0.1% Triton-X for 2 h. Samples used to compare differences (Imaris Bitplane Inc. Saint Paul MN) between PGS and glass influenced mRPC genetic expression were incubated with the primary antibodies:paired box gene 6 (Pax6) (Hybridoma Bank Iowa City IA) 1:20 Hairy and enhancer of split 1 (Hes1) (Chemicon) 1:200 Ki67 (Sigma St. Louis MO) 1:100 nestin (BD Biosciences San Jose CA) 1:200 SRY(sex determining region Y)-box2 (Sox2) (Chemicon Billerina CA) 1:200 and glial fibrillary acidic protein (GFAP) (Zymed San Francisco CA) (1:200). PGS-explant and transplant samples were incubated with the primary

Microscopic Technique
Confocal Microscopy

Cell Type(s)
PGS