Syntaxin 6 and Vti1b form a novel SNARE complex, which is upregulated in activated macrophages to facilitate exocytosis of TNF.

A key function of activated macrophages is to secrete proinflammatory cytokines such as TNF; however, the intracellular pathway and machinery responsible for cytokine trafficking and secretion is largely undefined. Here we show that individual SNARE proteins involved in vesicle docking and fusion are regulated at both gene and protein expression upon stimulation with the bacterial cell wall component lipopolysaccharide. Focusing on two intracellular SNARE proteins, Vti1b and syntaxin 6 (Stx6), we show that they are up-regulated in conjunction with increasing cytokine secretion in activated macrophages and that their levels are selectively titrated to accommodate the volume and timing of post-Golgi cytokine trafficking. In macrophages, Vti1b and syntaxin 6 are localized on intracellular membranes and are present on isolated Golgi membranes and on Golgi-derived TNF� vesicles budded in vitro. By immunoprecipitation, we find that Vti1b and syntaxin 6 interact to form a novel intracellular Q-SNARE complex. Functional studies using overexpression of full-length and truncated proteins show that both Vti1b and syntaxin 6 function and have rate-limiting roles in TNF� trafficking and secretion. This study shows how macrophages have uniquely adapted a novel Golgi-associated SNARE complex to accommodate their requirement for increased cytokine secretion.

A biphasic scaffold design combined with cell sheet technology for simultaneous regeneration of alveolar bone/periodontal ligament complex

This study describes the design of a biphasic scaffold composed of a Fused Deposition Modeling scaffold (bone compartment) and an electrospun membrane (periodontal compartment) for periodontal regeneration. In order to achieve simultaneous alveolar bone and periodontal ligament regeneration a cell-based strategy was carried out by combining osteoblast culture in the bone compartment and placement of multiple periodontal ligament (PDL) cell sheets on the electrospun membrane. In vitro data showed that the osteoblasts formed mineralized matrix in the bone compartment after 21 days in culture and that the PDL cell sheet harvesting did not induce significant cell death. The cell-seeded biphasic scaffolds were placed onto a dentin block and implanted for 8 weeks in an athymic rat subcutaneous model. The scaffolds were analyzed by μCT, immunohistochemistry and histology. In the bone compartment, a more intense ALP staining was obtained following seeding with osteoblasts, confirming the μCT results which showed higher mineralization density for these scaffolds. A thin mineralized cementum-like tissue was deposited on the dentin surface for the scaffolds incorporating the multiple PDL cell sheets, as observed by H&E and Azan staining. These scaffolds also demonstrated better attachment onto the dentin surface compared to no attachment when no cell sheets were used. In addition, immunohistochemistry revealed the presence of CEMP1 protein at the interface with the dentine. These results demonstrated that the combination of multiple PDL cell sheets and a biphasic scaffold allows the simultaneous delivery of the cells necessary for in vivo regeneration of alveolar bone, periodontal ligament and cementum. © 2012 Elsevier Ltd.

Evaluation of fibroin-based scaffolds for ocular tissue reconstruction

The epithelium of the corneolimbus contains stem cells for regenerating the corneal epithelium. Diseases and injuries affecting the limbus can lead to a condition known as limbal stem cell deficiency (LSCD), which results in loss of the corneal epithelium, and subsequent chronic inflammation and scarring of the ocular surface. Advances in the treatment of LSCD have been achieved through use of cultured human limbal epithelial (HLE) grafts to restore epithelial stem cells of the ocular surface. These epithelial grafts are usually produced by the ex vivo expansion of HLE cells on human donor amniotic membrane (AM), but this is not without limitations. Although AM is the most widely accepted substratum for HLE transplantation, donor variation, risk of disease transfer, and rising costs have led to the search for alternative biomaterials to improve the surgical outcome of LSCD. Recent studies have demonstrated that Bombyx mori silk fibroin (hereafter referred to as fibroin) membranes support the growth of primary HLE cells, and thus this thesis aims to explore the possibility of using fibroin as a biomaterial for ocular surface reconstruction. Optimistically, the grafted sheets of cultured epithelium would provide a replenishing source of epithelial progenitor cells for maintaining the corneal epithelium, however, the HLE cells lose their progenitor cell characteristics once removed from their niche. More severe ocular surface injuries, which result in stromal scarring, damage the epithelial stem cell niche, which subsequently leads to poor corneal re-epithelialisation post-grafting. An ideal solution to repairing the corneal limbus would therefore be to grow and transplant HLE cells on a biomaterial that also provides a means for replacing underlying stromal cells required to better simulate the normal stem cell niche. The recent discovery of limbal mesenchymal stromal cells (L-MSC) provides a possibility for stromal repair and regeneration, and therefore, this thesis presents the use of fibroin as a possible biomaterial to support a three dimensional tissue engineered corneolimbus with both an HLE and underlying L-MSC layer. Investigation into optimal scaffold design is necessary, including adequate separation of epithelial and stromal layers, as well as direct cell-cell contact. Firstly, the attachment, morphology and phenotype of HLE cells grown on fibroin were directly compared to that observed on donor AM, the current clinical standard substrate for HLE transplantation. The production, transparency, and permeability of fibroin membranes were also evaluated in this part of the study. Results revealed that fibroin membranes could be routinely produced using a custom-made film casting table and were found to be transparent and permeable. Attachment of HLE cells to fibroin after 4 hours in serum-free medium was similar to that supported by tissue culture plastic but approximately 6-fold less than that observed on AM. While HLE cultured on AM displayed superior stratification, epithelia constructed from HLE on fibroin maintained evidence of corneal phenotype (cytokeratin pair 3/12 expression; CK3/12) and displayed a comparable number and distribution of ÄNp63+ progenitor cells to that seen in cultures grown on AM. These results confirm the suitability of membranes constructed from silk fibroin as a possible substrate for HLE cultivation. One of the most important aspects in corneolimbal tissue engineering is to consider the reconstruction of the limbal stem cell niche to help form the natural limbus in situ. MSC with similar properties to bone marrow derived-MSC (BM-MSC) have recently been grown from the limbus of the human cornea. This thesis evaluated methods for culturing L-MSC and limbal keratocytes using various serum-free media. The phenotype of resulting cultures was examined using photography, flow cytometry for CD34 (keratocyte marker), CD45 (bone marrow-derived cell marker), CD73, CD90, CD105 (collectively MSC markers), CD141 (epithelial/vascular endothelial marker), and CD271 (neuronal marker), immunocytochemistry (alpha-smooth muscle actin; á-sma), differentiation assays (osteogenesis, adipogenesis and chrondrogenesis), and co-culture experiments with HLE cells. While all techniques supported to varying degrees establishment of keratocyte and L-MSC cultures, sustained growth and serial propagation was only achieved in serum-supplemented medium or the MesenCult-XF„¥ culture system (Stem Cell Technologies). Cultures established in MesenCult-XF„¥ grew faster than those grown in serum-supplemented medium and retained a more optimal MSC phenotype. L-MSC cultivated in MesenCult-XFR were also positive for CD141, rarely expressed £\-sma, and displayed multi-potency. L-MSC supported growth of HLE cells, with the largest epithelial islands being observed in the presence of L-MSC established in MesenCult-XF„¥ medium. All HLE cultures supported by L-MSC widely expressed the progenitor cell marker £GNp63, along with the corneal differentiation marker CK3/12. Our findings conclude that MesenCult-XFR is a superior culture system for L-MSC, but further studies are required to explore the significance of CD141 expression in these cells. Following on from the findings of the previous two parts, silk fibroin was tested as a novel dual-layer construct containing both an epithelium and underlying stroma for corneolimbal reconstruction. In this section, the growth and phenotype of HLE cells on non-porous versus porous fibroin membranes was compared. Furthermore, the growth of L-MSC in either serum-supplemented medium or the MesenCult-XFR culture system within fibroin fibrous mats was investigated. Lastly, the co-culture of HLE and L-MSC in serum-supplemented medium on and within fibroin dual-layer constructs was also examined. HLE on porous membranes displayed a flattened and squamous monolayer; in contrast, HLE on non-porous fibroin appeared cuboidal and stratified closer in appearance to a normal corneal epithelium. Both constructs maintained CK3/12 expression and distribution of £GNp63+ progenitor cells. Dual-layer fibroin scaffolds consisting of HLE cells and L-MSC maintained a similar phenotype as on the single layers alone. Overall, the present study proposed to create a three dimensional limbal tissue substitute of HLE cells and L-MSC together, ultimately for safe and beneficial transplantation back into the human eye. The results show that HLE and L-MSC can be cultivated separately and together whilst maintaining a clinically feasible phenotype containing a majority of progenitor cells. In addition, L-MSC were able to be cultivated routinely in the MesenCult-XF® culture system while maintaining a high purity for the MSC characteristic phenotype. However, as a serum-free culture medium was not found to sustain growth of both HLE and L-MSC, the combination scaffold was created in serum-supplemented medium, indicating that further refinement of this cultured limbal scaffold is required. This thesis has also demonstrated a potential novel marker for L-MSC, and has generated knowledge which may impact on the understanding of stromal-epithelial interactions. These results support the feasibility of a dual-layer tissue engineered corneolimbus constructed from silk fibroin, and warrant further studies into the potential benefits it offers to corneolimbal tissue regeneration. Further refinement of this technology should explore the potential benefits of using epithelial-stromal co-cultures with MesenCult-XF® derived L-MSC. Subsequent investigations into the effects of long-term culture on the phenotype and behaviour of the cells in the dual-layer scaffolds are also required. While this project demonstrated the feasibility in vitro for the production of a dual-layer tissue engineered corneolimbus, further studies are required to test the efficacy of the limbal scaffold in vivo. Future in vivo studies are essential to fully understand the integration and degradation of silk fibroin biomaterials in the cornea over time. Subsequent experiments should also investigate the use of both AM and silk fibroin with epithelial and stromal cell co-cultures in an animal model of LSCD. The outcomes of this project have provided a foundation for research into corneolimbal reconstruction using biomaterials and offer a stepping stone for future studies into corneolimbal tissue engineering.

An analytical tool that quantifies cellular morphology changes from three-dimensional fluorescence images

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for inclusion and exclusion of data points, and computer-aided pattern recognition to support the interpretation and findings of the analysis. It has become increasingly important to be able to measure fluorescence images constructed from three-dimensional (3D) datasets in order to be able to capture the complexity of cellular dynamics and understand the basis of cellular plasticity within biological systems. Sophisticated microscopy instruments have permitted the visualization of 3D fluorescence images through the acquisition of multispectral fluorescence images and powerful analytical software that reconstructs the images from confocal stacks that then provide a 3D representation of the collected 2D images. Advanced design-based stereology methods have progressed from the approximation and assumptions of the original model-based stereology(1) even in complex tissue sections(2). Despite these scientific advances in microscopy, a need remains for an automated analytic method that fully exploits the intrinsic 3D data to allow for the analysis and quantification of the complex changes in cell morphology, protein localization and receptor trafficking. Current techniques available to quantify fluorescence images include Meta-Morph (Molecular Devices, Sunnyvale, CA) and Image J (NIH) which provide manual analysis. Imaris (Andor Technology, Belfast, Northern Ireland) software provides the feature MeasurementPro, which allows the manual creation of measurement points that can be placed in a volume image or drawn on a series of 2D slices to create a 3D object. This method is useful for single-click point measurements to measure a line distance between two objects or to create a polygon that encloses a region of interest, but it is difficult to apply to complex cellular network structures. Filament Tracer (Andor) allows automatic detection of the 3D neuronal filament-like however, this module has been developed to measure defined structures such as neurons, which are comprised of dendrites, axons and spines (tree-like structure). This module has been ingeniously utilized to make morphological measurements to non-neuronal cells(3), however, the output data provide information of an extended cellular network by using a software that depends on a defined cell shape rather than being an amorphous-shaped cellular model. To overcome the issue of analyzing amorphous-shaped cells and making the software more suitable to a biological application, Imaris developed Imaris Cell. This was a scientific project with the Eidgenössische Technische Hochschule, which has been developed to calculate the relationship between cells and organelles. While the software enables the detection of biological constraints, by forcing one nucleus per cell and using cell membranes to segment cells, it cannot be utilized to analyze fluorescence data that are not continuous because ideally it builds cell surface without void spaces. To our knowledge, at present no user-modifiable automated approach that provides morphometric information from 3D fluorescence images has been developed that achieves cellular spatial information of an undefined shape (Figure 1). We have developed an analytical platform using the Imaris core software module and Imaris XT interfaced to MATLAB (Mat Works, Inc.). These tools allow the 3D measurement of cells without a pre-defined shape and with inconsistent fluorescence network components. Furthermore, this method will allow researchers who have extended expertise in biological systems, but not familiarity to computer applications, to perform quantification of morphological changes in cell dynamics.

Delta opioid-receptor function in the dorsal striatum plays a role in high levels of ethanol consumption in rats

Binge-like patterns of excessive drinking during young adulthood increase the propensity for alcohol use disorders (AUDs) later in adult life; however, the mechanisms that drive this are not completely understood. Previous studies showed that the δ-opioid peptide receptor (DOP-R) is dynamically regulated by exposure to ethanol and that the DOP-R plays a role in ethanol-mediated behaviors. The aim of this study was to determine the role of the DOP-R in high ethanol consumption from young adulthood through to late adulthood by measuring DOP-R-mediated [(35)S]GTPγS binding in brain membranes and DOP-R-mediated analgesia using a rat model of high ethanol consumption in Long Evans rats. We show that DOP-R activity in the dorsal striatum and DOP-R-mediated analgesia changes during development, being highest during early adulthood and reduced in late adulthood. Intermittent access to ethanol but not continuous ethanol or water from young adulthood leads to an increase in DOP-R activity in the dorsal striatum and DOP-R-mediated analgesia into late adulthood. Multiple microinfusions of naltrindole into the dorsal striatum or multiple systemic administration of naltrindole reduces ethanol consumption, and following termination of treatment, DOP-R activity in the dorsal striatum is attenuated. These findings suggest that DOP-R activity in the dorsal striatum plays a role in high levels of ethanol consumption and suggest that targeting the DOP-R is an alternative strategy for the treatment of AUDs.

The delta opioid receptor antagonist, SoRI-9409, decreases yohimbine stress-induced reinstatement of ethanol-seeking

A major problem in treating alcohol use disorders (AUDs) is the high rate of relapse due to stress and re-exposure to cues or an environment previously associated with alcohol use. Stressors can induce relapse to alcohol-seeking in humans or reinstatement in rodents. Delta opioid peptide receptors (DOP-Rs) play a role in cue-induced reinstatement of ethanol-seeking; however, their role in stress-induced reinstatement of ethanol-seeking is not known. The objective of this study was to determine the role of DOP-Rs in yohimbine-stress-induced reinstatement of ethanol-seeking. Male, Long-Evans rats were trained to self-administer 10% ethanol in daily 30-minute operant self-administration sessions using a FR3 schedule of reinforcement, followed by extinction training. Once extinction criteria were met, we examined the effects of the DOP-R antagonist, SoRI-9409 (0–5 mg/kg, i.p.) on yohimbine (2 mg/kg, i.p.) stress-induced reinstatement. Additionally, DOP-R-stimulated [35S]GTPS binding was measured in brain membranes and plasma levels of corticosterone (CORT) were determined. Pre-treatment with SoRI-9409 decreased yohimbine stress-induced reinstatement of ethanol-seeking but did not affect yohimbine-induced increases in plasma CORT levels. Additionally, yohimbine increased DOP-R-stimulated 35[S]GTPS binding in brain membranes of ethanol-trained rats, an effect that was inhibited by SoRI-9409. This suggests that the DOP-R plays an important role in yohimbine-stress-induced reinstatement of ethanol-seeking behavior, and DOP-R antagonists may be promising candidates for further development as a treatment for AUDs.

Stroma regulates increased epithelial lateral cell adhesion in 3D culture : a role for actin/cadherin dynamics

BACKGROUND: Cell shape and tissue architecture are controlled by changes to junctional proteins and the cytoskeleton. How tissues control the dynamics of adhesion and cytoskeletal tension is unclear. We have studied epithelial tissue architecture using 3D culture models and found that adult primary prostate epithelial cells grow into hollow acinus-like spheroids. Importantly, when co-cultured with stroma the epithelia show increased lateral cell adhesions. To investigate this mechanism further we aimed to: identify a cell line model to allow repeatable and robust experiments; determine whether or not epithelial adhesion molecules were affected by stromal culture; and determine which stromal signalling molecules may influence cell adhesion in 3D epithelial cell cultures. METHODOLOGY/PRINCIPAL FINDINGS: The prostate cell line, BPH-1, showed increased lateral cell adhesion in response to stroma, when grown as 3D spheroids. Electron microscopy showed that 9.4% of lateral membranes were within 20 nm of each other and that this increased to 54% in the presence of stroma, after 7 days in culture. Stromal signalling did not influence E-cadherin or desmosome RNA or protein expression, but increased E-cadherin/actin co-localisation on the basolateral membranes, and decreased paracellular permeability. Microarray analysis identified several growth factors and pathways that were differentially expressed in stroma in response to 3D epithelial culture. The upregulated growth factors TGFβ2, CXCL12 and FGF10 were selected for further analysis because of previous associations with morphology. Small molecule inhibition of TGFβ2 signalling but not of CXCL12 and FGF10 signalling led to a decrease in actin and E-cadherin co-localisation and increased paracellular permeability. CONCLUSIONS/SIGNIFICANCE: In 3D culture models, paracrine stromal signals increase epithelial cell adhesion via adhesion/cytoskeleton interactions and TGFβ2-dependent mechanisms may play a key role. These findings indicate a role for stroma in maintaining adult epithelial tissue morphology and integrity.

The structures of COPI-coated vesicles reveal alternate coatomer conformations and interactions

Transport between compartments of eukaryotic cells is mediated by coated vesicles. The archetypal protein coats COPI, COPII, and clathrin are conserved from yeast to human. Structural studies of COPII and clathrin coats assembled in vitro without membranes suggest that coat components assemble regular cages with the same set of interactions between components. Detailed three-dimensional structures of coated membrane vesicles have not been obtained. Here, we solved the structures of individual COPI-coated membrane vesicles by cryoelectron tomography and subtomogram averaging of in vitro reconstituted budding reactions. The coat protein complex, coatomer, was observed to adopt alternative conformations to change the number of other coatomers with which it interacts and to form vesicles with variable sizes and shapes. This represents a fundamentally different basis for vesicle coat assembly.

An in-vitro 3-D cell culture model for studying pathomechanisms in AMD

Purpose To develop a novel 3-D cell culture model with the view to studying the pathomechanisms underlying the development of age-related macular degeneration (AMD). Our central hypothesis is that the silk structural protein fibroin used in conjunction with cultured human cells can be used to mimic the structural relationships between the RPE and choriocapillaris in health and disease. Methods Co-cultures of human RPE cells (ARPE-19 cells grown in Miller’s medium) and microvascular endothelial cells (HMEC-1 cells grown in endothelial culture medium) were established on opposing sides of a synthetic Bruch’s membrane (3 microns thick) constructed from B mori silk fibroin. Cell attachment was facilitated by pre-coating the fibroin membrane with vitronectin (for ARPE-19 cells) and gelatin (for HMEC-1 cells) respectively. The effects of tropoelastin on attachment of ARPE-19 cells was also examined. Barrier function was examined by measurement of trans-epithelial resistance (TER) using a voltohmmeter (EVOM-2). The phagocytic activity of the synthetic RPE was tested using vitronectin-coated microspheres (2 micron diameter FluoSpheres). In some cultures, membrane defects were created by puncturing within a 24 G needle. The architecture of the synthetic tissue before and after wounding was examined by confocal microscopy after staining for ZO-1 and F-actin. Results The RPE layer of the 3D model developed a cobblestoned morphology (validated by staining for ZO-1 and F-actin), displayed barrier function (validated by measurement of TER) and demonstrated cytoplasmic uptake of vitronectin-coated microspheres. Attachment of ARPE-19 cells to fibroin was unaffected by tropoelastin. Microvascular endothelial cells attached well to the gelatin-coated surface of the fibroin membrane and remained physically separated from the overlaying RPE layer. The fibroin membranes were amenable to puncturing without collapse thus providing the opportunity to study transmembrane migration of the endothelial cells. Conclusions Synthetic Bruch’s membranes constructed from silk fibroin, vitronectin and gelatin, support the co-cultivation of RPE cells and microvascular endothelial cells. The resulting RPE layer displays functions similar to that of native RPE and the entire tri-layered structure displays potential to be used as an in vitro model of choroidal neovascularization.

The cultivation of human retinal pigment epithelial cells on Bombyx mori silk fibroin

We have presently evaluated membranes prepared from Bombyx mori silk fibroin (BMSF), for their potential use as a prosthetic Bruch’s membrane and carrier substrate for human retinal pigment epithelial (RPE) cell transplantation. Porous BMSF membranes measuring 3 μm in thickness were prepared from aqueous solutions (3% w/v) containing poly(ethylene oxide) (0.09%). The permeability coefficient for membranes was between 3 and 9 × 10-5 cm/s by using Allura red or 70 kDa FITC-dextran respectively. Average pore size (± sd) was 4.9 ± 2.3 µm and 2.9 ± 1.5 µm for upper and lower membrane surfaces respectively. Optimal attachment of ARPE-19 cells to BMSF membrane was achieved by pre-coating with vitronectin (1 µg/mL). ARPE-19 cultures maintained in low serum on BMSF membranes for approximately 8 weeks, developed a cobble-stoned morphology accompanied by a cortical distribution of F-actin and ZO-1. Similar results were obtained using primary cultures of human RPE cells, but cultures took noticeably longer to establish on BMSF compared with tissue culture plastic. These findings encourage further studies of BMSF as a substrate for RPE cell transplantation.

Intracellular trafficking and secretion of inflammatory cytokines

The secretion of cytokines by immune cells plays a significant role in determining the course of an inflammatory response. The levels and timing of each cytokine released are critical for mounting an effective but confined response, whereas excessive or dysregulated inflammation contributes to many diseases. Cytokines are both culprits and targets for effective treatments in some diseases. The multiple points and mechanisms that have evolved for cellular control of cytokine secretion highlight the potency of these mediators and the fine tuning required to manage inflammation. Cytokine production in cells is regulated by cell signaling, and at mRNA and protein synthesis levels. Thereafter, the intracellular transport pathways and molecular trafficking machinery have intricate and essential roles in dictating the release and activity of cytokines. The trafficking machinery and secretory (exocytic) pathways are complex and highly regulated in many cells, involving specialized membranes, molecules and organelles that enable these cells to deliver cytokines to often-distinct areas of the cell surface, in a timely manner. This review provides an overview of secretory pathways - both conventional and unconventional - and key families of trafficking machinery. The prevailing knowledge about the trafficking and secretion of a number of individual cytokines is also summarized. In conclusion, we present emerging concepts about the functional plasticity of secretory pathways and their modulation for controlling cytokines and inflammation.

Desalination by a solar thermal membrane distillation process

A solar thermal membrane distillation pilot plant was operated for over 70 days in field conditions. The pilot plant incorporated a single spiral wound permeate gap membrane distillation style of module. All energy used to operate the unit was supplied by solar hot water collectors and photovoltaic panels. The process was able to produce a distillate stream of product water with a conductivity less than 10 µS/cm. Feed water concentration varied from 2,400 µS/cm to 106,000 µS/cm. The process is expected to find application in the production of drinking water for remote island and arid regions without the consumption of electrical energy.

The effect of Fe doping on adsorption of CO2/N2within carbon nanotubes : a density functional theory study with dispersion corrections

An ab initio density functional theory (DFT) study with correction for dispersive interactions was performed to study the adsorption of N2 and CO2 inside an (8, 8) single-walled carbon nanotube. We find that the approach of combining DFT and van der Waals correction is very effective for describing the long-range interaction between N2/CO2 and the carbon nanotube (CNT). Surprisingly, exohedral doping of an Fe atom onto the CNT surface will only affect the adsorption energy of the quadrupolar CO2 molecule inside the CNT (20–30%), and not that of molecular N2. Our results suggest the feasibility of enhancement of CO2/N2 separation in CNT-based membranes by using exohedral doping of metal atoms.

Development of an ultra-thin fibroin membrane for RPE cell transplantation

Purpose: One of the challenges associated with cell-based therapies for repairing the retina is the development of suitable materials on which to grow and transplant retinal cells. Using the ARPE-19 cell line, we have previously demonstrated the feasibility of growing RPE-derived cells on membranes prepared from the silk protein fibroin. The present study was aimed at developing a porous, ultra-thin fibroin membrane that might better support development of apical-basal polarity in culture, and to extend this work to primary cultures of human RPE cells. Methods: Ultra-thin fibroin membranes were prepared using a highly polished casting table coated with Topas® (a cyclic olefin copolymer) and a 1:0.03 aqueous solution of fibroin and PEO (Mv 900 000 g/mol). Following drying, the membranes were water annealed to make them water-stable, washed in water to remove PEO, sterilised by treatment with 95% ethanol, and washed extensively in saline. Primary cultures containing human RPE cells were established from donor posterior eye cups and maintained in DMEM/F12 medium supplemented with 10% fetal bovine serum and antibiotics. First passage cultures were seeded onto fibroin membranes pre-coated with vitronectin and grown for 6 weeks in medium supplemented with 1% serum. Comparative cultures were established on porous 1.0 µm pore PET membrane (Millipore) and using ARPE-19 cells. Results: The fibroin membranes displayed an average thickness of 3 µm and contained numerous dimples/pore-like structures of up to 3-5 µm in diameter. The primary cultures predominantly contained pigmented epithelial cells, but mesenchymal cells (presumed fibroblasts) were also often present. Passaged cultures appeared to attach equally well to either fibroin or PET membranes. Over time cells on either material adopted a more cobblestoned morphology. Conclusions: Progress has been made towards developing a porous ultra-thin fibroin membrane that supports cultivation of RPE cells. Further studies are required to determine the degree of membrane permeability and RPE polarity.

Fibroin-based materials support cultivation of limbal stromal cells

Purpose: The silk protein fibroin provides a potential substrate for use in ocular tissue reconstruction. We have previously demonstrated that transparent membranes produced from fibroin support cultivation of human limbal epithelial cells (Tissue Eng A. 14(2008)1203-11). We presently extend this body of work to studies of human limbal stromal cell (HLS) growth on fibroin in the presence and absence of serum. Methods: Primary cultures of HLS cells were established in DMEM/F12 medium supplemented with either 10% fetal bovine serum (FBS) or 2% B27 supplement. Defined keratinocyte serum-free medium (DK-SFM, Invitrogen) was also tested. The resulting cultures were analysed by flow cytometry for expression of CD34, CD90, CD45, and CD141. Cultures grown under each condition were subsequently passaged either onto transparent fibroin membranes prepared from purified fibroin or within 3D scaffolds prepared from partially-solubilised fibroin. Results: HLS cultures were successfully established under each condition, but grew more slowly and passaged poorly in the absence of serum. Cultures grown in 10% FBS were <0.5% CD34+ (keratocytes) and >97% CD90+ (fibroblasts). Cultures established in 2% B27 formed floating spheres and contained >8% CD34+ cells and reduced CD90 expression. Cultures established in DK-SFM displayed traces of epithelial cell growth (CD141), but mostly consisted of CD90+ cells with <1% CD34+ cells. Cells of bone marrow lineage (CD45) were rarely observed under any conditions. Cultures grown in 10% FBS were able to adhere to and proliferate on silk fibroin 3-D scaffolds and transparent films while those grown serum-free could not. Adhesion of HLS cells to fibroin was initially poorer than that displayed on tissue culture plastic. Conclusions: HLS cultures containing cells of predominantly fibroblast lineage can be grown on fibroin-based materials, but this process is dependent upon additional ECM factors such as those provided by serum.