Elsevier

Biomaterials

Volume 33, Issue 16, June 2012, Pages 4110-4117
Biomaterials

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

https://doi.org/10.1016/j.biomaterials.2012.02.040Get rights and content

Abstract

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.

Introduction

Silk extracted from the cocoons of the domesticated silkworm Bombyx mori has been used in the manufacture of surgical sutures and more recently has been utilised as a source of materials for tissue engineering applications [1], [2]. In particular, fibroin, a fibrous protein responsible for the mechanical properties of silk fibres, can be readily purified and fashioned into a variety of structures that support the attachment and growth of numerous cell types of both epithelial as well as mesenchymal lineage. Its versatility has lead to this protein being considered as a biomaterial for reconstructing tissues found within the human eye [3]. Transparent membranes cast from aqueous solutions of silk fibroin have been successfully used as a substrate for corneal epithelial cells [4], [5], [6], [7], corneal fibroblasts [8], [9] and corneal endothelial cells [10]. The resulting phenotype of corneal cell cultures grown on fibroin membranes suggests that they may prove useful in restoring transparency and barrier function to the ocular surface [4]. This success has led us to investigate whether fibroin might also prove suitable as a biomaterial for reconstructing other ocular tissues and especially the retinal pigment epithelium.

The retinal pigment epithelium, or RPE, is a highly specialised monolayer of epithelial cells that supports the survival and function of photoreceptor cells through a variety of structural features and metabolic activities [11]. One of the key structural features of the RPE is the maintenance of tight epithelial cell junctions that restrict the movement of solutes between the retina and an adjacent, highly vascularised tissue known as the choroid. Movement of solutes is further impeded by a dense layer of extracellular matrix known as Bruch's membrane that incorporates RPE's basement membrane [12]. The RPE, assisted by Bruch's membrane, regulates the movement of nutrients and waste products between the outer retina and choroidal blood supply via specialised trans-epithelial transport functions.

A healthy RPE together with a normal Bruch's membrane are, not surprisingly, essential for maintaining the function of photoreceptor cells, and thus vision, throughout adult life. Nevertheless, Bruch's membrane undergoes a variety of biochemical and structural changes with age [12] that, in conjunction with loss of adjacent RPE cells, leads to age-related macular degeneration (AMD), the leading cause of blindness in developed countries [13]. While the underlying causes of AMD are complex, the final pathological changes often include loss of RPE cells and underlying choroid tissue. Therefore it is logical to consider one form of potential treatment is the transplantation of RPE cells associated with some form of prosthetic Bruch's membrane [14]. Ideally, materials used in construction of the prosthetic Bruch's membrane might also be used as a carrier for the transplanted RPE cells. A key challenge in this field of research is therefore to identify materials that support the fabrication of an RPE carrier substrate, while also displaying a level of permeability that is at least equal to that of Bruch's membrane.

In the present study we report critical data on the attachment, growth and morphology of human RPE cells grown on membranes prepared from B. mori silk fibroin (BMSF). In considering the importance of nutrient/waste exchange across the RPE/Bruch's membrane complex in vivo, we have utilised ultra-thin BMSF membranes measuring only a few microns in thickness. Permeability has been evaluated using membranes that have been rendered porous by casting BMSF in the presence low concentrations of poly(ethylene oxide) (PEO) which is later leached from the membrane by washing in water. Given that corneal cell attachment to BMSF is often improved in the presence of exogenous cell-adhesion factors [8], [10], we have examined the effect of ECM proteins found naturally within the retina and RPE-basement membrane on RPE cell attachment to BMSF. We have initially conducted our studies using the spontaneously immortalised human RPE cell line ARPE-19 since this is a well-established model for human RPE cell function in vitro [15]. These studies include morphological assessments of long-term ARPE-19 cultures (2 months) which are necessary to develop key morphological characteristics similar to those found in vivo including formation of tight junctional complexes and extension of microvilli from the apical surface. Finally, we report on the relative feasibility of establishing primary cultures of adult human RPE cells on BMSF compared with conventional tissue culture plastic.

Section snippets

Materials

B. mori cocoons were supplied by Tajima Shoji Co. Ltd. (Yokohama, Japan) with the pupae already removed. All chemical reagents were purchased from Sigma Aldrich (St. Louis, MO, USA) and were of A.R. grade unless otherwise indicated. Tissue culture reagents were purchased from Invitrogen (Carlsbad, CA, USA) and the tissue culture plastic plates and flasks were Iwaki brand (Asahi Glass Co. Ltd. Japan) unless otherwise indicated.

Preparation of ultra-thin porous BMSF membranes

Aqueous solutions of BMSF were prepared as described previously [4],

Characterisation of BMSF membranes used for RPE cells

We have previously demonstrated that 10–20 μm thick membranes prepared from BMSF support the growth of corneolimbal epithelial cells [4], but in considering the more intensive transport functions of the RPE we chose to presently evaluate ultra-thin (∼3 μm thick) membranes which additionally contained pores generated through casting in the presence of PEO. The resulting membranes appeared slightly opaque when held to light but were sufficiently transparent to enable direct observations of cell

Discussion

Silk fibroin has significant potential as a biomaterial owing to a relatively unique combination of physical and chemical properties [1]. We and other groups have recently established that these properties can be exploited for the purpose of generating corneal tissue equivalents [4], [6], [7], [9], [10]. Our present findings demonstrate that BMSF supports cultivation of retinal pigment epithelial (RPE) cells and might therefore provide a useful material to construct RPE tissue equivalents for

Conclusion

The thickness and permeability of BMSF membranes used in this study are consistent with the requirements of a prosthetic Bruch's membrane. Moreover, our findings confirm the feasibility of growing RPE cells on these BMSF membranes. This is an important finding, as an ideal material for constructing a prosthetic Bruch's membrane with attached RPE cells has yet to be found. BMSF is therefore a worthy candidate for exploration as a vehicle for RPE transplantation in pre-clinical trials of AMD

Acknowledgements

This work was supported by a grant received from Vision Australia administered through the Ophthalmic Research Institute of Australia (awarded to ASK, TVC and DGH) with supplementary funding from the National Health & Medical Research Council of Australia (awarded to DHG and TVC) and the Prevent Blindness Foundation, Queensland, Australia (supported through Viertel's Vision program). We also acknowledge the support of staff from the Queensland Eye Bank for facilitating access to donor eye used

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    Shadforth et al. have reported the preparation of Bombyx mori silk-fibroin films using such film casting techniques. Incorporation of poly(ethylene oxide) into silk-fibroin solutions was demonstrated to introduce porosity into the films, which were capable of being cast at a thickness of 3 µm, similar to that of human Bruch's membrane (Fig. 4.A) [78]. Preparations of biodegradable, non-porous polymer membranes have also been used as RPE cell sheets supports for in vivo implantation.

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