TERMIS-EU Abstracts

London, UK
September 4th-7th 2007
Published in Tissue Engineering (2007) 13(7)
Use of a rabbit cornea model for the development of a cell transfer system for limbal epithelial cells
Deshpande P, Bullett NA, Notara M, Daniels JT, Haddow DB, MacNeil S.
This work involves the development of a contact lens system for transferring laboratory expanded limbal epithelial cells for treatment of the cornea. The approach taken is to use a chemically defined engineered surface using plasma polymerisation technology to develop a coating which supports both the initial attachment of epithelial cells and their subsequent transfer onto the denuded cornea. To assist in this development we have established a rabbit organ culture model to examine transfer of cells onto the cornea. First we examined the culture of a human corneal epithelial cell line and primary rabbit limbal epithelial cells on a range of plasma coatings. Acrylic acid, allyl amine and allyl alcohol surfaces were synthesised at different power and flow rates. From these, the surface which best supported epithelial cell culture (both human and rabbit cells) was identified. Cells were then cultured on contact lenses coated with this surface. Rabbit corneal organ cultures with the intact epithelium were then denuded of epithelial cells. Lenses with cells were placed onto the cornea and kept in place for 4 days. Transfer of cells from lenses was examined by pre-staining cells with CellTracker™ Red CMTPX and also by subsequent staining of cells on the cornea with DAPI and phalloidin FITC. The results showed that the surface that best supported the epithelial cell culture was acrylic acid. Preliminary results using this model show that the primary rabbit limbal epithelial cells and the human cell line will transfer from the lens onto the cornea.
Improved technologies for translating culture of human melanocytes to the clinic
Eves P, Bullett NA, Haddow DB, Gawkrodger D, MacNeil S
While laboratory expanded autologous melanocytes have clearly been shown to be of benefit in the surgical treatment of patients with vitiligo, there is a need for a robust delivery and grafting strategy for patients. In this paper we present a convenient methodology for delivering cultured autologous melanocytes and keratinocytes from the laboratory to the patient which is low risk for the patient but also user friendly for the surgeon.
A chemically defined substrate (acrylic acid deposited by plasma polymerisation onto medical grade silicone backing dressing) was used to culture both melanocytes and keratinocytes in medium (Greens) currently used in the clinic (which contains fetal calf serum sourced from New Zealand), but also in a serum free alternative—M2. We demonstrate successful transfer of melanocyte:keratinocyte co-cultures from these carriers onto an in vitro human wound bed model. The transferred melanocytes were capable of achieving pigmentation and the keratinocytes rapidly provided an epithelial barrier layer.
In conclusion we have developed a pre-clinical methodology for the delivery of co-cultures of autologous melanocytes and keratinocytes using a flexible and user-friendly chemically defined carrier surface. This should facilitate translation of this work from the laboratory to the clinic to benefit those patients with stable vitiligo who could be candidates for surgical treatment.
Development of biocompatible, biodegradable electrospun scaffolds for tissue engineering of human skin
Blackwood K, Freeman CO, McKean R, Farthing P, Haycock JW, Ryan AJ, Brook I, MacNeil S.
Electrospun scaffolds show potential in becoming a synthetic substitute of dermis for skin tissue engineering. We report on the in vitro and in vivo degradation rates and biocompatibility of two lactide based electrospun scaffolds: poly-L-lactide (PLLA) and P(D,L)LA-co-polyglycolic acid (PGA) (50%, 75%, 85% PLLA by weight). Degradation in vitro was studied by light microscopy and SEM after incubating samples in Ringers solution at 37ºC for up to 52 days. Degradation and biocompatibility in vivo was studied by implanting scaffolds subcutaneously into the flanks of adult rats for up to six months. Scaffolds were removed, processed by routine histological methods and examined by light microscopy and SEM. PLLA showed no evidence of degradation in vitro after 52 days and scaffolds implanted in vivo were clearly visible after 6 months. Implanted scaffolds were also associated with the formation of foreign body giant cells and were infiltrated by fibroblast and endothelial cells after 4 weeks. Granulation tissue was also observed with some scar formation. In contrast, P(D,L)LA-co-PGA 50% scaffolds underwent complete degradation in vitro by 20 days, whereas P(D,L)LA-co-PGA 85% scaffold breakdown was slower. When 85% P(D,L)LA-co-PGA scaffolds were implanted, a foreign body giant cell response was observed together with the formation of granulation tissue, but importantly this response diminished in line with a reduction of scaffold fibre diameter, with complete degradation seen after 5 months. In conclusion P(D,L)LAco-PGA scaffolds show potential for skin tissue engineering as they are associated with cellular penetration, granulation tissue, limited scarring and have a controllable degradation rate.