Cultured allogeneic keratinocyte transplantation in the large white pig

Rubin, Philip (2002) Cultured allogeneic keratinocyte transplantation in the large white pig. MD thesis, University of Glasgow.

Abstract

Introduction: Banked cultured Langerhans cell (LC) free allogeneic keratinocytes, could be a readily available resource for the treatment of burns. The key questions that have not been resolved regarding the natural history of allotransplanted cultured keratinocytes, are when, how and why they are eliminated. Previous work at the Blond McIndoe Centre in a Large White pig model has demonstrated the survival of allotransplanted keratinocytes at three weeks in a ''chimeric" context (transplanting mixtures of autologous and allogeneic keratinocytes). The cultured cells were labelled by retroviral gene transfer to introduce a lacZnIs histochemically detectable marker. This work however, involved grafting onto de-epidermalised dermis that was shown to be capable of regenerating an epidermis without the aid of transplanted keratinocytes. Additionally, the keratinocyte labelling efficiency was low. The purpose of this study was to investigate the fate of cultured allogeneic transplanted keratinocytes, when they are the only source of epidermal tissue in a wound incapable of epidermal regeneration. To accomplish this, the aim was to develop a more efficient means of porcine keratinocyte labelling with the lacZnIs marker and to immunohistochemically characterise the cellular infiltrate with regard to allogeneic split skin graft rejection and compare this with the immune cell infiltrate associated with lacZnIs labelled LC-free cultured allogeneic keratinocytes grafted onto Integra(TM) (a non-regenerative dermal template). Methods: The Large White pig was used in view of the similarity of its skin architecture to humans. Retroviral gene transduction of cultured porcine keratinocytes was conducted with a MFGIacZnIs construct in the PT67amphotropic packaging cell line. Leucocyte phenotypes infiltrating porcine skin were characterised immunohistochemically using a panel of monoclonal antibodies specific to porcine antigens: CD1, CD3, CD4, CD5, CDS, CDS, CD25, CD45, SUKC3, SH/C6, SLA DP, SLA DP, SLA DR and igXLC with ABC staining. Porcine Jejunum was employed as a positive control. LC depletion with Anti-MHC II and complement vyas monitored using flow cytometry. MFGIacZnIs labelled LC-depleted cultured keratinocytes sheets and keratinocyte-dermal composites were auto and allografted onto Integra and onto deep fascia respectively. LacZnIs j3-galactosidase expression was detected using X-Gal staining of tissue biopsies at different time points up to 21 days following grafting and a reduced panel of monoclonal antibodies (omitting CDS, SLA DP and SLA DQ) was used to immunophenotype the infiltrate at these time points. Results: The new PTS7 producer line succeeded in achieving close to 100% of keratinocyte transduction with the MFG lacZ construct following three passages in a cell culture system containing irradiated retroviral producer cells and 3T3 fibroblasts as feeder cells. Stability was demonstrated with no apparent reduction in lacZnis expression by porcine keratinocytes following a further seven passages on irradiated STS fibroblasts. A reduction was demonstrated however, both in the number and size of colonies formed by keratinocytes following transduction. LCs were shown to be successfully eliminated from disaggregated epidermal cells using antibody and complement. We also demonstrated that overnight incubation results in up regulation of MHC class II antigen and increases the efficiency of LC elimination. As with retrovirally transduced keratinocytes a reduction was demonstrated however, both in the number and size of colonies formed by keratinocytes that had undergone treatment with antibody and complement. The differential distribution of leucocyte phenotypes as determined by the panel of antibodies within normal porcine skin was shown for the first time.

• Item Type: Thesis (MD)
• Qualification Level: Doctoral
• Additional Information: Adviser: Blond McIndoe
• Keywords: Medicine
• Date of Award: 2002
• Depositing User: Enlighten Team
• Unique ID: glathesis:2002-73761
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 14 Jun 2019 08:56
• Last Modified: 14 Jun 2019 08:56
• URI: https://theses.gla.ac.uk/id/eprint/73761