GMP-Compliant Production of Autologous Adipose-Derived Stromal Cells in the NANT 001 Closed Automated Bioreactor.

In recent years mesenchymal stromal cells (MSCs) have received a great deal of interest for the treatment of major diseases, but clinical translation and market authorization have been slow. This has been due in part to a lack of standardization in cell manufacturing protocols, as well as a lack of biologically meaningful cell characterization tools and release assays. Cell production strategies to date have involved complex manual processing in an open environment which is costly, inefficient and poses risks of contamination. The NANT 001 bioreactor has been developed for the automated production of small to medium cell batches for autologous use. This is a closed, benchtop system which automatically performs several processes including cell seeding, media change, real-time monitoring of temperature, pH, cell confluence and cell detachment. Here we describe a validation of the bioreactor in an environment compliant with current good manufacturing practice (cGMP) to confirm its utility in replacing standardized manual processing. Stromal vascular fraction (SVF) was isolated from lipoaspirate material obtained from healthy donors. SVF cells were seeded in the bioreactor. Cell processing was performed automatically and cell harvesting was triggered by computerized analysis of images captured by a travelling microscope positioned beneath the cell culture flask. For comparison, the same protocol was performed in parallel using manual methods. Critical quality attributes (CQA) assessed for cells from each process included cell yield, viability, surface immunophenotype, differentiation propensity, microbial sterility and endotoxin contamination. Cell yields from the bioreactor cultures were comparable in the manual and automated cultures and viability was >90% for both. Expression of surface markers were consistent with standards for adipose-derived stromal cell (ASC) phenotype. ASCs expanded in both automated and manual processes were capable of adipogenic and osteogenic differentiation. Supernatants from all cultures tested negative for microbial and endotoxin contamination. Analysis of labor commitment indicated considerable economic advantage in the automated system in terms of operator, quality control, product release and management personnel. These data demonstrate that the NANT 001 bioreactor represents an effective option for small to medium scale, automated, closed expansion of ASCs from SVF and produces cell products with CQA equivalent to manual processes.

An assessment of histological margins and recurrence of melanoma in situ.

BACKGROUND: Melanoma in situ (MIS) accounts for up to 27% of all melanomas. MIS has no metastatic potential and the aim should be to excise the lesion completely with a clear histological margin, although margin clearance remains undefined. We aimed to assess the relation of histological excision margins of MIS to recurrence and progression to invasive disease. METHODS: We analyzed all patients with MIS excised by wide local excision or staged excision in our institution over a 5-year period from December 2008 to January 2014 using a prospectively maintained database. Clinicopathologic details included patient demographics, anatomical site of lesion, melanoma subtype, histological excision margin, and recurrence. RESULTS: A total of 410 patients had MIS excised during this time, the majority of which were lentigo maligna subtype (79%). The average histological excision margin was 3.7 mm. The rate of recurrence was 2.2% (9/410), with a median follow-up of 23 months. Lentigo maligna had a similar rate of recurrence to non-lentigo MIS (2.3% vs 1.2%) (P = 0.69). The mean excision margin of those that recurred was 1.9 mm compared with an average of 3.8 mm in those that did not. The rate of recurrence of MIS with histological excision margin ≤3.00 mm was 3.8% compared with 0.5% in those with a histological margin >3.00 mm (P = 0.03). One case of MIS recurred as invasive disease. CONCLUSION: At institutions using wide local excision or staged excision for MIS, a histological margin of >3.0 mm is required to achieve a low recurrence rate.

The pinking shears: a novel tool for improving skin graft cosmesis.

SUMMARY: A significant aesthetic disadvantage to split skin grafts is the obvious transition between the graft and the normal skin. We report on a novel method to interrupt this transition point by using pinking shears, which are dressmaking scissors with saw-toothed blades that create a chevron pattern instead of a straight edge. We describe a case where the pinking shears were utilized on a split skin graft and Integra for reconstruction of the skin on a volar forearm. This technique allows for breaking-up of the transition point between the skin graft and normal skin and gives rise to an improved aesthetic outcome as the boundary is significantly less well-defined. This novel method shows promise and further study is certainly warranted.

Plastic surgical delivery systems for targeted gene therapy.

The expansion of gene therapy applications from inherited disorders to acquired conditions has been mirrored by an exponential rise in both experimental work and clinical trials. This review highlights current plastic surgical delivery systems and clinical applications for targeted gene therapy. We revisit some of the vectors used both experimentally and in clinical gene therapy trials, with an emphasis on developments in plastic surgical delivery systems resulting in improved targeting of therapeutic genes. In addition, we discuss a novel technique for the delivery of gene therapy using the ex vivo transduction of free flaps, developed in our laboratory. This delivery system achieves targeted high-level transgene expression with minimal demonstrable systemic toxicity. Advances in delivery systems are essential for translating basic research into clinical therapeutics.