Cytocentrifugation: a convenient and efficient method for seeding tendon-derived cells into monolayer cultures or 3-D tissue engineering scaffolds.

Tendon and ligament injuries are very common, requiring some 200,000 reconstructions per year in the USA. Autografting can be used to repair these but donor tissue is limited and harvesting leads to morbidity at the graft sites. Tissue engineering has been used to grow simple tissues such as skin, cartilage and bone and due to their low vascularity and simple structure, tendons should be ideal candidates for such an approach. Scaffolds are essential for tissue engineering as they provide structure and signals that regulate growth. However, they present a physical barrier to cell seeding with the majority of the cells congregating at the scaffold surface. To address this we used centrifugation to enhance penetration of tendon-derived cells to the centres of 3-D scaffolds. The process had no apparent deleterious effects on the cells and both plating efficiency and cell distribution improved. After attachment the cells continued to proliferate and deposit a collagenous matrix. Scaffold penetration was investigated using layers of Azowipes allowing the separation and examination of individual leaves. At relatively low g-forces, cells penetrated a stack of 6 Azowipes leaving cells attached to each leaf. These data suggest that cytocentrifugation improves the penetration and homogeneity of tendon derived cells in 3-D and monolayer cultures.

Establishing a transport protocol for the delivery of melanocytes and keratinocytes for the treatment of vitiligo.

We have previously developed a cell delivery and transfer technology for delivering autologous keratinocytes and melanocytes to patients with vitiligo. However, for this technology to benefit many patients geographically distant from the cell culture facility transportation issues need to be overcome. In this study we begin to investigate this by looking at what role surface chemistry and medium supplements, including fetal calf serum, CO2 gassing, and temperature, play in influencing cell viability. Cells were maintained on carriers for up to 48 h outside of a CO2 incubator at 37 °C and their subsequent ability to adhere and become organized into a new epithelium with appropriately located melanocytes was assessed. Consistently good viability and performance on an in vitro wound bed model was achieved by maintaining cells for 48 h adherent to a 20% acrylic acid coated carrier at lower (around 23 °C rather than 37 °C) temperatures in the medium preperfused with CO2 before transport. Under these circumstances fetal calf serum was not required. In summary, the surface chemistry of the transport substrate and an appropriately CO2 buffered medium at near room temperature can extend the effective performance life of these cultured cells to at least 48 h from when they leave standard incubator conditions.

Simplifying the delivery of melanocytes and keratinocytes for the treatment of vitiligo using a chemically defined carrier dressing.

Obtaining pigmentary function in autologous skin grafts is a current challenge for burn surgeons as is developing reliable robust grafting strategies for patients with vitiligo and piebaldism. In this paper, we present the development of a simple methodology for delivering cultured keratinocytes and melanocytes to the patient that is of low risk for the patient but also user friendly for the surgeon. In this study, we examined the ability of keratinocytes and melanocytes to transfer from potential cell carriers under different media conditions to an in vitro human wound bed model. The number of melanocytes transferred, their location within the neoepidermis, and their ability to pigment were evaluated as preclinical end points. Two inert substrates (polyvinyl chloride and silicone sheets) and three candidate plasma-polymerized coatings with controlled surface chemistry deposited on these substrates were explored. Two media for expansion of cells, Greens, currently used clinically (but which contains fetal calf serum), and a serum-free alternative, M2 (melanocyte medium), were explored. Reproducible transfer of physiologically relevant numbers of melanocytes capable of pigmentation from the coculture of melanocytes and keratinocytes was obtained using either Greens medium or M2 medium, and a silicone carrier pretreated with 20% carboxylic acid deposited by plasma polymerization.