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Introduction: The treatment of burn wounds and hypopigmentation conditions often require autologous transplantation of keratinocytes and melanocytes. Tracking transplanted cells to ascertain their contribution to tissue recapitulation presents a challenge. This study demonstrates a methodology based on passive staining with carboxyfluorescein hydroxysuccinimidyl ester (CFSE) that enables localization of cells in tissue sections to investigate the fate of transplanted cells in wound re-epithelialisation. Methods: Viability and migration of CFSE-stained keratinocytes and melanocytes were investigated using viability staining and scratch assays, while proliferation of cells was measured using flow cytometry. In addition, CFSE-stained keratinocytes and melanocytes were transplanted to a human ex vivo wound model, either in suspension, or with the aid of macroporous gelatine microcarriers. Wounds were analysed seven, 14 and 21 days post transplantation using cryosectioning and fluorescence microscopy. Sections from wounds with transplanted co-cultured keratinocytes and melanocytes were stained for pancytokeratin to distinguish keratinocytes. Results: CFSE-staining of keratinocytes and melanocytes did not affect the viability, migration or proliferation of the cells. Transplanted cells were tracked in ex vivo wounds for 21 days, illustrating that the staining had no effect on wound re-epithelialisation. In conclusion, this study presents a novel application of CFSE-staining for tacking transplanted primary human keratinocytes and melanocytes. [ABSTRACT FROM AUTHOR]

Adherent cells cultured in vitro must usually, at some point, be detached from the culture substrate. Presently, the most common method of achieving detachment is through enzymatic treatment which breaks the adhesion points of the cells to the surface. This comes with the drawback of deteriorating the function and viability of the cells. Other methods that have previously been proposed include detachment of the cell substrate itself, which risks contaminating the cell sample, and changing the surface energy of the substrate through thermal changes, which yields low spatial resolution and risks damaging the cells if they are sensitive to temperature changes. Here cell culture substrates, based on thin films of the ferroelectric polyvinylidene fluoride trifluoroethylene (PVDF-TrFE) co-polymer, are developed for electroactive control of cell adhesion and enzyme-free detachment of cells. Fibroblasts cultured on the substrates are detached through changing the direction of polarization of the ferroelectric substrate. The method does not affect subsequent adhesion and viability of reseeded cells. [ABSTRACT FROM AUTHOR]

Testing of irritant compounds has traditionally been performed on animals and human volunteers. Animal testing should always be restricted and for skin irritancy mice and rabbits hold poor predictive value for irritant potential in humans. Irritant testing on human volunteers is restricted by the duration subjects can be exposed, and by the subjectivity of interpreting the visual signs of skin irritation. We propose an irritant testing system using viable human full thickness skin with the loss of cell viability in the exposed skin area as end point measurement. Skin was exposed to sodium dodecyl sulfate (SDS) at 20% concentration by non-occluded topical exposure to establish a positive control response and subsequent test compounds were statistically compared with the 20% SDS response. Cell viability and metabolism were measured with 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The model presents correlation between increased concentration of SDS and decreased viability of cells in the exposed skin area (R2 = 0.76). We propose the model to be used for cytotoxicity testing of irritant compounds. With fully intact barrier function, the model comprises all cells present in the skin with quantifiable end point measurement. [ABSTRACT FROM AUTHOR]

Spatial control of cell detachment is potentially of great interest when selecting cells for clonal expansion and in order to obtain a homogeneous starting population of cells aimed for tissue engineering purposes. Here, selective detachment and cell sorting of human primary keratinocytes and fibroblasts is achieved using thin films of a conjugated polymer. Upon electrochemical oxidation, the polymer film swells, cracks, and finally detaches taking cells cultured on top along with it. The polymer can be patterned using standard photolithography to fabricate a cross-point matrix with polymer pixels that can be individually addressed and thus detached. Detachment occurs above a well-defined threshold of +0.7 V versus Ag/AgCl, allowing the use of a relatively simple and easily manufactured passive matrix-addressing configuration, based on a resistor network, to control the cell-sorting device. [ABSTRACT FROM AUTHOR]

Background: Chronic wounds are one of the greatest challenges for the healthcare system. Today, a plethora of dressings are used in the treatment of these wounds, each with specific influence on the wound environment. Due to differences in the permeability of the dressings the use will result in differences in the pH balance in the wound bed. However, little is known about how changes in the pH in the wound environment affect the different phases of the healing process. Aim: The aim of the present study was to investigate the effects of acidic pH on the regeneration phase by studying keratinocyte function in vitro and re-epithelialisation in an in vitro model of human skin. Results:In vitro assays showed reduced viability and migration rates in human keratinocytes when pH was lowered. Real time PCR revealed differential expression of genes related to wound healing and environmental impairment. Tissue culture showed no re-epithelialisation of wounds subjected to pH 5.0 and moderate re-epithelialisation at pH 6.0, compared to controls at pH 7.4. Conclusion: The results indicate that lowering pH down to pH 5.0 in wounds is counterproductive in aspect of keratinocyte function which is crucial for successful wound healing. [ABSTRACT FROM AUTHOR]

The possibility to use a suspended tridimensional matrix as scaffolding for re-epithelialization of in vitro cutaneous wounds was investigated with the aid of a human in vitro wound healing model based on viable full thickness skin. Macroporous gelatin microcarriers, CultiSpher-S, were applied to in vitro wounds and cultured for 21 days. Tissue sections showed incorporation of wound edge keratinocytes into the microcarriers and thicker neoepidermis in wounds treated with microcarriers. Thickness of the neoepidermis was measured digitally, using immunohistochemical staining of keratins as epithelial demarcation. Air-lifting of wounds enhanced stratification in control wounds as well as wounds with CultiSpher-S. Immunohistochemical staining revealed expression of keratin 5, keratin 10, and laminin 5 in the neoepidermal component. We conclude that the CultiSpher-S microcarriers can function as tissue guiding scaffold for re-epithelialization of cutaneous wounds. [ABSTRACT FROM AUTHOR]

Introduction: First aid performed by immediate responders can be the difference between life and death in the case of trauma with massive bleeding. To develop effective training programs to teach bleeding control to laypersons, it is important to be aware of beliefs and misconceptions people hold on bleeding and severity of bleeding situations.Method: A controlled study was conducted in which 175 American college students viewed 78 video clips of simulated bleeding injuries. The volume of blood present (between 0 and 1900 ml), rate of blood flow, and victim gender were systematically varied within participants. Participants were asked to rate injury severity, indicate the appropriate first aid action, and estimate the amount of time until death for the victim.Results: Though the Stop the Bleed® campaign recommends training laypeople to treat 165 ml of blood loss as life threatening, participants largely rated this volume of blood loss as minimal, mild, or moderate and estimated that the victim had just under one hour to live. Increased blood loss was associated with increased recommendations to use a tourniquet. However, in the 1900 ml conditions, participants still estimated that victims had around 22 minutes to live and approximately 15% recommended direct pressure as the intervention. Severity ratings and recommendations to use a tourniquet were also higher for the male victim than the female victim.Conclusions: Injury classification, intervention selection, and time to death-estimations revealed that training interventions should connect classifications of blood loss to appropriate action and focus on perceptions of how much time one has to respond to a bleeding. The study also revealed a gender related bias in terms of injury classification and first aid recommendations. Bleeding control training programs can be designed to address identified biases and misconceptions while building on existing knowledge and commonly used terminology. [ABSTRACT FROM AUTHOR]