In vitro studies with keloid fibroblasts frequently present contradictory results. This may occur because keloids present distinct genotypic and phenotypic characteristics in its different regions, such as the peripheral region in relation to the central region. We suggest an explant model for keloid fibroblasts harvesting, standardising the initial processing of keloid samples to obtain fragments from different regions, considering its biological differences, for primary cell culture. The different keloid regions were delimited and fragments were obtained using a 3-mm diameter punch. To remove fragments from the periphery, the punch was placed in one longitudinal line extremity, respecting the lesion borders. For the central region, it was placed in the intersection of lines at the level of the largest longitudinal and transversal axes, the other fragments being removed centrifugally in relation to the first one. Primary fibroblast culture was carried out by explant. Flow cytometry analysis showed cell cycle differences between the groups, confirming its different origins and biological characteristics. In conclusion, our proposed model proved itself efficient for keloid fibroblast isolation from specific regions and cultivation. Its simplicity and ease of execution may turn it into an important tool for studying the characteristics of the different keloid-derived fibroblasts in culture.

PURPOSE:There is a growing scientific interest in the plasticity and therapeutic potential of adipose-derived stem cells (ASCs), which are multipotent and abundant in adipose tissue and can differentiate in vitro into multiple lineages, including adipocytes, chondrocytes, osteoblasts, neural cells, endothelial cells and cardiomyocytes. The aim of this study was to isolate, cultivate and identify ASCs. METHODS: Human adipose precursor cells were obtained from subcutaneous abdominal tissue. Recently dispersed cells were separated by density centrifugation gradient, cultured and then analyzed. RESULTS: Human ASCs were able to replicate in our culture conditions. The cells maintained their phenotypes throughout the studied period on different passages confirming they suitability for in vitro cultivation. We also induced their adipogenic, osteogenic and chondrogenic differentiation, verifying their mesenchymal stem cells potentiality in vitro. Flow cytometry results showed that these cells expressed CD73, CD90 and CD105, (mesenchymal stem-cells markers), contrasting with the lack of expression of CD16, CD34 and CD45 (hematopoietic cells markers). RESUMO OBJETIVO:Há um interesse científico crescente na plasticidade e potencial terapêutico das células-tronco do tecido adiposo humano, células multipotentes e abundantes no tecido adiposo que podem se diferenciar in vitro em múltiplas linhagens celulares, incluindo adipócitos, condrócitos, osteoblastos, células neurais, endoteliais e cardiomiócitos. O objetivo deste estudo foi isolar, cultivar e identificar células-tronco do tecido adiposo humano. MÉTODOS: Células precursoras humanas do tecido adiposo foram obtidas de tecido abdominal subcutâneo. As células recém-dispersas foram separadas por gradiente de centrifugação por densidade, cultivadas e então analisadas. RESULTADOS: As células-tronco do tecido adiposo humano foram capazes de se replicar nas nossas condições de cultivo e mantiveram seu fenótipo em diferentes passagens durante o estudo, confirmando sua adequabilidade para cultivo in vitro. A diferenciação adipogênica, osteogênica e condrogênica também foi induzida, confirmando seu potencial de células-tronco mesenquimais in vitro. Os resultados de citometria de fluxo evidenciaram a expressão dos marcadores de células-tronco mesenquimais CD73, CD90 e CD105, contrastando com a falta de expressão dos marcadores de células hematopoiéticas CD16, CD34 e CD45. CONCLUSÃO: Foi possível isolar células-tronco do tecido adiposo humano por cultivo in vitro sem indução adipogênica, mantendo sua integridade funcional e altos níveis de proliferação. As células demonstraram potencial de diferenciação adipogênico, osteogênico e condrogênico in vitro. Descritores: Adipócitos. Células-Tronco Adultas. Células-Tronco Mesenquimais. Células-Tronco. Tecido Adiposo.Gaiba S et al.

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PURPOSE:To propose an experimental burn model in NIH-3T3 cell line. METHODS:Induction of thermal injury in cultures of mouse fibroblast -NIH-3T3-cell line and determination of cell viability by MTT and imunofluorescence. RESULTS:The heating of the Petri dish increased proportionally to the temperature of the base and the time of exposure to microwave.In this in vitro burn model, using the cell line NIH-3T3 was observed drastic cellular injury with significant changes in cell viability and activity. It showed drastically modified cell morphology with altered membrane, cytoskeleton and nucleus, and low cellularity compared to the control group. CONCLUSION:The burn model in vitro using the cell line NIH-3T3 was reproductive and efficient. This burn model was possible to determine significant changes in cell activity and decreased viability, with drastic change in morphology, cell lysis and death.

Purpose: To evaluate the effects of the adipose-derived stem cells (ADSC) in the viability of random skin flap in rats.Methods: Thirty five adult male Wistar rats (weight 250-300 g) were used. ADSC were isolated from adult male rats (n=5). ADSC were separated, cultured and then analyzed. A dorsal skin flap measuring 10x4 cm was raised and a plastic barrier was placed between the flap and its bed. After the surgical procedure, the animals were randomized into two groups (n=15 each group), group control and group ADSC. In all groups the procedures were performed immediately after the surgery. The percentage of flap necrosis was measured on the seventh postoperative day. Results:The ADSC were able to replicate in our culture conditions. We also induced their adipogenic, osteogenic and chondrogenic differentiation, verifying their mesenchymal stem cells potentiality in vitro. The results were statistically significant showing that the ADSC decreased the area of necrosis (p<0.05). Conclusion:The cells demonstrated adipogenic, osteogenic and chondrogenic differentiation potential in vitro. The administration of adipose-derived stem cells was effective to increase the viability of the random skin flaps in rats.

PURPOSE:To evaluate the viability of random pattern dorsal skin flaps in rats after injection of adipose-derived stem cells (ADSC). METHODS:Thirty five adult male Wistar EPM rats (weight 250-300 g) were distributed, at random, in two groups. I-Control (flap elevation with injection of saline solution) with fifteen animals and II-Experimental (flap elevation with injection of ADSC ) with fifteen animal. The ADSC were isolated from others five adult male rats. A dorsal skin flap measuring 10x4 cm was raised and a plastic barrier was placed between the flap and its bed in both groups and the injection (cells or saline solution) were perfomed immediately after the surgery. The percentage of flap necrosis was measured on the seventh postoperative day. RESULTS:The ADSC were able to replicate in our culture conditions. We also induced their adipogenic, osteogenic and chondrogenic differentiation to verify their mesenchymal stem cells potentiality in vitro. The results were statistically significant showing that the ADSC decreased the area of necrosis (p<0.05). CONCLUSIONS:The cells demonstrated adipogenic, osteogenic and chondrogenic differentiation potential in vitro. The administration of adipose-derived stem cells was effective to increase the viability of the random random pattern dorsal skin flaps in rats.

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