Development of experimental in vitro burn model

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.

Burn; fibroblast; cell line NIH-3T3; microwave; MTT; Confocal microscopy

Authorship

Ana Carolina Morais Fernandes

MD. Resident, Burn Care Unit, Plastic Surgery Division, Universidade Federal de São Paulo, São Paulo, SP, Brazil. Technical proceduresUniversidade Federal de São PauloBrazilSão Paulo, SP, BrazilMD. Resident, Burn Care Unit, Plastic Surgery Division, Universidade Federal de São Paulo, São Paulo, SP, Brazil. Technical procedures

Jerônimo Pereira de França

PhD, Associate Professor, Department of Biological Sciences, Universidade Estadual de Santa Cruz, Ilhéus-BA, Brazil. Scientific and intellectual content of the study, interpretation of data and critical revisionUniversidade Estadual de Santa CruzBrazilIlhéus, BA, BrazilPhD, Associate Professor, Department of Biological Sciences, Universidade Estadual de Santa Cruz, Ilhéus-BA, Brazil. Scientific and intellectual content of the study, interpretation of data and critical revision

Silvana Gaiba

PhD, Fellow Pos-PhD degree, Department of Biological Sciences, Universidade Estadual de Santa Cruz, Ilhéus-BA, Brazil. Technical procedures, acquisition and interpretation of data, manuscript writingUniversidade Estadual de Santa CruzBrazilIlhéus, BA, BrazilPhD, Fellow Pos-PhD degree, Department of Biological Sciences, Universidade Estadual de Santa Cruz, Ilhéus-BA, Brazil. Technical procedures, acquisition and interpretation of data, manuscript writing

Antonio Carlos Aloise

PhD, Fellow Pos-PhD degree, Plastic Surgery Division, Universidade Federal de São Paulo, São Paulo, SP, Brazil. Interpretation of data and critical revisionUniversidade Federal de São PauloBrazilSão Paulo, SP, BrazilPhD, Fellow Pos-PhD degree, Plastic Surgery Division, Universidade Federal de São Paulo, São Paulo, SP, Brazil. Interpretation of data and critical revision

Andrea Fernandes de Oliveira

PhD, Fellow PhD degree, Plastic Surgery Division, Universidade Federal de São Paulo, São Paulo, SP, Brazil. Interpretation of data and critical revisionUniversidade Federal de São PauloBrazilSão Paulo, SP, BrazilPhD, Fellow PhD degree, Plastic Surgery Division, Universidade Federal de São Paulo, São Paulo, SP, Brazil. Interpretation of data and critical revision

Andrea Aparecida de Fátima Souza Moraes

PhD, Department of Biological Sciences, Universidade Estadual de Santa Cruz, Ilhéus-BA, Brazil. Technical proceduresUniversidade Estadual de Santa CruzBrazilIlhéus, BA, BrazilPhD, Department of Biological Sciences, Universidade Estadual de Santa Cruz, Ilhéus-BA, Brazil. Technical procedures

Lucimar Pereira de França

Lydia Masako Ferreira

Head and Full Professor, Plastic Surgery Division, UNIFESP, Researcher 1A-CNPq, Director Medicine III-CAPES, Sao Paulo-SP, Brazil. Interpretation of data and critical revisionUNIFESPBrazilSao Paulo, SP, BrazilHead and Full Professor, Plastic Surgery Division, UNIFESP, Researcher 1A-CNPq, Director Medicine III-CAPES, Sao Paulo-SP, Brazil. Interpretation of data and critical revision

Correspondence: Lydia Masako Ferreira Disciplina de Cirurgia Plástica-UNIFESP Rua Napoleão de Barros, 715/4º andar 04042-002 Sao Paulo - SP Brasil Tel.: (55 11)5576-4118 Fax: (55 11)5571-6579 sandra.dcir@epm.br jeronimopf@gmail.com

SCIMAGO INSTITUTIONS RANKINGS

Figures

Figures (7)

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Figure 1
Mouse fibroblast cell line NIH-3T3 culture on culture dish.

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Figure 2
Heating of the base of glass to enabe the heat transfer to the cell culture dishes.

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Figure 3
Heat of the base of glass and heat transfer to the dishes with 3T3 line cell culture.

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Figure 4
Increase of the temperature of 3T3 culture dish following the transfer of heat from the base of glass to the culture dishes after a contact time of 30 seconds.

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Figure 5
A- NIH-3T3 cells 2h after incubation in MTT. B- Expanded image shows the formazan crystals.

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Figure 6
The effects of thermal injury on cell viability determined by MTT assay. A- Represents the MTT assay that determinated two graphics curves (two phases) with an inflection point for the delta temperature of 15 º C. B- Blue line, represents the first phase (up) and the statistic difference of the experimental groups compared to the control group. C- Red line, represents the second phase (down) and the statistic difference of experimental groups compared to the inflection point.

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Figure 7
Fluorescence microscopy image of NIH-3T3 cells control and after effects of thermal. Experimental Groups CG: (A1, A2) and microscopy with extended depth of field (B, B1 e B2) and after Heating of the base of glass plate to enabe to heat transfer to the glass botton culture dishes: (C e D). The cells were stained with CellMask(tm) Deep Red plasma membrane stain and location of the cell nuclei stained with DAPI (blue) and actin filaments making up the cytoskeleton labeled phalloidin conjugated to Alexa-Fluor 488 (green), overlap of images (A1, A2, C and D). DAPI staining was performed. Arrows (figure C and D) indicate the cells with DNA fragmentation and membrane blebbing. The star (figure A2) indicates pyknotic nuclei. Arrows (figure A2) indicate the cell division (mitosis) and microscopy with extended depth of field (B, B1 e B2). These images are representative of 5 replicates and 04 independent experiments.

Figure 1 Mouse fibroblast cell line NIH-3T3 culture on culture dish.

Figure 2 Heating of the base of glass to enabe the heat transfer to the cell culture dishes.

Figure 3 Heat of the base of glass and heat transfer to the dishes with 3T3 line cell culture.

Figure 4 Increase of the temperature of 3T3 culture dish following the transfer of heat from the base of glass to the culture dishes after a contact time of 30 seconds.

Figure 5 A- NIH-3T3 cells 2h after incubation in MTT. B- Expanded image shows the formazan crystals.

Figure 6 The effects of thermal injury on cell viability determined by MTT assay. A- Represents the MTT assay that determinated two graphics curves (two phases) with an inflection point for the delta temperature of 15 º C. B- Blue line, represents the first phase (up) and the statistic difference of the experimental groups compared to the control group. C- Red line, represents the second phase (down) and the statistic difference of experimental groups compared to the inflection point.

Figure 7 Fluorescence microscopy image of NIH-3T3 cells control and after effects of thermal. Experimental Groups CG: (A1, A2) and microscopy with extended depth of field (B, B1 e B2) and after Heating of the base of glass plate to enabe to heat transfer to the glass botton culture dishes: (C e D). The cells were stained with CellMask(tm) Deep Red plasma membrane stain and location of the cell nuclei stained with DAPI (blue) and actin filaments making up the cytoskeleton labeled phalloidin conjugated to Alexa-Fluor 488 (green), overlap of images (A1, A2, C and D). DAPI staining was performed. Arrows (figure C and D) indicate the cells with DNA fragmentation and membrane blebbing. The star (figure A2) indicates pyknotic nuclei. Arrows (figure A2) indicate the cell division (mitosis) and microscopy with extended depth of field (B, B1 e B2). These images are representative of 5 replicates and 04 independent experiments.

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