A modified hydrogel production protocol to decrease cellular content

We analyzed the effect of multiple decellularization processes by histological analysis, DNA quantification, and flow cytometry. Subsequently, we analyzed the most appropriate hydrogel concentration to minimize cytotoxicity on fibroblast culture and to maximize cell proliferation.

Results:

After the fourth decellularization, the DNA quantification showed the lowest DNA concentration (< 50 ng/mg). Histological analysis showed no cell components in the hydrogel. Moreover, hematoxylin and eosin showed a heterogeneous structure of collagen fibers. The best hydrogel concentration ranged from 3 to 25%, and there was no significant difference between the 24 hours and seven days.

Conclusions:

The process of hydrogel production was effective for removing cells and DNA elements. The best hydrogel concentration ranged from 3 to 25%.

Key words
Regenerative Medicine; Biocompatible Materials; Hydrogels; Skin, Artificial; Cells, Cultured; Fibroblasts

Authorship

Gabriela Catão Diniz Braga

Acquisition of data

Acquisition and analysis of data

Bachelor. Universidade de São Paulo – Discipline of Plastic Surgery, Microsurgery and Plastic Surgery Laboratory – School of Medicine – São Paulo (SP), Brazil.Universidade de São PauloBrazilSão Paulo, SP, BrazilBachelor. Universidade de São Paulo – Discipline of Plastic Surgery, Microsurgery and Plastic Surgery Laboratory – School of Medicine – São Paulo (SP), Brazil.

https://orcid.org/0000-0003-2234-7702

Cristina Pires Camargo ^**Corresponding author: cristinacamargo@usp.br | (55 11) 30620415

Conception to the study

Critical revision

PhD. Universidade de São Paulo – Discipline of Plastic Surgery, Microsurgery and Plastic Surgery Laboratory – School of Medicine – São Paulo (SP), Brazil.Universidade de São PauloBrazilSão Paulo, SP, BrazilPhD. Universidade de São Paulo – Discipline of Plastic Surgery, Microsurgery and Plastic Surgery Laboratory – School of Medicine – São Paulo (SP), Brazil.

https://orcid.org/0000-0002-3134-0003

Martin Conrad Harmsen

Conception to the study

Critical revision

PhD. Associate professor. University Medical Center Groningen – Laboratory for Cardiovascular Regenerative Medicine – Department of Pathology and Medical Biology – Hanzeplein 1, Netherlands.University Medical Center GroningenNetherlandsHanzeplein, NetherlandsPhD. Associate professor. University Medical Center Groningen – Laboratory for Cardiovascular Regenerative Medicine – Department of Pathology and Medical Biology – Hanzeplein 1, Netherlands.

https://orcid.org/0000-0002-7128-2741

Aristides Tadeu Correia

Acquisition and analysis of data

PhD. Universidade de São Paulo – Department of Cardiopneumology – Thoracic Surgery Research Laboratory – Heart Institute of School of Medicine – São Paulo (SP), Brazil.Universidade de São PauloBrazilSão Paulo, SP, BrazilPhD. Universidade de São Paulo – Department of Cardiopneumology – Thoracic Surgery Research Laboratory – Heart Institute of School of Medicine – São Paulo (SP), Brazil.

https://orcid.org/0000-0002-1195-6732

Sonia Souza

Acquisition and analysis of data

Bachelor. Universidade de São Paulo – Department of Cardiopneumology – Cardiovascular Surgery and Circulatory Physiopathology Laboratory – School of Medicine – São Paulo (SP), Brazil.Universidade de São PauloBrazilSão Paulo, SP, BrazilBachelor. Universidade de São Paulo – Department of Cardiopneumology – Cardiovascular Surgery and Circulatory Physiopathology Laboratory – School of Medicine – São Paulo (SP), Brazil.

https://orcid.org/0000-0001-6981-9039

Marilia Seelaender

Critical revision

PhD. Associate professor. Universidade de São Paulo – Department of Clinical Surgery – School of Medicine – São Paulo (SP), Brazil.Universidade de São PauloBrazilSão Paulo, SP, BrazilPhD. Associate professor. Universidade de São Paulo – Department of Clinical Surgery – School of Medicine – São Paulo (SP), Brazil.

https://orcid.org/0000-0002-9999-8020

Viviane Araujo Nunes

PhD. Associate professor. Universidade de São Paulo – Department of Biotechnology – School of Arts, Sciences and Humanities – São Paulo (SP), Brazil.Universidade de São PauloBrazilSão Paulo, SP, BrazilPhD. Associate professor. Universidade de São Paulo – Department of Biotechnology – School of Arts, Sciences and Humanities – São Paulo (SP), Brazil.

https://orcid.org/0000-0003-0861-4392

Jeniffer Farias dos Santos

Acquisition of data

PhD. Universidade de São Paulo – Department of Biotechnology – School of Arts, Sciences and Humanities – São Paulo (SP), Brazil.Universidade de São PauloBrazilSão Paulo, SP, BrazilPhD. Universidade de São Paulo – Department of Biotechnology – School of Arts, Sciences and Humanities – São Paulo (SP), Brazil.

https://orcid.org/0000-0002-2084-752X

Elida Adalgisa Neri

Acquisition of data

PhD. Universidade de São Paulo – Laboratory of Genetics and Molecular Cardiology – Heart Institute – School of Medicine – São Paulo (SP), Brazil.Universidade de São PauloBrazilSão Paulo, SP, BrazilPhD. Universidade de São Paulo – Laboratory of Genetics and Molecular Cardiology – Heart Institute – School of Medicine – São Paulo (SP), Brazil.

https://orcid.org/0000-0003-1121-5413

Iuri Cordeiro Valadão

Acquisition of data

https://orcid.org/0000-0001-8417-6836

Luiz Felipe Pinho Moreira

Conception to the study

Critical revision

PhD. Associate professor. Universidade de São Paulo – Department of Cardiopneumology, Cardiovascular Surgery and Circulatory Physiopathology Laboratory – School of Medicine – São Paulo (SP), Brazil.Universidade de São PauloBrazilSão Paulo, SP, BrazilPhD. Associate professor. Universidade de São Paulo – Department of Cardiopneumology, Cardiovascular Surgery and Circulatory Physiopathology Laboratory – School of Medicine – São Paulo (SP), Brazil.

https://orcid.org/0000-0003-0179-4976

Rolf Gemperli

Critical revision

PhD. Full professor. Universidade de São Paulo – Discipline of Plastic Surgery, Microsurgery and Plastic Surgery Laboratory – School of Medicine – São Paulo (SP) Brazil.Universidade de São PauloBrazilSão Paulo, SP, BrazilPhD. Full professor. Universidade de São Paulo – Discipline of Plastic Surgery, Microsurgery and Plastic Surgery Laboratory – School of Medicine – São Paulo (SP) Brazil.

https://orcid.org/0000-0002-4147-4478

Conflict of interest: Nothing to declare.

*Corresponding author: cristinacamargo@usp.br | (55 11) 30620415

SCIMAGO INSTITUTIONS RANKINGS

Figures

Figures (10)

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Figure 1
Porcine skin decellularization. (a) Porcine skin in natura. (b) Porcine skin triturated and incubated with reagents (trisol) during the decellularization process. (c) Decellularized and lyophilized extracellular matrix (n = 5).

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Figure 2
Hydrogel aspect from decellularized porcine skin. (a) Hydrogel at 4 °C in liquid state used in the experiments. (b) Hydrogel at 37 °C in a swollen state and used in cell culture experiments (n = 5).

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Figure 3
Conditioned culture medium with hydrogel. The content of two phases in the wells are: culture Dulbecco’s modified eagle medium (pink on the surface), and solid hydrogel (white on the bottom) (n = 5).

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Figure 4
Hydrogel stained with hematoxylin and eosin. (a and b) Hydrogel from in-natura skin with cellular nuclei stained in purple, and (c and d) hydrogel from decellularized skin without cellular nuclei.

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Figure 5
Cell counting in a decellularized hydrogel graph. Control sample mean: 34,6 cells/area. Decellularized sample mean: 0,7 cell/area.

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Figure 6
Hydrogel stained with picrosirius. (a and b) Hydrogel from in-natura skin with thicker and more condensed collagen fibers in comparison to hydrogel from decellularized skin, which (c and d) contain more spread collagen fibers.

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Figure 7
DNA quantification in decellularized porcine skin. First decellularization: control sample: mean = 3,549.67; decellularized sample: mean = 795.821; p > 0.05. Second decellularization: control sample: mean = 2,872.18, decellularized sample: mean = 223.761; p = 0.0122. Third decellularization: control sample: mean = 3,267.7, decellularized sample: mean = 232.3; p = 0.0126. Fourth decellularization: control sample: mean = 3,491.72, decellularized sample: mean = 41.06; p = 0.0126. P compared between all decellularization: first vs. second decellularization: p = 0.001; first vs. third decellularization: p = 0.0007; first vs. fourth decellularization: p < 0.0001; second vs. third decellularization: p > 0.05; second vs. fourt decellularization: p = 0.0008; third vs. fourth decellularization: p = 0.0014 (n = 5).

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Figure 8
Immunophenotyping of fibroblasts. Results of fibroblast’s immunophenotyping are represented in histogram, with 10,000 events per graph. Cells labeled with antibody anti-human (control) conjugated with fluorophorephycoerythrin (PE) (a, c, and e) or fluorescein isothiocyanate (FITC) (b, d, and f) have their population represented at left in the graph. Positive labels for fibroblast: (a) anti-CD73 conjugated with PE; (b) anti-CD29 conjugated with FITC; (c) anti-CD105 conjugated with PE; (d) anti-CD90 conjugated with FITC. Negative labels for fibroblast, but positive for endothelial cells: (e) CD-31 conjugated with peridinin chlorophyll protein complex (PerCP) and for hematopoietic cells in (f) anti-CD45 conjugated with FITC. (g) Cells labeled with propidium iodide, marker of dead cells. The left population represents live cells, and the right population represents dead cells (5.03%).

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Figure 9
Indirect cytotoxicity with culture medium with hydrogel (CMH) and fibroblasts. The assay was made with a conditioned CMH. CMH was conditioned for two different periods of time: 24 hours and seven days. The serial dilution refers to the percentage of CMH used during cell incubation – 100%: 100-μL CMH; 50%: 50-μL MCH + 50-μL Dulbecco’s modified eagle medium (DMEM); 25%: 25-μL + 75-μL DMEM; 12,5%: 12,5-μL CMH + 87,5-μL DMEM; 6,25%: 6,25-μL MHC + 93,75-μL DMEM; 3%: 3 μL-MHC + 97-μL DMEM; 0%: 100-μL DMEM; Control +: 100-μL DMEM;

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Figure 10
Tridimensional (3D) fibroblast cell culture in hydrogel. The control of this experiment (a-d) are fibroblast cultivated in monolayer. (a) Fibroblast in grayscale. (b) Cytoplasm labeled with Calcein AM. (c) Nucleus labeled with Hoechst. (d) Overlaid fluorescence of cytoplasm and nucleus. (e and f) The control of 3D fibroblasts in hydrogel in grayscale. (g and h) 3D fibroblast in hydrogel labeled with Calcein AM and Hoechst rendered images.

Figure 1 Porcine skin decellularization. (a) Porcine skin in natura. (b) Porcine skin triturated and incubated with reagents (trisol) during the decellularization process. (c) Decellularized and lyophilized extracellular matrix (n = 5).

Figure 2 Hydrogel aspect from decellularized porcine skin. (a) Hydrogel at 4 °C in liquid state used in the experiments. (b) Hydrogel at 37 °C in a swollen state and used in cell culture experiments (n = 5).

Figure 3 Conditioned culture medium with hydrogel. The content of two phases in the wells are: culture Dulbecco’s modified eagle medium (pink on the surface), and solid hydrogel (white on the bottom) (n = 5).

Figure 4 Hydrogel stained with hematoxylin and eosin. (a and b) Hydrogel from in-natura skin with cellular nuclei stained in purple, and (c and d) hydrogel from decellularized skin without cellular nuclei.

Magnification of 40x. Scale bar: 500 μm (n = 5).

Figure 5 Cell counting in a decellularized hydrogel graph. Control sample mean: 34,6 cells/area. Decellularized sample mean: 0,7 cell/area.

p < 0.0001 (n = 5).

Figure 6 Hydrogel stained with picrosirius. (a and b) Hydrogel from in-natura skin with thicker and more condensed collagen fibers in comparison to hydrogel from decellularized skin, which (c and d) contain more spread collagen fibers.

Magnification of 20x. Scale bar: 500 μm (n = 5).

Figure 7 DNA quantification in decellularized porcine skin. First decellularization: control sample: mean = 3,549.67; decellularized sample: mean = 795.821; p > 0.05. Second decellularization: control sample: mean = 2,872.18, decellularized sample: mean = 223.761; p = 0.0122. Third decellularization: control sample: mean = 3,267.7, decellularized sample: mean = 232.3; p = 0.0126. Fourth decellularization: control sample: mean = 3,491.72, decellularized sample: mean = 41.06; p = 0.0126. P compared between all decellularization: first vs. second decellularization: p = 0.001; first vs. third decellularization: p = 0.0007; first vs. fourth decellularization: p < 0.0001; second vs. third decellularization: p > 0.05; second vs. fourt decellularization: p = 0.0008; third vs. fourth decellularization: p = 0.0014 (n = 5).

Figure 8 Immunophenotyping of fibroblasts. Results of fibroblast’s immunophenotyping are represented in histogram, with 10,000 events per graph. Cells labeled with antibody anti-human (control) conjugated with fluorophorephycoerythrin (PE) (a, c, and e) or fluorescein isothiocyanate (FITC) (b, d, and f) have their population represented at left in the graph. Positive labels for fibroblast: (a) anti-CD73 conjugated with PE; (b) anti-CD29 conjugated with FITC; (c) anti-CD105 conjugated with PE; (d) anti-CD90 conjugated with FITC. Negative labels for fibroblast, but positive for endothelial cells: (e) CD-31 conjugated with peridinin chlorophyll protein complex (PerCP) and for hematopoietic cells in (f) anti-CD45 conjugated with FITC. (g) Cells labeled with propidium iodide, marker of dead cells. The left population represents live cells, and the right population represents dead cells (5.03%).

Figure 9 Indirect cytotoxicity with culture medium with hydrogel (CMH) and fibroblasts. The assay was made with a conditioned CMH. CMH was conditioned for two different periods of time: 24 hours and seven days. The serial dilution refers to the percentage of CMH used during cell incubation – 100%: 100-μL CMH; 50%: 50-μL MCH + 50-μL Dulbecco’s modified eagle medium (DMEM); 25%: 25-μL + 75-μL DMEM; 12,5%: 12,5-μL CMH + 87,5-μL DMEM; 6,25%: 6,25-μL MHC + 93,75-μL DMEM; 3%: 3 μL-MHC + 97-μL DMEM; 0%: 100-μL DMEM; Control +: 100-μL DMEM;

Control -: 100 μL dimethyl sulfoxide (DMSO). Dotted lines represent the positive control (n = 5).

Figure 10 Tridimensional (3D) fibroblast cell culture in hydrogel. The control of this experiment (a-d) are fibroblast cultivated in monolayer. (a) Fibroblast in grayscale. (b) Cytoplasm labeled with Calcein AM. (c) Nucleus labeled with Hoechst. (d) Overlaid fluorescence of cytoplasm and nucleus. (e and f) The control of 3D fibroblasts in hydrogel in grayscale. (g and h) 3D fibroblast in hydrogel labeled with Calcein AM and Hoechst rendered images.

Scale: x and y = 326 μm; z = 86 μm (n = 5).

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A modified hydrogel production protocol to decrease cellular content

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[1] Conflict of interest: Nothing to declare.