Isolation, characterization, and cryopreservation of collared peccary skin-derived fibroblast cell lines
Background Biobanking of cell lines is a promising tool of support for wildlife conservation. In particular, the ability to preserve fibroblast cell lines derived from collared peccaries is of significance as these wild mammals are unique to the Americas and play a large role in maintaining the ecos...
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PeerJ Inc.
2020-06-01
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| סדרה: | PeerJ |
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| גישה מקוונת: | https://peerj.com/articles/9136.pdf |
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| author | Alana Azevedo Borges Gabriela Pereira De Oliveira Lira Lucas Emanuel Nascimento Maria Valéria De Oliveira Santos Moacir Franco De Oliveira Alexandre Rodrigues Silva Alexsandra Fernandes Pereira |
| author_facet | Alana Azevedo Borges Gabriela Pereira De Oliveira Lira Lucas Emanuel Nascimento Maria Valéria De Oliveira Santos Moacir Franco De Oliveira Alexandre Rodrigues Silva Alexsandra Fernandes Pereira |
| author_sort | Alana Azevedo Borges |
| collection | DOAJ |
| description | Background Biobanking of cell lines is a promising tool of support for wildlife conservation. In particular, the ability to preserve fibroblast cell lines derived from collared peccaries is of significance as these wild mammals are unique to the Americas and play a large role in maintaining the ecosystem. We identified collared peccary fibroblasts by immunofluorescence and evaluated their morphology, growth and adherence capacity. Further, we monitored the viability and metabolic activity of the fibroblasts to determine the effects of passage number and cryopreservation on establishment of cell lines. Methods Skin biopsies were collected from the peripheral ear region from five adult animals in captivity. Initially, cells were isolated from fragments and cultured in the Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum and 2% antibiotic–antimycotic solution under a controlled atmosphere (38.5 °C, 5% CO2). We evaluated the maintenance of primary cells for morphology, adherence capacity of explants, explants in subconfluence, cell growth and absence of contamination. Moreover, we identified the fibroblast cells by immunofluorescence. Additionally, to evaluate the influence of the number of passages (first, third and tenth passage) and cryopreservation on establishment of cell lines, fibroblasts were analysed for the viability, metabolic activity, population doubling time (PDT), levels of reactive oxygen species (ROS), and mitochondrial membrane potential (ΔΨm). Results All explants (20/20) adhered to the dish in 2.4 days ± 0.5 with growth around the explants in 4.6 days ± 0.7, and subconfluence was observed within 7.8 days ± 1.0. Moreover, by morphology and immunocytochemistry analyses, cells were identified as fibroblasts which presented oval nuclei, a fusiform shape and positive vimentin staining. No contamination was observed after culture without antibiotics and antifungals for 30 days. While there was no difference observed for cell viability after the passages (first vs. third: P = 0.98; first vs. tenth: P = 0.76; third vs. tenth: P = 0.85), metabolic activity was found to be reduced in the tenth passage (23.2 ± 12.1%) when compared to that in the first and third passage (100.0 ± 24.4%, P = 0.006). Moreover, the cryopreservation did not influence the viability (P = 0.11), metabolic activity (P = 0.77), or PDT (P = 0.11). Nevertheless, a greater ΔΨm (P = 0.0001) was observed for the cryopreserved cells (2.12 ± 0.14) when compared to that in the non-cryopreserved cells (1.00 ± 0.05). Additionally, the cryopreserved cells showed greater levels of intracellular ROS after thawing (1.69 ± 0.38 vs. 1.00 ± 0.22, P = 0.04). Conclusions This study is the first report on isolation, characterization and cryopreservation of fibroblasts from collared peccaries. We showed that adherent cultures were efficient for obtaining fibroblasts, which can be used as donor cells for nuclei for species cloning and other applications. |
| first_indexed | 2024-03-09T08:09:57Z |
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| issn | 2167-8359 |
| language | English |
| last_indexed | 2024-03-09T08:09:57Z |
| publishDate | 2020-06-01 |
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| spelling | doaj.art-e25b31e6d2a44460a9ef2e6c468c5be52023-12-02T23:33:01ZengPeerJ Inc.PeerJ2167-83592020-06-018e913610.7717/peerj.9136Isolation, characterization, and cryopreservation of collared peccary skin-derived fibroblast cell linesAlana Azevedo Borges0Gabriela Pereira De Oliveira Lira1Lucas Emanuel Nascimento2Maria Valéria De Oliveira Santos3Moacir Franco De Oliveira4Alexandre Rodrigues Silva5Alexsandra Fernandes Pereira6Laboratory of Animal Biotechnology, Universidade Federal Rural do Semi-Árido, Mossoró, Rio Grande do Norte, BrazilLaboratory of Animal Biotechnology, Universidade Federal Rural do Semi-Árido, Mossoró, Rio Grande do Norte, BrazilLaboratory of Animal Biotechnology, Universidade Federal Rural do Semi-Árido, Mossoró, Rio Grande do Norte, BrazilLaboratory of Animal Biotechnology, Universidade Federal Rural do Semi-Árido, Mossoró, Rio Grande do Norte, BrazilLaboratory of Applied Animal Morphophysiology, Universidade Federal Rural do Semi-Árido, Mossoró, Rio Grande do Norte, BrazilLaboratory of Animal Germplasm Conservation, Universidade Federal Rural do Semi-Árido, Mossoró, Rio Grande do Norte, BrazilLaboratory of Animal Biotechnology, Universidade Federal Rural do Semi-Árido, Mossoró, Rio Grande do Norte, BrazilBackground Biobanking of cell lines is a promising tool of support for wildlife conservation. In particular, the ability to preserve fibroblast cell lines derived from collared peccaries is of significance as these wild mammals are unique to the Americas and play a large role in maintaining the ecosystem. We identified collared peccary fibroblasts by immunofluorescence and evaluated their morphology, growth and adherence capacity. Further, we monitored the viability and metabolic activity of the fibroblasts to determine the effects of passage number and cryopreservation on establishment of cell lines. Methods Skin biopsies were collected from the peripheral ear region from five adult animals in captivity. Initially, cells were isolated from fragments and cultured in the Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum and 2% antibiotic–antimycotic solution under a controlled atmosphere (38.5 °C, 5% CO2). We evaluated the maintenance of primary cells for morphology, adherence capacity of explants, explants in subconfluence, cell growth and absence of contamination. Moreover, we identified the fibroblast cells by immunofluorescence. Additionally, to evaluate the influence of the number of passages (first, third and tenth passage) and cryopreservation on establishment of cell lines, fibroblasts were analysed for the viability, metabolic activity, population doubling time (PDT), levels of reactive oxygen species (ROS), and mitochondrial membrane potential (ΔΨm). Results All explants (20/20) adhered to the dish in 2.4 days ± 0.5 with growth around the explants in 4.6 days ± 0.7, and subconfluence was observed within 7.8 days ± 1.0. Moreover, by morphology and immunocytochemistry analyses, cells were identified as fibroblasts which presented oval nuclei, a fusiform shape and positive vimentin staining. No contamination was observed after culture without antibiotics and antifungals for 30 days. While there was no difference observed for cell viability after the passages (first vs. third: P = 0.98; first vs. tenth: P = 0.76; third vs. tenth: P = 0.85), metabolic activity was found to be reduced in the tenth passage (23.2 ± 12.1%) when compared to that in the first and third passage (100.0 ± 24.4%, P = 0.006). Moreover, the cryopreservation did not influence the viability (P = 0.11), metabolic activity (P = 0.77), or PDT (P = 0.11). Nevertheless, a greater ΔΨm (P = 0.0001) was observed for the cryopreserved cells (2.12 ± 0.14) when compared to that in the non-cryopreserved cells (1.00 ± 0.05). Additionally, the cryopreserved cells showed greater levels of intracellular ROS after thawing (1.69 ± 0.38 vs. 1.00 ± 0.22, P = 0.04). Conclusions This study is the first report on isolation, characterization and cryopreservation of fibroblasts from collared peccaries. We showed that adherent cultures were efficient for obtaining fibroblasts, which can be used as donor cells for nuclei for species cloning and other applications.https://peerj.com/articles/9136.pdfBiological characterizationCellular conservationCryobankingWild mammals |
| spellingShingle | Alana Azevedo Borges Gabriela Pereira De Oliveira Lira Lucas Emanuel Nascimento Maria Valéria De Oliveira Santos Moacir Franco De Oliveira Alexandre Rodrigues Silva Alexsandra Fernandes Pereira Isolation, characterization, and cryopreservation of collared peccary skin-derived fibroblast cell lines PeerJ Biological characterization Cellular conservation Cryobanking Wild mammals |
| title | Isolation, characterization, and cryopreservation of collared peccary skin-derived fibroblast cell lines |
| title_full | Isolation, characterization, and cryopreservation of collared peccary skin-derived fibroblast cell lines |
| title_fullStr | Isolation, characterization, and cryopreservation of collared peccary skin-derived fibroblast cell lines |
| title_full_unstemmed | Isolation, characterization, and cryopreservation of collared peccary skin-derived fibroblast cell lines |
| title_short | Isolation, characterization, and cryopreservation of collared peccary skin-derived fibroblast cell lines |
| title_sort | isolation characterization and cryopreservation of collared peccary skin derived fibroblast cell lines |
| topic | Biological characterization Cellular conservation Cryobanking Wild mammals |
| url | https://peerj.com/articles/9136.pdf |
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