Morphological, Ultrastructural, and Immunocytochemical Characterization and Assessment of Puma (<i>Puma concolor</i> Linnaeus, 1771) Cell Lines After Extended Culture and Cryopreservation

Lira, Gabriela P.O.; Borges, Alana Azevedo; Nascimento, Matheus B.; Aquino, Leonardo Vitorino Costa de; Moura, Luiz F.M.P.; Silva, Herlon V. R.; Ribeiro, Leandro Rodrigues; Silva, Alexandre Rodrigues; Pereira, Alexsandra Fernandes

doi:10.1089/bio.2021.0117

Cited by 9 publications

(11 citation statements)

References 37 publications

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“…Skin tissue samples (1.0-2.0 cm 2 ) were obtained from the ear notch of three healthy adult puma, one female and two males, between 2 to 5 years old, at the Ecologic Park Ecopoint (Fortaleza, CE, Brazil) and Sargento Prata Municipal Zoo (Fortaleza, CE, Brazil). The skin samples were cultured, and three fibroblast lines were previously established ( Lira et al, 2022 ). Subsequently, cells from three lines frozen in 10% dimethyl sulfoxide (DMSO), 10% FBS, and 0.2 M sucrose were thawed, and 4 th and 5 th passage cells were used for this study.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…Cells were evaluated under an inverted microscope (Nikon TS100, Tokyo, Japan) for cell forms and cytoplasmic extensions ( Lira et al, 2022 ). Moreover, trypan blue exclusion assay determined the percentage of living cells ( Lira et al, 2022 ). Briefly, the cells were stained with 0.4% trypan blue in phosphate-buffered saline (PBS) and counted using a hemocytometer.…”

Section: Methodsmentioning

confidence: 99%

“…A minimum of 300 cells was counted using ImageJ software (National Institutes of Health, Bethesda, Maryland, USA) in every sample, and the percentage of cells recorded in four different groups: viable (V), early apoptotic (EA), late apoptotic (LA), and necrotic cells (N). With V: cells with a uniform light green nucleus; EA: cells in initial apoptosis, with a non-uniform green nucleus; LA: cells in late apoptosis, with a non-uniform bright orange nucleus, and N: necrotic cells, with a uniform orange nucleus ( Lira et al, 2022 ).…”

Section: Methodsmentioning

confidence: 99%

“…We have developed the first two steps in puma ( Puma concolor Linnaeus, 1781). Recently, we have established somatic tissue banks for the species ( Lira et al, 2021 ), as well as we have established cell lines for their use as nucleus donor cells for cloning ( Lira et al, 2022 ). Considering that there are variations between the synchronization methods of the cell cycle in G 0 /G 1 among species ( Młodawska et al, 2022 ), including even among felids ( Veraguas et al, 2017 ), we propose to evaluate different culture conditions for puma fibroblasts, aiming to use these cells in G 0 /G 1 .…”

Section: Introductionmentioning

confidence: 99%

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Full confluency, serum starvation, and roscovitine for inducing arrest in the G0/G1 phase of the cell cycle in puma skin-derived fibroblast lines

et al. 2023

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The puma population is constantly decreasing, and cloning by somatic cell nuclear transfer can be used to conserve the species. One of the factors determining the success of the development of cloned embryos is the cell cycle stage of the donor cells. We evaluated the effects of full confluency (~100%), serum starvation (0.5% serum), and roscovitine (15 µM) treatments on the cell cycle synchronization in G 0 /G 1 of puma skin-derived fibroblasts by flow cytometric analysis. Also, we assessed the effects of these synchronization methods on morphology, viability, and apoptosis levels using microscopy tools. The results showed that culturing the cells to confluence for 24 h (84.0%), 48 h (84.6%), and 72 h (84.2%) and serum starvation for 96 h (85.4%) yielded a significantly higher percentage of cells arrested in the G 0 /G 1 (P 0.05) phase than cells not subjected to any cell cycle synchronization method (73.9%). Nevertheless, while serum starvation reduced the percentage of viable cells, no difference was observed for the full confluence and roscovitine treatments (P 0.05). Moreover, roscovitine for 12 h (78.6%) and 24 h (82.1%) was unable to synchronize cells in G 0 /G 1 (P 0.05). In summary, full confluency induces puma fibroblast cell cycle synchronization at the G 0 /G 1 stage without affecting cell viability. These outcomes may be valuable for planning donor cells for somatic cell nuclear transfer in pumas.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Full confluency, serum starvation, and roscovitine for inducing arrest in the G0/G1 phase of the cell cycle in puma skin-derived fibroblast lines

et al. 2023

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Establishment and characterization of fibroblast lines from the northern tiger cat (Leopardus tigrinus, Schreber, 1775) during extended passage and cryopreservation

Viana,

de Oliveira,

Rodrigues

et al. 2024

Cell Biology International

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The establishment of fibroblast lines enables several applications from the formation of biobanks for the conservation of biodiversity to the use of these cells in physiological and toxicological assays. Considered a species vulnerable to extinction, the characterization of fibroblastic lines of northern tiger cat would contribute to its conservation. Therefore, we established and characterized fibroblasts derived from northern tiger cat during extended passage (third, seventh, and eleventh passages) and cryopreservation with regard to the morphology, viability, apoptotic classification, metabolism, proliferative activity, and oxidative stress by reactive oxygen species (ROS) levels and mitochondrial membrane potential (ΔΨm). Initially, we identified four dermal fibroblast lines by morphology, immunophenotyping, and karyotyping assays. In vitro culture after the third, seventh, and eleventh passages did not affect the viability, apoptotic classification, and ROS levels. Nevertheless, cells at seventh and eleventh passages featured a reduction in metabolism and an alteration in ΔΨm when compared to third passage cells. Additionally, cells at eleventh passage showed changes in the proliferative activity and morphology when compared to other passages. Regarding cryopreservation, no effect was observed on cryopreserved cells for morphology, viability, apoptotic classification, metabolism, and proliferative activity. Nevertheless, cryopreserved cells had alteration for ROS levels and ΔΨm. In summary, fibroblasts from northern tiger cat were affected by extended passage (seventh and eleventh passages) and cryopreservation. Adjustments to the in vitro culture and cryopreservation are necessary to reduce cellular oxidative stress caused by in vitro conditions.

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Comparison between concentration and type of intracellular cryoprotectants and the presence of sucrose for cryobanks of somatic cells derived from captive Pumas

et al. 2022

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The loss of wild biodiversity has prompted the development of cryobanks, such as those of somatic cells. This is the reality of Pumas, wild felids of ecological importance that suffer from anthropogenic actions, population decline, and subsequent loss of genetic diversity. Somatic cell banks are a strategy for conserving population diversity. We compared different concentrations and types of intracellular cryoprotectants (dimethyl sulfoxide, DMSO; ethylene glycol, EG) associated with 0.2 M of sucrose (SUC) in the cryopreservation of the somatic cells of captive Pumas. The cells were cryopreserved by slow freezing with different solutions containing Dulbecco's modified Eagle's medium with 10% fetal bovine serum and varying concentrations of DMSO and EG in the absence or presence of SUC. The cells were analyzed for morphological characteristics, viability, proliferative activity, metabolic activity, and apoptosis levels. Cells maintained similar fusiform morphology before and after cryopreservation. There was no difference in viability, regardless of the reduction in the concentration and type of intracellular cryoprotectants and sucrose. Similarly, proliferative activity, metabolic activity, and apoptosis levels were not altered by the composition of the cryoprotectants. In summary, we demonstrate that reducing the concentration of DMSO or EG ensures adequate cryopreservation of Puma somatic cells, regardless of the presence of SUC.

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Morphological, Ultrastructural, and Immunocytochemical Characterization and Assessment of Puma (Puma concolor Linnaeus, 1771) Cell Lines After Extended Culture and Cryopreservation

Cited by 9 publications

References 37 publications

Full confluency, serum starvation, and roscovitine for inducing arrest in the G0/G1 phase of the cell cycle in puma skin-derived fibroblast lines

Full confluency, serum starvation, and roscovitine for inducing arrest in the G0/G1 phase of the cell cycle in puma skin-derived fibroblast lines

Establishment and characterization of fibroblast lines from the northern tiger cat (Leopardus tigrinus, Schreber, 1775) during extended passage and cryopreservation

Comparison between concentration and type of intracellular cryoprotectants and the presence of sucrose for cryobanks of somatic cells derived from captive Pumas

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