Combination of ethylene glycol with sucrose increases survival rate after vitrification of somatic tissue of collared peccaries (Pecari tajacu Linnaeus, 1758)

Borges, Alana Azevedo; Neta, L. B. Queiroz; Santos, M. V. O.; Oliveira, Moacir Franco de; Silva, Alexandre Rodrigues; Pereira, Alexsandra Fernandes

doi:10.1590/1678-5150-pvb-5193

Cited by 9 publications

(11 citation statements)

References 42 publications

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“…Subconfluent cells were washed with PBS then trypsinized with a trypsin/EDTA solution (0.25%/0.2%) for 7 min and centrifuged at 600×g for 10 min. The supernatant was removed, the cell pellet was resuspended in culture medium, and the cell suspension was transferred to another dish for subculturing (Borges et al, 2018b). The medium was replaced with fresh medium every other day and the cells were monitored daily.…”

Section: Evaluation Of the Somatic Cells In Primary Cultures And Subcmentioning

confidence: 99%

“…Briefly, the cells were stained with 0.4% trypan blue in PBS and counted on a hemocytometer. Subsequently, the cells were also analyzed for a metabolic activity using the MTT assay, according to the method described by Borges et al (2018b). A concentration of 5.0 × 10 4 cells/mL from the first, third and tenth passages was grown in 12-well polystyrene plates treated for cell adhesion.…”

Section: Influence Of the Passage Number On The Quality Of Fibroblastmentioning

confidence: 99%

“…In this sense, studies related to the conservation of the collared peccary have been intensified, especially aimed at improving the techniques related to the preservation of somatic samples. Using this study, we established a culture condition for explants derived from the skin of adult collared peccaries (Santos et al, 2016) and developed a protocol for cryopreservation (Borges et al, 2017(Borges et al, , 2018a(Borges et al, , 2018b) and refrigeration of these explants (Queiroz Neta et al, 2018). In order to conduct the cloning experiments on this species by a somatic cell nuclear transfer, as well as to produce induced pluripotent cells, it is necessary to establish properly characterized cell lines.…”

Section: Introductionmentioning

confidence: 99%

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Isolation, characterization, and cryopreservation of collared peccary skin-derived fibroblast cell lines

Borges

Lira

Nascimento

et al. 2020

PeerJ

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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.

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Section: Evaluation Of the Somatic Cells In Primary Cultures And Subcmentioning

confidence: 99%

Section: Influence Of the Passage Number On The Quality Of Fibroblastmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Isolation, characterization, and cryopreservation of collared peccary skin-derived fibroblast cell lines

Borges

Lira

Nascimento

et al. 2020

PeerJ

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“…In a previous study, 18 we observed by quantitative histology that EG with sucrose resulted in a better conservation of skin tissues derived from collared peccaries; nevertheless, the effect of these cryopreservation solutions on cell recovery after in vitro culture has not yet been observed in the species. Thus, the aim of the current study was to compare the efficiency of various vitrification solutions for recovery of viable cells after in vitro culture of cryopreserved skin tissues derived from collared peccaries.…”

Section: Introductionmentioning

confidence: 82%

Influence of Cryopreservation Solution on the In Vitro Culture of Skin Tissues Derived from Collared Peccary (Pecari tajacu Linnaeus, 1758)

BorgesAlana

LiraGabriela

NascimentoLucas

et al. 2018

Biopreservation and Biobanking

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Skin vitrification is a promising and alternative tool for the conservation of biodiversity, especially for wild mammals, such as collared peccaries. Several factors can affect the success of this procedure, such as the cryoprotectant solution used. Therefore, this study was carried out to compare the efficiency of various vitrification solutions for recovery of viable cells after in vitro culture of cryopreserved skin tissues derived from the collared peccary, aiming to study the application in biobanking, where cellular use is not immediately required. Then, Dulbecco's modified Eagle's medium (DMEM) composed of 2.2 g/L sodium bicarbonate and 10% fetal bovine serum (FBS) was supplemented with 3.0 M ethylene glycol (EG) or 3.0 M dimethyl sulfoxide (DMSO) or 1.5 M EG plus 1.5 M DMSO with or without sucrose (SUC; 0.25 M) to produce six solutions for solid-surface vitrification. After warming, skin tissues were cultured in vitro and recovered cells were analyzed for morphology, adhesion, subconfluence, and proliferative activity for developing the growth curve and determining the population doubling time (PDT), and viability by Trypan Blue. The vitrification did not alter the ability of the tissues to adhere to the culture dish, as well as the day of all explants with cell growth, subconfluence samples, subconfluence total time, and PDT (p > 0.05). Moreover, independent of the cryoprotectant solution used, the vitrification altered the day of all attached explants (p < 0.05). Nevertheless, for viability after the first passage, only the EG-SUC (86.9%) and DMSO-SUC (91.4%) groups maintained viable cell recovery similar to the nonvitrified group (96.3%, p > 0.05). Additionally, for viability after the third passage, only the EG-SUC group maintained the cell quality (88.3%), when compared with the nonvitrified (97.8%, p > 0.05). In conclusion, DMEM with 10% FBS, 3.0 M EG, and 0.25 M sucrose was the most efficient solution for vitrifying collared peccary skin tissues, leading to the in vitro culture of viable cells.

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“…Embora esta técnica já tenha demonstrado resultados promissores, ainda pode levar a formação de cristais de gelo, e exige o uso de materiais e equipamentos de Research, Society and Development, v. 9, n. 8, e969986686, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i8.6686 valor econômico mais elevado (Borges et al, 2017;Praxedes et al, 2019). Já a vitrificação em superfície sólida, consiste no resfriamento abrupto do tecido sobre uma superfície metálica com a presença de nitrogênio líquido, e posterior armazenamento, sendo esta técnica a mais utilizada nos protocolos de criopreservação de pele (Borges et al, 2018), por ser uma técnica simples e de baixo custo.…”

Section: Introductionunclassified

Estudo comparativo de técnicas de criopreservação em tecido somático de Felis silvestres catus

Martins

Praxedes

Nascimento

et al. 2020

RSD

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O objetivo do presente trabalho foi comparar os efeitos da Congelação Lenta (CL) e da Vitrificação em Superfície Sólida (VSS) na criopreservação da pele auricular apical de gatos domésticos utilizando ferramentas histológicas. Fragmentos de pele obtidos a partir de seis gatos (Felis silvestres catus) foram processados e destinadas à criopreservação por CL e VSS, utilizando uma solução composta por Meio Essencial Mínimo Modificado por Dulbecco (DMEM) acrescida de dimetilsulfóxido (DMSO) a 1,5 M ou 3,0 M, para CL e VSS, respectivamente, além de 0,25 M de sacarose e 10% de soro fetal bovino (SFB). Após o aquecimento, as amostras foram destinadas ao processamento histológico para posterior realização das colorações com hematoxilina-eosina, tricrômico de Gômori, e coloração de Weigert, bem como foi realizada a marcação de regiões organizadoras de nucléolo (NORs) utilizando nitrato de prata. Na técnica de VSS, características como espessura da epiderme, derme e espessura total da pele, apresentaram valores similares ao grupo controle (p > 0,05). Além disso, a VSS mostrou-se eficiente na manutenção do número de células epidermais e percentual de fibras de colágenas. Conclui-se que a aplicação da técnica de VSS mostrou-se superior na manutenção histológica da pele auricular apical de gatos domésticos, auxiliando na geração de um banco somático da espécie.

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Combination of ethylene glycol with sucrose increases survival rate after vitrification of somatic tissue of collared peccaries (Pecari tajacu Linnaeus, 1758)

Cited by 9 publications

References 42 publications

Isolation, characterization, and cryopreservation of collared peccary skin-derived fibroblast cell lines

Isolation, characterization, and cryopreservation of collared peccary skin-derived fibroblast cell lines

Influence of Cryopreservation Solution on the In Vitro Culture of Skin Tissues Derived from Collared Peccary (Pecari tajacu Linnaeus, 1758)

Estudo comparativo de técnicas de criopreservação em tecido somático de Felis silvestres catus

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