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

Abstract

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.

Evaluating the Effects of Cryopreservation on the Viability and Gene Expression of Porcine-Ear-Skin Fibroblasts

“…Cell-type confirmation is an essential process before cryopreservation. In the present study, the cultures obtained from the ear skin grew with a typical morphology of fibroblasts and expressed intermediate filament protein vimentin ( Figure S1 and Figure 1 ), which confirmed that ear skin can be regarded as a common source of fibroblast separation [ 23 ]. The post-thawing cellular-viability measurement is a key step in the evaluation of the success of cryopreservation.…”

Effects of Incubation Time and Method of Cell Cycle Synchronization on Collared Peccary Skin-Derived Fibroblast Cell Lines

“…An appropriate cell synchronization protocol in G0/G1 is a crucial step in the development of cloning by SCNT in collared peccaries. Previously, our group developed somatic resource banks aiming at their application in the conservation of this species (Borges et al, 2017(Borges et al, , 2018a(Borges et al, , 2020aLira et al, 2020;Queiroz Neta et al, 2018a). With this new study, we have developed a suitable protocol for somatic cell synchronization, the last step in the preparation of karyoplasts, i.e., somatic nucleus donor cells.…”

“…The skin samples were cultured, and four fibroblast lines were previously established (Borges et al, 2020a). Subsequently, cells from four lines frozen in 10% DMSO, 10% FBS and 0.2 M sucrose were thawed, and 4 th and 5 th passage cells were used for this study.…”

“…This path has been developed since the establishment of the ideal environment for receptor oocytes (cytoplasts, Borges et al, 2018b), and the development of an efficient oocyte artificial activation protocol to provide the development of the reconstructed embryo (Borges et al, 2020b). In parallel, we established somatic tissue banks (Borges et al, 2017(Borges et al, , 2018aQueiroz Neta et al, 2018), somatic cell banks (Lira et al, 2020), and, recently, we established skin-derived fibroblasts lines (Borges et al, 2020a). Therefore, now, we have evaluated the different cell cycle synchronization protocols of these lines, aiming at a higher percentage of cells in G0/G1.…”

Strategies for the Establishment of Fibroblastic Lines for the Conservation of Wild Mammals

“…Panthera onca 10% DMSO, 10% FBS NI [4] Cerdocyon thous 10% DMSO, 50% FBS, 1% ATB-ATM NI [41] Mazama gouazoubira 10% DMSO > 80% [42] Elephas maximus 10% DMSO, 40% FBS > 95.5% [10] Pecari tajacu 10% DMSO, 10% FBS, 0.2 M sucrose > 74.5% [43] Dasyprocta leporina 10% DMSO, 10% FBS > 84.7% [36] Table 2.…”