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. 2024 Feb 22;14(5):686.
doi: 10.3390/ani14050686.

Establishment and Characterization of a Primary Fibroblast Cell Culture from the Amazonian Manatee (Trichechus inunguis)

Flávia Dos Santos Tavares  1 , Cesar Martins  2 , Flávia Karina Delella  2 , Luís Adriano Santos do Nascimento  3 , Angélica Lúcia Figueiredo Rodrigues  4 , Sávia Moreira  4 , Adauto Lima Cardoso  1   2   3 , Renata Coelho Rodrigues Noronha  1
Affiliations

Establishment and Characterization of a Primary Fibroblast Cell Culture from the Amazonian Manatee (Trichechus inunguis)

Flávia Dos Santos Tavares et al. Animals (Basel). .

Abstract

The vulnerable status of the Amazon manatee, Trichechus inunguis, indicates the need to seek measures to guarantee its conservation. In this context, the cultivation of cells in vitro is a strategy that should at least guarantee the preservation of their genetic material. Thus, we established for the first time a primary culture of Amazonian manatee fibroblasts (TINsf) from a skin biopsy of a young male. Karyotypic analysis of the 3rd, 7th, and 12th passages confirmed the taxonomic identity of the species T. inunguis (2n = 56/NF = 92) and indicated that this culture presents genomic stability. Gene and protein expression of vimentin at the 13th passage show the predominant presence of fibroblasts in TINsf. To test the cell line's responsiveness to materials and demonstrate a possible application of this culture, it was exposed to andiroba seed oil (ASO), and its viability and proliferative capacity were evaluated. ASO demonstrated toxic effects at the highest concentrations and longest exposure times tested, reproducing results observed in human cultures, indicating the applicability of TINsf in toxicological and biotechnological studies. After cryopreservation, the TINsf line maintained its proliferative potential, indicating the establishment of a new culture available for future studies.

Keywords: Amazon biobank; andiroba; aquatic mammals; conservation; cryopreservation; endangered species; sirenians.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
The Amazonian manatee Trichechus inunguis. (a,b) Photos of the sample analyzed in the present study; (c) Map indicating the site of collection at Santa Bárbara do Pará city, State of Pará, Brazil.
Figure 2
Figure 2
Characterization of the primary fibroblast cell culture from the Amazonian manatee Trichechus inunguis (TINsf). (a) The cell culture at the 3rd passage; scale bar = 100 μm. (b) Expression of vimentin as shown by immunofluorescence with anti-vimentin antibody in TINsf at the 13th passage; vimentin is shown in green and the nuclei are stained with DAPI in blue; scale bar = 100 μm (c) A dividing cell highlighted; scale bar = 10 μm. (d) A interphase cell highlighted; scale bar = 20 μm. (e) Transcription of vimentin gene as assessed by RT-PCR; L—DNA ladder, V—vimentin. (f) Cell growth curve; PDT—Population Doubling Time. (g) karyotype at the 3rd passage showing 2n = 56.
Figure 3
Figure 3
Effects of Andiroba seed oil (ASO) in the TINsf cell line. (a) Cell viability as measured by MTT assay showing the toxic effects of ASO from 1500 μg/mL. (b) Summary bar graph illustrating the percentage wound closure at the indicated time points during the scratch wound assay. (c) Representative images from in vitro scratch wound healing assays demonstrating that cell migration into the cell-free region (outlined) is significantly reduced in the presence of 150 μg/mL ASO when compared to the control. * p < 0.05; ** p < 0.005.
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