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Isolation and characterization of vascular endothelial cells derived from fetal tooth buds of miniature swine

Abstract

The aim of the present study was to isolate endothelial cells from tooth buds (unerupted deciduous teeth) of miniature swine. Mandibular molar tooth buds harvested from swine fetuses at fetal days 90–110 were cultured in growth medium supplemented with 15% fetal bovine serum in 100-mm culture dishes until the primary cells outgrown from the tooth buds reached confluence. A morphologically defined set of pavement-shaped primary cells were picked up manually with filter paper containing trypsin/ethylenediamine tetraacetic acid solution and transferred to a separate dish. A characterization of the cellular characteristics and a functional analysis of the cultured cells at passages 3 to 5 were performed using immunofluorescence, a reverse transcriptase polymerase chain reaction assay, a tube formation assay, and transmission electron microscopy. The isolated cells grew in a pavement arrangement and showed the characteristics of contact inhibition upon reaching confluence. The population doubling time was ~48 h at passage 3. As shown by immunocytostaining and western blotting with specific antibodies, the cells produced the endothelial marker proteins such as vascular endothelial cadherin, von Willebrand factor, and vascular endothelial growth factor receptor-2. Observation with time-lapse images showed that small groups of cells aggregated and adhered to each other to form tube-like structures. Moreover, as revealed through transmission electron microscopy, these adherent cells had formed junctional complexes. These endothelial cells from the tooth buds of miniature swine are available as cell lines for studies on tube formation and use in regenerative medical science.

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Acknowledgments

The authors would like to thank Mr. Takehiro Iwanaga (The Japan Farm CLAWN Institute) for collecting the miniature swine fetuses and Dr. Akihiro Ohyama (The Nippon Dental University) for his technical support. This work was supported by a Grant-in-Aid for Young Scientists (A) (No. 24689073 to T.N.) from the Japan Society for the Promotion of Science (JSPS), a Grant-in-Aid for Scientific Research (C) (No. 19592184 to M.N.) from JSPS, and the Science Research Promotion Fund (2008-2010 to T.N. and H.I.) from the Promotion and Mutual Aid Corporation for Private Schools of Japan.

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Correspondence to Masanori Nasu.

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Editor: T. Okamoto

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Time-lapse video of tube formation: Matrigel was poured in the central hollow of a glass bottom dish and gelled. Next, 3 × 105 cells were placed in the center of the glass and cultured using an incubation system for microscopes. During the culturing process, 60 photographs (one picture every 3 min) were taken and edited at 12 s by Windows Live Movie Maker. Many of the cells moved extensively at the start of the incubation. After 2–3 s (actual incubation time, ~30–45 min), they gathered and connected mutually within an alignment. Afterward, they presented a tube form, which subsequently formed a network and became like the structure of the meshes of a net. (WMV 18955 kb)

Time-lapse video of tube formation: Matrigel was poured in the central hollow of a glass bottom dish and gelled. Next, 3 × 105 cells were placed in the center of the glass and cultured using an incubation system for microscopes. During the culturing process, 60 photographs (one picture every 3 min) were taken and edited at 12 s by Windows Live Movie Maker. Many of the cells moved extensively at the start of the incubation. After 2–3 s (actual incubation time, ~30–45 min), they gathered and connected mutually within an alignment. Afterward, they presented a tube form, which subsequently formed a network and became like the structure of the meshes of a net. (WMV 18955 kb)

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Nasu, M., Nakahara, T., Tominaga, N. et al. Isolation and characterization of vascular endothelial cells derived from fetal tooth buds of miniature swine. In Vitro Cell.Dev.Biol.-Animal 49, 189–195 (2013). https://doi.org/10.1007/s11626-013-9584-6

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Keywords

  • Sus scrofa
  • Miniature pig
  • Tooth buds
  • Endothelial cells
  • Tube formation