1 March 2006 In Vitro Culture of Mesenchymal Lineage Cells Established from the Colonial Tunicate Botryllus primigenus
Kazuo Kawamura, Sae Takeoka, Show Takahashi, Takeshi Sunanaga
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Body trunks were isolated from juvenile zooids of the Japanese colonial tunicate Botryllus primigenus and cultured in vitro to establish tissue-specific cell lines. Epidermal cells from some explants spread and formed a flat sheet consisting of vacuolated cells. They then dissociated into single cells, and their growth stopped within two weeks. Continuously proliferating cells were established from four explants. After the 20th implantation, nuclear and mitochondrial DNAs were extracted from these cells. The nucleotide sequences of proliferating cell nuclear antigen (PCNA) and mitochondrial large ribosomal RNA (mtlrRNA) completely matched the PCNA and mtlrRNA taken from living colonies of B. primigenus; this shows that the four independently proliferating cells were indeed of the Botryllus origin. One cell line (Bp0306E10) comprised round-shaped cells with a diameter of 8–10 μm. These cells have been cultured in vitro with a doubling time of approximately 24 hours since June, 2003. The BrdU labeling index was approximately 2%. Monoclonal antibodies raised against the cultured cells recognized a 28 kDa polypeptide and stained free mesenchymal cells in vivo. G418-resistant subclonal cells could be established by introducing a tunicate retrotransposon loaded with the neomycin resistance gene into the cells by electroporation. This study is the first to succeed in producing a sustainable cell culture of Botryllus.

Kazuo Kawamura, Sae Takeoka, Show Takahashi, and Takeshi Sunanaga "In Vitro Culture of Mesenchymal Lineage Cells Established from the Colonial Tunicate Botryllus primigenus," Zoological Science 23(3), 245-254, (1 March 2006). https://doi.org/10.2108/zsj.23.245
Received: 28 October 2005; Accepted: 1 December 2005; Published: 1 March 2006
Cell culture
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