Ex vivo expansion of primary cells from limb tissue of Pleurodeles waltl.

Pleurodeles waltl comes to light as an animal model, especially in regeneration study, but in deep study on molecular mechanisms has been limited due to the absence of primary tissue cells for widely usage. Thus, we aimed to grow primary cells from limb tissue of P. waltl for in vitro experiments. Limb tissues were cut into small pieces and seeded as "explant" on culture dish coated with fibronectin and gelatin. Compared to the control without coating, both fibronectin and gelatin coating supports quicker outgrowth of cells from explant and faster cell adhesion, although fibronectin shows significant better performance than gelatin. Interestingly, doubling time of cells on fibronectin- and gelatin-coated surface is almost same (42.39 ± 2.79 h vs. 42.91 ± 3.69 h) and was not significantly differed from the non-coated condition (49.64 ± 3.63 h). The cryopreserved cells were successfully recovered and showed similar multiplying capacity like fresh cells. Senescent cells were barely detected even through long-term sub-culture (>15 passages). Moreover, enhanced fluorescence of MitoSOX™ Red in cells under H 2 O 2 exposure confirmed the respondence to chemical stimuli. Collectively, we are able to grow enough number of good quality cells from P. waltl limb tissue for in vitro experiments, and fibronectin coating provides the best biocompatible environment for cell outgrow and attachment. This article is protected by copyright. All rights reserved.