Development of in vitro organ culture techniques for differentiation and growth of cartilages and bones from teleost fish and comparisons with in vivo skeletal development

To examine molecular and cellular mechanisms of fish skeletogenesis, an in vitro organ culture system was developed to achieve effective maintenance, growth, and/or differentiation of embryonic skeletal tissues from Oryzias latipes (Japanese ricefish) and Brachydanio rerio (zebra‐fish). In vivo and in vitro skeletogenesis were compared. The techniques developed here open the way for descriptive and experimental in vitro analyses of fish skeletogenesis. The present study found that submerged organ culture using medium 199 or Leibovitz's L‐15, supplemented with fetal bovine serum and ascorbic acid, was most suitable. Supplementation with antibiotic‐antimycotics prevented microbial contamination, especially during explant preparation. Two types of inorganic membrane substrates did not produce different results from culture on plastic Falcon dishes; all sustained healthy embryonic skeletal explants. The present system sustained maintenance, growth, or de novo differentiation of embryonic skeletal tissues, when compared with in vivo formation and growth of the embryonic cranial skeletons in the two species. Already formed bones (parasphenoid, opercule and/or branchiostegal rays) and cartilages were successfully maintained and/or grew in culture. Both perichondral bone (around gill arches, hyosymplecticum, hyoid arch, Meckel's cartilage) and membrane bone (dentary) differentiated de novo, but did not mineralize. De novo hypertrophy of chondrocytes was observed in some cartilages as occurs in normal development. Although already formed teeth were maintained, de novo differentiation of teeth was not observed in vitro, suggesting that mandibular and gill perichondral bones may form independently of tooth formation.