• Storage and subsequent cryopreservation of liver cells of zebrafish

      Phonphaisan, Wirada (University of Bedfordshire, 2008)
      When gametes or embryos are not available or cannot be successfully cryopreserved, somatic cells should be considered for cryobanking of material from endangered fish species. Although the preservation of such material is not difficult, their collection and ultimate cryopreservation can present logistic difficulties; and initial storage is probably needed to retain viability before cryopreservation. The aim of this study was to develop a reliable method for initial storage and cryoprotectant treatment of liver cells that could be employed prior to the cryopreservation. Two bathing solutions were assessed for initial storage at 4°C or 6°C over a 48 hour storage period in chilling sensitivity studies. Medium C (lml L~15 supplemented with Hepes (5.958g1L), NaHC03 (0.42g/L), Gentamycine (lOOmglL), Amphotericine B (2.5mgIL), Lglutamine (0.374g/L) and Fetal bovine serum (10%)) and PBS were used as bathing solutions of the cells stored at 4°C or 6°C for up to 48 hours. Fluorescein diacetate propidium iodide staining was used to assess the viability after 12,24 and 48 hours of storage. Four cryoprotectants (CPAs) were investigated at different concentrations-DMSO (lM), MeOH (2M, 3M and 4M), PG (lM) and sucrose (0.05M and O.IM)and used to treat the cells for up to 30 minutes. Viability assessment was done using Trypan blue and Fluorescein diacetate - propidium iodide staining after 10, 20 or 30 minute exposure to CP As. Fresh and stored liver cells were frozen using slow cooling methods based on existing LIRANS procedures for cryopreservation of zebrafish blastomeres. The results indicated that exposure to 4·C PBS for 12, hours was an optimal condition for storage of liver cell prior to cryopreservation. In addition, results showed that cryopreservation at a slow freezing rate following 10 minute treatment of 2M methanol, allowed the recovery of more than 50% of the zebrafish liver cells.