FADILAH, Sarah Nurul (2018) Characteristic of In Vitro Osteochilus Vittatus (Val., 1842) Hepatopancreatic Cells Isolated Using 20% and 40% Percoll Gradient Centrifugation. Skripsi thesis, Universitas Jenderal Soedirman.

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Abstract

Fish hepatopancreas consists of various types of cells, for instance, hepatocyte and pancreatic cell. Isolation of a certain type of cells is needed in order to obtain the homogenous cells to be grown in vitro. The homogenous cell is important for several studies such as toxicity and xenobiotic studies. One of the common techniques for cell isolation is percoll gradient centrifugation. Therefore, the objectives of this research were to determine the gradient concentration suitable for bony lip barb (Osteochilus vittatus) hepatopancreas-origin cell isolation and to evaluate the characteristics and survival rate of hepatopancreas-origin cells after primary culture and the first subculture. The observed parameters include cell viability, density, confluency, proportion, and morphology. The hepatopancreas-origin cells were separated using percoll gradient centrifugation and each cell suspension was subjected to the experiment using CRD. The serum concentration consisted of 0%, 5%, 10%, and 15% were the treatments with four replicates were provided for each treatment. The viability, density, confluency, and proportion of the cells were analyzed using twoway ANOVA and post hoc test using Tukey’s HSD, while the cell type was morphologically described. The population doubling time (PDT) and population doubling level (PDL) were also calculated. The result showed that the 20% and 40% percoll concentration were not effective to isolate a particular type of hepatopancreatic cell such as hepatocytes. The cells isolated from 20% or 40% percoll gradient centrifugation also share the common characteristics. There were 4 types of cells identified in primary culture i.e. A cell (ovoid shape with high nucleus-cytoplasm ratio), C cell (round shape with a round nucleus), D cell (the cell and nucleus were round to ovoid shape), and F cell (irregular shape with bluish cytoplasm), but only C cell, D cell, and F cell were survived in the subculture. The cell confluency and density of primary culture and subculture were observed at the value of ≥82.50% and ≥0.45 x 10 8 -1 respectively. The value of PDL was ≥6.08 times and PDT was ≤23.47 hours observed in the primary culture and subculture. The cell survival in the culture supplemented with different concentration of FBS reach ≥99.41% either in primary culture or subculture. cell.ml

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