روشي ساده براي جداسازي سلول هاي بنيادي (نئوبلاست ها) از كرم هاي پلاناريا

A Simple Method for Isolation of Neoblasts from Planaria

Objective : Freshwater planarians were used as models for studying metazoan regene ration and stem cell biology. Here a simple , fast and high throughput method for extracting their stem cells (neoblasts) is represented. Materials and Methods: Specimens of the Dugesia sp with an average length of 18 mm were homogenized by a glass Dounce tissue grinder which contained about 1 ml of planarian saline solution. The extracted suspension was serially filtered by 60, 41, 30, 20 and 11 urn nylon meshes . In order to obtain purified neoblasts in the final suspension : this suspension has been compared with a cell suspension from 30 Gy irradiated worms . Hoechst 33342 was used to determine cells from non-cellular particles; methylene blue and propidiurn iodide were used to detect the number of dead cells in each extraction. Results: About 2.6-3 million cells were extracted from 10-12 worms. Flow cytometry analySIS showed about 83% of the extracted particles were cells. In suspensions from irradiated animal s. about 50% of the cells were absent, the final suspen sion contained about 62-66% neoblasts and about 17% non-cellular particles. When these extracts were treated with distilled water to dest roy the cells , only rabdites and chitinous spines of the parenchym a were observed in the extract. Conclusion: Results show that the purity of neoblasts in the final suspension is about 66%. Non-cellular particles have a carboh ydrate nature and, therefore, this extraction method is completely compatible with molecular (e.g. proteomics and transcriptomi cs) and cellu lar methods (e.g. neoblast culture ).