Document Type : Original Article
Stem Cells and Developmental Biology Department, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
Genetics Department, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran
Radiotherapy Department, Shohada Hospital, Tehran, Iran
Developmental Biology Department, University of Science and Culture, Tehran, Iran
Objective: Freshwater planarians were used as models for studying metazoan regeneration 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 μm 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 propidium 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 animals, about 50% of the cells were absent, the final suspension contained about 62-66% neoblasts and about 17% non-cellular particles. When these extracts were treated with distilled water to destroy the cells, only rabdites and chitinous spines of the parenchyma 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 carbohydrate nature and, therefore, this extraction method is completely compatible with molecular (e.g. proteomics and transcriptomics) and cellular methods (e.g. neoblast culture).