A Simple Method for Isolation of Neoblasts from Planaria

Objective: Freshwater planarians were used as models for studying metazoan regenerationand stem cell biology. Here a simple, fast and high throughput method for extracting theirstem cells (neoblasts) is represented.Materials and Methods: Specimens of the Dugesia sp with an average length of 18 mmwere homogenized by a glass Dounce tissue grinder which contained about 1 ml of planariansaline solution. The extracted suspension was serially filtered by 60, 41, 30, 20 and 11 μmnylon meshes. In order to obtain purified neoblasts in the final suspension; this suspensionhas been compared with a cell suspension from 30 Gy irradiated worms. Hoechst 33342was used to determine cells from non-cellular particles; methylene blue and propidium iodidewere 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 analysisshowed about 83% of the extracted particles were cells. In suspensions from irradiatedanimals, 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 distilledwater to destroy the cells, only rabdites and chitinous spines of the parenchyma wereobserved 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 iscompletely compatible with molecular (e.g. proteomics and transcriptomics) and cellularmethods (e.g. neoblast culture).