Planarian cell culture: a comparative review of methods and an improved protocol for primary cultures of neoblasts

To develop an improved method for preparing and cultivating planarian cells, several protocols published previously were compared with each other. Cells, and in particular neoblasts, proved remarkably resistant to hyposmotic conditions. However, survival periods depended critically on the content in nutrients and on osmotic conditions. Starting from an optimized method to disintegrate planarian tissues and prepare purified neoblast fractions, different media and additives were tried. Hyposmotic media and layers of extracellular matrix components enhanced the adhesion of neoblasts and favoured the formation of transient processes. Proteins in the medium supported long-term survival of neoblasts that retained a spherical shape. Eventually, an isosmotic medium was devised that supported the survival of neoblasts with a viability of 46\% on day 31 of primary cultures. With light microscopical techniques, no signs of differentiation were observed in these cultures. Mitoses were detected until the second day of cultivation. In contrast, cultures of total cells still displayed mitoses after 7 days of cultivation. Some guidelines are proposed for future research directed towards establishing permanent neoblast lines.