Enhanced cell contact and planarian tissue extract increase DNA replication and viability for neoblast cultivation.

The development of an in vitro culture system for planarian neoblasts remains at an early stage. Although modified neoblast enrichment method and basic culture conditions have been established, it is unclear which micro- and macro-environments can functionally improve neoblast behavior in vitro. Neoblasts tend to aggregate in culture, and when cultured as aggregates in the U-bottom plate or on feeder layers, they show increased DNA replication, potentially due to improved cell-cell interactions. At the macro-environment level, the addition of planarian extract to the medium reduces cell death, increases lipid droplet formation, and decreases the intracellular reactive oxygen species levels. An optimized culture condition has thus been established using U-bottom plates supplemented with planarian extract for the first 3 days, effectively incorporating cues of micro- and macro-environment. By modulating both micro- and macro-environmental factors, this study provides a foundation for further optimization at the metabolic and genetic levels in subsequent studies.