Enhanced Cell-Cell Contact and Planarian Tissue Extract increase DNA replication and cell 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 still unclear whether mimicking the in vivo micro- and macro-environments can functionally improve neoblast behavior in vitro. Neoblasts tend to aggregate in culture, but 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 that mimic the in vivo microenvironment. At the macroenvironment level, the addition of planarian extract to the culture medium reduces cell death, increases lipid droplet formation, and decreases the intracellular reactive oxygen species (ROS) levels. An optimized culture condition has thus been established using U-bottom plates supplemented with planarian extract for the first three days, effectively recapitulating aspects of both the in vivo microenvironment and macroenvironment. Although the culture period remains limited, this system enabled gene knockdown via dsRNA in culturing neoblasts. 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. RNA seq profiling of planarian SiRNeoblasts cultured in different test conditions. 5 experiments are included: experiment1, compare cultured cells in aggregates&non-aggregating cells at2 days, 3 replicates per group; experiment2, compare SiRNeoblasts cultured in aggrewell & flat conditions at 1 day, 3 replicates per group; experiment3, compare cells without culture and cultured in U-bottom plate & 100Pa PA gel & flat conditions at 1 day & 2 days & 3 days, 3 replicates per group except for PA_3d group (2 replicates); experiment4, compare cells without culture and cultured in HFF1 feeder & MEF feeder & flat conditions at 1 day & 3 days, 3 replicates per group; experiment5, compare cells without culture and cultured in U-bottom plate-extract & U-bottom plate+extract & flat -extract conditions at 1 day & 3 days & 7 days, 3 replicates per group.