An efficient and affordable laboratory method to produce and sustain high concentrations of microcystins by .

is a cosmopolitan cyanobacteria that continues to jeopardize freshwater ecosystem services by releasing the hepatotoxin microcystin, which can, in some cases, cause death to aquatic fauna and even humans. Currently, our abilities to understand the mechanisms of microcystin toxicology are limited by the lack of a method for producing high concentrations, which are central to large-scale and long-term research in natural systems. Here we present an efficient and affordable laboratory method to produce high concentrations of microcystins by a toxigenic strain of . Through batch culture studies, we yielded microcystins at concentrations that are environmentally relevant to freshwaters around the world (1-300 μg L), maintained these concentrations without resupplying fresh medium (further reducing costs), and utilized rate equations to model the relationship between the environmental conditions in the cultures and changes occurring within the cells. Our assessment suggests that steady production of microcystins depends on the availability of carbon throughout the experiment. Hence, we recommend the use of tissue culture treated flasks with a vented cap to ensure the production of microcystins is uninterrupted. This method demonstrates that microcystins can be produced in the laboratory at concentrations relevant to freshwater ecosystems. •The method demonstrates CPCC 300 is a reliable strain of freshwater cyanobacteria that can yield microcystins at environmentally relevant concentrations.•Validation showed CPCC 300 is resilient in carbon-limited situations and may respond to stress by shifting the ratio of microcystin congeners.•Cell culture flasks with vented caps -filled no more than 50 % of the flask volume to allow for sufficient air exchange- are an excellent and cost-effective approach to maintaining cell growth and producing microcystins at a range between 300 to 1200 μg L.