3.2. Co-Treatment with Colchicine and Cytochalasin B

For long-term selection, microalgal cells (1 × 10⁶/mL, 6-well plate, a total volume of 2 mL) were treated with colchicine (50 μg/mL, Sigma-Aldrich, Poznan, Poland) and cytochalasin B (3 μg/mL, Sigma-Aldrich, Poznan, Poland) (CC treatment) at 30 °C for 24 h with shaking (500 rpm/min) and continuous red and blue light (P.426103). Then, the cells were selected without colchicine and cytochalasin B for further sub-culturing in 6-well plates for 96 h and 250 mL flasks for 10 days. When the cell culture reached 5 × 10⁵ cells/mL, the cells (1–3 × 10⁴/mL, a total volume of 250 mL) were transferred to 2.5 L photobioreactors and grown in cell culture medium with CO₂ flow (8%), continuous red and blue light at 30 °C for 7 days. Typically, the cell density of 6 × 10⁶ cells/mL and 2 g of wet biomass per liter of culture were achieved at the end of the culture. To ensure that 24 h co-treatment with colchicine and cytochalasin B resulted in stable phenotype of increased cell size and DNA content compared to untreated cells, cells were additionally cultured without colchicine and cytochalasin B for 30 days and DNA levels were compared (time 0 and after 30 days) and cells were then propagated in 120 L bioreactors. During all selection steps, the cells were monitored using a light microscope (objectives 40× and 100×). The cell number and cell viability were monitored using a TC10™ automated cell counter and trypan blue staining (BioRad, Warsaw, Poland) and measurements of optical density (OD) using a TECAN microplate reader at 500 nm. After CC treatment and cell selection in 2.5 L bioreactors, twelve clones were considered for further analysis based on the most potent proliferative activity as judged by OD values (OD measurements at 500 nm using a microplate reader). Wet biomass was obtained after centrifugation of the cell culture (3000× g, 5 min) and dry mass was obtained using thin layer drying method using a moisture analyzer at 42 °C for 24 h.