5.2 Media optimization
Firstly, a preliminary screening of various factors was performed using the Plackett-Burman Design (PBD) method to identify the most influential variables. Subsequently, the Central Composite Design (CCD) method of Response Surface Methodology (RSM) was employed to optimize the levels of these significant variables. All experimental procedures were conducted in 2 mL volume wells within the Biolector XT Microbioreactor, each containing 1 mL of the respective culture media. Prior to the experiments, the media were prepared according to the specific design points and then inoculated with an overnight seed culture. The inoculated cells grew for 4 hours in each well, with constant agitation at 800 rpm, and maintained at 37°C. To facilitate these experiments, 48-well FlowerPlates (mp2-labs, Baesweiler, Germany) was used, with a working volume of 1000 µL. Throughout the study, real-time monitoring of growth kinetics was conducted by continuously measuring scattered light intensities. Additionally, the final insulin density, expressed in grams per liter, was determined using a UV/Vis spectrophotometer at a wavelength of 280 nm. To further analyze the results, SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) was performed.