FIGURE 1.

A Schematic workflow of the current study. 1) Strategies for the adaptation procedures of serum-free cultures of HEK293 cells by sequential and direct adaptations in Freestyle293 medium. The portion of serum is gradually reduced according to each step, described in Supplementary Table S1. Until reaching the 100% serum-free medium, cells were grown in adherent culture mode. During the proceeding of adaptation, backup cell samples were prepared in parallel under the same conditions as in previous step. 2) Establishment of final serum-free cultures for both adherent and suspension modes. The transition of adherent to suspension culture was performed at this stage. In total, 5 different culture systems were established (CGM; control growth medium culture, SFM Seq Ad; Serum-free medium sequential adapted adherent culture, SFM Dir Ad: Serum-free medium direct adapted adherent culture, SFM Seq Sus; Serum-free medium sequential adapted suspension culture, SFM Dir Sus; Serum-free medium direct adapted suspension culture), 3) After fully adapted serum-free culture, the growth of HEK293 cells were monitored in 5 different culture systems, and extracellular glucose and lactate in the medium were measured by NMR experiments. 4) Investigation of intracellular metabolic profiling. HEK293 cells under the exponential growth phase were harvested for the metabolic profiling analysis from 5 different culture systems. Adherent and suspension cells were extracted and targeted metabolomics based on mass spectrometry was applied to quantify the concentration of intracellular metabolites.