Optimization Of Culture Media For CHO Cells Based On DF12 Medium

In this work,the basal medium DF12 is optimized for CHO-S cells growth using a high-throughput biological reactor "TubeSpin" by cell counting and metabolism analysis.Compared with ProCH05 introduced by Lonza company,we finally obtained independently developed serm-free medium formulations.The results showed that different additives could not obviously promote the growth of CHO-S cells separately,where the highest increase in cell density could be less than 20%.Seven of the amino acids additives were analyzed in the orthogonal experiments,to have the most significant effects on the cell growth,and the cell density could be increased by 80%.Ferric citrate and zinc sulfate are substituted for transferrin and insulin,and the appropriate concentrations would be 18 mg/L of ferric citrate,1.2 mg/L of zinc sulfate and 28 ?g/L of sodium selenite.For achieving higher density,Plackett-Burman(PB)experiment of design was employed to optimize 13 components and the highest density reached 5.8·106 cells/mL.The stability of the final optimized medium was verified and the cost of medium could be controlled lower than 400 RMB/1000 mL.Most nutrients added in the medium could not be consumed absolutely in the cell metabolism by the cells,and the remaining materials may cause toxic metabolite.We found that the glutamine and glucose decreased with the increase of culturing time,while the free ammonia,lactic acid and pyruvic acid increased at first and decreased finally.The suitable osmotic pressure of medium is essential to the growth of animal cells.We determined the osmotic pressure of the cells by adding different concentrations materials in the basal medium DF12 to analyze the relationship between the osmotic pressure and the concentrations.The amino acids are important to the culture of CHO-S cells in the medium additives.The L-proline is soluble in water and ethanol,and is purified by recrystallization process.With solvent-out crystallization method,the crude L-proline was dissolved at 70?,then the granular activated carbon was added at 0.5%(the mass of the solute)at 350 rpm of stirring rate for 30min.After filtration,the solution was cooled down to 50?,anti-solvent acetone was pumped at 5mL/min.The final L-proline crystalline product was obtained via vacuum drying.