Optimization Of Serum-free Medium For Vero Cells And Application Of The Influenza Virus

Influenza is one of infectious diseasea which humanity has not effectively control,Influenza has a threat to human health and economy when popular in the word.Although there are antivial drugs,but drugs may induce drug-resistant strains.It is not suitable for a wide range to resistant to the influenza virus.The vaccine is an important method of prevention and control of influenza.Traditionally,influenza virus production often use chicken embryonic egg.However,chicken embryonic eggs may contain various microbiological contamination in the disadvantages.Anymore,this production process can’t mass production and has poor influenza pandemic emergency response capability. So it is important to develop satisfactory mammalian cell systems for vaccines production.African green monkey Vero cells are approved by the World Health Organization as a safe substrate foe vaccine production.It have been to proved safely to produce a variety of vaccine.When flu pandemic,Since large scale animal cell culture presents the core technology nowladays in the field of production of influenza vaccine,How to meet the demands for higher quality and effectivity of influenza vaccine has become the theme for its current and future development.Among this,the development of serum-free medium is one of the critical steps in process development of influenza vaccine and lead to influenza vaccine production at lower cost and higher efficency.So,in the present study statistical design methodlogies were utilized to development serum-free medium for Vero cells.First of all,a high throughput technology was established by sequential applic-ation of four statistical methodologies including Single factor experiment, Plackett-Burman,method of steepest ascent and Box-Behnken design.These study certainly lay the foundation for developing efficient process of medium optimization for Vero cells.By single factor experiment design to determine the added concentration range of insulin, transferrin, high-density lipoprotein, bovine serum albumin, epidermal growth factor, hyaluronic acid and sodium selenite in the subsequent experiment,Acoording to the result,the concentration added were as follow:insulin,3-6 ug/mL;transferrin.7.5-15 ug/mL;high-density lipoprotein,10-20 ug/mL;bovine serum albumin,2-4 ug/mL; epidermal growth factor,50-100 ng/mL;hyaluronic acid,15-30 mg/mL;sodium selenite 4-8 mmol/mL.Further, insulin, transferrin, high-density lipoprotein, bovine serum albumin,epidermal growth factor,hyaluronic acid and sodium selenite were selected to evaluate their effects on Vero cells growth based on the Plackett-Burman experimental.The results of statistical analyses revealed that insulin, bovine serum albumin has a significant positive effect on Vero cell growth (p<0.05);transferrin, epidermal growth factor, hyaluronic acid sodium have a positive impact (p>0.05);But high density lipoprotein and sodium selenite exists negative impact for the growth of Vero cell s.After by the steepest ascent experiment broaden the concentration range of insulin, transferrin, bovine serum albumin,There was a largest OD490 value of the measurement at insulin,5.4 ug/mL;transferrin,3.8 ug/mL;bovine serum albumin,3.6 mg/mL.Last,Box-Behnken design was adopted to search optimal levels of insulin, transferrin, bovine serum albumin for growth of Vero cells,The result were insulin,5.625 ug/mL;transferrin,14.034 ug/mL;bovine serum albumin,3.92 mg/mL, The optimial serum for Vero cells can support continuous culture of Vero cells.Further,the production conditions of H5N1 virus were optimized preliminarily with the serum-free medium.Since the the optimal conditions for H5N1 virus cultured in serum-free culture system were MOI=0.001,TPCK-trypsin= 1 ug/mL, and every 24 h adding initial concentration of TPCK-trypsin to the culture system.Under the optimial condition,there is no significant difference of HA and TCID50 of H5N1 virus between the optimial serum free medium and Gibco serum free medium,But are lower than in complete medium.The HA values in complete medium, optimial serum free medium and Gibco commercial serum free medium were respectively:512HAU/50uL、277.3HAU/50uL、149.3HAU/50uL;TCID50 values were respectively:108.375±0.25TCID50/mL、107.75±0.006TCID50/mL �'� 107.32±0.12TCID50/mL. Ithas proved optimial serum free medium system is suitable for influenza virus vaccine production,But There still has difference compared with the complete me dium cultivation system.it can be continued to optimize the serum free medium.