Ghasemi Nazanin, Bandehpour Mojgan, Ranjbari Javad
Department of Immunology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Iran J Pharm Res. 2019 Fall;18(Suppl1):146-156. doi: 10.22037/ijpr.2020.112322.13681.
Researchers add serum to a classical medium at concentrations of 5 to 10% (v/v) to grow cells culture media. Unfortunately, serum is a poorly defined culture medium component as its composition can vary considerably while serum-free cell culture media are an excellent alternative to standard serum-containing media and offer several major advantages. Advantages of using serum-free media include a lower risk of infectious agents, lower risk of interfering components, less contaminant, avoids ethical issues. According to previous studies insulin, selenium, transferrin and glucose are important component of serum that affect cell growth. In the present study, we optimized amount of these factors in order to serum free culture medium fabrication. Response surface methodology (RSM) was employed for optimization of key factors in serum free medium to enhance recombinant human GM-CSF (rhGM-CSF) production in CHO cell line. Four important process parameters including insulin concentration (0-2 g/L), transferrin concentration (0-1 g/L), selenium concentration (0-0.001 g/L) and glucose concentration (0-5 g/L) were optimized to obtain the best response of rhGM-CSF production using the statistical Box-Behnken design. The experimental data obtained were analyzed by analysis of variance (ANOVA) and fitted to a second-order polynomial equation using multiple regression analysis. Numerical optimization applying desirability function was used to identify the optimum conditions for maximum production of rhGM-CSF. The optimum conditions were found to be insulin concentration = 1.1 g/L, transferrin concentration = 0.545 g/L, selenium concentration = 0.000724 g/L and glucose = 1. 4 g/L. Maximum rhGM-CSF production was found to be 3.5 g/L.
研究人员在经典培养基中添加浓度为5%至10%（v/v）的血清来培养细胞培养基。不幸的是，血清是一种成分定义不明确的培养基成分，因为其组成可能有很大差异，而无血清细胞培养基是标准含血清培养基的绝佳替代品，并具有几个主要优点。使用无血清培养基的优点包括感染因子风险较低、干扰成分风险较低、污染物较少、避免伦理问题。根据先前的研究，胰岛素、硒、转铁蛋白和葡萄糖是影响细胞生长的血清重要成分。在本研究中，我们为了制备无血清培养基而优化了这些因素的用量。采用响应面法（RSM）优化无血清培养基中的关键因素，以提高CHO细胞系中重组人GM-CSF（rhGM-CSF）的产量。使用统计Box-Behnken设计优化了四个重要工艺参数，包括胰岛素浓度（0-2 g/L）、转铁蛋白浓度（0-1 g/L）、硒浓度（0-0.001 g/L）和葡萄糖浓度（0-5 g/L），以获得rhGM-CSF产量的最佳响应。通过方差分析（ANOVA）对获得的实验数据进行分析，并使用多元回归分析将其拟合到二阶多项式方程。应用期望函数的数值优化用于确定rhGM-CSF最大产量的最佳条件。发现最佳条件为胰岛素浓度 = 1.1 g/L、转铁蛋白浓度 = 0.545 g/L、硒浓度 = 0.000724 g/L和葡萄糖 = 1.4 g/L。发现rhGM-CSF的最大产量为3.5 g/L。
