狂犬病毒单克隆抗体CHO细胞灌流培养中细胞特异性灌流速率的研究

目的 研究灌流培养中,不同的细胞特异性灌流速率(cell specific perfusion rate, CSPR)对狂犬病毒单克隆抗体CHO细胞生长及抗体蛋白表达的影响,摸索适合本细胞株灌流培养的CSPR。方法 在标号为CSPR 1～5[CSPR1;0.02 nL/(细胞·d)、CSPR2:0.03 nL/(细胞·d)、CSPR3:0.04 nL/(细胞·d)、CSPR4:0.05 nL/(细胞·d)、CSPR5:0.06 nL/(细胞·d),每组3个重复]的15个TPP管中按100万个/mL的初始密度接种相同的CHO种子细胞;摇床中,以转速225 r/min、CO₂浓度5.0%、湿度80%和温度37℃的条件培养细胞;以后每天取细胞样品,分别检测活细胞密度(viable cell density, VCD)、细胞活率、葡萄糖浓度、乳酸浓度和渗透压。接种3 d起,每天分别从CSPR 1～5的各管中,按0.02～0.06 nL/(细胞·d)的CSPR计算所需要更换的细胞悬液体积,将各管中的细胞悬液离心,取出相应体积的上清并补入相同体积的新鲜培养液重悬细胞后,继续培...     

利用高通量生物反应器优化一种CHO细胞无血清培养基

研究以DMEM/F12(1:1 V/V)培养基为基础,添加不同添加剂优化一种适宜CHO DG44细胞生长的廉价培养基。以细胞密度和细胞活率为主要指标,对DMEM/F12(1:1 V/V)培养基进行了优化。通过正交试验和单因素试验筛选出了CHO DG44细胞生长的最佳培养基。正交试验结果表明添加8mg/L Insulin、10mg/L Transferrin、12mM Glutamine、9mg/L Ethanolamine、9mg/L Sodium selenite、0.5×Lipids、0.5×Vitamin,对细胞生长有较好促进作用,细胞密度从0.6×106 cells/mL上升到1.8×106 cells/mL。在此基础上添加2.5g/L Malt Peptone和2.5g/L YeastExtract可使细胞密度达到2.65×106 cells/mL,基本上达到商业培养基的培养效果,而成本降低了约60%。     

用于重组蛋白药物生产的CHO细胞无血清培养基的研究进展

哺乳动物表达系统因其具有类似于人源化细胞的翻译后修饰方式,已经成为重组蛋白药物生产的主要表达系统.中国仓鼠卵巢(Chinese hamster ovary, CHO)细胞是生产重组蛋白的理想哺乳动物细胞宿主,目前近70％批准上市的重组蛋白药物是由CHO细胞生产的.常规细胞培养所用的培养基需要补充血清才能正常生长,但血清...     

微囊化重组CHO细胞制备和培养条件的优化

微囊化重组基因细胞移植治疗肿瘤是一种新兴的肿瘤基因治疗方法,然而由于目前微囊化细胞规模化制备和培养技术还不成熟,阻碍了其在临床治疗中的推广与应用。以重组CHO细胞为模型,考察了不同的微囊制备和培养条件对微囊化细胞生长和内皮抑素表达的影响。实验表明,种子细胞所处的生长阶段和细胞接种密度对微囊化细胞生长和内皮抑素表达的影响较大,对数生长期的细胞进行包囊并且细胞接种密度为1×106~2×106cells/mL微囊时微囊内细胞生长良好、内皮抑素表达量高。微囊制备时间对细胞活性和内皮抑素表达也有较大的影响,制备时间延长对细胞的损伤增大,因此制备时间应控制在5h以内。生物微胶囊在制备过程中会造成细胞损伤,而体外培养是恢复细胞活性的良好方法,在培养过程中微囊接种量为5%时对细胞生长和内皮抑素表达有利。     

适合棉铃虫细胞HzAm1生长的培养基筛选及低血清驯化

昆虫细胞-杆状病毒系统是昆虫杀虫剂生产和医用外源基因表达的有效工具。昆虫细胞的无血清或低血清培养是十分必要的。从三种商业化的培养基TC-100、GRACE和IPL-41中筛选出了最适合棉铃虫细胞HzAm1生长的基础培养基TC-100。以该培养基为基础,将血清用量从常用的10%降至1%,同时补加一定量的水解乳蛋白以及酵母提取物等,对棉铃虫细胞HzAm1进行驯化培养,效果良好。     

Scale-up and optimization of an acoustic filter for 200 L/day perfusion of a CHO cell culture

Acoustic cell retention devices have provided a practical alternative for up to 50 L/day perfusion cultures but further scale-up has been limited. A novel temperature-controlled and larger-scale acoustic separator was evaluated at up to 400 L/day for a 10(7) CHO cell/mL perfusion culture using a 100-L bioreactor that produced up to 34 g/day recombinant protein. The increased active volume of this scaled-up separator was divided into four parallel compartments for improved fluid dynamics. Operational settings of the acoustic separator were optimized and the limits of robust operations explored. The performance was not influenced over wide ranges of duty cycle stop and run times. The maximum performance of 96% separation efficiency at 200 L/day was obtained by setting the separator temperature to 35.1 degrees C, the recirculation rate to three times the harvest rate, and the power to 90 W. While there was no detectable effect on culture viability, viable cells were selectively retained, especially at 50 L/day, where there was a 5-fold higher nonviable washout efficiency. Overall, the new temperature-controlled and scaled-up separator design performed reliably in a way similar to smaller-scale acoustic separators. These results provide strong support for the feasibility of much greater scale-up of acoustic separations.     

Long-term operation of depth filter perfusion systems (DFPS) for monoclonal antibody production using recombinant CHO cells: Effect of temperature,pH, and dissolved oxygen

Recombinant CHO cells of DG44 origin (CS*13-1.00), expressing a chimeric antibody against the S surface antigen of the Hepatitis B virus, were cultivated in single-stage and two-stage depth filter perfusion systems (DFPS) under varying temperature, pH, and oxygen tension conditions to determine their effects on recombinant antibody production. A long-term culture was carried out in a single-stage depth filter for 81 days, during which an occasional clog interrupted the experiment. However, this problem was solved via trypsin injection. The DFPS showed a steady production of monoclonal antibody at a concentration of 100∼150 mg/L. As the cultivation temperature was increased from 33 to 37°C, the monoclonal antibody (Mab) concentration increased from 80.33 to 133.47 mg/L. Likewise, the glucose uptake rate (GUR) and lactate production rate (LPR) also increased. With an increase in pH from 6.95 to 7.61, the Mab concentration increased from 61.64 to 94.31 mg/L. When the oxygen tension was increased from 60 to 80%, the Mab concentration increased from 93.78 to 128.30 mg/L.