多孔微载体无血清培养rCHO细胞生产u-PA

在30L搅拌式反应器中无血清培养分泌尿激酶型纤溶酶原激活剂(u-PA)的DNA重组CHO细胞,定期部分更换Cytopore多孔微载体,使生长在多孔微载体中的细胞不断更新繁殖,解决大规模细胞培养中的细胞凋亡问题。在91d连接换液培养过程中,细胞密度可维持在(1.3～2.6)×107/mL,活细胞比率维持在90%以上。在7.5L搅拌罐中培养细胞,利用外部周期性压力振荡刺激并结合载体更新技术,可减轻密度效应对细胞生长和表达的影响,在一定程度上提高细胞在高密度培养条件下的表达水平。在67d连续换液培养中,细胞最高密度为2.64×10⁷/mL,活细胞比率维持在95%以上。与稳压操作相比,利用周期变压刺激技术可提高产量10%～20%,且可降低葡萄糖厌氧代谢生成乳酸的转化率,利用4步纯化工艺,从含u-PA约135g的2100 L上清中获得约80guPA(单链比例约为90%)。     

纤维素多孔微载体的制备及其用于动物细胞培养

将纤维素铜氨溶液喷洒至-40℃的硅油：正己烷=1：4的冷冻液中形成含冰晶的微球,用-30℃、40%的H₂SO₄再生纤维素,并用EDAE盐酸盐修饰其表面,制成适合动物细胞培养的纤维素多孔微载体。利用该微载体培养能分泌尿激酶原(Pro-UK)的重组CHO细胞,在100mL搅拌瓶中换液培养25d,细胞最高密度为6.3×10⁶/mL,尿激酶原最高活性为2325IU/mL,共获28.7mg产品。之后转入1000mL搅拌瓶中培养,可观察到细胞可从种子微载体中自动转移到新微载体中生长繁殖直至所有微载体中都长有细胞。在25d二级培养中,细胞最高密度为7.3×10⁶/mL,尿激酶原最高活性为3108IU/mL,共获含353mg尿激酶原的上清13.7L。在培养后期换用无血清培养基培养,细胞生长及蛋白表达水平正常。     

细胞密度和营养供给对H1N1流感病毒产率的影响

探究细胞培养生产流感病毒过程中,高细胞密度引起低单位细胞病毒产率(Svy)的原因及找到解决方法。通过增加微载体浓度以及换液操作获得较高的细胞密度;考察了感染时细胞密度(CCI)和病毒感染用维持培养基对病毒产量和单位细胞病毒产率的影响。在12.6 g/L微载体培养过程中,通过换液操作,可获得高细胞密度,达到1.47×107 cells/m L。在CCI为1×107cells/m L条件时,选择合适的维持培养基,其Svy最高达到5.14×103 virions/cell,比同等条件下用DMEM维持培养基的Svy值提高了近一倍。高密度培养MDCK细胞生产流感病毒时,充分考虑不同阶段的培养条件和营养需求,可以提高细胞密度和单位细胞病毒产率,进而提高流感病毒的产量。该研究结果为工业化生产流感病毒疫苗提供了基础数据。     

微载体高密度培养Vero细胞的研究

微载体是动物细胞高密度培养的有效手段。首先在硅化的方瓶中对Cytodex 1、Cy-todex 3、Biosilon、Bellco Glass Microcarrier、CT-1、CT-3、MC-1、CT-28种国产和进口微载体进行了比较和筛选。确定以Biosilon作为Vero细胞高密度培养的首选微载体。用500mlWheaton搅拌瓶探索影响Vero细胞高密度培养的条件,表明50～60mg/ml的微载体浓度、1～2×10⁶/ml的细胞接种密度、适当的通气(95％O_2+5％CO₂)对该细胞的高密度培养具有重要意义。在200ml培养体积的Wheaton搅拌瓶中,微载体浓度为50～60mg/ml,细胞接种密度为9.24×10⁵/ml,搅拌速度为65～85r/min,经25d培养,Vero细胞密度可达2.34×10⁷/ml,表明50～60mg/ml的微载体浓度对培养细胞没有毒性。接着在1.5L CelliGen生物反应器中进行培养,细胞接种密度为4.98×10⁵/ml,培养体积为1.2L,日灌流量从0.20L逐渐加大到3.65L,经22d连接灌流培养,最终细胞密度可达2.05×10⁷/ml。     

应用生物反应器培养Vero细胞制备EV71病毒初探

目的应用生物反应器培养Vero细胞制备EV71病毒。方法以3 L生物反应器采用4 g/L、8 g/L Cytodex-1微载体培养比较Vero细胞比生长率,并以4 g/L微载体培养EV71病毒。结果 4 g/L微载体培养Vero细胞3～4 d微载体细胞密度达2.3×106/mL,按0.001的感染复数(MOI)接种EV71病毒,病毒收获液的滴度最高达7.90 lgPFU/mL,较静置培养平均高出0.92 lgPFU/mL。结论初步建立了3 L生物反应器微载体培养Vero细胞制备EV71病毒的工艺,为进一步放大生产规模奠定了基础。     

多孔微球固定化CHO工程细胞生产rt-PA的研究

以Cytopore多孔微球固定产重组组织型纤溶酶原激活剂(rtPA)CHO工程细胞株4B3,在2L搅拌式生物反应器用无血清培养基DF5S连续灌流培养。4B3细胞的最大活细胞密度和rtPA生产水平分别达到8.83×10⁶/mL和12473 IU/mL。含rtPA的4B3细胞培养上清经MPG吸附层析和Lysine-sepharose 4B亲和层析两步纯化,rt-PA的纯度达到98%。     

微载体浓度与细胞接种密度对ST细胞生长的影响

选择合适的微载体浓度、细胞接种密度以提高微载体利用率,优化微载体培养体系猪睾丸细胞(Swine testicle cells)的贴附生长与维持。使用DMEM补加10%血清、LSM(Low serum medium)两种培养基考察微载体浓度、细胞接种密度对细胞生长维持的影响,进而比较ST细胞在不同条件下对Cytodex1微载体的利用率。结果显示,使用LSM在T150方瓶中连续传代培养30d,平均比生长速率为0.626d~(0-1),是DMEM补加10%FBS培养基的1.15倍。选择10×10~5cells/mL细胞接种3g/LCytodex1搅拌瓶体系,最大细胞密度为38.3×10~5cells/mL,微载体利用率上升到58.8%。在灌注培养体系中培养ST细胞15d,最终细胞密度达到36.6×10~5cells/mL,扩增了13.6倍。微载体悬浮培养的使用一方面有利于ST细胞的贴附与生长,实现高密度生长,另一方面增加了微载体的使用成本,选择合适的微载体浓度、细胞接种密度,能够最大化利用微载体与培养基中的营养物质实现细胞的最优生长。     

MDCK细胞微载体培养条件优化研究

研究细胞接种量、搅拌转速和微载体浓度对MDCK细胞微载体培养时的影响,以合理优化MDCK细胞微载体培养最大增殖时期的最优条件,对疫苗和病毒分离具有重要意义,以期达到在疫苗和病毒分离领域提高生产效率。采用微载体培养MDCK细胞,在不同搅拌转速、微载体浓度和细胞接种量进行培养,每隔24 h取样计数,确定最优的培养条件。结果表明,细胞接种量在20个/球、搅拌转速45 r/min和载体浓度在2 g/L时,MDCK细胞的增殖较快,细胞密度较大,细胞的密度最大可达15.6×106个/mL,适合MDCK细胞增殖生长。     

牛肾细胞在两种不同载体中培养效果的比较

目的通过牛肾细胞在两种不同载体中培养效果的比较,为牛肾细胞在细胞工厂中规模化生产提供真实的、有力的支持。方法不同代次牛肾细胞在两种载体中经过相同培养条件进行培养。结果实验中原代牛肾细胞在细胞工厂接种密度为5.5×104/cm2左右,在15 L转瓶接种密度为9.0×104/cm2左右。一代牛肾细胞在细胞工厂接种密度为6.5×104/cm2左右,在15 L转瓶接种密度为10×104/cm2左右。二代牛肾细胞在细胞工厂接种密度为7.0×104/cm2左右,在15 L转瓶接种密度为14×104/cm2左右。两种载体中牛肾细胞生长状况均能达到培养要求。结论细胞工厂能在有限的空间内利用最大限度的培养表面培养牛肾细胞,不仅节约了传代前的细胞用量,而且提高了培养后的细胞产量。     

H1N1流感病毒在微载体培养MDCK细胞上增殖的研究

目的探索MDCK细胞在微载体上的培养条件,并研究H1N1型流感病毒在MDCK细胞上的增殖条件。方法在微载体上培养好MDCK细胞上用H1N1型流感病毒在不同的病毒感染复数(MOI)、胰酶浓度两个关键的病毒增殖条件进行流感病毒在细胞上的增殖研究。结果微载体质量浓度为6 g/L时,MDCK细胞培养密度可以达到4.5×106cells/mL。在MOI为0.05接种流感病毒,胰酶质量浓度4μg/mL,流感病毒在MDCK细胞上可获得较高的滴度。结论 MDCK细胞用微载体培养可以达到较高的细胞密度,可以作为规模化生产新型流感病毒疫苗的主要细胞基质进行进一步的研究。     

Effects of cell density and glucose and glutamine levels on the respiration rates of hybridoma cells

The effects of cell density as well as the concentration levels of glucose and glutamine on the specific respiration rate of a hybridoma cell line were investigated. The experimental oxygen consumption rate was found to be constant over a wide range of dissolved oxygen levels if the suspension medium contained glutamine. In glutamine-free medium, however, the rate of oxygen consumption decreased slowly with time.In a stationary flask batch culture, the specific respiration rate decreased from about 7 to 2.9 mumol/min per 10(9) cells as the cell density increased exponentially from 1 x 10(5) to 1.2 x 10(6)/mL. To isolate the effect of cell density, cells were re suspended in fresh culture medium so that nutrient concentrations were the same for all experiments. The specific respiration rate decreased with increasing cell density in the same manner as in the stationary flask culture, falling from 8 to 4 mumol/min per 10(9) cells as the cell density increased from 10(5) to 10(6) cells/mL, then declining to 2 mumol/min per 10(9) cells when the cell density reached 10(7) cells/mL.Cells suspended in Hanks balanced sale solution (HBSS) were used to elucidate the effect of glucose and glutamine levels on respiration. The addition of glucose in concentrations of 0.25, 0.50, and 0.75 g/L had no observable effect on the specific oxygen uptake rate; however, a glucose concentration of 1 g/L reduced the uptake rate by 22%. Glutamine in a concentration of 0.30 g/L increased the specific respiration rate in HBSS containing 0 and 1 g/L glucose by approximately 13%.     

Large-scale, high-density freezing of hybridomas and its application to high-density culture

Large-scale, high-density freezing of hybridomas was studied to apply frozen cells to start high-density culture. We showed here that hybridomas can be frozen at 1.5 x 10(8) cells/mL, without decrement in viability and proliferating activity. Blood transporting bags were used for large-scale freezing to store 25 mL of cell suspension with a cell density, 1.5 x 10(8)/mL. The number of cells stored in a bag (3.0 x 10(9) cells) was enough to start a high-density culture at a 10 times higher cell density (6.0 x 10(6) cells/mL) than normal inoculation, and the cells proliferated to 10(7) cells/mL within 2 days. These results indicate that the large-scale freezing method is useful for large-scale culture of mammalian cells.     

Culture of transgenic Drosophila melanogaster Schneider 2 cells in serum-free media based on TC100 basal medium

Requirements of eliminating animal proteins from cell culture have intensified in recent years, with the pressure of regulatory agencies related to biopharmaceuticals production. In this work, the substitution of fetal bovine serum by yeastolate and a soy hydrolysate (Hy Soy) for the culture of Drosophila melanogaster Schneider 2 cells transfected for the production of rabies virus G glycoprotein was evaluated. TC100 supplemented with glucose, glutamine, lipid emulsion and Pluronic F68 was employed as basal medium. Results show that yeastolate was more efficient on cell growth stimulation than Hy Soy. Cells adapted in medium formulation supplemented with 3 g/L yeastolate, 1% lipid emulsion, 10 g/L glucose, 3.5 g/L glutamine and 0.1% Pluronic F68 attained a maximum concentration of 10.7 x 10(6) cells/mL, with the expression of 9.4 ng/mL G glycoprotein.     

High-density transient gene expression in suspension-adapted 293 EBNA1 cells

Large-scale transient gene expression (TGE) in mammalian cells is an attractive method to rapidly produce recombinant proteins for pre-clinical studies, with some processes reported to reach 100 L. However, the yield remains low, hardly over 20 mg protein/L, mainly because the current TGEs have been performed at low cell density (approximately 5 x 10(5) cells/mL). In this study, the strategy to improve TGE focuses on facilitating transfection at high cell density. A high-density perfusion culture of 293 EBNA1 cells was established in 2-L bioreactor using Freestyle 293 expression medium (Invitrogen, Singapore) to grow the cells for transfection. Transfection was then carried out at 1 x 10(7) cells/mL using polyethylenimine (PEI) as DNA carrier, at the optimized conditions of 6 microg DNA/10(7) cells and 1:3 DNA to PEI mass ratio. During the post-transfection phase, 80.8 mg/L of the model protein, EPO was obtained at day 5.5 post-transfection (130 mg total EPO production) using a fed-batch culture mode. In comparison, perfusion cultures using an enriched SFM II medium resulted in a longer post-transfection production phase (8 days), and 227 mg of EPO was produced in 10.7 L medium, showing that high-density TGE enables the production of several hundreds of milligrams of protein in a 2 L bioreactor. In addition, a protocol for economical plasmid preparation based on anion exchange was also established to satisfy TGE's demand in terms of quality and quantity. To the best of our knowledge, this is the first report of transient transfections at a high cell density of up to 1 x 10(7) cells/mL.     

50L规模中试发酵重组核苷二磷酸激酶A工程菌

中试规模发酵重组人核苷二磷酸激酶A（rhNDPK-A）工程菌,并对表达产物进行纯化。摇瓶培养一级种子至合适密度,以10%比例接种二级种子培养基,在7L发酵罐中培养至OD600为9.6～10.5,然后转入80L发酵罐中进行补料分批培养,所得菌体裂解后,经离子交换层析和亲和层析两步纯化得重组蛋白制品。结果表明,50L培养液经过10h培养后,湿菌收量为1560 g/批,NDPK-A表达量为23.8%。另外,补料方式对发酵密度有明显影响。与单纯补加碳源相比,同时补加碳源和氮源可以显著提高菌体产量,但对目的蛋白表达量地提高不明显。在较优条件下,菌体产量为(2220.00±169.71) g/批,蛋白表达量为(22.00±0.42) %,纯化后重组蛋白得率为510mg/L。产物可溶、密度适中、工艺简便的中试发酵条件的建立为高得率、大规模制备重组rhNDPK-A奠定了基础。     

Cultivation of immortalized human hepatocytes HepZ on macroporous CultiSpher G microcarriers

Cultivation of the new immortalized hepatocyte cell line HepZ was performed with a 1:1 mixture of DMEM and Ham's F12 media containing 5% FCS. The cells were grown in their 40th passage in 100 mL and 1 L volumes in spinner flasks and in a bioreactor, respectively. For the production of adherently growing HepZ cells macroporous CultiSpher G gelatin microcarriers were used in various concentrations from 1 to 3 g/L. The cells were seeded in a density of 2 x 10(5) cells/mL when using a microcarrier concentration of 1 g/L and 5 x 10(5) cells/mL at a microcarrier concentration of 3 g/L. After 7 days of cultivation a maximum cell concentration of 4.5 x 10(6) cells/mL was obtained in the spinner culture using a microcarrier concentration of 1 g/L. With bubble-free aeration and daily medium exchange from day 7, 7.1 x 10(6) cells/mL were achieved in the bioreactor using a microcarrier concentration of 3 g/L. The cells exhibited a maximum specific growth rate of 0.84 per day in the spinner system and 1.0 per day in the bioreactor, respectively. During the growth phase the lactate dehydrogenase (LDH) activity rose slightly up to values of 200 U/L. At the end of cultivation the macroporous carriers were completely filled with cells exhibiting a spherical morphology whereas the hepatocytes on the outer surface were flat-shaped. Concerning their metabolic activity the cells predominantly consumed glutamine and glucose. During the growth phase lactate was produced up to 19.3 mM in the spinner culture and up to 9.1 mM in the bioreactor. Maximal oxygen consumption was 1950 nmol/(10(6) cells. day). HepZ cells resisted a 4-day long chilling period at 9.5 degrees C. The cytochrome P450 system was challenged with a pulse of 7 microgram/mL lidocaine at a cell density of 4.5 x 10(6) cells/mL. Five ng/mL monoethylglycinexylidide (MEGX) was generated within 1 day without phenobarbital induction compared to 26 ng/mL after a preceded three day induction period with 50 microgram/mL of phenobarbital indicating hepatic potency. Thus, the new immortalized HepZ cell line, exhibiting primary metabolic functions and appropriate for a mass cell cultivation, suggests its application for a bioartificial liver support system.     

利用盘基网柄菌表达可溶性人Fas配体

用PCR扩增从激活的人中性粒细胞中得到的编码可溶性Fas配体胞外区中第141个到第281个氨基酸的cDNA ,将其与hCG-β信号肽片段融合到质粒MB12neo中,随后导入到盘基网柄菌AX3细胞中,得到分泌性表达hFasL的重组菌AX3-H3。为提高shFasL的表达量,对质粒pMB12neo作了改造,得到衍生质粒pMB74。利用质粒pMB74克隆表达shFasL ,得到高通量表达shFasL的重组菌AX3_pLu8。在复杂培养基HL_5C中,重组菌的细胞密度可达(1.5～2 )×10⁷ mL ,AX3-H3及AX3_pLu8分泌的shFasL浓度分别为23.5 μg/L及206μg/L。利用合成培养基SIH培养重组菌AX3-H3及AX3-pLu8,细胞密度均达到(4～5)×10⁷m/L ,shFasL浓度则分别达到111μg/L和420μg/L。     

大肠杆菌BL21(DE3)磷酸转乙酰基酶缺陷变株的发酵研究

研究了E.coliBL21(DE3)及其磷酸转乙酰基酶(PTA)缺陷变株FR55发酵过程中菌体生长和有机酸产生情况,并以肿瘤坏死因子(TNF)为外源蛋白表达的模型考察了pta基因缺陷对外源蛋白表达的影响。在摇瓶培养条件下,pta变株TNF的表达水平比亲株提高了23%。在5L发酵罐中进行了补料分批培养试验,在不限制比生长速率的条件下pta变株能够以较长时间和较高比生长速率保持对数生长,最终达到32.5g(DCW)/L的菌密度,TNF的总表达量达2.8g/L;而在相同条件下,以BL21(DE3)为受体菌的对照组最高菌密度为19.5g(DCW)/L,TNF总表达量只有0.84g/L。表明pta变株对于提高工程菌外源蛋白的表达和实现高密度培养具有一定应用价值。分析了补料分批培养过程中发酵液有机酸组成和含量的动态变化情况,发现pta变株乙酸累积水平明显降低(为亲株乙酸累积水平的42%)的同时,其他几种有机酸(丙酮酸、乳酸、琥珀酸)的累积有显著增加的趋势,使发酵液中总有机酸浓度增加了123%,其中乳酸的累积是影响菌体进一步生长的主要因素。     

Effect of flow rate pattern on glucose-6-phosphate dehydrogenase synthesis in fed-batch culture of recombinant Saccharomyces cerevisiae

A strain of genetically modified Saccharomyces cerevisiae (S. cerevisiae) W303 181 was used to improve glucose-6-phosphate dehydrogenase (G6PDH) production in aerobic culture. Fed-batch cultures were carried out in a 5 L fermentor at variable values of the parameter K, namely, 0.2, 0.3, 0.5, 0.7, and 0.8 h(-)(1). The highest G6PDH production (1164 U/L) and specific activity (517 U/g(cell)) were obtained using the following conditions: glucose, 5.0 g/L; adenine, 8 microg/mL; histidine, 8 microg/mL; tryptophan, 8 microg/mL; temperature, 30 degrees C; inoculum, 1.28 g/L; pH, 5.7; agitation, 400 rpm; aeration, 2.2 vvm; and K, 0.2 h(-)(1). The exponential feeding pattern increased cell density (2.14 g/L), enzyme productivity (149.27), and biomass yield (0.18 g(glu)/g(cell)( )(mass)). The level of G6PDH in the genetically modified S. cerevisiae was approximately 4.1-fold higher than that found in a commercial strain.     

Oxygen mass transfer enhancement via fermentor headspace pressurization.

Bioreactor headspace pressurization represents an excellent means of enhancing oxygen mass transfer to a culture. This method is particularly effective in situations where stirring or vigorous aeration is difficult. Because it in itself introduces no undesirable hydrodynamic force, the proposed method is also attractive for cells susceptible to agitation and sparging. Experiments were first conducted in an ideal fermentor by sparging air into a sulfite solution free from extraneous microbial effects. An increased oxygen mass transfer rate resulting from pressurization led to a superior cell growth rate and a higher maximum cell density in both of the microbial systems studied: a bacterial (Escherichia coli) culture up to 2.72 bar and a fragile algal (Ochromonas malhamensis) culture with pressure programming. Applying pressurization increased the maximum dry cell weight from 1.47 g/L to 1.77 g/L in the E. coli culture and increased the maximum viable cell density from 4 x 10(7) cells/mL to 10(8) cells/mL in the algal culture. An additional advantage is that formation of undesirable products under oxygen limitation, e.g., acetic acid in the E. coli culture, can be suppressed. A significant (over 250%) improvement in the oxygen transfer rate can be achieved with existing fermentors with little modification as they are already designed to withstand reasonable pressure from autoclaving. This method is simple, clean, inexpensive, and easily implemented, and it can be applied alongside other existing methods of oxygen mass transfer enhancement.