Increased hGM-CSF production and secretion with Pluronic F-68 in transgenic<Emphasis Type="Italic">Nicotiana tabacum</Emphasis> suspension cell cultures

The effects of Pluronic F-68, a nonionic surfactant, on the production and secretion of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) in a transgenicNicotiana tabacum cell suspension culture were investigated in this study. The addition of Pluronic F-68 was shown to extend cell survival in the stationary phase, but had no influence on effective initial cell growth. With regard to production, it increased the level of extracellular hGM-CSF two-fold. This may be attributable not only to the enhanced expression level, but also to the improved permeability of the cell membrane due to the interaction between Pluronic F-68 and the cell membrane and cell wall. The effect of Pluronic F-68 on the production and secretion of hGM-CSF in a bioreactor was also evaluated. hGM-CSF production in the bioreactor after the addition of Pluronic F-68 proved more effective than in flask cultures.     

Modulating and optimizing Pluronic F-68 concentrations and feeding for intensified perfusion Chinese hamster ovary cell cultures

Perfusion culture is often performed with micro-sparger to fulfill the high oxygen demand from the densified cells. Protective additive Pluronic F-68 (PF-68) is widely used to mitigate the adverse effect in cell viability from micro-sparging. In this study, different PF-68 retention ratio in alternating tangential filtration (ATF) columns was found to be crucial for cell performance of different perfusion culture modes. The PF-68 in the perfusion medium was found retained inside the bioreactor when exchanged through ATF hollow fibers with a small pore size (50 kD). The accumulated PF-68 could provide sufficient protection for cells under micro-sparging. On the other hand, with large-pore-size (0.2 μm) hollow fibers, PF-68 could pass through the ATF filtration membranes with little retention, and consequently led to compromised cell growth. To overcome the defect, a PF-68 feeding strategy was designed and successfully verified on promoting cell growth with different Chinese hamster ovary (CHO) cell lines. With PF-68 feeding, enhancements were observed in both viable cell densities (20%–30%) and productivity (~30%). A threshold PF-68 concentration of 5 g/L for high-density cell culture (up to 100 × 10⁶ cells/mL) was also proposed and verified. The additional PF-68 feeding was not observed to affect product qualities. By designing the PF-68 concentration of perfusion medium to or higher than the threshold level, a similar cell growth enhancement was also achieved. This study systematically investigated the protecting role of PF-68 in intensified CHO cell cultures, shedding a light on the optimization of perfusion cultures through the control of protective additives.     

The fate of Pluronic F-68 in chondrocytes and CHO cells

The surfactant Pluronic F-68 (PF-68) is widely used in large-scale mammalian cell culture to protect cells from shear stress that arises from agitation and gas sparging. Several studies suggested that PF-68 is incorporated into the cell plasma membrane and could enter the cells, but without providing any direct evidence. The current study has examined this question for two cell types, one of pharmaceutical interest (CHO cells) and the other of biomedical interest (chondrocytes or cartilage cells). A fluorescent derivative of PF-68 was synthesized to detect and localize internalized Pluronic with culture time. PF-68 uptake by the cells was quantified and characterized. We clearly demonstrate that PF-68 enters the cells, and possibly accumulates in the endocytic pathway. CHO cells showed an average uptake of 11.7 +/- 6.7 (SEM) microg PF-68/10(6) cells while the uptake of chondrocytes was 56.0 +/- 10.9 (SEM) microg PF-68/10(6) cells, independently of the initial PF-68 concentration (between 0.01 and 0.2%, w/v) and of cell concentration (from 1 x 10(6) to 4 x 10(6) cells/mL). These uptake values were identical for both static and agitated culture conditions. Finally, we found that CHO cells are able to eliminate intracellular fluorescent PF-68 but chondrocytes are not. These results show that the uptake of PF-68 by the cells can severely affect PF-68 concentration in the culture medium and thus shear protection effect.     

Physiological responses of CHO cells to repetitive hydrodynamic stress

A majority of the previous investigations on the hydrodynamic sensitivity of mammalian cells have focused on lethal effects as determined by cell death or lysis. In this study, we investigated the effect of hydrodynamic stress on CHO cells in a fed‐batch process using a previously reported system which subjects cells to repetitive, high levels of hydrodynamic stress, quantified by energy dissipation rate (EDR). The results indicated that cell growth and monoclonal antibody production of the test cells were very resistant to the hydrodynamic stress. Compared to the control, no significant variation was observed at the highest EDR tested, 6.4 × 10⁶ W/m³. Most product quality attributes were not affected by intense hydrodynamic stress either. The only significant impact was on glycosylation. A shift of glycosylation pattern was observed at EDR levels at or higher than 6.0 × 10⁴ W/m³, which is two orders of magnitude lower than the EDR where physical cell damage, as measured by lactate dehydrogenase release, was observed. While not as extensively investigated, a second monoclonal antibody produced in a different CHO clone exhibited the same glycosylation change at an intensive EDR, 2.9 × 10⁵ W/m³. Conversely, a low EDR of 0.9 × 10² W/m³ had no effect on the glycosylation pattern. As 6.0 × 10⁴ W/m³, the lowest EDR that triggers the glycosylation shift, is about one order of magnitude higher than the estimated, maximum EDR in typically operated, large‐scale stirred tank bioreactors, further studies in a lower EDR range of 1 × 10³–6.0 × 10⁴ W/m³ are needed to assess the glycosylation shift effect under typical large‐scale bioreactor operation conditions. Follow‐up studies in stirred tanks are also needed to confirm the glycosylation shift effect and to validate the repetitive hydrodynamic stress model. Biotechnol. Bioeng. 2009;103: 1103–1117. © 2009 Wiley Periodicals, Inc.     

Shear-induced changes in membrane fluidity during culture of a fragile dinoflagellate microalga

The commonly used shear protective agent Pluronic F68 (PF68) was toxic to the marine dinoflagellate microalga Protoceratium reticulatum, but had a shear-protective effect on it at concentrations of ≤ 0.5 g L(-1) . Supplementation of P. reticulatum cultures with PF68 actually increased the fluidity of the cell membrane; therefore, the shear protective effect of PF68 could not be ascribed to reduced membrane fluidity, an explanation that has been commonly used in relation to its shear protective effect on animal cells. Data are reported on the membrane fluidity of P. reticulatum and its response to the presence of PF68 under sublethal and lethal turbulence regimens. The membrane fluidity was found to depend strongly on the level of lipoperoxides in the cells produced under lethal agitation.     

Influence of pluronic F68 on oxygen mass transfer

Pluronic F68 is one of the most used shear protecting additives in cell culture cultivations. It is well known from literature that such surface‐active surfactants lower the surface tension at the gas‐liquid interface, which influences the mass transfer. In this study, the effect of Pluronic F68 on oxygen mass transfer in aqueous solutions was examined. Therefore, the gassing in/gassing out method and bubble size measurements were used. At low concentrations of 0.02 g/L, a 50% reduction on mass transfer was observed for all tested spargers and working conditions. An explanation of the observed effects by means of Higbie's penetration or Dankwerts surface renewal theory was applied. It could be demonstrated that the suppressed movement of the bubble surface layer is the main cause for the significant drop down of the k_La‐values. For Pluronic F68 concentrations above 0.1 g/L, it was observed that it comes to changes in bubble appearance and bubble size strongly dependent on the sparger type. By using the bubble size measurement data, it could be shown that only small changes in mass transfer coefficient (k_L) take place above the critical micelle concentration. Further changes on overall mass transfer at higher Pluronic F68 concentrations are mainly based on increasing of gas holdup and, more importantly, by increasing of the surface area available for mass transfer. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1278–1288, 2013     

Stimulation of murine granulocyte macrophage-colony stimulating factor production by Pluronic F-68 and polyethylene glycol in transgenic Nicotiana tabacum cell culture

Pluronic F-68, PEG 8000, or PEG 20 000 added to cell suspension cultures of transgenic Nicotiana tabacum promoted cell growth and the production of the recombinant murine granulocyte macrophage-colony stimulating factor (mGM-CSF) in a 5-l stirred tank bioreactor. The specific growth rates were enhanced from 0.27 d^–1 to 0.47 d^–1, 0.37 d^–1 and 0.4 d^–1 when Pluronic F-68, PEG 8000, or PEG 20 000 was added, respectively. The maximum cell density was also increased most to 13.6 g l^–1 when Pluronic F-68 was added (11.3 g l^–1 in the control culture). In terms of mGM-CSF production, PEG 8000 gave the greatest stimulation and with 2 g PEG 8000 l^–1, mGM-CSF increased from 1.6 to 6.6 ng ml^–1.     

Pluronic和纤维素类对杂交瘤细胞保护特性的研究

利用鼠鼠杂交增2F7细胞(分泌IgG2a单抗)研究了Pluronic F-68、甲基纤维素、羧甲基纤维素及聚醚多元醇与杂交瘤细胞生物相容性及添加限制浓度;研究了添加浓度对葡萄糖的利用及氨的生成影响}在高速搅拌、高剪切力下考查添加剂的保护效果。结果表明,O·05—0·10％(w／V)Pluronic F-68、O．10—0．20％(w／V)甲基纤维素能较好地保护杂交瘤细胞;高浓度Pluronic F-68增加了葡萄糖的比消耗速率及氨的比生成速率;高浓度甲基纤维素增加了氨比生成速率;羧甲基纤维素添加浓度低于0．1％不影响细胞生长,也无保护作用,羧甲基纤维素不影响细胞对葡萄糖的比消耗速率,但增加了氨比生成速率,聚醚多元醇分解细胞。在1．5升GemlliGen生物反应器中,培养基添加0．10％Pluronic F-68、搅拌转速70r／min下细胞正常生长。     

Fluid-mechanical forces in agitated bioreactors reduce the CD13 and CD33 surface protein content of HL60 cells

Flow cytometry was used to examine the effect of hydrodynamic forces in surface aerated stirred tank bioreactors on the quantity of CD13 and CD33 surface proteins of Hl60 (human promyelocytic leukemia) cells. A step increase in agitation of the 2-L bioreactors from 80 to 400 rpm reduced the apparent growth rate and the average CD13 and CD33 content per HL60 cell. The effects on the two surface proteins were observed within 30-60 min following the increase in the agitation and preceded observed effects on cell growth by at least 10 h. Upon reduction of the agitation rate back to 80 rpm, the CD13 and CD33 content recovered (in ca. 10 h) for CD13 and ca. 29h for (CD33) to the levels of the control culture whose agitation rate was maintained at 80rpm. The CD13 and CD33 cell content was reduced even at agitation rates (270 rpm) that did not affect cell proliferation. Pluronic F68 (a commonly used shear protectant) had a protective effect on the CD33 content per cell of cultures subjected to hydrodynamic injury but no effect on the CD13 cell content. Possible bioprocessing and physiological implications of these findings are discussed (c) 1993 Wiley & Sons, Inc.     

动物细胞在鼓泡式生物反应器中的死亡速率

通过实验测定,证明生物反应器中细胞死亡速率与气体鼓泡速率成正比而与反应器体积成反比。实验发现气泡大小对细胞死亡速率具有两种作用,一种作用在于影响气泡表面积生成速率;另一种作用则在于影响细胞在气泡表面的吸附程度,其最佳直径为5mm左右。血清和Pluronic F68能显著降低细胞死亡速率,当Pluronic F68浓度达到0．1％时,kd趋于零。所有这些实验结果均与前文提出的生物反应器设计模型具有很好的一致性。     

Synergistic enhancement of protoplast growth by oxygenated perfluorocarbon and Pluronic F-68

Summary Cell suspension-derived protoplasts of albino Petunia hybrida were grown for 10 d at the interface between aqueous culture medium (KM8P) and an oxygenated (10 mbar for 15 min) perfluorocarbon liquid, perfluorodecalin. Protoplasts synthesised new cell walls and divided normally at the perfluorodecalin/culture medium interface, with a mean viability after 10 d of > 92.0%. The mean plating efficiency of protoplasts was elevated by 37% (P<0.05) following culture at the perfluorodecalin/medium interface, but was unaltered by perfluorodecalin or oxygen separately. The mean plating efficiency of protoplasts cultured at the interface was further increased to a maximium of 52% above control, in the presence of oxygenated perfluorodecalin and KM8P medium supplemented with the non-ionic, co-polymer surfactant, Pluronic F-68 at 0.01% (w/v). These findings demonstrate the effectiveness of oxygenated perfluorodecalin for promoting protoplast growth, by facilitating oxygen delivery. The finding that Pluronic F-68 further increased the plating efficiency of protoplasts cultured at the perfluorocarbon/aqueous interface suggests that these agents improve growth through separate, but cumulative, mechanisms.Abbreviations ATP adenosine triphosphate - PFCs perfluorochemicals - STP standard temperature and pressure     

Cell death from bursting bubbles: Role of cell attachment to rising bubbles in sparged reactors

Bursting bubbles are thought to be the dominant cause of cell death in sparged animal or insect cell cultures. Cells that die during the bubble burst can come from three sources: cells suspended near the bubble; cells trapped in the bubble lamella; and cells that attached to the rising bubble. This article examines cell attachment to rising bubbles using a model in which cell attachment depends on cell radius, bubble radius, and cell–bubble attachment time. For bubble columns over 1 m in height and without protective additives, the model predicts significant attachment for 0.5‐ to 3‐mm radius bubbles, but no significant attachment in the presence of protective additives. For bubble columns over 10 cm in height, and without protective additives, the model predicts significant attachment for 50‐ to 100‐μm radius bubbles, but not all protective additives prevent attachment for these bubbles. The model is consistent with three sets of published data and with our experimental results. Using hybridoma cells, serum‐free medium with antifoam, and 1.60 ± 0.05 mm (standard error) radius bubbles, we measured death rates consistent with cell attachment to rising bubbles, as predicted by the model. With 1.40 ± 0.05 mm (SE) radius bubbles and either 0.1% w/v Pluronic‐F68 or 0.1% w/v methylcellulose added to the medium, we measured death rates consistent with no significant cell attachment to rising bubbles, as predicted by the model. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 62: 468–478, 1999.     

Rheological properties of mammalian cell culture suspensions: Hybridoma and HeLa cell lines

Data on viscous (eta') and elastic (eta') components of the complex viscosity versus oscillatory angular frequency (0.01 to 4.0 rad/s) with increasing strains were obtained for hybridoma cell (62'D3) and HeLa cell (S3) suspensions in PBS at 0.9 (mL/mL) cell volume fraction using a Weissenberg rheogoniometer equipped with two parallel plate geometry at ambient temperature. Both cell suspensions exhibited shear thinning behavior. From the measured viscoelastic properties, the yield stress was calculated. Hybridoma cell suspension (15 mum as the mean diameter of cells) showed the yield stress at 550 dyne/cm(2) that was 1.8 times higher than the value of HeLa cell suspension (22 mum mean diameter) as measured at the oscillatory angular frequency, 4.0 rad/s. The apparent viscosities of HeLa cell suspension at four concentrations and varying steady shear rate were also determined using the Brookfield rotational viscometer. The yield stress to steady shear test was about 130 dyne/cm(2) for HeLa cell suspension at 0.9 (mL/mL) cell volume fraction. The apparent viscosity was in the range about 1 approximately 1000 Poise depending on the cell concentration and shear rate applied. A modified semiempirical Mooney equation, \documentclass{article}\pagestyle{empty}\begin{document}$ \eta = \eta _0 \exp [K\dot \gamma ;{ - \beta } \phi /(1 - K'\sigma \phi _c /D)] $\end{document} was derived based on the cell concentration, the cell morphology, and the steady shear rate. The beta, shear rate index, was estimated as 0.159 in the range of shear rate, 0.16 to 22.1 s(-1), for the cell volume fractions from 0.6 to 0.9 (mL/mL). In this study, the methods of determining the shear sensitivity and the viscous and the elastic components of mammalian cell suspensions are described under the steady shear field. (c) 1993 John Wiley & Sons, Inc.     

Physical modeling of animal cell damage by hydrodynamic forces in suspension cultures

Physical damage of animal cells in suspension culture, due to stirring and sparging, is coupled with complex metabolic responses. Nylon microcapsules, therefore, were used as a physical model to study the mechanisms of damage in a stirred bioreactor and in a bubble column. Microcapsule breaskage folowed first-order kinetices in all experiments Entrainment of bubbles into the liquid phase in the stirred bioreactor gave more microcapsule breakage. In the bubble column, the bubble bursting zone at gas-liquid interface was primarilu responsible for microcapsule breakage. The forces on the microcapsules were equivalent to an external pressure of approximately 4 x 10(4) N . m(-2), based on the critical microcapsule diameter for survival of 190 mum. A stable foam layer, however, was found to be effective in protecting microcapsules from damage. The microcapsule transport to the gas-liquid interface and entrainment into the foam phase was consistent with flotation by air bubbles. This result implies that additives and operation of bioreactors should be selected to minimize flotation of cells. (c) 1992 John Wiley & Sons, Inc.     

耐受性工程调控微生物细胞工厂胁迫抗性

微生物细胞工厂的生产效率是由菌株生长性能、产品合成能力和胁迫抗性共同决定的,其中增强微生物细胞工厂的胁迫抗性是关键.耐受性工程基于微生物细胞工厂抵御胁迫压力的应激反应机制,通过巩固壁膜屏障增强胁迫防御能力,加快应激反应提高损伤修复能力,创制耐受进化工具筛选鲁棒性增强的工业微生物.文中分析归纳了耐受性工程的调控策略,并展...     

Computer simulations of the energy dissipation rate in a fluorescence-activated cell sorter: Implications to cells

Fluorescence activated cell sorting, FACS, is a widely used method to sort subpopulations of cells to high purities. To achieve relatively high sorting speeds, FACS instruments operate by forcing suspended cells to flow in a single file line through a laser(s) beam(s). Subsequently, this flow stream breaks up into individual drops which can be charged and deflected into multiple collection streams. Previous work by Ma et al. (2002) and Mollet et al. (2007; Biotechnol Bioeng 98:772-788) indicates that subjecting cells to hydrodynamic forces consisting of both high extensional and shear components in micro-channels results in significant cell damage. Using the fluid dynamics software FLUENT, computer simulations of typical fluid flow through the nozzle of a BD FACSVantage indicate that hydrodynamic forces, quantified using the scalar parameter energy dissipation rate, are similar in the FACS nozzle to levels reported to create significant cell damage in micro-channels. Experimental studies in the FACSVantage, operated under the same conditions as the simulations confirmed significant cell damage in two cell lines, Chinese Hamster Ovary cells (CHO) and THP1, a human acute monocytic leukemia cell line.     

Ensuring oxygenation of carrot cell cultures in a Couette viscometer during investigation of shear effects

Mathematical simulation and experimental measurement of dissolved O₂ were performed for extended (up to 8 h) shear testing of Daucus carota (carrot) cell cultures in a conventional Couette viscometer (0.625 mm annulus). The results suggest O₂ depletion below critical levels for cell growth may occur. A novel design modification incorporating an O₂-permeable silicone-layer spun cast on a porous ceramic bowl was devised. It significantly improved oxygenation of the cell cultures, keeping dissolved O₂ near saturation.     

含羞草(Mimosa pudica L.)叶片匀浆悬浮液的滞后环

由于受力后叶子立即发生运动 ,含羞草是一个研究力对于生物细胞作用的良好模型。在以往的研究中 ,人们认为此种现象与受力后渗透压改变、离子通道被激活、细胞骨架的动态变化有关。该文旨在通过观察含羞草叶片和叶柄匀浆悬浮液的应力 切变率滞后环变化 ,揭示含羞草的力学性质。在用于比较的含羞草、叶下珠和猪骨骼肌匀浆悬浮液以及水 4个系统中 ,只有含羞草系统具有明显的逆时针滞后环轨迹 ,而其它的 3个系统均不存在。以上结果提示 ,在含羞草的匀浆悬浮液系统中 ,有一种或多种物质 (可能是蛋白质和细胞骨架 )在剪切应力作用过程中由颗粒状结构向网状结构转变 ,由无序结构向有序结构转变 ,由液体结构向黏弹性状态转变 ,而当力撤除以后再缓慢恢复。     

谷胱甘肽转移酶在植物抵抗非生物胁迫方面的角色

非生物胁迫因子如高盐、干旱、低温、重金属污染等严重影响植物的生长和繁殖。植物进化出一系列包括各种酶类物质的系统抵抗逆境所带来的氧化伤害。谷胱甘肽转移酶(glutathione S-transferase,GST,EC 2.5.1.18)是由多种功能的蛋白质组成的超家族,在植物遭受高盐、干旱、低温胁迫时,GSTs可清除活性氧,保护植物细胞膜结构和蛋白质活性。对谷胱甘肽转移酶在植物抵御非生物胁迫中的作用进行综述,为今后利用基因工程育种提供理论依据。