Effect of Pluronic F-68 on the mechanical properties of mammalian cells

The mechanical properties of TB/C3 hybridoma cells taken from a continuous culture were measured by micromanipulation. The culture conditions were constant except for the presence or absence of Pluronic F-68 in the medium. It was found that the mean bursting membrane tension and the mean elastic area compressibility modulus of the cells were significantly greater (60% and 120%, respectively) in a medium with 0.05% (w/u) Pluronic F-68 compared to that without Pluronic. Pluronic F-68 therefore affected the strength of the membranes when the cells were exposed to it for a long period of time, i.e., in culture. The short-term effect of Pluronic F-68 on cell strength was also tested by its addition at various levels up to 0.2% (w/v) immediately before the mechanical property measurements. The resulting cell strength depended on the Pluronic concentration, but a significant short-term effect could only be detected above a threshold of 0.1% (w/v). Previous reports on the effect of Pluronic F-68 on animal cell culture are evaluated in the light of these observations.     

Improved micropropagation of Populus spp. by Pluronic F-68

Stem explants and leaves (without petioles) excised from axenic shoots of Populus tremula cv. Ahle or P. tremula × tremuloides cv. Münden were cultured in the presence of the non-ionic, co-polymer surfactant, Pluronic F-68. Stem explants developed shoots within 10 d of culture and significant (p<0.05), but genotype-dependent, increases in total shoot fresh weight (maximum 2 × control) occurred in cultures supplemented with 0.001–0.1% Pluronic F-68 over a 72 d period. Similarly, increases in both fresh weight (up to 10-fold) and number of shoots per P. tremula × tremuloides leaf explant (5-fold maximum) over 60 d occurred with Pluronic F-68 at 0.001%.Abbreviations BAP 6-benzylamino purine - IBA indolebutyric acid - MS0 Murashige and Skoog medium [14] lacking growth regulators - NAA -naphthaleneacetic acid - WPM woody plant medium     

Interactions between animal cells and gas bubbles: The influence of serum and pluronic F68 on the physical properties of the bubble surface

We describe a method by which the degree of bubble saturation can be determined by measuring the velocity of single bubbles at different heights from the bubble source in pure water containing increasing concentrations of surfactants. The highest rising velocities were measured in pure water. Addition of surfactants caused a concentration-dependent and height-dependent decrease in bubble velocity; thus, bubbles are covered with surfactants as they rise, and the distance traveled until saturation is reached decreases with increased concentration of surfactant. Pluronic F68 is a potent effector of bubble saturation, 500 times more active than serum. At Pluronic F68 concentrations of 0.1% (w/v), bubbles are saturated essentially at their source. The effect of bubble saturation on the interactions between animal cells and gas bubbles was investigated by using light microscopy and a micromanipulator. In the absence of surfactants, bubbles had a killing effect on cells; hybridoma cells and Chinese hamster ovary (CHO) cells were ruptured when coming into contact with a bubble. Bubbles only partially covered by surfactants adsorbed the cells. The adsorbed cells were not damaged and they also could survive subsequent detachment. Saturated bubbles, on the other hand, did not show any interactions with cells. It is concluded that the protective effect of serum and Pluronic F68 in sparged cultivation systems is based on covering the medium-bubble interface with surfaceactive components and that cell death occurs either after contact of cells with an uncovered bubble or by adsorption of cells through partially saturated bubbles and subsequent transport of cells into the foam region. (c) 1994 John Wiley & Sons, Inc.     

Effects of Pluronic F-68 on callus growth and protoplast plating efficiency of Solanum dulcamara

Summary The effects of the non-ionic surfactant, Pluronic F-68, on the growth of callus and protoplasts from Solanum dulcamara L. have been studied. Growth of callus was stimulated by addition of 0.1% (w/v) commercial grade Pluronic to culture medium, whereas lower concentrations (0.01% w/v) had no corresponding effect. In contrast, higher concentrations (1.0% w/v) of Pluronic inhibited callus growth. The mean plating efficiency of protoplasts grown at different densities (15 days after plating) was increased up to 26% following culture with 0.1% (w/v) Pluronic, while 0.01% (w/v) Pluronic was ineffective. Mean protoplast plating efficiency decreased by up to 32% following culture with 1.0% (w/v) Pluronic.     

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     

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.     

Strategies for promoting division of cultured plant protoplasts: synergistic beneficial effects of haemoglobin (Erythrogen) and Pluronic F-68

Novel approaches, involving supplementation of aqueous culture medium with haemoglobin solution (Erythrogen), in the presence or absence of the copolymer surfactant, Pluronic F-68, have been evaluated to facilitate cellular oxygen availability to promote mitotic division. Cell-suspension-derived protoplasts of albino Petunia hybrida cv. Comanche were cultured for up to 45 days in KM8P medium containing 1:50–1:500 (vol:vol) Erythrogen. The mean initial protoplast plating efficiency after 9 days with 1:50 Erythrogen (18.5%) was significantly greater (P<0.05) than in untreated controls (11.3%). Supplementation of culture medium with 1:50 Erythrogen, together with 0.01% (wt/vol) Pluronic F-68, increased the mean plating efficiency after 9 days (24.4%) by 92% (P<0.05) over the control (12.7%). These treatments also produced increases in biomass of protoplast-derived cells up to 2.5-fold greater than control (P<0.01) over 80 days. Gassing the medium, containing 1:50 Erythrogen, with carbon monoxide abolished the increase in plating efficiency. There was no additional benefit of gassing Erythrogen-supplemented medium with 100% oxygen. The synergistic, beneficial effect of Erythrogen and Pluronic F-68 on protoplast division has implications for plant biotechnology utilising protoplasts. Received: 24 May 1996 / Revision received: 12 July 1996 / Accepted: 15 August 1996     

Pluronic F68 enhanced the conformational stability of salmon calcitonin in both aqueous solution and lyophilized solid form

The effects of different surfactants on the conformational stability and structural similarity of salmon calcitonin (sCT) in aqueous solution and lyophilized forms were investigated by using microscopic Fourier transform infrared (FTIR) spectroscopy with second-derivative spectral analysis. Six surfactants, HCO-60, sodium dodecyl sulfate (SDS), Tween 80, PEG 400, Pluronic 68, and F127 were selected. The sCT aqueous solution with or without different surfactants was, respectively, incubated at 40°C for up to 35 h. sCT films were casted on the CaF(2) plates and IR spectra were collected as a function of incubation time. Second derivative analysis showed that the native sCT having a major α-helical structure was gradually changed to the combination of α-helix, random coil, and β-sheet conformations in aqueous solution at 40°C. Similar conformational changes with delayed β-sheet formation were obtained for sCT after co-incubation with all the surfactants except Pluronic F68. When the native sCT was freeze-dried alone, a marked conformational alteration was found as illustrated by a poor spectral correlation coefficient (r) value of 0.823 as compared to that of the unlyophilized native sCT. This r value was significantly deviated from 1, strongly indicating the influence of lyophilization stress on the surfactant-free sCT. The r value for sCT after lyophilizing with HCO-60, Pluronic F127, PEG 400, or Pluronic F68 was >0.9, suggesting the possible stabilization of these surfactants in the lyophilization process. The sCT sample after lyophilizing with Pluronic F68 showed a highest r value (>0.968), indicating the most optimal stabilization effect of Pluronic F68 for sCT sample via lyophilization. Pluronic F68 was found to be the preferential surfactant for preventing the secondary structure changes in aqueous solution at 40°C as well as in lyophilized powder.     

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.     

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.     

The effect of Pluronic F-68 on hybridoma cells in continuous culture

Summary The comparative effects of Pluronic on cell growth of hybridomas were studied. The addition of 0.05% Pluronic decreased the steady-state cell number in continuous culture by about 12% compared to a non-supplemented culture. In short-term experiments, results demonstrated a gradual decrease in cell number with increasing concentration of Pluronic. Such growth inhibition was found to be a result of lowering the rate of DNA synthesis. Offprint requests to: M. Al-Rubeai     

Effects of Pluronic F-68 on plant cells in suspension culture

Summary The effects of the non-ionic surfactant, Pluronic F-68, on growth and structure ofSolanum dulcamara cells in suspension culture have been studied. Growth of cells, as measured by dry weight, was unaffected by low concentrations (0.01–1.0% w/v) of pluronic, while culture with higher concentrations (2.5–10.0%) resulted in cell death. It is suggested that low concentrations of pluronic may be valuable supplements in plant cell cultures to protect against mechanical damage and to manipulate membrane systems.     

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     

Protection mechanisms of freely suspended animal cells (CRL 8018) from fluid-mechanical injury. Viscometric and bioreactor studies using serum, pluronic F68 and polyethylene glycol

We use bioreactor and viscometric studies to examine the mechanism by which three additives, fetal bovine serum (FBS), pluronic F68, and polyethylene glycol (PEG), protect the freely suspend CRL-8018 cells from damage due to interactions with bubbles in agitated bioreactors. In bioreactor studies, the protective effect of an addictive could be due to either changes in the ability of the cell resist shear (biological mechanism) or to changes in the medium properties that effect the level or frequency of forces experienced by the cells (physical mechanism). Bioreactor studies show that protection by all three addictives occurs whether the cells are grown in the presence of the addictives (long exposure) or the addictives are added to medium after the cells were exposed to detrimental agitation intensity (short exposure). In the viscometric studies, exposure of cells to laminar shear in the absence of gas-liquid interfaces assesses only the ability of the cells to resist a constant level of shear in a medium with or without an additive. Viscometric studies show that prolonged exposure to FBS makes the cells more shera tolerant, but that short (30-120 min) exposure to FBS does not affect their shear tolerance. We thus conclude that the protective effect of FBS in bioreactors id of both physical and biological nature. The biological contribution is metabolic in nature rather than fast acting. Viscometric studies show that either long or short exposure of the cells to either F68 or PEG does not make the cells more shear tolerant. WE therefore conclude that the protective effect of F68 and PEG does not make the cells more shear tolerant. We therefore conclude that the protective effect of F68 and PEG in bioreactors is physical in nature.     

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.     

Effect of shear stress on expression of a recombinant protein by Chinese hamster ovary cells

A flow chamber was used to impart a steady laminar shear stress on a recombinant Chinese hamster ovary (CHO) cell line expressing human growth hormone (hGH). The cells were subjected to shear stress ranging from 0.005 to 0.80 N m(-2). The effect of shear stress on the cell specific glucose uptake, cell specific hGH, and lactate productivity rates were calculated. No morphological changes to the cells were observed over the range of shear stresses examined. When the cells were subjected to 0.10 N m(-2) shear in protein-free media without Pluronic F-68, recombinant protein production ceased with no change in cell morphology, whereas control cultures were expressing hGH at 0.35 microg/10(6 )cells/h. Upon addition of the shear protectants, Pluronic F-68 (0.2% [w/v]) or fetal bovine serum (1.0% [v/v] FBS), the productivity of the cells was restored. The effect of increasing shear stress on the cells in protein-free medium containing Pluronic F-68 was also investigated. Cell specific metabolic rates were calculated for cells under shear stress and for no-shear control cultures performed in parallel, with shear stress rates expressed as a percentage of those obtained for control cultures. Upon increasing shear from 0.005 to 0.80 N m(-2), the cell specific hGH productivity decreased from 100% at 0.005 N m(-2) to 49% at 0.80 N m(-2) relative to the no-shear control. A concurrent increase in the glucose uptake rate from 115% at 0.01 N m(-2) to 142% at 0.80 N m(-2), and decreased lactate productivity from 92% to 50%, revealed a change in the yield of products from glucose compared with the static control. It was shown that shear stress, at sublytic levels in medium containing Pluronic F-68, could decrease hGH specific productivity.     

Albumin handling by renal tubular epithelial cells in a microfluidic bioreactor

Epithelial cells in the proximal tubule of the kidney reclaim and metabolize protein from the glomerular filtrate. Proteinuria, an overabundance of protein in the urine, affects tubular cell function and is a major factor in the progression of chronic kidney disease. By developing experimental systems to study tubular protein handling in a setting that simulates some of the environmental conditions of the kidney tubule in vivo, we can better understand how microenviromental conditions affect cellular protein handling to determine if these conditions are relevant in disease. To this end, we used two in vitro microfluidic models to evaluate albumin handling by renal proximal tubule cells. For the first system, cells were grown in a microfluidic channel and perfused with physiological levels of shear stress to evaluate the effect of mechanical stress on protein uptake. In the second system, a porous membrane was used to separate an apical and basolateral compartment to evaluate the fate of protein following cellular metabolism. Opossum kidney (OK) epithelial cells were exposed to fluorescently labeled albumin, and cellular uptake was determined by measuring the fluorescence of cell lysates. Confocal fluorescence microscopy was used to compare uptake in cells grown under flow and static conditions. Albumin processed by the cells was examined by size exclusion chromatography (SEC) and SDS-PAGE. Results showed that cellular uptake and/or degradation was significantly increased in cells exposed to flow compared to static conditions. This was confirmed by confocal microscopy. Size exclusion chromatography and SDS-PAGE showed that albumin was broken down into small molecular weight fragments and excreted by the cells. No trace of intact albumin was detectable by either SEC or SDS-PAGE. These results indicate that fluid shear stress is an important factor mediating cellular protein handling, and the microfluidic bioreactor provides a novel tool to investigate this process.     

Ikarugamycin: A Natural Product Inhibitor of Clathrin‐Mediated Endocytosis

Ikarugamycin (IKA) is a previously discovered antibiotic, which has been shown to inhibit the uptake of oxidized low‐density lipoproteins in macrophages. Furthermore, several groups have previously used IKA to inhibit clathrin‐mediated endocytosis (CME) in plant cell lines. However, detailed characterization of IKA has yet to be performed. Consequently, we performed biochemistry and microscopy experiments to further characterize the effects of IKA on CME. We show that IKA has an IC₅₀ of 2.7 μm in H1299 cells and acutely inhibits CME, but not other endocytic pathways, in a panel of cell lines. Although long‐term incubation with IKA has cytotoxic effects, the short‐term inhibitory effects on CME are reversible. Thus, IKA can be a useful tool for probing routes of endocytic trafficking.      

Aerosol delivery of mammalian cells for tissue engineering

We developed a two fluid atomizer to spray mammalian cells suspended in phosphate buffered saline (PBS)-Pluronic F-127 polymer solutions. We used the device to spray suspensions of bovine articular chondrocytes and porcine tracheal epithelial cells, in either PBS or the polymer solutions, and studied their viability and growth kinetics. Under the operating conditions examined in this study, the cells had better than 70% viability after spraying, the growth rates were comparable to cells that were not sprayed, and the appearance of the cells at confluence was normal.