Foaming and media surfactant effects on the cultivation of animal cells in stirred and sparged bioreactors.

Foam formation and the subsequent cell damage/losses in the foam layer were found to be the major problems affecting cell growth and monoclonal antibody (MAb) production in stirred and sparged bioreactors for both serum-supplemented and serum-free media. Surfactants in the culture media had a profound effect on cell growth by changing both the properties of bubbles and the qualities of foam formed. Comparable cell growth and MAb production in sparged bioreactors and in stirred and surface-aerated control cultures were observed only in Pluronic F-68 containing culture media. In media devoid of Pluronic F-68, cells became more sensitive to direct bubble aeration in the presence of antifoam agent which was used to suppress foam formation. Compared with serum-supplemented medium, more severe cell damage effects were observed in serum-free medium. In addition, serum-free medium devoid of cells was partially degraded under continuous air sparging. The mechanism of this damage effect was not clear. Pluronic F-68 provided protective effect to cells but not to the medium. A theoretical model based on the surface active properties of Pluronic F-68 was proposed to account for its protective effect on cell growth. Optimum media surfactant composition in terms of maximum cell growth and minimum foam formation was proposed for stirred and sparged animal cell bioreactor.     

Effects of Pluronic F-68 on shoot regeneration from cultured jute cotyledons and on growth of transformed roots

The effects have been studied of the non-ionic surfactant, Pluronic F-68, on the growth in culture of jute (Corchorus capsularis L.) cotyledons with attached petioles, cotyledon explants and transformed roots. Supplementation of culture medium with 0.001–0.5% (w/v) of either commercial grade Pluronic F-68 or a purified fraction prepared by passage through silica gel, stimulated shoot production from the petioles of C. capsularis var. D154 and C134 cotyledons. This effect was most marked in C134, because of the failure of control cotyledons to produce shoots in the absence of Pluronic. Plants regenerated from Pluronic-treated cotyledons were morphologically normal. Growth of transformed roots of C. capsularis var. D154 was stimulated in medium supplemented with commercial grade or purified Pluronic F-68, with maximum increases in both fresh and dry weights with 0.1% (w/v) of the surfactant. Roots cultured in the presence of Pluronic F-68 could be maintained without sub-culture for up to 70 days, whereas roots cultured in the absence of Pluronic required subculture every 7 days, to prevent necrosis. Transformed roots also produced callus in the presence of 0.001–1.0% (w/v) of either commercial grade or purified Pluronic. The biotechnological implications of these results are discussed in relation to the potential value of non-ionic surfactants as growth-stimulating additives to plant culture media.Abbreviations NAA -naphthaleneacetic acid - BA 6-benzyladenine - IAA indole-3-acetic acid - MS Murashige & Skoog (1962)     

Evidence of Pluronic F-68 direct interaction with insect cells: impact on shear protection, recombinant protein, and baculovirus production*

Pluronic F-68 has been widely used to protect animal cells from hydrodynamic stress, but its mechanism of action is still debatable. Published evidence indicates that Pluronic F-68 interacts with cells, yet scarce information exists of its effect on recombinant protein and virus production by insect cells. In this work, the effect of Pluronic F-68 on production of recombinant baculovirus and rotavirus protein VP7 was determined. Evidence of Pluronic F-68 direct interaction with Sf-9 insect cells also was obtained. Maximum recombinant VP7 concentration and yield increased 10x, whereas virus production decreased by 20x, in spinner flask cultures with 0.05% (w/v) Pluronic F-68 compared to controls lacking the additive. No differences were observed in media rheology, nor kinetics of growth and infection (as inferred from cell size) between both cultures. Hence, Pluronic F-68 influenced cell physiology independently of its shear protective effect. Cells subjected to a laminar shear rate of 3000 s(-1) for 15 min, without gas/liquid interfaces, were protected by Pluronic F-68 even after its removal from culture medium. Furthermore, the protective action was immediate in vortexed cells. The results shown here indicate that Pluronic F-68 physically interacts with cells in a direct, strong, and stable mode, not only protecting them from hydrodynamic damage, but also modifying their capacity for recombinant protein and virus production.     

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.     

Pluronic F-68: an answer for shoot regeneration recalcitrance in microspore-derived <Emphasis Type="Italic">Brassica napus</Emphasis> embryos

Recalcitrance to tissue culture is observed in some genotypes of Brassica napus. Several studies have confirmed that Pluronic F-68 has growth-promoting effects on numerous tissue types. This work investigated the effect of the non-ionic surfactant Pluronic F-68 at four concentrations (0.1%, 0.25%, 0.5%, and 1% (w/v)) on the responsiveness of recalcitrant B. napus lines to tissue culture. Microspores from seven populations of B. napus were cultured on Nitsch and Nitsch medium with this compound. The embryos obtained were plated on solid B5 medium supplemented with zeatin for shoot induction. Pluronic F-68 had a highly significant effect on the proportion of shoot regeneration (P < 0.05) in some of the recalcitrant populations. However, no strong dose–response effect was observed. The estimated probability of a shoot occurring in the absence of Pluronic F-68 ranged from 0.04 to 0.31 depending on the genotype, while in the presence of Pluronic F-68, it ranged from 0.07 to 0.53, respectively.     

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.     

Pluronic F-68 Stimulates Growth of Solanum dulcamara in Culture

The effects have been studied of the non-ionic surfactant, PluronicF-68, on the growth of transformed roots, callus and protoplastsof Solanum dulcamara L. Root growth was stimulated by additionof 0001–005% (w/v) of freshly-prepared, commercial gradePluronic to culture medium, with maximum increases in root freshand dry weights at 001%. Higher concentrations (05–10%w/v) of freshly-prepared Pluronic inhibited growth. A Pluronicfraction, prepared by passage through silica-Amberlite resin,retarded root growth even at concentrations that were stimulatorywith the commercial preparation. Similarly, commercial gradePluronic solutions stored at 4C or 22C for 5 d (‘aged’)also inhibited root growth. Roots grew faster on Pluronic F-68-treatedmembrane rafts compared with growth on commercially-availablerafts; such growth enhancement was comparable to that seen inmedium supplemented with 001% (w/v) freshly-prepared commercialPluronic. Callus growth was also stimulated by the addition of freshly-prepared,commercial grade Pluronic F-68 to medium, with maximum increasesat 01% (w/v); in contrast, 10% (w/v) Pluronic was inhibitoryto callus growth. The mean plating efficiency (15 d after plating)of protoplasts cultured at densities of 01–2010⁵ cm_–3was increased up to 26% by 01% (w/v) Pluronic, while 10% wasinhibitory. Both root and callus soluble carbohydrates and proteinswere increased by exposure to freshly-prepared, commercial Pluronic.Similarly, the specific activities of malate dehydrogenase andacid phosphatase were increased in Pluronic F-68-treated callusand roots. The biotechnological implications of these resultsare discussed in relation to the potential value of non-ionicsurfactants as growth-stimulating additives to plant culturemedia. Key words: Solanum dulcamara, Pluronic F-68, surfactant, transformed roots, callus, protoplasts, malate dehydrogenase, acid phosphatase     

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.     

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.     

Image analysis assessments of perfluorocarbon- and surfactant-enhanced protoplast division

Image analysis has been used to assess the growth of cell suspension-derived protoplasts of Petunia hybrida cv. Comanche at an interface between aqueous culture medium (KM8P), supplemented with 0.01% (w/v) Pluronic F-68, and oxygenated (10 mbar; 10 min) perfluorodecalin. Protoplasts synthesised a new cell wall and entered normal mitotic division which was sustainable to the cell colony/callus stage. This process was accentuated by the collective and additive effects of oxygen, perfluorodecalin and surfactant media supplements. The mean area (mm³) of protoplast-derived cell colonies after 68 days of growth was increased 35 fold over control (media alone) in the presence of these combined treatments. The new cultural regime, leading to improved cell throughput from protoplasts, is discussed primarily in relation to the role of perfluorodecalin as a gas carrier and possible effects of Pluronic F-68 in stimulating cellular uptake of nutrients and/or growth regulators. Image analysis provides a novel and accurate approach to quantifying cell growth responses.Abbreviations dpi dots per inch - FPE final plating efficiency - IPE initial plating efficiency - KM Kao & Michayluk (1975) - PFC Perfluorocarbon - UM Uchimiya & Murashige (1974)     

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     

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     

Interface-mediated death of unconditioned Tetrahymena cells: effect of the medium composition

ABSTRACT We have previously shown that the cell death of Tetrahymena thermophila in low inocula cultures in a chemically-defined medium is not apoptotic. The death is caused by a cell lysis occurring at the medium-air interface and can be prevented by the addition of insulin or Pluronic F-68. Here, we report that cell death can also be caused by the medium. The specific effects of several medium constituents were tested in the presence and absence of an interface. Four of the 19 amino acids (arginine, aspartic acid, glutamic acid, and histidine in millimolar concentration) as well as Ca²⁺ (68 μM) and Mg²⁺ (2 mM) and trace metal ions (micromolar concentrations) are all sufficient to induce the interface-mediated death. The effect of the amino acids and the salt ions Ca²⁺ and Mg²⁺ can be abolished by the addition of insulin (10^-6 M) or Pluronic F-68 (0.01% w/v), whereas insulin/Pluronic F-68 only postpones the death induced by trace metal ions. On the basis of our findings, a new recipe for a chemically-defined medium has been formulated. Single cells can grow in this medium in the presence of medium-air interface without any supplements.     

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     

Effects of Pluronic F-68 on larval development of the hookworm,Necator americanus

Summary Eggs of the nematode,Necator americanus, were incubated at 28°C for up to 120h in medium (prepared from mouse faeces) supplemented with 0.4% (w/v) or 5% of either commercial grade or a silica-Amberlite purified fraction of the non-ionic surfactant, Pluronic F-68. Exposure to commercial grade or purified Pluronic at both concentrations delayed egg hatching. However, subsequent larval growth, as assessed by length of larva, buccal capsule, oesophagus and tail, was significantly (P<0.05) increased (maximally following exposure to 5.0% of the purified fraction). No further development occurred in larvae exposed to other Pluronic fractions tested after 48h, irrespective of concentration. These results suggest that Pluronic may be a valuable supplement in media used for the axenic culture of nematode larvae.     

Stimulation of multiple shoot regeneration from seedling leaves of Hypericum perforatum L. by pluronic F-68

The effects of the non-ionic surfactant, Pluronic F-68, on the growth of shoots regenerated from seedlings (14 days post-germination) of Hypericum perforatum L. were studied. The supplementation of agar-solidified medium with 0.001% (w/v) of Pluronic increased the mean fresh weight of the regenerants after 60 days by 40% and the mean number of plant regenerants recovered per seedling by 34%; a less pronounced increase in the number of regenerants occurred with 0.01% (w/v) of the surfactant. By contrast, the mean fresh weight of the regenerants cultured in the presence of 0.1% (w/v) Pluronic F-68 was 15% lower than untreated controls, although the mean number of regenerants per seedling remained unaltered. The growth of seedling leaf-derived Hypericum callus after 60 days was unaffected by all the concentrations of Pluronic tested. However, there was a tendency for callus cells grown in the presence of Pluronic to be more highly pigmented with anthocyanins. The cultivation of leaf explants with 0.001% or 0.01% (w/v) Pluronic did not affect either the mean fresh weight of the regenerants or the mean number of regenerants per explant. However, decreases in both the mean fresh weight and the mean number of regenerants (both 33.0% lower than the control) occurred following the cultivation with 0.1% (w/v) Pluronic.     

Pluronic Enhances the Robustness and Reduces the Cell Attachment of Mammalian Cells

The addition of the non-ionic surfactant, Pluronic F-68, to serum-free CHO cultures causes multi-functional effects that enhance cell yield in agitated cultures and reduce cell adhesion in stationary cultures. Three independent CHO cell lines were subjected to high liquid shear in assay systems that either included or excluded a liquid-gas interface. In the absence of Pluronic, there was a loss in cell viability in either assay system, although there was an intrinsic variability in sensitivity of the cell lines to shear damage. Supplementation with Pluronic prevented loss of cell viability, indicating protection in either a gas sparged or bubble-free environment. However, we found no evidence of long-term protection of cells once Pluronic was removed. Pluronic was capable of repairing trypsin-damaged cells as evidenced by enhanced growth, reduced membrane porosity, and improved robustness under liquid shear. The proportion of adherent cells was reduced to a minimal level by the presence of Pluronic although its effect was rapidly reversible with a high proportion (70%) of adherent cells observed within a few culture passages of its removal. The observed effects of Pluronic on these cultures are compatible with a mechanism in which the polymer forms a protective layer on the cell membrane, which has a significantly lower hydrophobicity.     

Influence of surfactants on growth and regeneration from mature internodal stem segments of sweet orange (<Emphasis Type="Italic">Citrus</Emphasis><Emphasis Type="Italic">sinensis</Emphasis>) cv. Hamlin

The development of a reliable shoot regeneration system for mature tissue of citrus is of major importance to accelerate the evaluation of commercial traits. Three non-ionic surfactants were evaluated independently in terms of their affects on the growth and regeneration of mature internodal stem segments of sweet orange cv. Hamlin in culture. Growth and shoot development of explants were influenced by type of surfactant added to the regeneration medium DBA3, its concentration and order of flush growth used for explant preparation. Supplementation of Pluronic F-68 at 0.001% (w/v) to the medium was the superior treatment resulting in significantly higher fresh weight gain of explant, improved mean number of shoots per explant and the percentage of explants giving shoots (33.5% from first flush) and shoot yield was twofold higher compared to treatments without surfactant (17%). Triton X-100 was the least responsive in terms of its affect on the growth and regeneration of stem segments but such shoots had a normal phenotype. Explants cultured on DBA3 medium containing Tween 20 exhibited growth and shoot yield similar to treatments without surfactant, but at concentrations 0.01–0.5% (v/v), the shoots became vitrified and failed to graft successfully in vivo. Growth and shoot yield of explants showed a general decline between flushes especially from second and third harvests. Shoots derived from stem segments which were cultured on media containing Pluronic F-68 and no surfactant had a higher survival rate (70–80%, respectively) compared to treatments using Triton X-100 at 0.001–0.1% (v/v) (33% survival). All acclimatized grafts exhibited typical mature wood characteristics and flowered 14–16 months after transfer to the greenhouse.     

Oxygen transfer properties of bubbles in animal cell culture media

Oxygen transfer rates were determined in a bubble aerated animal cell bioreactor. It was found that the oxygen transfer rates increased in the following order: large bubbles ( approximately 5 mm diameter) < intermediate bubbles ( approximately 1 mm diameter) < micron-sized bubbles ( approximately 100 mum diameter). Under certain conditions, the micron-sized bubbles were capable of achieving oxygen transfer rate up to 100 h(-1), a 10-20-fold higher transfer rate than the large bubbles. The effects of medium composition on oxygen transfer rates were different for the three ranges of bubbles studied. For the large bubbles, oxygen transfer rates decreased with increasing medium complexity. The lowest oxygen transfer rate was found in new-born calf serum (NBCS) and/or Pluronic F-68 supplemented media. For the intermediate and micron-sized bubbles, supplementation with NBCS into the culture media resulted in decreased oxygen transfer rate. However, further supplementation with Pluronic F-68 enhanced oxygen transfer rate greatly for both types of bubbles. The highest oxygen transfer rate was found for micron-sized bubbles in Pluronic F-68 supplemented media containing antifoam agent and NBCS.     

Effects of Pluronic F-68 on growth of transformed roots ofSolanum dulcamara

Summary The effects of the non-ionic surfactant, Pluronic F-68, on the growth of transformed roots ofSolanum dulcamara L. have been studied. Growth was stimulated by addition of low concentrations (0.001–0.1% w/v) of freshly-prepared commercial grade Pluronic to liquid culture medium, with maximum increases in root fresh and dry weights at 0.01% (w/v). In contrast, higher concentrations (0.25–1.00% w/v) of freshly-prepared Pluronic inhibited growth. Freshly-prepared purified Pluronic retarded root growth, even at concentrations that were stimulatory with the commercial preparation. Similarly, commercial grade Pluronic solutions stored at 4°C or 22°C for 5 days (aged) were inhibitory to root growth.