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 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.     

Sparged animal cell bioreactors: mechanism of cell damage and Pluronic F-68 protection.

Pluronic F-68 is a widely used protective agent in sparged animal cell bioreactors. In this study, the attachment-independent Spodoptera frugiperda Sf9 insect cell line was used to explore the mechanism of this protective effect and the nature of cell damage in sparged bioreactors. First, bubble incorporation via cavitation or vortexing was induced by increasing the agitation rate in a surface-aerated bioreactor; insect cells were rapidly killed under these conditions of the absence of polyols. Supplementing the medium with 0.2% (w/v) Pluronic F-68, however, fully protected the cells. Next, cell growth was compared in two airlift bioreactors with similar geometry but different sparger design; one of these bioreactors consisted of a thin membrane distributor, while the other consisted of a porous stainless steel distributor. The flow rates and bubble sizes were comparable in the two bioreactors. Supplementing the medium with 0.2% (w/v) Pluronic F-68 provided full protection to cells growing in the bioreactor with the membrane distributor but provided essentially no protection in the bioreactor with the stainless steel distributor. These results strongly suggest that cell damage can occur in the vicinity of the gas distributor. In addition, these results demonstrate that bubble size and gas flow rate are not the only important considerations of cell damage in sparged bioreactors. A model of cell death in sparged bioreactors is presented.     

Microscopic visualization of insect cell-bubble interactions. II: The bubble film and bubble rupture.

In this paper, the second in the series, the use of a microscopic, high-speed video system to study the interactions of two suspended insect cells strains, Trichoplusia ni (TN-368) and Spodoptera frugiperda (SF-9), with rupturing bubbles is reported. Events such as the adsorption of cells onto the bubble film and the mechanism of bubble rupture were observed. On the basis of these observations and the experimental and theoretical work of other researchers on bubble rupture and cell death as a result of sparging, it is proposed that cells are killed by the rapid acceleration of the bubble film after rupture and the high levels of shear stress in the boundary layer flow associated with bubble jet formation.     

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.     

Properties of a baculovirus mutant defective in the protein phosphatase gene.

Autographa california nuclear polyhedrosis virus (AcMNPV) contains a gene, ptp, encoding a protein tyrosine/serine phosphatase, BV-PTP. To investigate the biological function of ptp in the baculoviral replication cycle, we constructed a recombinant baculovirus, vPTPdel, in which the catalytically active site of BV-PTP was deleted. Although the vPTPdel mutant was viable in cell culture, it was partially defective in occluded virus production in SF-21 but not TN-368 cell lines. SF-21 cells infected with vPTPdel were heterogeneous in their ability to support occluded virus production. These results suggest that BV-PTP functions in a cell line-specific and possibly a cell cycle-specific fashion. The yield of occlusion bodies, infectivity (concentration of virus causing 50% mortality) and virulence (the time at which 50% of the cells died) of vPTPdel appeared to be normal in insect larvae. We identified a 35-kDa phosphoprotein as a potential target of the BV-PTP in SF-21 cells.     

DNA double-strand break repair in eukaryotic cell lines having radically different radiosensitivities

TN-368 lepidopteran insect cells are on the order of 100 times more resistant to the lethal effects of ionizing radiation than cultured mammalian cells. DNA double-strand breaks (DSB) are believed by many to be the critical molecular lesion leading to cell death. We have therefore compared the rejoining of DSB in TN-368 and V79 Chinese hamster cells. Cells were irradiated on ice with 137Cs gamma rays at a dose rate of 2.5 Gy/min, incubated for various periods of time, and assayed for DNA DSB using the method of neutral elution. The kinetics of DSB rejoining following a dose of 90.2 Gy is similar for both cell lines with 50% of the rejoining completed in about 12 min. Approximately 83 and 87% of the DSB are rejoined in the TN-368 and V79 cells, respectively, by 1 h postirradiation. However, no further rejoining occurs in the TN-368 cells through at least 6 h postirradiation, whereas approximately 92% of the DSB are rejoined in the V79 cells by 2 h postirradiation. Other studies (from 22.6 to 226 Gy) demonstrate that the amount of rejoining of DSB varies inversely with dose for both cell lines, but this relationship is not as pronounced for the TN-368 cells. In general, these findings do not support the hypothesis that unrejoined DNA DSB represent the critical molecular lesion responsible for cell death.     

Microscopic visualization of insect cell-bubble interactions. I: Rising bubbles, air-medium interface, and the foam layer.

Through the use of microscopic, high-speed video technology, the interactions of two suspended insect cell lines, Trichoplusia ni (TN-368) and Spodoptera frugiperda (SF-9), with air and oxygen bubbles were studied. Events such as cell-bubble attachment, cell-bubble collision, cell transport into the foam layer, and trapping of cells in the foam layer are presented and discussed. Based on these observations and those in a companion paper (Chalmers, J. J.; Bavarian, F. Biotechnol. Prog. 1991, following paper in this issue) and the experimental and theoretical work of other researchers, several mechanisms of cell damage as a result of sparging are presented.     

Development of quenching and washing protocols for quantitative intracellular metabolite analysis of uninfected and baculovirus-infected insect cells

Metabolomics refer to the global analysis of small molecule metabolites in a biological system, and can be a powerful tool to elucidate and optimize cellular processes, particularly when integrated into a systems biology framework. Determining the endometabolome in cultured animal cells is especially challenging, due to the conflicting demands for rapid quenching of metabolism and retention of membrane integrity, while cells are separated from the complex medium. The challenge is magnified in virus infected cells due to increased membrane fragility. This paper describes an effective methodology for quantitative intracellular metabolite analysis of the baculovirus–insect cell expression system, an important platform for the production of heterologous proteins and baculovirus-based biopesticides. These two applications were represented by Spodoptera frugiperda (Sf9) and Helicoverpa zea (HzAM1) cells infected with recombinant Autographa californica and wild-type Helicoverpa armigera nucleopolyhedroviruses (AcMNPV and HaSNPV), respectively. Specifically, an ice-cold quenching solution comprising 1.1% w/v NaCl and 0.2% w/v Pluronic® F-68 (NaCl + P) was found to be efficacious in preserving cell viability and minimizing cell leakage during quenching and centrifugation-based washing procedures (prior to extraction using cold 50% v/v acetonitrile). Good recoveries of intracellular adenosine triphosphate, total adenosine phosphates and amino acids were obtained after just one wash step, for both uninfected and infected insect cells. The ability to implement wash steps is critical, as insect cell media are metabolites-rich, while infected insect cells are much more fragile than their uninfected counterparts. Hence, a promising methodology has been developed to facilitate endometabolomic analysis of insect cell–baculovirus systems for bioprocess optimization.     

A Comparison of Survival and Repair of Uv-Induced DNA Damage in Cultured Insect VERSUS Mammalian Cells

Survival and unscheduled DNA synthesis (UDS) were measured in a cultured insect cell line, TN-368, and a cultured mammalian cell line, V-79-4, following exposure to several fluences of ultraviolet light. TN-368 cells were approximately seven times more resistant to the lethal effects of UV than V-79 cells, as determined by colony formation. The amount of UDS per unit amount of DNA is about the same in both cell types 4 hr after 10–50 J/m² UV irradiations.     

Differential requirements for baculovirus late expression factor genes in two cell lines.

A plasmid library of 18 late expression factor (LEF) genes (LEF library) from the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) supports transient expression from a late viral promoter in the SF-21 cell line, derived from Spodoptera frugiperda. We found, however, that this LEF library was unable to support expression from the same promoter in the TN-368 cell line, derived from Trichoplusia ni, which is also permissive for AcMNPV replication. To identify the additional factor(s) required for expression in TN-368 cells, we cotransfected the LEF library with clones representing portions of the AcMNPV genome not represented in the LEF library. A single additional gene was identified; this gene corresponded to ORF70 of the complete AcMNPV sequence and potentially encodes a 34-kDa cysteine-rich polypeptide. Because of its differential effect on late gene expression in the two cell lines, we renamed ORF70 hcf-1 (for host cell-specific factor 1). hcf-1 was involved in expression from reporter plasmids under late and very late but not early promoter control, indicating that it was also a LEF gene. Plasmid DNA replication assays indicated that HCF-1 was involved in virus origin-specific DNA replication in TN-368 cells. Three LEF genes, ie-2, lef-7, and p35, required for optimal virus origin-specific plasmid DNA replication or stability in SF-21 cells had little or no influence in TN-368 cells. Thus, as determined by transient-expression assays, cell line-specific and potentially host-specific factors are required for origin-specific DNA replication or stability.     

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.     

KLF2-dependent, shear stress-induced expression of CD59: a novel cytoprotective mechanism against complement-mediated injury in the vasculature

Complement activation may predispose to vascular injury and atherogenesis. The atheroprotective actions of unidirectional laminar shear stress led us to explore its influence on endothelial cell expression of complement inhibitory proteins CD59 and decay-accelerating factor. Human umbilical vein and aortic endothelial cells were exposed to laminar shear stress (12 dynes/cm(2)) or disturbed flow (+/- 5 dynes/cm(2) at 1Hz) in a parallel plate flow chamber. Laminar shear induced a flow rate-dependent increase in steady-state CD59 mRNA, reaching 4-fold at 12 dynes/cm(2). Following 24-48 h of laminar shear stress, cell surface expression of CD59 was up-regulated by 100%, whereas decay-accelerating factor expression was unchanged. The increase in CD59 following laminar shear was functionally significant, reducing C9 deposition and complement-mediated lysis of flow-conditioned endothelial cells by 50%. Although CD59 induction was independent of PI3-K, ERK1/2 and nitric oxide, an RNA interference approach demonstrated dependence upon an ERK5/KLF2 signaling pathway. In contrast to laminar shear stress, disturbed flow failed to induce endothelial cell CD59 protein expression. Likewise, CD59 expression on vascular endothelium was significantly higher in atheroresistant regions of the murine aorta exposed to unidirectional laminar shear stress, when compared with atheroprone areas exposed to disturbed flow. We propose that up-regulation of CD59 via ERK5/KLF2 activation leads to endothelial resistance to complement-mediated injury and protects from atherogenesis in regions of laminar shear stress.     

Enhanced survival by photoreactivation and liquid holding following UV damage of TN-368 insect cells

These studies demonstrate that the TN-368 lepidopteran insect cell line, which is extremely resistant to the lethal effects of ionizing radiation, is also quite resistant to 254-nm ultraviolet light. While resistance to ionizing radiation in TN-368 cells has been associated with superior DNA repair processes, previous findings have indicated no correlation between survival ability and amount of unscheduled DNA synthesis in response to ultraviolet light. The present studies were undertaken to define the TN-368 ultraviolet light survival response, the ability of the cells to repair UV-induced damage by photoreactivation, the capacity of the cells to undergo UV repair during liquid holding in the dark, and the relationship between photoreactivation and liquid-holding recovery. Survival was assayed by colony formation. 254-nm irradiations were performed using germicidal lamps and photoreactivation was accomplished using black lights. Photoreactivable sectors of UV damage at 50 and 10% survival are 0.65 and 0.68, respectively. Survival responses, both with and without photoreactivation, have a small initial shoulder followed by an exponential region, and finally the curves continue to decrease but with decreasing slope. F0, Fq, and extrapolation number for the exponential portion of the curves are 77.5 J/m2, 16.8 J/m2, and 1.7 for non-photoreactivated cells and 234 J/m2, 56.1 J/m2, and 1.7 for those exposed to photoreactivating light. In the primarily exponential survival region, the fluences required to produce equivalent levels of survival in photoreactivated cells range from approximately 10.8 to 23.3 times as great as cells receiving UV light alone. The maximum survival enhancement of cells maintained under liquid-holding conditions over cells plated immediately following 100-400 J/m2 irradiations appears to be about 2-fold and occurs at 3-6 h of holding. Photoreactivation alone has a greater enhancement of survival than when photoreactivation follows liquid holding, but when liquid holding follows photoreactivation, the enhancement surpasses that of photoreactivation alone.     

A cell sorting protocol for selecting high-producing sub-populations of Sf9 and High Five™ cells

Insect cell lines such as Sf9 and High Five™ have been widely used to produce recombinant proteins mostly by the lytic baculovirus vector system. We have recently established an expression platform in Sf9 cells using a fluorescence-based recombinase mediated cassette exchange (RMCE) strategy which has similar development timelines but avoids baculovirus infection. To expedite cell engineering efforts, a robust fluorescence-activated cell sorting (FACS) protocol optimized for insect cells was developed here. The standard sorting conditions used for mammalian cells proved to be unsuitable, resulting in post-sorting viabilities below 10% for both cell lines. We found that the extreme sensitivity to the shear stress displayed by Sf9 and High Five™ cells was the limiting factor, and using Pluronic F-68 in the cell suspension could increase post-sorting viabilities in a dose dependent manner. The newly developed protocol was then used to sort stable populations of both cell lines tagged with a DsRed-expressing cassette. Before sorting, the average fluorescence intensity of the Sf9 cell population was 3-fold higher than that of the High Five™ cell population. By enriching with the 10% strongest DsRed-fluorescent cells, the productivity of both cell populations could be successfully improved. The established sorting protocol potentiates the use of RMCE technology for recombinant protein production in insect cells.     

A serum-free medium for the culture of insect cells and production of recombinant proteins

Summary A low protein aqueous lipid supplement (Ex-Cyte VLE), in combination with pluronic polyol, is an effective replacement for fetal bovine serum for insect Sf-9 cells. Serum-free medium with lipid supplement and pluronic (SFM-LP) supported higher cell viability and maximum cell populations than serum-supplemented medium. No adaptation procedures are required when switching cells from serum-containing medium to SFM-LP, and growth rates remain constant during continued passages in SFM-LP. The amounts of recombinant proteins produced, which is the major use for the Sf-9 cells, are better or equal in SFM-LP compared to serum-supplemented medium. SFM-LP also supports growth of the TN-368 cell line but IPLB-SF-21AE or IZD-Mb0503 lines grow poorly in this medium.     

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     

Baculovirus p33 Binds Human p53 and Enhances p53-Mediated Apoptosis

In vertebrates, p53 participates in numerous biological processes including cell cycle regulation, apoptosis, differentiation, and oncogenic transformation. When insect SF-21 cells were infected with a recombinant of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) overexpressing human p53, p53 formed a stable complex with the product of the AcMNPV orf92, a novel protein p33. The interaction between p53 and p33 was further confirmed by immunoprecipitation studies. When individually expressed in SF-21 cells, human p53 localized mainly in the nucleus whereas baculovirus p33 displayed diffuse cytoplasmic staining and punctuate nuclear staining. However, coexpression of p33 with p53 resulted in exclusive nuclear localization of p33. In both SF-21 and TN-368 cells, p53 expression induced typical features of apoptosis including nuclear condensation and fragmentation, oligonucleosomal ladder formation, cell surface blebbing, and apoptotic body formation. Coexpression of p53 with a baculovirus inhibitor of apoptosis, p35, OpIAP, or CpIAP, blocked apoptosis, whereas coexpression with p33 enhanced p53-mediated apoptosis approximately twofold. Expression of p53 in SF-21 cells stably expressing OpIAP inhibited cell growth in the presence or absence of p33. Thus, human p53 can influence both insect cell growth and death and baculovirus p33 can modulate the death-inducing effects of p53.     

The role of the plasma membrane fluidity on the shear sensitivity of hybridomas grown under hydrodynamic stress

The role of the plasma membrane fluidity (PMF) on the shear sensitivity of HB-32 hybridomas to laminar fluid shear was investigated. Steady-state fluorescence anisotropy (r(s)) of the cationic fluorescent probe 1-[4-(trimethylamino) phenyl]-6-phenylhexa-1,3,5-triene, was used to evaluate the PMF of whole hybridoma cells. The PMF was manipulated by the addition of the anesthetic benzyl alcohol, by temperature changes and by cholesterol enrichment. The effect of these PMF modifying procedures on the shear sensitivity of HB-32 was assessed by exposing the cells to defined levels of laminar shear stress in a Couette flow device. Conditions that resulted in lower r(s) values (indicating higher PMF) yielded a more fragile cell. Batch cultivations supplemented with the shear protective agent Pluronic(R) F-68 showed higher values of r(s) compared to control experiments during various growth phases, suggesting that the protective mechanism of Pluronic F-68 relies on its ability to decrease the PMF through direct interaction with the plasma membrane. The protective mechanism of serum against turbulent fluid shear is also discussed from analysis of growth and death kinetics of agitated and static cultures at increasing serum levels. The results of this study show that the fluid state of the plasma membrane is important in determining the integrity of hybridomas when exposed to lethal shear levels. It is concluded that increasing membrane fluidity correlates with increasing shear sensitivity.