Microtubule-mediated and microtubule-independent transport of adenovirus type 5 in HEK293 cells

Adenovirus serotypes 2 and 5 are taken into cells by receptor-mediated endocytosis, and following release from endosomes, destabilized virions travel along microtubules to accumulate around the nucleus. The entry process culminates in delivery of the viral genome through nuclear pores. This model is based on studies with conventional cell lines, such as HeLa and HEp-2, but in HEK293 cells, which are routinely used in this laboratory because they are permissive for replication of multiple adenovirus serotypes, a different trafficking pattern has been observed. Nuclei of 293 cells have an irregular shape, with an indented region, and virions directly labeled with carboxyfluorescein accumulate in a cluster within that indented region. The clusters, which form in close proximity to the microtubule organizing center (MTOC) and to the Golgi apparatus, are remarkably stable; a fluorescent signal can be seen in the MTOC region up to 16 h postinfection. Furthermore, if cells are infected and then undergo mitosis after the cluster is formed, the signal is found at each spindle pole. Despite the sequestration of virions near the MTOC, 293 cells are no less sensitive than other cells to productive infection with adenovirus. Even though cluster formation depends on intact microtubules, infectivity is not compromised by disruption of microtubules with either nocodazole or colchicine, as determined by expression of an enhanced green fluorescent protein reporter gene inserted in the viral genome. These results indicate that virion clusters do not represent the infectious pathway and suggest an alternative route to the nucleus that does not depend on nocodazole-sensitive microtubules.     

Hyperosmotic pressure on HEK 293 cells during the growth phase, but not the production phase, improves adenovirus production

Hyperosmotic stress has been widely explored as a means of improving specific antibody productivity in mammalian cell cultures. In contrast, a decrease in cell-specific productivity of adenovirus production has been reported in several studies in which virus production in HEK 293 cell cultures was conducted under hyperosmotic conditions. However, production of viral vectors and, in particular, adenoviral vectors is the result of two consecutive phases: the growth phase and the virus production phase. In this study, the singular and combined effects of osmolality on the phases of cell growth and virus production were evaluated in culture media with osmolalities ranging from 250 to 410 mOsm. A two-factor, five-level full factorial design was used to investigate the effect of osmotic stress on cell physiology, as determined through the characterization of cell growth, cell metabolism, cell viability, cell cycle, cell RNA and total protein content, and total virus yield/cell-specific virus productivity. Overall, the results show that the growth of cells under hyperosmotic conditions induced favorable physiological states for viral production, and the specific virus productivity was improved by more than 11-fold when the medium's osmolality was increased from 250 to 410 mOsm during the cell growth phase. Both hypo- and hyperosmotic stresses in the virus production phase reduced virus productivity by as much as a factor of six. Optimal virus productivity was achieved by growing cells in media with an osmolality of 370 mOsm or greater, followed by a virus production phase at an osmolality of 290 mOsm. Compared to standard culture and production conditions in isotonic media, the shift from high to low osmolality between the two phases resulted in a two- to three-fold increase in virus yields. This hyperosmotic pressure effect on virus productivity was reproduced in five different commercial serum-free media.     

Stable high volumetric production of glycosylated human recombinant IFNalpha2b in HEK293 cells

Background

Mammalian cells are becoming the prevailing expression system for the production of recombinant proteins because of their capacity for proper protein folding, assembly, and post-translational modifications. These systems currently allow high volumetric production of monoclonal recombinant antibodies in the range of grams per litre. However their use for large-scale expression of cytokines typically results in much lower volumetric productivity.

Results

We have engineered a HEK293 cell clone for high level production of human recombinant glycosylated IFNα2b and developed a rapid and efficient method for its purification. This clone steadily produces more than 200 mg (up to 333 mg) of human recombinant IFNα2b per liter of serum-free culture, which can be purified by a single-step cation-exchange chromatography following media acidification and clarification. This rapid procedure yields 98% pure IFNα2b with a recovery greater than 70%. Purified IFNα2b migrates on SDS-PAGE as two species, a major 21 kDa band and a minor 19 kDa band. N-terminal sequences of both forms are identical and correspond to the expected mature protein. Purified IFNα2b elutes at neutral pH as a single peak with an apparent molecular weight of 44,000 Da as determined by size-exclusion chromatography. The presence of intramolecular and absence of intermolecular disulfide bridges is evidenced by the fact that non-reduced IFNα2b has a greater electrophoretic mobility than the reduced form. Treatment of purified IFNα2b with neuraminidase followed by O-glycosidase both increases electrophoretic mobility, indicating the presence of sialylated O-linked glycan. A detailed analysis of glycosylation by mass spectroscopy identifies disialylated and monosialylated forms as the major constituents of purified IFNα2b. Electron transfer dissociation (ETD) shows that the glycans are linked to the expected threonine at position 106. Other minor glycosylated forms and non-sialylated species are also detected, similar to IFNα2b produced naturally by lymphocytes. Further, the HEK293-produced IFNα2b is biologically active as shown with reporter gene and antiviral assays.

Conclusion

These results show that the HEK293 cell line is an efficient and valuable host for the production of biologically active and glycosylated human IFNα2b.     

Transient gene expression: recombinant protein production with suspension-adapted HEK293-EBNA cells

Transient gene expression (TGE) in mammalian cells at the reactor scale is becoming increasingly important for the rapid production of recombinant proteins. We improved a process for transient calcium phosphate-based transfection of HEK293-EBNA cells in a 1-3 L bioreactor volume. Cells were adapted to suspension culture using a commercially available medium (BioWhittaker, Walkersville, MD). Process parameters were optimized using a plasmid reporter vector encoding the enhanced green fluorescent protein (EGFP/CLONTECH, Palo Alto, CA, USA). Using GFP as a marker-protein, we observed by microscopic examination transfection efficiencies between 70-100%. Three different recombinant proteins were synthesized within a timeframe of 7 days from time of transfection to harvest. The first, a human recombinant IgG(1)-type antibody, was secreted into the supernatant of the cell culture and achieved a final concentration of >20 mg/L. An E. coli-derived DNA-binding protein remained intracellular, as expected, but accumulated to such a concentration that the lysate of cells, taken up into the entire culture volume, gave a concentration of 18 mg/L. The third protein, a transmembrane receptor, was expressed at 3-6 x 10(6) molecules/cell.     

Effects of elevated cytoplasmic calcium and protein kinase C on endoplasmic reticulum structure and function in HEK293 cells

In human embryonic kidney (HEK) cells stably transfected with green fluorescent protein targeted to the endoplasmic reticulum (ER), elevation of intracellular Ca2+ ([Ca2+]i) altered ER morphology, making it appear punctate. Electron microscopy revealed that these punctate structures represented circular and branched rearrangements of the endoplasmic reticulum, but did not involve obvious swelling or pathological fragmentation. Activation of protein kinase C with phorbol 12-myristate 13-acetate (PMA), prevented the effects of ionomycin on ER structure without affecting the elevation of [Ca2+]i. These results suggest that protein kinase C activation alters cytoplasmic or ER components underlying the effects of high [Ca2+]i on ER structure. Treatment of HEK cells with PMA also reduced the size of the thapsigargin-sensitive Ca2+ pool and inhibited Ca2+ entry in response to thapsigargin. Thus, protein kinase C activation has multiple actions on the calcium storage and signalling function of the endoplasmic reticulum in HEK cells: (1) reduced intracellular Ca2+ storage capacity, (2) inhibition of capacitative Ca2+ entry, and (3) protection of the endoplasmic reticulum against the effects of high [Ca2+]i.     

Effects of Wnt proteins on cell proliferation and apoptosis in HEK293 cells

Wnt proteins and Wnt signalings have been implicated in a variety of development and cell processes, while aberrant activation of Wnt signaling is linked to a range of cancers in many tissues. In this study, we used the HEK293 cell line to investigate the effects of Wnt3a and Wnt5a on proliferation and apoptosis in a serum starvation culture. After Wnt3a and Wnt5a proteins were expressed, they both promoted the proliferation of HEK293 cells under serum starvation. After 48h of serum starvation, both Wnt3a and Wnt5a inhibited serum starvation-induced apoptosis of HEK293 cells and continued up to 96h. We demonstrated that Wnt3a and Wnt5a can promote proliferation of HEK293 cells and inhibit serum starvation-induced apoptosis, which implies that Wnt3a and Wnt5a can maintain the survival of HEK293 cells under stress, and also provide a novel insight into the role of Wnt3a and Wnt5a and their related signalings in carcinogenesis.     

Influence of culture passages on growth kinetics and adenovirus vector production for gene therapy in monolayer and suspension cultures of HEK 293 cells

To characterize the changes in cell growth rate and adenovirus vector (AdV) production capability of 293 cells during culture passages, 293 cells obtained at the 31st culture passage from ATCC (293M #31) were maintained as a monolayer culture and 293 cells obtained at an unknown culture passage from Invitrogen (293S) were maintained as suspension culture. In monolayer culture, the specific growth rate () of 293M cells increased rapidly with culture passage up to passage 65 and thereafter became saturated. The of 293M passage 43 (#43) was 0.29 day^–1, while the average of 293M from #66 to #86 was 0.74±0.01 day^–1 (average ± standard deviation). It was also noted that the cells became smaller in size during early culture passages. AdV production was also influenced by the number of culture passages. The AdV titer in the culture of 293M #66 was ca. tenfold higher than that of 293M #44, resulting from both a higher cell concentration and a higher AdV titer per cell at #66. In contrast, the , cell size, and AdV production of 293S cells in suspension culture did not change significantly as the culture passage number increased up to #40. Taken together, the culture passage influenced cell growth and AdV production of 293M cells in monolayer culture, but not those of 293S cells in suspension culture.     

Functional properties of endogenous receptor- and store-operated calcium influx channels in HEK293 cells

Activation of phospholipase C (PLC)-mediated signaling pathways in non-excitable cells causes the release of calcium (Ca2+) from inositol 1,4,5-trisphosphate (InsP3)-sensitive intracellular Ca2+ stores and activation of Ca2+ influx via plasma membrane Ca2+ channels. The properties and molecular identity of plasma membrane Ca2+ influx channels in non-excitable cells is a focus of intense investigation. In the previous studies we used patch clamp electrophysiology to describe the properties of Ca2+ influx channels in human carcinoma A431 cell lines. Now we extend our studies to human embryonic kidney HEK293 cells. By using a combination of Ca2+ imaging and whole cell and single channel patch clamp recordings we discovered that: 1) HEK293 cells contain four types of plasma membrane Ca2+ influx channels: I(CRAC), Imin, Imax, and I(NS); 2) I(CRAC) channels are highly Ca2+-selective (P(Ca/Cs)>1000) and I(CRAC) single channel conductance is too small for single channel analysis; 3) Imin channels in HEK293 cells display functional properties identical to Imin channels in A431 cells, with single channel conductance of 1.2 pS for divalent cations, 10 pS for monovalent cations, and divalent cation selectivity P(Ba/K)=20; 4) Imin channels in HEK293 cells are activated by InsP3 and inhibited by phosphatidylinositol 4,5-bisphosphate, but store-independent; 5) when compared with Imin, Imax channels have higher conductance for divalent (17 pS) and monovalent (33 pS) cations, but less selective for divalent cations (P(Ba/K)=4), 6) Imax channels in HEK293 cells can be activated by InsP3 or by Ca2+ store depletion; 7) I(NS) channels are non-selective (P(Ba/K)=0.4) and display a single channel conductance of 5 pS; and 8) I(NS) channels are not gated by InsP3 but activated by depletion of intracellular Ca2+ stores. Our findings provide novel information about endogenous Ca2+ channels supporting receptor-operated and store-operated Ca2+ influx pathways in HEK293 cells.     

诱导表达p27对HEK293细胞生长代谢的影响

目的:研究诱导表达p27对HEK293细胞生长和代谢的影响。方法:将pTet-on载体和响应于Dox的p27诱导表达载体共转染HEK293细胞,随机挑选单克隆细胞株。以细胞周期分布和活细胞密度为主要观察指标,考察稳定转染的细胞在Dox诱导下的细胞生长;以Qglc、Qlac和Qgln为主要观察指标,考察转染细胞在Dox诱导下的细胞代谢。结果:p27基因的表达使HEK293细胞的增殖速度显著降低,G1期细胞比例显著升高,葡萄糖消耗和乳酸生产减少。结论:诱导表达p27基因是对HEK293细胞进行G1期阻滞的一种有效策略。     

诱导表达p27对HEK293细胞生长代谢的影响

目的：研究诱导表达p27对HEK293细胞生长和代谢的影响。方法：将pTet—on载体和响应于Dox的p27诱导表达载体共转染HEK293细胞,随机挑选单克隆细胞株。以细胞周期分布和活细胞密度为主要观察指标,考察稳定转染的细胞在Dox诱导下的细胞生长;以Qglc、Qlac和Qgln为主要观察指标,考察转染细胞在Dox诱导下的细胞代谢。结：p27基因的表达使HEK293细胞的增殖速度显著降低,G1期细胞比例显著升高,葡萄糖消耗和乳酸生产减少。结论：诱导表达p27基因是对HEK293细胞进行G1期阻滞的一种有效策略。     

293 cell cycle synchronisation adenovirus vector production

As the market requirements for adenovirus vectors (AdV) increase, the maximisation of the virus titer per culture volume per unit time is a key requirement. However, despite the fact that 293 cells can grow up to 8 × 10⁶ cell/mL in simple batch mode operations, for optimal AdV infection a maximum cell density of 1 × 10⁶ cell/mL at infection time has usually been utilized due to the so called “cell density effect”. In addition, AdV titer appears to be dependent upon cell cycle phase at the time of infection. To evaluate the dependence of AdV production upon cell cycle phase, 293 cells were chemically synchronised at each phase of the cell cycle; a 2.6‐fold increase on AdV cell specific titer was obtained when the percentage of cells at the S phase of the cell cycle was increased from 36 to 47%; a mathematical equation was used to relate AdV cell specific productivities with cell synchronisation at the S phase using this data. To avoid the use of chemical inhibitors, a temperature shift strategy was also used for synchronisation at the S phase. S phase synchronisation was obtained by decreasing the culture temperature to 31°C during 67 h and restoring it to 37°C during 72 h. By using this strategy we were able to synchronise 57% of the population in the S phase of the cell cycle obtaining an increase of 7.3‐fold on AdV cell specific titer after infection. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Low-glutamine fed-batch cultures of 293-HEK serum-free suspension cells for adenovirus production

Recent developments in gene therapy using adenoviral (Ad) vectors have fueled renewed interest in the 293 human embryonic kidney cell line traditionally used to produce these vectors. Low-glutamine fed-batch cultures of serum-free, suspension cells in a 5-L bioreactor were conducted. Our aim was to tighten the control on glutamine metabolism and hence reduce ammonia and lactate accumulation. Online direct measurement of glutamine was effected via a continuous cell-exclusion system that allows for aseptic, cell-free sampling of the culture broth. A feedback control algorithm was used to maintain the glutamine concentration at a level as low as 0.1 mM with a concentrated glucose-free feed medium. This was tested in two media: a commercial formulation (SFM II) and a chemically defined DMEM/F12 formulation. The fed-batch and batch cultures were started at the same glucose concentration, and it was not controlled at any point in the fed-batch cultures. In all cases, fed-batch cultures with double the cell density and extended viable culture time compared to the batch cultures were achieved. An infection study on the high density fed-batch culture using adenovirus-green fluorescent protein (Ad-GFP) construct was also done to ascertain the production capacity of the culture. Virus titers from the infected fed-batch culture showed that there is an approximately 10-fold improvement over a batch infection culture. The results have shown that the control of glutamine at low levels in cultures is sufficient to yield significant improvements in both cell densities and viral production. The applicability of this fed-batch system to cultures in different media and also infected cultures suggests its potential for application to generic mammalian cell cultures.     

Effect of re-feed strategies and non-ammoniagenic medium on adenovirus production at high cell densities

Recombinant adenoviruses became one of the vectors of choice for delivery and expression of foreign proteins for gene therapy and vaccination purposes. Nevertheless, the production of adenovirus is currently limited by the so-called "cell density effect", i.e., a drop in cell specific productivity concomitant with increased cell concentration at infection (CCI). This work describes the characterisation and optimisation of the infection process in order to improve recombinant adenovirus type 5 yields at high cell densities. For that purpose, 293 cells adapted to suspension were grown in 2l bioreactors and infected at different cell concentrations, using different re-feed strategies, while evaluating cell metabolism. The consumption of amino acids is enhanced during infection, although no amino acid limitation was detected for cells infected at concentrations in the range of 2 x 10(6)cell/ml, for which the highest volumetric productivity was obtained in batch mode. Conversely, infecting at cell concentrations in the range of 3 x10(6)cell/ml led to complete depletion of glucose, glutamine and threonine before the optimal harvesting time, a significant decrease in volumetric productivity being observed; the effect of amino acids and glucose addition at infection time on cell specific and volumetric productivity of adenovirus was assessed, no improvement on adenovirus production being achieved. The effect of ammonia, present in high concentrations at 3 x10(6)cell/ml, was evaluated and seem to be detrimental; an 1.8-fold increase on adenovirus volumetric productivity was obtained for infections performed at 3 x10(6)cell/ml when non-ammoniagenic medium was used.     

Immobilization of 293 cells using porous support particles for adenovirus vector production

Adenovirus vector production by anchorage-independent 293 cells immobilized using porous biomass support particles (BSPs) was investigated in static and shake-flask cultures for efficient large-scale production of adenovirus vectors for gene therapy applications. The density of cells immobilized within BSPs was evaluated by measuring their WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) reduction activity. In shake-flask culture, 293-F cells, which were adapted to serum-free suspension culture, were not successfully retained within reticulated polyvinyl formal (PVF) resin BSPs (2 × 2 × 2 mm cubes) with matrices of relatively small pores (pore diameter 60 μm). When the BSPs were coated with a cationic polymer polyethyleneimine, a high cell density of more than 10⁷ cells cm⁻³-BSP was achieved in both static and shake-flask cultures with regular replacement of the culture medium. After infection with an adenovirus vector carrying the enhanced green fluorescent protein gene (Ad EGFP), the specific Ad EGFP productivity of the immobilized cells was comparable to the maximal productivity of non-immobilized 293-F cells by maintaining favorable conditions in the culture environment.     

Transient transfection induces different intracellular calcium signaling in CHO K1 versus HEK 293 cells

For the controlled production of recombinant proteinsin mammalian cells by transient transfection, it maybe desirable not only to manipulate, but also todiagnose the expression success early. Here, weapplied laser scanning confocal microscopy to monitortransfection induced intracellular Ca²⁺responses. We compared Chinese hamster ovary (CHO K1)versus human embryo kidney (HEK) 293 cell lines, whichdiffer largely in their transfectability. An improvedcalcium phosphate transfection method was used for itssimplicity and its demonstrated upscale potential.Cytosolic Ca²⁺ signaling appeared to inverselyreflect the cellular transfection fate. Virtually allCHO cells exhibited asynchronous, cytosolicCa²⁺ oscillations, which peaked 4 h afteraddition of the transfecting solution. Yet, most ofthe HEK cells displayed a slow and continuousCa²⁺ increase over the time of transfection. CHOcells, when exposed to a transfection-enhancingglycerol shock, strongly downregulated their Ca²⁺response, including its oscillations. When treatedwith thapsigargin, a Ca²⁺ store depleting drug,the number of successfully transfected CHO cells was significantly reduced. Our result points tointracellular store release as a critical componentfor the transfection fate of CHO cells, and its early detection before product visualization.