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[Application of autologous and allogenic keratynocyte cultures in burn management].

First attempts to culture keratinocytes with intention of treating burn wounds started in the 19th century (Reverdin). Fast development of tissue engineering was connected to cell culture technology improvement in the fifties. Tissue culture of isolated keratinocytes was introduced by J.G. Rheinwald and H. Green. This method was modificated many times but is still used. Autologous keratinocytes culture was applied to burn management in 1981. Primary cell cultures became more popular in burn treatment. "Cell factories" and bioreactors are used to enhance proliferation to obtain huge amounts of cells in a short time. Cells are also seeded on matrixes made from glicosamonogycans, collagen, elastin or fibrin. These products are used as a skin substitutes.

Burns↗

Generation of a packaging cell line for prolonged large-scale production of high-titer HIV-1-based lentiviral vector.

BACKGROUND: A stable packaging cell line facilitates large-scale lentivirus vector manufacture. However, it has been difficult to produce clinical-scale HIV-1-based lentiviral vectors using a packaging cell line, in part due to toxicity of packaging genes, and gene silencing that occurs during the long culture period necessary for sequential addition of packaging constructs. METHODS: To avoid these problems, we developed a three-level cascade gene regulation system designed to remove tetracycline transactivator (tTA) from cytomegalovirus immediate early promoter (CMV)-controlled expression to reduce cytotoxicity from constitutive expression of tTA and leaky expression of packaging genes. We also performed a one-step integration of the three packaging plasmids to shorten the culture time for clonal selection. RESULTS: Although leaky expression of p24 and vector production still occurred despite the three-level regulation system, little cytotoxicity was observed and producer cells could be expanded for large-scale production. Producer cells yielded remarkably stable vector production over a period greater than 11 days with the highest titer 3.5 x 10(7) transducing units (TU)/ml and p24 300 ng/ml, yielding 2.2 x 10(11) TU and 1.8 milligram (mg) p24 from one cell factory. No replication-competent lentivirus (RCL) was detected. Long-term analysis demonstrated that, although the cells are genetically stable, partial gene silencing occurs after 2-3 months in culture; however, the one-step construct integration allowed prolonged vector production before significant gene silencing. Concentrated vector resulted in 90% transduction in CD4+ lymphocytes at 20 TU per cell. CD34+ progenitor cells were transduced at 41-46% efficiency, and long-term initiating culture (LTC-IC) was transduced at 45-51%. CONCLUSIONS: These results demonstrate for the first time HIV-1-based lentiviral vector production on the large scale using a packaging cell line.

Base Sequence↗

Propagation and assay of hepatitis A virus in vitro.

Ten strains of hepatitis A virus (HAV) originating from far distant geographical locations were adapted to growth in PLC/PRF/5 (human hepatoma derived and/or MRC-5 (human embryonic lung) cells. In the course of primary adaptation some of these strains exhibited a predilection for distinct cultural conditions such as type of host cell and temperature of incubation. With progressive passage, variant viruses with quite different requirements could be selected; yet, it proved impossible to isolate a virus which replicated equally well in both types of cells and at both 32 and 37 degrees C without at least one preceding passage under the new conditions. Analysis of the virus/cell relationship of well adapted HAV strains revealed that the replication cycle of HAV extends over about 24 h. Moreover, replication evidently passes from a state of active production of infectious virus to a phase during which hepatitis A antigen (HAAg) is synthesized and terminates in the state of persistent infection with markedly reduced synthetic activity. In all three phases replication of HAV is non-cytolytic and the vast majority of both infectious virus and of HAAg remains cell associated. The observations concerning the growth characteristics of HAV were used to develop two rapid in vitro assay systems for HAV infectivity (fluorescent focus assay and in situ RIA). Finally, the conditions for large scale production of infectious HAV and of HAAg in a cell factory system were analysed.

Adaptation, Physiological↗

Microencapsulation of cells producing therapeutic proteins: optimizing cell growth and secretion.

Microencapsulation of genetically engineered cells may have important applications as delivery systems for therapeutic proteins. However, optimization of the microcapsules with regard to mechanical stability, cell growth, and secretion of proteins is necessary in order to evaluate the future use of this delivery technology. We have explored the growth, survival, and secretion of therapeutic proteins from 293-EBNA cells producing endostatin (293 endo cells) and JJN3 myeloma cells producing hepatocyte growth factor (HGF) that have been embedded in various types of alginate capsules. Parameters that affect capsule integrity such as homogenous and inhomogenous gel cores and addition of an outer poly-L-lysine (PLL)-alginate coating were evaluated in relation to cell functions. When cells were encapsulated, the PLL layer was found to be absolutely required for the capsule integrity. The JJN3 and 293 endo cells displayed completely different growth and distribution patterns of live and dead cells within the microcapsules, as shown by 3D pictures reconstructed from images taken with confocal laser scanning microscopy (CLSM). Encapsulated JJN3 cells showed a bell-shaped growth and HGF secretion curve over a time period of 5 months. The 293 endo cells reached a plateau phase in growth after 23 days postencapsulation; however, after around 30 days a fraction of the microcapsules started to disintegrate. Microcapsule disintegration occurred with time irrespective of capsule and cell type, showing that alginate microcapsules possessing relatively high gel strength are not strong enough to keep proliferating cells within the microcapsules for prolonged time periods. Although this study shows that the stability of an alginate-based cell factory can be increased by a PLL-alginate coating, further improvement is necessary with regard to capsule integrity as well as controlling the cell growth before this technology can be used for therapy.

Alginates↗

Programmable in vivo mRNA circularization for enhanced gene expression in bacteria.

The minute-scale lifetime of mRNA strongly influences bacterial gene expression, whereas a robust and programmable approach to directly control the mRNA stability and topology remains elusive. Here, we develop CRESEnT (Circular RNA Expression for Stable and Enhanced Translation), a programmable in vivo mRNA circularization system based on a permuted intron-exon architecture to engineer mRNA topology. CRESEnT enables facile circularization of mRNA, which led to a substantial increase in protein expression across diverse promoters, RBS variants, genetic cargos, and bacterial hosts. Furthermore, application of CRESEnT to biosynthetic pathways increased the production of several value-added metabolites, demonstrating that mRNA circularization can be harnessed to improve the metabolic performance of microbial cell factories. Together, these results establish RNA topology engineering via circularization as a transformative axis for controlling bacterial gene expression and enhancing the functionality of microbial cells.

RNA, Messenger↗

Unraveling cadaverine toxicity effect to guide the engineering of robust strain.

End-product inhibition represents a major challenge in the microbial synthesis of various value-added chemicals. Cadaverine, a key monomer for polyamide synthesis, exhibits severe cytotoxicity, limiting its high-titer biosynthesis. Here, transcriptomic analysis and genome-wide library screening were integrated to systematically elucidate the cytotoxic mechanisms of cadaverine in Escherichia coli (E. coli) and identify beneficial genes for enhanced tolerance and overproduction. Transcriptomic analysis revealed that high concentrations of cadaverine disrupted cell membrane integrity and impaired oxidative phosphorylation, leading to redox imbalance and reactive oxygen species (ROS) accumulation. Subsequent genome-wide screening further confirmed these toxicity mechanisms and uncovered crucial cellular defense strategies. Functional validation highlighted the important role of NikR, UbiE, and YcbX in enhancing membrane integrity, restoring respiratory function and ROS homeostasis, or scavenging 6-N-hydroxylaminopurine (6-HAP) to prevent DNA damage. Among these, YcbX emerged as the most effective target for improving production. Consequently, we constructed a robust E. coli strain by implementing a dynamic regulation system for YcbX expression under cadaverine-responsive promoters, which significantly enhanced cadaverine biosynthesis to 87.2 g/L (a 46.8% enhancement). This work provides an in-depth understanding of cadaverine toxicity and tolerance, offering valuable targets and strategies for the rational design of high-performance microbial cell factories for diamines.

6-HAP clearance↗

A versatile adeno-associated virus vector producer cell line method for scalable vector production of different serotypes.

Application of adeno-associated virus (AAV) vector in large animal studies and clinical trials often requires high-titer and high-potency vectors. A number of currently used vector production methods, based on either transient transfection or helper virus infection of cell lines, have their advantages and limitations. We previously developed a 293-cell-based producer cell line method for high-titer and high-potency AAV2 vectors. Similar to several other methods, however, it requires multiple cloning steps for the vector and packaging plasmids and a two-step transfection and selection for stable cell lines. Here we report a simplified method with several key improvements and advantages: (1) a one-step cloning of AAV vector cassette into the serotype-specific packaging plasmid; (2) a single plasmid transfection and selection for stable AAV vector producer cell lines; (3) high vector yields of different serotypes, e.g., AAV2, 8, and 9, upon infection with an E1A/E1B-deleted helper adenovirus; (4) efficient packaging of both single-stranded and double-stranded (self-complementary) AAV vectors; and (5) efficient packaging of large AAV cassettes such as a mini-dystrophin vector (5.0 kb). All cell lines were stable with growth rates identical to the parental 293 cells. The vector yields were consistent among serotypes, with 5 × 10(13) to 8 × 10(13) vector genome particles per Nunc cell factory (equivalent to 40 15-cm plates). The vectors showed high potency for in vitro and in vivo transduction. In conclusion, the simple and versatile AAV producer cell line method can be useful for large scale AAV vector production in preclinical and clinical studies.

Biotechnology↗

Permeability issues in whole-cell bioprocesses and cellular membrane engineering.

Nutrient uptake and waste excretion are among the many important functions of the cellular membrane. While permitting nutrients into the cell, the cellular membrane system evolves to guide against noxious agents present in the environment from entering the intracellular milieu. The semipermeable nature of the membrane is at odds with biomolecular engineers in their endeavor of using microbes as cell factory. The cellular membrane often retards the entry of substrate into the cellular systems and prevents the product from being released from the cellular system for an easy recovery. Consequently, productivities of whole-cell bioprocesses such as biocatalysis, fermentation, and bioremediations are severely compromised. For example, the rate of whole-cell biocatalysis is usually 1-2 orders of magnitude slower than that of the isolated enzymes. When product export cannot keep pace with the production rate, intracellular product accumulation quickly leads to a halt of production due to product inhibition. While permeabilization via chemical or physical treatment of cell membrane is effective in small-scale process, large-scale implementation is problematic. Molecular engineering approach recently emerged as a much better alternative. Armed with increasingly sophisticated tools, biomolecular engineers are following nature's ingenuity to derive satisfactory solutions to the permeability problem. This review highlights these exciting molecular engineering achievements.

Biodegradation, Environmental↗

Manipulation of malic enzyme in Saccharomyces cerevisiae for increasing NADPH production capacity aerobically in different cellular compartments.

The yeast Saccharomyces cerevisiae is an attractive cell factory, but in many cases there are constraints related with balancing the formation and consumption of redox cofactors. In this work, we studied the effect of having an additional source of NADPH in the cell. In order to do this, two strains were engineered by overexpression of malic enzyme. In one of them, malic enzyme was overexpressed as its wild-type mitochondrial form, and in the other strain a short form lacking the mitochondrial targeting sequence was overexpressed. The recombinant strains were analyzed in aerobic batch and continuous cultivations, and the basic growth characteristics were generally not affected to a great extent, even though pleiotropic effects of the manipulations could be seen by the altered in vitro activities of selected enzymes of the central metabolism. Moreover, the decreased pentose-phosphate pathway flux and the ratios of redox cofactors showed that a net transhydrogenase effect was obtained, which can be directed to the cytosol or the mitochondria. This may find application in redirecting fluxes for improving specific biotechnological applications.

Aerobiosis↗

Nonperfused attachment systems for cell cultivation.

The pressing need for large-scale culture methods has prompted efforts to develop vessels which will accommodate the growth of large numbers of anchorage-dependent cells. The large-scale "cell factories" have been used to grow viruses for vaccine production, nucleic acid studies, and various cancer research projects. Two particular types of culture vessels used for large-scale production of anchorage-dependent cells were discussed and examples of their use in vaccine production given. Development of these large-scale culture systems has enabled the pharmaceutical companies to (1) meet the ever-increasing demands worldwide for vaccine, (2) employ a production process that produces a cost-efficient vaccine product in cell culture, and (3) produce large volumes of bulk vaccine at a single campaign, thus allowing multiple usage of a single production unit over the course of a year. The type of large-scale culture vessel used depends on the purpose of the culture and the type of cells that are to be grown in the vessel. The unit process vessel, with its multidisks, provides superior surface area, but because of the stainless steel housing, the cells cannot be monitored microscopically. Additionally, the unit process vessel can be equipped with a jacket for precise temperature regulation and thus eliminate the need for incubators. The roller bottles, on the other hand, allow microscopic monitoring of the growing cells but are limited in surface area available for growth, and the volume of harvest fluid obtainable from a bottle is very limited. It should be noted that handling of roller bottles is labor-intensive and requires many more manipulations and hence more personnel than employing unit process-type vessels for vaccine-manufacturing operations. The use of mass cultivation techniques in cancer research has also been of immense benefit. The production of large numbers of neoplastic cells has provided researchers with the raw materials to perform molecular biology experiments such as DNA sequencing, which required milligram quantities of cells before sufficient quantities of DNA could be isolated to allow sequencing studies. Additionally, the technique of mass cultivation also provided sufficient quantities of cells for inoculation into either nude mice or immunosuppressed hamsters in an attempt to determine the tumor induction capacity of the cultured cells. Additional characterization studies which require large numbers of cell, enzyme, and isoenzyme profiles can also be performed on cells grown in roller bottle cultures. There are numerous factors which limit the productivity of mass cultivation systems and the scientist should be aware of their existence.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

How reliable are thermodynamic feasibility statements of biochemical pathways?

The driving force for organo- or lithotrophic growth as well as for each step in the metabolic network is the Gibbs reaction energy. For each enzymatic step it must be negative. Thermodynamics contributes therefore to the in-silico description of living systems. It may be used for assessing the feasibility of a given pathway because it provides a further constraint for those pathways which are feasible from the point of view of mass balance calculations (metabolic flux analysis) and the genetic potential of an organism. However, when this constraint was applied to lactic acid fermentation according to a method proposed by Mavrovouniotis (1993a, ISMB 93:273-283) it turned out that an unrealistically wide metabolite concentration range had to be assumed to make this well-known glycolytic pathway thermodynamically feasible. During a search for the reasons of this surprising result the insufficient consideration of the activity coefficients was identified as main cause. However, it is shown in the present contribution that the influence of the activity coefficients on Gibbs reaction energy can be easily taken into account based on the intracellular ionic strength. The uncertainty of the tabulated equilibrium constants and of the apparent standard Gibbs energies derived from them was found to be the second most important reason for the erroneous result of the feasibility analysis. Deviations of intracellular pH from the standard value and bad estimations of currency metabolites, e.g., NAD(+) and NADH, were found to be of lesser importance but not negligible. The pH dependency of Gibbs reaction enthalpy was proved to be easily taken into account. Therefore, the application of thermodynamics for a better in-silico prediction of the behavior of living cell factories calls predominantly for better equilibrium data determined under well defined conditions and also for a more detailed knowledge about the intracellular ionic strength and pH value.

Cell Physiological Phenomena↗

Cloning and expression of alpha-amylase from the hyperthermophilic archaeon Pyrococcus woesei in the moderately halophilic bacterium Halomonas elongata.

An extracellular alpha-amylase gene from the hyperthermophilic archaeon Pyrococcus woesei has been cloned and sequenced. The 1.4-kb protein-coding sequence is identical to that of the corresponding alpha-amylase gene of the closely related species P. furiosus. By using a shuttle cloning vector for halophilic bacteria, the P. woesei alpha-amylase was expressed in the moderate halophile Halomonas elongata, under the control of a native H. elongata promoter. The hyperthermophilic amylase activity expressed in the halophilic host was recovered completely in the crude membrane fraction of cell homogenates, suggesting the formation of inclusion bodies or that the secretion machinery of H. elongata may fail to recognize and release the pyrococcal alpha-amylase to the extracellular medium. However, thermal stability, metal ion interactions, optimal temperature and pH values for the crude and purified recombinant alpha-amylase were comparable with those of the native pyrococcal enzyme. The P. woesei amylase activity expressed in H. elongata was consistently detected in the cells upon growth on a wide range of NaCl concentrations (0.7-2.5 mol l-1). To our knowledge, this is the first report on the expression of an archaeal gene (P. woesei alpha-amylase) in a moderate halophilic host which serves as a cell factory able to grow under extreme salt conditions and with very simple nutritional requirements.

Base Sequence↗

Partial purification and characterization of the soluble glutathione transferase isoenzymes from cultured Hep G2 cells.

Hep G2 cells, an established cell line derived from a human hepatoma, were mass cultured in a cell factory for the isolation of glutathione transferase isoenzymes. These were enriched by affinity chromatography and separated in an anionic and a cationic fraction. They were partially characterized by different kinetic and inhibition parameters. Three different subunits were observed. The results were compared with human liver data. It is concluded that Hep G2 cells can be considered as a valuable alternative tool for in vitro research of human liver phenomena, especially when toxicological interactions are investigated.

Carcinoma, Hepatocellular↗

Comparison of two apheresis systems for the collection of CD14+ cells intended to be used in dendritic cell culture.

BACKGROUND: Monocytes collected by leukapheresis are increasingly used for dendritic cell (DC) culture in cell factories suitable for DC vaccination in cancer. STUDY DESIGN AND METHODS: Using modified MNC programs on two apheresis systems (Cobe Spectra and Fresenius AS.TEC204), leukapheresis components collected from 84 patients with metastatic malignant melanoma and from 31 healthy male donors were investigated. MNCs, monocytes, RBCs, and platelets (PLTs) in donors and components were analyzed by cell counters, WBC differential counts, and flow cytometry. RESULTS: In 5-L collections, Astec showed better results regarding monocyte collection rates (11.0 vs. 7.4 x 10(6)/min, p = 0.04) and efficiencies (collection efficiency, 51.9 vs. 31.9%; p < 0.001). Both devices resulted in monocyte yields at an average of 1 x 10(9) (donors) and 2.5 x 10(9) (patients), whereas Astec components contained high residual RBCs. Compared to components with low residual PLTs, high PLT concentration resulted in higher monocyte loss (48 vs. 20%, p < 0.0001) before DC culture. CONCLUSION: The Astec is more efficient in 5-L MNC collections compared to the Spectra. Components with high residual PLTs result in high MNC loss by purification procedures. Thus, optimizing MNC programs is essential to obtain components with high MNC yields and low residual cells as prerequisite for high DC yields.

Adult↗

Stabilizing plasmid copy number to improve recombinant protein production.

The key objective for recombinant protein production in bacteria is the maximum exploitation of the cell factory's potential, whereby often strong expression vectors are used to increase product yield. If the metabolic load caused by recombinant expression exceeds the host's capacity, the system exhausts itself, resulting in a loss of protein yield. Excessive plasmid replication is observed after inducing recombinant gene expression, which greatly contributes to metabolic overload of the host cell. The transcriptional and translational machineries are extremely overstrained. By abolishing sequence homology between ColE1 RNA I/RNA II and tRNAs, we were able to restore the plasmid's replication control mechanisms and to keep the plasmid copy number constant throughout the culture process, thereby prolonging metabolic activity and productivity of the bacterial expression system. Because the bacterial host cell is not being exploited beyond its tolerable potential with this method, the constancy of the plasmid copy number level throughout the whole period of the bioprocess provides novel strategies for bioprocess optimization.

Bacterial Proteins↗

Immortalized feeders for the scale-up of human embryonic stem cells in feeder and feeder-free conditions.

Human embryonic stem cells (hESC) are pluripotent cells that proliferate indefinitely in culture, whilst retaining their capacity for differentiation into different cell types. However, hESC cultures require culture in direct contact with feeder cells or conditioned medium (CM) from feeder cells. The most common source of feeders has been primary mouse embryonic fibroblast (MEF). In this study, we immortalized a primary MEF line with the E6 and E7 genes from HPV16. The immortal line, DeltaE-MEF, was able to proliferate beyond 7-9 passages and has an extended lifespan beyond 70 passages. When tested for its ability to support hESC growth, it was found that hESC continue to maintain the undifferentiated morphology for >40 passages both in co-culture with DeltaE-MEF and in feeder-free cultures supplemented with CM from DeltaE-MEF. The cultures also continue to express the pluripotent markers, Oct-4, SSEA-4, Tra-1-60, Tra-1-81, alkaline phosphatase and maintain a normal karyotype. In addition, these hESC formed teratomas when injected into SCID mice. Lastly, we demonstrated the feasibility of scaling-up significant quantities of undifferentiated hESC (>10(8) cells) using DeltaE-MEF in cell factories. The results from this study suggest that immortalized feeders can provide a consistent and reproducible source of feeders for hESC expansion and research.

Animals↗

Safety, tolerability and immunogenicity of a formalin-inactivated hepatitis A vaccine (VAQTA) in rural Kentucky children.

This study evaluated the immunogenicity and safety/tolerability profile of an investigational formalin-inactivated hepatitis A virus vaccine (VAQTA; Merck Research Laboratories) in 150 seronegative healthy children, 4 to 12 years old. The vaccine was derived from virus grown in infected MRC-5 cells in either roller bottles or Nunc cell factories (Nunc, Denmark). Subjects were vaccinated intramuscularly in a two dose regimen initially and at 24 weeks: Group A (n = 50) with a 12-unit dose from a roller bottle lot; Group B (n = 50) with a 25-unit dose from another roller bottle lot; and Group C (n = 50) with a 25-unit dose from a Nunc cell lot. Sera for anti-hepatitis A virus antibodies were drawn 3 weeks before vaccination and 4, 24 and 28 weeks after the first dose. Seroconversion from < 10 mIU/ml to > or = 10 mIU/ml by modified HAVAB (Abbott Laboratories) was observed in 99% of subjects at week 4 and persisted in 100% of subjects at week 28 (4 weeks after the second dose). The ranges of geometric mean titers of anti-HAV for all subjects at weeks 4, 24 and 28 were 31 to 49, 51 to 79 and 7059 to 29,609 mIU/ml, respectively. The 12- and 25-unit dose levels of roller bottle yielded similar geometric mean titers. The rise in geometric mean titers after the booster dose was > 120-fold and was highest in the recipients of the 25-unit Nunc cell lot (P < 0.05 for Group C vs. B).(ABSTRACT TRUNCATED AT 250 WORDS)

Child↗