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Scaffold materials should favor cell attachment and proliferation, and provide designable 3D structures with appropriate mechanical strength. Collagen matrices have proven to be beneficial scaffolds for tissue regeneration. However, apart from small intestinal submucosa, they offer a limited mechanical strength even if crosslinking can enhance their mechanical properties. A more cell-friendly way to increase material strength is to combine synthetic polymer meshes with plastic compressed collagen gels. This work describes the potential of plastic compressed collagen poly(lactic acid-co-epsilon-caprolactone) (PLAC) hybrids as scaffolds for bladder tissue regeneration. Human bladder smooth muscle and urothelial cells were cultured on and inside collagen PLAC hybrids in vitro. Scaffolds were analyzed by electron microscopy, histology, immunohistochemistry, and AlamarBlue assay. Both cell types proliferated in and on the hybrid, forming dense cell layers on top after two weeks. Furthermore, hybrids were implanted subcutaneously in the backs of nude mice. Host cell infiltration, scaffold degradation, and the presence of the seeded bladder cells were analyzed. Hybrids showed a lower inflammatory reaction in vivo than PLAC meshes alone, and first signs of polymer degradation were visible at six months. Collagen PLAC hybrids have potential for bladder tissue regeneration, as they show efficient cell seeding, proliferation, and good mechanical properties. (C) 2011 Elsevier Ltd. All rights reserved.

Three synthesis lots of linear poly(ethyleneimine) (PEI) are compared to a fully hydrolyzed linear PEI (commercially available as PEI Max) regarding structure, polyplex formation with plasmid DNA, and transfection of suspension-adapted HEK-293E cells. PEI Max binds DNA more efficiently than the other PEIs, but it is the least effective in terms of transient recombinant protein yield. One PEI lot is fractionated by means of SEC. The fractions of high-$\overline {M} _{{\rm n}} $ PEI are the most efficient for complex formation and transfection. Nevertheless, the highest transient recombinant protein yields are achieved with unfractionated PEI. The results demonstrate that the polydispersity and charge density of linear PEI are important parameters for gene delivery to suspension-adapted HEK-293E cells.

Orbitally shaken bioreactors are an emerging alternative to stirred-tank bioreactors for large-scale mammalian cell culture, but their fluid dynamics is still not well defined. Among the theoretical and practical issues that remain to be resolved, the characterization of the liquid free surface during orbital shaking remains a major challenge because it is an essential aspect of gas transfer and mixing in these reactors. To simulate the fluid behavior and the free surface shape, we developed a numerical method based on the finite element framework. We found that the large density ratio between the liquid and the gas phases induced unphysical results for the free surface shape. We therefore devised a new pressure correction scheme to deal with large density ratios. The simulations operated with this new scheme gave values of wave amplitude similar to the ones measured experimentally. These simulations were used to calculate the shear stress and to study the mixing principle in orbitally shaken bioreactors. Copyright (C) 2012 John Wiley & Sons, Ltd.

Alzheimer's disease (AD)-associated gamma-secretase is a ubiquitously expressed multi-subunit protease complex embedded in the lipid bilayer of cellular compartments including endosomes and the plasma membrane. Although gamma-secretase is of crucial interest for AD drug discovery, its atomic structure remains unresolved. Gamma-secretase assembly and maturation is a multistep process, which includes extensive glycosylation on nicastrin (NCT), the only g-secretase subunit having a large extracellular domain. These posttranslational modifications lead to protein heterogeneity that likely prevents the three-dimensional (3D) crystallization of the protease complex. To overcome this issue, we have engineered a Chinese hamster ovary (CHO) cell line deficient in complex sugar modifications (CHO lec1) to overexpress the four subunits of g-secretase as a functional complex. We purified glycosylation-deficient g-secretase from this recombinant cell line (CL1-9) and fully glycosylated g-secretase from a recombinant CHO DG44-derived cell line (SS20). We characterized both complexes biochemically and pharmacologically in vitro. Interestingly, we found that the complex oligosaccharides, which largely decorate the extracellular domain of fully glycosylated NCT, are not involved in the proper assembly and maturation of the complex, and are dispensable for the specific generation, in physiological ratios, of the amyloid precursor protein (APP) cleavage products. In conclusion, we propose a novel bioengineering approach for the production of functional glycosylation-deficient g-secretase, which may be suitable for crystallization studies. We expect that these findings will contribute both to solving the high-resolution 3D structure of g-secretase and to structure-based drug discovery for AD. Biotechnol. Bioeng. 2015;112: 2516-2526. (c) 2015 Wiley Periodicals, Inc.

The aim of this study was to gain a better understanding of orbitally shaken bioreactors (OSRs) operated without controllers for pH and dissolved oxygen (DO) concentration. We used cylindrical OSRs with working volumes ranging from 250mL to 200L to determine that the volumetric mass transfer coefficient of oxygen (k(L)a) is a good predictor of the performance of OSRs at different scales. We showed that k(L)a values of 7-10hour(-1) were required to avoid DO limitations and to prevent conditions of low pH during the cultivation of CHO cells. Overall, cell cultures in probe-independent OSRs of different nominal volumes ranging from 250mL to 200L achieved similar cell densities, recombinant protein concentrations, and pH and DO profiles when having the same k(L)a. We conclude that k(L)a is a key parameter for probe-independent bioprocesses in OSRs and can be used as a scale-up factor for their operation.

Human alpha 1-antitrypsin (AAT) is an abundant acute phase glycoprotein expressing anti-protease and immunomodulatory activities, and is used as a biopharmaceutical to treat patients with inherited AAT deficiency. The pleiotropic properties of AAT provide a rationale for using this therapy outside of inherited AAT deficiency. Therapy with AAT is administrated intravenously, yet the alternative routes are being considered. To examine the putative transepidermal application of AAT we used epiCS (R), the 3D human epidermis equivalents reconstructed from human primary epidermal keratinocytes. We topically applied various concentrations of AAT protein with a constant volume of 50 mu l, prepared in Hank's balance solution, HBSS, to epiCS cultured under bas\al condition or when culture medium supplemented with 100 mu g/ml of a combined bacterial lipopolysaccharide (LPS) and peptidoglycan (PGN) mixture. AAT freely diffused across epidermis layers in a concentration and time-dependent manner. Within 18 h topically provided 0.2 mg AAT penetrated well the stratum corneum and localizes within the keratinocytes. The treatments with AAT did not induce obvious morphological changes and damages in keratinocyte layers. As expected, LPS/PGN triggered a strong pro-inflammatory activation of epiCS. AAT exhibited a limited capacity to neutralize the effect of LPS/PGN, but more importantly, it lowered expression of IL-18 and IL-8, and preserved levels of filaggrin, a key protein for maintaining the epidermal barrier integrity. Our findings suggest that the transepidermal route for delivering AAT is worthwhile to explore further. If successful, this approach may offer an easy-to-use therapy with AAT for skin inflammatory diseases.

Orbitally shaken bioreactors (OSRs) are commonly used for the cultivation of mammalian cells in suspension. Here we conducted a three-dimensional computational fluid dynamics (CFD) simulation to characterize the fluid field in the disposable 600-mL orbitally shaken bioreactor (OSR600), basically a cylindrical vessel with a conical bottom and a ventilated cap. The CFD models established for the OSR600 were validated by visual comparison of the liquid flow pattern, in an experimentally agitated OSR600. In the model, both shear stress and energy dissipation rate (Phi) were calculated to evaluate the hydrodynamic stress environment for cell cultivation. The highest values of shear stress and Phi were localized along the lower part of the conical vessel wall. The effect of filling volume and shaking speed on k(L)a, Phi and shear stress were also analyzed. An increase of the percentage of the liquid affected by higher shear stress and Phi was observed at filling volumes of 300 mL and 400 mL compared to lower filling volumes. This may be due to the twisted curvature at the base of the liquid wave under these conditions. In conclusion, the CFD model provided a means to characterize the fluid dynamics of the OSR600 under various operating conditions to help identify those most suitable for cell cultivation. (C) 2017 Elsevier B.V. All rights reserved.

For the treatment of severe COVID-19, supplementation with human plasma-purified alpha-1 antitrypsin (AAT) to patients is currently considered. AAT inhibits host proteases that facilitate viral entry and possesses broad anti-inflammatory and immunomodulatory activities. Researchers have demonstrated that an interaction between SARS-CoV-2 spike protein (S) and lipopolysaccharides (LPS) enhances pro-inflammatory responses in vitro and in vivo. Hence, we wanted to understand the potential anti-inflammatory activities of plasma-derived and recombinant AAT (recAAT) in a model of human total peripheral blood mononuclear cells (PBMCs) exposed to a combination of CHO expressed trimeric spike protein and LPS, ex vivo. We confirmed that cytokine production was enhanced in PBMCs within six hours when low levels of LPS were combined with purified spike proteins ("spike"). In the presence of 0.5 mg/mL recAAT, however, LPS/spike-induced TNF-alpha and IL-1 beta mRNA expression and protein release were significantly inhibited (by about 46-50%) relative to LPS/spike alone. Although without statistical significance, recAAT also reduced production of IL-6 and IL-8. Notably, under the same experimental conditions, the plasma-derived AAT preparation Respreeza (used in native and oxidized forms) did not show significant effects. Our findings imply that an early pro-inflammatory activation of human PBMCs is better controlled by the recombinant version of AAT than the human plasma-derived AAT used here. Considering the increasing clinical interest in AAT therapy as useful to ameliorate the hyper-inflammation seen during COVID-19 infection, different AAT preparations require careful evaluation.

BACKGROUND For several years, orbitally shaken cylindrical bioreactors (OSRs) have become a new approach for propagation of mammalian cells cultivated under suspension. With new commercial cell culture media available today, allowing exceptionally high cell densities, the identification of suitable operating conditions presents a special challenge. Balancing hydrodynamic stress and other factors in reactors, this is particularly true for gas transfer rates providing sufficient oxygen (O-2) to cultures that can attain ten-fold greater biomasses than just a decade ago. This challenge is more profound the larger the working volume of the reactor becomes. RESULT For 10-L working volume cylindrical OSRs, we established a computational fluid dynamics model to evaluate the impacts of different operational conditions. Both fluid velocity distributions and O-2 transfer coefficients (k(L)a) were calculated for different filling volumes and shaking speeds. Also, hydrodynamic stress parameters were investigated considering the most critical working conditions. CONCLUSION The fluid velocity decreased with larger working volumes and, as expected, was found to increase with higher shaking frequency. Gas transfer rates between 10 and 54 h(-1) were found in working volumes from 2.5 to 10 L and under shaking frequencies from 100-160 rpm. Critically assessing the obtained data, with no liquid-submerged gas supply, we conclude that an air flow into the head-space of a disposable bag appears to be satisfactory under certain operating conditions to support the culture of the most sensitive mammalian or insect cell cultures towards high cell density, presently in fed-batch cultures achieving

Several naturally occurring vertebrate transposable elements have been genetically modified to enable the transposition of recombinant genes in mammalian cells. We compared three transposonspiggyBac, Tol2, and Sleeping Beautyfor their ability to generate cell pools (polyclonal cultures of recombinant cells) and clonal cell lines for the large-scale production of recombinant proteins using Chinese hamster ovary cells (CHO-DG44) as the host. Transfection with each of the dual-vector transposon systems resulted in cell pools with volumetric yields of tumor necrosis factor receptor-Fc fusion protein (TNFR:Fc) that were about ninefold higher than those from cell pools generated by conventional plasmid transfection. On average, the cell pools had 10-12 integrated copies of the transgene per cell. In the absence of selection, the volumetric productivity of the cell pools decreased by 50% over a 2-month cultivation period and then remained constant. The average volumetric TNFR:Fc productivity of clonal cell lines recovered from cell pools was about 25 times higher than that of cell lines generated by conventional transfection. In 14-day fed-batch cultures, TNFR:Fc levels up to 900mg/L were obtained from polyclonal cell pools and up to 1.5g/L from clonal cell lines using any of the three transposons. (C) 2015 Wiley Periodicals, Inc.