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Treatment of peripheral nerve lesions remains a major challenge due to poor functional recovery; hence, ongoing research efforts strive to enhance peripheral nerve repair. In this study, we aimed to establish three-dimensional tissue-engineered bands of Büngner constructs by subjecting Schwann cells (SCs) embedded in fibrin hydrogels to mechanical stimulation. We show for the first time that the application of strain induces (i) longitudinal alignment of SCs resembling bands of Büngner, and (ii) the expression of a pronounced repair SC phenotype as evidenced by upregulation of BDNF, NGF, and p75 NTR . Furthermore, we show that mechanically aligned SCs provide physical guidance for migrating axons over several millimeters in vitro in a co-culture model with rat dorsal root ganglion explants. Consequently, these constructs hold great therapeutic potential for transplantation into patients and might also provide a physiologically relevant in vitro peripheral nerve model for drug screening or investigation of pathologic or regenerative processes., Competing Interests: The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: CH and ATW disclose a potential conflict of interest related to the research presented in this study. They have submitted a patent application for a technology that is related to a component used in the research described in this paper., (© The Author(s) 2024.)

Each process step in the manufacture of biological products requires expensive resources and reduces total process productivity. Since downstream processing of biologicals is the main cost driver, process intensification is a persistent topic during the entire product life cycle. We present here one approach for the intensification of bioprocesses by applying on-column virus inactivation using solvent/detergent (S/D) treatment during ion-exchange chromatography. The established purification process of a recombinant protein was used as a model to compare key process parameters (i.e., product yield, specific activity, impurity clearance) of the novel approach to the standard process protocol. Additional wash and incubation steps with and without S/D-containing buffers were introduced to ensure sufficient contact time to effectively eliminate enveloped viruses and to significantly decrease the amount of S/D reagents. Comparison of key process parameters demonstrated equivalent process performance. To assess the viral clearance capacity of the novel approach, XMuLV was spiked as model virus to the chromatographic load and all resulting fractions were analyzed by TCID 50 and RT-qPCR. Data indicates the inactivation capability of on-column virus inactivation even at 10% of the nominal S/D concentration, although the mechanism of viral clearance needs further investigation., Competing Interests: Declaration of competing interest All authors were employees of Baxalta Innovations GmbH (part of Takeda) or Takeda Manufacturing Austria AG at the time of this study. Andreas Flicker, Christian Fiedler, Maria R. Farcet, Martin Purtscher, and Thomas R. Kreil are Takeda stock owners. Daniel Polasek is currently employee of Octapharma Pharmazeutika Produktionsges.m.b.H (Oberlaaer Straβe 235, 1100 Vienna, Austria)., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Detection, quantification and monitoring of virus - host cell interactions are of great importance when evaluating the safety of pharmaceutical products. With the wide usage of viral based vector systems in combination with mammalian cell lines for the production of biopharmaceuticals, the presence of replication competent viral particles needs to be avoided and potential hazards carefully assessed. Consequently, regulatory agencies recommend viral clearance studies using plaque assays or TCID 50 assays to evaluate the efficiency of the production process in removing viruses. While plaque assays provide reliable information on the presence of viral contaminations, they are still tedious to perform and can take up to two weeks to finish. To overcome some of these limitations, we have automated, miniaturized and integrated the dual cell culture bioassay into a common lab-on-a-chip platform containing embedded electrical sensor arrays to enrich and detect infectious viruses. Results of our microfluidic single step assay show that a significant reduction in assay time down to 3 to 4 days can be achieved using simultaneous cell-based viral amplification, release and detection of cytopathic effects in a target cell line. We further demonstrate the enhancing effect of continuous fluid flow on infection of PG-4 reporter cells by newly formed and highly active virions by M. dunni cells, thus pointing to the importance of physical relevant viral-cell interactions.

Reengineering functional vascular networks in vitro remains an integral part in tissue engineering, since the incorporation of non-perfused tissues results in restricted nutrient supply and limited waste removal. Microfluidic devices are routinely used to mimic both physiological and pathological vascular microenvironments. Current procedures either involve the investigation of growth factor gradients and interstitial flow on endothelial cell sprouting alone or on the heterotypic cell-cell interactions between endothelial and mural cells. However, limited research has been conducted on the influence of flow on co-cultures of these cells. Here, we exploited the ability of microfluidics to create and monitor spatiotemporal gradients to investigate the influence of growth factor supply and elution on vascularization using static as well as indirect and direct flow setups. Co-cultures of human adipose-derived stem/stromal cells and human umbilical vein endothelial cells embedded in fibrin hydrogels were found to be severely affected by diffusion limited growth factor gradients as well as by elution of reciprocal signaling molecules during both static and flow conditions. Static cultures formed pre-vascular networks up to a depth of 4 mm into the construct with subsequent decline due to diffusion limitation. In contrast, indirect flow conditions enhanced endothelial cell sprouting but failed to form vascular networks. Additionally, complete inhibition of pre-vascular network formation was observable for direct application of flow through the hydrogel with decline of endothelial cell viability after seven days. Using finite volume CFD simulations of different sized molecules vital for pre-vascular network formation into and out of the hydrogel constructs, we found that interstitial flow enhances growth factor supply to the cells in the bulk of the chamber but elutes cellular secretome, resulting in truncated, premature vascularization.

We have developed a lab-on-a-chip system for continuous and non-invasive monitoring of microfluidic cell cultures using integrated high-frequency contactless impedance spectroscopy. Electrically insulated microfabricated interdigitated electrode structures were embedded into four individually addressable microchambers to reliably and reproducibly detect cell-substrate interactions, cell viability and metabolic activity. While silicon nitride passivated sensor substrates provided a homogeneous cell culture surface that minimized cell orientation along interdigitated electrode structures, the application of high-frequency AC fields reduced the impact of the 300 nm thick passivation layer on sensor sensitivity. The additional implementation of multivariate data analysis methods such as partial least square (PLS) for high-frequency impedance spectra provided unambiguous information on intracellular pathway activation, up and down-regulation of protein synthesis as well as global cellular stress responses. A comparative cell analysis using connective tissue fibroblasts showed that high-frequency contactless impedance spectroscopy and time-resolved quantification of IL-6 secretion using ELISA provided similar results following stimulation with circulating pro-inflammatory cytokines IL-1β and TNFα. The combination of microfluidics with contactless impedance sensing and time-resolved quantification of stress factor release will provide biologist with a new tool to (a) establish a variety of uniform cell culture surfaces that feature complex biochemistries, micro- and nanopatterns; and (b) to simultaneously characterize cell responses under physiologically relevant conditions using a complementary non-invasive cell analysis method.

Nanotechnology provides the tools to develop novel biosensors with improved performance, including sensitivity and response time that can be readily integrated into diagnostic devices. We have developed a miniaturized cell analysis platform to advance microfluidic cell cultures by combining two complementary, label-free and non-invasive cell analysis methods for the long-term monitoring of dynamic cell behavior. The novel dual-parameter cell-on-a-chip detects light scattering from adherent cells to provide information on cell numbers and intracellular granularity, while simultaneously performing impedance spectroscopy to monitor cell adhesion and cell-cell interaction. In the present work we have integrated spray-coated organic photodiode arrays with a lab-on-a-chip containing embedded interdigitated electrode structures to improve assay reproducibility, reliability and accuracy. We successfully demonstrate that the complementary cell chip technology can accurately detect cell numbers, clarify misleading results during cell-substance interaction assays, as well as the cytotoxicity screening of drug substances. The ability to precisely determine cell numbers within minutes constitutes a major step towards standardization.

The purpose of this study was to compare the influence of physiological and supraphysiological concentrations of 4-hydroxynonenal (HNE), a peroxidation product of omega-6-polyunsaturated fatty acids, on the proliferation of malignant CEM-NKR lymphatic leukaemia cells and normal human lymphocytes (HPBM). Furthermore the growth modulating effect of phytohemagglutinin (PHA) on both cell lines was examined. The effects of HNE were monitored 18 hours and 3 days after incubation, using two different DNA-synthesis assays ([3H]-thymidine- and BrdU- incorporation) and a mitochondrial dehydrogenases activity assay (MTT). On the one hand HNE showed dose-dependent effects on both of the cell lines, while on the other hand a clear difference between the response of CEM-NKR cells and HPBM cells, respectively, to HNE was observed. On CDM-NKR cells, both concentrations of HNE caused significant cytotoxic effects on DNA-synthesis as well as on mitochondrial activity, while in contrast, HNE did not show any significant toxicity to HPBM cells. After 3 days there was even a slight stimulation of DNA synthesis with the physiological concentration of HNE. Furthermore the presence of PHA in the culture medium increased the difference of the response of CEM-NKR and HPBM cells, respectively to HNE.

We have established a panel of human monoclonal antibodies against human immunodeficiency virus type 1 (HIV-1). The antibodies 2F5 and 2G12 have been identified to be two of the most potently in vitro neutralizing antibodies against HIV-1. Here we report on a further antibody, 4E10, of similar in vitro neutralizing potency. 4E10 binds to a novel epitope C terminal of the ELDKWA sequence recognized by 2F5, which has been so far the only described broadly neutralizing anti-gp41 antibody. Both 4E10 and 2F5 bind only weakly to infected cells compared with gp120-specific 2G12 and polyclonal anti-HIV-1 immunoglobulin (HIVIG), but show potent in vitro neutralizing properties. 4E10 neutralizes potently not only tissue culture-adapted strains but also primary isolates of different clades, including A, B, C, D, and E. Viruses that were found to be resistant to 2F5 were neutralized by 4E10 and vice versa; none of the tested isolates was resistant to both anti-gp41 antibodies. This confirms that the region recognized by 2F5 and 4E10 is essential for viral infectivity and may be important for vaccine design. Moreover, our results suggest that 4E10 should be further investigated for passive anti-HIV immunotherapy.

The human monoclonal antibody (humAb) 2F5 is a potent candidate for immunotherapy of HIV-1. The xenohybridoma derived humAb 2F5 is of IgG3 kappa isotype. Generally, IgG1 isotype has a longer half-life (beta-clearance) in humans than IgG3. Therefore the isotype was switched to an IgG1 by ligation of the 2F5 heavy chain variable region to an IgG1 constant region and expressed as IgG1 kappa in CHO cells. CHO clones have been established, which stably express humAb 2F5 at high levels. The specificity, affinity and in vitro function of both isotypes were identical., (Copyright 2000 John Wiley & Sons, Inc.)

Although typical primary isolates of human immunodeficiency virus type 1 (HIV-1) are relatively neutralization resistant, three human monoclonal antibodies and a small number of HIV-1(+) human sera that neutralize the majority of isolates have been described. The monoclonal antibodies (2G12, 2F5, and b12) represent specificities that a putative vaccine should aim to elicit, since in vitro neutralization has been correlated with protection against primary viruses in animal models. Furthermore, a neutralization escape mutant to one of the antibodies (b12) selected in vitro remains sensitive to neutralization by the other two (2G12 and 2F5) (H. Mo, L. Stamatatos, J. E. Ip, C. F. Barbas, P. W. H. I. Parren, D. R. Burton, J. P. Moore, and D. D. Ho, J. Virol. 71:6869-6874, 1997), supporting the notion that eliciting a combination of such specificities would be particularly advantageous. Here, however, we describe a small subset of viruses, mostly pediatric, which show a high level of neutralization resistance to all three human monoclonal antibodies and to two broadly neutralizing sera. Such viruses threaten antibody-based antiviral strategies, and the basis for their resistance should be explored.

Objective: To investigate whether variations of the conserved gp41 amino-acid sequence ELDKWA affect its binding or neutralization by monoclonal antibody (MAb) 2F5., Design and Methods: Neutralization assays were performed with primary isolates from different HIV-1 subtypes and the sequences corresponding to the 2F5 epitope region were analysed. Studies of MAb 2F5 peptide reactivity were performed by spot analysis, using peptides immobilized on cellulose. The frequency of emergence of neutralization-resistant virus variants was determined by immune selection experiments in the presence of MAb 2F5., Results: Primary isolates from clades A, B and E were neutralized by MAb 2F5. Neutralization sensitivity correlated with the presence of the LDKW motif. A K-to-N change in the core sequence was identified in a neutralization-resistant patient isolate. Neutralization resistant virus variants that were selected in the presence of MAb 2F5 were found to contain D-to-N, D-to-E, or K-to-N changes within the LDKW sequence. Neither in natural isolates nor in variants obtained under immune selection conditions in the laboratory were changes in the L and W positions observed. Studies of MAb 2F5 binding to variations of the ELDKWA peptide confirmed that the changes at the first and last positions did not significantly reduce binding capacity, whereas amino-acid changes from D to N, D to E, and K to N almost completely abrogated binding of MAb 2F5., Conclusion: Sequence analysis of a variety of primary isolates suggests that the major determinant of MAb 2F5 binding corresponds to the amino-acid sequence LDKW. Naturally occurring and in vitro selected neutralization-resistant viruses contained changes in the D and K positions of the ELDKWA motif.

We have established a hybridoma clone, designated 2F5, secreting a neutralizing human monoclonal antibody (MAb) specific for gp41 of human immunodeficiency virus type 1 (HIV-1). The epitope of MAb 2F5 was mapped to amino acid sequence Glu-Leu-Asp-Lys-Trp-Ala on the ectodomain of gp41. In this study different in vitro test systems were used to characterize the neutralizing properties of MAb 2F5. In syncytium inhibition assays, fusion inhibition experiments, and neutralization assays on different HIV-susceptible cells (H9, U937, and peripheral blood mononuclear cells) MAb 2F5 showed broad-spectrum neutralizing capacity against HIV-1 laboratory isolates IIIB, MN, RF, and SF2. In addition, primary isolates from AIDS patients were also neutralized.

A fast protein liquid chromatographic method for the preparative separation of the various isoproteins is described. Highly purified human monoclonal antibodies, recombinant human superoxide dismutase and human superoxide dismutase from erythrocytes were used as starting material. The isoproteins were separated by chromatofocusing on Mono P columns. A very narrow pH gradient was applied to achieve complete separation of the isoproteins. The prepurification steps and the pretreatment of the samples to achieve optimum resolution are described in detail. The method is also applicable to extremely basic monoclonal antibodies (pI = 9). The successful separation was checked by isoelectric focusing in immobilized pH gradients (Immobilines). The future of these methods is discussed, because for many different biochemical and biophysical investigations pure and homogeneous isoproteins are necessary.

Protein A Superose, protein G Sepharose fast flow and copolymerized hydroxyapatite were used for the purification of human monoclonal antibodies against HIV 1. Both desalted culture supernatant and a prepurified protein solution were used as starting materials. The different runs were compared with respect to yield and recovery of biological activity. The biological activity (specific reactivity) was checked by antigen enzyme-linked immunosorbent assay with recombinant antigen. The human monoclonal antibodies could not be selectively eluted from the hydroxyapatite but elution could be effected from the protein A Superose at pH 4.0 and from protein G at pH 3.0. The eluted immunoglobulin G was distributed over a broad pH range when protein G Superose was used. Biologically active material could be obtained from protein A Superose and protein G Sepharose fast flow.

Human monoclonal antibodies against the transmembrane protein gp41 of HIV-1 were isolated and purified on a pilot scale. A purification scheme was established for the production of human monoclonal antibodies on the gram scale. 50 1 of culture supernatant can be treated in one purification cycle. The hybridomas were mass cultured in an airlift fermenter. The culture broth was clarified by microfiltration and chromatographed on CM-Sepharose fast flow and protein A Superose. Scale up of the high performance affinity chromatography from 1 ml protein A Superose up to 40 ml is described. All desalting steps were performed by gel filtration on Sephadex G-25 coarse. The yield of the whole purification procedure is in the range of 50-60%. The purity is higher than 99.9%. DNA and reverse transcriptase could not be detected. The whole method is designed as a basis for scale up to industrial scale. Results from quality control assays have proven the validity of this approach.