Cell Bank Origin of MDCK Parental Cells Shapes Adaptation to Serum-Free Suspension Culture and Canine Adenoviral Vector Production.

Phenotypic variation in cultured mammalian cell lines is known to be induced by passaging and culture conditions. Yet, the effect these variations have on the production of viral vectors has been overlooked. In this work we evaluated the impact of using Madin-Darby canine kidney (MDCK) parental cells from American Type Culture Collection (ATCC) or European Collection of Authenticated Cell Cultures (ECACC) cell bank repositories in both adherent and suspension cultures for the production of canine adenoviral vectors type 2 (CAV-2). To further explore the differences between cells, we conducted whole-genome transcriptome analysis. ECACC's MDCK showed to be a less heterogeneous population, more difficult to adapt to suspension and serum-free culture conditions, but more permissive to CAV-2 replication progression, enabling higher yields. Transcriptome data indicated that this increased permissiveness is due to a general down-regulation of biological networks of innate immunity in ECACC cells, including apoptosis and death receptor signaling, Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling, toll-like receptors signaling and the canonical pathway of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling. These results show the impact of MDCK source on the outcome of viral-based production processes further elucidating transcriptome signatures underlying enhanced adenoviral replication. Following functional validation, the genes and networks identified herein can be targeted in future engineering approaches aiming at improving the production of CAV-2 gene therapy vectors.

Detection and Quantification of Label-Free Infectious Adenovirus Using a Switch-On Cell-Based Fluorescent Biosensor.

Reliable and fast viral detection and quantification protocols are a requirement for the advance of basic research and clinical approaches with wild type or recombinant viruses. However, available cell-based assays are either time-consuming or require labeled viral particles, which may alter virus biology or pose safety issues in clinical applications. Since adenoviruses constitute a major healthcare burden but also, when engineered, widely used vectors in vaccination and gene and oncolytic therapies, herein we developed a genetically encoded switch-on fluorescent biosensor consisting of a cyclized Green fluorescent protein-cVisensor-with an adenoviral protease cleavable site as a switch. After initial sensor optimization (35% increase in performance), whole-cell biosensors were established-by stably expressing cVisensor in mammalian cells-and used for live-cell monitoring of adenovirus infection as the intracellular biosensor is specifically activated by the viral protease. A rapid flow cytometry-based bioassay using cVisensor cells was established 48 h postinfection, showing an estimated limit of detection of 105 infectious particles/mL, in-line with previously reported flow cytometry assays requiring labeled virus, and significantly faster than standard plaque-forming assays requiring up to 14 days. cVisensor was also successfully applied in the detection of HIV-1 protease activity, validating its wider potential for the detection of other viruses. Overall, this work presents a fast and easy method for detection and quantification of label-free infectious virus, allowing the establishment of new biosensing platforms for basic research in virology and biotechnological applications of recombinant virus biopharmaceuticals.

CXCL6 is an important paracrine factor in the pro-angiogenic human cardiac progenitor-like cell secretome.

Studies in recent years have established that the principal effects in cardiac cell therapy are associated with paracrine/autocrine factors. We combined several complementary techniques to define human cardiac progenitor cell (CPC) secretome constituted by 914 proteins/genes; 51% of these are associated with the exosomal compartment. To define the set of proteins specifically or highly differentially secreted by CPC, we compared human mesenchymal stem cells and dermal fibroblasts; the study defined a group of growth factors, cytokines and chemokines expressed at high to medium levels by CPC. Among them, IL-1, GROa (CXCL1), CXCL6 (GCP2) and IL-8 are examples whose expression was confirmed by most techniques used. ELISA showed that CXCL6 is significantly overexpressed in CPC conditioned medium (CM) (18- to 26-fold) and western blot confirmed expression of its receptors CXCR1 and CXCR2. Addition of anti-CXCL6 completely abolished migration in CPC-CM compared with anti-CXCR2, which promoted partial inhibition, and anti-CXCR1, which was inefficient. Anti-CXCL6 also significantly inhibited CPC CM angiogenic activity. In vivo evaluation also supported a relevant role for angiogenesis. Altogether, these results suggest a notable angiogenic potential in CPC-CM and identify CXCL6 as an important paracrine factor for CPC that signals mainly through CXCR2.

A multi-organ chip co-culture of neurospheres and liver equivalents for long-term substance testing.

Current in vitro and animal tests for drug development are failing to emulate the systemic organ complexity of the human body and, therefore, often do not accurately predict drug toxicity, leading to high attrition rates in clinical studies (Paul et al., 2010). The phylogenetic distance between humans and laboratory animals is enormous, this affects the transferability of animal data on the efficacy of neuroprotective drugs. Therefore, many neuroprotective treatments that have shown promise in animals have not been successful when transferred to humans (Dragunow, 2008; Gibbons and Dragunow, 2010). We present a multi-organ chip capable of maintaining 3D tissues derived from various cell sources in a combined media circuit which bridges the gap in systemic and human tests. A steady state co-culture of human artificial liver microtissues and human neurospheres exposed to fluid flow over two weeks in the multi-organ chip has successfully proven its long-term performance. Daily lactate dehydrogenase activity measurements of the medium and immunofluorescence end-point staining proved the viability of the tissues and the maintenance of differentiated cellular phenotypes. Moreover, the lactate production and glucose consumption values of the tissues cultured indicated that a stable steady-state was achieved after 6 days of co-cultivation. The neurospheres remained differentiated neurons over the two-week cultivation in the multi-organ chip, proven by qPCR and immunofluorescence of the neuronal markers ßIII-tubulin and microtubule-associated protein-2. Additionally, a two-week toxicity assay with a repeated substance exposure to the neurotoxic 2,5-hexanedione in two different concentrations induced high apoptosis within the neurospheres and liver microtissues, as shown by a strong increase of lactate dehydrogenase activity in the medium. The principal finding of the exposure of the co-culture to 2,5-hexanedione was that not only toxicity profiles of two different doses could be discriminated, but also that the co-cultures were more sensitive to the substance compared to respective single-tissue cultures in the multi-organ-chip. Thus, we provide here a new in vitro tool which might be utilized to predict the safety and efficacy of substances in clinical studies more accurately in the future.

Novel scalable 3D cell based model for in vitro neurotoxicity testing: Combining human differentiated neurospheres with gene expression and functional endpoints.

There is an urgent need for new in vitro strategies to identify neurotoxic agents with speed, reliability and respect for animal welfare. Cell models should include distinct brain cell types and represent brain microenvironment to attain higher relevance. The main goal of this study was to develop and validate a human 3D neural model containing both neurons and glial cells, applicable for toxicity testing in high-throughput platforms. To achieve this, a scalable bioprocess for neural differentiation of human NTera2/cl.D1 cells in stirred culture systems was developed. Endpoints based on neuronal- and astrocytic-specific gene expression and functionality in 3D were implemented in multi-well format and used for toxicity assessment. The prototypical neurotoxicant acrylamide affected primarily neurons, impairing synaptic function; our results suggest that gene expression of the presynaptic marker synaptophysin can be used as sensitive endpoint. Chloramphenicol, described as neurotoxicant affected both cell types, with cytoskeleton markers' expression significantly reduced, particularly in astrocytes. In conclusion, a scalable and reproducible process for production of differentiated neurospheres enriched in mature neurons and functional astrocytes was obtained. This 3D approach allowed efficient production of large numbers of human differentiated neurospheres, which in combination with gene expression and functional endpoints are a powerful cell model to evaluate human neuronal and astrocytic toxicity.

Lentivirus production is influenced by SV40 large T-antigen and chromosomal integration of the vector in HEK293 cells.

Currently, lentiviral vectors for research and gene therapy are produced from 293-T cells that are transiently transfected with plasmids encoding the vector and helper functions. However, transiently transfected vectors as well as the presence of SV40 virus large T-antigen (T-Ag) cause serious technical and safety considerations. We aimed to exploit single copy integration sites in the HEK293 genome supporting lentiviral vector production. We found that lentiviral vectors result in minimal infectious particle production from single copy integrants in HEK293. Moreover, once this cell line harbors single copy integrations of lentiviral vectors, its ability to transiently produce lentiviral vectors becomes strongly impaired. T-Ag has a dramatic effect on virus production. Low levels of constitutive T-Ag expression can overcome the production restriction imposed by integrated lentiviral vectors copies. Interestingly, T-Ag does not exert its role at the level of transcriptional activity of the vector; rather, it seems to impose an indirect effect on the cell thereby enabling lentiviral vector production. Altogether, our study highlights the restrictions for integrated lentiviral vectors that are relevant for the establishment of stable and safe producer cell lines.

Stabilization of gammaretroviral and lentiviral vectors: from production to gene transfer.

BACKGROUND: The low stability of gammaretroviral and lentiviral vectors affects their production, making high quality clinical preparations a difficult goal to achieve. Recently, our laboratory has shown that the main inactivation mechanism for both these vectors is the loss of their capacity to perform reverse transcription. The present study aimed to increase the stability of gammaretroviral and lentiviral at 37 degrees C and at 4 degrees C. METHODS: Inactivation studies were performed with gammaretroviral and lentiviral vectors at 37 and 4 degrees C, with and without several stabilizing compounds. The residual viral infectivity and reverse transcription capacity of these samples were tested. RESULTS: The results obtained demonstrate that it is possible to increase the stability of reverse transcription and the infectivity stability of purified gammaretroviral vectors by adding recombinant human albumin (rHSA) to the storage buffer, both at 37 degrees C and at 4 degrees C. For lentiviral vectors, it was observed that further protection was needed. This was achieved by adding lipids to the storage buffer, using a mixture of lipoproteins and rHSA. The difference of stabilization between gammaretroviral and lentiviral vectors was validated by performing stabilization tests with vectors possessing different envelope proteins and produced by different cell lines. CONCLUSIONS: The presented study reveals that it is possible to increase the half-life of purified gammaretroviral and lentiviral vectors at 37 degrees C and at 4 degrees C, but the two vectors have different stabilization requirements: for retroviral vectors, the addition of rHSA is enough and, for lentiviral vectors, it is necessary to add both lipoproteins and rHSA. The increase of the stability of the reverse transcription process was shown to have a high impact with respect to the increase of the stability of infectivity.