Advances in cell culture: anchorage dependence

Anchorage-dependent cells are of great interest for various biotechnological applications. (i) They represent a formidable production means of viruses for vaccination purposes at very large scales (in 1000–6000 l reactors) using microcarriers, and in the last decade many more novel viral vaccines have been developed using this production technology. (ii) With the advent of stem cells and their use/potential use in clinics for cell therapy and regenerative medicine purposes, the development of novel culture devices and technologies for adherent cells has accelerated greatly with a view to the large-scale expansion of these cells. Presently, the really scalable systems—microcarrier/microcarrier-clump cultures using stirred-tank reactors—for the expansion of stem cells are still in their infancy. Only laboratory scale reactors of maximally 2.5 l working volume have been evaluated because thorough knowledge and basic understanding of critical issues with respect to cell expansion while retaining pluripotency and differentiation potential, and the impact of the culture environment on stem cell fate, etc., are still lacking and require further studies. This article gives an overview on critical issues common to all cell culture systems for adherent cells as well as specifics for different types of stem cells in view of small- and large-scale cell expansion and production processes.     

Quantitative proteomic analysis of lentiviral vectors using 2‐DE

Among the integrative gene therapy vectors developed to date, human immunodeficiency virus type 1 (HIV‐1)‐derived lentiviral vectors (LV) are distinguished by their capacity to infect both dividing and non‐dividing cells. Recombinant LV particles contain viral proteins necessary for their packaging, infectious and integrating functions. Like the parental HIV‐1 virus they are able to acquire various cellular proteins, but the number and localisation of these proteins are poorly characterised. In the present study we used 2‐DE followed by MALDI‐TOF to quantify the protein content of several types of vesicular stomatitis virus G‐pseudotyped LV including those that were extensively purified in the perspective of clinical gene therapy studies. A proteinase K treatment was used to distinguish between cellular proteins incorporated into virions (I‐proteins) and those co‐purified with vectors (C‐proteins). We found 10 C‐proteins and 18 I‐proteins associated with LV. Copy numbers for these core I‐proteins varied from 5 (AIP‐1/ALIX) to 280 (Cyclophilin A) per vector particle. Three novel I‐proteins, guanine nucleotide‐binding protein 2, L‐lactate dehydrogenase B chain and hnRNP core protein A1, were found. This study defines for the first time, the protein stoichiometry of infectious HIV‐1‐derived LV particles.     

Gene transduction and cell entry pathway of fiber-modified adenovirus type 5 vectors carrying novel endocytic peptide ligands selected on human tracheal glandular cells

Monolayers of cystic fibrosis transmembrane conductance regulator (CFTR)-deficient human tracheal glandular cells (CF-KM4) were subjected to phage biopanning, and cell-internalized phages were isolated and sequenced, in order to identify CF-KM4-specific peptide ligands that would confer upon adenovirus type 5 (Ad5) vector a novel cell target specificity and/or higher efficiency of gene delivery into airway cells of patients with cystic fibrosis (CF). Three different ligands, corresponding to prototypes of the most represented families of phagotopes recovered from intracellular phages, were designed and individually inserted into Ad5-green fluorescent protein (GFP) (AdGFP) vectors at the extremities of short fiber shafts (seven repeats [R7]) terminated by scissile knobs. Only one vector, carrying the decapeptide GHPRQMSHVY (abbreviated as QM10), showed an enhanced gene transduction of CF-KM4 cells compared to control nonliganded vector with fibers of the same length (AdGFP-R7-knob). The enhancement in gene transfer efficiency was not specific to CF-KM4 cells but was observed in other mammalian cell lines tested. The QM10-liganded vector was referred to as AdGFP-QM10-knob in its knobbed version and as AdGFP-QM10 in its proteolytically deknobbed version. AdGFP-QM10 was found to transduce cells with a higher efficiency than its knob-bearing version, AdGFP-QM10-knob. Consistent with this, competition experiments indicated that the presence of knob domains was not an absolute requirement for cell attachment of the QM10-liganded vector and that the knobless AdGFP-QM10 used alternative cell-binding domains on its capsid, including penton base capsomer, via a site(s) different from its RGD motifs. The QM10-mediated effect on gene transduction seemed to take place at the step of endocytosis in both quantitative and qualitative manners. Virions of AdGFP-QM10 were endocytosed in higher numbers than virions of the control vector and were directed to a compartment different from the early endosomes targeted by members of species C Ad. AdGFP-QM10 was found to accumulate in late endosomal and low-pH compartments, suggesting that QM10 acted as an endocytic ligand of the lysosomal pathway. These results validated the concept of detargeting and retargeting Ad vectors via our deknobbing system and redirecting Ad vectors to an alternative endocytic pathway via a peptide ligand inserted in the fiber shaft domain.     

Fusoselect: cell–cell fusion activity engineered by directed evolution of a retroviral glycoprotein

Membrane fusion plays a key role in many biological processes including vesicle trafficking, synaptic transmission, fertilization or cell entry of enveloped viruses. As a common feature the fusion process is mediated by distinct membrane proteins. We describe here ‘Fusoselect’, a universal procedure allowing the identification and engineering of molecular determinants for cell–cell fusion-activity by directed evolution. The system couples cell–cell fusion with the release of retroviral particles, but can principally be applied to membrane proteins of non-viral origin as well. As a model system, we chose a γ-retroviral envelope protein, which naturally becomes fusion-active through proteolytic processing by the viral protease. The selection process evolved variants that, in contrast to the parental protein, mediated cell–cell fusion in absence of the viral protease. Detailed analysis of the variants revealed molecular determinants for fusion competence in the cytoplasmic tail (CT) of retroviral Env proteins and demonstrated the power of Fusoselect.     

Characterization of trypsinogens 1 and 2 in two human pancreatic adenocarcinoma cell lines; CFPAC-1 and CAPAN-1.

Proteins with trypsin-like immunoreactivity (first detected by a specific immunoenzymatic assay) were isolated from CAPAN-1 and CFPAC-1 cell culture-conditioned media by chromatography on an immunoadsorbent prepared with a polyclonal antibody directed against trypsin 1. The adsorbed proteins were devoid of free trypsin activity but trypsin activity was present after enterokinase activation demonstrating that the immunoreactive trypsin present in cell supernatants corresponds to trypsinogens. When characterised by Western blotting using a monoclonal antibody directed against human trypsin 1 two protein bands corresponding to trypsinogen 1 (23 kDa) and trypsinogen 2 (25 kDa) gave a positive reaction. These results demonstrate the presence of trypsinogens 1 and 2 in CAPAN-1 and CFPAC-1 cells and in their culture-conditioned media.     

Quantifying Preferences and Responsiveness of Marine Zooplankton to Changing Environmental Conditions using Microfluidics

Global environmental change significantly affects marine species composition. However, analyzing the impact of these changes on marine zooplankton communities was so far mostly limited to assessing lethal doses through mortality assays and hence did not allow a direct assessment of the preferred conditions, or preferendum. Here, we use a microfluidic device to characterize individual behavior of actively swimming zooplankton, and to quantitatively determine their ecological preferendum. For the annelid zooplankton model Platynereis dumerilii we observe a broader pH preferendum than for the copepod Euterpina acutifrons, and reveal previously unrecognized sub-populations with different pH preferenda. For Platynereis, the minimum concentration difference required to elicit a response (responsiveness) is ~1 μM for H⁺ and ~13.7 mM for NaCl. Furthermore, using laser ablations we show that olfactomedin-expressing sensory cells mediate chemical responsiveness in the Platynereis foregut. Taken together, our microfluidic approach allows precise assessment and functional understanding of environmental perception on planktonic behaviour.