A quantitative approach for understanding small-scale human mesenchymal stem cell culture – implications for large-scale bioprocess development

Human mesenchymal stem cell (hMSC) therapies have the potential to revolutionise the healthcare industry and replicate the success of the therapeutic protein industry; however, for this to be achieved there is a need to apply key bioprocessing engineering principles and adopt a quantitative approach for large-scale reproducible hMSC bioprocess development. Here we provide a quantitative analysis of the changes in concentration of glucose, lactate and ammonium with time during hMSC monolayer culture over 4 passages, under 100% and 20% dissolved oxgen (dO₂), where either a 100%, 50% or 0% growth medium exchange was performed after 72h in culture. Yield coefficients, specific growth rates (h^-1) and doubling times (h) were calculated for all cases. The 100% dO₂ flasks outperformed the 20% dO₂ flasks with respect to cumulative cell number, with the latter consuming more glucose and producing more lactate and ammonium. Furthermore, the 100% and 50% medium exchange conditions resulted in similar cumulative cell numbers, whilst the 0% conditions were significantly lower. Cell immunophenotype and multipotency were not affected by the experimental culture conditions. This study demonstrates the importance of determining optimal culture conditions for hMSC expansion and highlights a potential cost savings from only making a 50% medium exchange, which may prove significant for large-scale bioprocessing.     

The use of charcoal in in vitro culture – A review

Activated charcoal is commonly used in tissue culture media. Its addition to culture medium may promote or inhibit in vitro growth, depending on species and tissues used. The effects of activated charcoal may be attributed to establishing a darkened environment; adsorption of undesirable/inhibitory substances; adsorption of growth regulators and other organic compounds, or the release of growth promoting substances present in or adsorbed by activated charcoal.     

Chemometric analysis of soy protein hydrolysates used in animal cell culture for IgG production – An untargeted metabolomics approach

Soy protein hydrolysates are used as the most cost effective medium supplement to enhance cell growth and recombinant protein productivity in cell cultures. Such hydrolysates contain diverse classes of compounds, such as peptides, carbohydrates and phenolic compounds. To identify if specific compounds dominate the functionality of hydrolysates in cell cultures, thirty samples of hydrolysates with different cell culture performances were analyzed for chemical composition using an untargeted metabolomics approach. Out of 410 detected compounds, 157 were annotated. Most of the remaining 253 compounds were identified as peptides, but could not be annotated exactly. All compounds were quantified relatively, based on their average signal intensities. The cell growth and total immunoglobulin (IgG) production, relative to the CD medium (100%), ranged from 148 to 438% and 117 to 283%, respectively. Using bootstrapped stepwise regression (BSR), the compounds with the highest inclusion frequency were identified. The most important compound, i.e. phenyllactate and ferulate explained 29% and 30% of the variance for cell growth and total IgG production, respectively. Surprisingly, all compounds identified in the BSR showed a positive correlation with cell growth and total IgG production. This knowledge can be applied to monitor the production and accumulation of these compounds during the production process of hydrolysates. Consequently, the processing conditions can be modulated to produce soy protein hydrolysates with enhanced and consistent cell culture performance.     

Oxidoreductases for the remediation of organic pollutants in water – a critical review

Water contamination by various recalcitrant organic aromatic compounds is an emerging environmental issue that is increasingly attracting the attention of environmental scientists. A great majority of these recalcitrant pollutants are industrial wastes, textile dyes, pharmaceuticals, hormones, and personal care products that are discharged into wastewater. Not surprisingly, various chemical, physical, and biological strategies have been proposed and developed to remove and/or degrade these pollutants from contaminated water bodies. Biological approaches, specifically using oxidoreductase enzymes (such as peroxidases and laccases) for pollutant degradation are a relatively new and a promising research area that has potential advantages over other methods due to their higher efficiency and the ease of handling. This review focuses on the application of different classes of oxidoreductase enzymes to degrade various classes of organic pollutants. In addition to classifying these enzymes based on structural differences, the major factors that can affect their remediation ability, such as the class of peroxidases employed, pH, molecular structure of the pollutant, temperature, and the presence of redox mediators are also examined and discussed. Interestingly, a literature survey combined with our unpublished data suggests that “peroxidases” are a very heterogeneous and diverse family of enzymes and have different pH profiles, temperature optima, thermal stabilities, requirements for redox mediators, and substrate specificities as well as varying detoxification abilities. Additionally, remediation of real-life polluted samples by oxidoreductases is also highlighted as well as a critical look at current challenges and future perspectives.     

Bioenergetic consequences in the interaction of mammalian cell and virus for the multiplication of recombinant vaccinia and expression of heterologous protein (β-hCG)

Summary Energy generation in vaccinia virus infected Vero cell is investigated. The specific ATP pool in infected cells increases in the range of 40–80%, in contrast to uninfected cells. Virus replication and -hCG expression are associated with the drop in ATP/ADP ratio from 7.0 to 2.5. High initial infection level leads to more energy generation and also more product formation.     

Laboratory-scale permeabilization of Escherichia coli cells for recovery of a small recombinant protein – Staphylokinase

The recovery of recombinant proteins includes a purification process that has to be compressed to a minimum of steps in order to get high yields with a low cost expenditure. A selective liberation of recombinant proteins by cell permeabilization leads to both a high product purity just in the beginning of the recovery process and to a simplification of the cell residue separation compared to the mechanical cell disruption. In case of the purification of the bacterial plasminogen activator Staphylokinase from E. coli cells, yields of 82% with a purity of 46% were attained by utilization of permeabilization by biomass freezing, resuspension in a Tris/EDTA-buffer and following micro-diafiltration. A recovery process without interruption (freezing) is possible due to the addition of guanidine-HCl and Triton X100 to the buffer. These methods were developed on a laboratory-scale.     

Organic acids in the rhizosphere – a critical review

Organic acids, such as malate, citrate and oxalate, have been proposed to be involved in many processes operating in the rhizosphere, including nutrient acquisition and metal detoxification, alleviation of anaerobic stress in roots, mineral weathering and pathogen attraction. A full assessment of their role in these processes, however, cannot be determined unless the exact mechanisms of plant organic acid release and the fate of these compounds in the soil are more fully understood. This review therefore includes information on organic acid levels in plants (concentrations, compartmentalisation, spatial aspects, synthesis), plant efflux (passive versus active transport, theoretical versus experimental considerations), soil reactions (soil solution concentrations, sorption) and microbial considerations (mineralization). In summary, the release of organic acids from roots can operate by multiple mechanisms in response to a number of well-defined environmental stresses (e.g., Al, P and Fe stress, anoxia): These responses, however, are highly stress- and plant-species specific. In addition, this review indicates that the sorption of organic acids to the mineral phase and mineralisation by the soil's microbial biomass are critical to determining the effectiveness of organic acids in most rhizosphere processes.     

Polyomavirus inactivation – A review

Polyomavirus inactivation has been studied since the 1950s when it became apparent that certain polio vaccines were contaminated with SV40. Relatively high temperatures (≥70 °C) are required to effect thermal inactivation of the polyomaviruses. The chemical inactivants that are effective (β-propiolactone, ethanol, sodium hydroxide, and formaldehyde) are those that have displayed efficacy for other small, non-enveloped viruses, such as the circoviruses. Low pH inactivation can be effective, especially at pH at or below 3 and at higher temperatures. Polyomaviruses are more resistant to UV-C irradiation than are other small non-enveloped viruses such as the parvoviruses and caliciviruses. The efficacy of photodynamic inactivation of polyomaviruses is very much dye-dependent, with toluidine blue, acridine orange, and methylene blue dyes being effective photosensitizers. Ionizing radiation can be effective, depending on the conditions employed and the inactivation matrix. Inactivation of the oncogenic properties of the polyomaviruses may require higher doses of inactivant than those required to inactivate infectivity. While the polyomaviruses are considered to be highly resistant to inactivation, the degree of resistance is dependent upon the specific approach under consideration. For certain approaches, such as UV-C and gamma-irradiation, the polyomaviruses appear to be more resistant than other small non-enveloped viruses.     

Host cell protein analysis in therapeutic protein bioprocessing – methods and applications

The analysis of host cell proteins (HCPs) is one of the most important analytical requirements during bioprocess development of therapeutic moieties. In this review, we focus on the comparison of different methods for the analysis of HCPs and how cell lines, fermentation conditions, and unit operations influence HCP distribution during the process chain. Current guidelines typically require reduction of HCPs to the ppm level, depending on the intended use, the route of administration of the product, and the production system. A range of immunospecific and non-specific methods are available that have been globally accepted by regulatory bodies. Immunospecific methods, such as ELISA, are simple to use in routine analysis and can quantify low levels of HCPs when specific antibodies are available. Non-specific methods are more complex; however, they provide a holistic view of the HCP profile and qualitative information of the composition of HCP in the sample. Different methods for the comparison of bioprocessing strategies during scale-up and purification development are compared herein. The methods include immunospecific methods, such as ELISA, western blot, and threshold, and non-specific methods, such as 2D-DIGE and 2D-HPLC combined with MS.     

Transient recombinant protein expression in a human amniocyte cell line: the CAP-T® cell system

The impact of transient gene expression approaches (TGE) on the rapid production of recombinant proteins is undisputed, despite that all efforts are currently relying on two host cell families only, namely HEK293 derivatives and CHO cell line(s). Yet, the increasing complexity of biological targets calls for more than two host cell types to meet the challenges of difficult‐to‐express proteins. For this reason, we evaluated the more recently established novel CAP‐T® cell line derived from human amniocytes for its performance and potential in transient gene expression. Upon careful analyses and adaptation of all process parameters we show here that indeed the CAP‐T® cells are extremely amenable to transient gene expression and recombinant protein production. Additionally, they possess inherent capabilities to express and secrete complex and difficult target molecules, thus adding an attractive alternative to the repertoire of existing host cell lines used in transient production processes. Biotechnol. Bioeng. 2012;109: 2250–2261. © 2012 Wiley Periodicals, Inc.     

Can aging be programmed? A critical literature review

The evolution of the aging process has long been a biological riddle, because it is difficult to explain the evolution of a trait that has apparently no benefit to the individual. Over 60 years ago, Medawar realized that the force of natural selection declines with chronological age because of unavoidable environmental risks. This forms the basis of the mainstream view that aging arises as a consequence of a declining selection pressure to maintain the physiological functioning of living beings forever. Over recent years, however, a number of articles have appeared that nevertheless propose the existence of specific aging genes; that is, that the aging process is genetically programmed. If this view were correct, it would have serious implications for experiments to understand and postpone aging. Therefore, we studied in detail various specific proposals why aging should be programmed. We find that not a single one withstands close scrutiny of its assumptions or simulation results. Nonprogrammed aging theories based on the insight of Medawar (as further developed by Hamilton and Charlesworth) are still the best explanation for the evolution of the aging process. We hope that this analysis helps to clarify the problems associated with the idea of programmed aging.     

MannDB – A microbial database of automated protein sequence analyses and evidence integration for protein characterization

Background  

MannDB was created to meet a need for rapid, comprehensive automated protein sequence analyses to support selection of proteins suitable as targets for driving the development of reagents for pathogen or protein toxin detection. Because a large number of open-source tools were needed, it was necessary to produce a software system to scale the computations for whole-proteome analysis. Thus, we built a fully automated system for executing software tools and for storage, integration, and display of automated protein sequence analysis and annotation data.     

Feeding studies take guts – critical review and recommendations of methods for stomach contents analysis in fish

Studies on the feeding ecology of fish are essential for exploring and contrasting trophic interactions and population and community dynamics within and among aquatic ecosystems. In this respect, many different methods have been adopted for the analysis of fish stomach contents. No consensus has, however, been reached for a standardised methodology despite that for several decades there has been an ongoing debate about which methodical approaches that should be preferred. Here, we critically review and scrutinise methods, addressing their strengths and weaknesses and emphasising inherent problems and possible pitfalls in their use. Although our critical assessment reveals that no completely ideal approach exists, appropriate and reliable procedures can be adopted through careful considerations and implementation. In particular, we advocate that different objectives require different methodical approaches and the choice of method should therefore be closely linked to the research questions that are addressed. For a standardisation of methods, we recommend a combination of the relative-fullness and presence–absence methods as the optimal approach for the commonly applied feeding studies addressing relative dietary composition in terms of prey diversity and abundance. Additionally, we recommend the gravimetric method for objectives related to the quantification of food consumption rates and the numerical method for prey selection studies. DNA-based dietary analysis provides a new and promising complementary approach to visual examination of stomach contents, although some technical challenges still exist. The suggested method standardisation facilitates comparisons across species, ecosystems and time and will enhance the applicability and benefits of fish feeding studies in trophic ecology research.     

Feeder cell density—A key parameter in human embryonic stem cell culture

Summary A key issue in human embryonic stem (ES) cell culture that has largely been ignored is the high degree of variability in the murine embryonic fibroblast (MEF) feeder cell density, which has been reported by different studies and protocols. Presumably, too low a feeder cell density would result in insufficient levels of secreted factors, extracellular matrix, and cellular contacts provided by the feeder cells for the maintenance of human ES cells in the undifferentiated state. Too high a feeder cell density, on the other hand, may result in a more rapid depletion of nutrients and oxygen within the in vitro culture milieu, as well as physically hinder the attachment and growth of ES colonies during serial passaging. Preliminary investigations by our group revealed that an elevated MEF cell density of 32,000 cells/cm², above the recommended value of 20,000 cells/cm², appeared to be highly detrimental to the attachment and growth of serially passaged ES colonies of the H9 line (WiCell Research Institute Inc., Wilmington, MA, USA). At the edge of ES colonies that have attached to the higher density feeder layer (32,000 cells/cm²), the ES cells appear to stack up to form a “bulge.” This was not observed under the recommended feeder cell density of 20,000 cells/cm². By contrast, other established ES cell lines are routinely propagated at much higher feeder densities of 60,000 to 70,000 cells/cm². This report briefly discusses the issue of MEF feeder cell density in relation to our preliminary observations, and the results of other studies.