Rapid prototyping as a tool for manufacturing bioartificial livers

Rapid prototyping (RP) technologies are a set of manufacturing processes that can produce very complex structures directly from computer-aided design models without structure-specific tools or knowledge. These technologies might eventually enable the manufacture of human livers to create functional substitutes for treating liver failure or dysfunctionality. However, the approaches used currently face many challenges, such as the complex branched vascular and bile ductular systems and the variety of cell types, matrices and regulatory factors involved in liver development. Here, we discuss the challenges and provide evidence for the usefulness of RP in overcoming them.     

The program Proteocat as a tool for planning of proteomic experiments

ProteoCat is a computer program that has been designed to help researchers in the planning of large-scale proteomic experiments. The central part of this program is the unit of hydrolysis simulation that supports 4 proteases (trypsin, lysine C, endoproteinases Asp-N and GluC). For peptides obtained after virtual hydrolysis or loaded from data files a number of properties important in mass-spectrometric experiments can be calculated and predicted; the resultant data can be analyzed or filtered (to reduce a set of peptides). The program is using new and improved modifications of own earlier developed methods for pI prediction, which can be also predicted by means of popular pKa scales proposed by other reseachers. The algorithm for prediction of peptide retention time has been realized similarly to the algorithm used in the SSRCalc program. Using ProteoCat it is possible to estimate the coverage of amino acid sequences of analyzed proteins under defined limitation on peptides detection, as well as the possibility of assembly of peptide fragments with user-defined minimal sizes of “sticky” ends. The program has a graphical user interface, written on JAVA and available at http://www.ibmc.msk.ru/LPCIT/ProteoCat.     

A tool for modeling strategic decisions in cell culture manufacturing

The development of a prototype tool for modeling manufacturing in a biopharmaceutical plant is discussed. A hierarchical approach to modeling a manufacturing process has been adopted to confer maximum user flexibility. The use of this framework for assessing the impact of manufacturing decisions on strategic technical and business indicators is demonstrated via a case study. In the case study, which takes the example of a mammalian cell culture process delivering a therapeutic for clinical trials, the dynamic modeling tool indicates how manufacturing options affect the demands on resources and the associated manufacturing costs. The example illustrates how the decision-support software can be used by biopharmaceutical companies to investigate the effects of working toward different strategic goals on the cost-effectiveness of the process, prior to committing to a particular option.     

Embryonic stem cells: a promising tool for cell replacement therapy

Embryonic stem (ES) cells are revolutionizing the field of developmental biology as a potential tool to understand the molecular mechanisms occurring during the process of differentiation from the embryonic stage to the adult phenotype. ES cells harvested from the inner cell mass (ICM) of the early embryo can proliferate indefinitely in vitro while retaining the ability to differentiate into all somatic cells. Emerging results from mice models with ES cells are promising and raising tremendous hope among the scientific community for the ES-cell based cell replacement therapy (CRT) of various severe diseases. ES cells could potentially revolutionize medicine by providing an unlimited renewable source of cells capable of replacing or repairing tissues that have been damaged in almost all degenerative diseases such as diabetes, myocardial infarction and Parkinson's disease. This review updates the progress of ES cell research in CRT, discusses about the problems encountered in the practical utility of ES cells in CRT and evaluates how far this approach is successful experimentally.     

Embryonic stem-cell culture as a tool for developmental cell biology

The cell biology of the early processes of mammalian embryogenesis, such as germ-layer formation, has been technically challenging to study owing to the size and accessibility of mammalian embryos. Embryonic stem cells, which can generate the three germ layers in vitro, are useful for studying embryogenesis at the cellular level. So, how can the study of embryonic stem cells and their differentiation provide a deeper understanding of the cell biology of early development?     

Abundance and sensitivity of beneficials in deciduous fruit as a decision tool for selectivity testing

Thirty-two selectivity tests on apple trees were evaluated in order to determine the best timing for selectivity tests based on the predominance of various beneficial groups and their sensitivity to pesticides during the season. In the testing method used the whole insect populations of apple trees were measured. For the purpose of this evaluation the numbers of beneficial species were aggregated at the family/order level (Anthocoridae, Miridae, Nabidae, Coccinellidae, Hymenoptera and Araneae, respectively). Logarithmically transformed figures of the species counts in azinphos-methyl- and water-treated trees, plotted over the observation period, demonstrated that the months of June to August are the most suitable ones for carrying out selectivity tests on fruit trees. The sensitivity of the various beneficial groups were obtained by comparing the logits of the mortality rates of azinphos-methyl and water by weighted t -tests. The sensitivity did not vary greatly over the testing season from June to October for all groups except Miridae during the second half of the season. This can be explained by the predominance of the less sensitive adults ready for hibernation.     

The use of p53 as a tool for human cancer therapy

The p53 tumor suppressor is a central component of a system that reinforces genetic stability of somatic cells in animals and humans. Inactivation of this gene occurs virtually in every cancer case, which eliminates results in further rapid accumulation of additional mutations leading to progression of a cancer cell toward more malignant phenotype. The mechanisms of p53 inhibition in cancer include point mutations leading to accumulation of inactive protein, deletion of the whole gene, or its portion, alteration in the genes involved in regulation of activity of p53, and defects in the genes controlled by p53. In addition, oncogenic viruses encode specialized proteins that are entitled to modify p53 functions in order to provide optimal condition for replication of viral genome. These viral proteins play central role in viral carcinogenesis, including 95% of cases of cervical carcinoma in women. The approacheas to restoration of p53 activity depend on particular type of alteration within the p53 pathway. In some cases an effective mean would be introduction of exogenous p53, particularly with the use of adenoviral vectors. There are also approaches in development that target reactivation of mutant proteins, or suppress natural inhibitors of p53. The review summarizes various schemes for therapy and prevention of cancer that are based on our knowledge of the p53 gene functions. Potential usefulness of the approaches for practical applications is discussed.     

Hybrid spheroids as a tool for prediction of radiosensitivity in tumor therapy

Optimization in radiotherapy may be conceivably achieved by individualized treatment regimens. For this, the radiosensitivity of the tumor cells to be treated must be known. A method is presented to show that the effect of radiation on tumor cells in spheroids can be quantitatively evaluated without complicated cell determinations of spheroid composition. This evaluation is based on the dynamics of inactivation of the colony forming ability of whole spheroids composed chiefly of non-transformed diploid fibroblasts and a minority of HeLa "test" cells. Here, spheroids of identical composition, but of different sizes are inactivated proportional to their sizes, thus obviating the need for tedious single cell procedures. The use of spheroids of different sizes permits the deduction of dose-effect relationships, and the innate radiosensitivity of tumors cells. This is a novel method for measuring the radio and chemosensitivity of tumors in primary culture, i.e. cells directly isolated from tumors.     

Mesenchymal stem cells as tool for antitumor therapy

The development of antitumor preparations with low toxicity and high selectivity of action is one of the top priorities of cancer gene therapy. Mesenchymal stem cells possess natural tropism towards tumors, a property that makes it possible to use them as vehicles for the targeted delivery of therapeutic genes to tumors of various etiologies. At present, genes that encode enzymes (cytosine deaminase, thymidine kinase, carboxyl esterase), cytokines (IL-2, IL-4, IL-12, and IFN-β), and apoptosis inducing factors (TRAIL) are used as therapeutic genes. Mesenchymal stem cells, as demonstrated using experimental models of tumors of various etiologies, as well as animals with metastases in brain and lungs, are able to successfully deliver therapeutic genes into tumors and produce a significant antitumor effect. However, to effectively use this therapeutic strategy in a clinical setting, a number of technical problems must be solved.     

Mathematical modeling as a tool for investigating cell cycle control networks

Although not a traditional experimental "method," mathematical modeling can provide a powerful approach for investigating complex cell signaling networks, such as those that regulate the eukaryotic cell division cycle. We describe here one modeling approach based on expressing the rates of biochemical reactions in terms of nonlinear ordinary differential equations. We discuss the steps and challenges in assigning numerical values to model parameters and the importance of experimental testing of a mathematical model. We illustrate this approach throughout with the simple and well-characterized example of mitotic cell cycles in frog egg extracts. To facilitate new modeling efforts, we describe several publicly available modeling environments, each with a collection of integrated programs for mathematical modeling. This review is intended to justify the place of mathematical modeling as a standard method for studying molecular regulatory networks and to guide the non-expert to initiate modeling projects in order to gain a systems-level perspective for complex control systems.     

A fungicide-responsive kinase as a tool for synthetic cell fate regulation

Engineered biological systems that precisely execute defined tasks have major potential for medicine and biotechnology. For instance, gene- or cell-based therapies targeting pathogenic cells may replace time- and resource-intensive drug development. Engineering signal transduction systems is a promising, yet presently underexplored approach. Here, we exploit a fungicide-responsive heterologous histidine kinase for pathway engineering and synthetic cell fate regulation in the budding yeast Saccharomyces cerevisiae. Rewiring the osmoregulatory Hog1 MAPK signalling system generates yeast cells programmed to execute three different tasks. First, a synthetic negative feedback loop implemented by employing the fungicide-responsive kinase and a fungicide-resistant derivative reshapes the Hog1 activation profile, demonstrating how signalling dynamics can be engineered. Second, combinatorial integration of different genetic parts including the histidine kinases, a pathway activator and chemically regulated promoters enables control of yeast growth and/or gene expression in a two-input Boolean logic manner. Finally, we implemented a genetic ‘suicide attack’ system, in which engineered cells eliminate target cells and themselves in a specific and controllable manner. Taken together, fungicide-responsive kinases can be applied in different constellations to engineer signalling behaviour. Sensitizing engineered cells to existing chemicals may be generally useful for future medical and biotechnological applications.     

DNA interpolyelectrolyte complexes as a tool for efficient cell transformation.

A tool was developed for enhancement of plasmid penetration into an intact cell, based on increasing DNA hydrophobicity via inclusion into a soluble interpolyelectrolyte complex (IPC) with polycations. The characteristics of formation of DNA IPC with synthetic polycations [poly(N-ethyl-4-vinylpyridinium)bromide (PVP) and PVP modified with 3% of N-cetyl-4-vinylpyridinium units (PVP-C)] were studied using ultracentrifugation and polyacrylamide gel electrophoresis methods. The conditions were established under which the mixing of DNA and polycation aqueous solutions results in the self-assembly of soluble IPC species. Incorporation of DNA into IPC results in the enhancement of DNA binding with isolated Bacillus subtilis membranes. A considerable increase in the efficiency of transformation of B. subtilis cells with pBC16 plasmid resulted from incorporation of the plasmid into the IPC with PVP and CVP.     

Electrical stimulation as a biomimicry tool for regulating muscle cell behavior

There is a growing need to understand muscle cell behaviors and to engineer muscle tissues to replace defective tissues in the body. Despite a long history of the clinical use of electric fields for muscle tissues in vivo, electrical stimulation (ES) has recently gained significant attention as a powerful tool for regulating muscle cell behaviors in vitro. ES aims to mimic the electrical environment of electroactive muscle cells (e.g., cardiac or skeletal muscle cells) by helping to regulate cell-cell and cell-extracellular matrix (ECM) interactions. As a result, it can be used to enhance the alignment and differentiation of skeletal or cardiac muscle cells and to aid in engineering of functional muscle tissues. Additionally, ES can be used to control and monitor force generation and electrophysiological activity of muscle tissues for bio-actuation and drug-screening applications in a simple, high-throughput, and reproducible manner. In this review paper, we briefly describe the importance of ES in regulating muscle cell behaviors in vitro, as well as the major challenges and prospective potential associated with ES in the context of muscle tissue engineering.     

Hollow fiber modules as a tool for enzyme and cell immobilization

Owing to their properties, hollow fiber modules are attractive carriers for the immobilization of biocatalysts. Various systems and modes of operation are summarized and discussed.     

Cyclodextrins as a useful tool for bioconversions in plant cell biotechnology

The application of cyclodextrins as precursor solubilizers in biotechnological processes, in which plant cells are involved, is new. In this paper the possibilities for cyclodextrin facilitated bioconversions by freely suspended and/or immobilized plant cells or plant enzymes are demonstrated. After complexation with -cyclodextrin, the phenolic steroid 17-estradiol could be ortho-hydroxylated into a catechol, mainly 4-hydroxyestradiol, by a phenoloxidase from in vitro grown cells of Mucuna pruriens. By complexation with -cyclodextrin the solubility of the steroid increased from almost insoluble to 660 M. In addition, by complexation with -cyclodextrin, a solution of 3 mM coniferyl alcohol could be fed to cell cultures of Podophyllum hexandrum in order to enhance the accumulation of podophyllotoxin. Finally, the glucosylation of podophyllotoxin by cell cultures derived from Linum flavum was investigated. Four cyclodextrins: -cyclodextrin, -cyclodextrin, hydroxypropyl--cyclodextrin and dimethyl--cyclodextrin were used to improve the solubility of podophyllotoxin. Dimethyl--cyclodextrin met our needs the best and the solubility of podophyllotoxin could be enhanced from 0.15 to 1.92 mM. Podophyllotoxin--d-glucoside was formed at a rate of 0.51 mmol l^-1 suspension per day by the L. flavum cells growing in the presence of 1.35 mM podophyllotoxin, complexed with dimethyl--cyclodextrin.Abbreviations DW dry weight - E2 17-estradiol - FW fresh weight - PCV packed cell volume     

Longitudinal two-dimensional gas chromatography mass spectrometry as a non-destructive at-line monitoring tool during cell manufacturing identifies volatile features correlative to cell product quality

Background aimsCell therapies have emerged as a potentially transformative therapeutic modality in many chronic and incurable diseases. However, inherent donor and patient variabilities, complex manufacturing processes, lack of well-defined critical quality attributes and unavailability of in-line or at-line process or product analytical technologies result in significant variance in cell product quality and clinical trial outcomes. New approaches for overcoming these challenges are needed to realize the potential of cell therapies.MethodsHere the authors developed an untargeted two-dimensional gas chromatography mass spectrometry (GC×GC-MS)-based method for non-destructive longitudinal at-line monitoring of cells during manufacturing to discover correlative volatile biomarkers of cell proliferation and end product potency.ResultsSpecifically, using mesenchymal stromal cell cultures as a model, the authors demonstrated that GC×GC-MS of the culture medium headspace can effectively discriminate between media types and tissue sources. Headspace GC×GC-MS identified specific volatile compounds that showed a strong correlation with cell expansion and product functionality quantified by indoleamine-2,3-dioxygenase and T-cell proliferation/suppression assays. Additionally, the authors discovered increases in specific volatile metabolites when cells were treated with inflammatory stimulation.ConclusionsThis work establishes GC×GC-MS as an at-line process analytical technology for cell manufacturing that could improve culture robustness and may be used to non-destructively monitor culture state and correlate with end product function.