Mammalian Cell Culture

The ethanolic extract of propolis, especially the Brazilian green type, is widely and mainly used for therapeutic purposes despite the lack of knowledge about its effects and its cellular mode of action. This type of propolis, derived from Baccharis dracunculifolia (alecrim-do-campo), has been extensively commercialized and the consumers use it to enhance health. This work aimed to assess the genotoxic/mutagenic and antigenotoxic/antimutagenic potential of the ethanolic extracts of Brazilian green propolis and of B. dracunculifolia, on mammalian cells. It was not observed genotoxic and mutagenic effect by both extracts. After evaluate the exposure of the cells to each extract with a recognized mutagen, simultaneously, the results showed a significant reduction on DNA damage. The experiment carried out with a pre-incubation period was more effective than without incubation test, showing that the tested extracts were able to inactivate the mutagen before it could react with the DNA.

Cell culture is one of the technique of biological science research that lead to rapid and exiting discovery in many fields such as virology, immunology, cytology, cytochemistry, toxicology and molecular biology. This technique make us able grow cells outside of their natural environment (in vitro). In this technique, we can control and monitor the cell’s physicochemical environment such as  pH and temperature. In this practice, MCF-7 (human breast cancer cell lines) which are continuous (non-primary culture) is used.

Process intensification strives for more efficient conversion of raw materials into products while minimizing resource usage. In the biopharma industry, typical intensification gains include increased plant capacities, reduced raw material costs, smaller facilities and improved sustainability. Rapid growth combined with increasingly diverse and challenging molecule formats in the biopharma sector necessitate strategies for fast development of highly productive and cost-efficient processes. Using monoclonal antibody production in CHO cells as example, we present intensification techniques and process sequences that deliver synergistic benefits like increasing space-time yields up to 10-fold, shortening production runs by 30%, or saving numerous days in cell expansion. Combining highly productive perfusion with continuous downstream purification promises improved yields and production economics, especially for advanced, often labile molecules. Predictive scale-down models demonstrated here for selecting intensification-ready cell lines and for rapid setup of intensified processes are essential to realize such benefits broadly in the industry. They enable testing and quick tailoring of intensification alternatives like high-inoculation fed-batch or perfusion to suit molecule needs, facility space, and financial conditions. Since many techniques also readily extend to other protein biotherapeutics, intensification can be regarded as key pillar enabling fast, cost-efficient development and production of biopharma products in the coming years.

Tissue engineering is a newly emerging biomedical technology, which aids and increases the repair and regeneration of deficient and injured tissues. It employs the principles from the fields of materials science, cell biology, transplantation, and engineering in an effort to treat or replace damaged tissues. Tissue engineering and development of complex tissues or organs, such as heart, muscle, kidney, liver, and lung, are still a distant milestone in twenty-first century. Generally, there are four main challenges in tissue engineering which need optimization. These include biomaterials, cell sources, vascularization of engineered tissues, and design of drug delivery systems. Biomaterials and cell sources should be specific for the engineering of each tissue or organ. On the other hand, angiogenesis is required not only for the treatment of a variety of ischemic conditions, but it is also a critical component of virtually all tissue-engineering strategies. Therefore, controlling the dose, location, and duration of releasing angiogenic factors via polymeric delivery systems, in order to ultimately better mimic the stem cell niche through scaffolds, will dictate the utility of a variety of biomaterials in tissue regeneration. This review focuses on the use of polymeric vehicles that are made of synthetic and/or natural biomaterials as scaffolds for three-dimensional cell cultures and for locally delivering the inductive growth factors in various formats to provide a method of controlled, localized delivery for the desired time frame and for vascularized tissue-engineering therapies.

A strategy for adaptive control and energetic optimization of aerobic fermentors was implemented, with both air flow and agitation speed as manipulated variables. This strategy is separable in its components: control, optimization, estimation. We optimized parameter’s estimation (from the usual KLa correlation) using sinusoidal excitation of air flow and agitation speed. We have implemented parameter’s estimation trough recursive least squares algorithm with forgetting factor. We carried separate essays on control, optimization and estimation algorithms. We carried our essays using an original computational simulation environment, with noise and delay generating facilities for data sampling and filtering.

Our results show the convergence and robustness of the estimation algorithm used, improved with use of both forgetting factor and KLa dead-band facilities. Control algorithm used in our work compares favorably with PID using the integrated area criteria for deviation between oxygen molarity and critical molarity (set point). Optimization algorithm clearly reduces energetic consumption, respecting critical molarity. Integration of control, optimization and adaptive algorithms was implemented, but future work is needed for stability. Methods were defined and implemented for stability improvement. We have implemented data acquisition and computer manipulation of air flow and agitation speed for actual fermentors.

Proteína de fusión con direccionamiento de antígenos vacunales a células presentadoras de antígeno y sus aplicaciones. Se presenta una construcción génica que comprende operativamente unidas al menos una secuencia nucleotídica (A) que codifica para un polipéptido de SEQ ID Nº 1 que presenta una región que reconoce a la cadena b del antígeno DR de la Clase II presente en la superficie de las células presentadoras del antígeno, y una secuencia nucleotídica (B) que codifica para un antígeno vacunal de interés. Además se refiere a vectores recombinantes útiles para la expresión de la construcción génica de la presente, células y plantas transgénicas transformadas o transfectadas con dichos vectores, proteínas de fusión codificadas por la construcción génica presentada y vacunas que comprendan dichas proteínas de fusión.
[Patente P080101073]

Vero cells are nowadays widely used in the production of human vaccines. They are considered as one of the most productive and flexible continuous cell lines available for vaccine manufacturing. However, these cells are anchorage dependent, which greatly complicates upstream processing and process scale-up. Moreover, there is a recognized need to reduce the costs of vaccine manufacturing to develop vaccines that are affordable worldwide. The use of cell lines adapted to suspension growth contributes to reach this objective. The current work describes the adaptation of Vero cells to suspension culture in different serum free media according to multiple protocols based on subsequent passages. The best one that relies on cell adaption to IPT-AFM an in-house developed animal component free medium was then chosen for further studies. Besides, as aggregates have been observed, the improvement of IPT-AFM composition and mechanical dissociation were also investigated. In addition to IPT-AFM, three chemically defined media (CD293, Hycell CHO and CD-U5) and two serum free media (293SFMII and SFM4CHO) were tested to set up a serum free culture of the suspensionadapted Vero cells (VeroS) in shake flasks. Cell density levels higher than 2 Â 10 6 cells/mL were obtained in the assessed conditions. The results were comparable to those obtained in spinner culture of adherent Vero cells grown on Cytodex 1 microcarriers. Cell infection with LP-2061 rabies virus strain at an MOI (Multiplicity of Infection) of 0.1 and a cell density of 8 ± 0.5 Â 10 5 cells/mL resulted in a virus titer higher than 10 7 FFU/mL in all media tested. Nevertheless, the highest titer equal to 5.2 ± 0.5 Â 10 7 FFU/mL, was achieved in IPT-AFM containing a reduced amount of Ca ++ and Mg ++. Our results demonstrate the suitability of the obtained VeroS cells to produce rabies virus at a high titer, and pave the way to develop VeroS cells bioreactor process for rabies vaccine production.

The application of fluorescence excitation-emission matrix (EEM) spectroscopy to the quantitative analysis of complex, aqueous solutions of cell culture media components was investigated. These components, yeastolate, phytone, recombinant human insulin, eRDF basal medium, and four different chemically defined (CD) media, are used for the formulation of basal and feed media employed in the production of recombinant proteins using a Chinese Hamster Ovary (CHO) cell based process. The comprehensive analysis (either identification or quality assessment) of these materials using chromatographic methods is time consuming and expensive, and not suitable for high throughput quality control. The use of EEM in conjunction with multi-way chemometric methods provided a rapid, non-destructive analytical method suitable for the screening of large numbers of samples. Here we used multiway robust principal component analysis (MROBPCA) in conjunction with n-way partial least-squares-discriminant analysis (NPLS-DA) to develop a robust routine for both the identification and quality evaluation of these important cell culture materials. These methods are applicable to a wide range of complex mixtures since they do not rely on any predetermined compositional or property information, thus making them potentially very useful for sample handling, tracking, and quality assessment in biopharmaceutical industries.

Porous bioresorbable polymers have been widely used as scaffolds in tissue engineering. Most of the bioresorbable scaffolds are aliphatic polyesters and the methods employed to prepare the porous morphology may vary. This work describes and evaluates the in vitro degradation of porous and dense scaffolds of poly(e-caprolactone) (PCL) and poly(D,L-lactic acid-co-glycolic acid) (50/50) (PLGA50) prepared by particulate  leaching-melt compression process. Biological evaluation was carried out using osteoblast cell cultures. The results showed an autocatalytic effect on the dense samples. Osteoblasts presented intermediate adhesion and the cell morphology on the surface of these materials was dispersed, which indicated a good interaction of the cells with the surface and the material.

ClanTox (classifier of animal toxins) was developed for identifying toxin-like candidates from complete proteomes. Searching mammalian proteomes for short toxin-like proteins (coined TOLIPs) revealed a number of overlooked secreted short proteins with an abundance of cysteines throughout their sequences. We applied bioinformatics and data-mining methods to infer the function of several top predicted candidates. We focused on cysteine-rich peptides that adopt the fold of the three-finger proteins (TFPs). We identified a cluster of duplicated genes that share a structural similarity with elapid neurotoxins, such as α-bungarotoxin. In the murine proteome, there are about 60 such proteins that belong to the Ly6/uPAR family. These proteins are secreted or anchored to the cell membrane. Ly6/uPAR proteins are associated with a rich repertoire of functions, including binding to receptors and adhesion. Ly6/uPAR proteins modulate cell signaling in the context of brain functions and cells of the innate immune system. We postulate that TOLIPs, as modulators of cell signaling, may be associated with pathologies and cellular imbalance. We show that proteins of the Ly6/uPAR family are associated with cancer diagnosis and malfunction of the immune system.

Capturing cell-secreted extracellular matrix (ECM) proteins through cooperative binding with high specificity and affinity is an important function of native tissue matrices during both tissue homeostasis and repair. However, while synthetic hydrogels, such as those based on poly(ethylene glycol) (PEG), are often proposed as ideal materials to deliver human mesenchymal stem cells (hMSCs) to sites of injury to enable tissue repair, they do not have this capabilitya capability that would enable cells to actively remodel their local extracellular microenvironment and potentially provide the required feedback control for more effective tissue genesis. In this work, we detail a methodology that engenders poly-(ethylene glycol) (PEG)-based two-dimensional substrates and three-dimensional porous hydrogels with the ability to capture desired extracellular matrix (ECM) proteins with high specificity. This " encoded " ECM protein capture is achieved by decorating the PEG-based materials with protein binding peptides (PBPs) synthesized to be specific in their binding of fibronectin, laminin, and collagen I, which are not only the most omnipresent ECM proteins in human tissues but, as we confirmed, are also secreted to differing extents by hMSCs under in vitro maintenance conditions. By encapsulating hMSCs into these PBP-functionalized hydrogels, and culturing them in protein-free maintenance media, we demonstrate that these PBPs not only actively recruit targeted ECM proteins as they are secreted from hMSCs but also retain them to much higher levels compared to nonfunctionalized gels. This novel approach thus enables the fabrication of encoded surfaces and hydrogels that capture cell-secreted proteins, with high specificity and affinity, in a programmable manner, ready for applications in many bioengineering applications, including bioactive surface coatings, bioassays, stem cell culture, tissue engineering, and regenerative medicine.

Objective: This study assesses phytoconstituents of Calamus tenuis Roxb. (CT) and further studies its cytotoxic potential in two carcinoma cells lines. Method: Phytochemical extraction was done using hexane, ethyl acetate and methanol as extraction solvents. Cytotoxic potential was assessed via MTT assay against A549 and MCF-7 cell lines. Results: Hexane extract was rich in saponins and steroids whereas, ethyl acetate extract recorded presence of steroids only. These fractions were not able to induce any cytotoxic effect on the said cell lines. However, methanolic extract containing carbohydrates, saponins, flavonoids, steroids, tannins and glycosides accounted for significant cytotoxic activity. Conclusion: This study is the first to investigate the phytoconstituents of Calamus tenuis Roxb. shoot and its subsequent cytotoxicity on carcinoma cells that implies towards its anticancer potential.

Strategies for expanding the sensor space of designer receptors are urgently needed to tailor cell-based therapies to respond to any type of medically relevant molecules. Here, we describe a universal approach to designing receptor scaffolds that enables antibody-specific molecular input to activate JAK/STAT, MAPK, PLCG or PI3K/Akt signaling rewired to transgene expression driven by synthetic promoters. To demonstrate its scope, we equipped the GEMS (generalized extracellular molecule sensor) platform with antibody fragments targeting a synthetic azo dye, nicotine, a peptide tag and the PSA (prostate-specific antigen) biomarker, thereby covering inputs ranging from small molecules to proteins. These four GEMS devices provided robust signaling and transgene expression with high signal-to-noise ratios in response to their specific ligands. The sensitivity of the nicotine- and PSA-specific GEMS devices matched the clinically relevant concentration ranges, and PSA-specific GEMS were able to detect pathological PSA levels in the serum of patients diagnosed with prostate cancer.

The goal of this project is to develop and utilize a microfluidic device to isolate and enrich tumor cells circulating in peripheral blood. Circulating tumor cells (CTCs) have been used as biomarkers for cancer diagnostics and prognosis. However, the rarity of these cells (roughly 1 in a billion) poses a technological challenge to sort and concentrate them. CTCs hold potential sources for early detection, characterization, monitoring of cancers, and development of personalized treatment. The project is currently funded by the US National Cancer Institute (NCI) of the National Institutes of Health (NIH).

Inflammatory episodes observed in multiple sclerosis are characterized by perivascular infiltration of central nervous system (CNS) tissue by circulating leucocytes directed by chemokines and activation of both microglia and astrocytes. An in vitro approach, using a three-dimensional aggregate culture system derived from foetal rat brain tissue, is being taken to study the effect of inflammatory mediators on CNS cells. These aggregates model the brain in vivo in terms of glial and neuronal development and cell-cell interactions. Aggregates cultures were treated with lipopolysaccharide (LPS) and the pro-inflammatory cytokines tumour necrosis factor-alpha (TNF-α) and interferon-gamma (IFNγ). The effect of these treatments on the expression of a range of chemokine receptors (CCR1, CCR2, CCR5, CXCR3 and CX3CR1) and other markers of cell activation (MHC class I and II, CR3) was determined at the mRNA level by semiquantitative RT-PCR and at the protein level by confocal microscopy. Rat peritoneal macrophages were loaded with a fluorescent cell-tracer and their ability to infiltrate inflamed aggregates treated with IFNγ was analysed by FACS and confocal microscopy.

The identification and characterization of many biological substructures at high resolution requires the use of electron microscopy (EM) technologies. Scanning electron microscopy (SEM) allows the resolution of cellular structures to approximately 3 nm and has facilitated the direct visualization of macromolecular structures, such as nuclear pore complexes (NPCs), which are essential for nucleo-cytoplasmic molecular trafficking. However, SEM generates only static images of fixed samples and therefore cannot give unambiguous information about protein dynamics. The investigation of active processes and analysis of protein dynamics has greatly benefited from the development of molecular biology techniques whereby vectors can be generated and transfected into tissue culture cells for the expression of specific proteins tagged with a fluorescent moiety for real-time light microscopy visualization. As light microscopy is limited in its powers of resolution relative to electron microscopy, it has been important to adapt a protocol for the processing of samples for real-time imaging by conventional light microscopy with protein labels that can also be identified by SEM. This allows correlation of dynamic events with high resolution molecular and structural identification. This method describes the use of GFP for tracking the dynamic distribution of NPC components in real-time throughout the cell cycle and for high resolution immuno-SEM labeling to determine localization at the nanometer level.

Post the industrial revolution, there has been a rapid development in almost all the fields of science and technology, providing a better standard of living. We the humans in the quest for a luxurious life have either knowingly or unknowingly brought about disturbances in the environmental homeostasis. Environmental pollution, climate change, outbreak of life threatening diseases, and crop loss are some of the hardships being faced by the mankind due to developmental operations. There is a need of strong scientific and logical interventions or approach to amend for the loss that did occur in the name of developments. In such a scenario, biotechnology offers a good option for sustainable development of the humans and their environment. The book in its current form discusses the possible interventions that can be carried out employing biotechnology for betterment of human life.
With no prior experience of editing a book, making this dream come true took more than a year, lot of patience, and hard work. Finally, it was a nice learning experience that would surely help me in my future projects. I would like to thank the contributors in the form of students, scholars, scientists, and professors for helping me in this journey with their valuable time.
The book offers scientific knowledge on various topics which includes environment, microbiology, diseases, immunology, in-vitro fertilization, pharmacy, food technology, and many more. I am quitte assured that the book will serve the interest of the scientific society as well as students and common masses. I hereby thus present this book titled – ―Biotechnological interventions in current scientific scenario: Way forward to a healthy future‖ to the open public.

Biomaterials prepared from polyesters of lactic acid and glycolic acid, or a mixture of the two, degrade in the presence of water into the naturally occurring metabolites, lactic acid and glycolic acid. While the lactic acid degradation product that is released from biomaterials is well tolerated by the body, lactic acid can influence the metabolic function of cells; it can serve as an energy substrate for cells, and has been shown to have antioxidant properties. Neural precursor cells, a cell population of considerable interest as a source of cells for neural tissue regeneration strategies, generate a high amount of reactive oxygen species, and when associated with a degradable biomaterial, may be impacted by released lactic acid. In this work, the effect of lactic acid on a neural cell population containing proliferative neural precursor cells was examined in monolayer culture. Lactic acid was found to scavenge exogenously added free radicals produced in the presence of either hydrogen peroxide or a photoinitiator (I2959) commonly utilized in the preparation of photopolymerizable biomaterials. In addition to its effect on exogenously added free radicals, lactic acid reduced intracellular redox state, increased the proliferation of the cell population, and modified the cell composition. The findings of this study provide insight into the role that lactic acid plays naturally on developing neural cells and are also of interest to biomaterials scientists that are focused on the development of degradable lactic-acid-based polymers for cell culture devices. The effect of lactic acid on other cell populations may differ and should be characterized to best understand how cells function in degradable cell culture devices.

Monitoring mammalian cell culture with UV-vis spectroscopy has not been widely explored. The aim of this work was to calibrate Partial Least Squares (PLS) models from off-line UV-vis spectral data in order to predict some nutrients and metabolites, as well as viable cell concentrations for mammalian cell bioprocess using phenol red in culture medium. The BHK-21 cell line was used as a mammalian cell model. Spectra of samples taken from batches performed at different dissolved oxygen concentrations (10, 30, 50, and 70% air saturation), in two bioreactor configurations and with two strategies to control pH

Precise control of gene expression with small-molecular or physical inducers has been a central aim of synthetic biology in recent decades, and has led, for example, to dramatic improvements in the production of protein therapeutics. However, the number of inputs for inducible promoters and gene circuits is still limited, and new orthogonal inducers are needed to support the construction of more complex programmable systems in the fields of bioengineering and biocomputing. Here, two new gene switches inducible by the antibiotic simocyclinone D8 (SD8) and the flavonoid luteolin in mammalian cells are presented. Streptomyces antibioticus Tü 6040 has evolved the DNA gyrase inhibitor SD8, which, through the bacterial TetR-like transcriptional repressor SimR, also regulates the multidrug efflux pump SimX. Taking advantage of SimR, as well as the luteolin-binding TetR-like transcriptional repressor EmrR from Sinorhizobium meliloti, we optimized the amounts of trans-fected genes and DNA operator sites to engineer highly effective, orthogonal transcriptional OFF-as well as ON-switches triggered by SD8 and/or luteolin. We confirmed that SD8 and luteolin are not cytotoxic at the concentrations required for switching, and demonstrated the functionality of these gene switches in a range of biotechnologically relevant cell lines. These switches were combined to generate OR and AND Boolean logic gates, and we confirmed their modularity by the addition of the vanillic acid-responsive transcriptional repressor VanR to generate a three-input AND gate. These additions to the panoply of inducers available for synthetic biology are expected to facilitate advances in the fields of biocomputing, biopharmaceu-tical manufacturing, and biomedicine.

Synthetic biology, the synthesis of engineering and biology, has rapidly matured and has dramatically increased the complexity of artificial gene circuits in recent years. The deployment of intricate synthetic gene circuits in mammalian cells requires the establishment of very precise and orthogonal control of transgene expression. In this chapter, we describe methods of modulating the expression of transgenes at the transcriptional level. Using cAMP-response element-binding protein (CREB)-dependent promoters as examples, a tool for the precise tuning of gene expression by using different core promoters and by varying the binding affinity of transcription factor operator sites is described.

Since their discovery in the 1980s and 1990s, the human protein lysine acetyltransferase encoded by the paralogous p300 and CBP genes has received much interest. p300/CBP functions in regulating the expression of genes controlling several basic cellular processes, such as proliferation and homeostasis, and plays a role in a variety of human diseases, particularly solid tumors.

Numerous natural product and synthetic inhibitors of the acetyltransferase activity have been identified and used to generate a crystal structure of the active site, probe the p300/CBP enzyme kinetics, and interrogate p300/CBP cellular functions. These studies revealed that acetyl-CoA binds in a tunnel enclosed by a unique loop, while the substrate protein transiently associates with an acidic patch, following a hit-and-run kinetic mechanism. p300/CBP acetylates histones as well as other proteins including other epigenetic enzymes and transcription factors. Studies with inhibitor compounds in cells and animals have confirmed that the p300/CBP acetylation activity has roles in diverse functions including cell migration and invasion, maintenance of the differentiated state, tau-mediated neurodegeneration, and learning and memory.

We describe an in vitro culture process that
uses 100-lm diameter poly(ethylene terephthalate) monofilaments
as contact guidance of human umbilical vein endothelial
cells (HUVECs) to orient the development of
microvessels in a 3D environment. Untreated fibers, distanced
either by 0.05, 0.1, 0.15, or 0.2 mm were first covered
with HUVECs and then sandwiched between two
layers of fibrin gel containing HUVECs. After 2 and 4
days of culture, cell connections and microvessels were
evaluated. Cell connections formed massively along and
in between adjacent fibers that were distanced by 0.05
and 0.1 mm, whereas with fibers separated by larger distances,
connections were rare. After 4 days of culture, the
optimum fiber-to-fiber distance to form microvessels was
0.1 mm. This study reveals that polymer fibers embedded
in gel can be used as guides to direct the microvascularization
process, with potential applications in cancer and
cardiovascular research and tissue engineering.

Yeastolate or yeast extract which are hydrolysates produced by autolysis of yeast, are often employed as a raw material in the media used for industrial mammalian cell culture. The source and quality of yeastolate can significantly affect cell growth and production, however, analysis of these complex biologically-derived materials is not straightforward. The best current method, liquid chromatography-mass spectrometry (LC-MS) is time-consuming and requires extensive expertise. This study describes the use of surface-enhanced Raman scattering (SERS) and fluorescence excitation-emission matrix (EEM) spectroscopy coupled with robust principal component analysis (ROBPCA) for the rapid and facile characterization and discrimination of yeast extracts in aqueous solution. SERS using silver colloids generates time-dependant signals where adenine is the strongest contributor, and the spectra are stable and reproducible (< ~3%) at 180 minutes after mixing. Combining this spectral behavior with chemometric methods enables SERS to be used for discriminating between different yeastolate sources, for assessing lot-to-lot variability, and potentially to monitor storage-induced compositional changes. Fluorescence EEM combined with multiway ROBPCA also provides a rapid and inexpensive method for discrimination of yeastolate, yielding very similar results in terms of sample discrimination to that obtained by SERS. However, the EEM data does not provide the same level of chemical information as provided by the SERS. Thus the combination of the two methodologies has the potential to be extremely useful in biopharmaceutical manufacturing for the rapid characterization and screening of biogenic hydrolysates from animal or plant sources.

Capitalizing on the ability of mammalian cells to conduct complex post-translational modifications, most protein therapeutics are currently produced in cell culture systems. Addition of a signal peptide to the product protein enables its accumulation in the cell culture supernatant, but separation of the product from endogenously secreted proteins remains costly and labor-intensive. We considered that global downregulation of translation of non-product proteins would be an efficient strategy to minimize downstream processing requirements. Therefore, taking advantage of the ability of mammalian protein kinase R (PKR) to switch off most cellular translation processes in response to infection by viruses, we fused a caffeine-inducible dimerization domain to the catalytic domain of PKR. Addition of caffeine to this construct results in homodimerization and activation of PKR, effectively rewiring rapid global translational downregulation to the addition of the stimulus in a dose-dependent manner. Then, to protect translation of the target therapeutic, we screened viral and cellular internal ribosomal entry sites (IRESes) known or suspected to be resistant to PKR-induced translational stress. After choosing the best-in-class Seneca valley virus (SVV) IRES, we additionally screened for IRES transactivation factors (ITAFs) as well as for supplementary small molecules to further boost the production titer of the product protein under conditions of global translational downregulation. Importantly, the residual global translation activity of roughly 10% under maximal downregulation is sufficient to maintain cellular viability during a production timeframe of at least five days. Standard industrially used adherent as well as suspension-adapted cell lines transfected with this synthetic biology-inspired Protein Kinase R-Enhanced Protein Production (PREPP) system could produce several medicinally relevant protein therapeutics, such as the blockbuster drug rituximab, in substantial quantities and with significantly higher purity than previous culture technologies. We believe incorporation of such purity-by-design technology in the production process will alleviate downstream processing bottlenecks in future biopharmaceutical manufacturing.

Programming cellular behavior using trigger-inducible gene switches is integral to synthetic biology. Although significant progress has been achieved in trigger-induced transgene expression, side-effect-free remote control of transgenes continues to challenge cell-based therapies. Here, utilizing a caffeine-binding single-domain antibody we establish a caffeine-inducible protein dimerization system, enabling synthetic transcription factors and cell-surface receptors that enable transgene expression in response to physiologically relevant concentrations of caffeine generated by routine intake of beverages such as tea and coffee. Coffee containing different caffeine concentrations dose-dependently and reversibly controlled transgene expression by designer cells with this caffeine-stimulated advanced regulators (C-STAR) system. Type-2 diabetic mice implanted with microencapsulated, C-STAR-equipped cells for caffeine-sensitive expression of glucagon-like peptide 1 showed substantially improved glucose homeostasis after coffee consumption compared to untreated mice. Biopharmaceutical production control by caffeine, which is non-toxic, inexpensive and only present in specific beverages, is expected to improve patient compliance by integrating therapy with lifestyle.