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Advances in mechanistic knowledge of human neurological disorders have been hindered by the lack of adequate human in vitro models. Three-dimensional (3D) cellular models displaying higher biological relevance are gaining momentum; however, their lack of robustness and scarcity of analytical tools adapted to three dimensions hampers their widespread implementation. Herein we show that human midbrain-derived neural progenitor cells, cultured as 3D neurospheres in stirred culture systems, reproducibly differentiate into complex tissue-like structures containing functional dopaminergic neurons, as well as astrocytes and oligodendrocytes. Moreover, an extensive toolbox of analytical methodologies has been adapted to 3D neural cell models, allowing molecular and phenotypic profiling and interrogation. The generated neurons underwent synaptogenesis and elicit spontaneous Ca 2+ transients. Synaptic vesicle trafficking and release of dopamine in response to depolarizing stimuli was also observed. Under whole-cell current-and-voltage clamp, recordings showed polarized neurons ( V m =−70 mV) and voltage-dependent potassium currents, which included A-type-like currents. Glutamate-induced currents sensitive to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-D-aspartate antagonists revealed the existence of functional glutamate receptors. Molecular and phenotypic profiling showed recapitulation of midbrain patterning events, and remodeling toward increased similarity to human brain features, such as extracellular matrix composition and metabolic signature. We have developed a robust and reproducible human 3D neural cell model, which may be extended to patient-derived induced pluripotent stem cells, broadening the applicability of this model.

Engineering human brain organoids with floating scaffolds enhances the maturity and reproducibility of cortical tissue structure. Three-dimensional cell culture models have either relied on the self-organizing properties of mammalian cells 1 , 2 , 3 , 4 , 5 , 6 or used bioengineered constructs to arrange cells in an organ-like configuration 7 , 8 . While self-organizing organoids excel at recapitulating early developmental events, bioengineered constructs reproducibly generate desired tissue architectures. Here, we combine these two approaches to reproducibly generate human forebrain tissue while maintaining its self-organizing capacity. We use poly(lactide-co-glycolide) copolymer (PLGA) fiber microfilaments as a floating scaffold to generate elongated embryoid bodies. Microfilament-engineered cerebral organoids (enCORs) display enhanced neuroectoderm formation and improved cortical development. Furthermore, reconstitution of the basement membrane leads to characteristic cortical tissue architecture, including formation of a polarized cortical plate and radial units. Thus, enCORs model the distinctive radial organization of the cerebral cortex and allow for the study of neuronal migration. Our data demonstrate that combining 3D cell culture with bioengineering can increase reproducibility and improve tissue architecture.

Knowledge of the effect of foods on gut microbiota composition and functionality is expanding. To isolate the effect of single foods and/or single nutrients (i.e., fiber, polyphenols), this protocol describes an in vitro batch fermentation procedure to be carried out after an in vitro gastrointestinal digestion. Therefore, this is an extension of the previous protocol described by Brodkorb et al. (2019) for studying in vitro digestion. The current protocol uses an oligotrophic fermentation medium with peptone and a high concentration of fecal inoculum from human fecal samples both to provide the microbiota and as the main source of nutrients for the bacteria. This protocol is recommended for screening work to be performed when many food samples are to be studied. It has been used successfully to study gut microbiota fermentation of different foodstuffs, giving insights into their functionality, community structure or ability to degrade particular substances, which can contribute to the development of personalized nutrition strategies. The procedure does not require a specific level of expertise. The protocol takes 4–6 h for preparation of fermentation tubes and 20 h for incubation. This extension of a previous in vitro digestion protocol provides a subsequent in vitro batch fermentation stage that is carried out afterward to enable investigation of the effect of food on the gut microbiome.

Cortical neurons are rapidly derived from human pluripotent stem cells using a cocktail of small molecules. Considerable progress has been made in converting human pluripotent stem cells (hPSCs) into functional neurons. However, the protracted timing of human neuron specification and functional maturation remains a key challenge that hampers the routine application of hPSC-derived lineages in disease modeling and regenerative medicine. Using a combinatorial small-molecule screen, we previously identified conditions to rapidly differentiate hPSCs into peripheral sensory neurons. Here we generalize the approach to central nervous system (CNS) fates by developing a small-molecule approach for accelerated induction of early-born cortical neurons. Combinatorial application of six pathway inhibitors induces post-mitotic cortical neurons with functional electrophysiological properties by day 16 of differentiation, in the absence of glial cell co-culture. The resulting neurons, transplanted at 8 d of differentiation into the postnatal mouse cortex, are functional and establish long-distance projections, as shown using iDISCO whole-brain imaging. Accelerated differentiation into cortical neuron fates should facilitate hPSC-based strategies for disease modeling and cell therapy in CNS disorders.

Hyaluronan is widely used in cosmetics and pharmaceutics. Development of robust and safe cell factories and cultivation approaches to efficiently produce hyaluronan is of many interests. Here, we describe the metabolic engineering of Corynebacterium glutamicum and application of a fermentation strategy to manufacture hyaluronan with different molecular weights. C. glutamicum is engineered by combinatorial overexpression of type I hyaluronan synthase, enzymes of intermediate metabolic pathways and attenuation of extracellular polysaccharide biosynthesis. The engineered strain produces 34.2 g L −1 hyaluronan in fed-batch cultures. We find secreted hyaluronan encapsulates C. glutamicum , changes its cell morphology and inhibits metabolism. Disruption of the encapsulation with leech hyaluronidase restores metabolism and leads to hyper hyaluronan productions of 74.1 g L −1 . Meanwhile, the molecular weight of hyaluronan is also highly tunable. These results demonstrate combinatorial optimization of cell factories and the extracellular environment is efficacious and likely applicable for the production of other biopolymers. Bioproduction of hyaluronan needs increases in yield and greater diversity of the molecular weights. Here, the author increases hyaluronan production and diversifies the molecular weights through engineering the hyaluronan biosynthesis pathway and disruption of Corynebacterium glutamicum encapsulation caused by secreted hyaluronan.

There is currently much interest in fucoxanthin due to its broad beneficial health effects. The major commercial source of fucoxanthin is marine seaweed, which has many shortcomings, and has thus restricted its large-scale production and more diversified applications. In this study, growth characteristics and fucoxanthin accumulation were evaluated to explore the potential of the marine diatom Nitzschia laevis in fucoxanthin production. The results suggested that heterotrophic culture was more effective for cell growth, while the mixotrophic culture was favorable for fucoxanthin accumulation. A two-stage culture strategy was consequently established. A model of exponential fed-batch culture led to a biomass concentration of 17.25 g/L. A mix of white and blue light significantly increased fucoxanthin content. These outcomes were translated into a superior fucoxanthin productivity of 16.5 mg/(L·d), which was more than 2-fold of the best value reported thus far. The culture method established herein therefore represents a promising strategy to boost fucoxanthin production in N. laevis, which might prove to be a valuable natural source of commercial fucoxanthin.

Development of a human embryonic stem cell (hESC)-based therapy for type 1 diabetes will require the translation of proof-of-principle concepts into a scalable, controlled, and regulated cell manufacturing process. We have previously demonstrated that hESC can be directed to differentiate into pancreatic progenitors that mature into functional glucose-responsive, insulin-secreting cells in vivo. In this study we describe hESC expansion and banking methods and a suspension-based differentiation system, which together underpin an integrated scalable manufacturing process for producing pancreatic progenitors. This system has been optimized for the CyT49 cell line. Accordingly, qualified large-scale single-cell master and working cGMP cell banks of CyT49 have been generated to provide a virtually unlimited starting resource for manufacturing. Upon thaw from these banks, we expanded CyT49 for two weeks in an adherent culture format that achieves 50-100 fold expansion per week. Undifferentiated CyT49 were then aggregated into clusters in dynamic rotational suspension culture, followed by differentiation en masse for two weeks with a four-stage protocol. Numerous scaled differentiation runs generated reproducible and defined population compositions highly enriched for pancreatic cell lineages, as shown by examining mRNA expression at each stage of differentiation and flow cytometry of the final population. Islet-like tissue containing glucose-responsive, insulin-secreting cells was generated upon implantation into mice. By four- to five-months post-engraftment, mature neo-pancreatic tissue was sufficient to protect against streptozotocin (STZ)-induced hyperglycemia. In summary, we have developed a tractable manufacturing process for the generation of functional pancreatic progenitors from hESC on a scale amenable to clinical entry.

The general approach to industrial production of monoclonal antibodies is fed-batch culture using Chinese Hamster Ovary (CHO) cells. Perfusion culture is also attracting attention as a next-generation culture method. In these culture methods, optimization of amino acid and glucose concentration in the culture medium is essential, and influences cell proliferation, viability, productivity, and monoclonal antibody quality. Further, the maintenance of optimal nutrient levels – by avoiding both depletion and accumulation – is crucial. This study aimed to develop a dynamic feeding strategy based on specific indicators to maintain optimal amino acid and glucose concentrations. Multivariate correlation analysis confirmed a strong relationship between nutrient consumption and viable cell density (VCD). Regression analysis was used to establish a regression model to estimate amino acid and glucose consumption based on VCD. Using this model, the nutrient composition of feed media for both fed-batch and perfusion cultures was adjusted, and a dynamic feeding strategy guided by VCD was evaluated. The observed nutrient concentration trends closely matched the model’s predictions, confirming that VCD is a reliable indicator for implementing dynamic feeding. In both fed-batch and perfusion cultures, the VCD-guided dynamic feeding strategy enables the maintenance of multiple amino acids and glucose at target concentrations.

Advances in the ethanol fermentation process are essential to improving the performance of bioethanol production. Fed-batch fermentation is a promising approach to increase the final ethanol titer, which benefits the recovery in the bioethanol industry’s downstream process. However, the development of feeding strategies, a crucial control variable in the fed-batch approach, is limited. Thus, in the present work, different modes of substrate delivery—fixed feeding, adapted feeding—were investigated in fed-batch cultures of Saccharomyces cerevisiae in a 5-L bioreactor. Evolved gas production, which was positively correlated with glucose consumption, was used to adjust the sugar feed rate in fed-batch fermentations under an adapted feeding strategy. The adapted feeding strategy enhanced ethanol productivity by 21% compared to the fixed feeding strategy, in which the sugar feed rate was stable, and the ethanol titer reached 91 g/L (~ 11.5%, v/v) at the end of fermentation. Moreover, cell biomass accumulation and cell growth rate were significantly improved when using the adapted feeding strategy. The effect of nitrogen availability on the performance of the adapted feeding strategy was further explored using a low-nitrogen content medium. The results showed that, even under low nitrogen feeding conditions (N/C = 0.046:10), the adapted feeding strategy maintained the same ethanol productivity as nitrogen-rich medium feeding. Overall, these results suggest that sugar delivery with low nitrogen content using the adapted feeding strategy could help reduce medium costs and improve the productivity of current facilities in the ethanol industry.Future work will integrate adapted feeding strategies with other fermentation approaches to improve ethanol production. Graphical abstract Key points • Novel continuous sugar delivery was developed for fed-batch ethanol fermentation. • The adapted feeding strategy improved ethanol productivity by 21%. • The final ethanol concentration reached 91 g/L (11.5%, v/v) with no residual sugar.

Adeno-associated virus (AAV) has shown great promise as a gene therapy vector in multiple aspects of preclinical and clinical applications. Many developments including new serotypes as well as self-complementary vectors are now entering the clinic. With these ongoing vector developments, continued effort has been focused on scalable manufacturing processes that can efficiently generate high-titer, highly pure, and potent quantities of rAAV vectors. Utilizing the relatively simple and efficient transfection system of HEK293 cells as a starting point, we have successfully adapted an adherent HEK293 cell line from a qualified clinical master cell bank to grow in animal component-free suspension conditions in shaker flasks and WAVE bioreactors that allows for rapid and scalable rAAV production. Using the triple transfection method, the suspension HEK293 cell line generates greater than 1 × 105 vector genome containing particles (vg)/cell or greater than 1 × 1014 vg/l of cell culture when harvested 48 hours post-transfection. To achieve these yields, a number of variables were optimized such as selection of a compatible serum-free suspension media that supports both growth and transfection, selection of a transfection reagent, transfection conditions and cell density. A universal purification strategy, based on ion exchange chromatography methods, was also developed that results in high-purity vector preps of AAV serotypes 1–6, 8, 9 and various chimeric capsids tested. This user-friendly process can be completed within 1 week, results in high full to empty particle ratios (>90% full particles), provides postpurification yields (>1 × 1013 vg/l) and purity suitable for clinical applications and is universal with respect to all serotypes and chimeric particles. To date, this scalable manufacturing technology has been utilized to manufacture GMP phase 1 clinical AAV vectors for retinal neovascularization (AAV2), Hemophilia B (scAAV8), giant axonal neuropathy (scAAV9), and retinitis pigmentosa (AAV2), which have been administered into patients. In addition, we report a minimum of a fivefold increase in overall vector production by implementing a perfusion method that entails harvesting rAAV from the culture media at numerous time-points post-transfection.