Mapping the Mouse Cell Atlas by Microwell-Seq.

Single-cell RNA sequencing (scRNA-seq) technologies are poised to reshape the current cell-type classification system. However, a transcriptome-based single-cell atlas has not been achieved for complex mammalian systems. Here, we developed Microwell-seq, a high-throughput and low-cost scRNA-seq platform using simple, inexpensive devices. Using Microwell-seq, we analyzed more than 400,000 single cells covering all of the major mouse organs and constructed a basic scheme for a mouse cell atlas (MCA). We reveal a single-cell hierarchy for many tissues that have not been well characterized previously. We built a web-based "single-cell MCA analysis" pipeline that accurately defines cell types based on single-cell digital expression. Our study demonstrates the wide applicability of the Microwell-seq technology and MCA resource.

Architectures of Lipid Transport Systems for the Bacterial Outer Membrane.

How phospholipids are trafficked between the bacterial inner and outer membranes through the hydrophilic space of the periplasm is not known. We report that members of the mammalian cell entry (MCE) protein family form hexameric assemblies with a central channel capable of mediating lipid transport. The E. coli MCE protein, MlaD, forms a ring associated with an ABC transporter complex in the inner membrane. A soluble lipid-binding protein, MlaC, ferries lipids between MlaD and an outer membrane protein complex. In contrast, EM structures of two other E. coli MCE proteins show that YebT forms an elongated tube consisting of seven stacked MCE rings, and PqiB adopts a syringe-like architecture. Both YebT and PqiB create channels of sufficient length to span the periplasmic space. This work reveals diverse architectures of highly conserved protein-based channels implicated in the transport of lipids between the membranes of bacteria and some eukaryotic organelles.

Comprehensive stable-isotope tracing of glucose and amino acids identifies metabolic by-products and their sources in CHO cell culture.

Mammalian cell culture processes are widely utilized for biotherapeutics production, disease diagnostics, and biosensors, and hence, should be optimized to support robust cell growth and viability. However, toxic by-products accumulate in cultures due to inefficiencies in metabolic activities and nutrient utilization. In this study, we applied comprehensive 13C stable-isotope tracing of amino acids and glucose to two Immunoglobulin G (IgG) producing Chinese Hamster Ovary (CHO) cell lines to identify secreted by-products and trace their origins. CHO cells were cultured in media formulations missing a single amino acid or glucose supplemented with a 13C-tracer of the missing substrate, followed by gas chromatography-mass spectrometry (GC-MS) analysis to track labeled carbon flows and identify by-products. We tracked the sources of all secreted by-products and verified the identity of 45 by-products, majority of which were derived from glucose, leucine, isoleucine, valine, tyrosine, tryptophan, methionine, and phenylalanine. In addition to by-products identified previously, we identified several metabolites including 2-hydroxyisovaleric acid, 2-aminobutyric acid, L-alloisoleucine, ketoisoleucine, 2-hydroxy-3-methylvaleric acid, desmeninol, and 2-aminobutyric acid. When added to CHO cell cultures at different concentrations, certain metabolites inhibited cell growth while others including 2-hydroxy acids, surprisingly, reduced lactate accumulation. In vitro enzymatic analysis indicated that 2-hydroxy acids were metabolized by lactate dehydrogenase suggesting a possible mechanism for lowered lactate accumulation, e.g., competitive substrate inhibition. The 13C-labeling assisted metabolomics pipeline developed and the metabolites identified will serve as a springboard to reduce undesirable by-products accumulation and alleviate inefficient substrate utilization in mammalian cultures used for biomanufacturing and other applications through altered media formulations and pathway engineering strategies.

Small-Molecule Regulators for Gene Switches to Program Mammalian Cell Behaviour.

Synthetic or natural small molecules have been extensively employed as trigger signals or inducers to regulate engineered gene circuits introduced into living cells in order to obtain desired outputs in a controlled and predictable manner. Here, we provide an overview of small molecules used to drive synthetic-biology-based gene circuits in mammalian cells, together with examples of applications at different levels of control, including regulation of DNA manipulation, RNA synthesis and editing, and protein synthesis, maturation, and trafficking. We also discuss the therapeutic potential of these small-molecule-responsive gene circuits, focusing on the advantages and disadvantages of using small molecules as triggers, the mechanisms involved, and the requirements for selecting suitable molecules, including efficiency, specificity, orthogonality, and safety. Finally, we explore potential future directions for translation of these devices to clinical medicine.

Elucidating uptake and metabolic fate of dipeptides in CHO cell cultures using 13C labeling experiments and kinetic modeling.

The rapidly growing market of biologics including monoclonal antibodies has stimulated the need to improve biomanufacturing processes including mammalian host systems such as Chinese Hamster Ovary (CHO) cells. Cell culture media formulations continue to be enhanced to enable intensified cell culture processes and optimize cell culture performance. Amino acids, major components of cell culture media, are consumed in large amounts by CHO cells. Due to their low solubility and poor stability, certain amino acids including tyrosine, leucine, and phenylalanine can pose major challenges leading to suboptimal bioprocess performance. Dipeptides have the potential to replace amino acids in culture media. However, very little is known about the cleavage, uptake, and utilization kinetics of dipeptides in CHO cell cultures. In this study, replacing amino acids, including leucine and tyrosine by their respective dipeptides including but not limited to Ala-Leu and Gly-Tyr, supported similar cell growth, antibody production, and lactate profiles. Using 13C labeling techniques and spent media studies, dipeptides were shown to undergo both intracellular and extracellular cleavage in cultures. Extracellular cleavage increased with the culture duration, indicating cleavage by host cell proteins that are likely secreted and accumulate in cell culture over time. A kinetic model was built and for the first time, integrated with 13C labeling experiments to estimate dipeptide utilization rates, in CHO cell cultures. Dipeptides with alanine at the N-terminus had a higher utilization rate than dipeptides with alanine at the C-terminus and dipeptides with glycine instead of alanine at N-terminus. Simultaneous supplementation of more than one dipeptide in culture led to reduction in individual dipeptide utilization rates indicating that dipeptides compete for the same cleavage enzymes, transporters, or both. Dipeptide utilization rates in culture and cleavage rates in cell-free experiments appeared to follow Michaelis-Menten kinetics, reaching a maximum at higher dipeptide concentrations. Dipeptide utilization behavior was found to be similar in cell-free and cell culture environments, paving the way for future testing approaches for dipeptides in cell-free environments prior to use in large-scale bioreactors. Thus, this study provides a deeper understanding of the fate of dipeptides in CHO cell cultures through an integration of cell culture, 13C labeling, and kinetic modeling approaches providing insights in how to best use dipeptides in media formulations for robust and optimal mammalian cell culture performance.

Assessment of the three-test genetic toxicology battery for groundwater metabolites.

The two-test in vitro battery for genotoxicity testing (Ames and micronucleus) has in the majority of cases replaced the three-test battery (as two-test plus mammalian cell gene mutation assay) for the routine testing of chemicals, pharmaceuticals, cosmetics, and agrochemical metabolites originating from food and feed as well as from water treatment. The guidance for testing agrochemical groundwater metabolites, however, still relies on the three-test battery. Data collated in this study from 18 plant protection and related materials highlights the disparity between the often negative Ames and in vitro chromosome aberration data and frequently positive in vitro mammalian cell gene mutation assays. Sixteen of the 18 collated materials with complete datasets were Ames negative, and overall had negative outcomes in in vitro chromosome damage tests (weight of evidence from multiple tests). Mammalian cell gene mutation assays (HPRT and/or mouse lymphoma assay (MLA)) were positive in at least one test for every material with this data. Where both MLA and HPRT tests were performed on the same material, the HPRT seemed to give fewer positive responses. In vivo follow-up tests included combinations of comet assays, unscheduled DNA synthesis, and transgenic rodent gene mutation assays, all gave negative outcomes. The inclusion of mammalian cell gene mutation assays in a three-test battery for groundwater metabolites is therefore not justified and leads to unnecessary in vivo follow-up testing.

Modeling of bioprocess pre-stages for optimization of perfusion profiles and increased process understanding.

Improving bioprocess efficiency is important to reduce the current costs of biologics on the market, bring them faster to the market, and to improve the environmental footprint. The process intensification efforts were historically focused on the main stage, while intensification of pre-stages has started to gain attention only in the past decade. Performing bioprocess pre-stages in the perfusion mode is one of the most efficient options to achieve higher viable cell densities over traditional batch methods. While the perfusion-mode operation allows to reach higher viable cell densities, it also consumes large amount of medium, making it cost-intensive. The change of perfusion rate during a process (perfusion profile) determines how much medium is consumed, thereby running a process in optimal conditions is key to reduce medium consumption. However, the selection of the perfusion profile is often made empirically, without full understanding of bioprocess dynamics. This fact is hindering potential process improvements and means for cost reduction. In this study, we propose a process modeling approach to identify the optimal perfusion profile during bioprocess pre-stages. The developed process model was used internally during process development. We could reduce perfused medium volume by 25%-45% (project-dependent), while keeping the difference in the final cell within 5%-10% compared to the original settings. Additionally, the model helps to reduce the experimental workload by 30%-70% and to predict an optimal perfusion profile when process conditions need to be changed (e.g., higher seeding density, change of operating mode from batch to perfusion, etc.). This study demonstrates the potential of process modeling as a powerful tool for optimizing bioprocess pre-stages and thereby guiding process development, improving overall bioprocess efficiency, and reducing operational costs, while strongly reducing the need for wet-lab experiments.

Enhancing cell-based therapies with synthetic gene circuits responsive to molecular stimuli.

Synthetic biology aims to contribute to the development of next-generation patient-specific cell-based therapies for chronic diseases especially through the construction of sophisticated synthetic gene switches to enhance the safety and spatiotemporal controllability of engineered cells. Indeed, switches that sense and process specific cues, which may be either externally administered triggers or endogenous disease-associated molecules, have emerged as powerful tools for programming and fine-tuning therapeutic outputs. Living engineered cells, often referred to as designer cells, incorporating such switches are delivered to patients either as encapsulated cell implants or by infusion, as in the case of the clinically approved CAR-T cell therapies. Here, we review recent developments in synthetic gene switches responsive to molecular stimuli, spanning regulatory mechanisms acting at the transcriptional, translational, and posttranslational levels. We also discuss current challenges facing clinical translation of cell-based therapies employing these devices.

Model-based control system design to manage process parameters in mammalian cell culture for biopharmaceutical manufacturing.

To enhance the robustness and flexibility of biopharmaceutical manufacturing, a paradigm shift toward methods of continuous processing, such as perfusion, and fundamental technologies for high-throughput process development are being actively investigated. The continuous upstream process must establish an advanced control strategy to ensure a "State of Control" before operation. Specifically, feedforward and feedback control must address the complex fluctuations that occur during the culture process and maintain critical process parameters in appropriate states. However, control system design for industry-standard mammalian cell culture processes is still often performed in a laborious trial-and-error manner. This paper provides a novel control approach in which controller specifications to obtain desired control characteristics can be determined systematically by combining a culture model with control theory. In the proposed scheme, control conditions, such as PID parameters, can be specified mechanistically based on process understanding and control requirements without qualitative decision making or specific preliminary experiments. The effectiveness of the model-based control algorithm was verified by control simulations assuming perfusion Chinese hamster ovary culture. As a tool to assist in the development of control strategies, this study will reduce the high operational workload that is a serious problem in continuous culture and facilitate the digitalization of bioprocesses.

Relationship of Cytotoxic and Antimicrobial Effects of Triphenylphosphonium Conjugates with Various Quinone Derivatives.

Quinone derivatives of triphenylphosphonium have proven themselves to be effective geroprotectors and antioxidants that prevent oxidation of cell components with participation of active free radicals - peroxide (RO2·), alkoxy (RO·), and alkyl (R·) radicals, as well as reactive oxygen species (superoxide anion, singlet oxygen). Their most studied representatives are derivatives of plastoquinone (SkQ1) and ubiquinone (MitoQ), which in addition to antioxidant properties also have a strong antibacterial effect. In this study, we investigated antibacterial properties of other quinone derivatives based on decyltriphenylphosphonium (SkQ3, SkQT, and SkQThy). We have shown that they, just like SkQ1, inhibit growth of various Gram-positive bacteria at micromolar concentrations, while being less effective against Gram-negative bacteria, which is associated with recognition of the triphenylphosphonium derivatives by the main multidrug resistance (MDR) pump of Gram-negative bacteria, AcrAB-TolC. Antibacterial action of SkQ1 itself was found to be dependent on the number of bacterial cells. It is important to note that the cytotoxic effect of SkQ1 on mammalian cells was observed at higher concentrations than the antibacterial action, which can be explained by (i) the presence of a large number of membrane organelles, (ii) lower membrane potential, (iii) spatial separation of the processes of energy generation and transport, and (iv) differences in the composition of MDR pumps. Differences in the cytotoxic effects on different types of eukaryotic cells may be associated with the degree of membrane organelle development, energy status of the cell, and level of the MDR pump expression.

Completely noninvasive multi-analyte monitoring system for cell culture processes.

Although online monitoring of dissolved O2, pH, and dissolved CO2 is critical in bioprocesses, nearly all existing technologies require some level of direct contact with the cell culture environment, posing risks of contamination. This study addresses the need for an accurate, and completely noninvasive technique for simultaneous measurement of these analytes. A "non-contact" technique for simultaneous monitoring of dissolved O2, pH, and dissolved CO2 was developed. Instead of direct contact with the culture media, the measurements were made through permeable membranes via either a sampling port in the culture vessel wall or a flow cell. The efficacy of the "non-contact" technique was validated in Escherichia coli (E.coli), Chinese hamster ovary (CHO) culture processes, and dynamic environments created by sparging gases in cell culture medium. The measurements obtained through the developed techniques were comparable to those obtained through control methods. The noninvasive monitoring system can offer accurate, and contamination-minimized monitoring of critical process parameters including dissolved O2, pH, and dissolved CO2. These advancements will enhance the control and optimization of cell culture processes, promising improved cell culture performance.

Purification of In Vivo or In Vitro-Assembled RNA-Protein Complexes by RNA Centric Methods.

Throughout their entire life cycle, RNAs are associated with RNA-binding proteins (RBPs), forming ribonucleoprotein (RNP) complexes with highly dynamic compositions and very diverse functions in RNA metabolism, including splicing, translational regulation, ribosome assembly. Many RNPs remain poorly characterized due to the challenges inherent in their purification and subsequent biochemical characterization. Therefore, developing methods to isolate specific RNA-protein complexes is an important initial step toward understanding their function. Many elegant methodologies have been developed to isolate RNPs. This chapter describes different approaches and methods devised for RNA-specific purification of a target RNP. We focused on general methods for selecting RNPs that target a given RNA under conditions favourable for the copurification of associated factors including RNAs and protein components of the RNP.

Incorporation of photosynthetically active algal chloroplasts in cultured mammalian cells towards photosynthesis in animals.

Chloroplasts are photosynthetic organelles that evolved through the endosymbiosis between cyanobacteria-like symbionts and hosts. Many studies have attempted to isolate intact chloroplasts to analyze their morphological characteristics and photosynthetic activity. Although several studies introduced isolated chloroplasts into the cells of different species, their photosynthetic activities have not been confirmed. In this study, we isolated photosynthetically active chloroplasts from the primitive red alga Cyanidioschyzon merolae and incorporated them in cultured mammalian cells via co-cultivation. The incorporated chloroplasts retained their thylakoid structure in intracellular vesicles and were maintained in the cytoplasm, surrounded by the mitochondria near the nucleus. Moreover, the incorporated chloroplasts maintained electron transport activity of photosystem II in cultured mammalian cells for at least 2 days after the incorporation. Our top-down synthetic biology-based approach may serve as a foundation for creating artificially photosynthetic animal cells.

Regulation of PKD2 channel function by TACAN.

Autosomal dominant polycystic kidney disease is caused by mutations in the membrane receptor PKD1 or the cation channel PKD2. TACAN (also termed TMEM120A), recently reported as an ion channel in neurons for mechanosensing and pain sensing, is also distributed in diverse non-neuronal tissues, such as kidney, heart and intestine, suggesting its involvement in other functions. In this study, we found that TACAN is in a complex with PKD2 in native renal cell lines. Using the two-electrode voltage clamp in Xenopus oocytes, we found that TACAN inhibits the channel activity of PKD2 gain-of-function mutant F604P. TACAN fragments containing the first and last transmembrane domains interacted with the PKD2 C- and N-terminal fragments, respectively. The TACAN N-terminus acted as a blocking peptide, and TACAN inhibited the function of PKD2 by the binding of PKD2 with TACAN. By patch clamping in mammalian cells, we found that TACAN inhibits both the single-channel conductance and the open probability of PKD2 and mutant F604P. PKD2 co-expressed with TACAN, but not PKD2 alone, exhibited pressure sensitivity. Furthermore, we found that TACAN aggravates PKD2-dependent tail curvature and pronephric cysts in larval zebrafish. In summary, this study revealed that TACAN acts as a PKD2 inhibitor and mediates mechanosensitivity of the PKD2-TACAN channel complex. KEY POINTS: TACAN inhibits the function of PKD2 in vitro and in vivo. TACAN N-terminal S1-containing fragment T160X interacts with the PKD2 C-terminal fragment N580-L700, and its C-terminal S6-containing fragment L296-D343 interacts with the PKD2 N-terminal A594X. TACAN inhibits the function of the PKD2 channel by physical interaction. The complex of PKD2 with TACAN, but not PKD2 alone, confers mechanosensitivity.

A genome-scale dynamic constraint-based modelling (gDCBM) framework predicts growth dynamics, medium composition and intracellular flux distributions in CHO clonal variations.

Optimizing mammalian cell growth and bioproduction is a tedious task. However, due to the inherent complexity of eukaryotic cells, heuristic experimental approaches such as, metabolic engineering and bioprocess design, are frequently integrated with mathematical models of cell culture to improve biological process efficiency and find paths for improvement. Constraint-based metabolic models have evolved over the last two decades to be used for dynamic modelling in addition to providing a linear description of steady-state metabolic systems. Formulation and implementation of the underlying optimization problems require special attention to the model's performance and feasibility, lack of defects in the definition of system components, and consideration of optimal alternate solutions, in addition to processing power limitations. Here, the time-resolved dynamics of a genome-scale metabolic network of Chinese hamster ovary (CHO) cell metabolism are shown using a genome-scale dynamic constraint-based modelling framework (gDCBM). The metabolic network was adapted from a reference model of CHO genome-scale metabolic model (GSMM), iCHO_DG44_v1, and dynamic restrictions were imposed to its exchange fluxes based on experimental results. We used this framework for predicting physiological changes in CHO clonal variants. Because of the methodical creation of the components for the flux balance analysis optimization problem and the integration of a switch time, this model can generate sequential predictions of intracellular fluxes during growth and non-growth phases (per hour of culture time) and transparently reveal the shortcomings in such practice. As a result of the differences exploited by various clones, we can understand the relevance of changes in intracellular flux distribution and exometabolomics. The integration of various omics data into the given gDCBM framework, as well as the reductionist analysis of the model, can further help bioprocess optimization.

Cell factory engineering: Challenges and opportunities for synthetic biology applications.

The production of high-quality recombinant proteins is critical to maintaining a continuous supply of biopharmaceuticals, such as therapeutic antibodies. Engineering mammalian cell factories presents a number of limitations typically associated with the proteotoxic stress induced upon aberrant accumulation of off-pathway protein folding intermediates, which eventually culminate in the induction of apoptosis. In this review, we will discuss advances in cell engineering and their applications at different hierarchical levels of control of the expression of recombinant proteins, from transcription and translational to posttranslational modifications and subcellular trafficking. We also highlight challenges and unique opportunities to apply modern synthetic biology tools to the design of programmable cell factories for improved biomanufacturing of therapeutic proteins.

Leveraging mathematical models for optimizing filter utility at manufacturing scale.

In the production of biopharmaceuticals depth filters followed by sterile filters are often employed to remove residual cell debris present in the feed stream. In the back drop of a global pandemic, supply chains associated with the production of biopharmaceuticals have been constrained. These constraints have limited the available amount of depth filters for the manufacture of biologics. This has placed manufacturing facilities in a difficult position having to choose between running processes with reduced number of depth filters and risking a failed batch or the prospect of plants going into temporary shutdown until the depth filter resources are replenished. This communication describes a modeling based method that leverages manufacturing scale filtration data to predict the depth filter performance with a reduced number of filters and an increased operational flux. This method can be used to quantify the acceptable level of area reduction before which the filtration process performance is affected. This enables facilities to manage their filter inventory avoiding potential plant shutdowns and reduces the risks of negative depth filter performance.

Enhancing CHO cell productivity through a dual selection system using Aspg and Gs in glutamine free medium.

The dominant method for generating Chinese hamster ovary (CHO) cell lines that produce high titers of biotherapeutic proteins utilizes selectable markers such as dihydrofolate reductase (Dhfr) or glutamine synthetase (Gs), alongside inhibitory compounds like methotrexate or methionine sulfoximine, respectively. Recent work has shown the importance of asparaginase (Aspg) for growth in media lacking glutamine-the selection medium for Gs-based selection systems. We generated a Gs/Aspg double knockout CHO cell line and evaluated its utility as a novel dual selectable system via co-transfection of Gs-Enbrel and Aspg-Enbrel plasmids. Using the same selection conditions as the standard Gs system, the resulting cells from the Gs/Aspg dual selection showed substantially improved specific productivity and titer compared to the standard Gs selection method, however, with reduced growth rate and viability. Following adaptation in the selection medium, the cells improved viability and growth while still achieving ~5-fold higher specific productivity and ~3-fold higher titer than Gs selection alone. We anticipate that with further optimization of culture medium and selection conditions, this approach would serve as an effective addition to workflows for the industrial production of recombinant biotherapeutics.

Recombinant production of antibody antigen-binding fragments with an N-terminal human growth hormone tag in mammalian cells.

Antigen-binding fragments (Fabs) of antibodies are both key biopharmaceuticals and valuable tools for basic life science. To streamline the production of diverse Fabs by capitalizing on standard and highly optimized protein production protocols, we here explore a method to prepare recombinant Fabs as secreted fusion proteins with an N-terminal human growth hormone domain and an octa-histidine tag. These tagged Fabs can be purified with standard immobilized metal chelate affinity chromatography. We first demonstrated Fab overproduction using the rat monoclonal antibody NZ-1. Optimization of linker residues enabled the complete removal of the tags by TEV protease, leaving only two additional residues at the N-terminus of the heavy chain. We purified NZ-1 Fab at ∼4 µg/mL of culture supernatant and further confirmed that the NZ-1 Fab from the fusion protein maintained its native fold and binding affinity for target cell-surface antigens. We also showed that several other Fabs of mouse IgG1s, the major subclass in mice, could be produced with the same procedure. Our preparation method can provide greater flexibility in functional and structural modifications of target Fabs because specialized purification techniques are not necessary.

Expression of mammalian cell entry genes in clinical isolates of M. tuberculosis and the cell entry potential and immunological reactivity of the Rv0590A protein.

Mammalian cell entry (mce) operons play a vital role in cell invasion and survival of M. tuberculosis. Of the mce genes, the function of Rv0590A is still unknown. The present study was performed to investigate the function and immunogenic properties of the protein Rv0590A. Human leukemia monocytic cell line (THP-1) derived macrophages were infected with M. tuberculosis H37Rv at 3, 6, and 24 h of infection. The maximum colony forming units (CFU) were observed at 6 h (p < 0.005), followed by 3 h after infection. M. tuberculosis H37Rv and clinical isolates representative of Delhi/CAS, EAI, Beijing, Haarlem and Euro-American-superlineage were included in the study for expression analysis of mce1A, mce2A, mce3A, mce4A, and Rv0590A genes. Maximum upregulation of all mce genes was observed at 3 h of infection. All the five clinical isolates and H37Rv upregulated Rv0590A at various time points. Macrophage infection with M. tuberculosis H37Rv-overexpressing Rv0590A gene showed higher intracellular CFU as compared to that of wild-type H37Rv. Further, purified Rv0590A protein stimulated the production of TNFα, IFNγ, and IL-10 in macrophages. Thus, Rv0590A was found to be involved in cell invasion and showed good immunological response.