RESUMEN
There is a growing interest in perfusion or continuous processes to achieve higher productivity of biopharmaceuticals in mammalian cell culture, specifically Chinese hamster ovary (CHO) cells, towards advanced biomanufacturing. These intensified bioprocesses highly require concentrated feed media in order to counteract their dilution effects. However, designing such condensed media formulation poses several challenges, particularly regarding the stability and solubility of specific amino acids. To address the difficulty and complexity in relevant media development, the biopharmaceutical industry has recently suggested forming dipeptides by combining one from problematic amino acids with selected pairs to compensate for limitations. In this study, we combined one of the lead amino acids, L-tyrosine, which is known for its poor solubility in water due to its aromatic ring and hydroxyl group, with glycine as the partner, thus forming glycyl-L-tyrosine (GY) dipeptide. Subsequently, we investigated the utilization of GY dipeptide during fed-batch cultures of IgG-producing CHO cells, by changing its concentrations (0.125 × , 0.25 × , 0.5 × , 1.0 × , and 2.0 ×). Multivariate statistical analysis of culture profiles was then conducted to identify and correlate the most significant nutrients with the production, followed by in silico model-guided analysis to systematically evaluate their effects on the culture performance, and elucidate metabolic states and cellular behaviors. As such, it allowed us to explain how the cells can more efficiently utilize GY dipeptide with respect to the balance of cofactor regeneration and energy distribution for the required biomass and protein synthesis. For example, our analysis results uncovered specific amino acids (Asn and Gln) and the 0.5 × GY dipeptide in the feed medium synergistically alleviated the metabolic bottleneck, resulting in enhanced IgG titer and productivity. In the validation experiments, we tested and observed that lower levels of Asn and Gln led to decreased secretion of toxic metabolites, enhanced longevity, and elevated specific cell growth and titer. KEY POINTS: ⢠Explored the optimal Tyr dipeptide for the enhanced CHO cell culture performance ⢠Systematically analyzed effects of dipeptide media by model-guided approach ⢠Uncovered synergistic metabolic utilization of amino acids with dipeptide.
Asunto(s)
Aminoácidos , Técnicas de Cultivo Celular por Lotes , Cricetinae , Animales , Cricetulus , Células CHO , Medios de Cultivo/química , Técnicas de Cultivo Celular por Lotes/métodos , Aminoácidos/metabolismo , Tirosina , Dipéptidos , Inmunoglobulina G , Simulación por ComputadorRESUMEN
Proposed herein is a systematic media design framework that combines multivariate statistical approaches with in silico analysis of a genome-scale metabolic model of Chinese hamster ovary cell. The framework comprises sequential modules including cell culture and metabolite data collection, multivariate data analysis, in silico modeling and flux prediction, and knowledge-based identification of target media components. Two monoclonal antibody-producing cell lines under two different media conditions were used to demonstrate the applicability of the framework. First, the cell culture and metabolite profiles from all conditions were generated, and then statistically and mechanistically analyzed to explore combinatorial effects of cell line and media on intracellular metabolism. As a result, we found a metabolic bottleneck via a redox imbalance in the TCA cycle in the poorest growth condition, plausibly due to inefficient coenzyme q10-q10h2 recycling. Subsequent in silico simulation allowed us to suggest q10 supplementation to debottleneck the imbalance for the enhanced cellular energy state and TCA cycle activity. Finally, experimental validation was successfully conducted by adding q10 in the media, resulting in increased cell growth. Taken together, the proposed framework rationally identified target nutrients for cell line-specific media design and reformulation, which could greatly improve cell culture performance.
Asunto(s)
Técnicas de Cultivo de Célula , Modelos Biológicos , Animales , Células CHO , Simulación por Computador , Cricetinae , Cricetulus , Medios de CultivoRESUMEN
Filopodia have a key role in sensing both chemical and mechanical cues in surrounding extracellular matrix (ECM). However, quantitative understanding is still missing in the filopodial mechanosensing of local ECM stiffness, resulting from dynamic interactions between filopodia and the surrounding 3D ECM fibers. Here we present a method for characterizing the stiffness of ECM that is sensed by filopodia based on the theory of elasticity and discrete ECM fiber. We have applied this method to a filopodial mechanosensing model for predicting directed cell migration toward stiffer ECM. This model provides us with a distribution of force and displacement as well as their time rate of changes near the tip of a filopodium when it is bound to the surrounding ECM fibers. Aggregating these effects in each local region of 3D ECM, we express the local ECM stiffness sensed by the cell and explain polarity in the cellular durotaxis mechanism.
Asunto(s)
Movimiento Celular/fisiología , Simulación por Computador , Matriz Extracelular/fisiología , Modelos Biológicos , Fenómenos Biomecánicos , Adhesión Celular , Citoesqueleto/fisiología , Elasticidad , Adhesiones Focales , SeudópodosRESUMEN
Focusing on intracellular targets, we propose a new cell separation technique based on a nanoneedle array (NNA) device, which allows simultaneous insertion of multiple needles into multiple cells. The device is designed to target and lift ("fish") individual cells from a mixed population of cells on a substrate using an antibody-functionalized NNA. The mechanics underlying this approach were validated by force analysis using an atomic force microscope. Accurate high-throughput separation was achieved using one-to-one contacts between the nanoneedles and the cells by preparing a single-cell array in which the positions of the cells were aligned with 10,000 nanoneedles in the NNA. Cell-type-specific separation was realized by controlling the adhesion force so that the cells could be detached in cell-type-independent manner. Separation of nestin-expressing neural stem cells (NSCs) derived from human induced pluripotent stem cells (hiPSCs) was demonstrated using the proposed technology, and successful differentiation to neuronal cells was confirmed.
Asunto(s)
Anticuerpos Inmovilizados/química , Separación Celular/instrumentación , Nanoestructuras/química , Agujas , Animales , Línea Celular , Diseño de Equipo , Células HeLa , Humanos , Células Madre Pluripotentes Inducidas/citología , Células MCF-7 , Ratones , Células 3T3 NIH , Nanoestructuras/ultraestructura , Células-Madre Neurales/citología , Análisis de Matrices Tisulares/instrumentaciónRESUMEN
The dynamics of filopodia interacting with the surrounding extracellular matrix (ECM) play a key role in various cell-ECM interactions, but their mechanisms of interaction with the ECM in 3D environment remain poorly understood. Based on first principles, here we construct an individual-based, force-based computational model integrating four modules of 1) filopodia penetration dynamics; 2) intracellular mechanics of cellular and nuclear membranes, contractile actin stress fibers, and focal adhesion dynamics; 3) structural mechanics of ECM fiber networks; and 4) reaction-diffusion mass transfers of seven biochemical concentrations in related with chemotaxis, proteolysis, haptotaxis, and degradation in ECM to predict dynamic behaviors of filopodia that penetrate into a 3D ECM fiber network. The tip of each filopodium crawls along ECM fibers, tugs the surrounding fibers, and contracts or retracts depending on the strength of the binding and the ECM stiffness and pore size. This filopodium-ECM interaction is modeled as a stochastic process based on binding kinetics between integrins along the filopodial shaft and the ligands on the surrounding ECM fibers. This filopodia stochastic model is integrated into migratory dynamics of a whole cell in order to predict the cell invasion into 3D ECM in response to chemotaxis, haptotaxis, and durotaxis cues. Predicted average filopodia speed and that of the cell membrane advance agreed with experiments of 3D HUVEC migration at r(2) > 0.95 for diverse ECMs with different pore sizes and stiffness.
Asunto(s)
Adhesión Celular/fisiología , Movimiento Celular/fisiología , Matriz Extracelular/fisiología , Mecanotransducción Celular/fisiología , Modelos Biológicos , Seudópodos/fisiología , Animales , Simulación por Computador , Módulo de Elasticidad/fisiología , Humanos , Estrés MecánicoRESUMEN
We report here a method for controlling cell adhesion, allowing simple yet accurate cell detachment from the substrate, which is required for the establishment of new cytometry-based cell processing and analyzing methods. A biocompatible anchor for membrane (BAM) was conjugated with bovine serum albumin (BSA) to produce a cell-anchoring agent (BAM-BSA). By coating polystyrene substrates with a mixture of BAM-BSA and BSA, controlled suppression of the substrate's adhesive properties was achieved. Hook-shaped nanoneedles were used to pick up cells from the substrate, while recording the cell-substrate adhesion force, using an atomic force microscope (AFM). Due to the lipid bilayer targeting property of BAM, the coated surface showed constant adhesion forces for various cell lines, and controlling the BAM-BSA/BSA ratio enabled tuning of the adhesion force, ranging from several tens of nano-Newtons down to several nano-Newtons. Optimized tuning of the adhesion force also enabled the detachment of cells from BAM-BSA/BSA-coated dishes, using a shear flow. Moreover, the method was shown to be noncell type specific and similar results were observed using four different cell types, including nonadherent cells. The attenuation of cell adhesion was also used to enable the collection of single cells by capillary aspiration. Thus, this versatile and relatively simple method can be used to control the adhesion of various cell types to substrates.
Asunto(s)
Materiales Biocompatibles/química , Albúmina Sérica Bovina/química , Animales , Bovinos , Adhesión Celular , Membrana Celular , Células Cultivadas , Ratones , Estructura Molecular , Células 3T3 NIHRESUMEN
Designing and selecting cell culture media along with their feeding are a key strategy to maximize culture performance in biopharmaceutical processes. However, the sensitivity of mammalian cells to their culture environment necessitates specific nutritional requirements for their growth and the production of high-quality proteins such as antibodies, depending on the cell lines and operational conditions employed. In this regard, previously we developed a data-driven and in-silico model-guided systematic framework to investigate the effect of growth media on Chinese hamster ovary (CHO) cell culture performance, allowing us to design and reformulate basal media. To expand our exploration for media development research, we evaluated two chemically defined feed media, A and B, using a monoclonal antibody-producing CHO-K1 cell line in ambr15 bioreactor runs. We observed a significant impact of the feed media on various aspects of cell culture, including growth, longevity, viability, productivity, and the production of toxic metabolites. Specifically, the concentrated feed A was inadequate in sustaining prolonged cell culture and achieving high titers when compared to feed B. Within our framework, we systematically investigated the major metabolic bottlenecks in the tricarboxylic acid cycle and relevant amino acid transferase reactions. This analysis identified target components that play a crucial role in alleviating bottlenecks and designing highly productive cell cultures, specifically the addition of glutamate to feed A and asparagine to feed B. Based on our findings, we reformulated the feeds by adjusting the amounts of the targeted amino acids and successfully validated the effectiveness of the strategy in promoting cell growth, life span, and/or titer.
Asunto(s)
Anticuerpos Monoclonales , Técnicas de Cultivo de Célula , Cricetinae , Animales , Cricetulus , Células CHO , Aminoácidos/metabolismo , Medios de Cultivo/químicaRESUMEN
Retinol and conjugated linoleic acid (CLA) have previously been shown to have an important role in gene expression and various cellular processes, including differentiation, proliferation and cell death. In this study we have investigated the effect of retinol and CLA, both individually and in combination, on the intracellular cytoskeleton, focal adhesions (FAs) and the nanomechanical properties of 3T3 fibroblasts. We observed a dose-dependent decrease in the formation of FAs following treatment with either compound, which was directly correlated to an increase in cell height (>30%) and a decrease in the measured Young's modulus (approximately 28%). Furthermore, treatments with both compounds demonstrated an increased effect and led to a reduction of >70% in the average number of FAs per cell and a decrease of >50% in average cell stiffness. These data reveal that retinol and CLA disrupt FA formation, leading to an increase in cell height and a significant decrease in stiffness. These results may broaden our understanding of the interplay between cell nanomechanics and cellular contact with the external microenvironment, and help to shed light on the important role of retinoids and CLA in health and disease.
Asunto(s)
Fenómenos Biomecánicos/efectos de los fármacos , Adhesiones Focales/metabolismo , Ácidos Linoleicos Conjugados/farmacología , Vitamina A/farmacología , Vitaminas/farmacología , Células 3T3 , Animales , Forma de la Célula/efectos de los fármacos , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Relación Dosis-Respuesta a Droga , Ratones , Microscopía de Fuerza Atómica , Microscopía ConfocalRESUMEN
Efficient and rapid delivery of macromolecule probes, such as quenchbodies and other large biomarkers that cannot readily pass through the plasma membrane, is necessary for live-cell imaging and other intracellular analyses. We present here an alternative, simple method for delivery of macromolecules into live cells. In this method, which we term here mechanoporation, a nanoneedle array is used for making transient pores in the plasma membrane to allow access of desired macromolecules into thousands of live cells, simultaneously. This rapid, 3-step method facilitates an efficient delivery by adding macromolecules into the medium, inserting nanoneedles into the cells and oscillating the nanoneedle array, a process that takes no more than 5 min in total. In addition, we demonstrate here how this method can repeatedly and reproducibly deliver molecules into specifically-selected locations on a given cell culture dish. The results presented here show how this unique mechanoporation method enables rapid and high-throughput bio-macromolecule delivery and live-cell imaging.
Asunto(s)
Permeabilidad de la Membrana Celular , Rastreo Celular/métodos , Sustancias Macromoleculares/farmacocinética , Animales , Técnicas de Cultivo de Célula/instrumentación , Técnicas de Cultivo de Célula/métodos , Dextranos/farmacocinética , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/farmacocinética , Células HEK293 , Humanos , Ratones , Células 3T3 NIH , Nanoestructuras , Agujas , Análisis de la Célula Individual/métodosRESUMEN
Delivery of biomolecules with use of nanostructures has been previously reported. However, both efficient and high-throughput intracellular delivery has proved difficult to achieve. Here, we report a novel material and device for the delivery of biomacromolecules into live cells. We attribute the successful results to the unique features of the system, which include high-aspect-ratio, uniform nanoneedles laid across a 2D array, combined with an oscillatory feature, which together allow rapid, forcible and efficient insertion and protein release into thousands of cells simultaneously.
Asunto(s)
Técnicas de Transferencia de Gen/instrumentación , Nanoestructuras/química , Silicio/química , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Células HEK293 , Humanos , Integrasas/genética , Integrasas/metabolismo , Agujas , Plásmidos/genética , Plásmidos/metabolismo , Análisis de Matrices Tisulares/instrumentaciónRESUMEN
We present here an alternative, force-based measurement method for the detection of intracellular cytoskeletal proteins in the live cell. High aspect ratio nanoneedles of 200 nm in diameter were functionalized with anti-tubulin antibodies and inserted, using an atomic force microscope (AFM), into live NIH3T3 cells, without affecting cell viability. Force curves were recorded during insertion and evacuation of nanoneedles from the cells, and used to analyse intracellular interactions of the nanoneedles with the microtubule cytoskeleton during evacuation from the cell. Disruption of microtubules led to a correlated time-dependent decrease in the measured intracellular binding forces, pointing to the high-sensitivity and high-specificity of this detection method. This analytical technique allows for real-time evaluation of the microtubule network in the live cell, without the need to use potentially harmful molecular markers as do conventional detection methods, and may prove beneficial in the diagnosis and investigation of cytoskeleton-associated diseases.
Asunto(s)
Anticuerpos/química , Microtúbulos/metabolismo , Técnicas de Sonda Molecular/instrumentación , Nanocables , Tubulina (Proteína)/metabolismo , Animales , Ratones , Microscopía de Fuerza Atómica , Células 3T3 NIH , Nanotecnología , Estrés MecánicoRESUMEN
Mechanical stress affects various aspects of cell behavior, including cell growth, morphology, differentiation, and genetic expression. Here, we describe a method to quantify the intracellular mechanical response to an extracellular mechanical perturbation, specifically the displacement of mitochondria. A combined fluorescent-atomic force microscope (AFM) was used to simultaneously produce well-defined nanomechanical stimulation to a living cell while optically recording the real-time displacement of fluorescently labeled mitochondria. A single-particle tracking (SPT) approach was then applied in order to quantify the two-dimensional displacement of mitochondria in response to local forces.
Asunto(s)
Mitocondrias/metabolismo , Nanotecnología , Animales , Ratones , Microscopía de Fuerza Atómica , Células 3T3 NIHRESUMEN
An atomic force microscope probe can be formed into an ultra-sharp cylindrical shape (a nanoneedle) using micro-fabrication techniques such as focused ion beam etching. This nanoneedle can be effectively inserted through the plasma membrane of a living cell to not only access the cytosol, but also to penetrate through the nuclear membrane. This technique shows great potential as a tool for performing intranuclear measurements and manipulations. Repeated insertions of a nanoneedle into a live cell were previously shown not to affect cell viability. However, the effect of nanoneedle insertion on the nucleus and nuclear components is still unknown. DNA is the most crucial component of the nucleus for proper cell function and may be physically damaged by a nanoneedle. To investigate the integrity of DNA following nanoneedle insertion, the occurrence of DNA double-strand breaks (DSBs) was assessed. The results showed that there was no chromosomal DNA damage due to nanoneedle insertion into the nucleus, as indicated by the expression level of γ-H2AX, a molecular marker of DSBs.
Asunto(s)
Núcleo Celular , Cromosomas , Roturas del ADN de Doble Cadena , ADN , Microscopía de Fuerza Atómica/instrumentación , Nanoestructuras , Membrana Celular/metabolismo , Núcleo Celular/genética , Núcleo Celular/efectos de la radiación , Supervivencia Celular , Cromosomas/genética , Cromosomas/efectos de la radiación , ADN/efectos de la radiación , Roturas del ADN de Doble Cadena/efectos de la radiación , Rayos UltravioletaRESUMEN
A cell diagnosis technique was developed, which uses an Atomic Force Microscope (AFM) and an ultra-thin AFM probe sharpened to a diameter of 200 nm (nanoneedle). Due to the high aspect ratio of the nanoneedle, it was successfully inserted into a living cell without affecting its viability. Furthermore, by functionalizing the nanoneedle with specific antibodies and measuring the unbinding forces ('fishing forces') during evacuation of the nanoneedle from the cell, it was possible to measure specific mechanical interactions between the antibody-functionalized nanoneedle and the intracellular contents of the cell. In this study, an anti-actin-antibody-functionalized nanoneedle was used to evaluate the actin cytoskeleton state in living cells. To examine the effect of cytoskeleton condition on the measured fishing forces, the cytoskeleton-disrupting drugs cytochalasin D (cytD) and Y-27632 were used, showing a marked decrease in the measured fishing forces following incubation with either of the drugs. Furthermore, the technique was used to measure the time course changes in a single cell during incubation with cytD, showing a gradual time-dependent decrease in fishing forces. Even minute doses of the drugs, the effects of which were hardly evident by optical and fluorescence methods, could be clearly detected by the measurement of nanoneedle-protein fishing forces, pointing to the high sensitivity of this detection method. This technique may prove beneficial for the evaluation of cytoskeleton conditions in health and disease, and for the selection of specific cells according to their intracellular protein contents, without the need for introduction of marker proteins into the cell.
Asunto(s)
Citoesqueleto de Actina/fisiología , Técnicas Biosensibles/instrumentación , Inmunoensayo/instrumentación , Micromanipulación/instrumentación , Microscopía de Fuerza Atómica/instrumentación , Nanotecnología/instrumentación , Agujas , Animales , Anticuerpos/inmunología , Módulo de Elasticidad/fisiología , Diseño de Equipo , Análisis de Falla de Equipo , Ratones , Células 3T3 NIH , Resistencia a la Tracción/fisiologíaRESUMEN
Patterning of semiconducting polymers on surfaces is important for various applications in nanoelectronics and nanophotonics. However, many of the approaches to nanolithography that are used to pattern inorganic materials are too harsh for organic semiconductors, so research has focused on optical patterning and various soft lithographies. Surprisingly little attention has been paid to thermal, thermomechanical and thermochemical patterning. Here, we demonstrate thermochemical nanopatterning of poly(p-phenylene vinylene), a widely used electroluminescent polymer, by a scanning probe. We produce patterned structures with dimensions below 28 nm, although the tip of the probe has a diameter of 5 microm, and achieve write speeds of 100 microm s(-1). Experiments show that a resolution of 28 nm is possible when the tip-sample contact region has dimensions of approximately 100 nm and, on the basis of finite-element modelling, we predict that the resolution could be improved by using a thinner resist layer and an optimized probe. Thermochemical lithography offers a versatile, reliable and general nanopatterning technique because a large number of optical materials, including many commercial crosslinker additives and photoresists, rely on chemical mechanisms that can also be thermally activated.
RESUMEN
Mechanical stress affects and regulates many aspects of the cell, including morphology, growth, differentiation, gene expression and apoptosis. In this study we show how mechanical stress perturbs the intracellular structures of the cell and induces mechanical responses. In order to correlate mechanical perturbations to cellular responses, we used a combined fluorescence-atomic force microscope (AFM) to produce well defined nanomechanical perturbations of 10 nN while simultaneously tracking the real-time motion of fluorescently labelled mitochondria in live cells. The spatial displacement of the organelles in response to applied loads demonstrates the highly dynamic mechanical response of mitochondria in fibroblast cells. The average displacement of all mitochondrial structures analysed showed an increase of approximately 40%, post-perturbation ( approximately 160 nm in comparison to basal displacements of approximately 110 nm). These results show that local forces can produce organelle displacements at locations far from the initial point of contact (up to approximately 40 microm). In order to examine the role of the cytoskeleton in force transmission and its effect on mitochondrial displacements, both the actin and microtubule cytoskeleton were disrupted using Cytochalasin D and Nocodazole, respectively. Our results show that there is no significant change in mitochondrial displacement following indentation after such treatments. These results demonstrate the role of the cytoskeleton in force transmission through the cell and on mitochondrial displacements. In addition, it is suggested that care must be taken when performing mechanical experiments on living cells with the AFM, as these local mechanical perturbations may have significant structural and even biochemical effects on the cell.