Exploring metabolic effects of dipeptide feed media on CHO cell cultures by in silico model-guided flux analysis.

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.

Debottlenecking and reformulating feed media for improved CHO cell growth and titer by data-driven and model-guided analyses.

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.

Data-driven and model-guided systematic framework for media development in CHO cell culture.

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.

Single beam low frequency 2D Raman spectroscopy.

Low frequency Raman spectroscopy resolves the slow vibrations resulting from collective motions of molecular structures. This frequency region is extremely challenging to access via other multidimensional methods such as 2D-IR. In this paper, we describe a new scheme which measures 2D Raman spectra in the low frequency regime. We separate the pulse into a spectrally shaped pump and a transform-limited probe, which can be distinguished by their polarization states. Low frequency 2D Raman spectra in liquid tetrabromoethane are presented, revealing coupling dynamics at frequencies as low as 115 cm-1. The experimental results are supported by numerical simulations which replicate the key features of the measurement. This method opens the door for the deeper exploration of vibrational energy surfaces in complex molecular structures.

Light focusing through scattering media via linear fluorescence variance maximization, and its application for fluorescence imaging.

We demonstrate focusing and imaging through a scattering medium without access to the fluorescent object by using wavefront shaping. Our concept is based on utilizing the spatial fluorescence contrast which naturally exists in the hidden target object. By scanning the angle of incidence of the illuminating laser beam and maximizing the variation of the detected fluorescence signal from the object, as measured by a bucket detector at the front of the scattering medium, we are able to generate a tightly focused excitation spot. Thereafter, an image is obtained by scanning the focus over the object within the memory effect range. The requirements for applicability of the method and the comparison with speckle-correlation based focusing methods are discussed.

Simplified approach to low-frequency coherent anti-Stokes Raman spectroscopy using a sharp spectral edge filter.

Coherent anti-Stokes Raman scattering (CARS) has found wide applications in biomedical research. Compared with alternatives, single-beam CARS is especially attractive at low frequencies. Yet, currently existing schemes necessitate a relatively complicated setup to perform high-resolution spectroscopy. Here we show that the spectral sharp edge formed by an ultra-steep long-pass filter is sufficient for performing CARS spectroscopy, simplifying the system significantly. We compare the sensitivity of the presented methodology with available counterparts both theoretically and experimentally. Importantly, we show that this method, to the best of our knowledge, is the simplest and most suitable for vibrational imaging and spectroscopy in the very low-frequency regime (<200 cm-1).

Observation of Stimulated Hawking Radiation in an Optical Analogue.

The theory of Hawking radiation can be tested in laboratory analogues of black holes. We use light pulses in nonlinear fiber optics to establish artificial event horizons. Each pulse generates a moving perturbation of the refractive index via the Kerr effect. Probe light perceives this as an event horizon when its group velocity, slowed down by the perturbation, matches the speed of the pulse. We have observed in our experiment that the probe stimulates Hawking radiation, which occurs in a regime of extreme nonlinear fiber optics where positive and negative frequencies mix.

Mode conversion via wavefront shaping.

In recent years, wavefront shaping has been utilized to control and correct distorted light for enhancing a bright spot, generation of a Bessel beam or darkening a complete area at the output of a scattering system. All these outcomes can be thought of as enhancing a particular mode of the output field. In this letter, we study the relation between the attainable enhancement factor, corresponding to the efficiency of mode conversion, and the field distribution of the target mode. Working in the limit of a thin diffuser enables not only a comparison between experimental and simulated results, but also allows for derivation of an analytic formula. These results shed light on the ability to use a scattering medium as a mode converter and on the relationship between the desired shape and the efficiency.

Impulsive Raman spectroscopy via precision measurement of frequency shift with low energy excitation.

Stimulated Raman scattering (SRS) has recently become useful for chemically selective bioimaging. It is usually measured via modulation transfer from the pump beam to the Stokes beam. Impulsive stimulated Raman spectroscopy, on the other hand, relies on the spectral shift of ultrashort pulses as they propagate in a Raman active sample. This method was considered impractical with low energy pulses since the observed shifts are very small compared to the excitation pulse bandwidth, spanning many terahertz. Here we present a new apparatus, using tools borrowed from the field of precision measurement, for the detection of low-frequency Raman lines via stimulated-Raman-scattering-induced spectral shifts. This method does not require any spectral filtration and is therefore an excellent candidate to resolve low-lying Raman lines (<200 cm-1), which are commonly masked by the strong Rayleigh scattering peak. Having the advantage of the high repetition rate of the ultrafast oscillator, we reduce the noise level by implementing a lock-in detection scheme with a wavelength shift sensitivity well below 100 fm. This is demonstrated by the measurement of low-frequency Raman lines of various liquid samples.

Computational modeling of three-dimensional ECM-rigidity sensing to guide directed cell migration.

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.

A New Cell Separation Method Based on Antibody-Immobilized Nanoneedle Arrays for the Detection of Intracellular Markers.

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.

Weakly diverging to tightly focused Gaussian beams: a single set of analytic expressions: continuation-symmetric beams.

The always diverging-converging laser beams, more rigorously referred to as Gaussian beams, are part of many physical and electro-optical systems. Obviously, a single set of analytic expressions describing these beams in a large span of divergence-convergence angles at the focal plane, and at any distance away from the focal plane, will prove very handy. We have recently published three such analytic sets, one set for linearly polarized beams and two sets for radially polarized beams. However, our published analytic set for linearly polarized beams describes nonsymmetric electric-magnetic field components. Specifically, the strong transverse magnetic field component does not become elliptic at very large divergence angles as it should be, and the other transverse magnetic component, indeed very weak, is missing altogether. Here we present an analytic set of expressions symmetrically describing linearly polarized Gaussian beams. The symmetry applies to the x-electric y-magnetic components and vice versa and to the two electric-magnetic z-components. An important property of the presented set of expressions is power conservation. That is, the electromagnetic power crossing a plane transverse to the propagation direction in a unit time is conserved. Power conservation assures beam description accuracy at any axial distance. The presented analytic expressions, although not strictly satisfying Maxwell's equations, describe Gaussian beams with very reasonable accuracy from low divergence angles up to divergence angles as large as 0.8 rad in a medium with refractive index of 1.5, i.e., up to a NA of 1.1. These expressions should then readily assist in the design of practically all laser-related systems and in the research of diverse physics and electro-optic fields.

Quantum correlation enhanced super-resolution localization microscopy enabled by a fibre bundle camera.

Despite advances in low-light-level detection, single-photon methods such as photon correlation have rarely been used in the context of imaging. The few demonstrations, for example of subdiffraction-limited imaging utilizing quantum statistics of photons, have remained in the realm of proof-of-principle demonstrations. This is primarily due to a combination of low values of fill factors, quantum efficiencies, frame rates and signal-to-noise characteristic of most available single-photon sensitive imaging detectors. Here we describe an imaging device based on a fibre bundle coupled to single-photon avalanche detectors that combines a large fill factor, a high quantum efficiency, a low noise and scalable architecture. Our device enables localization-based super-resolution microscopy in a non-sparse non-stationary scene, utilizing information on the number of active emitters, as gathered from non-classical photon statistics.

Revealing true coupling strengths in two-dimensional spectroscopy with sparsity-based signal recovery.

Two-dimensional (2D) spectroscopy is used to study the interactions between energy levels in both the field of optics and nuclear magnetic resonance (NMR). Conventionally, the strength of interaction between two levels is inferred from the value of their common off-diagonal peak in the 2D spectrum, which is termed the cross peak. However, stronger diagonal peaks often have long tails that extend into the locations of the cross peaks and alter their values. Here, we introduce a method for retrieving the true interaction strengths by using sparse signal recovery techniques and apply our method in 2D Raman spectroscopy experiments.

Weakly diverging to tightly focused Gaussian beams: a single set of analytic expressions.

Analytic expressions describing all vector components of Gaussian beams, linearly polarized as well as radially polarized, are presented. These simple expressions, to high powers in divergence angle, were derived from a single-component vector potential. The vector potential itself, as in the 1979 work of Davis [Phys. Rev. A19, 1177 (1979)PLRAAN1050-294710.1103/PhysRevA.22.1159], was approximated by the first two terms of an infinite series solution of the Helmholtz equation. The expressions presented here were formulated to emphasize the dependence of the amplitude of the various field components on the beam's divergence angle. We show that the amplitude of the axial component of a linearly polarized Gaussian beam scales as the divergence angle squared, whereas the amplitude of the cross-polarized component of a linearly polarized Gaussian beam scales as the divergence angle to the fourth power. Weakly diverging Gaussian beams as well as strongly focused Gaussian beams can be described by exactly the same set of mathematical expressions, up to normalization constant. For a strongly focused linearly polarized Gaussian beam, the ellipticity of the dominant electric field component, typically calculated by the Debye-Wolf integral, is reproduced. For yet higher accuracy, terms with higher powers in divergence angle are presented, but the inclusion of these terms is limited to low divergence angles and short axial distances.

Mechanoporation of living cells for delivery of macromolecules using nanoneedle array.

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.

Oscillating high-aspect-ratio monolithic silicon nanoneedle array enables efficient delivery of functional bio-macromolecules into living cells.

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.

Ensemble-Averaged Quantum Correlations between Path-Entangled Photons Undergoing Anderson Localization.

We measure ensemble-averaged quantum correlations of path-entangled photons, propagating in a disordered lattice and undergoing Anderson localization. These result in intriguing patterns, which show that quantum interference leads to unexpected dependencies of the location of one particle on the location of the other. These correlations are shared between localized and nonlocalized components of the two-photon wave function, and, moreover, yield information regarding the nature of the disorder itself. Such effects cannot be reproduced with classical waves, and are undetectable without ensemble averaging.

Second and third harmonic waves excited by focused Gaussian beams.

Harmonic generation by tightly-focused Gaussian beams is finding important applications, primarily in nonlinear microscopy. It is often naively assumed that the nonlinear signal is generated predominantly in the focal region. However, the intensity of Gaussian-excited electromagnetic harmonic waves is sensitive to the excitation geometry and to the phase matching condition, and may depend on quite an extended region of the material away from the focal plane. Here we solve analytically the amplitude integral for second harmonic and third harmonic waves and study the generated harmonic intensities vs. focal-plane position within the material. We find that maximum intensity for positive wave-vector mismatch values, for both second harmonic and third harmonic waves, is achieved when the fundamental Gaussian is focused few Rayleigh lengths beyond the front surface. Harmonic-generation theory predicts strong intensity oscillations with thickness if the material is very thin. We reproduced these intensity oscillations in glass slabs pumped at 1550nm. From the oscillations of the 517nm third-harmonic waves with slab thickness we estimate the wave-vector mismatch in a Soda-lime glass as Δk(H)= -0.249µm(-1).

Cell Invasion Dynamics into a Three Dimensional Extracellular Matrix Fibre Network.

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.