Nanoplasmonic Avidity-Based Detection and Quantification of IgG Aggregates.

Production of therapeutic monoclonal antibodies (mAbs) is a complex process that requires extensive analytical and bioanalytical characterization to ensure high and consistent product quality. Aggregation of mAbs is common and very problematic and can result in products with altered pharmacodynamics and pharmacokinetics and potentially increased immunogenicity. Rapid detection of aggregates, however, remains very challenging using existing analytical techniques. Here, we show a real-time and label-free fiber optical nanoplasmonic biosensor system for specific detection and quantification of immunoglobulin G (IgG) aggregates exploiting Protein A-mediated avidity effects. Compared to monomers, IgG aggregates were found to have substantially higher apparent affinity when binding to Protein A-functionalized sensor chips in a specific pH range (pH 3.8-4.0). Under these conditions, aggregates and monomers showed significantly different binding and dissociation kinetics. Reliable and rapid aggregate quantification was demonstrated with a limit of detection (LOD) and limit of quantification (LOQ) of about 9 and 30 µg/mL, respectively. Using neural network-based curve fitting, it was further possible to simultaneously quantify monomers and aggregates for aggregate concentrations lower than 30 µg/mL. Our work demonstrates a unique avidity-based biosensor approach for fast aggregate analysis that can be used for rapid at-line quality control, including lot/batch release testing. This technology can also likely be further optimized for real-time in-line monitoring of product titers and quality, facilitating process intensification and automation.

Process integrated biosensors for real-time monitoring of antibodies for automated affinity purification.

Therapeutic monoclonal antibodies (mAbs) provide new means for treatments of a wide range of diseases and comprise a large fraction of all new approved drugs. Production of mAbs is expensive compared to conventional drug production, primarily due to the complex processes involved. The affinity purification step is dominating the cost of goods in mAb manufacturing. Process intensification and automation could reduce costs, but the lack of real-time process analytical technologies (PAT) complicates this development. We show a specific and robust fiber optical localized surface plasmon resonance (LSPR) sensor technology that is optimized for in-line product detection in the effluent in affinity capture steps. The sensor system comprises a flow cell and a replaceable sensor chip functionalized with biorecognition elements for specific analyte detection. The high selectivity of the sensor enable detection of mAbs in complex sample matrices at concentrations below 2.5 µg mL-1. In place regeneration of the sensor chips allowed for continuous monitoring of multiple consecutive chromatographic separation cycles. Excellent performance was obtained at different purification scales with flow rates up to 200 mL min-1. This sensor technology facilitates efficient column loading, optimization, and control of chromatography systems, which can pave the way for continuous operation and automation of protein purification steps.

Realization of user-friendly bioanalytical tools to quantify and monitor critical components in bio-industrial processes through conceptual design.

This minireview suggests a conceptual and user-oriented approach for the design of process monitoring systems in bioprocessing. Advancement of process analytical techniques for quantification of critical analytes can take advantage of basic conceptual process design to support reasoning, reconsidering and ranking solutions. Issues on analysis in complex bio-industrial media, sensitivity and selectivity are highlighted from users' perspectives. Meeting challenging analytical demands for understanding the critical interplay between the emerging bioprocesses, their biomolecular complexity and the needs for user-friendly analytical tools are discussed. By that, a thorough design approach is suggested based on a holistic design thinking in the quest for better analytical opportunities to solve established and emerging analytical needs.

Dynamic peptide-folding mediated biofunctionalization and modulation of hydrogels for 4D bioprinting.

Hydrogels are used in a wide range of biomedical applications, including three-dimensional (3D) cell culture, cell therapy and bioprinting. To enable processing using advanced additive fabrication techniques and to mimic the dynamic nature of the extracellular matrix (ECM), the properties of the hydrogels must be possible to tailor and change over time with high precision. The design of hydrogels that are both structurally and functionally dynamic, while providing necessary mechanical support is challenging using conventional synthesis techniques. Here, we show a modular and 3D printable hydrogel system that combines a robust but tunable covalent bioorthogonal cross-linking strategy with specific peptide-folding mediated interactions for dynamic modulation of cross-linking and functionalization. The hyaluronan-based hydrogels were covalently cross-linked by strain-promoted alkyne-azide cycloaddition using multi-arm poly(ethylene glycol). In addition, a de novo designed helix-loop-helix peptide was conjugated to the hyaluronan backbone to enable specific peptide-folding modulation of cross-linking density and kinetics, and hydrogel functionality. An array of complementary peptides with different functionalities was developed and used as a toolbox for supramolecular tuning of cell-hydrogel interactions and for controlling enzyme-mediated biomineralization processes. The modular peptide system enabled dynamic modifications of the properties of 3D printed structures, demonstrating a novel route for design of more sophisticated bioinks for four-dimensional bioprinting.

Cell-Based Assays Using Differentiated Human Induced Pluripotent Cells.

This chapter describes the requirements and preconditions for using human induced pluripotent cell lines in assay development within the pharmaceutical industry. The joint collaborative effort between academic and pharma partners within the StemBANCC consortium which enabled the implementation of iPSC-derived cellular models for drug discovery is highlighted. This large collaborative scientific network has successfully derived a significant number of well-characterized patient-specific iPSC lines and established disease-relevant cellular assays, both of which are requirements for enabling pharmaceutical companies to develop more efficacious and safer medicines.

Evaluating the Effect of Drug Compounds on Cardiac Spheroids Using the Cardiac Cell Outgrowth Assay.

The ideal cell culture model should mimic the cell physiology and the mechanical and the chemical cues that are present in specific tissues and organs, within a convenient high-throughput format. A possible key feature for such models is to recapture the cell polarity, the interactions between cells, and the interactions between the cells and the elastic extracellular matrix (ECM) by orienting the cells in a three-dimensional (3D) matrix. A common method to create 3D cell environments is to let the cells aggregate into spheroids with a diameter of around 200 µm. A major challenge for 3D cell cultures is to perform quick and easy imaging of the dense cell population, especially noninvasively. This protocol explains how to take advantage of the number of cells growing out from cell spheroids over time as a readout of the effect of a drug. The assay is compatible with standard imaging techniques and can be performed noninvasively using light microscopy or as a complement to other fluorescent imaging assays.

Fabrication of a Microfluidic Cell Culture Device Using Photolithographic and Soft Lithographic Techniques.

Photolithography and soft lithography are two common methods for fabrication of microfluidic cell culture devices. Well-defined microstructures are created by exposing a photoresist to UV-light under a photolithographic mask in which the intended patterns are transparent. The subsequent cross-linking of UV-exposed photoresist generates a reusable master that serves as a template for an elastomer, commonly polydimethylsiloxane (PDMS), that reciprocally recaptures the structures of the master in an optically clear and oxygen-permeable rubber-like material. Connections to the cell culture-containing channels of the device for perfusion of culture medium can be established by inserting tubing through the elastomer. In this protocol, the basic steps for making a standard microfluidic device for cell-based assays from photolithography and soft lithography techniques are outlined.

Using a Microfluidic Device for Culture and Drug Toxicity Testing of 3D Cells.

Microfluidic devices provide convenient assays tools for testing cell cultures in three-dimensional (3D) formats. These devices have significant potential for establishing assays that are better at predicting drug toxicity and efficacy effects compared to assays in conventional two-dimensional (2D) cultures. Microfluidic cell culture devices consist of perfused cell culture chambers with inlets and outlet for seeding, culturing, sampling, and assaying the cells. This protocol describes how to prepare and seed cells in a microfluidic cell culture device for drug toxicity testing on cells in a 3D structure. The protocol exemplifies the use of a basic microfluidic device with HepG2 hepatoma cells but can be transferred and optimized for other cells and cell types, including iPSC-derived tissue and organ cells.

Effect of inoculum density on human-induced pluripotent stem cell expansion in 3D bioreactors.

OBJECTIVE: For optimized expansion of human-induced pluripotent stem cells (hiPSCs) with regards to clinical applications, we investigated the influence of the inoculum density on the expansion procedure in 3D hollow-fibre bioreactors. MATERIALS AND METHODS: Analytical-scale bioreactors with a cell compartment volume of 3 mL or a large-scale bioreactor with a cell compartment volume of 17 mL were used and inoculated with either 10 × 106 or 50 × 106 hiPSCs. Cells were cultured in bioreactors over 15 days; daily measurements of biochemical parameters were performed. At the end of the experiment, the CellTiter-Blue® Assay was used for culture activity evaluation and cell quantification. Also, cell compartment sections were removed for gene expression and immunohistochemistry analysis. RESULTS: The results revealed significantly higher values for cell metabolism, cell activity and cell yields when using the higher inoculation number, but also a more distinct differentiation. As large inoculation numbers require cost and time-extensive pre-expansion, low inoculation numbers may be used preferably for long-term expansion of hiPSCs. Expansion of hiPSCs in the large-scale bioreactor led to a successful production of 5.4 × 109 hiPSCs, thereby achieving sufficient cell amounts for clinical applications. CONCLUSIONS: In conclusion, the results show a significant effect of the inoculum density on cell expansion, differentiation and production of hiPSCs, emphasizing the importance of the inoculum density for downstream applications of hiPSCs. Furthermore, the bioreactor technology was successfully applied for controlled and scalable production of hiPSCs for clinical use.

Model-based monitoring of industrial reversed phase chromatography to predict insulin variants.

Downstream processing in the manufacturing biopharmaceutical industry is a multistep process separating the desired product from process- and product-related impurities. However, removing product-related impurities, such as product variants, without compromising the product yield or prolonging the process time due to extensive quality control analytics, remains a major challenge. Here, we show how mechanistic model-based monitoring, based on analytical quality control data, can predict product variants by modeling their chromatographic separation during product polishing with reversed phase chromatography. The system was described by a kinetic dispersive model with a modified Langmuir isotherm. Solely quality control analytical data on product and product variant concentrations were used to calibrate the model. This model-based monitoring approach was developed for an insulin purification process. Industrial materials were used in the separation of insulin and two insulin variants, one eluting at the product peak front and one eluting at the product peak tail. The model, fitted to analytical data, used one component to simulate each protein, or two components when a peak displayed a shoulder. This monitoring approach allowed the prediction of the elution patterns of insulin and both insulin variants. The results indicate the potential of using model-based monitoring in downstream polishing at industrial scale to take pooling decisions.

Online measurement of oxygen enables continuous noninvasive evaluation of human-induced pluripotent stem cell (hiPSC) culture in a perfused 3D hollow-fiber bioreactor.

For clinical and/or pharmaceutical use of human-induced pluripotent stem cells (hiPSCs), large cell quantities of high quality are demanded. Therefore, we combined the expansion of hiPSCs in closed, perfusion-based 3D bioreactors with noninvasive online monitoring of oxygen as culture control mechanism. Bioreactors with a cell compartment volume of 3 or 17 ml were inoculated with either 10 × 106 or 50 × 106 cells, and cells were expanded over 15 days with online oxygen and offline glucose and lactate measurements being performed. The CellTiter-Blue® Assay was performed at the end of the bioreactor experiments for indirect cell quantification. Model simulations enabled an estimation of cell numbers based on kinetic equations and experimental data during the 15-day bioreactor cultures. Calculated oxygen uptake rates (OUR), glucose consumption rates (GCR), and lactate production rates (LPR) revealed a highly significant correlation (p < 0.0001). Oxygen consumption, which was measured at the beginning and the end of the experiment, showed a strong culture growth in line with the OUR and GCR data. Furthermore, the yield coefficient of lactate from glucose and the OUR to GCR ratio revealed a shift from nonoxidative to oxidative metabolism. The presented results indicate that oxygen is equally as applicable as parameter for hiPSC expansion as glucose while providing an accurate real-time impression of hiPSC culture development. Additionally, oxygen measurements inform about the metabolic state of the cells. Thus, the use of oxygen online monitoring for culture control facilitates the translation of hiPSC use to the clinical setting.

Fabrication of modular hyaluronan-PEG hydrogels to support 3D cultures of hepatocytes in a perfused liver-on-a-chip device.

Liver cell culture models are attractive in both tissue engineering and for development of assays for drug toxicology research. To retain liver specific cell functions, the use of adequate cell types and culture conditions, such as a 3D orientation of the cells and a proper supply of nutrients and oxygen, are critical. In this article, we show how extracellular matrix mimetic hydrogels can support hepatocyte viability and functionality in a perfused liver-on-a-chip device. A modular hydrogel system based on hyaluronan and poly(ethylene glycol) (HA-PEG), modified with cyclooctyne moieties for bioorthogonal strain-promoted alkyne-azide 1, 3-dipolar cycloaddition (SPAAC), was developed, characterized, and compared for cell compatibility to hydrogels based on agarose and alginate. Hepatoma cells (HepG2) formed spheroids with viable cells in all hydrogels with the highest expression of albumin and urea in alginate hydrogels. By including an excess of cyclooctyne in the HA backbone, azide-modified cell adhesion motifs (linear and cyclic RGD peptides) could be introduced in order to enhance viability and functionality of human induced pluripotent stem cell derived hepatocytes (hiPS-HEPs). In the HA-PEG hydrogels modified with cyclic RGD peptides hiPS-HEPs migrated and grew in 3D and showed an increased viability and higher albumin production compared to when cultured in the other hydrogels. This flexible SPAAC crosslinked hydrogel system enabled fabrication of perfused 3D cell culture of hiPS-HEPs and is a promising material for further development and optimization of liver-on-a-chip devices.

A Microbore Tubing Based Spiral for Multistep Cell Fractionation.

Cells were separated with the aid of a multistep spiral fractionation device, utilizing hydrodynamic forces in a spiral tubing. The spiral was fabricated using "off-the-shelf" microbore tubing, allowing for cheap and fast prototyping to achieve optimal cell separation. As a first step, a model system with 20 and 40 µm beads was used to demonstrate the effectiveness of the multistep separation device. With an initial purity of 5%, a separation purity of 83% was achieved after a two-step separation with the addition of 0.1% polyethylene glycol (PEG)-8000. Next, doxorubicin-resistant polyploid giant breast cancer cells (MDA-MB-231) were separated from doxorubicin-sensitive monoploid small breast cancer cells in the same fashion as the beads, resulting in a purity of around 40%, while maintaining a cell viability of more than 90%. Combined with basic cell analytical methods, the hydrodynamic separation principle of the device could be envisaged to be useful for a variety of cell fractionation needs in cell biology and in clinical applications.

Conceptual Design of Micro-Bioreactors and Organ-on-Chips for Studies of Cell Cultures.

Engineering design of microbioreactors (MBRs) and organ-on-chip (OoC) devices can take advantage of established design science theory, in which systematic evaluation of functional concepts and user requirements are analyzed. This is commonly referred to as a conceptual design. This review article compares how common conceptual design principles are applicable to MBR and OoC devices. The complexity of this design, which is exemplified by MBRs for scaled-down cell cultures in bioprocess development and drug testing in OoCs for heart and eye, is discussed and compared with previous design solutions of MBRs and OoCs, from the perspective of how similarities in understanding design from functionality and user purpose perspectives can more efficiently be exploited. The review can serve as a guideline and help the future design of MBR and OoC devices for cell culture studies.

A Cardiac Cell Outgrowth Assay for Evaluating Drug Compounds Using a Cardiac Spheroid-on-a-Chip Device.

Three-dimensional (3D) models with cells arranged in clusters or spheroids have emerged as valuable tools to improve physiological relevance in drug screening. One of the challenges with cells cultured in 3D, especially for high-throughput applications, is to quickly and non-invasively assess the cellular state in vitro. In this article, we show that the number of cells growing out from human induced pluripotent stem cell (hiPSC)-derived cardiac spheroids can be quantified to serve as an indicator of a drug’s effect on spheroids captured in a microfluidic device. Combining this spheroid-on-a-chip with confocal high content imaging reveals easily accessible, quantitative outgrowth data. We found that effects on outgrowing cell numbers correlate to the concentrations of relevant pharmacological compounds and could thus serve as a practical readout to monitor drug effects. Here, we demonstrate the potential of this semi-high-throughput “cardiac cell outgrowth assay” with six compounds at three concentrations applied to spheroids for 48 h. The image-based readout complements end-point assays or may be used as a non-invasive assay for quality control during long-term culture.

On-line soft sensing in upstream bioprocessing.

This review provides an overview and a critical discussion of novel possibilities of applying soft sensors for on-line monitoring and control of industrial bioprocesses. Focus is on bio-product formation in the upstream process but also the integration with other parts of the process is addressed. The term soft sensor is used for the combination of analytical hardware data (from sensors, analytical devices, instruments and actuators) with mathematical models that create new real-time information about the process. In particular, the review assesses these possibilities from an industrial perspective, including sensor performance, information value and production economy. The capabilities of existing analytical on-line techniques are scrutinized in view of their usefulness in soft sensor setups and in relation to typical needs in bioprocessing in general. The review concludes with specific recommendations for further development of soft sensors for the monitoring and control of upstream bioprocessing.

Developing organ-on-a-chip concepts using bio-mechatronic design methodology.

Mechatronic design is an engineering methodology for conceiving, configuring and optimising the design of a technical device or product to the needs and requirements of the final user. In this article, we show how the basic principles of this methodology can be exploited for in vitro cell cultures-often referred to as organ-on-a-chip devices. Due to the key role of the biological cells, we have introduced the term bio-mechatronic design, to highlight the complexity of designing a system that should integrate biology, mechanics and electronics in the same device structure. The strength of the mechatronic design is to match the needs of the potential users to a systematic evaluation of overall functional design alternative. It may be especially attractive for organs-on-chips where biological constituents such as cells and tissues in 3D settings and in a fluidic environment should be compared, screened and selected. Through this approach, design solutions ranked to customer needs are generated according to specified criteria, thereby defining the key constraints of the fabrication. As an example, the bio-mechatronic methodology is applied to a liver-on-a-chip based on information extrapolated from previous theoretical and experimental knowledge. It is concluded that the methodology can generate new fabrication solutions for devices, as well as efficient guidelines for refining the design and fabrication of many of today's organ-on-a-chip devices.

A Microfluidic Bioreactor for Toxicity Testing of Stem Cell Derived 3D Cardiac Bodies.

Modeling tissues and organs using conventional 2D cell cultures is problematic as the cells rapidly lose their in vivo phenotype. In microfluidic bioreactors the cells reside in microstructures that are continuously perfused with cell culture medium to provide a dynamic environment mimicking the cells natural habitat. These micro scale bioreactors are sometimes referred to as organs-on-chips and are developed in order to improve and extend cell culture experiments. Here, we describe the two manufacturing techniques photolithography and soft lithography that are used in order to easily produce microfluidic bioreactors. The use of these bioreactors is exemplified by a toxicity assessment on 3D clustered human pluripotent stem cells (hPSC)-derived cardiomyocytes by beating frequency imaging.

Stem cell derived in vivo-like human cardiac bodies in a microfluidic device for toxicity testing by beating frequency imaging.

Beating in vivo-like human cardiac bodies (CBs) were used in a microfluidic device for testing cardiotoxicity. The CBs, cardiomyocyte cell clusters derived from induced pluripotent stem cells, exhibited typical structural and functional properties of the native human myocardium. The CBs were captured in niches along a perfusion channel in the device. Video imaging was utilized for automatic monitoring of the beating frequency of each individual CB. The device allowed assessment of cardiotoxic effects of drug substances doxorubicin, verapamil and quinidine on the 3D clustered cardiomyocytes. Beating frequency data recorded over a period of 6 hours are presented and compared to literature data. The results indicate that this microfluidic setup with imaging of CB characteristics provides a new opportunity for label-free, non-invasive investigation of toxic effects in a 3D microenvironment.