Multiplexed bioassays, in which multiple analytes of interest are probed in parallel within a single small volume, have greatly accelerated the pace of biological discovery. Bead-based multiplexed bioassays have many technical advantages, including near solution-phase kinetics, small sample volume requirements, many within-assay replicates to reduce measurement error, and, for some bead materials, the ability to synthesize analytes directly on beads via solid-phase synthesis. To allow bead-based multiplexing, analytes can be synthesized on spectrally encoded beads with a 1:1 linkage between analyte identity and embedded codes. Bead-bound analyte libraries can then be pooled and incubated with a fluorescently-labeled macromolecule of interest, allowing downstream quantification of interactions between the macromolecule and all analytes simultaneously via imaging alone. Extracting quantitative binding data from these images poses several computational image processing challenges, requiring the ability to identify all beads in each image, quantify bound fluorescent material associated with each bead, and determine their embedded spectral code to reveal analyte identities. Here, we present a novel open-source Python software package (the mrbles analysis package) that provides the necessary tools to: (1) find encoded beads in a bright-field microscopy image; (2) quantify bound fluorescent material associated with bead perimeters; (3) identify embedded ratiometric spectral codes within beads; and (4) return data aggregated by embedded code and for each individual bead. We demonstrate the utility of this package by applying it towards analyzing data generated via multiplexed measurement of calcineurin protein binding to MRBLEs (Microspheres with Ratiometric Barcode Lanthanide Encoding) containing known and mutant binding peptide motifs. We anticipate that this flexible package should be applicable to a wide variety of assays, including simple bead or droplet finding analysis, quantification of binding to non-encoded beads, and analysis of multiplexed assays that use ratiometric, spectrally encoded beads.

Transient, regulated binding of globular protein domains to Short Linear Motifs (SLiMs) in disordered regions of other proteins drives cellular signaling. Mapping the energy landscapes of these interactions is essential for deciphering and perturbing signaling networks but is challenging due to their weak affinities. We present a powerful technology (MRBLE-pep) that simultaneously quantifies protein binding to a library of peptides directly synthesized on beads containing unique spectral codes. Using MRBLE-pep, we systematically probe binding of calcineurin (CN), a conserved protein phosphatase essential for the immune response and target of immunosuppressants, to the PxIxIT SLiM. We discover that flanking residues and post-translational modifications critically contribute to PxIxIT-CN affinity and identify CN-binding peptides based on multiple scaffolds with a wide range of affinities. The quantitative biophysical data provided by this approach will improve computational modeling efforts, elucidate a broad range of weak protein-SLiM interactions, and revolutionize our understanding of signaling networks.

Protein-peptide interactions are essential for cellular responses. Despite their importance, these interactions remain largely uncharacterized due to experimental challenges associated with their measurement. Current techniques (e.g. surface plasmon resonance, fluorescence polarization, and isothermal calorimetry) either require large amounts of purified material or direct fluorescent labeling, making high-throughput measurements laborious and expensive. In this report, we present a new technology for measuring antibody-peptide interactions in vitro that leverages spectrally encoded beads for biological multiplexing. Specific peptide sequences are synthesized directly on encoded beads with a 1: 1 relationship between peptide sequence and embedded code, thereby making it possible to track many peptide sequences throughout the course of an experiment within a single small volume. We demonstrate the potential of these bead-bound peptide libraries by: (1) creating a set of 46 peptides composed of 3 commonly used epitope tags (myc, FLAG, and HA) and single amino-acid scanning mutants; (2) incubating with a mixture of fluorescently-labeled anti-myc, anti-FLAG, and anti-HA antibodies; and (3) imaging these bead-bound libraries to simultaneously identify the embedded spectral code (and thus the sequence of the associated peptide) and quantify the amount of each antibody bound. To our knowledge, these data demonstrate the first customized peptide library synthesized directly on spectrally encoded beads. While the implementation of the technology provided here is a high-affinity antibody/protein interaction with a small code space, we believe this platform can be broadly applicable to any range of peptide screening applications, with the capability to multiplex into libraries of hundreds to thousands of peptides in a single assay.

Microfluidics, the science of engineering fluid streams at the micrometer scale, offers unique tools for creating and controlling gradients of soluble compounds. Gradient generation can be used to recreate complex physiological microenvironments, but is also useful for screening purposes. For example, in a single experiment, adherent cells can be exposed to a range of concentrations of the compound of interest, enabling high-content analysis of cell behaviour and enhancing throughput. In this study, we present the development of a microfluidic screening platform where, by means of diffusion, gradients of soluble compounds can be generated and sustained. This platform enables the culture of adherent cells under shear stress-free conditions, and their exposure to a soluble compound in a concentration gradient-wise manner. The platform consists of five serial cell culture chambers, all coupled to two lateral fluid supply channels that are used for gradient generation through a source-sink mechanism. Furthermore, an additional inlet and outlet are used for cell seeding inside the chambers. Finite element modeling was used for the optimization of the design of the platform and for validation of the dynamics of gradient generation. Then, as a proof-of-concept, human osteosarcoma MG-63 cells were cultured inside the platform and exposed to a gradient of Cytochalasin D, an actin polymerization inhibitor. This set-up allowed us to analyze cell morphological changes over time, including cell area and eccentricity measurements, as a function of Cytochalasin D concentration by using fluorescence image-based cytometry.

An increasing number of orthopedic and craniomaxillofacial surgeries is one of the consequences of the current progressive population aging. Availability of a patient's own bone, the gold standard in bone regeneration, is limited, and therefore novel approaches are needed. Tissue engineering, both growth factor and cell based, is a promising alternative; however, its complexity makes the transfer from laboratory to the clinic a long and difficult one. Synthetic biomaterials form an interesting alternative to autogaft because they are available in large quantities off-the-shelf and relatively easy to adapt to various applications. Particularly, calcium phosphate-based synthetic bone graft substitutes such as calcium phosphate (CaP) ceramics are of great interest, as they resemble chemical composition of bone mineral and are therefore not only biocompatible but also bioactive. Biological performance of CaP ceramics in bone regeneration is generally inferior to that of autograft, as their effect on bone cell function and bone tissue formation is less pronounced. Improving the biological performance of bone graft substitutes, while retaining their synthetic character, is a challenge. Use of inorganic compounds, which are present in bone as trace elements and known to be involved in processes related to bone formation and remodeling, as additives to synthetic bone graft substitutes is a potentially interesting approach to improving their biological performance. In this chapter, five inorganic additives are described: three cations, namely, zinc, copper, and strontium, and two anions, namely, fluoride and carbonate. First, their role in bone metabolism and processes related to bone formation and resorption is discussed. Then, a number of methods are described by which these elements can be incorporated into CaP ceramics. Their effect on ceramic properties, as well as their direct or indirect effect on bone formation and resorption processes in vitro and in vivo, is also discussed. Finally, some conclusions and future perspectives are given for further research into synthetic bone graft substitutes.

We present here a screening method based on a microfluidic platform, which can generate four orthogonal and overlapping concentration gradients of soluble compounds over a monolayer of cells, in combination with automated and in situ image analysis, for use in regenerative medicine research. The device includes a square chamber in which cells are grown, and four independent supply channels along the sides of the chamber, which are connected through an array of small diffusion channels. Compounds flown through the supply channels diffuse through diffusion channels into the chamber to create a gradient over the cell culture area. Further, the chamber is connected to two channels intended for introduction of cells and in situ staining. In this study, the dimensions of the different channels were optimized through finite element modeling to yield stable gradients, and two designs were used with gradients spanning 2.9–2.4 μM and 3.4–2.0 μM. Next, overlapping gradients were generated using four rhodamine‐derived fluorescent dyes, and imaged using confocal microscopy. Finally, the platform was applied to assess the concentration‐dependent response of an osteoblastic cell line exposed to a hypoxia‐mimicking molecule phenanthroline, using an in situ fluorescent staining assay in combination with image analysis, applicable to closed microfluidic devices. The on‐chip assay yielded results comparable to those observed in conventional culture, where a range of concentrations was tested in independent microwells. In the future, we intend to use this method to complement or replace current research approaches in screening soluble compounds for regenerative medicine, which are often based on one‐sample‐for‐one‐experiment principle.

We present a microtiter plate-sized standalone chip holder for precise control of physiological conditions inside closed microfluidic cell culture systems, made from gas-impermeable materials. Specifically, we demonstrate the suitability of the holder to support cell growth in a glass chip, to allow time-lapse imaging of live cells and the creation of a hypoxic environment, all relevant for applications in regenerative medicine research.

The aim of regenerative medicine is to restore or establish normal function of damaged tissues or organs. Tremendous efforts are placed into development of novel regenerative strategies, involving (stem) cells, soluble factors, biomaterials or combinations thereof, as a result of the growing need caused by continuous population aging. To satisfy this need, fast and reliable assessment of (biological) performance is sought, not only to select the potentially interesting candidates, but also to rule out poor ones at an early stage of development. Microfluidics may provide a new avenue to accelerate research and development in the field of regenerative medicine as it has proven its maturity for the realization of high-throughput screening platforms. In addition, microfluidic systems offer other advantages such as the possibility to create in vivo-like microenvironments. Besides the complexity of organs or tissues that need to be regenerated, regenerative medicine brings additional challenges of complex regeneration processes and strategies. The question therefore arises whether so much complexity can be integrated into microfluidic systems without compromising reliability and throughput of assays. With this review, we aim to investigate whether microfluidics can become widely applied in regenerative medicine research and/or strategies.

We propose a microfluidics-based cell-culture platform for the screening of trace elements in bone tissue engineering. With this platform, it is possible to create four stable, independent and perpendicular diffusion-based concentration gradients in a single square chamber, in which cells are cultured. This allows examining the combined effect of four compounds in a single assay.