High-Scale 3D-Bioprinting Platform for the Automated Production of Vascularized Organs-on-a-Chip.

3D bioprinting possesses the potential to revolutionize contemporary methodologies for fabricating tissue models employed in pharmaceutical research and experimental investigations. This is enhanced by combining bioprinting with advanced organs-on-a-chip (OOCs), which includes a complex arrangement of multiple cell types representing organ-specific cells, connective tissue, and vasculature. However, both OOCs and bioprinting so far demand a high degree of manual intervention, thereby impeding efficiency and inhibiting scalability to meet technological requirements. Through the combination of drop-on-demand bioprinting with robotic handling of microfluidic chips, a print procedure is achieved that is proficient in managing three distinct tissue models on a chip within only a minute, as well as capable of consecutively processing numerous OOCs without manual intervention. This process rests upon the development of a post-printing sealable microfluidic chip, that is compatible with different types of 3D-bioprinters and easily connected to a perfusion system. The capabilities of the automized bioprint process are showcased through the creation of a multicellular and vascularized liver carcinoma model on the chip. The process achieves full vascularization and stable microvascular network formation over 14 days of culture time, with pronounced spheroidal cell growth and albumin secretion of HepG2 serving as a representative cell model.

A high-precision wound healing assay based on photosensitized culture substrates.

Quantitative assessment of cell migration in vitro is often required in fundamental and applied research from different biomedical areas including wound repair, tumor metastasis or developmental biology. A collection of assays has been established throughout the years like the most widely used scratch assay or the so-called barrier assay. It is the principle of these assays to introduce a lesion into an otherwise confluent monolayer in order to study the migration of cells from the periphery into this artificial wound and determine the migration rate from the time necessary for wound closure. A novel assay makes use of photosensitizers doped into a polystyrene matrix. A thin layer of this composite material is coated on the bottom of regular cell culture ware showing perfect biocompatibility. When adherent cells are grown on this coating, resonant excitation of the photosensitizer induces a very local generation of 1O2, which kills the cells residing at the site of illumination. Cells outside the site of illumination are not harmed. When excitation of the photosensitizer is conducted by microscopic illumination, high-precision wounding in any size and geometry is available even in microfluidic channels. Besides proof-of-concept experiments, this study gives further insight into the mechanism of photosensitizer-mediated cell wounding.

Contraction pressure analysis using optical imaging in normal and MYBPC3-mutated hiPSC-derived cardiomyocytes grown on matrices with tunable stiffness.

Current in vivo disease models and analysis methods for cardiac drug development have been insufficient in providing accurate and reliable predictions of drug efficacy and safety. Here, we propose a custom optical flow-based analysis method to quantitatively measure recordings of contracting cardiomyocytes on polydimethylsiloxane (PDMS), compatible with medium-throughput systems. Movement of the PDMS was examined by covalently bound fluorescent beads on the PDMS surface, differences caused by increased substrate stiffness were compared, and cells were stimulated with ß-agonist. We further validated the system using cardiomyocytes treated with endothelin-1 and compared their contractions against control and cells incubated with receptor antagonist bosentan. After validation we examined two MYBPC3-mutant patient-derived cell lines. Recordings showed that higher substrate stiffness resulted in higher contractile pressure, while beating frequency remained similar to control. ß-agonist stimulation resulted in both higher beating frequency as well as higher pressure values during contraction and relaxation. Cells treated with endothelin-1 showed an increased beating frequency, but a lower contraction pressure. Cells treated with both endothelin-1 and bosentan remained at control level of beating frequency and pressure. Lastly, both MYBPC3-mutant lines showed a higher beating frequency and lower contraction pressure. Our validated method is capable of automatically quantifying contraction of hiPSC-derived cardiomyocytes on a PDMS substrate of known shear modulus, returning an absolute value. Our method could have major benefits in a medium-throughput setting.

Investigating Optimal Time Step Intervals of Imaging for Data Quality through a Novel Fully-Automated Cell Tracking Approach.

Computer-based fully-automated cell tracking is becoming increasingly important in cell biology, since it provides unrivalled capacity and efficiency for the analysis of large datasets. However, automatic cell tracking's lack of superior pattern recognition and error-handling capability compared to its human manual tracking counterpart inspired decades-long research. Enormous efforts have been made in developing advanced cell tracking packages and software algorithms. Typical research in this field focuses on dealing with existing data and finding a best solution. Here, we investigate a novel approach where the quality of data acquisition could help improve the accuracy of cell tracking algorithms and vice-versa. Generally speaking, when tracking cell movement, the more frequent the images are taken, the more accurate cells are tracked and, yet, issues such as damage to cells due to light intensity, overheating in equipment, as well as the size of the data prevent a constant data streaming. Hence, a trade-off between the frequency at which data images are collected and the accuracy of the cell tracking algorithms needs to be studied. In this paper, we look at the effects of different choices of the time step interval (i.e., the frequency of data acquisition) within the microscope to our existing cell tracking algorithms. We generate several experimental data sets where the true outcomes are known (i.e., the direction of cell migration) by either using an effective chemoattractant or employing no-chemoattractant. We specify a relatively short time step interval (i.e., 30 s) between pictures that are taken at the data generational stage, so that, later on, we may choose some portion of the images to produce datasets with different time step intervals, such as 1 min, 2 min, and so on. We evaluate the accuracy of our cell tracking algorithms to illustrate the effects of these different time step intervals. We establish that there exist certain relationships between the tracking accuracy and the time step interval associated with experimental microscope data acquisition. We perform fully-automatic adaptive cell tracking on multiple datasets, to identify optimal time step intervals for data acquisition, while at the same time demonstrating the performance of the computer cell tracking algorithms.

An acoustically-driven biochip - impact of flow on the cell-association of targeted drug carriers.

The interaction of targeted drug carriers with epithelial and endothelial barriers in vivo is largely determined by the dynamics of the body fluids. To simulate these conditions in binding assays, a fully biocompatible in vitro model was developed which can accurately mimic a wide range of physiological flow conditions on a thumbnail-format cell-chip. This acoustically-driven microfluidic system was used to study the interaction characteristics of protein-coated particles with cells. Poly(D,L-lactide-co-glycolide) (PLGA) microparticles (2.9 +/- 1 microm) were conjugated with wheat germ agglutinin (WGA-MP, cytoadhesive protein) or bovine serum albumin (BSA-MP, non-specific protein) and their binding to epithelial cell monolayers was investigated under stationary and flow conditions. While mean numbers of 1500 +/- 307 mm(-2) WGA-MP and 94 +/- 64 mm(-2) BSA-MP respectively were detected to be cell-bound in the stationary setup, incubation at increasing flow velocities increasingly antagonized the attachment of both types of surface-modified particles. However, while binding of BSA-MP was totally inhibited by flow, grafting with WGA resulted in a pronounced anchoring effect. This was indicated by a mean number of 747 +/- 241 mm(-2) and 104 +/- 44 mm(-2) attached particles at shear rates of 0.2 s(-1) and 1 s(-1) respectively. Due to the compactness of the fluidic chip which favours parallelization, this setup represents a highly promising approach towards a screening platform for the performance of drug delivery vehicles under physiological flow conditions. In this regard, the flow-chip is expected to provide substantial information for the successful design and development of targeted micro- and nanoparticulate drug carrier systems.

Advanced 2D/3D cell migration assay for faster evaluation of chemotaxis of slow-moving cells.

Considering the essential role of chemotaxis of adherent, slow-moving cells in processes such as tumor metastasis or wound healing, a detailed understanding of the mechanisms and cues that direct migration of cells through tissues is highly desirable. The state-of-the-art chemotaxis instruments (e.g. microfluidic-based devices, bridge assays) can generate well-defined, long-term stable chemical gradients, crucial for quantitative investigation of chemotaxis in slow-moving cells. However, the majority of chemotaxis tools are designed for the purpose of an in-depth, but labor-intensive analysis of migratory behavior of single cells. This is rather inefficient for applications requiring higher experimental throughput, as it is the case of e.g. clinical examinations, chemoattractant screening or studies of the chemotaxis-related signaling pathways based on subcellular perturbations. Here, we present an advanced migration assay for accelerated and facilitated evaluation of the chemotactic response of slow-moving cells. The revised chemotaxis chamber contains a hydrogel microstructure-the migration arena, designed to enable identification of chemotactic behavior of a cell population in respect to the end-point of the experiment. At the same time, the assay in form of a microscopy slide enables direct visualization of the cells in either 2D or 3D environment, and provides a stable and linear gradient of chemoattractant. We demonstrate the correctness of the assay on the model study of HT-1080 chemotaxis in 3D and on 2D surface. Finally, we apply the migration arena chemotaxis assay to screen for a chemoattractant of primary keratinocytes, cells that play a major role in wound healing, being responsible for skin re-epithelialization and a successful wound closure. In direction of new therapeutic strategies to promote wound repair, we identified the chemotactic activity of the epithelial growth factor receptor (EGFR) ligands EGF and TGFα (transforming growth factor α).

An acoustically driven microliter flow chamber on a chip (muFCC) for cell-cell and cell-surface interaction studies.

A novel method for pumping very small volumes of liquid by using surface acoustic waves is employed to create a microfluidic flow chamber on a chip. It holds a volume of only a few mul and its planar design provides complete architectural freedom. This allows for the reconstruction of even complex flow scenarios (e.g. curvatures, bifurcations and stenosis). Addition of polymer walls to the planar fluidic track enables cell culturing on the chip surface and the investigation of cell-cell adhesion dynamics under flow. We demonstrate the flexibility of the system for application in many areas of microfluidic investigations including blood clotting phenomena under various flow conditions and the investigation of different stages of cell adhesion.

A computational framework for particle and whole cell tracking applied to a real biological dataset.

Cell tracking is becoming increasingly important in cell biology as it provides a valuable tool for analysing experimental data and hence furthering our understanding of dynamic cellular phenomena. The advent of high-throughput, high-resolution microscopy and imaging techniques means that a wealth of large data is routinely generated in many laboratories. Due to the sheer magnitude of the data involved manual tracking is often cumbersome and the development of computer algorithms for automated cell tracking is thus highly desirable. In this work, we describe two approaches for automated cell tracking. Firstly, we consider particle tracking. We propose a few segmentation techniques for the detection of cells migrating in a non-uniform background, centroids of the segmented cells are then calculated and linked from frame to frame via a nearest-neighbour approach. Secondly, we consider the problem of whole cell tracking in which one wishes to reconstruct in time whole cell morphologies. Our approach is based on fitting a mathematical model to the experimental imaging data with the goal being that the physics encoded in the model is reflected in the reconstructed data. The resulting mathematical problem involves the optimal control of a phase-field formulation of a geometric evolution law. Efficient approximation of this challenging optimal control problem is achieved via advanced numerical methods for the solution of semilinear parabolic partial differential equations (PDEs) coupled with parallelisation and adaptive resolution techniques. Along with a detailed description of our algorithms, a number of simulation results are reported on. We focus on illustrating the effectivity of our approaches by applying the algorithms to the tracking of migrating cells in a dataset which reflects many of the challenges typically encountered in microscopy data.

Planar chip device for PCR and hybridization with surface acoustic wave pump.

We have developed a microfluidic device operating at a planar surface instead of a closed channel network. The fluid is transported in single droplets using surface acoustic waves (SAW) on a piezoelectric LiNbO(3) substrate. The surface of the piezo is chemically structured to induce high contact angles of the droplets or enclose areas where the liquid can wet the substrate. Combining the SAW technique with thin film resistance heaters, a biological analysis chip with integrated DNA amplification by PCR and hybridization was designed. To prevent evaporation of the PCR reagents at high temperatures the sample is enclosed in droplets of mineral oil. On this chip the SAW resolves dried primers, shifts the oil capped liquid between the two heaters and mixes during hybridization. The chip is able to perform a highly sensitive, fast and specific PCR with a volume as low as 200 nl. During the temperature cycles an online monitoring of the DNA concentration is feasible with an optical unit, providing a sensitivity of 0.1 ng. After PCR the product is moved to the second heater for the hybridization on a spotted DNA array. With our chip we were able to detect a single nucleotide polymorphism (SNP) responsible for the Leiden Factor V syndrome from human blood.

Nano- and pico-dispensing of fluids on planar substrates using SAW.

The increasing interest in miniaturization of biological and chemical experiments or assays demands precise metering of the smallest amounts of reagents, e.g., on a planar substrate. Very sophisticated spotting systems can nowadays produce arrays of many thousands of different substances on an area of a few square inches. Such micro arrays and the technology behind them have become an important tool in genomic expression assays, proteomic applications, and even in the field of combinatoric chemistry. We present a technique to dispense the smallest amounts of fluids in the form of either simple spots or more complicated microarrays, where we use surface acoustic waves in combination with a predetermined surface chemistry. In addition to a detailed description of the technique, several examples of applications are presented.

Supported Membranes Meet Flat Fluidics: Monitoring Dynamic Cell Adhesion on Pump-Free Microfluidics Chips Functionalized with Supported Membranes Displaying Mannose Domains.

In this paper we demonstrate the combination of supported membranes and so-called flat microfluidics, which enables one to manipulate liquids on flat chip surfaces via "inverse piezoelectric effect". Here, an alternating external electric field applied to the inter-digital transducers excites a surface acoustic wave on a piezoelectric substrate. Employing lithographic patterning of self-assembled monolayers of alkoxysilanes, we successfully confine a free-standing, hemi-cylindrical channel with the volume of merely 7 µL . The experimentally determined maximum flow velocity scales linearly with the acoustic power, suggesting that our current setup can drive liquids at the speed of up to 7 cm/s (corresponding to a shear rate of 280 s-1) without applying high pressures using a fluidic pump. After the establishment of the functionalization of fluidic chip surfaces with supported membranes, we deposited asymmetric supported membranes displaying well-defined mannose domains and monitored the dynamic adhesion of E.Coli HB101 expressing mannose-binding receptors. Despite of the further technical optimization required for the quantitative analysis, the obtained results demonstrate that the combination of supported membranes and flat fluidics opens a large potential to investigate dynamic adhesion of cells on biofunctional membrane surfaces with the minimum amount of samples, without any fluidic pump.

A novel µ-fluidic whole blood coagulation assay based on Rayleigh surface-acoustic waves as a point-of-care method to detect anticoagulants.

A universal coagulation test that reliably detects prolonged coagulation time in patients, irrespective of the anticoagulant administered, has not been available to date. An easily miniaturised, novel µ-fluidic universal coagulation test employing surface acoustic waves (SAW) is presented here. SAW was employed to instantly mix and recalcify 6 µl citrated whole blood and image correlation analysis was used to quantify clot formation kinetics. The detection of clinically relevant anticoagulant dosing with old anticoagulants (unfractionated heparin, argatroban) and new anticoagulants (dabigatran, rivaroxaban) has been tested and compared to standard plasma coagulation assays. The applicability of this novel method has been confirmed in a small patient population. Coagulation was dose-proportionally prolonged with heparin, argatroban, dabigatran, and rivaroxaban, comparable to standard tests. Aspirin and clopidogrel did not interfere with the SAW-induced clotting time (SAW-CT), whereas the strong GPIIb/IIIa-inhibitor abciximab did interfere. Preliminary clinical data prove the suitability of the SAW-CT in patients being treated with warfarin, rivaroxaban, or dabigatran. The system principally allows assessment of whole blood coagulation in humans in a point-of-care setting. This method could be used in stroke units, emergency vehicles, general and intensive care wards, as well as for laboratory and home testing of coagulation.