Enumeration and gentle sorting of immune cells on chip, key to next generation advanced therapies in outpatient setting.

BACKGROUND: A thorough understanding of immune-oncology and molecular medicine has been vital in the development of cell therapeutics. At the basis of this translational research and its future implementation into a medicinal product, lies the availability of pure and viable cell populations. Currently, FACS and magnetic bead isolation are successfully used but suffer to fulfill all requirements. FACS is costly and difficult to upscale due to the limitation of shear stress, especially fragile, cells can handle. Therefore, magnetic bead isolation is often used as it is gentler, but it lacks the multiparametric aspect to isolate more complex cellular profiles. AIMS: We aim to develop a versatile technology able of multi marker detection and isolation of complex cell types with high purity, viability and throughput. METHODS: We have developed a gentle sorting mechanism based on a jet flow created by micro vapor bubbles, enabling a closed microfluidic cell isolation platform capable of multiparametric sorting with high viability, purity and throughput. In this work we compared the purity, recovery and viability of sorted CD4+ CD14- cells to magnetic isolation, most often used for other cell manufacturing approaches. Futhermore, we cultured the sorted cells of both isolation strategies and compared their growth curve and expression of activation-induced IL2 and IFN-γ. RESULTS: We demonstrate that this tool can achieve a pure population of CD4+ CD14- cells with high viability after sorting without compromising the recovery. On top of the viability also the growth and activation potential of sorted cells is unhampered by comparison to the benchmark gentle magnetic isolation. CONCLUSIONS: Our technology allows for the development of a compact system which sets it apart from other efforts intended to create automated cell therapeutic solutions.

Durability of the bubble-jet sorter enables high performance bio sample isolation.

Sorting cells while maintaining their viability for further processing or analysis is an essential step in a variety of biological processes ranging from early diagnostics to cell therapy. Sorting techniques such as fluorescence-activated cell sorting (FACS) have evolved considerably and provide standard ways of sorting. Nevertheless, the search for compact, integrated, efficient, and high throughput microfluidic sorting platforms continues due to challenges such as cost, cell viability, and biosafety. In our previous work, we introduced a technology with the potential to become such a platform: the bubble-jet sorter. It is a silicon-based sorter chip relying on cell deflection through micro vapor bubble formation. In this work, we present a new version of the sorter chip that emphasizes durability and continuous sorting operation. To characterize the sorter, we first focus on the technical performance and show a sorter lifetime that repeatedly exceeds 80 million actuation cycles. In addition, we show continuous operation at high firing rates, but also discuss limitations due to heat buildup. In a second step, we present continuous sorting runs of millions of beads and CD3 positive T cells at rates surpassing 1000 sorting events per second, while maintaining high purity (>90%) and recovery (>85%). Dedicated viability tests show that the gentle sorting process maintains cell viability in this closed, aerosol-free device. The remarkable combination of high lifetime, sorting rate, and sorting efficiency, along with the potential for on-chip parallelization show the promise of this technology to meet the growing demand for large-scale sample isolation in drug and immunotherapy development.

On-chip flow cytometer using integrated photonics for the detection of human leukocytes.

Differentiation between leukocyte subtypes like monocytes and lymphocytes is essential for cell therapy and research applications. To guarantee the cost-effective delivery of functional cells in cell therapies, billions of cells must be processed in a limited time. Yet, the sorting rates of commercial cell sorters are not high enough to reach the required yield. Process parallelization by using multiple instruments increases variability and production cost. A compact solution with higher throughput can be provided by multichannel flow cytometers combining fluidics and optics on-chip. In this work, we present a micro-flow cytometer with monolithically integrated photonics and fluidics and demonstrate that both the illumination of cells, as well as the collection of scattered light, can be realized using photonic integrated circuits. Our device is the first with sufficient resolution for the discrimination of lymphocytes and monocytes. Innovations in microfabrication have enabled complete integration of miniaturized photonic components and fluidics in a CMOS-compatible wafer stack. In combination with external optics, the device is ready for the collection of fluorescence using the on-chip excitation.

Use of high frequency electrorotation to identify cytoplasmic changes in cells non-disruptively.

In this paper we demonstrate how the use of frequencies ranging from 50 kHz to 5 GHz in the analysis of cells by electrorotation can open the path to the identification of differences not detectable by conventional set-ups. Earlier works usually reported electrorotation devices operating below 20 MHz, limiting the response obtained to properties associated with the cell membrane. Those devices are thus unable to resolve the physiological properties in the cytoplasm. We used microwave-based technology to extend the frequency operation to 5 GHz. At high frequencies (from tens of MHz to GHz), the electromagnetic signal passes through the membrane and allows probing the cytoplasm. This enables several applications, such as cell classification, and viability analysis. Additionally, the use of conventional microfabrication techniques reduces the cost and complexity of analysis, compared to other non-invasive methods. We demonstrated the potential of this set-up by identifying two different populations of T-lymphocytes not distinguishable through visual assessment. We also assessed the effect of calcein on cell cytoplasmic properties and used it as a controlled experiment to demonstrate the possibility of this method to detect changes happening predominantly in the cytoplasm.

Optimizing mRNA transfection on a high-definition electroporation microelectrode array results in 98% efficiency and multiplexed gene delivery.

Spatially resolved transfection, intracellular delivery of proteins and nucleic acids, has the potential to drastically speed up the discovery of biologically active cargos, for instance for the development of cell therapies or new genome engineering tools. We recently demonstrated the use of a high-density microelectrode array for the targeted electrotransfection of cells grown on its surface, a process called High-Definition Electroporation (HD-EP). We also developed a framework based on Design of Experiments to quickly establish optimized electroporation conditions across five different electrical pulse parameters. Here, we used this framework to optimize the transfection efficiency of primary fibroblasts with a mCherry-encoding mRNA, resulting in 98% of the cells expressing the desired fluorescent protein without any sign of cell death. That transfection yield is the highest reported so far for electroporation. Moreover, varying the pulse number was shown to modulate the fluorescence intensity of cells, indicating the dosage-controlled delivery of mRNA and protein expression. Finally, exploiting the single-electrode addressability of the microelectrode array, we demonstrated spatially resolved, high efficiency, sequential transfection of cells with three distinct mRNAs. Since the chip can be easily redesigned to feature a much large number of electrodes, we anticipate that this methodology will enable the development of dedicated screening platforms for analysis of mRNA variants at scale.

Electric field gradient focusing with electro-osmotic flow to reduce analyte dispersion: Concept and numerical investigation.

In proteomics, the need to precisely examine the protein compounds of small samples, requires sensitive analytical methods which can separate and enrich compounds with high precision. Current techniques require a minimal analysis time to obtain satisfactory compound separation where longer analysis time means better separation of compounds. But, molecular diffusion will create broadening of the separated compound bands over time, increasing the peak width, and thus reducing the resolution and the enrichment. Electric field gradient focusing (EFGF) is a separation technique, in which proteins are simultaneously separated and enriched by balancing a gradient electrostatic force with a constant hydrodynamic drag force. Because of this balance, analytes are continuously pushed back to their focusing point, limiting the time-dependent peak broadening due to molecular diffusion. Current EFGF techniques are however still suffering from peak broadening because of flow-profile inhomogeneities. In this paper, we propose to use AC electro-osmotic flow (AC EOF) to create a homogeneous flow in EFGF. The interference between the electric field gradient and the AC EOF was thoroughly analysed and the concept was validated using numerical simulations. The results show that a plug flow is obtained on top of a small, distorted boundary layer. While applying different DC electric fields in the electrolyte, a constant flow velocity can be obtained by including a DC offset to the electrodes generating the AC EOF. The plug flow is then maintained over the whole separation channel length, while an electric field gradient is applied. This way, the flow-induced contribution to peak broadening can be minimized in EFGF devices. By modelling the separation of green fluorescent protein (GFP) and R-Phycoerythrin (R-PE), it was shown that the peak width of separated compounds can be reduced and that the separation resolution can be improved, compared to current EFGF methods.

A Microfluidics Approach for Ovarian Cancer Immune Monitoring in an Outpatient Setting.

Among cancer diagnoses in women, ovarian cancer has the fifth-highest mortality rate. Current treatments are unsatisfactory, and new therapies are highly needed. Immunotherapies show great promise but have not reached their full potential in ovarian cancer patients. Implementation of an immune readout could offer better guidance and development of immunotherapies. However, immune profiling is often performed using a flow cytometer, which is bulky, complex, and expensive. This equipment is centralized and operated by highly trained personnel, making it cumbersome and time-consuming. We aim to develop a disposable microfluidic chip capable of performing an immune readout with the sensitivity needed to guide diagnostic decision making as close as possible to the patient. As a proof of concept of the fluidics module of this concept, acquisition of a limited immune panel based on CD45, CD8, programmed cell death protein 1 (PD1), and a live/dead marker was compared to a conventional flow cytometer (BD FACSymphony). Based on a dataset of peripheral blood mononuclear cells of 15 patients with ovarian cancer across different stages of treatment, we obtained a 99% correlation coefficient for the detection of CD8+PD1+ T cells relative to the total amount of CD45+ white blood cells. Upon further system development comprising further miniaturization of optics, this microfluidics chip could enable immune monitoring in an outpatient setting, facilitating rapid acquisition of data without the need for highly trained staff.

Amplification Efficiency and Template Accessibility as Distinct Causes of Rain in Digital PCR: Monte Carlo Modeling and Experimental Validation.

Partitions in digital PCR (dPCR) assays do not reach the detection threshold at the same time. This heterogeneity in amplification results in intermediate endpoint fluorescence values (i.e., rain) and misclassification of partitions, which has a major impact on the accuracy of nucleic acid quantification. Rain most often results from a reduced amplification efficiency or template inaccessibility; however, exactly how these contribute to rain has not been described. We developed and experimentally validated an analytical model that mechanistically explains the relationship between amplification efficiency, template accessibility, and rain. Using Monte Carlo simulations, we show that a reduced amplification efficiency leads to broader threshold cycle (Ct) distributions that can be fitted using a log-normal probability distribution. From the fit parameters, the amplification efficiency can be calculated. Template inaccessibility, on the other hand, leads to a different rain pattern, in which a distinct exponential tail in the Ct distribution can be observed. Using our model, it is possible to determine if the amplification efficiency, template accessibility, or another source is the main contributor of rain in dPCR assays. We envision that this model will facilitate and speed up dPCR assay optimization and provide an indication for the accuracy of the assay.

High-definition electroporation: Precise and efficient transfection on a microelectrode array.

Intracellular delivery is critical for a plethora of biomedical applications, including mRNA transfection and gene editing. High transfection efficiency and low cytotoxicity, however, are often beyond the capabilities of bulk techniques and synonymous with extensive empirical optimization. Moreover, bulk techniques are not amenable to large screening applications. Here, we propose an expeditious workflow for achieving optimal electroporation-based intracellular delivery. Using the multiplexing ability of a high-definition microelectrode array (MEA) chip, we performed a sequence of carefully designed experiments, multiple linear regression modelling and validation to obtain optimal conditions for on-chip electroporation of primary fibroblasts. Five electric pulse parameters were varied to generate 32 different electroporation conditions. The effect of the parameters on cytotoxicity and intracellular delivery could be evaluated with just two experiments. Most successful electroporation conditions resulted in no cell death, highlighting the low cytotoxicity of on-chip electroporation. The resulting delivery models were then used to achieve dosage-controlled delivery of small molecules, delivery of Cas9-GFP single-guide RNA complexes and transfection with an mCherry-encoding mRNA, resulting in previously unreported high-efficiency, single-cell transfection on MEAs: cells expressed mCherry on 81% of the actuated electrodes, underscoring the vast potential of CMOS MEA technology for the transfection of primary cells.

Rigorous analysis of the axial acoustic radiation force on a spherical object for single-beam acoustic tweezing applications.

Acoustic tweezers are increasingly utilized for the contactless manipulation of small particles. This paper provides a theoretical model demonstrating the acoustic manipulation capabilities of single-beam acoustic transducers. Analytical formulas are derived for the acoustic radiation force on an isotropic spherical object of arbitrary size, centered on a circular piston, simply supported and clamped radiator in an inviscid fluid. Using these results, the existence of a negative axial force pulling the object closer to the radiator is revealed and explored. These findings offer further insight into the feasibility of trapping objects in the near-field of a single-beam acoustic transducer. The calculations illustrate the trapping capabilities of the different emitters as a function of radiator size, particle size, and distance from the source and highlight the impact of radiator boundary conditions. Manipulation of a cell-like fluid sphere in water and an expanded polystyrene sphere in air are studied in more detail with results that are validated through finite element analysis. The developed theoretical model allows fast evaluation of acoustic radiation forces which could aid in the development of relatively simple and inexpensive contactless manipulation solutions.

Synthetic Antiferromagnetic Gold Nanoparticles as Bimodal Contrast Agents in MRI and CT-An Experimental In Vitro and In Vivo Study.

The use of multimodal contrast agents can potentially overcome the intrinsic limitations of individual imaging methods. We have validated synthetic antiferromagnetic nanoparticles (SAF-NPs) as bimodal contrast agents for in vitro cell labeling and in vivo cell tracking using magnetic resonance imaging (MRI) and computed tomography (CT). SAF-NP-labeled cells showed high contrast in MRI phantom studies (r2* = 712 s-1 mM-1), while pelleted cells showed clear contrast enhancement in CT. After intravenous SAF-NP injection, nanoparticles accumulated in the liver and spleen, as visualized in vivo by significant MRI contrast enhancement. Intravenous injection of SAF-NP-labeled cells resulted in cell accumulation in the lungs, which was clearly detectable by using CT but not by using MRI. SAF-NPs proved to be very efficient cell labeling agents for complementary MRI- and CT-based cell tracking. Bimodal monitoring of SAF-NP labeled cells is in particular of interest for applications where the applied imaging methods are not able to visualize the particles and/or cells in all organs.

Assessment of the Theranostic Potential of Gold Nanostars-A Multimodal Imaging and Photothermal Treatment Study.

Gold nanoparticles offer the possibility to combine both imaging and therapy of otherwise difficult to treat tumors. To validate and further improve their potential, we describe the use of gold nanostars that were functionalized with a polyethyleneglycol-maleimide coating for in vitro and in vivo photoacoustic imaging (PAI), computed tomography (CT), as well as photothermal therapy (PTT) of cancer cells and tumor masses, respectively. Nanostar shaped particles show a high absorption coefficient in the near infrared region and have a hydrodynamic size in biological medium around 100 nm, which allows optimal intra-tumoral retention. Using these nanostars for in vitro labeling of tumor cells, high intracellular nanostar concentrations could be achieved, resulting in high PAI and CT contrast and effective PTT. By injecting the nanostars intratumorally, high contrast could be generated in vivo using PAI and CT, which allowed successful multi-modal tumor imaging. PTT was successfully induced, resulting in tumor cell death and subsequent inhibition of tumor growth. Therefore, gold nanostars are versatile theranostic agents for tumor therapy.

Fast compressive lens-free tomography for 3D biological cell culture imaging.

We present a compressive lens-free technique that performs tomographic imaging across a cubic millimeter-scale volume from highly sparse data. Compared with existing lens-free 3D microscopy systems, our method requires an order of magnitude fewer multi-angle illuminations for tomographic reconstruction, leading to a compact, cost-effective and scanning-free setup with a reduced data acquisition time to enable high-throughput 3D imaging of dynamic biological processes. We apply a fast proximal gradient algorithm with composite regularization to address the ill-posed tomographic inverse problem. Using simulated data, we show that the proposed method can achieve a reconstruction speed ∼10× faster than the state-of-the-art inverse problem approach in 3D lens-free microscopy. We experimentally validate the effectiveness of our method by imaging a resolution test chart and polystyrene beads, demonstrating its capability to resolve micron-size features in both lateral and axial directions. Furthermore, tomographic reconstruction results of neuronspheres and intestinal organoids reveal the potential of this 3D imaging technique for high-resolution and high-throughput biological applications.

Pixel super-resolution for lens-free holographic microscopy using deep learning neural networks.

Lens-free holographic microscopy (LFHM) provides a cost-effective tool for large field-of-view imaging in various biomedical applications. However, due to the unit optical magnification, its spatial resolution is limited by the pixel size of the imager. Pixel super-resolution (PSR) technique tackles this problem by using a series of sub-pixel shifted low-resolution (LR) lens-free holograms to form the high-resolution (HR) hologram. Conventional iterative PSR methods require a large number of measurements and a time-consuming reconstruction process, limiting the throughput of LFHM in practice. Here we report a deep learning-based PSR approach to enhance the resolution of LFHM. Compared with the existing PSR methods, our neural network-based approach outputs the HR hologram in an end-to-end fashion and maintains consistency in resolution improvement with a reduced number of LR holograms. Moreover, by exploiting the resolution degradation model in the imaging process, the network can be trained with a data set synthesized from the LR hologram itself without resorting to the HR ground truth. We validated the effectiveness and the robustness of our method by imaging various types of samples using a single network trained on an entirely different data set. This deep learning-based PSR approach can significantly accelerate both the data acquisition and the HR hologram reconstruction processes, therefore providing a practical solution to fast, lens-free, super-resolution imaging.

Development and validation of a glass-silicon microdroplet-based system to measure sulfite concentrations in beverages.

Sulfite is often added to beverages as an antioxidant and antimicrobial agent. In fermented beverages, sulfite is also naturally produced by yeast cells. However, sulfite causes adverse health effects in asthmatic patients and accurate measurement of the sulfite concentration is therefore very important. Current sulfite analysis methods are time- and reagent-consuming and often require costly equipment. Here, we present a system allowing sensitive, ultralow-volume sulfite measurements based on a reusable glass-silicon microdroplet platform on which microdroplet generation, addition of enzymes through chemical-induced emulsion destabilization and pillar-induced droplet merging, emulsion restabilization, droplet incubation, and fluorescence measurements are integrated. In a first step, we developed and verified a fluorescence-based enzymatic assay for sulfite by measuring its analytical performance (LOD, LOQ, the dynamic working range, and the influence of salts, colorant, and sugars) and comparing fluorescent microplate readouts of fermentation samples with standard colorimetric measurements using the 5,5'-dithiobis-(2-nitrobenzoic acid) assay of the standard Gallery Plus Beermaster analysis platform. Next, samples were analyzed on the microdroplet platform, which also showed good correlation with the standard colorimetric analysis. Although the presented platform does not allow stable reinjection of droplets due to the presence of a tight array of micropillars at the fluidics entrances to prevent channel clogging by dust, removing the pillars, and integrating miniaturized pumps and optics in a future design would allow to use this platform for high-throughput, automated, and portable screening of microbes, plant, or mammalian cells. Graphical abstract ᅟ.

Fast and robust Fourier domain-based classification for on-chip lens-free flow cytometry.

The development of portable haematology analysers receives increased attention due to their deployability in resource-limited or emergency settings. Lens-free in-line holographic microscopy is one of the technologies that is being pushed forward in this regard as it eliminates complex and expensive optics, making miniaturisation and integration with microfluidics possible. On-chip flow cytometry enables high-speed capturing of individual cells in suspension, giving rise to high-throughput cell counting and classification. To perform a real-time analysis on this high-throughput content, we propose a fast and robust framework for the classification of leukocytes. The raw data consists of holographic acquisitions of leukocytes, captured with a high-speed camera as they are flowing through a microfluidic chip. Three different types of leukocytes are considered: granulocytes, monocytes and T-lymphocytes. The proposed method bypasses the reconstruction of the holographic data altogether by extracting Zernike moments directly from the frequency domain. By doing so, we introduce robustness to translations and rotations of cells, as well as to changes in distance of a cell with respect to the image sensor, achieving classification accuracies up to 96.8%. Furthermore, the reduced computational complexity of this approach, compared to traditional frameworks that involve the reconstruction of the holographic data, allows for very fast processing and classification, making it applicable in high-throughput flow cytometry setups.

High spatial resolution nanoslit SERS for single-molecule nucleobase sensing.

Solid-state nanopores promise a scalable platform for single-molecule DNA analysis. Direct, real-time identification of nucleobases in DNA strands is still limited by the sensitivity and the spatial resolution of established ionic sensing strategies. Here, we study a different but promising strategy based on optical spectroscopy. We use an optically engineered elongated nanopore structure, a plasmonic nanoslit, to locally enable single-molecule surface enhanced Raman spectroscopy (SERS). Combining SERS with nanopore fluidics facilitates both the electrokinetic capture of DNA analytes and their local identification through direct Raman spectroscopic fingerprinting of four nucleobases. By studying the stochastic fluctuation process of DNA analytes that are temporarily adsorbed inside the pores, we have observed asynchronous spectroscopic behavior of different nucleobases, both individual and incorporated in DNA strands. These results provide evidences for the single-molecule sensitivity and the sub-nanometer spatial resolution of plasmonic nanoslit SERS.

Reflective lens-free imaging on high-density silicon microelectrode arrays for monitoring and evaluation of in vitro cardiac contractility.

The high rate of drug attrition caused by cardiotoxicity is a major challenge for drug development. Here, we developed a reflective lens-free imaging (RLFI) approach to non-invasively record in vitro cell deformation in cardiac monolayers with high temporal (169 fps) and non-reconstructed spatial resolution (352 µm) over a field-of-view of maximally 57 mm2. The method is compatible with opaque surfaces and silicon-based devices. Further, we demonstrated that the system can detect the impairment of both contractility and fast excitation waves in cardiac monolayers. Additionally, the RLFI device was implemented on a CMOS-based microelectrode array to retrieve multi-parametric information of cardiac cells, thereby offering more in-depth analysis of drug-induced (cardiomyopathic) effects for preclinical cardiotoxicity screening applications.

Accurate label-free 3-part leukocyte recognition with single cell lens-free imaging flow cytometry.

Three-part white blood cell differentials which are key to routine blood workups are typically performed in centralized laboratories on conventional hematology analyzers operated by highly trained staff. With the trend of developing miniaturized blood analysis tool for point-of-need in order to accelerate turnaround times and move routine blood testing away from centralized facilities on the rise, our group has developed a highly miniaturized holographic imaging system for generating lens-free images of white blood cells in suspension. Analysis and classification of its output data, constitutes the final crucial step ensuring appropriate accuracy of the system. In this work, we implement reference holographic images of single white blood cells in suspension, in order to establish an accurate ground truth to increase classification accuracy. We also automate the entire workflow for analyzing the output and demonstrate clear improvement in the accuracy of the 3-part classification. High-dimensional optical and morphological features are extracted from reconstructed digital holograms of single cells using the ground-truth images and advanced machine learning algorithms are investigated and implemented to obtain 99% classification accuracy. Representative features of the three white blood cell subtypes are selected and give comparable results, with a focus on rapid cell recognition and decreased computational cost.

Supercritical Angle Fluorescence Characterization Using Spatially Resolved Fourier Plane Spectroscopy.

Most fluorescent immunoassays require a wash step prior to read-out due to the otherwise overwhelming signal of the large number of unbound (bulk) fluorescent molecules that dominate over the signal from the molecules of interest, usually bound to a substrate. Supercritical angle fluorescence (SAF) sensing is one of the most promising alternatives to total internal reflection fluorescence for fluorescence imaging and sensing. However, detailed experimental investigation of the influence of collection angle on the SAF surface sensitivity, i.e., signal to background ratio (SBR), is still lacking. In this Letter, we present a novel technique that allows to discriminate the emission patterns of free and bound fluorophores simultaneously by collecting both angular and spectral information. The spectrum was probed at multiple positions in the back focal plane using a multimode fiber connected to a spectrometer and the difference in intensity between two fluorophores was used to calculate the SBR. Our study clearly reveals that increasing the angle of SAF collection enhances the surface sensitivity, albeit at the cost of decreased signal intensity. Furthermore, our findings are fully supported by full-field 3D simulations.