Optimizing cell therapy by sorting cells with high extracellular vesicle secretion.

Critical challenges remain in clinical translation of extracellular vesicle (EV)-based therapeutics due to the absence of methods to enrich cells with high EV secretion. Current cell sorting methods are limited to surface markers that are uncorrelated to EV secretion or therapeutic potential. Here, we utilize a nanovial technology for enrichment of millions of single cells based on EV secretion. This approach is applied to select mesenchymal stem cells (MSCs) with high EV secretion as therapeutic cells for improving treatment. The selected MSCs exhibit distinct transcriptional profiles associated with EV biogenesis and vascular regeneration and maintain high levels of EV secretion after sorting and regrowth. In a mouse model of myocardial infarction, treatment with high-secreting MSCs improves heart functions compared to treatment with low-secreting MSCs. These findings highlight the therapeutic importance of EV secretion in regenerative cell therapies and suggest that selecting cells based on EV secretion could enhance therapeutic efficacy.

Defining T cell receptor repertoires using nanovial-based binding and functional screening.

The ability to selectively bind to antigenic peptides and secrete effector molecules can define rare and low-affinity populations of cells with therapeutic potential in emerging T cell receptor (TCR) immunotherapies. We leverage cavity-containing hydrogel microparticles, called nanovials, each coated with peptide-major histocompatibility complex (pMHC) monomers to isolate antigen-reactive T cells. T cells are captured and activated by pMHCs inducing the secretion of effector molecules including IFN-γ and granzyme B that are accumulated on nanovials, allowing sorting based on both binding and function. The TCRs of sorted cells on nanovials are sequenced, recovering paired αß-chains using microfluidic emulsion-based single-cell sequencing. By labeling nanovials having different pMHCs with unique oligonucleotide-barcodes and secretions with oligo-barcoded detection antibodies, we could accurately link TCR sequences to specific targets and rank each TCR based on the corresponding cell's secretion level. Using the technique, we identified an expanded repertoire of functional TCRs targeting viral antigens with high specificity and found rare TCRs with activity against cancer-specific splicing-enhanced epitopes.

Associating growth factor secretions and transcriptomes of single cells in nanovials using SEC-seq.

Cells secrete numerous bioactive molecules that are essential for the function of healthy organisms. However, scalable methods are needed to link individual cell secretions to their transcriptional state over time. Here, by developing and using secretion-encoded single-cell sequencing (SEC-seq), which exploits hydrogel particles with subnanolitre cavities (nanovials) to capture individual cells and their secretions, we simultaneously measured the secretion of vascular endothelial growth factor A (VEGF-A) and the transcriptome for thousands of individual mesenchymal stromal cells. Our data indicate that VEGF-A secretion is heterogeneous across the cell population and is poorly correlated with the VEGFA transcript level. The highest VEGF-A secretion occurs in a subpopulation of mesenchymal stromal cells characterized by a unique gene expression signature comprising a surface marker, interleukin-13 receptor subunit alpha 2 (IL13RA2), which allowed the enrichment of this subpopulation. SEC-seq enables the identification of gene signatures linked to specific secretory states, facilitating mechanistic studies, the isolation of secretory subpopulations and the development of means to modulate cellular secretion.

Single-cell sorting based on secreted products for functionally defined cell therapies.

Cell therapies have emerged as a promising new class of "living" therapeutics over the last decade and have been particularly successful for treating hematological malignancies. Increasingly, cellular therapeutics are being developed with the aim of treating almost any disease, from solid tumors and autoimmune disorders to fibrosis, neurodegenerative disorders and even aging itself. However, their therapeutic potential has remained limited due to the fundamental differences in how molecular and cellular therapies function. While the structure of a molecular therapeutic is directly linked to biological function, cells with the same genetic blueprint can have vastly different functional properties (e.g., secretion, proliferation, cell killing, migration). Although there exists a vast array of analytical and preparative separation approaches for molecules, the functional differences among cells are exacerbated by a lack of functional potency-based sorting approaches. In this context, we describe the need for next-generation single-cell profiling microtechnologies that allow the direct evaluation and sorting of single cells based on functional properties, with a focus on secreted molecules, which are critical for the in vivo efficacy of current cell therapies. We first define three critical processes for single-cell secretion-based profiling technology: (1) partitioning individual cells into uniform compartments; (2) accumulating secretions and labeling via reporter molecules; and (3) measuring the signal associated with the reporter and, if sorting, triggering a sorting event based on these reporter signals. We summarize recent academic and commercial technologies for functional single-cell analysis in addition to sorting and industrial applications of these technologies. These approaches fall into three categories: microchamber, microfluidic droplet, and lab-on-a-particle technologies. Finally, we outline a number of unmet needs in terms of the discovery, design and manufacturing of cellular therapeutics and how the next generation of single-cell functional screening technologies could allow the realization of robust cellular therapeutics for all patients.

Single Cell Mechanotype and Associated Molecular Changes in Urothelial Cell Transformation and Progression.

Cancer cell mechanotype changes are newly recognized cancer phenotypic events, whereas metastatic cancer cells show decreased cell stiffness and increased deformability relative to normal cells. To further examine how cell mechanotype changes in early stages of cancer transformation and progression, an in vitro multi-step human urothelial cell carcinogenic model was used to measure cellular Young's modulus, deformability, and transit time using single-cell atomic force microscopy, microfluidic-based deformability cytometry, and quantitative deformability cytometry, respectively. Measurable cell mechanotype changes of stiffness, deformability, and cell transit time occur early in the transformation process. As cells progress from normal, to preinvasive, to invasive cells, Young's modulus of stiffness decreases and deformability increases gradually. These changes were confirmed in three-dimensional cultured microtumor masses and urine exfoliated cells directly from patients. Using gene screening and proteomics approaches, we found that the main molecular pathway implicated in cell mechanotype changes appears to be epithelial to mesenchymal transition.

A ferrobotic system for automated microfluidic logistics.

Automated technologies that can perform massively parallelized and sequential fluidic operations at small length scales can resolve major bottlenecks encountered in various fields, including medical diagnostics, -omics, drug development, and chemical/material synthesis. Inspired by the transformational impact of automated guided vehicle systems on manufacturing, warehousing, and distribution industries, we devised a ferrobotic system that uses a network of individually addressable robots, each performing designated micro-/nanofluid manipulation-based tasks in cooperation with other robots toward a shared objective. The underlying robotic mechanism facilitating fluidic operations was realized by addressable electromagnetic actuation of miniature mobile magnets that exert localized magnetic body forces on aqueous droplets filled with biocompatible magnetic nanoparticles. The contactless and high-strength nature of the actuation mechanism inherently renders it rapid (~10 centimeters/second), repeatable (>10,000 cycles), and robust (>24 hours). The robustness and individual addressability of ferrobots provide a foundation for the deployment of a network of ferrobots to carry out cross-collaborative logistics efficiently. These traits, together with the reconfigurability of the system, were exploited to devise and integrate passive/active advanced functional components (e.g., droplet dispensing, generation, filtering, and merging), enabling versatile system-level functionalities. By applying this ferrobotic system within the framework of a microfluidic architecture, the ferrobots were tasked to work cross-collaboratively toward the quantification of active matrix metallopeptidases (a biomarker for cancer malignancy and inflammation) in human plasma, where various functionalities converged to achieve a fully automated assay.

Detection of EGFR Mutations in cfDNA and CTCs, and Comparison to Tumor Tissue in Non-Small-Cell-Lung-Cancer (NSCLC) Patients.

Lung cancer is the leading cause of cancer-related mortality worldwide. Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) therapies, based on the evaluation of EGFR mutations, have shown dramatic clinical benefits. EGFR mutation assays are mainly performed on tumor biopsies, which carry risks, are not always successful and give results relevant to the timepoint of the assay. To detect secondary EGFR mutations, which cause resistance to 1st and 2nd generation TKIs and lead to the administration of a 3rd generation drug, effective and non-invasive monitoring of EGFR mutation status is needed. Liquid biopsy analytes, such as circulating tumor cells (CTCs) and circulating tumor DNA (cfDNA), allow such monitoring over the course of the therapy. The aim of this study was to develop and optimize a workflow for the evaluation of cfDNA and CTCs in NSCLC patients all from one blood sample. Using Vortex technology and EntroGen ctEGFR assay, EGFR mutations were identified at 0.5 ng of DNA (∼83 cells), with a sensitivity ranging from 0.1 to 2.0% for a total DNA varying from 25 ng (∼4 CTCs among 4000 white blood cells, WBCs) to 1 ng (∼4 CTCs among 200 WBCs). The processing of plasma-depleted-blood provided comparable capture recovery as whole blood, confirming the possibility of a multimodality liquid biopsy analysis (cfDNA and CTC DNA) from a single tube of blood. Different anticoagulants were evaluated and compared in terms of respective performance. Blood samples from 24 NSCLC patients and 6 age-matched healthy donors were analyzed with this combined workflow to minimize blood volume needed and sample-to-sample bias, and the EGFR mutation profile detected from CTCs and cfDNA was compared to matched tumor tissues. Despite the limited size of the patient cohort, results from this non-invasive EGFR mutation analysis are encouraging and this combined workflow represents a valuable means for informing therapy selection and for monitoring treatment of patients with NSCLC.

Microfluidic-Based Approaches in Targeted Cell/Particle Separation Based on Physical Properties: Fundamentals and Applications.

Cell separation is a key step in many biomedical research areas including biotechnology, cancer research, regenerative medicine, and drug discovery. While conventional cell sorting approaches have led to high-efficiency sorting by exploiting the cell's specific properties, microfluidics has shown great promise in cell separation by exploiting different physical principles and using different properties of the cells. In particular, label-free cell separation techniques are highly recommended to minimize cell damage and avoid costly and labor-intensive steps of labeling molecular signatures of cells. In general, microfluidic-based cell sorting approaches can separate cells using "intrinsic" (e.g., fluid dynamic forces) versus "extrinsic" external forces (e.g., magnetic, electric field, etc.) and by using different properties of cells including size, density, deformability, shape, as well as electrical, magnetic, and compressibility/acoustic properties to select target cells from a heterogeneous cell population. In this work, principles and applications of the most commonly used label-free microfluidic-based cell separation methods are described. In particular, applications of microfluidic methods for the separation of circulating tumor cells, blood cells, immune cells, stem cells, and other biological cells are summarized. Computational approaches complementing such microfluidic methods are also explained. Finally, challenges and perspectives to further develop microfluidic-based cell separation methods are discussed.

Intelligent image-activated cell sorting 2.0.

The advent of intelligent image-activated cell sorting (iIACS) has enabled high-throughput intelligent image-based sorting of single live cells from heterogeneous populations. iIACS is an on-chip microfluidic technology that builds on a seamless integration of a high-throughput fluorescence microscope, cell focuser, cell sorter, and deep neural network on a hybrid software-hardware data management architecture, thereby providing the combined merits of optical microscopy, fluorescence-activated cell sorting (FACS), and deep learning. Here we report an iIACS machine that far surpasses the state-of-the-art iIACS machine in system performance in order to expand the range of applications and discoveries enabled by the technology. Specifically, it provides a high throughput of ∼2000 events per second and a high sensitivity of ∼50 molecules of equivalent soluble fluorophores (MESFs), both of which are 20 times superior to those achieved in previous reports. This is made possible by employing (i) an image-sensor-based optomechanical flow imaging method known as virtual-freezing fluorescence imaging and (ii) a real-time intelligent image processor on an 8-PC server equipped with 8 multi-core CPUs and GPUs for intelligent decision-making, in order to significantly boost the imaging performance and computational power of the iIACS machine. We characterize the iIACS machine with fluorescent particles and various cell types and show that the performance of the iIACS machine is close to its achievable design specification. Equipped with the improved capabilities, this new generation of the iIACS technology holds promise for diverse applications in immunology, microbiology, stem cell biology, cancer biology, pathology, and synthetic biology.

Computational cytometer based on magnetically modulated coherent imaging and deep learning.

Detecting rare cells within blood has numerous applications in disease diagnostics. Existing rare cell detection techniques are typically hindered by their high cost and low throughput. Here, we present a computational cytometer based on magnetically modulated lensless speckle imaging, which introduces oscillatory motion to the magnetic-bead-conjugated rare cells of interest through a periodic magnetic force and uses lensless time-resolved holographic speckle imaging to rapidly detect the target cells in three dimensions (3D). In addition to using cell-specific antibodies to magnetically label target cells, detection specificity is further enhanced through a deep-learning-based classifier that is based on a densely connected pseudo-3D convolutional neural network (P3D CNN), which automatically detects rare cells of interest based on their spatio-temporal features under a controlled magnetic force. To demonstrate the performance of this technique, we built a high-throughput, compact and cost-effective prototype for detecting MCF7 cancer cells spiked in whole blood samples. Through serial dilution experiments, we quantified the limit of detection (LoD) as 10 cells per millilitre of whole blood, which could be further improved through multiplexing parallel imaging channels within the same instrument. This compact, cost-effective and high-throughput computational cytometer can potentially be used for rare cell detection and quantification in bodily fluids for a variety of biomedical applications.

Enhanced In Vivo Delivery of Stem Cells using Microporous Annealed Particle Scaffolds.

Delivery to the proper tissue compartment is a major obstacle hampering the potential of cellular therapeutics for medical conditions. Delivery of cells within biomaterials may improve localization, but traditional and newer void-forming hydrogels must be made in advance with cells being added into the scaffold during the manufacturing process. Injectable, in situ cross-linking microporous scaffolds are recently developed that demonstrate a remarkable ability to provide a matrix for cellular proliferation and growth in vitro in three dimensions. The ability of these scaffolds to deliver cells in vivo is currently unknown. Herein, it is shown that mesenchymal stem cells (MSCs) can be co-injected locally with microparticle scaffolds assembled in situ immediately following injection. MSC delivery within a microporous scaffold enhances MSC retention subcutaneously when compared to cell delivery alone or delivery within traditional in situ cross-linked nanoporous hydrogels. After two weeks, endothelial cells forming blood vessels are recruited to the scaffold and cells retaining the MSC marker CD29 remain viable within the scaffold. These findings highlight the utility of this approach in achieving localized delivery of stem cells through an injectable porous matrix while limiting obstacles of introducing cells within the scaffold manufacturing process.

Functional profiling of circulating tumor cells with an integrated vortex capture and single-cell protease activity assay.

Tumor cells are hypothesized to use proteolytic enzymes to facilitate invasion. Whether circulating tumor cells (CTCs) secrete these enzymes to aid metastasis is unknown. A quantitative and high-throughput approach to assay CTC secretion is needed to address this question. We developed an integrated microfluidic system that concentrates rare cancer cells >100,000-fold from 1 mL of whole blood into ∼50,000 2-nL drops composed of assay reagents within 15 min. The system isolates CTCs by size, exchanges fluid around CTCs to remove contaminants, introduces a matrix metalloprotease (MMP) substrate, and encapsulates CTCs into microdroplets. We found CTCs from prostate cancer patients possessed above baseline levels of MMP activity (1.7- to 200-fold). Activity of CTCs was generally higher than leukocytes from the same patient (average CTC/leukocyte MMP activity ratio, 2.6 ± 1.5). Higher MMP activity of CTCs suggests active proteolytic processes that may facilitate invasion or immune evasion and be relevant phenotypic biomarkers enabling companion diagnostics for anti-MMP therapies.

Evaluation of PD-L1 expression on vortex-isolated circulating tumor cells in metastatic lung cancer.

Metastatic non-small cell lung cancer (NSCLC) is a highly fatal and immunogenic malignancy. Although the immune system is known to recognize these tumor cells, one mechanism by which NSCLC can evade the immune system is via overexpression of programmed cell death ligand 1 (PD-L1). Recent clinical trials of PD-1 and PD-L1 inhibitors have returned promising clinical responses. Important for personalizing therapy, patients with higher intensity staining for PD-L1 on tumor biopsies responded better. Thus, there has been interest in using PD-L1 tumor expression as a criterion for patient selection. Currently available methods of screening involve invasive tumor biopsy, followed by histological grading of PD-L1 levels. Biopsies have a high risk of complications, and only allow sampling from limited tumor sections, which may not reflect overall tumor heterogeneity. Circulating tumor cell (CTC) PD-L1 levels could aid in screening patients, and could supplement tissue PD-L1 biopsy results by testing PD-L1 expression from disseminated tumor sites. Towards establishing CTCs as a screening tool, we developed a protocol to isolate CTCs at high purity and immunostain for PD-L1. Monitoring of PD-L1 expression on CTCs could be an additional biomarker for precision medicine that may help in determining response to immunotherapies.

Separation of cancer cells using vortical microfluidic flows.

Label-free separation of viable cancer cells using vortical microfluidic flows has been introduced as a feasible cell collection method in oncological studies. Besides the clinical importance, the physics of particle interactions with the vortex that forms in a wall-confined geometry of a microchannel is a relatively new area of fluid dynamics. In our previous work [Haddadi and Di Carlo, J. Fluid. Mech. 811, 436-467 (2017)], we have introduced distinct aspects of inertial flow of dilute suspensions over cavities in a microchannel such as breakdown of the separatrix and formation of stable limit cycle orbits for finite size polystyrene particles. In this work, we extend our experiments to address the engineering-physics of cancer cell entrapment in microfluidic cavities. We begin by studying the effects of the channel width and device height on the morphology of the vortex, which has not been discussed in our previous work. The stable limit cycle orbits of finite size cancer cells are then presented. We demonstrate effects of the separatrix breakdown and the limit cycle formation on the operation of the cancer cell separation platform. By studying the flow of dilute cell suspensions over the cavities, we further develop the notion of the cavity capacity and the relative rate of cell accumulation as optimization criteria which connect the device geometry with the flow. Finally, we discuss the proper placement of multiple cavities inside a microchannel for improved cell entrapment.

Continuous and Quantitative Purification of T-Cell Subsets for Cell Therapy Manufacturing Using Magnetic Ratcheting Cytometry.

T-cell-based immunotherapies represent a growing medical paradigm that has the potential to revolutionize contemporary cancer treatments. However, manufacturing bottlenecks related to the enrichment of therapeutically optimal T-cell subpopulations from leukopak samples impede scale-up and scale-out efforts. This is mainly attributed to the challenges that current cell purification platforms face in balancing the quantitative sorting capacity needed to isolate specific T-cell subsets with the scalability to meet manufacturing throughputs. In this work, we report a continuous-flow, quantitative cell enrichment platform based on a technique known as ratcheting cytometry that can perform complex, multicomponent purification targeting various subpopulations of magnetically labeled T cells directly from apheresis or peripheral blood mononuclear cell (PBMC) samples. The integrated ratcheting cytometry instrument and cartridge demonstrated enrichment of T cells directly from concentrated apheresis samples with a 97% purity and an 85% recovery of magnetically tagged cells. Magnetic sorting of different T-cell subpopulations was also accomplished on chip by multiplexing cell surface targets onto particles with differing magnetic strengths. We believe that ratcheting cytometry's quantitative capacity and throughput scalability represents an excellent technology candidate to alleviate cell therapy manufacturing bottlenecks.

Optofluidic time-stretch quantitative phase microscopy.

Innovations in optical microscopy have opened new windows onto scientific research, industrial quality control, and medical practice over the last few decades. One of such innovations is optofluidic time-stretch quantitative phase microscopy - an emerging method for high-throughput quantitative phase imaging that builds on the interference between temporally stretched signal and reference pulses by using dispersive properties of light in both spatial and temporal domains in an interferometric configuration on a microfluidic platform. It achieves the continuous acquisition of both intensity and phase images with a high throughput of more than 10,000 particles or cells per second by overcoming speed limitations that exist in conventional quantitative phase imaging methods. Applications enabled by such capabilities are versatile and include characterization of cancer cells and microalgal cultures. In this paper, we review the principles and applications of optofluidic time-stretch quantitative phase microscopy and discuss its future perspective.

Enzyme-Free Nucleic Acid Amplification Assay Using a Cellphone-Based Well Plate Fluorescence Reader.

Nucleic acids, DNA and RNA, provide important fingerprint information for various pathogens and have significant diagnostic value; however, improved approaches are urgently needed to enable rapid detection of nucleic acids in simple point-of-care formats with high sensitivity and specificity. Here, we present a system that utilizes a series of toehold-triggered hybridization/displacement reactions that are designed to convert a given amount of RNA molecules (i.e., the analyte) into an amplified amount of signaling molecules without any washing steps or thermocycling. Fluorescent probes for signal generation were designed to consume products of the catalytic reaction in order to push the equilibrium and enhance the assay fold amplification for improved sensitivity and reaction speed. The system of toehold-assisted reactions is also modeled to better understand its performance and capabilities, and we empirically demonstrate the success of this approach with two analytes of diagnostic importance, i.e., influenza viral RNA and a micro RNA (miR-31). We also show that the amplified signal permits using a compact and cost-effective smartphone-based fluorescence reader, an important requirement toward a nucleic-acid-based point-of-care diagnostic system.

Elastomeric sensor surfaces for high-throughput single-cell force cytometry.

As cells with aberrant force-generating phenotypes can directly lead to disease, cellular force-generation mechanisms are high-value targets for new therapies. Here, we show that single-cell force sensors embedded in elastomers enable single-cell force measurements with ~100-fold improvement in throughput than was previously possible. The microtechnology is scalable and seamlessly integrates with the multi-well plate format, enabling highly parallelized time-course studies. In this regard, we show that airway smooth muscle cells isolated from fatally asthmatic patients have innately greater and faster force-generation capacity in response to stimulation than healthy control cells. By simultaneously tracing agonist-induced calcium flux and contractility in the same cell, we show that the calcium level is ultimately a poor quantitative predictor of cellular force generation. Finally, by quantifying phagocytic forces in thousands of individual human macrophages, we show that force initiation is a digital response (rather than a proportional one) to the proper immunogen. By combining mechanobiology at the single-cell level with high-throughput capabilities, this microtechnology can support drug-discovery efforts for clinical conditions associated with aberrant cellular force generation.

Probing Cell Adhesion Profiles with a Microscale Adhesive Choice Assay.

In this work, we introduce, to our knowledge, a new set of adhesion-based biomarkers for characterizing mammalian cells. Mammalian cell adhesion to the extracellular matrix influences numerous physiological processes. Current in vitro methods to probe adhesion focus on adhesive force to a single surface, which can investigate only a subcomponent of the adhesive, motility, and polarization cues responsible for adhesion in the 3D tissue environment. Here, we demonstrate a method to quantify the transhesive properties of cells that relies on the microscale juxtaposition of two extracellular matrix-coated surfaces. By multiplexing this approach, we investigate the unique transhesive profiles for breast cancer cells that are adapted to colonize different metastatic sites. We find that malignant breast cancer cells readily transfer to new collagen I surfaces, and away from basement membrane proteins. Integrins and actin polymerization largely regulate this transfer. This tool can be readily adopted in cell biology and cancer research to uncover, to our knowledge, novel drivers of adhesion (or de-adhesion) and sort cell populations based on complex phenotypes with physiological relevance.

Microscale Laminar Vortices for High-Purity Extraction and Release of Circulating Tumor Cells.

Circulating tumor cells (CTCs) are disseminated tumor cells that reflect the tumors of origin and can provide a liquid biopsy that would potentially enable noninvasive tumor profiling, treatment monitoring, and identification of targeted treatments. Accurate and rapid purification of CTCs holds great potential to improve cancer care but the task remains technically challenging. Microfluidic isolation of CTCs within microscale vortices enables high-throughput and size-based purification of rare CTCs from bodily fluids. Collected cells are highly pure, viable, and easily accessible, allowing seamless integration with various downstream applications. Here, we describe how to fabricate the High-Throughput Vortex Chip (Vortex-HT) and to process diluted whole blood for CTC collection. Lastly, immunostaining and imaging protocols for CTC classification and corresponding CTC image galleries are reported.