Scalable mesenchymal stem cell enrichment from bone marrow aspirate using deterministic lateral displacement (DLD) microfluidic sorting.

The growing interest in regenerative medicine has opened new avenues for novel cell therapies using stem cells. Bone marrow aspirate (BMA) is an important source of stromal mesenchymal stem cells (MSCs). Conventional MSC harvesting from BMA relies on archaic centrifugation methods, often leading to poor yield due to osmotic stress, high centrifugation force, convoluted workflow, and long experimental time (∼2-3 hours). To address these issues, we have developed a scalable microfluidic technology based on deterministic lateral displacement (DLD) for MSC isolation. This passive, label-free cell sorting method capitalizes on the morphological differences between MSCs and blood cells (platelets and RBCs) for effective separation using an inverted L-shaped pillar array. To improve throughput, we developed a novel multi-chip DLD system that can process 2.5 mL of raw BMA in 20 ± 5 minutes, achieving a 2-fold increase in MSC recovery compared to centrifugation methods. Taken together, we envision that the developed DLD platform will enable fast and efficient isolation of MSCs from BMA for effective downstream cell therapy in clinical settings.

Multiplexed Single-Cell Leukocyte Enzymatic Secretion Profiling from Whole Blood Reveals Patient-Specific Immune Signature.

Enzymatic secretion of immune cells (leukocytes) plays a dominant role in host immune responses to a myriad of biological triggers, including infections, cancers, and cardiovascular diseases. Current tools to probe these leukocytes inadequately profile these vital biomarkers; the need for sample preprocessing steps of cell lysis, labeling, washing, and pipetting inevitably triggers the cells, changes its basal state, and dilutes the individual cell secretion in bulk assays. Using a fully integrated system for multiplexed profiling of native immune single-cell enzyme secretion from 50 µL of undiluted blood, we eliminate sample handling. With a total analysis time of 60 min, the integrated platform performs six tasks of leukocyte extraction, cell washing, fluorescent enzyme substrate mixing, single-cell droplet making, droplet incubation, and real-time readout for leukocyte secretion profiling of neutrophil elastase, granzyme B, and metalloproteinase. We calibrated the device, optimized the protocols, and tested the leukocyte secretion of acute heart failure (AHF) patients at admission and predischarge. This paper highlights the presence of single-cell enzymatic immune phenotypes independent of CD marker labeling, which could potentially elucidate the innate immune response states. We found that patients recovering from AHF showed a corresponding reduction in immune-cell enzymatic secretion levels and donor-specific enzymatic signatures were observed, which suggests patient-to-patient heterogeneous immune response. This platform presents opportunities to elucidate the complexities of the immune response from a single drop of blood and bridge the current technological, biological, and medical gap in understanding immune response and biological triggers.

Label-Free Biophysical Markers from Whole Blood Microfluidic Immune Profiling Reveal Severe Immune Response Signatures.

Disease manifestation and severity from acute infections are often due to hyper-aggressive host immune responses which change within minutes. Current methods for early diagnosis of infections focus on detecting low abundance pathogens, which are time-consuming, of low sensitivity, and do not reflect the severity of the pathophysiology appropriately. The approach here focuses on profiling the rapidly changing host inflammatory response, which in its over-exuberant state, leads to sepsis and death. A 15-min label-free immune profiling assay from 20 µL of unprocessed blood using unconventional L and Inverse-L shaped pillars of deterministic lateral displacement microfluidic technology is developed. The hydrodynamic interactions of deformable immune cells enable simultaneous sorting and immune response profiling in whole blood. Preliminary clinical study of 85 donors in emergency department with a spectrum of immune response states from healthy to severe inflammatory response shows correlation with biophysical markers of immune cell size, deformability, distribution, and cell counts. The speed of patient stratification demonstrated here has promising impact in deployable point-of-care systems for acute infections triage, risk management, and resource allocation at emergency departments, where clinical manifestation of infection severity may not be clinically evident as compared to inpatients in the wards or intensive care units.

Microfluidic label-free bioprocessing of human reticulocytes from erythroid culture.

In vitro erythroid cultures from human hematopoietic stem cells produce immature red blood cells (RBCs) called reticulocytes, which are important for RBCs production, and are widely used in scientific studies of malaria pathology, hematological diseases and protein translation. However, in vitro reticulocyte cultures contain expelled cell nuclei and erythroblasts as undesirable by-products and current purification methods such as density gradient centrifugation and fluorescence-activated cell sorting (FACS) are not optimal for integrated bioprocessing and downstream therapeutic applications. Developments in Dean flow fractionation (DFF) and deterministic lateral displacement (DLD) microfluidic sorting methods are ideal alternatives due to label-free size sorting, throughput scalability and low manufacturing cost. DFF sorting of reticulocytes from whole erythroid culture showed a 2.4-fold increase in cell recovery compared to FACS albeit with a lower purity; DLD sorting showed comparable cell recovery and purity with FACS using an inverse-L pillar structure to emphasize size and deformability sorting of reticulocytes. The viability and functional assurance of purified reticulocytes showed conserved cell deformability and supported the propagation of malaria parasites. Collectively, our study on label-free RBCs isolation represents a significant technical advancement towards developing in vitro generated viable human RBCs, opening opportunities for close-loop cell manufacturing, downstream therapeutic and research purposes.

Fluorescent label-free quantitative detection of nano-sized bioparticles using a pillar array.

Disease diagnostics requires detection and quantification of nano-sized bioparticles including DNA, proteins, viruses, and exosomes. Here, a fluorescent label-free method for sensitive detection of bioparticles is explored using a pillar array with micrometer-sized features in a deterministic lateral displacement (DLD) device. The method relies on measuring changes in size and/or electrostatic charges of 1 µm polymer beads due to the capture of target bioparticles on the surface. These changes can be sensitively detected through the lateral displacement of the beads in the DLD array, wherein the lateral shifts in the output translates to a quantitative measurement of bioparticles bound to the bead. The detection of albumin protein and nano-sized polymer vesicles with a concentration as low as 10 ng mL-1 (150 pM) and 3.75 µg mL-1, respectively, is demonstrated. This label-free method holds potential for point-of-care diagnostics, as it is low-cost, fast, sensitive, and only requires a standard laboratory microscope for detection.

Advancements in microfluidics for nanoparticle separation.

Nanoparticles have been widely implemented for healthcare and nanoscience industrial applications. Thus, efficient and effective nanoparticle separation methods are essential for advancement in these fields. However, current technologies for separation, such as ultracentrifugation, electrophoresis, filtration, chromatography, and selective precipitation, are not continuous and require multiple preparation steps and a minimum sample volume. Microfluidics has offered a relatively simple, low-cost, and continuous particle separation approach, and has been well-established for micron-sized particle sorting. Here, we review the recent advances in nanoparticle separation using microfluidic devices, focusing on its techniques, its advantages over conventional methods, and its potential applications, as well as foreseeable challenges in the separation of synthetic nanoparticles and biological molecules, especially DNA, proteins, viruses, and exosomes.

Asymmetrical Deterministic Lateral Displacement Gaps for Dual Functions of Enhanced Separation and Throughput of Red Blood Cells.

Deterministic lateral displacement (DLD) method for particle separation in microfluidic devices has been extensively used for particle separation in recent years due to its high resolution and robust separation. DLD has shown versatility for a wide spectrum of applications for sorting of micro particles such as parasites, blood cells to bacteria and DNA. DLD model is designed for spherical particles and efficient separation of blood cells is challenging due to non-uniform shape and size. Moreover, separation in sub-micron regime requires the gap size of DLD systems to be reduced which exponentially increases the device resistance, resulting in greatly reduced throughput. This paper shows how simple application of asymmetrical DLD gap-size by changing the ratio of lateral-gap (GL) to downstream-gap (GD) enables efficient separation of RBCs without greatly restricting throughput. This method reduces the need for challenging fabrication of DLD pillars and provides new insight to the current DLD model. The separation shows an increase in DLD critical diameter resolution (separate smaller particles) and increase selectivity for non-spherical RBCs. The RBCs separate better as compared to standard DLD model with symmetrical gap sizes. This method can be applied to separate non-spherical bacteria or sub-micron particles to enhance throughput and DLD resolution.

Real-time modulated nanoparticle separation with an ultra-large dynamic range.

Nanoparticles exhibit size-dependent properties which make size-selective purification of proteins, DNA or synthetic nanoparticles essential for bio-analytics, clinical medicine, nano-plasmonics and nano-material sciences. Current purification methods of centrifugation, column chromatography and continuous-flow techniques suffer from particle aggregation, multi-stage process, complex setups and necessary nanofabrication. These increase process costs and time, reduce efficiency and limit dynamic range. Here, we achieve an unprecedented real-time nanoparticle separation (51-1500 nm) using a large-pore (2 µm) deterministic lateral displacement (DLD) device. No external force fields or nanofabrication are required. Instead, we investigated innate long-range electrostatic influences on nanoparticles within a fluid medium at different NaCl ionic concentrations. In this study we account for the electrostatic forces beyond Debye length and showed that they cannot be assumed as negligible especially for precise nanoparticle separation methods such as DLD. Our findings have enabled us to develop a model to simultaneously quantify and modulate the electrostatic force interactions between nanoparticle and micropore. By simply controlling buffer solutions, we achieve dynamic nanoparticle size separation on a single device with a rapid response time (<20 s) and an enlarged dynamic range (>1200%), outperforming standard benchtop centrifuge systems. This novel method and model combines device simplicity, isolation precision and dynamic flexibility, opening opportunities for high-throughput applications in nano-separation for industrial and biological applications.

DLD pillar shape design for efficient separation of spherical and non-spherical bioparticles.

Particle sorting methods in microfluidic platforms are gaining momentum for various biomedical applications. Bioparticles are found in different shapes and sizes. However, conventional separation techniques are mainly designed for separation of spherical particles. Thus, there is a need to develop new methods for effective separation of spherical and non-spherical bioparticles for various applications. Deterministic lateral displacement (DLD) microfluidic methods have become popular for high separation resolution, simplicity, and predictability. However, shape sorting in the DLD separation methods is not well researched. Recently, we explored this area and found that pillar shapes in DLD significantly affect bioparticle separation. In this work, we designed a group of different pillar shapes with protrusions and groove structures with the hypothesis that pillar protrusions will induce particle rotation while pillar grooves will confine the particle rotational movement in a directed path for effective separation in a DLD pillar array. Using combinations of protrusions and grooves, 3-dimensional spherical particles, 2-dimensional planar disc-shaped red blood cells and 1-dimensional rod-shaped bacteria were separated and two interesting phenomena were observed. Firstly, the arrangement of pillar protrusions and grooves induces inertial movements, enhancing the separation of spherical particles. Secondly, non-spherical particles experience dominant rotational movements due to the protrusions and grooves which help in changing their orientations. This gives an opportunity to perform efficient separation based on the desired orientation (the longest dimension of the particles) by restricting or containing their movement within a specific DLD path.

Rotational separation of non-spherical bioparticles using I-shaped pillar arrays in a microfluidic device.

Most bioparticles, such as red blood cells and bacteria, are non-spherical in shape. However, conventional microfluidic separation devices are designed for spherical particles. This poses a challenge in designing a separation device for non-spherical bioparticles, as the smallest dimension of the bioparticle has to be considered, which increases fabrication challenges and decreases the throughput. If current methods do not take into account the shape of non-spherical bioparticles, the separation will be inefficient. Here, to address this challenge, we present a novel technique for the separation of red blood cells as a non-spherical bioparticle, using a new I-shaped pillar arrays design. It takes the shape into account and induces rotational movements, allowing us to leverage on the largest dimension, which increases its separation size. This technique has been used for 100% separation of red blood cells from blood samples in a focused stream, outperforming the conventional pillar array designs.