Blood unit segments accurately represent the biophysical properties of red blood cells in blood bags but not hemolysis.

BACKGROUND: The biophysical properties of red blood cells (RBCs) provide potential biomarkers for the quality of donated blood. Blood unit segments provide a simple and nondestructive way to sample RBCs in clinical studies of transfusion efficacy, but it is not known whether RBCs sampled from segments accurately represent the biophysical properties of RBCs in blood bags. STUDY DESIGN AND METHODS: RBCs were sampled from blood bags and segments every two weeks during 8 weeks of storage at 4°C. RBC deformability was measured by deformability-based sorting using the microfluidic ratchet device in order to derive a rigidity score. Standard hematological parameters, including mean corpuscular volume (MCV), red cell distribution width (RDW), mean cell hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and hemolysis were measured at the same time points. RESULTS: Deformability of RBCs stored in blood bags was retained over 4 weeks storage, but a progressive loss of deformability was observed at weeks 6 and 8. This trend was mirrored in blood unit segments with a strong correlation to the blood bag data. Strong correlations were also observed between blood bag and segment for MCV, MCHC, and MCH but not for hemolysis. CONCLUSION: RBCs sampled from blood unit segments accurately represent the biophysical properties of RBCs in blood bags but not hemolysis. Blood unit segments provide a simple and nondestructive sample for measuring RBC biophysical properties in clinical studies.

Lossless immunocytochemistry using photo-polymerized hydrogel thin-films.

Immunocytochemistry (ICC), or immunofluorescence microscopy, is an essential biological technique for phenotyping cells in both research and diagnostic applications. Standard ICC methods often do not work well when the cell sample contains a small number of cells (<10 000) because of the significant cell loss that occurs during washing, staining, and centrifugation steps. Cell loss is particularly relevant when working with rare cells, such as circulating tumor cells, where such losses could significantly bias experimental outcomes. In order to eliminate cell loss in ICC protocols, we present a method to encapsulate the cell sample in a photo-polymerized hydrogel thin-film. The hydrogel thin-film is permeable to antibodies and other ICC reagents, thereby allowing the use of standard ICC protocols without modification. The cell sample is physically constrained by the hydrogel at the bottom surface of a standard (unmodified) imaging microtiter plate, thereby enabling the acquisition of high-quality micrographs regardless of the properties of the cell sample or staining reagents. Furthermore, while standard ICC requires several centrifugation steps during staining and washing, our hydrogel encapsulation method requires only a single centrifugation step. This property greatly reduces the time required to perform ICC protocols and is more compatible with robotic platforms. In this study, we show that standard ICC and Cytospin protocols are extremely lossy (>70% loss) when the sample contains less than 10 000 cells, while encapsulating the cells using a permeable hydrogel thin-film results in a lossless ICC process.

Cytotoxic mediators in paradoxical HIV-tuberculosis immune reconstitution inflammatory syndrome.

Tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) frequently complicates combined antiretroviral therapy and antituberculosis therapy in HIV-1-coinfected tuberculosis patients. The immunopathological mechanisms underlying TB-IRIS are incompletely defined, and improved understanding is required to derive new treatments and to reduce associated morbidity and mortality. We performed longitudinal and cross-sectional analyses of human PBMCs from paradoxical TB-IRIS patients and non-IRIS controls (HIV-TB-coinfected patients commencing antiretroviral therapy who did not develop TB-IRIS). Freshly isolated PBMC stimulated with heat-killed Mycobacterium tuberculosis H37Rv (hkH37Rv) were used for IFN-γ ELISPOT and RNA extraction. Stored RNA was used for microarray and RT-PCR, whereas corresponding stored culture supernatants were used for ELISA. Stored PBMC were used for perforin and granzyme B ELISPOT and flow cytometry. There were significantly increased IFN-γ responses to hkH37Rv in TB-IRIS, compared with non-IRIS PBMC (p = 0.035). Microarray analysis of hkH37Rv-stimulated PBMC indicated that perforin 1 was the most significantly upregulated gene, with granzyme B among the top five (log2 fold difference 3.587 and 2.828, respectively), in TB-IRIS. Downstream experiments using RT-PCR, ELISA, and ELISPOT confirmed the increased expression and secretion of perforin and granzyme B. Moreover, granzyme B secretion reduced in PBMC from TB-IRIS patients during corticosteroid treatment. Invariant NKT cell (CD3(+)Vα24(+)) proportions were higher in TB-IRIS patients (p = 0.004) and were a source of perforin. Our data implicate the granule exocytosis pathway in TB-IRIS pathophysiology. Further understanding of the immunopathogenesis of this condition will facilitate development of specific diagnostic and improved therapeutic options.

Continuous Flow Deformability-Based Separation of Circulating Tumor Cells Using Microfluidic Ratchets.

Circulating tumor cells (CTCs) offer tremendous potential for the detection and characterization of cancer. A key challenge for their isolation and subsequent analysis is the extreme rarity of these cells in circulation. Here, a novel label-free method is described to enrich viable CTCs directly from whole blood based on their distinct deformability relative to hematological cells. This mechanism leverages the deformation of single cells through tapered micrometer scale constrictions using oscillatory flow in order to generate a ratcheting effect that produces distinct flow paths for CTCs, leukocytes, and erythrocytes. A label-free separation of circulating tumor cells from whole blood is demonstrated, where target cells can be separated from background cells based on deformability despite their nearly identical size. In doping experiments, this microfluidic device is able to capture >90% of cancer cells from unprocessed whole blood to achieve 10(4) -fold enrichment of target cells relative to leukocytes. In patients with metastatic castration-resistant prostate cancer, where CTCs are not significantly larger than leukocytes, CTCs can be captured based on deformability at 25× greater yield than with the conventional CellSearch system. Finally, the CTCs separated using this approach are collected in suspension and are available for downstream molecular characterization.

Reduced deformability of parasitized red blood cells as a biomarker for anti-malarial drug efficacy.

BACKGROUND: Malaria remains a challenging and fatal infectious disease in developing nations and the urgency for the development of new drugs is even greater due to the rapid spread of anti-malarial drug resistance. While numerous parasite genetic, protein and metabolite biomarkers have been proposed for testing emerging anti-malarial compounds, they do not universally correspond with drug efficacy. The biophysical character of parasitized cells is a compelling alternative to these conventional biomarkers because parasitized erythrocytes become specifically rigidified and this effect is potentiated by anti-malarial compounds, such as chloroquine and artesunate. This biophysical biomarker is particularly relevant because of the mechanistic link between cell deformability and enhanced splenic clearance of parasitized erythrocytes. METHODS: Recently a microfluidic mechanism, called the multiplexed fluidic plunger that provides sensitive and rapid measurement of single red blood cell deformability was developed. Here it was systematically used to evaluate the deformability changes of late-stage trophozoite-infected red blood cells (iRBCs) after treatment with established clinical and pre-clinical anti-malarial compounds. RESULTS: It was found that rapid and specific iRBC rigidification was a universal outcome of all but one of these drug treatments. The greatest change in iRBC rigidity was observed for (+)-SJ733 and NITD246 spiroindolone compounds, which target the Plasmodium falciparum cation-transporting ATPase ATP4. As a proof-of-principle, compounds of the bisindole alkaloid class were screened, where cladoniamide A was identified based on rigidification of iRBCs and was found to have previously unreported anti-malarial activity with an IC50 lower than chloroquine. CONCLUSION: These results demonstrate that rigidification of iRBCs may be used as a biomarker for anti-malarial drug efficacy, as well as for new drug screening. The novel anti-malarial properties of cladoniamide A were revealed in a proof-of-principle drug screen.

Role of the interleukin 10 family of cytokines in patients with immune reconstitution inflammatory syndrome associated with HIV infection and tuberculosis.

BACKGROUND: The interleukin 10 (IL-10) family comprises cytokines structurally related to IL-10 that share signaling receptors that have conserved signaling cascades. The immunopathogenesis of immune reconstitution inflammatory syndrome (IRIS) in patients with human immunodeficiency virus (HIV) infection and tuberculosis remains incompletely understood. We hypothesized that a deficiency of IL-10 and its homologs may contribute to the immunopathology of IRIS in these patients. METHODS: We performed a case-control analysis involving patients with HIV infection and tuberculosis who had IRIS at clinical presentation (tuberculosis-IRIS) and similar patients with HIV infection and tuberculosis who did not develop tuberculosis-IRIS (non-IRIS). Peripheral blood mononuclear cells (PBMCs) were cultured in the presence or absence of heat-killed Mycobacterium tuberculosis for 6 and 24 hours. Messenger RNA was analyzed by quantitative reverse transcription polymerase chain reaction analysis. Cytokine concentrations in serum were also determined. RESULTS: Cultures of PBMCs stimulated with M. tuberculosis for 24 hours yielded higher IL-10 and interleukin 22 (IL-22) transcript levels for tuberculosis-IRIS patients, compared with non-IRIS patients. Analysis of corresponding serum samples showed significantly higher concentrations of IL-10 and IL-22 in tuberculosis-IRIS patients, compared with non-IRIS patients. CONCLUSIONS: IL-10 and IL-22 were differentially induced in PBMCs from tuberculosis-IRIS patients after in vitro stimulation, and higher concentrations of their corresponding proteins were detected in serum (in vivo). The higher levels of IL-10 observed in this study may represent a compensatory antiinflammatory response during tuberculosis-IRIS. The elevated levels of IL-22 suggest an association between this cytokine and immunopathology during tuberculosis-IRIS.

Biophysical profiling of red blood cells from thin-film blood smears using deep learning.

Microscopic inspection of thin-film blood smears is widely used to identify red blood cell (RBC) pathologies, including malaria parasitism and hemoglobinopathies, such as sickle cell disease and thalassemia. Emerging research indicates that non-pathologic changes in RBCs can also be detected in images, such as deformability and morphological changes resulting from the storage lesion. In transfusion medicine, cell deformability is a potential biomarker for the quality of donated RBCs. However, a major impediment to the clinical translation of this biomarker is the difficulty associated with performing this measurement. To address this challenge, we developed an approach for biophysical profiling of RBCs based on cell images in thin-film blood smears. We hypothesize that subtle cellular changes are evident in blood smear images, but this information is inaccessible to human expert labellers. To test this hypothesis, we developed a deep learning strategy to analyze Giemsa-stained blood smears to assess the subtle morphologies indicative of RBC deformability and storage-based degradation. Specifically, we prepared thin-film blood smears from 27 RBC samples (9 donors evaluated at 3 storage time points) and imaged them using high-resolution microscopy. Using this dataset, we trained a convolutional neural network to evaluate image-based morphological features related to cell deformability. The prediction of donor deformability is strongly correlated to the microfluidic scores and can be used to categorize images into specific deformability groups with high accuracy. We also used this model to evaluate differences in RBC morphology resulting from cold storage. Together, our results demonstrate that deep learning models can detect subtle cellular morphology differences resulting from deformability and cold storage. This result suggests the potential to assess donor blood quality from thin-film blood smears, which can be acquired ubiquitously in clinical workflows.

Auto-amplification and spatial propagation of neutrophil extracellular traps.

The release of cellular DNA as neutrophil extracellular traps (NETs) plays a pivotal role in the immune response to pathogens by physically entrapping and killing microbes. NET release occurs at a greater frequency within neutrophil clusters and swarms, indicating a potential for collective behavior. However, little is known about how dense clustering of cells influences the frequency of NET release. Using an image-based assay for NETosis in nanowells, we show that the frequency of NETosis increases with cell density. We then co-incubate NETotic neutrophils with naïve neutrophils and find that NETotic neutrophils can induce secondary NETosis in naïve neutrophils in a cell density-dependent manner. Further mechanistic studies show that secondary NETosis is caused by a combination of DNA and protein factors. Finally, we immobilize NETotic neutrophils in a plaque, and then place the plaque near naïve neutrophils to characterize the spatial propagation of secondary NETosis. We find that secondary NETosis from naïve neutrophils increases over time, but remains spatially restricted to the periphery of the plaque. Together, we show that NETosis is an auto-amplified process, but that the spatial propagation of NET release is strictly regulated.

Single cell glucose-stimulated insulin secretion assay using nanowell-in-microwell plates.

Pancreatic ß cells secrete insulin in response to elevated levels of glucose. Stem cell derived ß (SCß) cells aim to replicate this glucose-stimulated insulin secretion (GSIS) function, but current preparations cannot provide the same level of insulin as natural ß cells. Here, we develop an assay to measure GSIS at the single cell level to investigate the functional heterogeneity of SCß cells and donor-derived islet cells. Our assay involves randomly depositing single cells and insulin capture microbeads in open-top nanowells (40 × 40 × 55 µm3) fabricated on glass-bottom imaging microwell plates. Insulin secreted from single cells is captured on microbeads and then stained using a detection antibody. The nanowell microstructure limits diffusion of secreted insulin. The glass substrate provides an optically flat surface for quantitative microscopy to measure the concentration of secreted insulin. We used this approach to measure GSIS from SCß cells and donor-derived islet cells after 15 minutes exposure to 3.3 mM and 16.7 mM glucose. Both cell types exhibited significant GSIS heterogeneity, where elite cells (<20%) produced the majority of the secreted insulin (55-78%). This assay provides an immediate readout of single cell glucose-stimulated insulin secretion in a flexible well plate-based format.

HIV-1 infection alters CD4+ memory T-cell phenotype at the site of disease in extrapulmonary tuberculosis.

HIV-1-infected people have an increased risk of developing extrapulmonary tuberculosis (TB), the immunopathogenesis of which is poorly understood. Here, we conducted a detailed immunological analysis of human pericardial TB, to determine the effect of HIV-1 co-infection on the phenotype of Mycobacterium tuberculosis (MTB)-specific memory T cells and the role of polyfunctional T cells at the disease site, using cells from pericardial fluid and blood of 74 patients with (n = 50) and without (n = 24) HIV-1 co-infection. The MTB antigen-induced IFN-γ response was elevated at the disease site, irrespective of HIV-1 status or antigenic stimulant. However, the IFN-γ ELISpot showed no clear evidence of increased numbers of antigen-specific cells at the disease site except for ESAT-6 in HIV-1 uninfected individuals (p = 0.009). Flow cytometric analysis showed that CD4+ memory T cells in the pericardial fluid of HIV-1-infected patients were of a less differentiated phenotype, with the presence of polyfunctional CD4+ T cells expressing TNF, IL-2 and IFN-γ. These results indicate that HIV-1 infection results in altered phenotype and function of MTB-specific CD4+ T cells at the disease site, which may contribute to the increased risk of developing TB at all stages of HIV-1 infection.

Corticosteroid-modulated immune activation in the tuberculosis immune reconstitution inflammatory syndrome.

RATIONALE: HIV-tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) is an immunopathological reaction to mycobacterial antigens induced by antiretroviral therapy. Prednisone reduces morbidity in TB-IRIS, but the mechanisms are unclear. OBJECTIVES: To determine the effect of prednisone on the inflammatory response in TB-IRIS (antigen-specific effector T cells, cytokines, and chemokines). METHODS: Blood was taken from participants in a randomized placebo-controlled trial of prednisone for TB-IRIS, at 0, 2, and 4 weeks. Participants received prednisone at a dosage of 1.5 mg/kg/day for 2 weeks followed by 0.75 mg/kg/day for 2 weeks, or placebo at identical dosages. MEASUREMENTS AND MAIN RESULTS: Analyses included IFN-γ enzyme-linked immunospot (ELISPOT), reverse transcription-polymerase chain reaction on peripheral blood mononuclear cells after restimulation with heat-killed Mycobacterium tuberculosis, Luminex multiplex cytokine analysis of corresponding tissue culture supernatants, and Luminex multiplex cytokine analysis of serum. Fifty-eight participants with TB-IRIS (31 receiving prednisone, 27 receiving placebo) were included. In serum, significant decreases in IL-6, IL-10, IL-12 p40, tumor necrosis factor-α, IFN-γ, and IFN-γ-induced protein-10 concentrations during prednisone, but not placebo, treatment were observed. No differences in ELISPOT responses comparing prednisone and placebo groups were shown in response to ESAT-6 (early secreted antigen target-6), Acr1, Acr2, 38-kD antigen, or heat-killed H37Rv M. tuberculosis. Purified protein derivative ELISPOT responses increased over 4 weeks in the prednisone group and decreased in the placebo group (P = 0.007). CONCLUSIONS: The beneficial effects of prednisone in TB-IRIS appear to be mediated via suppression of predominantly proinflammatory cytokine responses of innate immune origin, not via a reduction of the numbers of antigen-specific T cells in peripheral blood.

Data for assessing red blood cell deformability from microscopy images using deep learning.

Red blood cell (RBC) deformability is a vital biophysical property that dictates the ability of these cells to repeatedly squeeze through small capillaries in the microvasculature. This capability is known to differ between individuals and degrades due to natural aging, pathology, and cold storage. There is great interest in measuring RBC deformability because this parameter is a potential biomarker of RBC quality for use in blood transfusions. Measuring this property from microscopy images would greatly reduce the effort required to acquire this information, as well as improve standardization across different centers. This dataset consists of live cell microscopy images of RBC samples from 10 healthy donors. Each RBC sample is sorted into fractions based on deformability using the microfluidic ratchet device. Each deformability fraction is imaged in microwell plates using a Nikon CFI S Plan Fluor ELWD 40 × objective and a Nikon DS-Qi2 CMOS camera on a Nikon Ti-2E inverted microscope. This data could be reused to develop deep learning algorithms to associate live cell images with cell deformability.

Degradation of red blood cell deformability during cold storage in blood bags.

Red blood cells (RBCs) stored in blood bags develop a storage lesion that include structural, metabolic, and morphologic transformations resulting in a progressive loss of RBC deformability. The speed of RBC deformability loss is donor-dependent, which if properly characterized, could be used as a biomarker to select high-quality RBC units for sensitive recipients or to provide customized storage timelines depending on the donor. We used the microfluidic ratchet device to measure the deformability of red blood cells stored in blood bags every 14 days over a span of 56 days. We observed that storage in blood bags generally prevented RBC deformability loss over the current standard 42-day storage window. However, between 42 and 56 days, the deformability loss profile varied dramatically between donors. In particular, we observed accelerated RBC deformability loss for a majority of male donors, but for none of the female donors. Together, our results suggest that RBC deformability loss could be used to screen for donors who can provide stable RBCs for sensitive transfusion recipients or to identify donors capable of providing RBCs that could be stored for longer than the current 42-day expiration window.

Technologies for measuring red blood cell deformability.

Human red blood cells (RBCs) are approximately 8 µm in diameter, but must repeatedly deform through capillaries as small as 2 µm in order to deliver oxygen to all parts of the body. The loss of this capability is associated with the pathology of many diseases, and is therefore a potential biomarker for disease status and treatment efficacy. Measuring RBC deformability is a difficult problem because of the minute forces (∼pN) that must be exerted on these cells, as well as the requirements for throughput and multiplexing. The development of technologies for measuring RBC deformability date back to the 1960s with the development of micropipette aspiration, ektacytometry, and the cell transit analyzer. In the past 10 years, significant progress has been made using microfluidics by leveraging the ability to precisely control fluid flow through microstructures at the size scale of individual RBCs. These technologies have now surpassed traditional methods in terms of sensitivity, throughput, consistency, and ease of use. As a result, these efforts are beginning to move beyond feasibility studies and into applications to enable biomedical discoveries. In this review, we provide an overview of both traditional and microfluidic techniques for measuring RBC deformability. We discuss the capabilities of each technique and compare their sensitivity, throughput, and robustness in measuring bulk and single-cell RBC deformability. Finally, we discuss how these tools could be used to measure changes in RBC deformability in the context of various applications including pathologies caused by malaria and hemoglobinopathies, as well as degradation during storage in blood bags prior to blood transfusions.

See-N-Seq: RNA sequencing of target single cells identified by microscopy via micropatterning of hydrogel porosity.

Single cell RNA sequencing has the potential to elucidate transcriptional programs underlying key cellular phenotypes and behaviors. However, many cell phenotypes are incompatible with indiscriminate single cell sequencing because they are rare, transient, or can only be identified by imaging. Existing methods for isolating cells based on imaging for single cell sequencing are technically challenging, time-consuming, and prone to loss because of the need to physically transport single cells. Here, we developed See-N-Seq, a method to rapidly screen cells in microwell plates in order to isolate RNA from specific single cells without needing to physically extract each cell. Our approach involves encapsulating the cell sample in a micropatterned hydrogel with spatially varying porosity to selectively expose specific cells for targeted RNA extraction. Extracted RNA can then be captured, barcoded, reverse transcribed, amplified, and sequenced at high-depth. We used See-N-Seq to isolate and sequence RNA from cell-cell conjugates forming an immunological synapse between T-cells and antigen presenting cells. In the hours after synapsing, we found time-dependent bifurcation of single cell transcriptomic profiles towards Type 1 and Type 2 helper T-cells lineages. Our results demonstrate how See-N-Seq can be used to associate transcriptomic data with specific functions and behaviors in single cells.

Assessing red blood cell deformability from microscopy images using deep learning.

Red blood cells (RBCs) must be highly deformable to transit through the microvasculature to deliver oxygen to tissues. The loss of RBC deformability resulting from pathology, natural aging, or storage in blood bags can impede the proper function of these cells. A variety of methods have been developed to measure RBC deformability, but these methods require specialized equipment, long measurement time, and highly skilled personnel. To address this challenge, we investigated whether a machine learning approach could be used to predict donor RBC deformability based on morphological features from single cell microscope images. We used the microfluidic ratchet device to sort RBCs based on deformability. Sorted cells are then imaged and used to train a deep learning model to classify RBC based image features related to cell deformability. This model correctly predicted deformability of individual RBCs with 81 ± 11% accuracy averaged across ten donors. Using this model to score the deformability of RBC samples was accurate to within 10.4 ± 6.8% of the value obtained using the microfluidic ratchet device. While machine learning methods are frequently developed to automate human image analysis, our study is remarkable in showing that deep learning of single cell microscopy images could be used to assess RBC deformability, a property not normally measurable by imaging. Measuring RBC deformability by imaging is also desirable because it can be performed rapidly using a standard microscopy system, potentially enabling RBC deformability studies to be performed as part of routine clinical assessments.

Multiplexed end-point microfluidic chemotaxis assay using centrifugal alignment.

A fundamental challenge to multiplexing microfluidic chemotaxis assays at scale is the requirement for time-lapse imaging to continuously track migrating cells. Drug testing and drug screening applications require the ability to perform hundreds of experiments in parallel, which is not feasible for assays that require continuous imaging. To address this limitation, end-point chemotaxis assays have been developed using fluid flow to align cells in traps or sieves prior to cell migration. However, these methods require precisely controlled fluid flow to transport cells to the correct location without undesirable mechanical stress, which introduce significant set up time and design complexity. Here, we describe a microfluidic device that eliminates the need for precise flow control by using centrifugation to align cells at a common starting point. A chemoattractant gradient is then formed using passive diffusion prior to chemotaxis in an incubated environment. This approach provides a simple and scalable approach to multiplexed chemotaxis assays. Centrifugal alignment is also insensitive to cell geometry, enabling this approach to be compatible with primary cell samples that are often heterogeneous. We demonstrate the capability of this approach by assessing chemotaxis of primary neutrophils in response to an fMLP (N-formyl-met-leu-phe) gradient. Our results show that cell alignment by centrifugation offers a potential avenue to develop scalable end-point multiplexed microfluidic chemotaxis assays.

Image-based phenotyping of disaggregated cells using deep learning.

The ability to phenotype cells is fundamentally important in biological research and medicine. Current methods rely primarily on fluorescence labeling of specific markers. However, there are many situations where this approach is unavailable or undesirable. Machine learning has been used for image cytometry but has been limited by cell agglomeration and it is currently unclear if this approach can reliably phenotype cells that are difficult to distinguish by the human eye. Here, we show disaggregated single cells can be phenotyped with a high degree of accuracy using low-resolution bright-field and non-specific fluorescence images of the nucleus, cytoplasm, and cytoskeleton. Specifically, we trained a convolutional neural network using automatically segmented images of cells from eight standard cancer cell-lines. These cells could be identified with an average F1-score of 95.3%, tested using separately acquired images. Our results demonstrate the potential to develop an "electronic eye" to phenotype cells directly from microscopy images.

Monolithic hydrogel nanowells-in-microwells enabling simultaneous single cell secretion and phenotype analysis.

Cytokine secretion is a form of cellular communication that regulates a wide range of biological processes. A common approach for measuring cytokine secretion from single cells is to confine individual cells in arrays of nanoliter wells (nanowells) fabricated using polydimethylsiloxane. However, this approach cannot be easily integrated in standard microwell plates in order to take advantage of high-throughput infrastructure for automated and multiplexed analysis. Here, we used laser micropatterning to fabricate monolithic hydrogel nanowells inside wells in a microwell plate (microwells) using polyethylene glycol diacrylate (PEGDA). This approach produces high-aspect ratio nanowells that retain cells and beads during reagent exchange, enabling simultaneous profiling of single cell secretion and phenotyping via immunostaining. To limit contamination between nanowells, we used methylcellulose as a media additive to reduce diffusion distance. Patterning nanowells monolithically in microwells also dramatically increases density, providing ∼1200 nanowells per microwell in a microwell plate. Using this approach, we profiled IL-8 secretion from single MDA-MB-231 cells, which showed significant heterogeneity. We further profiled the polarization of THP-1 cells into M1 and M2 macrophages, along with their associated IL-1ß and CCL-22 secretion profiles. These results demonstrate the potential to use this approach for high-throughput secretion and phenotype analysis on single cells.