Obtaining an enriched and phenotypically pure cell population from heterogeneous cell mixtures is important for diagnostics and biosensing. Existing techniques such as fluorescent-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) require preincubation with antibodies (Ab) and specialized equipment. Cell immunopanning removes the need for preincubation and can be done with no specialized equipment. The majority of the available antibody-mediated analyte capture techniques require a modification to the Abs for binding. In this work, no antibody modification is used because we take advantage of the carbohydrate chain in the Fc region of Ab. We use boronic acid as a cross-linker to bind the Ab to a modified surface. The process allows for functional orientation and cleavable binding of the Ab. In this study, we created an immunoaffinity matrix on polystyrene (PS), an inexpensive and ubiquitous plastic. We observed a 37% increase in Ab binding compared with that of a passive adsorption approach. The method also displayed a more consistent antibody binding with 17 times less variation in Ab loading among replicates than did the passive adsorption approach. Surface topography analysis revealed that a dextran coating reduced nonspecific antibody binding. Elemental analysis (XPS) was used to characterize the surface at different stages and showed that APBA molecules can bind upside-down on the surface. While upside-down antibodies likely remain functional, their elution behavior might differ from those bound in the desired way. Cell capture experiments show that the new surface has 43% better selectivity and 2.4-fold higher capture efficiency compared to a control surface of passively adsorbed Abs. This specific surface chemistry modification will allow the targeted capture of cells or analytes with the option of chemical detachment for further research and characterization.

Silk fibroin hydrogels are extensively explored for tissue engineering and regenerative medicine as an artificial extracellular matrix (ECM) that can support tissue growth. However, the nanometer pore size of hydrogels limits adequate cell, tissue, and vascular infiltration. Microgel scaffolds are an emerging class of microporous biomaterials formed by annealing small microscale hydrogels (microgels) into a 3D construct. In this work, silk microgels are generated using a microfluidic device that allows tuning of the microgel diameter (100-400 mu m) and are stabilized via visible light-initiated photo-crosslinking of native tyrosine residues in silk. Microgels are then covalently annealed using silk solution as glue and the same cytocompatible visible light-initiated crosslinking to form microgel scaffolds. Unlike the nano-porosity of bulk photo-crosslinked silk hydrogels, the microgel scaffolds have an average pore diameter of 29 +/- 17 or 192 +/- 81 mu m depending on the microgel size, with enhanced mechanical properties compared to bulk hydrogels. This microporosity supports enhanced cell spreading and proliferation in vitro and increases scaffold remodeling in vivo, encouraging improved tissue infiltration and matrix deposition. The microgel size and material format also affect inflammatory responses in vivo. This work demonstrates that silk microgels and microgel scaffolds are promising candidates for tissue engineering and regenerative medicine applications. To address limited cell, tissue, and vascular infiltration associated with nano-porous hydrogels, spherical silk microgels of tunable diameter and annealed microgel scaffolds are developed using photo-initiated crosslinking of native tyrosines in silk. Microgel scaffolds has micro-scale porosity and supported improved cell, tissue, and blood vessel infiltration in vitro and in vivo, and microgel properties modulated inflammatory responses to silk implants. image

Adoptive cell therapy using patient-derived chimeric receptor antigen (CAR) T cells redirected against tumor cells has shown remarkable success in treating hematologic cancers. However, wider accessibility of cellular therapies for all patients is needed. Manufacture of patient-derived CAR T cells is limited by prolonged lymphopenia in heavily pre-treated patients and risk of contamination with tumor cells when isolating T cells from patient blood rich in malignant blasts. Donor T cells provide a good source of immune cells for adoptive immunotherapy and can be used to generate universal off-the-shelf CAR T cells that are readily available for administration into patients as required. Genome editing tools such as TALENs and CRISPR-Cas9 and non-gene editing methods such as short hairpin RNA and blockade of protein expression are currently used to enhance CAR T cell safety and efficacy by abrogating non-specific toxicity in the form of graft versus host disease (GVHD) and preventing CAR T cell rejection by the host.

Precursors of the adult hematopoietic system arise from the aorta-gonad-mesonephros (AGM) region shortly after the embryonic circulation is established. Here, we develop a microfluidic culture system to mimic the primitive embryonic circulation and address the hypothesis that circulatory flow and shear stress enhance embryonic blood development. Embryonic (HOXA+) hematopoiesis was derived from human pluripotent stem cells and induced from mesoderm by small-molecule manipulation of TGF-β and WNT signaling (SB/CHIR). Microfluidic and orbital culture promoted the formation of proliferative CD34+RUNX1C-GFP+SOX17-mCHERRY+ precursor cells that were released into the artificial circulation from SOX17+ arterial-like structures. Single-cell transcriptomic analysis delineated extra-embryonic (yolk sac) and HOXA+ embryonic blood differentiation pathways. SB/CHIR and circulatory flow enhance hematopoiesis by the formation of proliferative HOXA+RUNX1C+CD34+ precursor cells that differentiate into monocyte/macrophage, granulocyte, erythrocyte, and megakaryocyte progenitors.

Microfluidic chips provide versatile tools to mimic the biological effect of blood flow on pluripotent stem cells (PSC). This paper presents methods for the use of microfluidics to model embryonic circulation using differentiated PSC. Pulsatile circulatory flow is created with a microfluidics device with pressure-driven microvalves and ventricles. Silicone rubber devices are cast from moulds manufactured using standard and 3D laser lithography. The surface chemistry is modified to support the growth of human umbilical vein endothelial cells and pluripotent stem cells. Pulsatile circulatory fluid flow can be applied at specific stages of cell differentiation with direct observation of cellular responses by time-lapse fluorescent microscopy.•Replicable manufacturing protocol of lab scale microfluidic device generating pulsatile fluid flow mimicry embryonic blood circulation.•Integration of human cell lines on microfluidic chip.

Besides cardiomyocytes, the heart contains numerous interstitial cell types, including cardiac fibroblasts, endothelial cells, immune (myeloid and lymphoid) cells, and mural cells (pericytes and vascular smooth muscle cells), which play key roles in heart repair, regeneration, and disease. We recently published a comprehensive map of cardiac stromal cell heterogeneity and flux in healthy and infarcted hearts using single-cell RNA sequencing (scRNA-seq) (Farbehi et al., 2019). Here, we describe the FACS (Fluorescent Activated Cell Sorting)-based method used in that study for isolation of two cardiac cell fractions from adult mouse ventricles: the total interstitial cell population (TIP; non-cardiomyocytes) and enriched (Pdgfra-GFP+) cardiac fibroblasts.

Red blood cell (RBC) shape change under static and dynamic shear stress has been a source of interest for at least 50 years. High-speed time-lapse microscopy was used to observe the rate of deformation and relaxation when RBCs are subjected to periodic shear stress and deformation forces as they pass through an obstacle. We show that red blood cells are reversibly deformed and take on characteristic shapes not previously seen in physiological buffers when the maximum shear stress was between 2.2 and 25 Pa (strain rate 2200 to 25,000 s−1). We quantify the rates of RBC deformation and recovery using Kaplan–Meier survival analysis. The time to deformation decreased from 320 to 23 milliseconds with increasing flow rates, but the distance traveled before deformation changed little. Shape recovery, a measure of degree of deformation, takes tens of milliseconds at the lowest flow rates and reached saturation at 2.4 s at a shear stress of 11.2 Pa indicating a maximum degree of deformation was reached. The rates and types of deformation have relevance in red blood cell disorders and in blood cell behavior in microfluidic devices.

Hydrogel materials which respond to changes in temperature are widely applicable for injectable drug delivery or tissue engineering applications. Here, we report the unsual heat-induced gelation behaviour of a low molecular weight gelator based on an Fmoc-hexapeptide, Fmoc-GFFRGD. We show that Fmoc-GFFRGD forms kinetically stable fibres when mixed with divalent cations (e.g. Ca2+). Gelation of the mixture occurs upon heating of the mixture which enables electrostatic screening by the divalent cations and hydrophobic collapse of the fibres to give a self-supporting hydrogel network that shows good biocompatibility with L929 fibroblast cells. This work highlights a unique mechanism to initiate heat-induced gelation which should find opportunities as a gelation trigger for injectable hydrogels or fundamental self-assembly applications.

Factorial analysis of the interactions between three hydrogel-forming peptides based on three different biological motifs, namely, fibronectin: Fmoc-GFFRGD, collagen: Fmoc-GFFGER, and laminin: Fmoc-DDIKVAV, was conducted through rheology and live cell imaging using L929 fibroblasts. Gels were formed from each of these three peptide gelators alone and in various combinations. Cellular growth was tracked for the first 48 h in time-lapse movies by counting fluorescent nuclei and segmenting the cell area. The correlation between cell growth and the gel structure was characterized by linear regression analysis. While all peptide combinations showed good biocompatibility, the single-component Fmoc-DDIKVAV gel had the most positive effect on cell growth, while Fmoc-GFFRGD was the least biocompatible and had the lowest growth rate and cell area. Linear regression modeling demonstrated possible negative and positive interactions between Fmoc-GFFRGD*Fmoc-DDIKVAV and Fmoc-GFFRGD*Fmoc-GFFGER, respectively. No correlation was observed between gel stiffness and cellular growth. However, an increase in the strain crossover point for the elastic and loss moduli was associated with greater cell proliferation. This may indicate that elastic gels that store the work of cell deformation during cytokinesis are required for proliferation.

Correction for 'Non-reversible heat-induced gelation of a biocompatible Fmoc-hexapeptide in water' by Jonathan P. Wojciechowski et al., Nanoscale, 2020, 12, 8262-8267, DOI: .

Endothelialisation of implantable vascular grafts and stents is directed by the adsorbed protein layer. Chemical and mechanical cues sensed at the biomaterial surface by endothelial cells determine their attachment, survival and proliferation. Given the multiplicity of possible interactions we describe the novel application of live cell imaging, factorial analysis and single molecule imaging to investigate higher order interactions between surface chemistry and adsorbed proteins influencing endothelial cell (EC) adhesion dynamics. EC fates were tracked by time-lapse imaging of cell contact area on plasma polymer modified surfaces. The combinatorial effect of plasma polymer chemistry and adsorbed proteins was characterised by factorial experimental design and analysis. Single molecule imaging and counting was used for the first time to quantify binding of fluorescently labelled albumin to plasma polymerized surfaces: Modified glass surfaces with thin plasma polymer coatings rich in primary amine groups had a high affinity for albumin (-2, 650 molecules/iim2) while plasma polymer surfaces functionalised with hydroxyl groups had very low levels of albumin binding (22-30 molecules/ m2). Plasma polymer coatings rich in primary amine groups also promoted endothelial cell adhesion and was superior to tissue culture plastic. An interaction between albumin, heparin and fibronectin promoted adhesion to amine plasma polymer coated glass, while hydroxyl plasma polymer coated glass prevented EC attachment. Tracking cell-specific interactions with the adsorbed protein layer by time-lapse microscopy is more predictive of in vivo cellular responses to biomaterials compared to studies that only measure protein adsorption.

TrackPad is an open-source, biologist-friendly, graphical user interface for tracking and annotating single-cell lineage fates from time-lapse movies. The software applies cross-correlative, template-matching (CCTM) to track each cell using transmitted or fluorescent images. Machine tracking is supervised by the investigator, who can manually annotate cell fates such as division, death, or changes in cell phenotype. Search parameters that minimise user interventions and tracking time are selected by simulating tracking using verified tracking data as a ground truth. Investigators who use this software can produce high-quality annotated cell trajectories and division pedigrees to understand how intrinsic and extrinsic factors influence cell states and fates.

Background & Aim Chimeric antigen receptor engineered T cells (CAR T) have shown clinical success especially in acute lymphoblastic leukemia. Innate Natural Killer (NK) cells bear functional resemblance to T cells, but display a safer cytokine profile, immediate availability and no risk of GvHD. CAR NK are thus an attractive alternative to CAR T, especially in an allogeneic setting. Our study aims to investigate the cytolytic activity of NK and GD2CAR-NK cells against GD2 positive neuroblastoma (NB), either alone or in combination with histone deacetylase inhibitor (HDACi) and Programmed death 1 (PD-1) blocker. Methods, Results & Conclusion We have shown NK cells expanded from mononuclear cells by co-culture with irradiated K562-mbIL15-41BBL cells exhibited robust cytotoxicity against NB cells. HDACi (selective Class I inhibitor entinostat and pan-inhibitor panobinostat) up-regulated surface expression of NKG2D ligands MIC A/B & ULBPs on NB cells and significantly increased NK-mediated killing in NB cell lines and patient-derived-xenograft (PDX) samples. Immune checkpoint blockade via neutralizing antibodies against PD1/PDL1 axis (upregulated during NB and NK co-culture), when used in conjunction with HDACi, further improved efficiency of NK-mediated killing. In NB subcutaneous and metastatic xenograft model, adoptive NK cell therapy significantly prolonged event free survival, an effect further enhanced by treatment with both HDACi and PD1 blocker. Retrovirally transduced GD2CAR-NK cells significantly increased killing against GD2-positive NB cells as compared to control NK cells. Live cell imaging showed robust infiltration of GD2CAR-NK cells into 3D tumor spheroids, significantly reduced growth rate and able to destroy tumor spheroid. In a metastatic xenograft model, GD2CAR-NK treated mice showed significantly lower bioluminescence signal 11 days post tumor inoculation compared to control NK cells. However, although lower signal persisted in GD2CAR-NK group, significance was lost by day 18. Persistence of NK cells in mice was detected for one time point and no expansion observed. Taken together, our data suggest that NK cells mediate efficient killing of NB cells, enhanced by use of HDACi and PD1/PD-L1 blocker. GD2CAR-NK further enhance killing of target cells in vitro and although transiently, slowed tumor progression in vivo. Further investigation to improve transduction efficiency and NK/GD2CAR-NK cell expansion and persistence, with aim to enhance efficacy of NK/GD2CAR-NK cell therapy in vivo. Chimeric antigen receptor engineered T cells (CAR T) have shown clinical success especially in acute lymphoblastic leukemia. Innate Natural Killer (NK) cells bear functional resemblance to T cells, but display a safer cytokine profile, immediate availability and no risk of GvHD. CAR NK are thus an attractive alternative to CAR T, especially in an allogeneic setting. Our study aims to investigate the cytolytic activity of NK and GD2CAR-NK cells against GD2 positive neuroblastoma (NB), either alone or in combination with histone deacetylase inhibitor (HDACi) and Programmed death 1 (PD-1) blocker. We have shown NK cells expanded from mononuclear cells by co-culture with irradiated K562-mbIL15-41BBL cells exhibited robust cytotoxicity against NB cells. HDACi (selective Class I inhibitor entinostat and pan-inhibitor panobinostat) up-regulated surface expression of NKG2D ligands MIC A/B & ULBPs on NB cells and significantly increased NK-mediated killing in NB cell lines and patient-derived-xenograft (PDX) samples. Immune checkpoint blockade via neutralizing antibodies against PD1/PDL1 axis (upregulated during NB and NK co-culture), when used in conjunction with HDACi, further improved efficiency of NK-mediated killing. In NB subcutaneous and metastatic xenograft model, adoptive NK cell therapy significantly prolonged event free survival, an effect further enhanced by treatment with both HDACi and PD1 blocker. Retrovirally transduced GD2CAR-NK cells significantly increased killing against GD2-positive NB cells as compared to control NK cells. Live cell imaging showed robust infiltration of GD2CAR-NK cells into 3D tumor spheroids, significantly reduced growth rate and able to destroy tumor spheroid. In a metastatic xenograft model, GD2CAR-NK treated mice showed significantly lower bioluminescence signal 11 days post tumor inoculation compared to control NK cells. However, although lower signal persisted in GD2CAR-NK group, significance was lost by day 18. Persistence of NK cells in mice was detected for one time point and no expansion observed.

This study aims to characterise the effect of fluid shear stress on human embryonic stem cell (hESC)-derived haematopoiesis and vasculogenesis by applying microfluidics, live cell imaging and single cell RNA sequencing. AGM-like haematopoietic cultures developed by Ng et al. were generated by manipulating WNT and TGFs signalling during mesoderm specification. The effect of pulsatile circulatory flow was studied in a microfluidic dynamic culture system, using SOX17 (Cherry)/RUNX1c(GFP) hESC line to read out arterial endothelial and haematopoietic differentiation, respectively. Microdevices generated cardiac-like, pulsatile flow in a circulatory culture system with a volume of 2–3µL. AGM-like haematopoietic development was observed by time-lapse imaging on chip for more than two weeks. We observed cells entering the circulation from the adherent layer, and the release of lightly tethered SOX17+ cells into the circulation. In parallel bulk differentiation culture using an orbital mixer to mimic the effect of wall shear stress, single cell RNA seq was performed on 8800 single cells at day 18 of culture. Control treatments were static culture and drug vehicle. Hierarchical cluster analysis identified 18 clusters belonging to erythroid-megakaryocytic, cardiovascular and myeloid differentiation pathways. We identified HOXA expressing mesenchymal cells in AGM cultures. Shear treatment promoted proliferative MYB, RUNX1, CD31 expressing blood progenitors, a reduced proportion of unipotent erythroid and megakaryocytic lineages, and increase numbers of myeloid and bipotent megakaryocyte-erythroid progenitors. The production of smooth muscle and cardiomyocytes was promoted by shear in non-AGM culture. This study demonstrates the feasibility of modelling human embryonic blood formation using microfluidic technology.

Droplet based scRNA-seq systems such as Drop-seq, inDrop and Chromium 10X have been the catalyst for the wide adoption of high-throughput scRNA-seq technologies in the research laboratory. In order to understand the capabilities of these systems to deeply interrogate biology; here we provide a practical guide through all the steps involved in a typical scRNA-seq experiment. Through comparing and contrasting these three main droplet based systems (and their derivatives), we provide an overview of all critical considerations in obtaining high quality and biologically relevant data. We also discuss the limitations of these systems and how they fit into the emerging field of Genomic Cytometry.

Cells are dynamic biological systems that interact with each other and their surrounding environment. Understanding how cell extrinsic and intrinsic factors control cell fate is fundamental to many biological experiments. However, due to transcriptional heterogeneity or microenvironmental fluctuations, cell fates appear to be random. Individual cells within well-defined subpopulations vary with respect to their proliferative potential, survival, and lineage potency. Therefore, methods to quantify fate outcomes for heterogeneous populations that consider both the stochastic and deterministic features of single-cell dynamics are required to develop accurate models of cell growth and differentiation. To study random versus deterministic cell behavior, one requires a probabilistic modelling approach to estimate cumulative incidence functions relating the probability of a cell's fate to its lifetime and to model the deterministic effect of cell environment and inheritance, i.e., nature versus nurture. We have applied competing risks statistics, a branch of survival statistics, to quantify cell fate concordance from cell lifetime data. Competing risks modelling of cell fate concordance provides an unbiased, robust statistical modelling approach to model cell growth and differentiation by estimating the effect of cell extrinsic and heritable factors on the cause-specific cumulative incidence function.

Besides cardiomyocytes (CM), the heart contains numerous interstitial cell types which play key roles in heart repair, regeneration and disease, including fibroblast, vascular and immune cells. However, a comprehensive understanding of this interactive cell community is lacking. We performed single-cell RNA-sequencing of the total non-CM fraction and enriched (Pdgfra-GFP+) fibroblast lineage cells from murine hearts at days 3 and 7 post-sham or myocardial infarction (MI) surgery. Clustering of >30,000 single cells identified >30 populations representing nine cell lineages, including a previously undescribed fibroblast lineage trajectory present in both sham and MI hearts leading to a uniquely activated cell state defined in part by a strong anti-WNT transcriptome signature. We also uncovered novel myofibroblast subtypes expressing either pro-fibrotic or anti-fibrotic signatures. Our data highlight non-linear dynamics in myeloid and fibroblast lineages after cardiac injury, and provide an entry point for deeper analysis of cardiac homeostasis, inflammation, fibrosis, repair and regeneration.