Surface Modification of Polystyrene with Boronic Acid for Immunoaffinity-Based Cell Enrichment.

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.

Mimicry of embryonic circulation enhances the hoxa hemogenic niche and human blood development.

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-ß 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.

Formation of an adherent hematopoietic expansion culture using fucoidan.

Expansion of transplantable cord blood (CB) progenitors using a stroma requires provision of an exogenous cell source because of the low frequency of stromal precursor cells in CB. A simpler approach from a clinical regulatory perspective would be to provide synthetic extracellular matrix. The aim of this study was to characterize the effect on hematopoietic cell culture of fucoidan. The modulation of cytokine-driven hematopoietic cell expansion by fucoidan was investigated using two-level fractional and full factorial experimental designs. Mobilized peripheral blood (PB) CD34(+) cells were grown over 10 days in various combinations of FL, SCF, TPO, G-CSF, and SDF-1. Cultures were analyzed by immunophenotype. The effect of fucoidan on the divisional recruitment of CD34(+) cells was studied by CFDA-SE division tracking. Fucoidan was adsorbed by polystyrene to tissue culture plates and promoted formation of an adherent hematopoietic culture. Factorial design experiments with mobilized PB-CD34(+) cells showed that fucoidan reduced the production of CD34(+) cells and CD34(+)CXCR4(+) ratio but did not affect the production of monocytic, granulocytic, or megakaryocytic cells. The inhibitory effect of fucoidan on expansion of CB-CD34(+) cells was greater than mobilized PB. Division tracking analysis showed that CD34(+) cell generation times were lengthened by fucoidan. Fucoidan binds growth factors via their heparin-binding domain. The formation of an adherent hematopoietic culture system by fucoidan is most likely mediated by the binding of L-selectin and integrin-αMß2 on myeloids. Fucoidan deserves further investigation as glycan scaffold that is suitable for immobilization of other matrix molecules thought to comprise blood stem cell niche.

Multi-type branching models to describe cell differentiation programs.

Cell proliferation and differentiation is described by a multi-type branching process, a probability model that defines the inheritance of cell type. Cell type is defined by (i) a repression index related to the time required for S-phase entry and (ii) phenotype as determined by cell markers and division history. The inheritance of cell type is expressed as the expected number and type of progeny cells produced by a mother cell given her type. Expressions for the expected number and type of cells produced by a multi-cellular (bulk culture) system are derived from the general model by making the simplifying assumption that cell generation times are independent. The multi-type Smith-Martin model (MSM) makes the further assumption that cell generation times are lag-exponentially distributed with phenotype transitions occurring just before entry into S-phase. The inheritance-modified MSM (IMSM) model includes the influence of generation time memory so that mother and daughter generation times are correlated. The expansion of human cord blood CD34(+) cells by haematopoietic growth factors was division tracked in bulk culture using carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE). The MSM model was fitted to division tracking data to identify cell cycle length, and the rates of CD34 antigen down-regulation and apoptosis. The IMSM model was estimated for mouse granulocyte-macrophage progenitors using live cell imaging data. Multi-type branching models describe cell differentiation dynamics at both single- and multi-cell scales, providing a new paradigm for systematic analysis of stem and progenitor cell development.

The method to generate pulsatile circulatory fluid flow using microfluidics.

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.

Microfluidic Obstacle Arrays Induce Large Reversible Shape Change in Red Blood Cells.

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.

FACS Enrichment of Total Interstitial Cells and Fibroblasts from Adult Mouse Ventricles.

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.

Correction: Non-reversible heat-induced gelation of a biocompatible Fmoc-hexapeptide in water.

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: .

Non-reversible heat-induced gelation of a biocompatible Fmoc-hexapeptide in water.

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.

Molecular engineering of the cellulosome complex for affinity and bioenergy applications.

The cellulosome complex has evolved to degrade plant cell walls and, as such, combines tenacious binding to cellulose with diverse catalytic activities against amorphous and crystalline cellulose. Cellulolytic microorganisms provide an extensive selection of domains; those with affinity for cellulose, cohesins and their dockerin binding partners that define cellulosome stoichiometry and architecture, and a range of catalytic activities against carbohydrates. These robust domains provide the building blocks for molecular design. This review examines how protein modules derived from the cellulosome have been incorporated into chimaeric proteins to provide biosynthetic tools for research and industry. These applications include affinity tags for protein purification, and non-chemical methods for immobilisation and presentation of recombinant protein domains on cellulosic substrates. Cellulosomal architecture provides a paradigm for design of enzymatic complexes that synergistically combine multiple catalytic subunits to achieve higher specific activity than would be obtained using free enzymes. Multimeric enzymatic complexes may have industrial applications of relevance for an emerging carbon economy. Biocatalysis will lead to more efficient utilisation of renewable carbon-fixing energy sources with the added benefits of reducing chemical waste streams and reliance on petroleum.

Droplet-based single cell RNAseq tools: a practical guide.

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.

Single-cell expression profiling reveals dynamic flux of cardiac stromal, vascular and immune cells in health and injury.

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.

High-fidelity replication of thermoplastic microneedles with open microfluidic channels.

Development of microneedles for unskilled and painless collection of blood or drug delivery addresses the quality of healthcare through early intervention at point-of-care. Microneedles with submicron to millimeter features have been fabricated from materials such as metals, silicon, and polymers by subtractive machining or etching. However, to date, large-scale manufacture of hollow microneedles has been limited by the cost and complexity of microfabrication techniques. This paper reports a novel manufacturing method that may overcome the complexity of hollow microneedle fabrication. Prototype microneedles with open microfluidic channels are fabricated by laser stereolithography. Thermoplastic replicas are manufactured from these templates by soft-embossing with high fidelity at submicron resolution. The manufacturing advantages are (a) direct printing from computer-aided design (CAD) drawing without the constraints imposed by subtractive machining or etching processes, (b) high-fidelity replication of prototype geometries with multiple reuses of elastomeric molds, (c) shorter manufacturing time compared to three-dimensional stereolithography, and (d) integration of microneedles with open-channel microfluidics. Future work will address development of open-channel microfluidics for drug delivery, fluid sampling and analysis.

A rapid co-culture stamping device for studying intercellular communication.

Regulation of tissue development and repair depends on communication between neighbouring cells. Recent advances in cell micro-contact printing and microfluidics have facilitated the in-vitro study of homotypic and heterotypic cell-cell interaction. Nonetheless, these techniques are still complicated to perform and as a result, are seldom used by biologists. We report here development of a temporarily sealed microfluidic stamping device which utilizes a novel valve design for patterning two adherent cell lines with well-defined interlacing configurations to study cell-cell interactions. We demonstrate post-stamping cell viability of >95%, the stamping of multiple adherent cell types, and the ability to control the seeded cell density. We also show viability, proliferation and migration of cultured cells, enabling analysis of co-culture boundary conditions on cell fate. We also developed an in-vitro model of endothelial and cardiac stem cell interactions, which are thought to regulate coronary repair after myocardial injury. The stamp is fabricated using microfabrication techniques, is operated with a lab pipettor and uses very low reagent volumes of 20 µl with cell injection efficiency of >70%. This easy-to-use device provides a general strategy for micro-patterning of multiple cell types and will be important for studying cell-cell interactions in a multitude of applications.

Scrutinizing calcium flux oscillations in T lymphocytes to deduce the strength of stimulus.

The capture and activation of individual T cells on functionalised surfaces enables real-time analyses of the magnitude and rhythm of intracellular calcium release. Application of Haarlet transformations generate a calcium flux 'threshold', with the frequency of the 'threshold crossings' correlating with the strength of the original T cell stimulus. These findings represent a new method to evaluate graduations in T cell activation in real time, and at a single-cell level.

Treatment-induced cell cycle kinetics dictate tumor response to chemotherapy.

Chemotherapy fails to provide durable cure for the majority of cancer patients. To identify mechanisms associated with chemotherapy resistance, we identified genes differentially expressed before and after chemotherapeutic treatment of breast cancer patients. Treatment response resulted in either increased or decreased cell cycle gene expression. Tumors in which cell cycle gene expression was increased by chemotherapy were likely to be chemotherapy sensitive, whereas tumors in which cell cycle gene transcripts were decreased by chemotherapy were resistant to these agents. A gene expression signature that predicted these changes proved to be a robust and novel index that predicted the response of patients with breast, ovarian, and colon tumors to chemotherapy. Investigations in tumor cell lines supported these findings, and linked treatment induced cell cycle changes with p53 signaling and G1/G0 arrest. Hence, chemotherapy resistance, which can be predicted based on dynamics in cell cycle gene expression, is associated with TP53 integrity.

Cardiac-like flow generator for long-term imaging of endothelial cell responses to circulatory pulsatile flow at microscale.

In vitro models of circulatory hemodynamics are required to mimic the microcirculation for study of endothelial cell responses to pulsatile shear stress by live cell imaging. This study reports the design, fabrication and characterisation of a microfluidic device that generates cardiac-like flow in a continuous culture system with a circulatory volume of only 2-3 µL. The device mimics a single chamber heart, with the following cardiac phases: (1) closure of the ventricle inlet valve, (2) contraction of the ventricle (systole) followed by opening of the outlet valve and (3) relaxation of the ventricle (diastole) with opening of the inlet valve whilst the outlet valve remains closed. Periodic valve states and ventricular contractions were actuated by microprocessor controlled pneumatics. The time-dependent velocity-field was characterised by micro-particle image velocimetry (µ-PIV). µ-PIV observations were used to help tune electronic timing of valve states and ventricular contractions for synthesis of an arterial pulse waveform to study the effect of pulsatile shear stress on bovine artery endothelial cells (BAECs). BAECs elongated and aligned with the direction of shear stress after 48 h of exposure to a pulsatile waveform with a maximum shear stress of 0.42 Pa. The threshold for BAECs alignment and elongation under steady (non-pulsatile) flow reported by Kadohama et al. (2006) is 0.7-1.4 Pa. These cells respond to transient shear stress because the time average shear stress of the pulse waveform to generate this morphological response was only 0.09 Pa, well below the steady flow threshold. The microfluidic pulse generator can simulate circulatory hemodynamics for live cell imaging of shear-induced signalling pathways.

Microwell perfusion array for high-throughput, long-term imaging of clonal growth.

Continuous cell tracking by time-lapse microscopy has led to detailed study of cell differentiation pathways using single cell fate maps. There are a multitude of cell fate outcomes, so hundreds of clonal division histories are required to measure these stochastic branching processes. This study examines the principle of condensing cell imaging information into a relatively small region to maximize live cell imaging throughput. High throughput clonal analysis of non-adherent cells by continuous live cell tracking was possible using a microwell perfusion array with an internal volume of 16 µl and 600 microwells at the base. This study includes examination of biocompatibility of buffer systems, connecting tubing, cell culture substrates, and media degradation. An intermittent perfusion protocol was selected for long-term time-lapse imaging of KG1a cells in the microwell array; 1500 clones were simultaneously cultured and scanned every 3 min at 100 × magnifications for 6 days. The advantages of perfusion microwell culture are continuous long-term cell tracking, higher cell imaging throughput, and greater control over cell microenvironment. Microwell devices facilitate high throughput analysis of cell lineage development and measurement of the probability distribution for cell life events such as mitosis.

Hollow-fibre affinity cell separation.

The developing fields of cell and tissue engineering will require cost-effective technologies for delivery of cells to patients. Hollow-fibre affinity cell separation is a monoclonal antibody-based cell separation process whereby monoclonal antibody (ligand) is immobilised onto a stationary substrate, the luminal surface of a parallel array of hollow fibres. Deposited cells are fractionated on the basis of adhesion strength using hollow fibre geometry that generates a well-defined shear stress for cell recovery. In this chapter we present the biophysical basis for the process of ligand-mediated cell adhesion and relate this to the performance of affinity cell separation. We also discuss the hydrodynamics of hollow fibre arrays and the various approaches for modifying polymer substrates with protein ligands. One of the major limiting factors for large-scale epitope selective cell separation will be the prohibitive cost of these affinity processes. Hollow fibre systems offer the promise of providing flexibility and scalability for many of these applications.

Analysis of cell differentiation by division tracking cytometry.

We propose a quantitative method to characterize growth and differentiation dynamics of multipotent cells from time series carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE) division tracking data. The dynamics of cell proliferation and differentiation was measured by combining (CFDA-SE) division tracking with phenotypic analysis. We define division tracking population statistics such as precursor cell frequency, generation time and renewal rate that characterize growth of various phenotypes in a heterogeneous culture system. This method is illustrated by study of the divisional recruitment of cord blood CD34(+) cells by hematopoietic growth factors. The technical issue of assigning the correct generation number to cells was addressed by employing high-resolution division tracking methodology and daily histogram analysis. We also quantified division-tracking artifacts such as CFDA-SE degeneration and cellular auto-fluorescence. Mitotic activation of cord blood CD34(+) cells by cytokines commenced after 2 days of cytokine stimulation. Mean generation number increased linearly thereafter, and it was conclusively shown that CD34(+) cells cycle slower than CD34(-) cells. Generation times for CD34(+) and CD34(-) cells were 24.7 +/- 0.8 h and 15.1 +/- 0.9 h (+/-SD, n = 5), respectively. The 20-fold increase in CD34(+) cell numbers at Day 6 could be attributed to a high CD34(+) cell renewal rate (91% +/- 2% per division). Although cultures were initiated with highly purified CD34(+) cells (approximately 96%), CD34(-) numbers had expanded rapidly by Day 6. This rapid expansion could be explained by their short generation time as well as a small fraction of CD34(+) cells (approximately 5%) that differentiated into CD34(-) cells. Multitype division tracking provides a detailed analysis of multipotent cell differentiation dynamics.