A Neoglycoprotein-Immobilized Fluorescent Magnetic Bead Suspension Multiplex Array for Galectin-Binding Studies

Carbohydrate-protein conjugates have diverse applications. They have been used clinically as vaccines against bacterial infection and have been developed for high-throughput assays to elucidate the ligand specificities of glycan-binding proteins (GBPs) and antibodies. Here, we report an effective process that combines highly efficient chemoenzymatic synthesis of carbohydrates, production of carbohydrate-bovine serum albumin (glycan-BSA) conjugates using a squarate linker, and convenient immobilization of the resulting neoglycoproteins on carboxylate-coated fluorescent magnetic beads for the development of a suspension multiplex array platform. A glycan-BSA-bead array containing BSA and 50 glycan-BSA conjugates with tuned glycan valency was generated. The binding profiles of six plant lectins with binding preference towards Gal and/or GalNAc, as well as human galectin-3 and galectin-8, were readily obtained. Our results provide useful information to understand the multivalent glycan-binding properties of human galectins. The neoglycoprotein-immobilized fluorescent magnetic bead suspension multiplex array is a robust and flexible platform for rapid analysis of glycan and GBP interactions and will find broad applications.

Interaction of Different Charged Polymers with Potassium Ions and Their Effect on the Yield Stress of Highly Concentrated Glass Bead Suspensions

The interaction of different charged polymers, namely anionic polycarboxylate superplasticizer (PCE) and neutral polyethylene glycol (PEG) with potassium ions, and their effect on the yield stress of highly concentrated glass bead suspension (GBS), were studied under different concentrations of potassium ions ([K+]). It was found that, compared to the neutral PEG, the negatively charged PCE can be adsorbed on glass beads (GB), and then decreases the yield stress of GBS. The increasing concentration of free polymer in the interstitial liquid phase with the increased polymer dosage leads to the higher yield stress of GBS, which may be caused by the higher depletion force. In addition, this effect is also related to the charge density of the polymer and the [K+] in the solution. Along with the increase in [K+], the yield stress of GBS increases significantly with the addition of PCE, but this cannot be observed with PEG, which indicates that potassium ions can interact with negatively charged PCE instead of the neutral PEG. At last, the interparticle forces between two single GB with adsorbed PCE in solutions containing [K+] and PCE were measured by colloidal probe atomic force microscopy to better understand the interaction of the charged polymer with counterions.

GMP manufacture of Regulatory T cells (Tregs) to facilitated reduced reliance on immunosuppressive drugs following renal transplantation

Seetha Abdul Wahab, Matthew Brook, Giovanna Lombardi, Joanna Hester, Paul Harden, Fadi Issa Due to significant side effects, it is desirable to be able to reduce patient’s dependency on immunosuppressive drugs after organ transplantation. Personalised Treg therapy offers a promising route to facilitating a reduction in immunosuppression. The TWO Study employs a GMP-compliant manufacturing process to produce autologous Tregs for therapy. GMP is a quality management system that entails production and quality control of advanced therapy medicinal products (ATMPs) ensuring that ATMPs are consistently produced per clinical trial authorisation or product specification. This work is undertaken at a purpose-built GMP facility at Guy’s Hospital, London. Patient samples of peripheral blood are received from Oxford and then volume reduced aseptically to 40 mls using Sepax. CD8+cells are eliminated using CliniMACS CD8 reagent. CliniMACS CD25 reagent is then added to isolate CD25+ cells. Isolated cells are expanded using Expact Treg bead suspension kit and Rapamycin. Cells are fed every two days and subsequently re-stimulated twice at five feeding cycle intervals or until the required cell number is reached. The cells are then pooled and centrifuged at 1000xg. The resulting pellet is re-suspended and the Expact expansion beads removed using CliniMACS tubing set LS. Finally, the cell product is formulated in CryoStor CS-10 to a maximum dose concentration of 10x106 cells/kg patient body weight. At the same time, a back-up dose is prepared as well as reference and QC samples. These are all cooled to -80oC at c.1oC/min and stored in liquid nitrogen vapour phase until dispatch. As of end 2019, three patient samples have been processed. Tregs were purified and propagated to 194.4, 282.1, and 183.4-fold expansion yielding 3.5x109, 2.2x109, and 3.1x109 cells respectively. Samples 1 and 2 have been released for treatment after phenotyping and testing for suppression activity, sterility, and contamination with aerobic/anaerobic pathogens and mycoplasma. The third product is scheduled to be released for treatment in May 2020.

Controlled Encapsulation of Micron-Sized Beads in a Droplet Based on Pulse Inertia Force Driving of Micro-Fluids

Loading drops with discrete objects, such as particles and cells, is often necessary when performing chemical and biological assays in microfluidic devices. The vast majority of reported encapsulating methods of particles into monodisperse picolitre droplets are based on micro-fluidic chip using the standard soft lithography technique are necessary. This paper presents a new approach, not based on micro-fluidic chip, for encapsulating particles into droplets actuated by microfluidic pulse inertia force. The polystyrene bead suspension can be ejected out of a tapered glass capillary in mineral oil drop by drop actuated by an enough pulse inertia force which is produced by a hollow PZT stack. The polystyrene beads will be randomly encapsulated in monodisperse picolitre droplets. The tapered glass capillary has the advantages of good chemical resistance, low friction, easy to manufacture and low cost and is suitable for chemical and biological analysis. The minimum size of the spherical droplets can reach 12 μm in diameter and about 1 picolitre in volume. The percentage of the droplets with single 5 μm-diameter polystyrene bead can reach 40% when the droplet size is 40 μm and the concentration of the bead suspension is 1×107 beads per milliliter. The experiment result can be applied in droplet-based single cell encapsulating and analyzing.