Bead Generator Research Articles

3D printed alginate bead generator for high-throughput cell culture.

Alginate hydrogel beads are a common platform for generating 3D cell cultures in biomedical research. Simple methods for bead generation using a manual pipettor or syringe are low-throughput and produce beads showing high variability in size and shape. To address these challenges, we designed a 3D printed bead generator that uses an airflow to cleave beads from a stream of hydrogel solution. The performance of the proposed alginate bead generator was evaluated by changing the volume flow rates of alginate (QAlg) and air (QA), the diameter of device nozzle (d) and the concentration of alginate gel solution (C). We identified that the diameter of beads (D = 0.9 -2.8mm) can be precisely controlled by changing QA and d. Also the bead generation frequency (f) can be tuned by changing QAlg. Finally, we demonstrated that viability and biological function (pericellular matrix deposition) of chondrocytes were not adversely affected by high f using this bead generator. Because 3D printing is becoming a more accessible technique, our unique design will allow greater access to average biomedical research laboratories, STEM education and industries in cost- and time-effective manner.

Characterization of collagen type I/tannic acid beads as a cell scaffold

Breast cancer is the most commonly diagnosed cancer among women worldwide. Surgical removal of tumors is often necessary and many patients suffer complications due to subsequent breast reconstruction. A safe and effective breast reconstructive material is needed for patients recovering from surgical removal of small breast cancer tumors. Our lab has developed injectable collagen/tannic acid beads seeded with patient-derived preadipocytes for regeneration of healthy breast tissue in patients post-lumpectomy. Previous research indicates that the inclusion of tannic acid in the matrix imparts an anticancer property. This research seeks to determine the variables needed to control collagen/tannic acid bead diameter and seeded cell attachment, which are essential to proper bead implantation and function. We found that as tannic acid concentration increases within the beads, cell attachment decreases. Bead diameter is controlled by bead generator voltage, solution osmolality, the degree of cell attachment, and tannic acid concentrations. Higher voltages resulted in significant decrease in bead diameter. Collagen/tannic acid beads decreased in diameter when placed in solutions of increasing osmolality. Higher degrees of cell attachment across the surface of the beads were associated with a significant decrease in diameter. In beads made with high concentrations of tannic acid, bead diameter was found to decrease. Collagen/TA beads are a promising subdermal tissue regenerative matrix with anticancer activity as an alternative to simple lipofilling in breast reconstructive procedures. This study was conducted to better understand the properties of collagen/TA beads in order to improve injection efficacy and tissue regenerative activity.

Rotating-liquid-based hydrogel bead generator

Hydrogel beads are widely used in various applications, but producing such beads often requires complicated devices. Instead, we propose an easy-to-adopt, cost effective, open source hydrogel bead generator. This generator consists of two modules. The first module rotates two immiscible liquids in rigid body motion: mineral oil as the continuous phase (CP) liquid on top, and a hydrogel cross-linking (CL) liquid at bottom. The second module injects a hydrogel pre-polymer solution as the dispersed phase (DP) liquid into the rotating CP liquid. As the DP liquid flows out of a syringe needle, its drops are pinched off by the shear force from the CP liquid, and move with the CP liquid while settling down. When the drops enter the CL liquid, they become hydrogel beads. Experiments using water and mineral oil showed that the size of produced drops could be controlled by adjusting the flow speed of the CP and DP liquids. A demonstration using alginate showed that the proposed generator could successfully create alginate gel beads of uniform size and shape.

Alginate Microspheres: The Innovative Approaches to Production of the Microbeads/Micro-Particles

Multiparticulate dosage form is pharmaceutical formulations where the active substance is in the form of a number of small independent subunit such as, granules, microparticle, beads, and microspheres. Multiparticulate drug delivery systems are specifically suitable for achieving delayed and sustained release oral formulations with a minimum risk of dose dumping, local irritation, systemic toxicity, and variation in rate of bioavailability as they are less dependent on gastrointestinal transit time and offers many advantages over unit particulate dosage forms. Microbeads are small, sphere shaped, systematically produced free-flowing properties, semi-spherical solid units. Microbeads are the agglomerates of about size ranging from 0.5 mm to 1.0 mm, obtained from various starting materials and utilizing different innovative techniques. A blend of multiparticulate can also be used to provide the desired drug release profile. These round shaped particles produced by mostly principle of atomization technique.Among various different innovative techniques Vibration technology, Electrostatic bead generator, Jetcutter technology, electrostatic forces, dropping method and a mechanical cutting device are few of many. In current article it describes about the total methods involved and their working principles with diagrams were clearly illustrated. Now a days sodium alginate beads were designed by these technologies mostly in relative to microencapsulation techniques.
 Keywords: Atomization, Vibrating technology, Jetcutter device, Electrostatic bead generator Dropping method and electro-spray technology

Comparison of different cationic polymers efficacy in fabrication of alginate multilayer microcapsules

In past decades, alginate-based multilayer microcapsules have been given important attention in various pharmaceutical investigations. Alginate-poly l lysine-alginate (APA) is studied the most. Due to the similarity between the structure of polyethyleneimine (PEI) and poly-L-lysine (PLL) and also lower price of PEI than PLL, this study was conducted to compare the efficacy of linear (LPEI) and branch (BPEI) forms of PEI with PLL as covering layers in fabrication of microcapsules. The microcapsules were fabricated using electrostatic bead generator and their shape/size, surface roughness, mechanical strength, and interlayer interactions were also investigated using optical microscopy, AFM, explosion test and FTIR, respectively. Furthermore, cytotoxicity was evaluated by comparing the two anionic final covering layers alginate (Alg) and sodium cellulose sulphate (NCS) using MTT test. BPEI was excluded from the rest of the study due to its less capacity to strengthen the microcapsules and also the aggregation of the resultant alginate-BPEI-alginate microcapsules, while LPEI showed properties similar to PLL. MTT test also showed that NCS has no superiority over Alg as final covering layer. Therefore, it is concluded that, LPEI could be considered as a more cost effective alternative to PLL and a promising subject for future studies.

Microencapsulated Cells for Cancer Therapy.

The microencapsulation of different types of cells that are able to produce therapeutic factors is being investigated for the treatment of several human diseases. Most efforts are focused on chronic and degenerative diseases as this strategy could become an alternative to some commonly used parenteral treatments that need to be repeatedly administered. But, this approach has also been investigated in the field of oncology with the aim of providing immunomodulatory antibodies that are able to enhance the patient's inherent immune response against the tumor. These kind of treatments would provide the patient with the therapeutic drug produced in situ, de novo, and in a sustained way, making the therapy more comfortable.Although different devices are nowadays available to produce cell-enclosing alginate-microcapsules, here, we describe the most important steps and advices in order to fabricate alginate-poly-L-lysine-alginate microcapsules containing hybridoma cells for cancer management using an electrostatic bead generator, and how to evaluate the viability of those cells over the time.

Alginate Used to Develop Bio-Inspired Composite Microparticles for Bone Regeneration

This work describes the synthesis of composite particles for bone regeneration. The developed method is based on a bio-inspired approach starting from the composition, properties and hierarchical organization of bone extracellular matrix (ECM). The composite material was designed as spherical particles able to ensure interconnected porosity through their assembling inside the bone defect. nanohydroxyapatite was generated in the organic matrix in order to mimic the composition and the organization of the mineral phase in bone tissue. A collagen derivative was used as main natural polymer of the organic matrix of the composite material, in order to mimic the composition of bone ECM. Alginate represents the second organic polymer due to its recognized capacity to easily generate spherical particles in divalent cations solutions. The combination of this polysaccharide with the two previously mentioned components simultaneously responds the architectural and compositional constraints of this approach. The particles were obtained using a modular electrostatic bead generator developed in our laboratory. Briefly, a solution containing the biopolymers was extruded into a mineralisation solution. Spherical particles with diameters of about 500 μm were obtained. They were characterised by scanning electron microscopy, X-ray diffraction, stability in aqueous media. The obtained results confirmed the importance of alginate to generate spherical particles and the potential of such materials to successfully serve the targeted application.

In Vitro Osteogenic Induction of Bone Marrow Stromal Cells with Encapsulated Gene-Modified Bone Marrow Stromal Cells and In Vivo Implantation for Orbital Bone Repair

Osteogenic induction with either growth factors or genetic modification has limitations due to the short half-life and cost of the former, or safety concerns regarding the latter. The objective of this study was to employ a microcapsulation technique to separate genetically modified and nonmodified bone marrow stromal cells (BMSCs) to establish a cost-effective and biosafe osteogenic induction methodology with functional evaluation in vitro and in vivo in a canine model. Autologous BMSCs were isolated and transduced with adenoviral vectors containing either BMP-2 or vascular endothelial growth factor (VEGF) or were dual transduced followed by encapsulation in alginate microcapsules using an electrostatic bead generator. After cocultured with encapsulated cells, normal autologous BMSCs were analyzed for osteogenic differentiation and seeded onto tricalcium phosphate (TCP) scaffolds for in vivo implantation to repair orbital wall bone defects (12 mm in diameter) in a canine model. In vitro assays showed that the expression of the transduced genes was significantly upregulated, with significantly more transduced proteins released from the transduced cells compared with control cells. Importantly, examination of the BMSCs induced by soluble factors released from the encapsulated cells revealed a significant upregulation of expression of osteogenic markers Runx2, BSP, OPN, and OCN in dual-transduction or induction groups. In addition, dual transduction and induction resulted in the highest increase of alkaline phosphatase activity and mineralization compared with other experimental groups. In vivo assays using CT, micro-CT, and histology further supported the qPCR and western blot findings. In conclusion, encapsulation of genetically modified BMSCs was able to release a sufficient amount of BMP-2 and VEGF, which effectively induced osteogenic differentiation of normal-cultured BMSCs and demonstrated bone repair of the orbital wall defect after implantation with β-TCP in vivo.

A three-dimensional microfluidic approach to scaling up microencapsulation of cells

Current applications of the microencapsulation technique include the use of encapsulated islet cells to treat Type 1 diabetes, and encapsulated hepatocytes for providing temporary but adequate metabolic support to allow spontaneous liver regeneration, or as a bridge to liver transplantation for patients with chronic liver disease. Also, microcapsules can be used for controlled delivery of therapeutic drugs. The two most widely used devices for microencapsulation are the air-syringe pump droplet generator and the electrostatic bead generator, each of which is fitted with a single needle through which droplets of cells suspended in alginate solution are produced and cross-linked into microbeads. A major drawback in the design of these instruments is that they are incapable of producing sufficient numbers of microcapsules in a short-time period to permit mass production of encapsulated and viable cells for transplantation in large animals and humans. We present in this paper a microfluidic approach to scaling up cell and protein encapsulations. The microfluidic chip consists of a 3D air supply and multi-nozzle outlet for microcapsule generation. It has one alginate inlet and one compressed air intlet. The outlet has 8 nozzles, each having 380 micrometers inner diameter, which produce hydrogel microspheres ranging from 500 to 700 μm in diameter. These nozzles are concentrically surrounded by air nozzles with 2 mm inner diameter. There are two tubes connected at the top to allow the air to escape as the alginate solution fills up the chamber. A variable flow pump 115 V is used to pump alginate solution and Tygon® tubing is used to connect in-house air supply to the air channel and peristaltic/syringe pump to the alginate chamber. A pressure regulator is used to control the flow rate of air. We have encapsulated islets and proteins with this high throughput device, which is expected to improve product quality control in microencapsulation of cells, and hence the outcome of their transplantation.

Polyelectrolyte microcapsules with entrapped multicellular tumor spheroids as a novel tool to study the effects of photodynamic therapy

In the current study, semi-permeable alginate-oligochitosan microcapsules for multicellular tumor spheroids (MTS) generation were elaborated and tested, to estimate a response of the microencapsulated MTS (MMTS) to photodynamic therapy (PDT). The microcapsules (mean diameter 600 μm) with entrapped human breast adenocarcinoma MCF-7 cells were obtained using an electrostatic bead generator, and MMTS were generated by in vitro long-term cell cultivation. The formed MMTS were incubated in Chlorin e6 photosensitizer solution and then irradiated using 650-nm laser light. The cell viability was measured by MTT-assay in 24 h after irradiation, and histological analysis was performed. The proposed MTS-based model was found to be more resistant to the PDT than the two-dimensional monolayer cell culture model. Thus, MMTS could be considered as a promising three-dimesional in vitro model to estimate the doses of drugs or parameters for PDT in vitro before carrying out preclinical tests.

Alginate Microencapsulation Technology for the Percutaneous Delivery of Adipose-Derived Stem Cells

Autologous fat is the ideal soft-tissue filler; however, its widespread application is limited because of variable clinical results and poor survival. Engineered fillers have the potential to maximize survival. Alginate is a hydrogel copolymer that can be engineered into spheres of <200 μm, thus facilitating mass transfer, allowing for subcutaneous injection, and protecting cells from shearing forces. Alginate powder was dissolved in saline, and adipose-derived stem cells (ADSCs) were encapsulated (1 million cells/mL) in alginate using an electrostatic bead generator. To assess effects of injection on cell viability, microspheres containing ADSCs were separated into 2 groups: the control group was decanted into culture wells and the injection group was mixed with basal media and injected through a 21-gauge needle into culture wells. Microbeads were cultured for 3 weeks, and cell number and viability were measured weekly using electron and confocal microscopy. To assess effects of percutaneous injection in vivo, twenty-four male nude mice were randomly separated into 2 groups and injected with either empty microcapsules or ADSC-laden microcapsules. Mice were harvested at 1 and 3 months, and the implants were examined microscopically to assess bead and cell viability. A flow rate of 5 mL/h and an electrostatic potential of 7 kV produced viable ADSC-laden microbeads of <200 μm. There were no differences in bead morphology and ADSC viability between microcapsules placed versus injected into tissue culture plates for up to 3 weeks. Microspheres implanted in a nude mouse model show durability up to 3 months with a host response around each individual sphere. ADSCs remained viable and showed signs of mitosis. ADSCs can be readily cultured, encapsulated, and injected in alginate microspheres. Stem cells suspended in alginate microspheres survive in vivo and are seen to replicate in vitro.

In vitro evaluation and modification of pectinate gel beads containing trimethyl chitosan, as a multi-particulate system for delivery of water-soluble macromolecules to colon

Pectinate beads containing trimethyl chitosan chloride (TMC) as an absorption enhancer were prepared using Coomassie Brilliant Blue G 250 (CB) as a relatively high molecular weight water-soluble model drug. Effects of different formulation variables, such as cross-linker type, cross-linking time, cross-linker concentration, TMC: pectin ratio, pectin concentration and voltage of the bead generator, were assessed on in vitro bead characteristics by release and swelling–erosion studies. The bead formulation was optimized by factorial design. Some measures were taken to improve the bead characteristics and prolong their integrity during the gastrointestinal transit, such as biomineralization of the beads or coating them with high-methoxy pectin (PHM) or Eudragit L30-D 55 (EU). Possible CB-TMC complexation was investigated by Job's Plot method. The suitable system was obtained by coating the optimized core formulation with PHM or EU. TMC was found to form a complex with CB as a model anionic drug. Therefore, TMC-drug interactions can be used to modify the release characteristics of dosage forms.

The role of capsule composition and biologic responses in the function of transplanted microencapsulated islets of Langerhans.

The role of capsule composition and biologic responses in the function of transplanted microencapsulated islets of langerhans

Controlling the size of alginate gel beads by use of a high electrostatic potential

The effect of several parameters on the size of alginate beads produced by use of an electrostatic potential bead generator was examined. Parameters studied included needle diameter, electrostatic potential, alginate solution flow rate, gelling ion concentration and alginate concentration and viscosity, as well as alginate composition. Bead size was found to decrease with increasing electrostatic potential, but only down to a certain level. Minimum bead size was reached at between 2-4 kV/cm for the needles tested. The smallest alginate beads produced (using a needle with inner diameter 0.18 mm) had a mean diameter of ~300 #181;m. Bead size was also found to be dependent upon the flow rate of the fed alginate solution. Increasing the gelling ion concentration resulted in a moderate decrease in bead size. The concentration and viscosity of the alginate solution also had an effect on bead size as demonstrated by an increased bead diameter when the concentration or viscosity was increased. This effect was primarily an effect of the viscosity properties of the solution, which led to changes in the rate of droplet formation in the bead generator. Lowering the flow rate of the alginate solution could partly compensate for the increase in bead size with increased viscosity. For a constant droplet size, alginates with a low G block content (F GG #44 0.20) resulted in ~30% smaller beads than alginates with a high G block content (F GG #44 0.60). This is explained as a result of differences in the shrinking properties of the beads.

Accumulation and effects of benzo(a)pyrene on cytochrome P450 1A in waterborne exposed and intraperitoneal injected juvenile turbot ( Scophthalmus maximus)

Juvenile turbot ( Scophthalmus maximus) were exposed to benzo(a)pyrene (BaP) for 14 d using a glass bead generator flow-through system. Exposure was followed by a recovery period of 16 d. The highest BaP concentration in the edible portion of the fish, 16.5 ± 4.3 μg BaP/kg, was observed on the first day. Then concentrations dropped following first-order kinetics. BaP was below detection level at the end of the experiment. A statistically significant increase in bile fluorescence was observed from day 9 until the end of the experiment, suggesting the elimination of BaP metabolites by this route. No significant differences between control and exposed fish in EROD activity and CYP1A concentration, measured by immunodetection method, were observed. Intraperitoneal injection of 2.5 mg BaP/kg in juvenile turbot induced EROD activity. Under waterborne experimental conditions, bile fluorescence was observed to be a more sensitive biomarker of BaP exposure than EROD activity and CYP1A measurement.

Dissolved organic matter mediated aquatic transport of chlorinated dioxins

The bioavailability and environmental fate of extremely hydrophobic environmental contaminants such as chlorinated dioxins is linked to their solubility characteristics in water. Solubilities of three chlorinated dioxins, viz., 1,2,3,7-T 4CDD, 1,2,3,4,7-P 5CDD, and 1,2,3,4,7,8-H 6CDD, were determined in pure water using a glass bead generator column technique, and their enhanced solubilities in the presence of several dissolved humic fractions quantified at 20, 30 and 40°C. The strengths of these interactions between chlorodioxins and the dissolved humic substances, viz., a fulvic acid, a humic acid, and Aldrich humic acid, were examined using simple thermodynamic calculations. A new partition/association coefficient, K oc (mobile) is defined.