PMC:4996398 / 27879-34615 JSONTXT

{"project":"2_test","denotations":[{"id":"27600217-6776628-69475937","span":{"begin":166,"end":168},"obj":"6776628"},{"id":"27600217-8516335-69475938","span":{"begin":5687,"end":5689},"obj":"8516335"},{"id":"27600217-16373911-69475939","span":{"begin":5690,"end":5692},"obj":"16373911"},{"id":"27600217-19549731-69475940","span":{"begin":5693,"end":5695},"obj":"19549731"},{"id":"27600217-14531571-69475941","span":{"begin":6285,"end":6287},"obj":"14531571"},{"id":"27600217-11135548-69475942","span":{"begin":6288,"end":6290},"obj":"11135548"},{"id":"27600217-11559558-69475943","span":{"begin":6291,"end":6293},"obj":"11559558"},{"id":"27600217-20842001-69475944","span":{"begin":6388,"end":6390},"obj":"20842001"},{"id":"27600217-24142904-69475945","span":{"begin":6391,"end":6393},"obj":"24142904"},{"id":"27600217-24626259-69475946","span":{"begin":6420,"end":6422},"obj":"24626259"},{"id":"27600217-20690708-69475947","span":{"begin":6423,"end":6425},"obj":"20690708"}],"text":"3.1. Beads\nThe technique to immobilize cells, particularly pancreatic islet cells, in calcium alginate matrices was developed by Lim and Sun at the end of the 1970s [13]. By coating the alginate gel bead with polycations like poly-L-lysine, poly-L-ornithine, or chitosan, the strength of the surface coating as well as the capsule porosity can be controlled (Figure 6) [78].\nFigure 6 Encapsulation of cells with alginate. (From FMC internal archive.) One important characteristic of alginates is their very limited inherent cell adhesion and cellular interaction. This is an advantage for cell encapsulation applications, but can be a disadvantage for tissue engineering applications. However, alginate can be modified by the addition of cell attachment peptides or other biologically active molecules (see Section 2.3.1).\nEncapsulation in an alginate hydrogel has been shown to be a rapid, non-toxic, and versatile method for immobilization of macromolecules and cells. The creation of artificial organs by encapsulating cells or tissue is under study for treatment of a variety of diseases such as Parkinson’s disease, liver failure, hypocalcemia as well as chronic pain and, perhaps the most well-known example, an artificial pancreas for the treatment of diabetes (encapsulated pancreatic islets).\nMost methods for encapsulation of cells or tissue in alginate gels basically involve two main steps. The first step is the formation of an internal phase where the alginate solution containing biological materials is dispersed into small droplets. In the second step, droplets are solidified by gelling, or by forming a membrane at the droplet surface.\nBead size is one of the most important parameters of alginate gel beads and capsules in biomedical applications. The appropriate size will often be a compromise. The bead itself must be large enough to contain the biological material. Larger beads are also easier to handle during washing or other treatments. In many applications involving cells, the cells should be homogeneously distributed within the internal capsular matrix. When generating beads the desired mean size and acceptable size distribution should be accounted for. The size of the beads is mainly controlled by regulating the formation of the droplet.\nDroplet size is dependent upon several factors: the size of the material to be immobilized or encapsulated (i.e., single cells or cell aggregates such as pancreatic islets), the technique used to generate droplets (i.e., pipette or syringe, coaxial air flow, electrostatic generator, jet-cutter, etc.), the viscosity of the alginate solution, and the rate of alginate flow. Generally, for biomedical applications, droplet size is regulated to give a gelled bead having a diameter of \u003c200–1000 µm. Per unit volume, smaller beads yield a larger surface area to transplant volume, a ratio that results in enhanced survival of tissue due to better nutritional and oxygen supply. Various techniques can be used to form droplets, as described in more detail by Dulieu et al. [79]. These include: • Extrusion through a needle: Beads can be made by dripping an alginate solution from a syringe with appropriate diameter needle directly into a gelling bath. While this method does not require any instrumentation, the size and size distribution of the produced beads are difficult to control.• Coaxial air or liquid flow: The coaxial air jet system is a simple way of generating small beads (down to around 400 µm), although the size distribution will normally be larger as compared to an electrostatic system. In this system, a coaxial air stream is used to pull droplets from a needle tip into a gelling bath (Figure 7).• Electrostatic potential: An electrostatic potential can be used to pull droplets from a needle tip into a gelling bath. The primary effect on droplet formation by the electrostatic potential is to direct charged molecules to the surface of the droplet to counteract surface tension. Using this type of instrument, beads below 200 µm and with a small size distribution may be generated. The desired bead size is obtained simply by adjusting the voltage (electrostatic potential) of the instrument. The principle for making smaller beads by electrostatic potential bead generators is shown in Figure 7.• Vibrating capillary jet breakage: A vibrating nozzle generates drops from a pressurized vessel.• Rotating capillary jet breakage: Bead generation is achieved by cutting a solid jet of fluid coming out of a nozzle by means of a rotating cutting device. The fluid is cut into cylindrical segments that then form beads due to surface tension while falling into a gelling bath.\nFigure 7 Principle of electrostatic (left) and coaxial air flow (right) bead generators [80]. Encapsulation of living cells, a form of cell immobilization, is one application for producing artificial organs and cell therapy constructs. Here, cells are mixed with alginate (osmolality is adjusted) and then the alginate + cell solution is dripped (or extruded) into a bath of calcium chloride (Figure 6). Since the ionic cross-linking reaction is instantaneous, living cells are entrapped inside an alginate hydrogel bead. The porosity of the alginate bead is such that oxygen and nutrients can diffuse into the gel while cell products, such as proteins, can diffuse out of the gel. The hydrogel is, however, porous barrier to antibodies and immune cells such as macrophages. Furthermore, it is possible to implant his alginate hydrogel ”biofactory” into an animal or man where the implant can act as a continuous production system for, for example, insulin. Implantation studies into animals and diabetic patients [81,82,83] have shown that long-term functionality.\nSome examples of encapsulated cells are shown in Figure 8. These photomicrographs are not of the same magnification. The panel on the left shows encapsulated genetically engineered murine 3T3 cells. Encapsulated Human Embryonal Kidney (HEK) 293 cells are shown in the center panel. Pancreatic islets encapsulated in alginate gel beads are shown in the right panel. Cells producing the anti‑angiogenic protein endostatin have been encapsulated in ultrapure alginate and implanted into the brains of dogs being treated for spontaneous brain cancer [84,85,86]. Many other cell types have been immobilized in alginate such as adipose-derived stem cells [87,88], mesenchymal stem cells [89,90], and chondrocytes [91,92].\nFigure 8 Examples of encapsulated cells in alginate. (From FMC internal archive.) Clinical cell therapy applications of alginate immobilized cells such as the treatment of Parkinson’s disease as well as diabetes are available at the web sites of Living Cell Technologies [93,94]."}