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Alginate cell microencapsulation implies the immobilization of cells within a polymeric membrane that allows the bidirectional diffusion of nutrients and oxygen inside the microcapsules and the release of waste and therapeutic molecules outside them. This technology has been applied to several cell types and it has been extensively described with pancreatic islets. However, other cells such as myoblasts are being currently studied and showing high interest. Moreover, different systems and approaches have been developed for cell encapsulation such as electrostatic extrusion and Flow focusing technology. When Flow focusing technology is applied for myoblast encapsulation, several factors should be considered, such as the pressure, the flow of the system, or the diameter size of the nebulizer, which will determine the final diameter size and shape of the microcapsules containing the myoblasts. Finally, viability of encapsulated myoblasts needs to be assessed before further studies are performed.

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.

The present work describes the formulation of Eudragit(®) L30 D-55 microparticles (MP) alone or with mucoadhesive agents, alginate or Carbopol(®), as an approach for the development of an oral cholera vaccine. In the first part, a spray drying technique was optimized for microparticle elaboration, obtaining a microparticle size ranging from 7 to 9 μm with high encapsulation efficiencies. Moreover, gastro resistant properties and Vibrio cholerae (VC) antigenicity were maintained, but for Eudragit(®)-Carbopol(®) microparticles which showed low antigenicity values, ≈25%. Next, a stability study was performed following ICH Q1 A (R2) guidelines, i.e. 25°C-60% relative humidity (RH) for 12 months, and 30°C-65% RH and 40°C-75% RH for 6 months. Upon storage, microparticle size changed slightly, 1 μm for Eudragit(®)-alginate MPs and 0.36 μm for Eudragit(®)MP. However, gastro resistance and antigenicity values were kept in an acceptance range. In the third stage of this work, in vivo experiments were performed. The immune response evoked was measured by means of vibriocidal titer quantification, observing that Eudragit(®)-alginate MPs were able to induce stronger immune responses, comparable to the free VC. Therefore, microencapsulation of VC by spray drying could be proposed as an approach to a cold chain free and effective oral cholera vaccine., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The field of cell microencapsulation is advancing rapidly. Particle size plays a critical role in terms of biocompatibility and limits decisively its applicability. Producing reduced size microcapsules involves broadening the possibilities to employ this technology in the treatment of many disorders. Nervous system diseases (NSD) represent a clear example of that. This work describes the feasibility of reducing the size of alginate-poly-L-lysine-alginate (APA) microcapsules up to 100 μm in a highly monodisperse way using the novel Flow Focusing technique. C(2)C(12) myoblasts genetically engineered to express the triple reporter gene thymidine kinase-green fluorescent protein-luciferase (TGL) and secrete vascular endothelial growth factor soluble receptor 2 (VEGFR2, also known as KDR) were encapsulated for further characterization. Resulting new particles were assayed in vitro to explore whether their functionality might be affected due to the physicochemical changes arising from such dramatic size reduction. Not only were negative effects at this level not noticed in terms of cell viability, cell proliferation and KDR secretion, but once again the suitability of APA microcapsules was also reinforced against other microcapsule designs. Furthermore, the fully viable and functional biosystems were successfully administered in the intravitreous space of rats, where the activity of encapsulated cells was monitoring over 3 weeks., (Copyright © 2011. Published by Elsevier B.V.)

Bovine serum albumin (BSA) loaded calcium alginate microparticles (MPs) produced in this study by a w/o emulsification and external gelation method exhibited spherical and fairly smooth and porous morphology with 1.052 ± 0.057 µm modal particle size. The high permeability of the calcium alginate hydrogel lead to a potent burst effect and too fast protein release. To overcome these problems, MPs were coated with polycations, such as chitosan, poly-L-lysine and DEAE-dextran. Our results demonstrated that coated MPs showed slower release and were able to significantly reduce the release of BSA in the first hour. Therefore, this method can be applied to prepare coated alginate MPs which could be an optimal system for the controlled release of biotherapeutic molecules. Nevertheless, further studies are needed to optimize delivery properties which could provide a sustained release of proteins.

Alginates are the most employed biomaterials for cell encapsulation due to their abundance, easy gelling properties and apparent biocompatibility. However, as natural polymers different impurities including endotoxins, proteins and polyphenols can be found in their composition. Several purification protocols as well as different batteries of assays to prove the biocompatibility of the alginates in vitro have been recently developed. However, little is known about how the use of alginates with different purity grade may affect the host immune response after their implantation in vivo. The present paper investigates the long-term functionality and biocompatibility of murine erythropoietin (EPO) secreting C2C12 cells entrapped in microcapsules elaborated with alginates with different properties (purity, composition and viscosity). Results showed that independently of the alginate type employed, the animals presented elevated hematocrit levels until day 130, remaining at values between 70-87%. However, histological analysis of the explanted devices showed higher overgrowth around non-biomedical grade alginate microcapsules which could be directly related with higher impurity content of this type of alginate. Although EPO delivery may be limited by the formation of a fibrotic layer around non-biomedical grade alginate microcapsules, the high EPO secretion of the encapsulated cells together with the pharmacodynamic behaviour and the angiogenic and immune-modulatory properties of EPO result in no direct correlation between the biocompatibility of the alginate and the therapeutic response obtained.

In the past, it has been proven that by properly adjusting the molecular mass of the oligochitosan samples, it is possible to optimize the formation of rigid, biocompatible capsules with semipermeable membranes under physiological conditions. In this study, the feasibility of four oligochitosan samples, with varying molar masses (M(n) in range 3-5 kDa), as biocompatible coatings of alginate/Ca capsules was investigated. By selection of appropriate depolymerization and purification methods we obtained oligochitosan samples that appeared to be noncytotoxic for C(2)C(12) myoblasts and did not influence the mammalian cell metabolism especially in relative short time during the process of capsule formation. Furthermore, oligochitosans can be used as a tool to reduce the membrane cut-off of the alginate capsules. However, such reduction, as well as mechanical resistance of formed microcapsules, depend on MM of the cationic polysaccharide and the chemical composition of the alginate (mannuronic/guluronic acid ratio). Here, we address that the use of low molar mass chitosan (< 5000 g/mol) permits the formation of mechanical stable capsules at physiological pH, which represents a strong advantage over other chitosan-based chemistries., (Copyright 2005 Wiley Periodicals, Inc.)

Biocompatibility of biomaterials and biomaterial-based medical devices is a critical issue for the long-term function on multiple therapeutic systems. In the past few years, there has been an increasing interest in producing more biocompatible biomaterials and in developing novel assays to analyze the quality of the products. In this study, a battery of in vitro techniques to assess the biocompatibility of alginates with different compositions and purities and alginate-based microcapsules is presented. Study of the protein and polyphenol content of the alginates revealed clear differences between the nonpurified and the purified alginates. A similar behavior was observed when the mitogenic activity and the tumor necrosis factor-alphasecretion induced by the alginates were assessed. Interestingly, when the latter two techniques were adapted to evaluate the different alginate microcapsules, a correlation with the results obtained for the alginate samples was observed. These results reinforce the idea of using the full battery of assays here reported to screen alginates and alginate-based microcapsules before implantation.

Mechanical stability, uniformity of size, complete encapsulation of cells and optimal microenvironment are major challenges in the design and development of microcapsules for cell immobilisation purposes. In this work, a novel microcapsule chemistry based on polyelectrolyte complexation between alginate and poly(methylene-co-guanidine) (PMCG) is presented. We have characterised the effect of PMCG concentration and time of exposure on microcapsule diameter and membrane thickness, selecting a PMCG concentration of 0.5% (v/v) and an exposure time of 1 min as optimal parameters for a correct coating. Afterwards, the mechanically most resistant alginate-PMCG-alginate (A-PMCG-A) microcapsule type was chosen according to two different stability studies. Beads with a solid core and an inhomogeneous internal configuration resulted in stronger microcapsules. Further, the selected A-PMCG-A beads presented both an increased stability compared to classical Ca(2+)/alginate and alginate-poly-L-lysine-alginate (APA) microcapsules, and had an adequate microenvironment for cell viability. This new chemistry allows the controlled adjustment of microcapsule size and wall thickness, offering new alternatives for cell transplantation.

Cell microencapsulation has emerged as a promising therapeutic strategy to treat a wide range of diseases. The optimisation of this technology depends on several critical issues such as the careful selection of the cell line, the controlled manufacture of microcapsules and the suitable adaptation of the construct design to the selected cell line. In this work, we studied the behavior of hybridoma cells once enclosed in solid and liquefied core alginate-agarose beads. Results show that hybridoma cells presented a better growing pattern and improved their viability and antibody production within liquefied beads. However, when these beads were evaluated with a compression resistance study, they were found to be mechanically more fragile than solid ones. To address this problem, we entrapped non-autologous cells (BHK fibroblast and C2C12 myoblast) in solid alginate-agarose beads and observed that they showed an improved growing profile and prolonged their viability up to 70 days in comparison to the 15 days seen for the hybridoma cells.

The biocompatibility of alginate-PLL-alginate (APA) microcapsules has been evaluated with respect to impurity levels. The impurity content of three different alginates (a raw high M-alginate, a raw high G-alginate and a purified high G-alginate) has been determined and the in vivo antigenic response of APA beads made with each alginate assessed. Results show that purification of the alginate not only reduces the total amount of impurities (63% less in polyphenols, 91.45% less in endotoxins and 68.5% less in protein in relation to raw high M-alginate), but also avoids an antibody response when microcapsules of this material are implanted in mice. In contrast, raw alginates produced a detectable antibody response though the differences in their impurity content. Consequently, this work revealed that purity of the alginate rather than their chemical composition, is probably of greater importance in determining microcapsule biocompatibility.

Alginate-PLL microcapsules containing BCG were prepared by emulsification/internal gelation of an alginate solution dispersed within a vegetable oil containing lecithins as emulsifiers. The lecithins studied were soy bean lecithin at 0.1, 0.5, 1 and 2% concentration; and dried egg yolk lecithin at 0.1, 0.25, 0.5 and 1%. The microcapsule particle size showed a dependence upon the amount and type of lecithin added to the oil. Increasing the emulsifying agent concentration was found to reduce particle size, from 50.9 microm obtained when lecithins were not used in the emulsification step, to 13.9 microm obtained when 1% dried egg yolk lecithin was employed. The encapsulated BCG was identified by the Difco TB stain set K, followed by observation under optical microscopy. Once prepared, microcapsules were freeze-dried using 5% trehalose as cryoprotectant in order to preserve their stability upon storage. The stability of the microcapsules was assayed over 12 months at room temperature, finding that alginate-PLL microcapsules were stable up to 6 months. Moreover, in the case of microcapsules prepared with lecithins, a significant increase in particle size was observed, from 16.9 microm at the beginning of the study to 25.2 microm at 12 months storage.

A biocompatible emulsification method for microencapsulation of live cells and enzymes within a calcium alginate matrix applied to Bacillus Calmette-Guérin (BCG) has been developed. Small-diameter alginate beads (microcapsules) were formed via internal gelation of an alginate solution emulsified within vegetable oil. Five different oils (sesame, sweet almond, perhydrosqualene, camomile and jojoba) were used. The rheological analysis of the oils showed a Newtonian behaviour, with viscosities = 30.0, 37.7, 51.2, 59.3 and 67.1 mPa.s for perhydrosqualene, jojoba, camomile, sesame and sweet almond oil respectively. The particle size of the microcapsules obtained ranged from 30.3 microns for the microcapsules prepared with sweet almond oil to 57.0 microns for those made with perhydrosqualene. The mean particle diameter obtained was found to be dependent on the viscosity of the oil employed, according to the equation: phi (micron) = 76.6-0.628 eta (mPa.s) (r2 = 0.943). The encapsulated BCG was identified by the Difco TB stain set K, followed by observation under optical microscopy. Freeze-drying of the microcapsules was carried out to ensure their stability during storage. Two batches of microcapsules (those prepared with sesame and jojoba oil) and four types of cryoprotectors (glucose, trehalose, mannitol and sorbitol), at three concentration levels (5, 10 and 20% w/v) were studied. The parameters evaluated were particle size, physical appearance, reconstitution of lyophilizates and microscopical evaluation. For both batches of microcapsules the best results were obtained with trehalose 5%, showing particle sizes of 42.1 microns in the case of the microcapsules prepared with sesame oil, and of 45.3 microns for those prepared with jojoba.