The potential of using cell microencapsulation in successful clinical applications can be realized only if several requirements encountered during the development process are optimized such as the use of an appropriate
biocompatible polymer to form the mechanically and chemically stable semi-permeable matrix, production of uniformly sized microcapsules, use of an appropriate immune-compatible polycations cross-linked to the encapsulation polymer to stabilized the capsules, selection of a suitable cell type depending on the situation.
Biomaterials The use of the best biomaterial depending on the application is crucial in the development of drug delivery systems and tissue engineering. The polymer
alginate is very commonly used due to its early discovery, easy availability and low cost but other materials such as cellulose sulphate,
collagen,
chitosan,
gelatin and
agarose have also been employed.
Alginate Several groups have extensively studied several natural and synthetic polymers with the goal of developing the most suitable biomaterial for cell microencapsulation. Extensive work has been done using alginates which are regarded as the most suitable biomaterials for cell microencapsulation due to their abundance, excellent biocompatibility and
biodegradability properties. Alginate is a natural polymer which can be extracted from seaweed and
bacteria with numerous compositions based on the isolation source. and an abnormal cell growth while some have demonstrated that
alginate with high-G content lead to an even higher cell overgrowth Even ultrapure alginates may contain
endotoxins, and
polyphenols which could compromise the biocompatibility of the resultant cell microcapsules. It has been shown that even though purification processes successfully lower endotoxin and polyphenol content in the processed alginate, it is difficult to lower the protein content Another approach to increasing the biocompatibility of the membrane biomaterial is through surface modification of the capsules using
peptide and protein molecules which in turn controls the proliferation and rate of differentiation of the encapsulated cells. One group that has been working extensively on coupling the amino acid sequence Arg-Gly-Asp (RGD) to alginate hydrogels demonstrated that the cell behavior can be controlled by the RGD density coupled on the alginate gels. Alginate microparticles loaded with myoblast cells and functionalized with RGD allowed control over the growth and differentiation of the loaded cells. Another vital factor that controls the use of cell microcapsules in clinical applications is the development of a suitable immune-compatible polycation to coat the otherwise highly porous alginate beads and thus impart stability and immune protection to the system.
Poly-L-lysine is the most commonly used polycation but its low biocompatibility restricts the successful clinical use of these PLL formulated microcapsules which attract inflammatory cells thus inducing necrosis of the loaded cells. Studies have also shown that alginate-PLL-alginate (APA) microcapsules demonstrate low mechanical stability and short term durability. Thus several research groups have been looking for alternatives to PLL and have demonstrated promising results with poly-L-ornithine and poly(methylene-co-guanidine) hydrochloride by fabricating durable microcapsules with high and controlled mechanical strength for cell encapsulation. Several groups have also investigated the use of
chitosan which is a naturally derived polycation as a potential replacement for PLL to fabricate alginate-chitosan (AC) microcapsules for cell delivery applications. However, studies have also shown that the stability of this AC membrane is again limited and one group demonstrated that modification of this alginate-chitosan microcapsules with
genipin, a naturally occurring iridoid glucoside from gardenia fruits, to form genipin cross-linked alginate-chitosan (GCAC) microcapsules could augment stability of the cell loaded microcapsules. This ability allows chitosan to control distribution of cells inside the polymeric system. Thus, Type-I collagen obtained from animal tissues is now successfully being used commercially as tissue engineered biomaterial for multiple applications. Collagen has also been used in nerve repair and bladder engineering.
Gelatin Gelatin is prepared from the denaturation of
collagen and many desirable properties such as
biodegradability, biocompatibility, non-immunogenity in physiological environments, and easy processability make this polymer a good choice for tissue engineering applications. It is used in engineering tissues for the skin, bone and cartilage and is used commercially for skin replacements.
Chitosan Chitosan is a polysaccharide composed of randomly distributed β-(1-4)-linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit). It is derived from the N-deacetylation of
chitin and has been used for several applications such as
drug delivery, space-filling implants and in wound dressings. However, one drawback of this polymer is its weak mechanical properties and is thus often combined with other polymers such
collagen to form a polymer with stronger mechanical properties for cell encapsulation applications.
Agarose Agarose is a
polysaccharide derived from seaweed used for nanoencapsulation of cells and the cell/agarose suspension can be modified to form microbeads by reducing the temperature during preparation. However, one drawback with the microbeads so obtained is the possibility of cellular protrusion through the
polymeric matrix wall after formation of the capsules.
Cellulose Sulphate Cellulose sulphate is derived from cotton and, once processed appropriately, can be used as a biocompatible base in which to suspend cells. When the poly-anionic cellulose sulphate solution is immersed in a second, poly-cationic solution (e.g. pDADMAC), a semi-permeable membrane is formed around the suspended cells as a result of gelation between the two poly-ions. Both mammalian cell lines and bacterial cells remain viable and continue to replicate within the capsule membrane in order to fill-out the capsule. As such, in contrast to some other encapsulation materials, the capsules can be used to grow cells and act as such like a mini-bioreactor. The biocompatible nature of the material has been demonstrated by observation during studies using the cell-filled capsules themselves for implantation as well as isolated capsule material. Capsules formed from cellulose sulphate have been successfully used, showing safety and efficacy, in clinical and pre-clinical trials in both humans and animals, primarily as anti-cancer treatments, but also exploring possible uses for gene therapy or antibody therapies. Using cellulose sulphate it has been possible to manufacture encapsulated cells as a pharmaceutical product at large scale and fulfilling Good Manufacturing Process (cGMP) standards. This was achieved by the company
Austrianova in 2007.
Biocompatibility The use of an ideal high quality biomaterial with the inherent properties of biocompatibility is the most crucial factor that governs the long term efficiency of this technology. An ideal biomaterial for cell encapsulation should be one that is totally
biocompatible, does not trigger an immune response in the host and does not interfere with cell
homeostasis so as to ensure high cell viability. However, one major limitation has been the inability to reproduce the different biomaterials and the requirements to obtain a better understanding of the chemistry and biofunctionality of the biomaterials and the
microencapsulation system. of the encapsulated cells. One study proposed the use of
zeta potential which measures the
electric charge of the microcapsule as a means to predict the interfacial reaction between microcapsule and the surrounding tissue and in turn the biocompatibility of the delivery system.
Microcapsule permeability A fundamental criterion that must be established while developing any device with a
semi-permeable membrane is to adjust the permeability of the device in terms of entry and exit of molecules. It is essential that the cell microcapsule is designed with uniform thickness and should have a control over both the rate of molecules entering the capsule necessary for cell viability and the rate of
therapeutic products and waste material exiting the capsule membrane. Immunoprotection of the loaded cell is the key issue that must be kept in mind while working on the permeability of the encapsulation membrane as not only immune cells but also
antibodies and
cytokines should be prevented entry into the microcapsule which in fact depends on the pore size of the biomembrane. Several groups have been dedicated towards the study of membrane permeability of cell microcapsules and although the role of permeability of certain essential elements like oxygen has been demonstrated, the permeability requirements of each cell type are yet to be determined. Sodium Citrate is used for degradation of alginate beads after encapsulation of cells. In order to determine viability of the cells or for further experimentation. Concentrations of approximately 25mM are used to dissolve the alginate spheres and the solution is spun down using a centrifuge so the sodium citrate can be removed and the cells can be collected.
Mechanical strength and durability It is essential that the microcapsules have adequate membrane strength (mechanical stability) to endure physical and
osmotic stress such as during the exchange of nutrients and waste products. The microcapsules should be strong enough and should not rupture on implantation as this could lead to an immune rejection of the encapsulated cells. However, further research proved that the GCAC microcapsules possess a higher mechanical stability as compared to APA microcapsules for oral delivery applications. Martoni et al. were experimenting with bacteria-filled capsules that would be taken by mouth to reduce serum cholesterol. The capsules were pumped through a series of vessels simulating the human GI tract to determine how well the capsules would survive in the body. Extensive research into the mechanical properties of the biomaterial to be used for cell microencapsulation is necessary to determine the durability of the microcapsules during production and especially for in vivo applications where a sustained release of the therapeutic product over long durations is required. van der Wijngaart et al. is a machine used to test • shear rate • shear strength • consistency coefficient • flow behavior index •
Viscometer - shear strength testing
Microcapsule Generation Microfluidics Droplet-based microfluidics can be used to generate microparticles with repeatable size. and in general the diameter of the cell loaded microcapsules should be between 350-450 μm so as to enable effective diffusion across the semi-permeable membrane.
Cell choice The cell type chosen for this technique depends on the desired application of the cell microcapsules. The cells put into the capsules can be from the patient (
autologous cells), from another donor (allogeneic cells) or from other species (xenogeneic cells). and after much debate several groups have concluded that studies should involve the use of allogeneic instead of xenogeneic cells. Depending on the application, the cells can be genetically altered to express any required protein. However, enough research has to be carried out to validate the safety and stability of the expressed gene before these types of cells can be used. This technology has not received approval for clinical trial because of the high immunogenicity of cells loaded in the capsules. They secrete
cytokines and produce a severe
inflammatory reaction at the implantation site around the capsules, in turn leading to a decrease in viability of the encapsulated cells. One promising approach being studied is the administration of anti-inflammatory drugs to reduce the immune response produced due to administration of the cell loaded microcapsules. Another approach which is now the focus of extensive research is the use of
stem cells such as mesenchymal stem cells for long term cell microencapsulation and cell therapy applications in hopes of reducing the immune response in the patient after implantation. Another issue which compromises long term viability of the microencapsulated cells is the use of fast proliferating cell lines which eventually fill up the entire system and lead to decrease in the
diffusion efficiency across the semi-permeable membrane of the capsule. A solution to this could be in the use of cell types such as
myoblasts which do not proliferate after the microencapsulation procedure. ==Non-therapeutic applications==