User:Afshankhan1/Cell Microencapsulation

Cell microencapsulation technology involves immobilization of the cells within a polymeric semi-permeable membrane that permits the bidirectional diffusion of molecules, such as, the influx of oxygen, nutrients, growth factors etc essential for cell metabolism and the outward diffusion of waste products and therapeutic proteins. At the same time, the semi-permeable nature of the membrane prevents immune cells and antibodies from destroying the encapsulated cells regarding them as foreign invaders.

The main motive of cell encapsulation technology is to overcome the existing problem of graft rejection in tissue engineering applications and in turn inhibit the need for chronic administration of immunosuppressive drugs given to patients to reduce side-effects after an organ transplant.



History
The concept of encapsulating cells in polymer membranes was first attempted in 1933 by Bisceglie who demonstrated that tumor cells in a polymer structure transplanted into pig abdominal cavity remained viable for a long period without being rejected by the immune system.

Thirty years later in 1964, the idea of encapsulating cells within ultra thin polymer membrane microcapsules so as to provide immunoprotection to the cells was then proposed by Dr. T.M.S Chang who introduced the term artificial cells to define this concept of bioencapsulation. He suggested that these artificial cells produced via drop method not only protected the encapsulated cells from immunorejection but also provided a high surface-to-volume relationship enabling good mass transfer of oxygen and nutrients. Twenty years later, this approach was successfully put to practice in small animal models when alginate-polylysine-alginate[APA] microcapsules immobilizing xenograft islet cells were developed. The study demonstrated that when these microencapsulated islets were implanted into diabetic rats, the cells remained viable and controlled glucose levels for several weeks.

Cell Microencapsultion as a tool for tissue engineering and regenerative medicine
Questions could arise as to why the technique of encapsulation of cells is even required when therapeutic products could just be injected at the site. An important reason for this is that the encapsulated cells would provide a source of sustained continuous release of ‘de novo’ therapeutic products for longer durations at the site of implantation. Another advantage of cell microencapsulation technology is that it allows the loading of non-human and genetically modified cells into the polymer matrix when the availability of donor cells is limited. Microencapsulation is a valuable technique for local, regional and oral delivery of therapeutic products as it can be implanted into numerous tissue types and organs. For the prolonged drug delivery at the treatment site, implantation of these drug loaded artificial cells would be more cost effective in comparison to direct drug delivery. Moreover, the prospect of implanting artificial cells with similar chemical composition in several patients irrespective of their leukocyte antigen could again enable reduction in costs.

Key parameters of cell microencapsulation technology
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 an ideal biomaterial depending on the application is a crutial factor in the development of drug delivery systems for tissue engineering. Several biomaterials have been used for cell microencapsulation applications such as alginate, collagen, chitosan, gelatin and agrose, with alginate  being one of the most commonly used polymers for cell microencapsulation technology.

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, that can be extracted from seaweed and bacteria, with numerous compositions based on the isolation source. However, depending on the composition, there still lies uncertainty and debate over the characteristics such as biocompatibility and stability of the alginate capsules.

Some researchers believe that alginates with high-M content could produce an inflammatory response and an abnormal cell growth while some have demonstrated that alginate with high-G content lead to an even higher cell overgrowth and inflammatory reaction in vivo as compared to intermediate-G alginates. A key issue that is a matter of concern before alginate(even ultrapure alginates) can be used in clinical applications, is the presence of endotoxins, proteins and polyphenols in the polymer, compromising 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 and the purification processes could in turn modify the properties of the biomaterial. Thus, it is essential that, an effective purification process is designed so as to remove all the contaminants from alginate before it can be successfully used in clinical applications.

Modification and functionalization of alginate
Researchers have also been able to develop alginate microcapsules using an altered form of alginate with enhanced biocompatibility and higher resistance to osmotic swelling. 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, 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(PLL) is the most commonly used polycation, but its low biocompatibility restricts the successful clinical use of PLL formulated microcapsules, which attract inflammatory cells and thus induce 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 a group demonstrated that modification of these alginate-chitosan microcapsules with genipin, a naturally occurring iridoid glucosid from gardenia fruits, to form genipin cross-linked alginate-chitosan (GCAC) microcapsules could augment stability of the cell loaded microcapsules.



Collagen
Collage, a major protein component of the ECM, provides support to tissues like skin, cartilage, bones, blood vessels and ligaments and is thus considered a model scaffold or matrix for tissue engineering due to its properties of biocompatibility, biodegradability and ability to promote cell binding. 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. However, despite its popularity, its immunogenicity has reduced the applications of collagen and gelatin is used as an alternative.

Gelatin
Gelatin is prepared from the denaturation of collagen and its 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 even being used commercially for skin replacements.

Chitosan
Chitosan is a cationic polymer produced 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 and is used for nanoencapsulation of cells. The cell/agrose suspension can be modified to form microbeads by reducing the temperature during preparation. However, one drawback with the microbeads obtained is the possibility of cellular protrusion through the polymeric matrix wall.

Biocompatibility
The use of an ideal high quality biomaterial, with the inherent property of biocompatibility, is the most crucial factor that governs the long term efficiency of this technology. An ideal biomaterial for cell encapsulation is one that is totally biocompatible, does not trigger an immune response in the host and does not interfere with cell homeostasis thus ensuring 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. Several studies demonstrate that surface modification of these cell containing microparticles allows control over the growth and differentiation of the encapsulated cells.

One study proposed the use of zeta potential, which measures the electric charge of the microcapsule, 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 cells, is a 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. It has been shown that since different cell types have different metabolic requirements, depending on the cell type being encapsulated, the permeability of the membrane has to be optimized. 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.

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. For instance, in the case of xeno-transplantation, a tighter more stable membrane would be required in comparison to allo-transplantation. Also, while investigating the potential of using APA microcapsules loaded with bile salt hydrolase (BSH) overproducing active Lactobacillus plantarum 80 cells, in a simulated gastro intestinal tract model for oral delivery applications, the mechanical integrity and morphology of the microcapsules was evaluated. It was shown that APA microcapsules could potentially be used in the oral delivery of live BSH active bacterial cells. However, further research proved that the GCAC microcapsules possess a higher mechanical stability as compared to APA microcapsules for oral delivery applications. Thus, 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.



Microcapsule size
The diameter of the microcapsules is an important factor that influences both the immune response towards the cell microcapsules as well as the mass transport across the capsule membrane. Studies show that the cellular response to smaller capsules is much lesser as compared to larger capsules and, in general, the diameter of the cell loaded microcapsules should be between 300-400 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. The sources for cell immobilization can be from the patient (autologous cells) or from another donor (allogeneic cells) or from other species (xenogeneic cells). The use of autologous cells in microencapsulation therapy is limited by the availability of these cells and even though xenogeneic cells are easily accessible, danger of possible transmission of viruses, especially porcine endogenous retrovirus to the patient restricts their clinical application, 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.

One of the major reasons why this technology has not received approval for clinical trial is due to the high immunogenicity of the loaded cells which 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 by the 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 the use of cell types such as myoblasts which do not proliferate after the microencapsulation procedure.

Non-Therapeutic Applications
Probiotics are increasingly being used in numerous dairy products such as ice cream, milk powders, yoghurts, frozen dairy desserts and cheese due to their important health benefits. But, low viability of probiotic bacteria in the food still remains a major hurdle. The pH, dissolved oxygen content, titratable acidity, storage temperature, species and strains of associative fermented dairy product organisms and concentration of lactic and acetic acids are some of the factors that greatly affect the probiotic viability in the product. As set by Food and Agriculture Organization (FAO) of the United Nations and the World Health Organization (WHO), in order to be considered a health food with probiotic addition, the product should contain at least 106-107 cfu per gram of viable probiotic bacteria. It is necessary that the bacterial cells remain stable and healthy in the manufactured product, are sufficiently viable while moving through the upper digestive tract and are able to provide positive effects upon reaching the intestine of the host.

Cell microencapsulation technology has successfully been applied in the food industry for the encapsulation of live probiotic bacteria cells to increase viability of the bacteria during processing of dairy products and for targeted delivery to the gastrointestinal tract.

Microencapsulated probiotics have also been used in non-dairy products, such as TheresweetTM,used as a replacement to the artificial sweeteners currently available.

Diabetes
The potential of using bioartificial pancreas, for treatment of diabetes mellitus, based on encapsulating islet cells within a semi permeable membrane is extensively being studied by scientists. It is envisaged that the implantation of these encapsulated cells would help to overcome the use of immunosuppressive drugs and also allow the use of xenograft cells thus obviating the problem of donor shortage. The use of microencapsulation would protect the islet cells from immune rejection as well as allow the use of animal cells or genetically modified insulin-producing cells. It is hoped that development of these islet encapsulated microcapsules could prevent the need for the insulin injections needed several times a day by type 1 diabetic patients. The Edmonton protocol involves implantation of human islets extracted from cadaveric donors and has shown improvements towards the treatment of type 1 diabetics who are prone to hypoglycemic unawareness. However, the two major hurdles faced in this technique are the limited availability of donor organs and the need for immunosuppresents to prevent an immune response in the patient’s body.

The first attempt towards developing bioartificial pancreas was demonstrated in 1980 by Lim et al where xenograft islet cells were encapsulated inside alginate Poly-l-lysine microcapsules and showed significant in vivo results for several weeks.

The polymers used for islet microencapsulation are alginate, chitosan , poly(ethylene glycol) (PEG) , agrose , sodium cellulose sulfate and water insoluble polyacrylates with alginate and PEG being commonly used polymers. With successful in vitro studies being performed using this technique, significant work in clinical trials using microencapsulated human islets is being carried out. In 2003, the use of alginate/PLO microcapsules containing islet cells for pilot phase-1 clinical trials was permitted to be carried out at the University of Perugia by the Italian Ministry of Health. In another study, the potential of clinical application of PEGylation and low doses of the immunosuppressant cyclosporine A were evaluated. The trial which began in 2005 by Novocell, now forms the phase I/II of clinical trials involving implantation of islet allografts into the subcutaneous site. However, there have been controversial studies involving human clinical trials where Living Cell technologies Ltd demonstrated the survival of functional xenogeneic cells transplanted without immunosuppressive medication for 9.5 years. However, the trial received harsh criticism from the International Xenotransplantation Association as being risky and premature. However, even though clinical trials are under way, several major issues such as biocompatibility and immunoprotection need to be overcome.

Cancer
The use of cell encapsulated microcapsules towards the treatment of several forms of cancer has shown great potential. One approach undertaken by researchers is through the implantation of microcapsules containing genetically modified cytokine secreting cells. An example of this was demonstrated by Cirone et al. when genetically modified IL-2 cytokine secreting  non-autologous mouse myoblasts implanted into mice showed a delay in the tumor growth with an increased rate of survival of the animals. However, the efficiency of this treatment was brief due to an immune response towards the implanted microcapsules. Another approach to cancer suppression is through the use of angiogenesis inhibitors to prevent the release of growth factors which lead to the spread of tumors. The effect of implanting microcapsules loaded with xenogenic cells genetically modified to secrete endostatin, an antiangiogenic drug which causes apoptosis in tumor cells, has been extensively studied. However, this method of local delivery of microcapsules was not feasible in the treatment of patients with many tumors or in metastasis cases and has led to recent studies involving systemic implantation of the capsules.

In 1998, a murine model of pancreatic cancer was used to study the effect of implanting genetically modified cytochrome P450 expressing feline epithelial cells encapsulated in cellulose sulfate polymers for the treatment of solid tumors. The approach demonstrated for the first time the application of enzyme expressing cells to activate chemotherapeutic agents. On the basis of these results, an encapsulated cell therapy product ,NovaCaps was tested in a phaseI/II clinical trial for the treatment of pancreatic cancer in patients and has recently been designated by the European medicines agency (EMEA) as an orphan drug in Europe.

These studies show the promising potential application of cell microcapsules towards the treatment of cancers. However, solutions to issues such as immune response leading to inflammation of the surrounding tissue at the site of capsule implantation have to be researched in detail before more clinical trials are possible.

Heart Diseases
Numerous studies have been dedicated towards the development of effective methods to enable cardiac tissue regeneration in patients after ischemic heart disease. An emerging approach to answer the problems related to ischemic tissue repair is though the use of stem cell-based therapy. However, the actual mechanism due to which this stem cell-based therapy has regenerative effects on cardiac function is still under investigation. Even though numerous methods have been studied for cell administration, the efficiency of the number of cells retained in the beating heart after implantation is still very low. A promising approach to overcome this problem is through the use of cell microencapsulation therapy which has shown to enable a higher cell retention as compared to the injection of free stem cells into the heart.

Another strategy to improve the impact of cell based encapsulation technique towards cardiac regenerative applications is through the use of genetically modified stem cells capable of secreting angiogenic factors such as vascular endothelial growth factor (VEGF) which stimulate neovascularization and restore perfusion in the damaged ischemic heart. An example of this is shown in the study by Zang et al. where genetically modified xenogeneic CHO cells expressing VEGF were encapsulated in alginate-Poly-l-lysine-alginate microcapsules and implanted into rat myocardium. It was observed that the encapsulation protected the cells from an immunorespone for three weeks and also led to an improvement in the cardiac tissue post-infarction due to increased angiogenesis.