Cell encapsulation

Cell microencapsulation technology basically 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 which are given to patients to reduce side-effects after an organ transplant.

History

The concept of encapsulating cells in immunoprotective membranes was first proposed in 1933 by Bisceglie who demonstrated that tumor cells 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 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.

Need/Advantages of cell microencapsulation technology

Questions could arise as to why the technique of encapsulation of cells is even required when therapeutic products could be encapsulated and injected at the required 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.[7]These microcapsules can also be administered into various types of tissues and organs which make this technique an appropriate choice for local, regional and oral delivery of the therapeutic products. In the case of long term delivery of therapeutic agents at the treated site, it is more cost effective to implant these artificial cells instead of direct administration of the therapeutic. Furthermore, the possibility of implanting artificial cells with the same chemical composition in numerous patients irrespective of their human leukocyte antigen again helps to reduce overall costs.

Requirements 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.

Biocompatibility

The most crucial issue governing the long term success of this technology is the use of an appropriate high quality material which is totally biocompatible and neither triggers an immune response in the host nor interferes with cell homeostasis so as to ensure that encapsulated cells remain viable for long durations. 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.

A study by De Vos et al demonstrated that measurement of the electric charge of the microcapsule surface through zeta potential can be used to predict the interfacial reaction between the microcapsule material and the surrounding tissue and in turn the biocompatibility of the system.

Alginate for cell encapsulation

With the aim to develop the most suitable biomaterial for cell microencapsulation, several natural and synthetic polymers have been studied. 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. Alginates are natural polymers that can be extracted from seaweeds and bacterium with different compositions depending on the source of isolation. However, there exists debate regarding the biocompatibility, stability and permeability of alginate capsules with respect to alginate composition. Alginate is a linear copolymer with homopolymeric blocks of (1-4)-linked β-D-mannuronate (M) and α-L-guluronate (G) residues covalently linked together in different sequences or blocks. Some scientists believe that the high-M alginates produce an inflammatory response and cause a cellular overgrowth while other studies show that high-G alginates produced a more severe cellular overgrowth and inflammatory reaction in vivo as compared to intermediate-G alginates.[36]

A key issue that is a matter of concern so as to allow the use alginate( even ultrapure alginates) towards clinical applications is the presence of endotoxins, proteins 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 and the purification processes could in turn modify the properties of the biomaterial. Thus it is essential that a 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

Studies have lead to the development of an enzymatically modified alginate to produce alginate microcapsules with improved biocompatibility and a high 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 and functionalized with RGD and allowed control over the growth and differentiation of the loaded cells.[12,37] 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 microcapsules demonstrate low mechanical stability. Thus several research groups have been looking for alternatives to PLL and have demonstrated promising results with poly-l-ornithineand poly(methylene-co-guanidine) hydrochloride by fabricating durable microcapsules with high and controlled mechanical strength for cell encapsulation.

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.

Studies have indicated that different cell types have varied metabolic requirements and hence the optimal permeability of the membrane depends on the cells enclosed in the membrane. 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. 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 in vivo applications especially 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.[40]

Cell choice

The cell type chosen for this technique depends on the desired application of the cell microcapsules. The cell sources available for immobilization can be obtained either from the patient (autologous cells), 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. The cells to be encapsulated can also be genetically modified to express any protein required based on the application. 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 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.

Therapeutic Applications

Diabetes

One of most common applications of cell microencapsulation is the transplantation of encapsulated pancreatic islets for treatment of diabetes mellitus. 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 type1 diabetics who are prone to hypoglycemic unawareness. However, the two major hurdles faced in this technique are the limited availability of donor organs and with the need for immunosuppresents to prevent an immune response in the patient’s body.

Several studies have been dedicated towards the development of bioartificial pancreas involving the immobilization of islets of langerhans inside polymeric capsules.[54-56] The first attempt towards this aim 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. [57] 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 polymers used for islet microencapsulation are alginate[58], chitosan[59], poly(ethylene glycol) (PEG) [60], agrose[61], sodium cellulose sulfate[62] and water insoluble polyacrylates[63] 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 approved to be conducted at the university of Perugia by the Italian Ministry of Health.[23] 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. [66] 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. [67] However, the trial received harsh criticism from the International Xenotransplantation Association as being risky and premature.[68] However, even though clinical trials are under way, several major issues such as biocompatibility and immunoprotection need to be overcome.[69]

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 nonautologous 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. Two research groups have studied the effect of implanting microcapsules loaded with xenogenic cells genetically modified to secrete endostatin, a potent antiangiogenic drug which causes apoptosis in tumor cells. 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[77] and has recently been designated by the European medicines agency (EMEA) as an orphan drug in Europe.[78]

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.[79] 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. [83] 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.