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IMMUNOISOLATED CELL TRANSPLANTATION BY MICROENCAPSULATION FOR THE TREATMENT OF LIVER FAILURE

1.      IMMUNOISOLATION

1.1  The Basis of Immunoisolated Cell Transplantation

A number of encapsulation systems have been developed and refined during the past several years in which living cells can be separated from the immune system of the body by a synthetic, selectively permeable membrane. The membrane allows the free exchange of nutrients, oxygen and biotherapeutic substances between the blood or plasma and the encapsulated cells, whereas high molecular weight substances such as immunocytes, antibodies and other transplant rejection effector mechanisms are excluded. These systems may also modulate the bidirectional diffusion of antigens, cytokines and other immunological moieties based on the chemical characteristics of the membrane and matrix support. Encapsulated cell technology offers a solutilon to the problem of donor organ supply, not only by potentially allowing the transplantation of cells and tissues without immunosuppression, but also by permitting use of materials isolated from animals [1].

In immunoisolated cell therapy, xenogeneic or allogeneic cells are contained within a medical device fabricated from carefully selected biomaterials. Xenogeneic cells are those transplanted across species barriers, whereas allograft cells are those transplanted within the same species. The device containing the living cells are then transplanted into a host. The aim of the transplanted foreign cells or tissues is to replace host function in the host tissue due to disease or degeneration [2, 3 ].

Immunoisolation of the transplanted cells is accompolished by selectively permeable membranes. These membranes are formulated “to allow small molecules such as nutrients to freely permeate into the encapsulated environment while hindering larger molecules and cells of the host immune system” (Fig.1). By using this technique the cells from one species can be transplanted into a discordant host species without immune rejection or with  the use of immunosuppressive drugs in case of graft rejection.

The use of xenogeneic cells or tissues greatly enlarges the available donor pool. Indeed, pharmacological immunosuppression is effective in preventing hyper-acute rejection of allograft tissue, but however, there is no way to get rid of the rejection of xenograft transplants. Even in the case of allograft organ transplantation (managed with an immunosuppressive drug regimen), there exists “restricted effectiveness” and half of all the transplanted organs fail within five years [4]. In addition, for disorders such as Parkinson’s and Huntington’s Diseases, the most appropriate allograft tissue is obtained from human fetal tissue (creating ethical barriers). Indeed, animal and human studies using adrenal and neural fetal tissue, as well as studies using genetically altered cell lines are still going on. Another study is currently under investigation, which focuses on transplantation of dopamine-secreting cells encapsulated in semi-permeable polymeric membranes [5].

1.2 Application Areas of Encapsulated Cell Devices

The application of encapsulated cell devices has proven most promising in the continuous delivery of biomolecules to specific sites. Previously, the area was claimed by polymer drug delivery sytems, indwelling catheters and pumps. Some drawbacks of these delivery methods include: frequent refilling and protein stability at body temperature (pumps); restricted drug loading, limited polymer choice and protein stability at body temperature (polymer release systems); and risk of infection (in dwelling catheters). However, in recent years, the use of transplanted living cells pumping out active factors directly at the site has proven an emergent technology [6].

Cell Encapsulation for the Treatment of Central Nervous System (CNS) Disorders:

Cell encapsulation strategy is also used for the treatment of neurological diseases when transplanting cells into the central nervous system (CNS) in order to deliver neuroactive molecules. For this respect, encapsulated cell implants have been transplanted in models of Parkinson’s disease, Alzheimer’s disease, Huntington’s disease and chronic pain syndrome. The use of encapsulated cells of drug delivery to the CNS has several advantages over other routes of administration. First of all, the systemic drug delivery to the CNS is blocked by the blood-brain barrier and this could be overcome by using the encapsulated cell therapy during which the devices are implanted directly at the target site for neurodegenerative diseases (the brain or spinal cord space).

In addition, living cells are ideally suited to the delivery of therapeutic proteins that are labile or have support half-lives and thus cannot be readily delivered through any other means. Recent advances have identified a number of specific neurotrophic factors critical for neuronal development, survival and function. Delivery of these factors holds much promise in the treatment of chronic neurodegenerative diseases by slowing or halting neuronal degeneration. However, the challenge has proven to be stable delivery of these labile proteins via conventional delivery methods. Encapsulated cell lines secreting these factors may provide a long-term solution to the delivery of these factors safely and efficiently to the CNS [5-7].

An emerging new area of  treatment is in the field of degenerative diseases affecting the eye (i.e. glaucoma and retinitis pigmentosa). Many of these diseases affect the retina, a part of the CNS that has a blood-retina barrier similar to the blood-brain barrier in that it must be bypassed for successful delivery of therapeutic factors.

Cell Encapsulation for Treatment of Diabetes Mellitus and Liver Failure:

Athough the encapsulated cell strategy is not feasible for whole organ transplantation, small masses of allogeneic or xenogeneic [8, 9] tissues have been transplanted to replace organ function (i.e. in the treatment of diabetes through islet cell encapsulation and in the treatment of liver dysfunction through hepatocyte cell encapsulation).

The hormone, insulin, is actually lifesaving for Type 1 (insuin-dependent) diabetess. Several experiments with small and large diabetic animal models, as well as human study, have shown that normo-glycemia may indeed be restored following implantation of encapsulated allo- or xenogeneic islets. Indeed, both of these treatments require immunosuppressive drugs and donor supply remains limited. Also, the large-scale isolation of animal islets under the required conditions of purity and sterility poses enormous technical problems. Thus, transformed cells, which exhibit secretory characteristics similar to those of islets and which can be amplified in culture under well-defined conditions, offer an atractive alternative [7, 10, 11].

While whole organ liver transplantation has been relied upon to treat liver failure, the gap between organ demand and supply has only widened in recent years. Thus, the use of liver cells has been explored for both extracorporeal and implanted therapies. There is a large ongoing effort in the extracorporeal liver field that may ultimately prove fruitful due to the large tissue mass required to support liver function. For liver therapy, hepatocytes or liver tumor cell lines are transplanted to serve as a bridge to transplantation, or to supplement the liver function until the diseased liver can either recover or regenerate. Typically the encapsulated cells are transplanted into the spleen or the peritoneal cavity [6].

In addition to these, the replacement organ functions are also evaluated for bioartificial pituary, parathyroid and thymus.

1.3  Device Configurations for Encapsulated Cell Therapy

Encapsulation is generally divided into two categories: micro and macro, each with some benefits and drawbacks (Table 1). Indeed, the most common geometric configurations of the immunoisolatory implanted devices are: intravascular tubular implants, hollow fibers, flat disk-shaped implants and microspheres (Fig. 2). The first three implant geometries are classified as macrocapsules-characterized by their size (0.5-1.5 mm inner diameter dimensions, length dimension ~ 1-10 cm) and internal capacity (thousands to millions of cells). Macrocapsules have sufficient volume capacity so only one or a few need to be implanted. Intravascular implants are simply macrocapsules connected directly to the host’s circulatory system where blood flows through the hollow fiber shunt that typically spans an artery and a vein. The cells are contained in a sheath on the outside of the tubular shunt [6].

Microencapsulation involves immobilizing cells in a thin, spherical membrane. The resulting microspheres or microcapsules are small (0.2-0.8 mm in diameter) beads, each holding only a few cells. Most applications require thousands of beads to provide the necessary cell number for a therapeutic dose [6]. The small size , thin wall, and spherical shape of microcapsules are optimal for diffusion, and, therefore, allow good viability of the encapsulated tissue and kinetics of response. The capsulesare, however, mechanically fragile and difficult to retrieve once implanted.

On the other hand, macroencasulation involves filling a hollow, cylindrical, permselective membrane with cells or tissue and then sealing the ends to form a capsule. The polymers most often used for macroencapsulation are mechanically more stable and the wall of the resultant capsule is generally thicker than in microencapsulation. So, this technique gives greater long term stability to the implant. However, the thicker wall and the larger diameter of the capsule can impair diffusion, threatening the viability of the tissue and slowing the release kinetics of the therapeutic factors. Macroencapsulated tissue can be removed from the recipient with a minor surgical procedure and replaced if necessary [5].

Table 1. Advantages and Disadvantages of Transplantation of Unencapsulated, Micro- and Macroencapsulated Cells [5].

The encapsulated cell device configurations are mainly composed of three basic components:

a permselective membrane,

an internal matrix and

the living cells.

Fig. 2 Most common configurations for encapsulated cell therapy [6].

Indeed, proper engineering of the component materials and the properties to fit the requirements of encapsulated cell and recipient host is a need for the success of the devices. Table 2 lists the critical factors that should be considered when selecting a biomaterial for an implantable cell-containing device.

Table 2. Factors determining the characteristics of a biomaterial for cell encapsulation [6].

2.      Membrane Materials for Microencapsulation

It is actually the choice of the membrane material and the formulation method which determine the resulting transport, morphology and strength characteristics of the device.. Transport properties must provide immunoisolation, maintain sufficient nutritional flux for the cells and alllow efflux of therapeutic molecules to the host. Membrane morphology describes the microgeometry of the wall structure and the topography of the outer surface. The outer surface for morphology greatly influences the host reaction to the implant since it serves as the host-implant interface. Membrane strength characteristics are often described in terms of material mechanical properties.

Membranes for microcapsules and macrocapsules have been formulated from a variety of materials. Microcapsules typically utilize hydrogel-based [5, 12-15], materials whereas macrocapsule membranes are commonly engineered from synthetic thermoplastic polymers. A list of membrane materials that have been used successfully to encapsulate cells is shown in table 2 (for microcapsules).

Existing Polymeric Biomaterials as Membrane Materials:

Polymers used as biomaterials can be naturally ocurring, synthetic or a combination of both. The characteristics of the main groups of polymeric material according to their origin, properties and principal fields application can be summarized as follows. Naturally derived polymers are abundant and usually biodegradable. Their principle disadvantage lies in the development of reproducible production methods, because their structural complexity often renders modification and purification difficult [6].

Synthetic polymers are available in a wide variety of compositions readily adjusted properties. Processing, copolymerization and blending provide simultaneous means of optimizing a polymer’s mechanical characteristics and its diffusive and biological properties. The primary difficulty is the general lack of biocompatibility of the majority of synthetic materials  (although poly(ethylene oxide) (PEO) and poly(lactic-co-glycolic acid) are notable exceptions. Synthetic polymers are therefore often associated with inflammattory reactions, which limit their use to solid, unmoving, impermeable devices [6].

Table 3. Materials used for microcapsules [6]

2.1  Hydrogel Membranes

Hydrogels are  a class of synthetic or natural polymer materials that swell in water but do not dissolve. Hydrogels have been widely used as materials for immunoisolation devices because of their long implant history [6]. For cell encapsulation devices, hydrogels are frequently used as the microencapsulation membrane and as the matrix component.

The polyelectrolyte hydrogel-based membranes were pioneered by Chang [1967, cited in 6] in the artificial cell field. This type of microcapsule is composed of an interfacial film formed between two polyelectrolytes of opposite charge.

Requirements for microcapsule materials include: biocompatibility, ease of processing into beads, sterilizability, long term biostability and lack of harsh chemicals or temperatures needed for synthesis or crosslinking. This last requirement is essential since microcapsules are formulated in the presence of living cells. Hydrogels are particularly conductive to microcapsule fabrication since they can be readily formed into beads, have a demonstrated biocompatibility record and are transparent (allowing easy visualization of encapsulated cells). Many naturally derived hydrogels can be formed with relatively gentle processing steps not detrimental to cell viability [6].

Formulation of a successful microcapsule depends on the proper balance between sufficient nutrient permeability to support viability (favoring thin walls) and sufficient mechanical integrity to withstand handling and minimizing defects (favoring thicker walls). Indeed, alginate has been the most common material employed for microcapsule fabrication [6]. A typical process for alginate based microcapsule formulation is shown in figure 3.

Fig. 3 Microencapsulation process- typical process for cells encapsulated in microspheres comprised of calcium-crosslinked alginate [6].

Alginate, a negatively charged polysaccharide based hydrogel derived from seeweed, can either be used alone, or in conjugation with positively charged polylysine polyelectrolyte complexes. Alginate itself may be ionically crosslinked by divalent cations such as calcium and barium. In this technique cells are mixed with a dilute (1-3%) alginate solution and droplets may be crosslinked in a 1% CaCl₂  bath for ~ 5 min.. If desired, the droplets may then be interfacially precipitated in a (0.05-0.1%) poly-L-lysine bath to form an outer skinned layer [6].

Since the early 1980s, several groups have successfully used the technique to transplant microencapsulated islets into the diabetic mouse model. In several of these experiments, glycemic control achieved for relatively long periods (months to one year) without immunosuppresion. In the 1990s, however, microencapsulation has emerged in the areas of hepatocyte, growth hormone replacement, adrenal and thyroid tissue transplantation using alginate based microcapsules. Dixit [1996] and others have implanted alginate-polylysine-alginate microencapsulated hepatocytes into the Gunn rat (a model for congenital metabolic liver disease) [16, 17]. Long term studies showed animals with transplanted hepatocytes had a significant decrease in serum bilirubin levels versus controls. Microcapsules were implanted into the peritoneal cavity. Alinate-polylysine-alginate microcapsules have also been used to encapsulate mouse cells genetically engineered to secrete human growth hormone (hGH) [18].

2.2  Thermoplastic Membranes

Several polyacrylates (including PMMA, HEMA-MMA) were developed to improve biocompatibility and potentially circumvent instability problems posed by water soluble problems. These polymers have the potential drawback of lower permeability of water soluble nutrients and the added challenge of maintaing cell viability in the presence of non-aqueous solvents.

These capsules have been produced by coextruding a solution of HEMA-MMA in polyethylene glycol with cells. Some success has been attained in vitro and in vivo with islet, CHO [19] and PC-12 cells [20]. However, maintenance of  long term cell viability in these non-aqueous polymer microcapsules remains untested.

3. Liver Cell Transplantation

3.1 The Raionale of Hepatocyte Transplantation

Liver cell transplantation has been developed for the treatment of acute and chronic liver failure, as well as for curing inherited metabolic disorders. For the treatment of inherited metabolic deficiencies, the normal genes can be introduced simply by transplanting normal hepatocytes from allogeneic donors. This, however, would require immunosuppression for the prevention of allograft rejection. Similar issues are also involved in using primary hepatocytes for the treatment of liver failure. Alternatively, hepatocytes can be harvested surgically from an affected individual with an inherited metabolic disease. These cells can be transduced in culture with a therapeutic gene and transplanted back into the subject. [21, 22]. Because the source is autologous, immunosuppression can be avoided. However, new methods are being explored for preventing allograft rejection, without the need for immunosuppression. In rodents, the host immune system has been tolerized to allotypic histocompatibility antigens by intrathymic inoculation, oral tolerization or interference with T cell-antigen presenting cell costimulation. In another approach, the donor cells could be modified by transferring immunomodulatory genes [23].

Studies in experimental animals and in humans show that transplanted primary human hepatocytes can engraft and function in structurally normal lives on a long term basis [24]. Hepatocytes introduced into the portal vein or injection into the splenic pulp translocate and integrate into normal liver chords with remarkable rapidity. Whether cirrhotic livers can provide suitable homes for transplanted cells is not yet clear. In transplanting hepatocytes into architecturally normal or abnormal livers, it is important to understand how many cells are needed for achieving the therapeutic goal and how many cells can be transplanted safely at one time. These issues are important not only in the transplantation of normal, untreated hepatocytes, but also for designing liver directed ex vivo gene therapy [23].

A major drawback against broad application of hepatocyte transplantation is the worldwide shortage of donor organs. Despite some recent advances, the current methods of primary hepatocyte culture do not permit large scale proliferation of human hepatocytes in culture. In certain conditions, such as inherited tyrosinemia type I (fumarylacetoacetate hydrolase deficiency) [25], progressive intrahepatic cholestasis type III (defect of MDR3) [26], and Wilson’s disease, the transplanted cells have a survival advantage, and a small number of hepatocytes may simultaneously repopulate the liver.

In other cases, however, there will be no specific proliferative pressure on the transplanted cells, which are therefore, not expected to increase in number out of proportion to host mutant cells. In these cases, modification of host liver cells by drugs, irradiation, or gene transfer may be necessary to provide specific growth advantage to the transplanted cells. At this time, it is not clear whether fetal liver cells or liver cell progenitors will have an advantage over adult hepatocytes in repopulating the liver [23].

3.2  Advances in the Treatment of Liver Failure by Using Immunoisolated Systems

Alternative locations of hepatocyte transplantation have been investigated, including transplantation to the spleen, pancreas, lungs, kidneys, subcutaneous tissue, muscle and thymus. Transplantation of isolated hepatocytes into parenchymatic organs/tissues has its complications, e.g. restriction in number and volume of implanted cells, bleeding within the organ and potential organ dysfunction. Injection of microcarrier attached hepatocytes into the peritoneal cavity has been designed to facilitate operation and to allow implantation of larger amouunts of cells. Intraperitoneal transplantation of hepatocytes may promote the inflammatory response and bacterial infections. So, this may cause events like peritonitis, abdominal sepsis and adhesion formation. Following intraperitoneal hepatocyte trasplantation, most of the transplanted cells diffuse on the peritoneal membrane, forming clusters. In order to overcome these complications, scientists tried to find out new alternatives and for this respect they’ve encapsulated hepatocytes in polymer devices and implanted the devices [ 6, 17, 19, 27]

There are many important properties that should be incorporated into the development of hepatocyte transplantation. Primarily, they are anchorage dependent cells and require an insoluble extracellular matrix for survival, reorganization, proliferation and function. Second, hepatocytes are highly metabolic cells and require rapid access to oxygen and nutrient supply. Finally, hepatocytes have a tremendous regenerative capacity with hepatotrophic stimulation in vivo. Based on these characteristics, hepatocyte transplantation using synthetic, highly porous, biodegradable polymer scaffolds is a promising approach for this respect  [28].

4. Advantages of Immunoisolated Cell Transplantation Devices

The development of encapsulation systems containing either bioengineered or primary isolates of living mammalian cells that produce specific bioactive agents offers enormous potential for treating human disease. Indeed, this helped to overcome the classical immune rejection of transplanted cells and tissues.

In addition, the use of encapsulated cells for gene therapy holds a big promise as the technology is attempting to keep pace with recent molecular biology developments. Biomaterials and polymers in particular have an enourmous role in cell encapsulation technology. I think, for the future, the development of new biomaterials that contain the unique biological requirements of these cell encapsulation systems are vital for further advancements in this technology.

Cellular medicine involving the replacement of diseased and defective cells and tissues is destined to occupy in increasingly important role in the future treatment of patients. Knowledge of the immune system at the molecular level has facilitated the development of strategies for overcoming the rejection of allografted cells, tissues and solid organs. Unfortunately, lack of availability of human organs and requirements for generalized immunosuppression have contributed significantly to patient morbidity and mortality. Covering all these issues, encapsulated cell technology offers a means for overcoming these limitations by enabling transplantation of cells and tissues from animal sources potentially without immunosuppression.

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