5.09
The Artificial Organ: Cell Encapsulation
Y Zhang, W Yu, G Lv, J Zhu, W Wang, and X Ma, Chinese Academy of Sciences, Dalian, China
X Liu, Dalian University, Dalian, China
© 2011 Elsevier B.V. All rights reserved.
5.09.1
Introduction
5.09.2
Materials of Encapsulation
5.09.2.1
Alginate
5.09.2.2
Chitosan
5.09.2.3
Collagen
5.09.2.4
Agarose
5.09.2.5
Polyethylene Glycol
5.09.3
Properties of the Microcapsules
5.09.3.1
Permeability and Mass Transport
5.09.3.2
Microcapsule Mechanical Stability
5.09.3.3
Biocompatibility
5.09.4
Applications of Encapsulated Cells
5.09.4.1
Encapsulated Primary Cells for Artificial Organ Research
5.09.4.2
Encapsulated Genetically Modified Cells for Gene Therapy
5.09.4.3
Encapsulated Stem Cells for Tissue Engineering and Regenerative Medicine
5.09.5
Conclusions and Future Considerations
Acknowledgments
References
Glossary
biocompatibility The ability of a material to perform
with an appropriate host response in a specific
application.
mechanical stability The property of the microcapsule
that resists the mechanical shear force.
permeability The property of the mass that passes the
membrane of microcapsule.
99
100
100
101
101
101
102
102
102
103
104
105
105
106
109
110
110
111
primary cells The cells taken directly from living tissue
(e.g., biopsy material) and established for growth
in vitro.
stem cells The cells that possess the ability to renew
themselves through mitotic cell division and
differentiate into a diverse range of specialized cell
types.
5.09.1 Introduction
Currently, a large number of people worldwide suffer from disease or damage of organs, and the only definite therapeutic strategy
for these diseases is partial- or whole-organ transplantation, but immune rejection is the major risk in this transplantation
technology [1]. The transplantation of allogeneic or isogeneic organ or tissue cells always stimulates strong immune response of
the recipient, which means that the recipient will have to take an immunosuppressor to avoid organ or cell rejection during the
lifetime. However, these drugs have significant toxicity that may give rise to other complications, for example, the risk of infection or
cancer occurrence. Over the last 30 years, researchers have developed many devices and methods to prevent or decrease the usage of
immune-modulating drugs [2]. In 1964, Chang proposed the idea of using microcapsules with polymer membrane, which were
used as artificial organs, to protect encapsulated cells from the attack of the host immune system [3].
The encapsulated artificial organ generally contains cells or cell clusters within a biocompatible semi-permeable membrane, and
this membrane allows the bidirectional diffusion of small molecules, such as nutrients, metabolites, and therapeutic drugs, but
prevents antibody and immunocyte from getting into the microcapsule so as to avoid host immune rejection, thereby permitting the
encapsulated cells to survive for a longer period, and to secrete and deliver the therapeutic drugs continuously (Figure 1). Moreover,
cell encapsulation allows the transplantation of nonhuman cells, which could be considered as an alternative to the limited supply
of donor tissue [4]. So far, encapsulated cells have been extensively used in the research of artificial organs because of the simplicity
of construction and the flexibility for modifying key components. This flexibility allows researchers to optimize the key parameters,
such as wall thickness [5], size [6], and membrane compactness [7], to fulfill the significant nutrient and oxygen diffusion demand
of the encapsulated cells in implantation site. Therefore, the encapsulated cells are a promising approach to organ damage repair
99
100
Enabling Technologies
Membrane
Antibody or inflammatory cells
Nutrients
Cells
Metabolites
Therapeutic drugs
Figure 1 The microencapsulated artificial organs generally contain cells within a biocompatible semi-permeable membrane and this membrane allows
the bidirectional diffusion of small molecules, such as nutrients, metabolites, and therapeutic drugs, and but prevents antibodies and immunocytes from
getting into the microcapsules.
and disease therapy. This article attempts to provide the summary of cell-encapsulation technology during the last few years and to
present the developments of the technology as a promising strategy for biomedical purpose.
5.09.2 Materials of Encapsulation
The accomplishment of immune-isolation process of the encapsulated cell often depends on the proper biomaterials and the
properties of microcapsule to fit the requirements of functional cells and recipient. The biocompatible materials applied to design
microcapsule must allow for long-term survival of the encapsulated cell, and the polymer used for transplantation must form a
membrane with enough strength and properly selective permeability. Furthermore, it is essential that the microcapsule should have
adequate mechanical stability to prevent the entry of the antibody or immune cells. In the research of the encapsulation design,
many types of natural and synthetic polymers have been explored. The synthetic materials include biodegradable poly(lactic acid–
co-glycolic acid) (PLGA) [8, 9], nonbiodegradable methyl methacrylate, and derivatives such as hydroxyethyl methacrylate–methyl
methacrylate (HEMA–MMA) [10, 11]. Although the synthetic materials display many advantages, such as chemical stability, facile
variance of composition, and structure to meet the application requirement, the unsatisfactory biocompatibility still limits their
potential clinical applications [12]. For example, islet cells entrapped in PLGA microcapsules produced lower insulin yields than
nonencapsulated cells, which may be due to the toxic effect of organic solvents used during the encapsulation process [13]. The
natural materials include proteins (such as collagen [14] and gelatin [15]) and polysaccharides (such as alginate [16] and
chitosan [17]). At present, the natural materials have become the preferred materials used in encapsulation systems because they
always show low/nontoxicity, low immunogenicity, and good biocompatibility.
5.09.2.1
Alginate
Sodium alginate is a natural polysaccharide extracted from brown algae. It consists of two linked anionic monomers,
β-D-mannuronic acid (M) and α-L-guluronic acid (G) residues. The polymer structure is composed of homopolymeric regions of
G units (G blocks) and M units (M blocks), interspersed with regions of mixed monomers (MG blocks) [18]. The sodium alginate
can transform into hydrogel when it encounters divalent cations such as Ca2+, and it holds more than 98% of water inside the
hydrogel. This is important for the maintenance of bioactivity by providing an aqueous environment to the encapsulated cells. It is
the G residues that bind with Ca2+ during the formation of the hydrogel [19]. The G residues of alginate chain become folded and
stacked under the bond interaction, which causes the structure transformation of adjacent alginate chains from random coils to
ordered ribbon-like structure. This entanglement of alginate chains finally contributes to the hydrogel with a three-dimensional
(3D) net structure [20]. Many researches showed that the mechanical strength of the microcapsule can be raised by increasing the G
and the length of the G blocks in the alginate [21]. The M block is not important for the cross-linking of the alginate gel, and its
contribution to the gel is not clear. Besides, the M component has immunogenic property [22, 23], and it may evoke immune
reactions if the M content in the alginate is more than 85%. Some studies had modified alginate with epimerases to transform M
block into G block, which transformed the microcapsule to be more elastic and compact, less permeable, more stable under
physiological conditions [24, 25], and better biocompatible [26]. Some researchers improved the mechanical stability of micro­
capsule by preparing inhomogeneous microcapsule. The homogeneity of the microcapsule refers to the uniformly distributed
The Artificial Organ: Cell Encapsulation
101
alginate throughout the microcapsule. The preparation strategy of inhomogeneous microcapsule is to remove the sodium ions or
add an osmolyte, such as mannitol, to the gelling bath [27]. The modifications of the alginate matrix have shown many advantages
over the basic alginate, for example, the microcapsule prepared by modified alginate with covalently conjugated oligopeptides of a
sequence of RGD (arginine, glycine, and aspartic acid) can improve muscle regeneration after implanted in mice while compared to
the nonmodified one [28–30]. The C2C12 skeletal myoblasts grown in RGD-modified alginate hydrogel display improved adhesion
and proliferation, and the cells have fused into fibrils and expressed differentiation markers [31].
An important limitation of alginate microcapsule in clinical approach is that alginate cannot be degraded after implantation. To
overcome this limitation, a biodegradable alginate polymer was synthesized by partial oxidation with oxidant, and the degradation
time of alginate hydrogel can be controlled by the oxidation degree [32]. Li et al. have achieved a low-molecular-weight alginate by
H2O2 treatment, and the treated alginate led to a decrease of the chain length and the formation of aldehyde groups being by
reducing the end of C-1. The cross-linked alginate scaffold prepared with the obtained alginate showed faster degradation rate than
that from the unmodified alginate, which was due to the lower molecular weight of oxidized alginate and the formed aldehyde
groups being susceptible to hydrolysis. These results suggest that hydrogen peroxide-oxidized alginate can be used in biodegradable
tissue engineering and drug delivery [33].
Though alternative materials have been proposed to design the microcapsule in the last years, alginate is still the most widely
employed material for cell encapsulation. In in vitro studies, the microcapsule with other materials was not proved to be better than
the alginate-based ones, and few in vivo data are available to evaluate the benefits and costs of these microcapsules. For these
reasons, alginate is still considered the most suitable material for cell-encapsulation technology.
5.09.2.2
Chitosan
Chitosan is a natural polysaccharide comprised of glucosamine monomer [34]. It is a product of partially deacetylated chitin, which
is extracted and isolated from abundant shells of crustacea such as lobster, prawn, shrimps, and crab [35]. Chitosan is actually a
copolymer composed of D-glucosamine and N-acetyl-D-glucosamine linked by β(1–4)-glycosidic bond and so it is difficult to
achieve complete deacetylation. The ratio of the two units varies according to the degree of deacetylation (DD) and reflects the
percentage of primary amino groups along the molecular chain. It shows that there are more primary amino groups in chitosan
molecule chain if the DD is higher. Chitosan is degraded predominantly by lysozyme through hydrolysis of the acetylated residue in
implantation site of the recipient [36], and higher DD leads to longer degradation times [37, 38]. Chitosan is soluble in weakly
acidic solutions, and it precipitates out of solution when the pH is above 6.3. When dissolved in acid, the primary amino groups of
D-glucosamine along the molecular chains are protonated, and it can easily complex with polyanions such as alginate. Moreover, the
positively charged chitosan can be cross-linked with negatively charged ions to form a hydrogel, which has a broad application in
the areas of wound healing [39], plastic surgery, and dental implants [40].
Many studies demonstrated that alginate/chitosan microcapsules have exhibited improved biocompatibility and mechanical
strength [41]. Being one of the biomaterials widely used in biomedical field [42], chitosan has been reported to be a biocompatible,
biodegradable, and nontoxic polysaccharide [43]. Chitosan can be readily modified because it has abundant amino and hydroxyl
groups. Hong et al. [44] modified chitosan by grafting methacrylic acid to create a cross-linkable polymer to enhance its water
solubility at physiological pH, and this cross-linked chitosan hydrogel could be readily degraded in the presence of lysozyme and
showed signs of degradation in the presence of chondrocytes without exogenous delivery of the lysozyme. Chitosan can also be
modified with RGD, and the modified chitosan can promote myoblast cell attachment and growth [45].
5.09.2.3
Collagen
Collagen is the major substantial component of natural extracellular matrix (ECM), and it performs important functions on cell
growth, metabolism, and differentiation. The collagen consists of three polypeptide α-chains, which can form about 300-nm long
triple helical structure [46]. In the fibril-forming collagen, the α-chains consist of uniform –Gly–X–Y– triplets, in which the –X–
position is frequently occupied by proline and the –Y– position by hydroxyproline. Some studies showed that the presence of
hydroxyproline residues is essential for the stability of collagens [47, 48].
Collagen has been widely used to simulate natural ECM in cell culture and entrap cells as an encapsulation matrix in artificial
organ therapy. In the artificial liver researches, collagen hydrogel is the widely used biomaterial. The hepatocyte function has been
preserved in the 3D collagen sandwich conformation, whereas cells cultured in a monolayer rapidly lose their function [49].
Collagen has also been used as a matrix for recombinant baby hamster kidney encapsulation to secrete nerve growth factor (NGF)
for the treatment of Alzheimer’s disease. Some studies report a 3D collagen microsphere culture system for glial cell line – derived
neurotrophic factor (GDNF)-secreting HEK293 cells, and this system provides a physiologically relevant 3D environment for the cell
growth. Therefore, the collagen–cell microsphere system has the potential to be used as a controlled proliferation technology in
biopharmaceutical manufacture of mass production of therapeutic proteins [50].
5.09.2.4
Agarose
Agarose, a marine-based polysaccharide, is the gelling component of agar extracted from red seaweeds. It is an alternating
copolymer of β-1,3-linked D-galactose and α-1,4-linked 3,6-anhydro-α-L-galactose residues [51, 52]. Agarose is a thermally gelling
102
Enabling Technologies
polymer; when the temperature is under 35 °C, the gelling process occurs because the infinite network of 3D agarose fibers is
formed. The networks of agarose hydrogel disassemble when the temperature is above 85 °C, and the gelling temperature is
controlled by the agarose concentration [53]. Agarose can be utilized to encapsulate mammalian cells because of the temperaturesensitive water solubility property.
As an encapsulation material, agarose offers some advantages, such as better controlled microcapsule quality and more stable
microcapsule membrane in vivo. Iwata had reported that the islet viability can maintain 32 days in xenograft therapy and even longer
than 100 days in allograft mice [54, 55]. Recently, a multilayered modified agarose microcapsule has been developed for cell
therapy, and it has shown to be more promising than agarose-only one for xenograft transplantation [56]. Karoubi et al. developed a
single-cell agarose hydrogel microcapsule, and the result demonstrated that agarose microcapsules may be a promising cell-delivery
system because it can maintain the survival of transplanted cells by ensuring the balance of mass transfer and metabolic demand of
the transplanted cells, and by minimizing mechanical injury [57].
5.09.2.5
Polyethylene Glycol
Polyethylene glycol (PEG) is a homopolymer of ethylene glycol with the general formula H(OCH2CH2)nOH. It is nontoxic,
odorless, neutral, lubricating, nonvolatile, nonirritating, and is easily soluble in water and many organic solvents [58] and hence
has many applications in pharmaceuticals and medications as solvent, dispensing agent, ointment and suppository base, and tablet
excipient.
Recently, PEG-based hydrogel has been proposed as a particularly promising material for encapsulated cell therapy, because it is
biocompatible, nontoxic, nonimmunogenic, and hydrophilic [59]. PEG hydrogel has the potential to decrease the immunogenicity
of the implanted tissue because it is bioinert. Moreover, the very tight mesh size of PEG hydrogel upon polymerization may isolate
donor cells from the host immune cells [60] and, thus, can minimize the major cause of acute transplant rejection [61]. Another
characteristic of PEG hydrogel is that its stiffness can be easily controlled by altering the molecular weight of PEG so as to match the
stiffness of organ and tissue [62]. Degradable PEG hydrogel can also be used as new tissue-regenerating materials because it has the
ability to be degraded over time and eventually leaves no trace in the patient [63]. Sawhney et al. have synthesized the graft
copolymer of polylysine and methoxy-PEG (mPEG) and used it to enhance the biocompatibility of alginate microcapsules [64].
Furthermore, the studies with PEG–polylysine copolymer-coated microcapsule have shown that the coating can eliminate the
fibrous tissue growth. Chitosan and PEG coatings are employed along with a mild glutaraldehyde (GA) treatment to design the
encapsulated red blood cells, and the results suggest that mammalian cells can be conveniently encapsulated within alginate–PEG/
chitosan system to develop artificial organs, and this microcapsule has better strength, flexibility, and biocompatibility [65].
5.09.3 Properties of the Microcapsules
The prerequisite for the successful application of encapsulation is the match of microcapsule properties, and performance of
encapsulated cells via the microcapsule properties will adapt to the particular application of encapsulated cells. In recent research,
many microcapsule properties have been evaluated in detail. The permeability and mass transport, mechanical strength, biocom­
patibility are the most important factors among these properties.
5.09.3.1
Permeability and Mass Transport
The assessment of the microcapsule permeability is very important because the survival of encapsulated cells will ultimately depend
on the optimal microcapsule mass diffusion characteristics. The prerequisite in designing a microcapsule device with a semi­
permeable membrane is to adjust its permeability in terms of the entry and exit of molecules. In recent years, there are many works
that have studied the microcapsule membrane transport characteristics, and many techniques have been designed to suffice the
transport properties of different types of microcapsules [66]. The mass transport of microcapsule operates in diffusive mode, and it
is driven by a concentration gradient across the membrane. The overall strategy in membrane processing is the formation of a highly
selective membrane, that is, a membrane with high diffusive permeability for the low-molecular-weight nutrient and low diffusive
permeability for the high-molecular-weight immunoglobulin. The appropriate membrane should efficaciously control both the
molecular weight cutoff (MWCO) and the diffusion rate of the molecules for cell survival, as well as the metabolic efficacy.
Generally, the process of various molecules transported across a membrane, characterized as the membrane permeability, is
governed by both the thermodynamic parameter known as the equilibrium partition coefficient and the kinetic parameter
known as the diffusion coefficient. At present, the membrane permeability commonly used for encapsulated cells has been rated
in the 50 000–100 000 Da MWCO, so it is available to protect the cells from the immune rejection of recipient [67].
The main factors determining the rate of diffusion include the solute type and size, interactions between solute and membrane,
and the membrane thickness. Although the high surface-to-volume ratio provided by encapsulation can considerably improve mass
transport property [68, 69], the relatively large size of conventional microcapsules, typically 400–800 μm in diameter, continues to
impose transport limitations [70]. Diffusion restrictions generally result in a dramatic reduction of nutrient, particularly oxygen
transfer limitation can ultimately lead to necrosis of the encapsulated cell or tissue. Many experimental evidence and mathematical
models demonstrate that oxygen concentration decreased radially within spherical devices due to the oxygen consumption by the
The Artificial Organ: Cell Encapsulation
103
encapsulated cells [71–73]. If oxygen levels are insufficient at the site of transplantation, cell density will be reduced to minimize
hypoxia of centrally located cells in the device diameter with larger diameter. Even sublethal levels of hypoxia can also have
deleterious effects on adenosine triphosphate (ATP)-dependent cell functions, such as insulin secretion [74], and may also induce
expression of inflammatory mediators [75]. Moreover, many cells of interest for encapsulation applications, such as liver cells and
pancreatic islet cells, have high oxygen demand rates. So, oxygen transport is an important factor to achieve the success of
implantation therapy. Many studies showed that oxygen transport was usually not controlled by the gel microstructure, but limited
by the thickness of the encapsulation motif. The distance of approximately 1 mm from the innermost cell to the O2 source
(i.e., blood or cell culture medium) is typically the upper tolerated limit [76]. Some studies enhanced oxygen transport by adding
the hemoglobin-based oxygen carriers into the inner of microcapsules. Despite research efforts having acquired higher transport
efficiency, O2 transport is still one of the major limitations in maintaining cell viability and functionality. At present, a novel
method is proposed to improve oxygen supply by adding perfluorocarbon into microcapsule. Perfluorocarbon is a class of
compounds that are biocompatible and biologically inert, which has a high capacity for dissolved gases [77]. The results demon­
strated that the HepG2 liver cell viability and metabolic functionality had statistically significant improvements and could sustain
over extended time period. The analysis showed that the improvement in cellular function and growth was the result of increased
oxygen supply by the addition of perfluorocarbon.
Effective cell-based therapy often relies on the ability of transplanted cell to respond to physiological stimuli in a
concentration- and time-dependent manner [78]. Usually, cells in the center of the device will experience a given solute
concentration at a later time than those on the periphery, thereby leading to a lag in response time [79]. The delayed in vitro
insulin secretion in response to step changes in glucose has been observed in a variety of different microcapsules [80], and
decreasing microcapsule size has been shown to minimize this delay [81]. Therefore, the microcapsule with small diameter is
suitable for the cell encapsulation therapy [82].
5.09.3.2
Microcapsule Mechanical Stability
At present, almost all microcapsule membranes are formed by complexation of the positively charged groups, such as primary
amino groups of poly(L-lysine) or chitosan, and the negatively charged ones, such as carboxyl group of alginate, under electrostatic
interaction. This membrane is weak from a mechanical standpoint, and this weak link limits the microcapsule strength. Therefore,
how to increase the mechanical strength is very important to improve the durability of implantation. The microcapsule membrane
can chronically hold integrity in implantation site if the microcapsule membrane has strong mechanical stability.
Since the mechanical stability of microcapsule is a limiting factor for in vivo applications [83], numerous studies have
clarified the mechanical properties of microcapsules and developed many technologies to improve the microcapsule strength
[84, 85]. It has been known that many factors, such as materials property, microcapsule size, membrane thickness, processing
method, pore volume fraction, polymer molecular weight, geometrical shape of microcapsule, implant site, implant duration,
and host reaction, can affect microcapsule strength. Although mechanical property of microcapsules has been recognized as a
limiting factor for in vivo applications, quantitative value of microcapsule strength is considerably lacking [86]. In some studies,
the thickness of microcapsule membrane has been engineered and used as an index of microcapsule strength [87]. However,
this method does not completely reflect the membrane characteristics. A simple way has been put forward to quantitatively
evaluate the microcapsule strength for the accurate examination of microcapsule durability. This method is to subject the
microcapsules to a well-defined shear flow, and the fraction of fractured microcapsules can be used as a simple index of
mechanical durability [88]. The mechanical stress experienced by the microcapsule depends on the shear rate and the viscosity
of the fluid in this system. Although it is simple, this method can be used as a screening tool to quantitatively evaluate
mechanical properties in designing microcapsule preparation process. At present, many methods have been developed to
improve the microcapsule strength, such as increase of coating time [89], extent of surface modification [90], choice of
polymeric additives to capsule graft components [85], alginate beads made from cross-linked barium ions [91], and coating
the microbeads with alternating layers of different polyanions and polycations. Some studies showed that the cell encapsula­
tion matrix must possess sufficient compressibility, tensility, and shear strength to ensure their integrity in vivo, for example, the
mechanical strength of the capsule can be raised by increasing the G content and the length of the G blocks of alginate because
alginate hydrogel with high G possesses more compact net structure and stronger toughness [92]. Lanza et al. studied the
mechanical stability of standard alginate–polylysine–alginate (APA) by varying alginate concentrations from 0.75% to 1.5% to
produce different microspheres [93]. The results showed that a better in vivo mechanical stability of microcapsules was observed
when alginate concentration was 1.5%; almost all of the microcapsules can be recovered from the animals and had remained
intact for the 1-year duration of the implantation. One of the methods to improve the mechanical stability and durability of
microcapsules is the modification to the chemical composition of the membrane, such as replacing poly-L-L-ysine (PLL) with
poly-L-ornithine (PLO) to form microcapsule membrane [94]. In another study, both weak polyanion (alginate) and strong
polyanion (cellulose sulfate) can complex with polycation (polymethylene-co-guanidine) to provide high and controlled
mechanical strength and microcapsule durability [95]. Another strategy is to incorporate an inorganic silica-based polycation
into the microcapsule matrix to improve mechanical stability, which has been successfully tested with rat islets in vitro and
in vivo [96]. Coradin et al. designed a composite alginate–poly-L-lysine microcapsule with the coating of sodium silicate, which
showed greater resistance to fracture than the alginate–poly-L-lysine–alginate microcapsule. Thus, alginate–inorganic composites
may open a promising route for new biocomposite design and biotechnology applications [97].
104
5.09.3.3
Enabling Technologies
Biocompatibility
Another important element to the success of implantation therapy is that the microcapsule membrane must possess the better
biocompatibility in implantation site. Biocompatibility means the encapsulated cells can be used to perform the function of
treatment and restore or replace any tissue or organ without causing immune reaction against it [98]. At present, biocompatibility
includes three parts: the reaction of the encapsulated cells to the polymer, the reaction of the recipient to the microcapsule (foreign
body reaction), and the reaction of the recipient to the encapsulated cells and therapeutic protein [99]. When the cells have been
encapsulated into microcapsule, the membrane can protect them from the mechanical damage or immune rejection of host; on the
other hand, the cells can also affect the microcapsule state due to the rapid proliferation if the encapsulated cells are an immortal cell
line, so it is considerably important to study the proliferation situation of encapsulated cells, especially in vivo study. The major
obstacle of cell encapsulation therapy is fibrotic overgrowth on microcapsule surface. The fibrotic overgrowth is induced by foreign
body and inflammatory reaction because the materials used for encapsulation are not completely inert [100]. The fibrotic over­
growth is a complex process after microcapsule implantation. First, it leads to chronic inflammatory response induced by foreign
body entrance/substance, and then the development of granulation tissue leads to the formation of a fibrotic tissue. The fibrotic
tissue on microcapsule surface is largely composed of collagen, macrophages, fibroblasts, and few capillaries [101]. As a result of
this fibrous tissue overgrowth, the diffusion of nutrients, oxygen, hormones, and waste products through the microcapsule is
blocked, and the encapsulated cells would quickly lose the viability and functionality because of hypoxia, starvation, and the
secretion of nitric oxide by the stimulated macrophage [102]. Though the immunoisolation membrane of microcapsule can prevent
monocytes and lymphocytes from destroying the encapsulated cells, the immune reactions against the microcapsule often occur.
Several studies reported that the recombinant proteins secreted by encapsulated cells can also induce the host to produce
immunoglobulins against the encapsulated material [103, 104].
As the immune response of host to encapsulated cells is very important to receive implantation success, what are the pathways of
immunoisolated cells rejection? In allograft immunity, because immune mechanism mainly consists of direct engagement of T
lymphocyte with donor cells, the physical isolation provided by the microcapsules would prevent cell-to-cell contact between the
encapsulated cells and the host immune system, thereby facilitating the immunological acceptance of the graft. In xenograft
immunity, although microcapsules would prevent antibodies or complement fractions into the microcapsule by the membrane’s
semi-permeative property, some small proteins or enzymes would easily cross the microcapsular membrane and trigger a significant
inflammatory cell reaction. These inflammatory cells can release the small cytokines, nitric oxide, and free oxygen radicals to
severely damage the encapsulated cells [105].
It is believed that the host immune reaction or biocompatibility to immunoisolation device is influenced by microcapsule size,
shape, surface morphology, chemical surface composition, biological characteristics of encapsulated cells, and the implantation
procedure [23, 106–109]. Many researchers have studied the influence of alginate composition on the immune responses, which
showed that alginate with high mannuronic acid content could activate macrophages in vivo, resulting in fibroblast proliferation and
eventual fibrotic overgrowth, while high G alginate had lower ability of inducing the antibody reaction [110]. Therefore, alginate
composition of less than 35% of M is suitable for cell encapsulation therapy. Another important issue to affect the immune response
of host is the diameter of the microcapsule. Sakai et al. studied the ability of different diameters of agarose microcapsules in inducing
the immune reaction of host; the results showed that the immune reaction to the smaller microcapsule was much lower than that to
the larger one [111]. Some reports suggest that the purity of alginate is the vital factor for the success of implantation therapy
[112, 113]. Specifically, reduction in endotoxin content and elimination of proteins and phenolic-like compounds are essential to
decrease the immune reaction.
Although all the microcapsules can induce immune reaction, the intensity of fibrotic response can vary greatly from individual to
individual, even within the same species [114]. Therefore, the specific microcapsules well tolerated in small animals must be tested
in large animal models before clinical application. Some authors concluded that the experiment results in rodent model have no
directional effect in human islet implantation; even it is significantly different from human recipients. Although pigs are metabo­
lically similar to humans, the fibrotic response of encapsulated cells is potentially different in pig models. Dufrane et al. studied the
impact of implantation sites on the biocompatibility of alginate-encapsulated pig islets. In this article, the adult pig islets
encapsulated in alginate were implanted into either abdominal cavity, subcutaneous tissue, or under the kidney capsule. Three
days after implantation, no significant difference for encapsulated pig islets was observed in terms of microcapsule biocompatibility
and islet functionality in peritoneum, kidney capsule, or subcutaneous tissue. However, between days 5 and 30 after transplanta­
tion, implanted microcapsules from abdominal cavity demonstrated a higher degree of broken microcapsules and microcapsules
with severe cellular overgrowth than microcapsules removed from subcutaneous tissue and kidney capsule. This was associated with
a significant reduction of islet viability, insulin content, and insulin secretion. Therefore, subcapsular and subcutaneous spaces of
kidney are more appropriate implantation sites than the peritoneum [115]. Another study showed that the central nervous system
(CNS) is a suitable site for implantation due to the specific immunologic status, and the immune responses in the CNS are mainly
cellular, and the alginate provides an immunoisolated membrane against cell-mediated (lymphocytes, natural killer cells, or
microglia) destruction of the implantation cells [116].
From a materials standpoint, the conventional strategy to improve biocompatibility involves the development of material
surface chemistry and morphology that can minimize host inflammatory fibrotic response. At present, it is a considerably difficult
and complicated task in abolishing the immune reject response. Although it is relatively easy to prevent the passage of cytotoxic
cells, macrophages, and other larger cellular immune molecules including high-molecular-weight antibodies through the
The Artificial Organ: Cell Encapsulation
105
semi-permeable membrane, a potentially more serious problem is the blockage of humoral immune components such as low­
molecular-weight cytokines as well as tissue antigens secreted by the cells inside the membrane. In recent years, many researchers
have put in considerable effort into new biomaterials, coatings, and surface treatments of microcapsule in an attempt toward
development of an inert or invisible implant that ideally has little host interaction (low protein binding, low tissue adhesion, and
little vascularization).
5.09.4 Applications of Encapsulated Cells
As an immune protection device, encapsulation of live cells has been widely studied to eliminate the problems associated with
immune rejection during allogenic and xenogenic transplantation. The therapy of encapsulated cells offers important advantages
compared with the therapy of peptides or proteins; it can not only maintain the chemical stability of the therapeutic product but
also improve the efficiency of the therapeutic drug [82]. At present, the major application of encapsulated cell therapy includes three
parts: (1) encapsulated primary cells for artificial organ research; (2) encapsulated recombinant cells for gene therapy research; and
(3) encapsulated stem cell for tissue-engineering research and regenerative therapy.
5.09.4.1
Encapsulated Primary Cells for Artificial Organ Research
At present, the age-related diseases have rapidly developed with the increase of old-age population, and these diseases are often
closely tied with deficient or subnormal metabolic and secretary cell functions. These degenerative and disabling disorder diseases
included diabetes mellitus, Parkinson’s disease, hemophilia, hypoparathyroidism, chronic pain, kidney or hepatic failure. The
appropriate therapeutic strategy for these diseases is organ or tissue transplantation, but the current desperate shortage of donor
organs does not meet the demand. Therefore, that the encapsulated cells supply the host with regulated and/or continuous delivery
of therapeutic product is a promising method. Cell encapsulation technology has been currently developed to create bioartificial
organs aiming at the treatment of several human diseases, such as diabetes [117], kidney failure [118], anemia [119], dwarfism
[120], Parkinson’s disease [121], and amyotrophic lateral sclerosis (ALS) [122].
In diabetes, the glucose concentration increases by the partial or whole deficient of islet function. It is a global disease with an
incidence rate of about 3–5% of the population, and 150 million people around the world suffer from diabetes mellitus at present
according to the statistics of the WHO, and this figure will be twofold by 2025 [123]. Today, the treatment strategy for diabetes is the
injection of exogenous insulin, whole-organ pancreas transplantation, and artificial and bioartificial pancreas. Among these
methods, bioartificial pancreas is a promising therapeutic strategy because it can avoid the use of immunosuppressive drugs and
provides moment-to-moment glucose homeostasis consistent with a well-functioning pancreas. The idea of using alginate micro­
capsules for the immunoprotection of transplanted islet cells was proposed by Chang in 1964 [3]. The transplantation of
encapsulated islets for the treatment of diabetes mellitus has been the most common application of cell encapsulation technology.
Until recent, many highly experienced research groups have made plenty of contribution for demonstrating the main challenges of
cell encapsulation technology. Paul de Vos’s studies showed that microcapsules prepared by alginates are biocompatible and stable
in vivo up to 2 years after implantation. The microcapsule did not interfere with islet function and could effectively protect the islets
against immune rejection of host as illustrated by the prolonged survival of the graft [124]. According to the promising results
obtained in allotransplantation and xenotransplantation approaches [125, 126], a pilot clinical trial had recently been initiated by
Calafiore et al. [127, 128] Encapsulated human islets were implanted into 10 non-immunosuppressed patients with type 1 diabetes.
Data of two patients indicated that pancreatic islets remained metabolically active. Mallett et al. developed a novel method to purify
and/or modify commercially available alginate and encapsulated the islet to demonstrate the direct effect of alginate purification on
long-term metabolic function of islet. Comparing the in vivo outcomes of encapsulated islets preparing with pre-modification and
post-modification alginate provided us greater insight into how alginate could be manipulated to be better suitable for use in
transplantation therapy. Recipients of purified alginate microcapsule exhibited a 105-day graft survival rate of 90.5% versus 69.2%
for recipients of unpurified alginate; recipients implanting purified alginate microcapsule showed improved blood glucose levels
and oral glucose challenge over recipients implanting unpurified alginate microcapsule. Furthermore, islets encapsulated in purified
alginate microcapsule demonstrated dramatically reduced fabric overgrowth and insulin secretory activity far superior to that of
islets in unpurified alginate microcapsule. Therefore, it can be concluded that microencapsulation with purified alginate can
improve the survival and metabolic function of encapsulated islets [129].
Huntington’s disease (HD) is an incurable neurodegenerative genetic disorder, which can affect muscle coordination and some
cognitive functions, typically becoming noticeable in middle age. It is the most common genetic cause of abnormal involuntary
writhing movements called chorea [130]. Emerich et al. developed an alginate-based encapsulation system to entrap the choroid
plexus and implanted this device to the brain in a primate model of HD for an appropriate delivery of neurotrophic factors. The
results showed that the implantation of choroid plexus significantly protected striatal neurons, which confirm that the encapsulated
choroid plexus might be useful for preventing the degeneration of neurons in HD [131].
Jeon et al. proposed that encapsulated cells could be used to the treatment of chronic neuropathic pain [132]. It was clear that the
bovine-derived adrenal chromaffin cells could synthesize and secrete the pain-reducing neuroactive compounds including catecho­
lamines, opioid peptides, and other neuroactive substances. Using the immune protective property of microcapsule, the adrenal
chromaffin cell implanted into the subarachnoid space of the spinal cord could continuously survive and stably secrete
106
Enabling Technologies
pain-reducing neuroactive compounds to relieve the chronic pain syndrome. The results showed that it took remarkable analgesic
effects in a rat model of acute and chronic pain while nonencapsulated cell transplantation did not alleviate the pain because of
immunological rejection [133]. Kim et al. encapsulated bovine adrenal chromaffin cells with APA microcapsule and implanted
intrathecally in a rat using the neuropathic pain model. The results showed that the intrathecal implant of encapsulated adrenal
chromaffin cells might be a useful method for treating chronic pain [134]. In our lab, the encapsulated bovine adrenal medulla
chromaffin cells had been implanted in the subarachnoid space of 22 cancer patients with severe chronic pain, requiring a large
amount of anodyne. Ninety percent of the patients experienced pain relief within 1 week after transplantation, among which 85%
showed considerably alleviated pain. The visual analogue score (VAS) pain index reduced, on average, from level 9 to level 2, and
the need for anodyne was eliminated. After one or two transplantations, three patients stopped anodyne for up to 300 days with
improved life quality [135].
Parkinson’s disease is one of the major neurodegenerative disorders of middle-aged and old-aged people, and the principal
pathology underlying these symptoms is a progressive loss of dopaminergic neurons in the substantia nigra (SN) and a concomitant
reduction of dopamine (DA) level in the striatum [136]. Using the immunoprotection property of microcapsule, the encapsulated
DA-secreting cells, such as bovine adrenal chromaffin cell, can replace the dead dopaminergic neurons in the SN to secrete DA for
the treatment of Parkinson’s disease [137]. Xue et al. encapsulated bovine adrenal chromaffin cells within APA membranes, and
encapsulated bovine adrenal chromaffin cell as well as unencapsulated cell and empty microcapsule were grafted into the brain of
hemiparkinsonian rats with 6-hydroxydopamine (6-OHDA) lesion. Apomorphine-induced rotational behavior of the host animals
and the survival of the grafted bovine adrenal chromaffin cell were examined after transplantation. The animals receiving
encapsulated bovine adrenal chromaffin cells showed a significant decrease (17.6–35.6%) in apomorphine-induced rotation
after 1 week of post-implantation and remained stable for a 10-month test period. Analysis of fluorescent histochemistry further
revealed that encapsulation increased the survival of bovine adrenal chromaffin cell with only a minimum host reaction for up to 10
months of post-transplantation while the unencapsulated bovine adrenal chromaffin cell had died at this time and accompanied by
a large inflammatory response. The reduction of apomorphine-induced rotation was correlated with the survival of bovine adrenal
chromaffin cell in the host brain. These data indicated that the encapsulation of bovine adrenal chromaffin cell in APA membrane
could reduce the host immune response to the xenograft and prolong the viability of the grafted cell [138]. In our laboratory, bovine
adrenal chromaffin cell or rat neuroendocrine PC-12 cell had been encapsulated in microcapsule with an average size of 200 μm in
diameter and were implanted into the striatum of 6-OHDA-lesioned monkeys or rats by injection using a stereotactic technique.
Following the implantation of encapsulated cell, the semi-parkinsonian symptoms were improved and showed a decline in
rotational asymmetry for up to 48 months, while only a temporary improvement was detected in the group of the nonencapsulated
cell and no change was observed in the group of empty microcapsule. DA and its metabolites were found completely depleted in the
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned side versus the normal side of the neo-striatum in the semiparkinsonian monkeys and the extracellular levels of DA in the right putamen increased markedly after the transplantation of
encapsulated bovine adrenal chromaffin cell as compared to pretransplantation (Figure 2) [135].
Severe liver disease is very often life threatening and dramatically diminishes quality of life. Effective artificial liver support
system should be capable of carrying out the liver’s essential processes, such as synthetic and metabolic functions, detoxification,
and excretion. Encapsulated hepatocyte therapy may be an alternative to the implantation of the whole liver, and making good use
and development of this technique may provide a clinical application for treating liver failure patients. Encapsulated porcine
hepatocyte with alginate–poly-L-lysine–alginate membranes had been transplanted in a mouse model of fulminant liver failure to
study the treatment efficacy. In vitro, the encapsulated hepatocyte treatment could maintain the metabolic functions, such as
albumin and urea synthesis, and drug catabolism. In vivo, comparing nonencapsulated hepatocytes cell treatment, the survival rate
of the animals treated with encapsulated hepatocyte cells increased from 25% to 75%. These results indicated that porcine
hepatocyte can be successfully encapsulated and transplanted into xenogeneic recipients with liver failure and sustain liver
metabolic functions [139]. Aoki et al. had encapsulated the hepatocyte into the collagen matrix microcapsule and sodium
alginate–poly-L-lysine–sodium alginate microcapsule and implanted them into the intraperitoneal area of hepatectomized rats to
investigate the encapsulated xenogeneic hepatocyte growth, protein secretion, and survival time of rats. Results showed that survival
times of rat with the encapsulated hepatocyte treatment were significantly prolonged, and blood ammonia levels remained lower in
the encapsulated hepatocyte treatment group than the nonencapsulated hepatocytes treatment group. Hematoxylin and eosin
staining (HE)-stained microcapsules indicated many viable hepatocytes without any lymphocyte infiltrates in them. Therefore,
encapsulated xenogeneic hepatocyte with alginate–poly-L-lysine–alginate ultrathin layer could avoid an adverse immune reaction,
and intraperitoneal transplantation could prolong the survival time of rats with only 4% of total liver volume and maintain
ammonia metabolism at the same level as the allogeneic hepatocyte [140]. Hamazaki et al. investigated the metabolic activity of
hepatocyte spheroids encapsulated with agarose hydrogel in vitro and the effect of encapsulated hepatocyte spheroids intraperito­
neal transplantation on survival of the 90% hepatectomized rats for preparing future xeno-hepatocyte spheroids transplantation.
Results showed that encapsulated hepatocyte spheroids had better metabolic activity in vitro. Survival rates of 90% hepatectomized
rats were improved by the intraperitoneal transplantation of encapsulated hepatocyte spheroids [141].
5.09.4.2
Encapsulated Genetically Modified Cells for Gene Therapy
Genetic engineering has emerged as a promising strategy for cell-based therapies, but genetically modified cell often arises from an
allogeneic or xenogeneic source and might also require protection from the host immune system. Encapsulation of recombinant cell
The Artificial Organ: Cell Encapsulation
(a)
107
(b)
(c)
12
*
PreTx
PostTx
9
#
pg µl–1
5
#
4
#
3
2
1
0
p-DA
c-DA
p-DOPAC c-DOPAC
Figure 2 The microencapsulated bovine adrenal chromaffin cells for the therapy of the monkeys with semi-parkinsonian: (a) the monkey before
implantation; (b) the monkey after cell implantation, who could move his arms freely and feed himself; and (c) secretion of dopamine and its derivatives.
represents a novel alternative nonviral approach to gene therapy in which therapeutic protein is sustainable and in the long term
delivered by encapsulated recombinant cells. Encapsulation of recombinant cell within a semi-permeable membrane allows the
bidirectional diffusion of nutrients, oxygen, and metabolites but prevents substances with high molecular weight, such as
antibodies, and immunocytes from entering microcapsules. Because the microcapsule may protect the cells from host immune
rejection, it can increase the efficiency of exogenous gene expression, reduce the need for frequent injection, and circumvent the
problems of toxicity, limited half-lives, and variation in circulating levels of protein drugs. Compared to other forms of gene
therapy, this approach has several advantages. For example, the semi-permeable membrane of microcapsule protects cells from host
immune rejection, increases the efficiency of gene transfer, and reduces the need for frequent injection. Furthermore, the risks of
unintentional viral infection and insertional mutagenesis due to vectors integrating their DNA into the host DNA are avoided since
the recombinant cells are enclosed in microcapsules [142]. At present, this strategy of cell microencapsulation has been used
preferentially to the treatment of many diseases such as anemia [143], dwarfism [144, 145], hemophilia B [146, 103], kidney [147]
and liver failure [148], and pituitary [149], and CNS insufficiencies [150, 151].
Because the drug can, with difficulty, reach the specific targets in the brain and CNS, cardiovascular disorders have posed an
important challenge to develop controlled drug delivery device for maintaining sustained drug level. Recently, a phase I trial had
been completed by Sieving et al. [152], where a 6-month period delivery of ciliary neurotrophic factor (CNTF) by encapsulated cells
derived from human retinal pigment epithelial cell line (ARPE-19) was achieved when implanted into human eyes. Regarding
cardiovascular disorders, a myocardial infarction rat model was evaluated using encapsulated Chinese hamster ovary (CHO) cells
delivering rat vascular endothelial growth factor (rat VEGF) [153]. A 21-day in vivo study was achieved where anti-CHO antibody
titers were found to be significantly lower to the control group, thus suggesting a novel alternative strategy for therapeutic
angiogenesis in ischemic heart disease. Other interesting therapeutic applications employing genetically engineered cell included
108
Enabling Technologies
the encapsulated recombinant human amniotic epithelial cell to treat mucopolysaccharidosis type VII (MPSVII) [154]. The
β-glucuronidase could be measured after 7 days of encapsulated recombinant cells’ implantation in mice. It showed that the
encapsulated recombinant cell was an effective strategy for the treatment of MPSVII. Some research groups had studied the longterm in vivo delivery of encapsulated genetically engineered erythropoietin (Epo)-producing C2C12 myoblasts in allogeneic and
syngeneic mice model [155, 156]. High and constant hematocrit levels were maintained during the study period after implantation
of cell-loaded microcapsule and without implementation of immunosuppressive protocol. Moreover, the subcutaneous space was
found to be a more suitable site than abdominal cavity which is the usually performed site due to the formation of a fully rich
vasculature across the microcapsule and the pharmacodynamic behavior and the immune-modulatory properties of Epo. Therefore,
this alternative technology could avoid the repeated weekly injections currently practiced in anemic patients. Jianping et al.
proposed a gene therapy strategy based on the implantation of encapsulated secreting defective factor IX (FIX) cells as a highly
desirable alternative treatment. The results showed that the cell within microcapsule still maintained viability after 60 days of
implantation. The human FIX (hFIX) level was 170 ng ml−1 in the circulating system of non-obese diabetic/severe combined
immunodeficiency (NOD/SCID) mice after implantation and it remained detectable for 1 month. Moreover, the delivered hFIX
indicated higher biological activity, and the activated partial thromboplastin time (APTT) was reduced from 94 s before treatment to
78–80 s and the tail bleeding time decreased from 15 min to 1.5–7 min after treatment. Therefore, the encapsulated human primary
myoblast could secrete functional hFIX. Furthermore, implantation of encapsulated human primary myoblast could partially correct
the phenotype of hemophilia B mice, supporting the feasibility of this gene therapy approach for hemophilia B [157]. Chang et al.
encapsulated mouse Ltk cells transfected with the human growth hormone (hGH) gene in immunoprotective alginate microcapsules
and implanted these microcapsules into allogeneic mice. The results demonstrated that the hGH could be measured after 2 weeks of
implantation, and the concentration of hGH in circulation is 0.1–1.5 ng ml−1 serum; moreover, mice implanted with the nonencapsulated transfected cells did not demonstrate significant level of circulating hGH. The survival of the genetic engineering cell and
the persistent expression of the recombinant protein were verified when the microcapsules were retrieved periodically, and the results
demonstrated that the encapsulated cell could remain viable, proliferative, and productive of hGH even after 111 days of implantation
[158].
Another important application field of encapsulated genetic engineering cell is cancer therapy. At present, some interesting
approaches have been carried out including the encapsulation of engineered cells to deliver sustained level of interleukin-6
(a pleiotropic cytokine that plays a central role in hepatic function and response) in an animal model of hepatocellular carcinoma
(HCC) [159] and require the better treatment efficacy. Shi et al. entrapped the genetically modified cells that secrete fusion protein
RM4-TNFa into microcapsule to treat cancer, and the implantation of the encapsulated cell had led to significant tumor regression
[160]. In addition, the encapsulated engineered cell can be stored frozen and ready to use in future patients, thus enabling
encapsulation-based gene therapy by a simple, routine treatment. Read et al. reported that encapsulated 293-endo cell could
long-term express endostatin in rats, and 70% of cells in microcapsule were alive after 4 months of implantation [161]. Rats with
BT4C cells glioma that received implantation of encapsulated 293-endo cells survived significantly longer than the control rats, and
some rats became long-term survivors. Joki et al. assessed the effect of local delivery of the angiogenesis inhibitor endostatin on
human glioma cell line (U-87MG) xenografts [162]. The results showed that human endostatin released from the microcapsule
brought about a 67.2% inhibition of bovine capillary endothelial (BCE) proliferation. Furthermore, secreted human endostatin
(hES) was able to inhibit tube formation in KDR/PAE cells (porcine aortic endothelial (PAE) cells stably transfected with KDR, a
tyrosine kinase) treated with conditioned U-87MG medium. A single local injection of encapsulated endostatin-secreting cells in a
nude mouse model resulted in a 72.3% reduction in subcutaneous U87 xenografts weight 21 days post-treatment. A combination of
immunotherapy with angiostatic therapy was investigated by treating B16-F0/neu melanoma-bearing mice with intraperitoneally
implanted, encapsulated genetically modified mouse myoblasts (C2C12) to deliver angiostatin and an interleukin 2 fusion protein
(sFvIL-2). The combination treatment resulted in improved survival, delayed tumor growth, and increased histological indices of
antitumor activity (apoptosis and necrosis). In addition to improved efficacy, the combination treatment also ameliorated some of
the undesirable side effects from the individual treatments that had led to the previous failure of the single treatments, for example,
inflammatory response to IL-2 or vascular mimicry due to angiostatin. Moreover, the combination of immuno- and antiangiogenic
therapies delivered by immunoisolated cells was superior to individual treatments for anti-umorigenesis activity, not only because
of their known mechanisms of action but also because of the protection against the adverse side effects of the single treatments.
Neutrophil-driven inflammation against microcapsule delivering immunotherapy was subdued by angiostatin, while tumor
endothelial cell apoptosis driven by angiostatin was enhanced by interleukin 2. Thus, strategy of encapsulation delivering multiple
antitumor recombinant molecules could improve the therapeutic efficacy of tumor [163]. In our lab, we have microencapsulated
endostatin over-expressing CHO (CHO-endo) cells and implanted the microcapsules into a tumor-bearing mice encapsulation for
gene therapy. Tumors of the mice treated with microencapsulated CHO-endo cells showed a dramatic delay in the model to study
the feasibility and efficiency of micro encapsulation for gene therapy. At day 27, the volume of the treatment group was
1211.7 � 113.4 mm3, while that of the control group was 3604.8 � 119.6 mm3. The growth of tumor in the microencapsulated
CHO-endo cells was inhibited 66.4% as compared to control (P < 0.05). The suppression of tumor growth by the microencapsu­
lated CHO-endo cells was corroborated with the improved survival of the treated animals. While only 40% of control animals
survived, 80% of the animals treated with microencapsulated CHO-endo cells were still alive after 27 days of tumor cell injection.
Therefore, the microencapsulation-based in vivo culture method offers a safe, highly efficient, and low-cost anti-angiogenesis
approach to tumor therapy. Combined with surgery, chemotherapy, and radiotherapy, the inhibition of tumor angiogenesis can
enhance anti-tumor efficacy, improve survival, and prolong span of the treatment in patients with a poor prognosis (Figure 3) [82].
The Artificial Organ: Cell Encapsulation
(a)
109
(b)
4000
100
80
3000
Survival (%)
Tumor volume (mm3)
3500
2500
2000
1500
1000
60
40
20
500
0
0
0
5
10
15
20
Time (days)
25
0
30
5
10
15
20
Time (days)
25
30
(c)
Control
Treatment
Blood vessel
Blood vessel
Endostatin concentration (ng ml–1)
(d)
30
25
20
15
10
5
0
0
5
10
15
Time (days)
20
25
30
Figure 3 Antiangiogenesis therapy of melanoma, using microencapsulated CHO-endo cells implanted in the peritoneal cavity of mice: (a) inhibition of
tumor growth; (b) increase in animal survival; (c) H&E staining of tumor tissue (�200) and (d) endostatin concentration in blood; scale = 50 μm.
Significant differences were obtained between the microencapsulated CHO-pac cell group (control) (open circles, n =10) and the microencapsulated CHO­
endo cell-treated group (solid circles, n = 10) (p < 0.05).
5.09.4.3
Encapsulated Stem Cells for Tissue Engineering and Regenerative Medicine
Currently, encapsulation of stem cells is an area of increasing interest in tissue replacement therapy, and it may be a
promising therapeutic strategy to tissue or organ deficient. The main objective of the encapsulated stem cells is to maintain
the stem cells’ undifferentiated state before implantation and direct their differentiation after implantation in the host-specific
tissue or organ.
Reliable control over the process of stem cell differentiation is a major challenge in moving stem cell-based therapy forward. The
composition of the ECM is known to play an important role in modulating differentiation. Batorsky et al. developed a system to
encapsulate adult human mesenchymal stem cells (hMSCs) within spherical 3D microenvironments using a microcapsule consist­
ing of a mixture of collagen I and agarose polymers. The results showed that cell viability post-encapsulation reached to 90% and
remained at this level for 8 days in vitro. Fluorescent staining of the actin cytoskeleton revealed that hMSC spreading increased with
increasing collagen concentration. This system of producing 3D microenvironments of defined matrix composition therefore offers
a way to control cell–matrix interactions and thereby guide hMSC differentiation [164]. Liu et al. implanted the encapsulated bone
110
Enabling Technologies
marrow mesenchymal stem cells into the spleen of 90% hepatectomized (PH) rat, and the survival rate of rat was 91% in day 14 of
implantation, while the survival rate was 21% in 90% hepatectomized rats and 25% for those receiving free MSC transplantation.
Unlike free MSCs, the encapsulated MSCs were retained in the spleen and their hepatotrophic factors can continue to drain directly
into the liver without dilution resulting in improved hepatic regeneration. In addition, the MSCs differentiated into hepatocyte-like
cells in the spleen as an ectopic liver support [165].
Embryonic stem (ES) cell is being widely investigated as a promising source of therapeutically useful cells with their
proliferative, renewable, and pluripotent capacities. Certain aspects of the stem cell microenvironment (or niche), including
intrinsic or extrinsic factors, play critical roles in regulating the fate of ES cell [166, 167]. However, the undifferentiating
proliferation in vitro and controlled ES cell differentiation into specific tissue cell are challenging. Xiuli Wang reported the
feasibility of using encapsulation technology to study the interaction between ES cell and their tissue niche. ES cell growth,
viability, and differentiation were evaluated in vitro when they were enclosed in solid or liquefied core APA microcapsule. In
comparison with those microcapsules with solid core, the liquefied microcapsule provided a more suitable culture environment
for the growth of ES cell. Typical markers for the undifferentiated ES cell, such as alkaline phosphatase (AP), stage-specific
embryonic antigen 1 (SSEA-1), and Oct-4 gene, were also tracked by immunochemistry and reverse transcriptase polymerase
chain reaction (RT-PCR) and expression of markers remained higher over 2 weeks of culture in vitro. Magyar et al. demonstrated
the differentiation ability of APA-encapsulated embryonic body and further demonstrated the feasibility of using APA
encapsulation system to the direct differentiation of stem cells [168]. The development of ES cell therapeutic strategies for
hepatic disorders is the identification and establishment of a controllable hepatic differentiation strategy. In order to address
this issue, Maguire et al. established an alginate encapsulation technology which provided an approach to modulate the
differentiation process through changes in key encapsulation parameters. A wide array of hepatocyte-specific markers is
expressed by differentiated cells within an alginate bead microenvironment during a 23-day of culture, such as urea and
albumin secretion, glycogen storage, and cytochrome P450 transcription factor activity. In addition, the results also demon­
strated that cellular aggregation was integral to the control of differentiation within the microcapsule environment and this
process was mediated by the E-cadherin protein. The temporal expression of surface E-cadherin and hepatocyte functional
expression occur concomitantly, and both cellular aggregation and albumin synthesis were blocked in the presence of anti-E­
cadherin immunoglobulin [169]. Fang et al. examined the differentiating potential of embryoid-body cell derived from ES cell
into hepatocyte in alginate microbead containing exogenous growth factors in vitro. Results showed that embryoid-body cell
could maintain cell viability in alginate microbead in vitro, and the differentiated cell expressed several hepatocyte genes
including alpha-fetoprotein (AFP), albumin (ALB), Cyp7a1, CK18, transthyretin (TTR), and tyrosine aminotransferase (TAT)
and produced ALB and urea in alginate microbeads, and the expression of ALB and CK18 proteins could continue to day 14.
Therefore, this technology may develop scalable stem cell differentiation strategies for bioartificial livers and hepatocyte
transplantation [170]. Liu et al. studied the ammonia removal capacity of encapsulated hepatocyte and bone marrow stem
cell in in vitro culture, and the hyperbilirubinemia removal efficacy of encapsulated hepatocyte and bone marrow stem cell in
Gunn rats after transplantation. The results showed that the ammonia removal capacity was maintained longer in the different
ammonia concentration media in encapsulated hepatocyte and bone marrow cell culture. In in vivo transplantation experiment,
the plasma bilirubin level with encapsulated hepatocyte and bone marrow stem cell transplantation treatment were signifi­
cantly lower than those in alone encapsulated hepatocyte transplantation during the period of 3–10 weeks posttransplantation. Therefore, the encapsulated hepatocyte and bone marrow cell could improve hepatocyte function of both
ammonia removal in in vitro culture and bilirubin decrease in in vivo transplantation [171].
5.09.5 Conclusions and Future Considerations
Encapsulated cells offer a useful method to preserve long-term viability and function of cells for transplantation therapy. Though cell
may remain viable in a given matrix, material properties must be well controlled to facilitate the desired metabolic functionality, so
material purification and modification to improve the biocompatibility and strength of microcapsule is one of the challenges of
encapsulated cell therapy. Another challenge of encapsulated cell is how to solve pivatal problem on the large scale preparation of
microcapsule, such as maintaining a controlled environment, standard discipline, and rigorous quality control. This step will be
essential for allowing cell encapsulation technology to enter human clinical trials, thereby becoming a real clinical therapeutic strategy.
Although the encapsulated artificial cells have taken some development to date, we believe that this technology might see
exciting improvement in the next few decades. With continuing advances in genetics, materials science, pharmaceutical technology,
biology, and chemical engineering, this technology will become a realistic proposal for clinical application in the future.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (grants 20236040, 20736006, 30472102,
20806080, 20876018, 20906088, 30970885, and 10979050); the National Basic Research Program of China (grants
2002CB713804 and 2005CB522702); the Hi-Tech Research and Development (863) Program of China (grants 2001AA326020,
2003AA205110, and 2006AA02A140); and The National Key Sci-Tech Special Project of China (2008ZX10002-019).
The Artificial Organ: Cell Encapsulation
111
References
[1] Silva AI, de Matos AN, Brons IG, and Mateus M (2006) An overview on the development of a bio-artificial pancreas as a treatment of insulin-dependent diabetes mellitus.
Medicinal Research Reviews 26(2): 181–222.
[2] Hunkeler D (1999) Bioartificial organs transplanted from research to reality. Nature Biotechnology 17(4): 335–336.
[3] Chang TMS (1964) Semipermeable microcapsules. Science 146(364): 524–525.
[4] Orive G, Gascon AR, Hernandez RM, et al. (2003) Cell microencapsulation technology for biomedical purposes: Novel insights and challenges. Trends in Pharmaceutical
Sciences 24(5): 207–210.
[5] Zonervan GJ, Hoppen HJ, Pennings AJ, et al. (1992) Design of a poly urethane membrane for the encapsulation of islets langerhans. Biomaterials 13(3): 136–144.
[6] Sugiura S, Oda T, Aoyagi Y, et al. (2007) Microfabricated airflow nozzle for microencapsulation of living cells into 150 micrometer microcapsules. Biomedical
Microdevices 9(1): 91–99.
[7] Persico P, Carfagna C, Danicher L, and Frere Y (2005) Polyamide microcapsules containing jojoba oil prepared by inter-facial polymerization. Journal of
Microencapsulation 22(5): 471–486.
[8] Morris W, Steinhoff MC, and Russell PK (1994) Potential of polymer microencapsulation technology for vaccine innovation. Vaccine 12(1): 5–11.
[9] Isobe M, Yamazaki Y, Oida S, et al. (1996) Bone morphogenetic protein encapsulated with a biodegradable and biocompatible polymer. Journal of Biomedical Materials Research
32(3): 433–438.
[10] Tse M, Uludag H, Sefton MV, and Chang PL (1996) Secretion of recombinant proteins from hydroxyethyl methacrylate-methyl methacrylate capsules. Biotechnology and
Bioengineering 51(3): 271–280.
[11] Zhou Y, Sun T, Chan M, et al. (2005) Scalable encapsulation of hepatocytes by electrostatic spraying. Journal of Biotechnology 117(1): 99–109.
[12] Wang W, Liu XD, Xie YB, et al. (2006) Microencapsulation using natural polysaccharides for drug delivery and cell implantation. Journal of Materials Chemistry 16
(32): 3252–3267.
[13] Abalovich A, Jatimliansky C, Diegex E, et al. (2001) Pancreatic islets microencapsulation with polylactide-co-glycolide. Transplantation Proceeding 33(1–2): 1977–1979.
[14] Lee M, Lo AC, Cheung PT, et al. (2009) Drug carrier systems based on collagen-alginate composite structures for improving the performance of GDNF-secreting HEK293 cells.
Biomaterials 30(6): 1214–1221.
[15] Li XY, Chen XG, Cha DS, et al. (2009) Microencapsulation of a probiotic bacteria with alginate-gelatin and its properties. Journal of Microencapsulation 26(4): 315–324.
[16] Zhang XL, Wang W, Xie YB, et al. (2006) Proliferation, viability, and metabolism of human tumor and normal cells cultured in microcapsule. Applied Biochemistry and
Biotechnology 134(1): 61–76.
[17] Qi WT, Ma J, Yu, et al. (2006) Behavior of microbial growth and metabolism in alginate-chitosan-alginate (ACA) microcapsules. Enzyme and Microbial Technology 38(5): 697–704.
[18] Johnson FA, Craig DQM, and Mercer AD (1997) Characterization of the block structure and molecular weight of sodium alginates. Journal of Pharmacy and
Pharmacology 49(7): 639–643.
[19] Devos P, Wolters GHJ, Fritschy WM, and Vanschilfgaarde R (1993) Obstacles in the application of microencapsulation in islet transplantation. International Journal of Artificial
Organs 16(4): 205–212.
[20] Morris ER, Rees DA, Thom D, and Boy J (1978) Chiroptical and stoichiometric evidence of a specific, primary dimerization process in alginate gelation. Carbohydrate Research
66: 145–154.
[21] Thu B, Smidsrod O, and SkjakBraek G (1996) Alginate gels: Some structure-function correlations relevant to their use as immobilization matrix for cells. Immobilized Cells:
Basics and Applications 11: 19–30.
[22] Otterlei M, Ostgaard K, Skjakbraek G, et al. (1991) Induction of cytokine production from human monocytes stimulated with alginate. Journal of Immunotherapy 10(4): 286–291.
[23] Kulseng B, Skjak-Braek G, Ryan L, et al. (1999) Transplantation of alginate microcapsules: Generation of antibodies against alginates and encapsulated porcine islet-like cell
clusters. Transplantation 67(7): 978–984.
[24] Strand BL, Morch YA, Syvertsen KR, et al. (2003) Microcapsules made by enzymatically tailored alginate. Journal of Biomedical Materials Research: Part A. 64A(3): 540–550.
[25] Morch YA, Donati I, Strand BL, and Skjak-Braek G (2007) Molecular engineering as an approach to design new functional properties of alginate. Biomacromolecules
8(9): 2809–2814.
[26] King A, Strand B, Rokstad AM, et al. (2003) Improvement of the biocompatibility of alginate/poly- L-lysine/alginatemicrocapsules by the use of epimerized alginate as a coating.
Journal of Biomedical Materials Research: Part A 64(3): 533–539.
[27] Skjak-Braek G, Grasdalen H, and Smidsrod O (1989) Inhomogeneous polysaccharide ionic gels. Carbohhydrate Polymers 10(1): 31–54.
[28] Rowley JA and Mooney DJ (2002) Alginate type and RGD density control myoblast phenotype. Journal of Biomedical Materials Research 60(2): 217–223.
[29] Hill E, Boontheekul T, and Mooney DJ (2006) Regulating activation of transplanted cells controls tissue regeneration. Proceedings of National Academy Sciences of the United
States of America 103(8): 2494–2499.
[30] Hill E, Boontheekul T, and Mooney DJ (2006) Designing scaffolds to enhance transplanted myoblast survival and migration. Tissue Engineering 12(5): 1295–1304.
[31] Rowley JA and Mooney DJ (2002) Alginate type and RGD density control myoblast phenotype. Journal of Biomedical of Materials Research 60(2): 217–223.
[32] Bouhadir, KH, Lee KY, Alsberg E, et al. (2001) Degradation of partially oxidized alginate and its potential application for tissue engineering. Biotechnology Progress 17(5): 945–950.
[33] Li XX, Xu AH, Xie HG, et al. (2010) Preparation of low molecular weight alginate by hydrogen peroxide depolymerization for tissue engineering. Carbohydrate Polymers
79(3): 660–664.
[34] Chandy T and Sharma C (1990) Chitosan as a biomaterial. Biomaterials Artificial Cells and Artificial Organs 18(1): 1–24.
[35] Illum L, Jabbal-Gill I, Hinchcliffe M, et al. (2001) Chitosan as a novel nasal delivery system for vaccines. Advanced Drug Delivery Reviews 51(1–3): 81–96.
[36] Li J, Du YM, and Liang HB (2007) Influence of molecular parameters on the degradation of chitosan by a commercial enzyme. Polymer Degradation and Stability
92(3): 515–524.
[37] Mao JS, Cui YL, Wang XH, et al. (2004) A preliminary study on chitosan and gelatin polyelectrolyte complex cytocompatibility by cell cycle and apoptosis analysis. Biomaterials
25 (18): 3973–3981.
[38] Ren DW, Yi HF, Wang W, and Ma XJ (2005) The enzymatic degradation and swelling properties of chitosan matrices with different degrees of N-acetylation. Carbohydrate
Research 340(15): 2403–2410.
[39] Bartone F and Adickes E (1988) Chitosan: Effects on wound healing in urogenital tissue: Preliminary report. Journal of Urology 140(5): 1134–1137.
[40] Muzzarelli R (1994) In vivo biochemical significance of chitin-based medical items. In: Dumitriu S (ed.) Polymeric Biomaterials, pp. 179–197. New York, NY: Marcel Dekker.
[41] Bartkowiak A and Hunkeler D (1999) New microcapsules based on oligoelectrolyte complexation. Annals of the New York Academy of Sciences 875: 36–45.
[42] Illum L (1998) Chitosan and its use as a pharmaceutical excipient. Pharmaceutical Research 15(9): 1326–1331.
[43] Janes KA, Calvo P, and Alonso MJ (2001) Polysaccharide colloidal particles as delivery systems for macromolecules. Advanced Drug Delivery Reviews 47(1): 83–97.
[44] Hong Y, Mao ZW, Wang HL, et al. (2006) Covalently crosslinked chitosan hydrogel formed at neutral pH and body temperature. Journal of Biomedical Materials Research: Part A
79A(4): 913–922.
[45] Yeo Y, Geng WL, Ito T, et al. (2007) Photocrosslinkable hydrogel for myocyte cell culture and injection. Journal of Biomedical Materials Research: Part B 81B(2): 312–322.
[46] Jenkins CL and Raines RT (2002) Insights on the conformational stability of collagen. Natural Product Reports 19(1): 49–59.
[47] Rosenbloom J, Harsch M, and Jimenez S (1973) Hydroxyproline content determines the denaturation temperature of chick tendon collagen. Archives of Biochemistry and
Biophysics 158(2): 478–484.
112
Enabling Technologies
[48] Berg RA and Prockop DJ (1973) The thermal transition of a non-hydroxylated form of collagen. Evidence for a role for hydroxyproline in stabilizing the triple-helix of collagen.
Biochemical and Biophysical Research Communications 52(1): 115–120.
[49] Colette SR, Ajay K, Surendra PB, and Prabhas VM (2000) Control of hepatocyte function on collagen foams: Sizing matrix pores toward selective induction of 2-D and 3-D cellular
morphogenesis. Biomaterials 21: 783–793.
[50] Wong HL, Wang MX, Cheung PT, et al. (2007) A 3D collagen microsphere culture system for GDNF-secreting HEK293 cells with enhanced protein productivity. Biomaterials 28
(35): 5369–5380.
[51] Normand V, Lootens DL, Amici E, et al. (2000) New insight into agarose gel mechanical properties. Biomacromolecules 1(4): 730–738.
[52] Gin H, Dupuy B, Baquey C, et al. (1987) Agarose encapsulation of islets of Langerhans: Reduced toxicity in vitro. Journal of Microencapsulation 4(3): 239–242.
[53] Arnott SA, Fulmer A, and Scott WE (1974) The agarose double helix and its function in agarose gel structure. Journal of Molecular Biology 90(2): 269–284.
[54] Iwata H, Kobayashi K, Takagi T, et al. (1994) Feasibility of agarose microbeads with xenogeneic islets as a bioartificial pancreas. Journal of Biomedical Materials
Research 28(8): 1003–1011.
[55] Yang H, Iwata H, Shimizu H, et al. (1994) Comparative-studies of in vitro and in vivo function of 3 different shaped bioartificial pancreases mad of agarose hydrogel. Biomaterials
15(2): 113–120.
[56] Tun T, Inoue K, Hayashi H, et al. (1996) A newly developed three-layer agarose microcapsule for a promising biohybrid artificial pancreas: Rat to mouse xenotransplantation. Cell
Transplantation 5(5 Supplement 1): S59–S63.
[57] Karoubi G, Ormiston ML, Stewart DJ, and Courtman DW (2009) Single-cell hydrogel encapsulation for enhanced survival of human marrow stromal cells. Biomaterials
30(29): 5445–5455.
[58] Chienchi L and Kristi SA (2009) PEG Hydrogels for the controlled release of biomolecules in regenerative. Medicine Pharmaceutical Research 6(3): 631–643.
[59] Mahoney MJ and Anseth KS (2006) Three-dimensional growth and function of neural tissue in degradable polyethylene glycol hydrogels. Biomaterials 27(10): 2265–2274.
[60] Wilson JT and Chaikof EL (2008) Challenges and emerging technologies in the immunoisolation of cells and tissues. Advanced Drug Delivery Reviews 60(2): 124–145.
[61] Modo M, Rezaie P, Heuschling P, et al. (2002) Transplantation of neural stem cells in a rat model of stroke: Assessment of short-term graft survival and acute host immunological
response. Brain Research 958(1): 70–82.
[62] Miller K, Chinzei K, Orssengo G, and Bednarz P (2000) Mechanical properties of brain tissue in vivo: Experiment and computer simulation. Journal of Biomechanics
33(11): 1369–1376.
[63] Sawhney AS, Chandrashekhar PP, and Hubbell JA (1993) Bioerodible hydrogels based on photopolymerized poly(ethylene glycol)-co-poly(alphahydroxyacid) diacrylate
monomers. Macromolecules 26(4): 581–594.
[64] Sawhney AS, Pathak CP, and Hubbell JA (1993) Interficial photopolymerization of poly(ethylene glycol)-based hydrogels upon alginate poly(L-lysine) microcapsules for enhanced
biocompatibility. Biomaterials 14(13): 1008–1016.
[65] Chandy T, Mooradian DL, and Rao GHR (1999) Evaluation of modified alginate–chitosan –polyethylene glycol microcapsules for cell encapsulation. Artificial Organ 23(10): 894–903.
[66] Dionne BC, Li RH, Bell W, et al. (1996) Transport characterization of membranes for immunoisolation. Biomaterials 17(3): 257–266.
[67] Christenson L, Dionne K, and Lysaught M (1993) Biomedical Application of Immobilized Cells: Fundamentals of Animal Cell Encapsulation, pp. 7–41. Boca Raton, FL:
CRC Press.
[68] Wang T, Lacik I, Brissova M, et al. (1997) An encapsulation system for the immunoisolation of pancreatic islets. Nature Biotechnology 15(4): 358–362.
[69] de Vos P and Marchetti P (2002) Encapsulation of pancreatic islets for transplantation in diabetes: The untouchable islets. Trends in Molecular Medicine 8(8): 363–366.
[70] Wilson JT and Chaik EL (2008) Challenges and emerging technologies in the immunoisolation of cells and tissues. Advanced Drug Delivery Reviews 60(2): 124–140.
[71] Avgoustiniatos ES and Colton CK (1997) Effect of external oxygen mass transfer resistances on viability of immunoisolated tissue. Annals of New York Academy of
Sciences 831: 145–167.
[72] Dulong JL and Legallais C (2007) A theoretical study of oxygen transfer including cell necrosis for the design of a bioartificial pancreas. Biotechnology and
Bioengineering 96: 990–998.
[73] Gross JD, Constantinidis I, and Sambanis A (2007) Modeling of encapsulated cell systems. Journal of Theoretical Biology 24(1): 4500–4510.
[74] Dionne KE, Colton CK, and Yarmush ML (1993) Effect of hypoxia on insulin secretion by isolated rat and canine islets of Langerhans. Diabetes 42: 12–21.
[75] Linn T, Schmitz J, Hauck-Schmalenberger I, et al. (2006) Ischaemia is linked to inflammation and induction of angiogenesis in pancreatic islets. Clinical and Experimental
Immunology 144: 179–187.
[76] Colton CK (1996) Engineering challenges in cell encapsulation technology. Trends in Biotechnology 14: 158–167.
[77] Johnson AS, Fisher RJ, Weir GC, and Colton CK (2009) Oxygen consumption and diffusion in assemblages of respiring spheres: Performance enhancement of a bioartificial
pancreas. Chemical Engineering Science 64: 4470–4487.
[78] Lee MK and Bae YH (2000) Cell transplantation for endocrine disorders. Advanced Drug Delivery Reviews 42: 103–120.
[79] Tziampazis E and Sambanis A (1995) Tissue engineering of a bioartificial pancreas: Modeling the cell environment and device function. Biotechnology Progress 11: 115–126.
[80] de Haan BJ, Faas MM, and de Vos P (2003) Factors influencing insulin secretion from encapsulated islets. Cell Transplantation 12: 617–625.
[81] Canaple L, Rehor A, and Hunkeler D (2002) Improving cell encapsulation through size control. Journal of Biomaterials Science, Polymer Edition 13: 783–796.
[82] Ying Zhang, Wei Wang, Yubing Xie, et al. (2007) In vivo culture of encapsulated endostatin-secreting Chinese hamster ovary cells for systemic tumor inhibition. Human Gene
Therapy 18: 474–481.
[83] Soon-Shiong P (1999) Treatment of type I diabetes using encapsulated islets. Advanced Drug Delivery Reviews 35: 259.
[84] Lee CS and Chu IM (1997) Characterization of modified alginate–poly-L-lysine microcapsules. Artificial Organs 21: 1002–1006.
[85] Hsu YL and Chu IM (1992) Poly(ethyleneimine)-reinforced liquid-core capsules for the cultivation of hybridoma cells. Biotechnology and Bioengineering 40: 1300–1308.
[86] Matthew HW, Salley SO, Peterson WD, and Klein MD (1993) Complex coacervate microcapsules for mammalian cell culture and artificial organ development. Biotechnology
Progress 9: 510–519.
[87] Lacik I, Brissova M, Anilkumar AV, et al. (1998) New capsule with tailored properties for the encapsulation of living cells. Journal of Biomedical Materials Research
39: 52–60.
[88] Peirone M, Ross CJD, Hortelano G, et al. (1998) Encapsulation of various recombinant mammalian cell types in different alginate microcapsules. Journal of Biomedical Materials
Research 42: 587–596.
[89] Wang FF, Wu CR, and Wang YJ (1992) Preparation and application of poly(vinylamine)/alginate microcapsules to culturing of a mouse erythroleukemia cell line. Biotechnology
and Bioengineering 40: 1115–1118.
[90] Kung IM, Wang FF, Chang YC, and Wang YJ (1994) Surface modification of alginate/poly(L-lysine) microcapsular membranes with poly(ethylene glycol) and poly(vinyl alcohol).
Biomaterials 16: 649–655.
[91] Cui YX, Shakesheff KM, and Adams, G (2006) Encapsulation of RIN-m5F cells within Ba2+ cross-linked alginate beads affects proliferation and insulin secretion. Journal of
Microencapsulation 23(6): 663–676.
[92] Thu B, Bruheim P, Espevik T, et al. (1996) Alginate polycation microcapsules. II. Some functional properties. Biomaterials 17(11): 1069–1079.
[93] Lanza RP, Jackson R, Sullivan A, et al. (1999) Xenotransplantation of cells using biodegradable microcapsules. Transplantation 67: 1105–1111.
[94] Darrabie MD, Kendall WF, and Opara EC (2005) Characteristics of poly-L-ornithine-coated alginate microcapsules. Biomaterials 26 (34): 6846–6852.
[95] Wang T, Lacik I, Brissova M, et al. (1997) An encapsulation system for the immunoisolation of pancreatic islets. Nature Biotechnology 15: 358–362.
[96] Sakai S, Ono T, Ijima H, and Kawakami (2001) Synthesis and transport characterization of alginate/aminopropyl-silicate/alginate microcapsule: Application to bioartificial
pancreas. Biomaterials 22: 2827–2834.
The Artificial Organ: Cell Encapsulation
113
[97] Coradin T, Nassif N, and Livage J (2003) Silica-alginate composites for microencapsulation. Applied Microbiology and Biotechnology 61: 429–434.
[98] Rihova B (1996) Biocompatibility of biomaterials: Hemocompatibility, immunocompatibility and biocompatibility of solid polymeric materials and soluble targetable polymeric
carriers. Advanced Drug Delivery Reviews 21: 157–176.
[99] Visted T, Bjerkvig R, and Enger PO (2001) Cell encapsulation technology as a therapeutic strategy for CNS malignancies. Neuro-oncology 3(3): 201–210.
[100] Rebecca HL (1998) Materials for immunoisolated cell transplantation. Advanced Drug Delivery Reviews 33: 87–109.
[101] Bunger CM, Tiefenbach B, Jahnke A, et al. (2005) Deletion of the tissue response against alginate-PLL capsules by temporary release of co-encapsulated steroids. Biomaterials
26: 2353–2360.
[102] De Vos P, De Haan BJ, Wolters GH, et al. (1997) Improved biocompatibility but limited graft survival after purification of alginate for microencapsulation of pancreatic islets.
Diabetologia 40: 262–270.
[103] Hortelano G, Al-Hendy A, Ofosu FA, and Chang PL (1996) Delivery of human factor IX in mice by encapsulated recombinant myoblasts: A novel approach towards allogeneic gene
therapy of hemophilia B. Blood 87: 5095–5103.
[104] Siebers U, Horcher A, Brandhorst H, et al. (1999) Analysis of the cellular reaction towards microencapsulated xenogeneic islets after intraperitoneal transplantation. Journal of
Molecular Medicine 77: 215–218.
[105] Anderson JM (1988) Inflammatory response to implants. ASAIO Transplantation 34: 101–107.
[106] Orive G, Tam SK, Pedraz JL, and Halle JP (2006) Biocompatibility of alginate-poly-L-lysine microcapsules for cell therapy. Biomaterials 27: 3691–3700.
[107] Orive G, Carcaboso AM, Hernandez RM, et al. (2005) Biocompatibility evaluation of different alginates and alginate-based microcapsules. Biomacromolecules 6: 927–931.
[108] Lin YH, Liang HF, Chung CK, et al. (2005) Physically crosslinked alginate/N,O-carboxymethyl chitosan hydrogels with calcium for oral delivery of protein drugs.
Biomaterials 26: 2105–2113.
[109] Vandenberg GW, Drolet C, Scott SL, and de la Noüe J (2001) Factors affecting protein release from alginate–chitosan coacervate microcapsules during production and gastric/
intestinal simulation. Journal of Controlled Release 77: 297–307.
[110] Zimmermann U, Klock G, Federlin K, et al. (1992) Production of mitogen-contamination free alginates with variable ratios of mannuronic acid to guluronic acid by free flow
electrophoresis. Electrophoresis 13: 269–274.
[111] Sakai S, Mu CJ, Kawabata K, et al. (2006) Biocompatibility of subsieve-size capsules versus conventional-size microcapsules. Journal of Biomedical Materials Research: Part A
78A(2): 394–398.
[112] Hasse C, Zielke A, Klöck G, et al. (1998) Amitogenic alginates: Key to first clinical application of microencapsulation technology. World Journal of Surgery 22: 659–665.
[113] van Schilfgaarde R and de Vos P (1999) Factors influencing the properties and performance of microcapsules for immunoprotection of pancreatic islets. Journal of Molecular
Medicine 77: 199–205.
[114] Weber CJ, Hagler M, Konieczny B, et al. (1995) Encapsulated islet iso-, allo-, and xenografts in diabetic NOD mice. Transplantation Proceedings 27: 3308–3311.
[115] Dufrane D, van Steenberghe M, Goebbels RM, et al. (2006) The influence of implantation site on the biocompatibility and survival of alginate encapsulated pig islets in rats.
Biomaterials 27: 3201–3208.
[116] Read TA, Stensvaag V, Vindenes H, et al. (1999) Cells encapsulated in alginate: A potential system for delivery of recombinant proteins to malignant brain tumours. International
Journal of Developmental Neuroscience 17: 653–663.
[117] Lim F and Sun AM (1980) Microencapsulated islets as bioartificial endocrine pancreas. Science 210: 908–910.
[118] Cieslinski, DA and Humes HD (1994) Tissue engineering of a bioartificial kidney. Biotechnology and Bioengineering 43: 678–681.
[119] Koo J and Chang TMS (1993) Secretion of erythropoietin from microencapsulated rat kidney cells. International Journal of Artificial Organs 16: 557–560.
[120] Chang PL, Shen N, and Westcott AJ (1993) Delivery of recombinant gene products with microencapsulated cells in vivo. Human Gene Therapy 4: 433–440.
[121] Colton CK (1995) Implantable biohybrid artificial organs. Cell Transplantation 4: 415–436.
[122] Colton CK (1996) Engineering challenges in cell encapsulation technology. Trends in Biotechnology 14: 158–167.
[123] Murua A, Portero A, Orive G, et al. (2008) Cell microencapsulation technology: Towards clinical application. Journal of Controlled Release 132: 76–83.
[124] de Vos P, de Haan B, and Van Schilfgaarde R (1997) Effect of the alginate composition on the biocompatibility of alginate-polylysine microcapsules. Biomaterials 18: 273–278.
[125] Dufrane D, Goebbels RM, Saliez A, et al. (2006) Six-month survival of microencapsulated pig islets and alginate biocompatibility in primates: Proof of concept. Transplantation
81: 1345–1353.
[126] Black SP, Constantinidis I, Cui H, et al. (2006) Immune responses to an encapsulated allogeneic islet β-cell line in diabetic NOD mice. Biochemical and Biophysical Research
Communications 340: 236–243.
[127] Calafiore R, Basta G, Luca G, et al. (2006) Standard technical procedures for microencapsulation of human islets for graft into nonimmunosuppressed patients with Type 1
diabetes mellitus. Transplantation Proceedings 38: 1156–1157.
[128] Calafiore R, Basta G, Luca G, et al. (2006) Microencapsulated pancreatic islet allografts into nonimmunosuppressed patients with type 1 diabetes: First two cases.
Diabetes Care 29: 137–138.
[129] Mallett AG and Korbutt GS (2009) Alginate modification improves long-term survival and function of transplanted encapsulated islets. Tissue Engineering Part A
15(6): 1301–1309.
[130] Rubinsztein DC and Carmichael J (2003) Huntington’s disease: Molecular basis of neurodegeneration. Expert Reviews in Molecular Medicine 5(20): 1–21.
[131] Emerich DF, Thanos CG, Goddard M, et al. (2006) Extensive neuroprotection by choroid plexus transplants in excitotoxin lesioned monkeys. Neurobiology of Disease 23:
471–480.
[132] Jeon Y, Kwak K, Kim S, et al. (2006) Intrathecal implants of microencapsulated xenogenic chromaffin cells provide a long-term source of analgesic substances. Transplantation
Proceedings 38: 3061–3065.
[133] Buchser E, Goddard M, Heyd B, et al. (1996) Immunoisolated xenogeneic chromaffin cell therapy for chronic pain – Initial clinical experience. Anesthesiology 85:
1005–1012.
[134] Kim YM, Jeon YH, Jin GC, et al. (2005) In vivo biocompatibility of alginate-PLL microcapsules with chromaffin cells for the alleviation of chronic pain. Key Engineering Materials
277: 62–66.
[135] Wang W, Liu XD, Xie YB, et al. (2006) Microencapsulation using natural polysaccharides for drug delivery and cell implantation. Journal of Materials Chemistry 16: 3252–3267.
[136] Jankovic J (2008) Parkinson’s disease: Clinical features and diagnosis. Journal of Neurology Neurosurgery and Psychiatry 79(4): 368–376.
[137] Aebischer P, Goddard M, Signore A, and Timpson R (1994) Functional recovery in hemiparkinsonian primates transplanted with polymer-encapsulated PC12 cells. Experimental
Neurology 126: 151–158.
[138] Xue YL, Gao JM, Xi ZF, et al. (2001) Microencapsulated bovine chromaffin cell xenografts into hemiparkinsonian rats: A drug-induced rotational behavior and histological
changes analysis. Artificial Organs 25(2): 131–135.
[139] Mei J, Sgroi A, Mai G, et al. (2009) Improved survival of fulminant liver failure by transplantation of microencapsulated cryopreserved porcine hepatocytes in mice. Cell
Transplantation 18(1): 101–110.
[140] Aoki K, Hakamada K, Seino K, et al. (2000) Development of a bioartificial liver using microencapsulated xenohepatocytes. New Directions for Cellular and Organ Transplantation
1210: 55–59.
[141] Hamazaki K, Doi Y, and Koide N (2002) Microencapsulated multicellular spheroid of rat hepatocytes transplanted intraperitoneally after 90% hepatectomy.
Hepato-Gastroenterology 48: 1514–1516.
[142] Ying Zhang, Wei Wang, Jing Zhou, et al. (2007) Tumor anti-angiogenic gene therapy with microencapsulated recombinant CHO cells. Annals of Biomedical Engineering
35(4): 605–614.
114
Enabling Technologies
[143] Koo J and Chang TMS (1993) Secretion of erythropoietin from microencapsulated rat kidney cells: Preliminary results. International Journal of Artificial Organs 16: 557–560.
[144] Al-Hendy A, Hortelano G, Tannenbaum GS, and Chang PL (1995) Allogeneic somatic gene therapy: Correction of growth hormone deficiency in dwarf mice with
microencapsulated non-autologous myoblasts. Human Gene Therapy 6: 165–175.
[145] Chang PL, Shen N, and Westcott AJ (1993) Delivery of recombinant gene products with microencapsulated cells in vivo. Human Gene Therapy 4: 433–440.
[146] Van Raamsdonk JM, Ross CJD, Potter MA, et al. (2002) Treatment of hemophilia B in mice with nonautologous somatic gene therapeutics. Journal of Laboratory and Clinical
Medicine 139: 35–42.
[147] Cieslinski DA and Humes HD (1994) Tissue engineering of a bioartificial kidney. Biotechnology and Bioengineering 43: 678–681.
[148] Aoki T, Jin ZH, Nishino N, et al. (2005) Intraspenic transplantation of encapsulated hepatocytes decreases mortality and improves liver functions in fulminant hepatic failure from
90% partial hepatectomy in rats. Transplantation 79: 783–790.
[149] Aebischer P, Russell PC, Christenson L, et al. (1986) A bioartificial parathyroid. ASAIO Transactions/American Society for Artificial Internal Organs 32: 134–137.
[150] Barsoum SC, Milgram W, Mackay W, et al. (2003) Delivery of recombinant gene product to canine brain with the use of microencapsulation. Journal of Laboratory and Clinical
Medicine 142: 399–413.
[151] Visted T, Bjerkvig R, and Enger PE (2001) Cell encapsulation technology as a therapeutic strategy for CNS malignancies. Neuro-oncology 3: 201–210.
[152] Sieving PA, Caruso RC, Tao W, et al. (2006) Ciliary neurotrophic factor (CNTF) for human retinal degeneration: Phase I trial of CNTF delivered by encapsulated cell intraocular
implants. Proceedings of the National Academy Sciences of the United States of America 103: 3896–3901.
[153] Zhang H, Zhu SJ, Wang W, et al. (2008) Transplantation of microencapsulated genetically modified xenogeneic cells augments angiogenesis and improves heart function. Gene
Therapy 15: 40–48.
[154] Nakama H, Ohsugi K, Otsuki T, et al. (2006) Encapsulation cell therapy for mucopolysaccharidosis type VII using genetically engineered immortalized human amniotic epithelial
cells. Tohoku Journal of Experimental Medicine 209: 23–32.
[155] Murua A, de Castro M, Orive G, et al. (2007) In vitro characterization and in vivo functionality of erythropoietin-secreting cells immobilized in alginate-poly-L-lysine-alginate
microcapsules. Biomacromolecules 8: 3302–3307.
[156] Ponce S, Orive G, Hernandez RM, et al. (2006) In vivo evaluation of EPO-secreting cells immobilized in different alginate-PLL microcapsules. Journal of Controlled
Release 116: 28–34.
[157] Wen JP, Xu N, Li NA, et al. (2007) Encapsulated human primary myoblasts deliver functional hFIX in hemophilic mice. Journal Gene Medicine 9(11): 1002–1010.
[158] Chang PL, Shen N, and Westcott AJ (1993) Delivery of recombinant gene products with microencapsulated ells in vivo. Human Gene Therapy 4: 433–440.
[159] Moran DM, Koniaris LG, Jablonski EM, et al. (2006) Microencapsulation of engineered cells to deliver sustained high circulating levels of interleukin-6 to study hepatocellular
carcinoma progression. Cell Transplantation 15: 785–798.
[160] Shi MQ, Hao SG, Quereshi M, et al. (2005) Significant tumor regression induced by microencapsulation of recombinant tumor cells secreting fusion protein. Cancer Biotherapy
and Radiopharmaceuticals 20(3): 260–266.
[161] Read TA, Sorensen DR, Mahesparan R, et al. (2001) Local endostatin treatment of gliomas by gene therapy with microencapsulated recombinant CHO cells. Nature Biotechnology
19: 29–34.
[162] Joki T, Machluf M, Atala A, et al. (2001) Continuous release of endostatin from microencapsulated engineered cells for tumor therapy. Nature Biotechnology 19: 35–39.
[163] Cirone P, Bourgeois JM, Shen F, and Chang PL (2004) Combined immunotherapy and antiangiogenic therapy of cancer with microencapsulated cells. Human Gene Therapy 15
(10): 945–959.
[164] Batorsky A, Liao JH, Lund AW, et al. Encapsulation of adult human mesenchymal stem cells within collagen-agarose microenvironments. Biotechnology and Bioengineering
92(4): 492–500.
[165] Liu ZC and Chang TMS (2009) Preliminary study on intrasplenic implantation of artificial cell bioencapsulated stem cells to increase the survival of 90% hepatectomized rats.
Artificial Cells Blood Substitutes and Biotechnology 37(1): 53–55.
[166] Bienaime C, Barbotin JN, and Nava-Saucedo JE (2003) How to build an adapted and bioactive cell microenvironment? A chemical interaction study of the structure of Ca-alginate
matrices and their repercussion on confined cells. Journal of Biomedical Materials Research: Part A 67A: 376–388.
[167] Fuchs E, Tumbar T, and Guasch G (2004) Socializing with the neighbors: Stem cells and their niche. Cell 116: 769–778.
[168] Magyar JP, Nemir M, Ehler E, et al. (2001) Mass Production of embryoid bodies in microbeads. Annals of the New York Academy of Sciences 944: 135–143.
[169] Maguire T, Davidovich AE, Wallenstein EJ, et al. (2007) Control of hepatic differentiation via cellular aggregation in an alginate microenvironment. Biotechnology and
Bioengineering 98(3): 631–644.
[170] Fang S, Qiu YD, Mao L, et al. (2007) Differentiation of embryoid-body cells derived from embryonic stem cells into hepatocytes in alginate microbeads in vitro. Acta
Pharmaceutica Sinica 28(2): 1924–1930.
[171] Liu ZC and Chang TMS (2003) Coencapsulation of hepatocytes and bone marrow stem cells: In vitro conversion of ammonia and in vivo lowering of bilirubin in hyperbilirubemia
Gunn rats. International Journal of Artificial Organs 26(6): 491–497.