Table Of ContentBIOMATERIALS FOR CELL IMMOBILIZATION
A look at carrier design
KATHRYN W. RIDDLE AND DAVID J. MOONEY
University of Michigan, Chemical Engineering, 2300 Hayward, 3074
H.H. Dow Ann Arbor, MI48105 - Fax: 734-763-0459
Email: [email protected]
1. Introdnction
There are several major biomedical applications where the transplantation of
immobilized cells is being employed to restore, maintain or improve tissue function.
These strategies can be split into two main categories: the replacement of biochemical
function only or the replacement of structurally functional tissue. As only chemical
communication (e.g., diffusion of molecules) is required in the former, it is possible to
deliver cells encapsulated in a nanoporous, immunoisolatory polymer membrane. The
membranes is constructed such that there are pores large enough to allow for nutrients,
waste and the bioactive factor to diffuse but not large enough as to allow immune cells
to attack the cells within [1]. This strategy has mainly been employed to temporarily or
permanently replace biochemical functions of the liver [2,3], pancreas [4,5], and
provide local protein delivery in neurological disorders [6]. The second major strategy
involves entrapping cells on a micro or macroporous polymer scaffold and promoting
the formation of a new tissue that is structurally and functionally integrated with the
surrounding tissue. The scaffold is constructed with a biocompatible material that
degrades over time to leave only the integrated tissue in its place. Researchers have
attempted to use this strategy with a variety of tissues, including skin [7,8,9], liver
[10,11,12], pancreas [13], cornea [14], blood vessels [15,16], cartilage [17,18], heart
[19], and bone [20,21].
The biomaterial component of these therapies must provide the appropriate mass
transport properties, membrane or scaffold stability and desirable cellular interactions
depending on the location and desired function of the implant. In the case of nanoporous
immunoisolated strategies, the main goal of the biomaterial is to provide a barrier to the
host defences while allowing the essential molecules to diffuse through (Figure 1). In
cases where cells are seeded onto a micro or macroporous biomaterial and then
implanted, the main function of the biomaterial is to provide cells with a synthetic
15
V. Nedovic and R. Willaert (eds.), Fundamentals ofC ell Immobilisation Biotechnology, 15-32.
© 2004 Kluwer Academic Publishers.
Kathryn W. Riddle and David J. Mooney
extracellular matrix (ECM) that directs multiple cell functions, including cell
proliferation, migration and gene expression (Figure 2).
This chapter will provide an overview of carrier design of biomaterials for cell
immobilization. For the purposes of this chapter, only applications involving
mammalian cells will be discussed. A brief overview of cell sourcing is followed by a
section on applications of nano-, micro- and macroporous materials, and the common
materials used for these applications are described in the last section.
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Figure 1. Cartoon ofa nanoporous immunoisolated device.
Polymer
Figure 2. Cartoon ofa macroporous scaffold seeded with cells.
2. Cell sourcing
There are three main sources of cells for cell immobilization biotechnology: autologous
(obtained from the same individual for whom the cells are intended), allogeneic
(obtained from an individual of the same species for whom the cells are intended), and
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Biomaterials for cell immobilization
xenogeneic (obtained from a species different from which the cells are intended). The
appropriate source of cells for a specific application depends on many factors, including
functional requirements, availability, ease of collection, processing and storage, and
economic factors.
Autologous cells potentially offer the most immunocompatible source of cells, and
for this reason are frequently used for reconstruction of structural tissues [22]. However,
the time required and costs incurred to generate sufficient numbers of these cells from a
tissue biopsy is often a deterrent to their application. Specifically, the cells must
typically be significantly expanded within a short (e.g., days to weeks) time frame
before returning to the patient [23]. In addition, the scarcity of healthy tissue from
which to harvest the cells or an inability to expand specific cell types in culture, as with
hepatocytes or ~-islets, can make other cell sourcing options necessary.
An attractive alternative to autologous cells in many situations is to instead use
allogeneic cells. These cells can potentially be expanded in large batches for treating
many patients and stored before they are needed [24]. Each batch can be screened for
safety and function, decreasing the cost associated with patient-by-patient screening of
autologous cells. In addition, genetically modified immortalized cells or cell lines can
be extensively studied and characterized for use in cell immobilization strategies [25].
Standardized allogeneic cell sources may also allow the construction of complex tissues
that could otherwise be prohibitively expensive in time, money, and effort if they were
to be produced by custom manufacture from autologous sources [24]. However, the use
of allogeneic cell therapy may require some form of immunoprotection to prevent
rejection by the host. For this reason, allogeneic cells have mainly been used in
applications where they will eventually be replaced by native cells (e.g., skin
replacement), or in immunoisolatory devices that are intended to provide a solely
biochemical function. In addition, the limited availability of certain cell types and/or an
inability to expand in culture, limits this cell source in certain applications (e.g., treating
diabetes with ~-islets).
Xenogeneic cells are highly attractive for replacement of biochemical functions in
certain situations, as they are available in unlimited supply. Two of the most widely
researched applications for xenogeneic cell transplantation are the replacement of liver
and pancreatic function [3,4,26,27]. Autologous and allogeneic cells from these organs
are scarce and difficult to expand in vitro, leading to great difficulty in identifying good
human sources for these strategies [28]. A main challenge in the application of
xenogeneic cells is rejection by the host immune system. To overcome these difficulties,
these cells must be encapsulated within nanoporous membranes that protect them from
rejection. Xenogeneic cells tend to elicit a greater immune response from the host than
allogeneic cells in these applications, possibly due to the release of antigenic products
from deteriorated and dying cells within the device in both cases [29]. Immunoisolated
xenogeneic cells may be useful for both implantation in the body, and as a component
of extracorporeal tissue support systems [30].
3. Material applications
Applications for nano, micro and macroporous materials are widely varied from
temporary replacement of biochemical function to formation of structurally functional
17
Kathryn W. Riddle and David J. Mooney
tissues that are completely integrated with the surrounding host tissue. Nanoporous
materials are utilized when it is desired to replace a biochemical function of a tissue by
entrapping viable cells within a semi-permeable membrane that prevents cells from
being recognized and therefore destroyed by the host immune system [31]. This
characteristic is especially useful when allogeneic or xenogeneic cells are used in this
strategy. Microporous and macroporous scaffolds provide a synthetic extracellular
matrix (ECM) for attachment and proliferation of anchorage dependant cells. These
types of scaffolds can be formed using natural or synthetic biomaterials, and have been
investigated for the replacement and repair of many tissue types. In all cell
encapsulation strategies, it is generally accepted that an appropriate biocompatible
material must be isolated or synthesized, and manufactured into the desired shape and
dimensions [32].
3.1. NANOPOROUS
Potential applications of nanoencapsulation technology include replacement of the
biochemical functions of major organs and transplantation of engineered cells for gene
therapy [29]. In many of these situations, the scarcity of healthy autologous tissue from
which to harvest cells requires the use of xenogeneic and allogeneic cells requiring
immunoisolation. Pancreatic function may potentially be replaced with transplanted
porcine islets of Langerhans [5,26] and have been widely investigated as a treatment for
diabetes. Similarly, porcine hepatocytes [2,27] have been investigated for temporary
replacement of liver function. Other disorders that have been addressed by this method
include chronic pain [33,34], hypocalcaemia [35], dwarfism [36], anaemia [37,38], and
haemophilia [39,40].
A major limitation to this form of therapy is the limited number of cells that can be
delivered, and thus a limited dose of the therapeutic factor secreted by the cells. In some
cases, the sheer number of cells needed to replace the function of the damaged organ
makes this form of therapy impractical. For example, it has been estimated that
approximately 7x109 hepatocytes are required to adequately replace the function of the
liver [41]. Considering that the average hepatocyte is approximately 25 microns in
diameter [42] and that the average hollow fibre nanoporous device has a diameter of
approximately three millimetres [43], it would take an implant 8 meters in length to
accommodate the number of cells necessary to adequately replace liver function. In
many of these cases implantation of the device is impractical and it is preferable to
pursue extracorporeal strategies reviewed elsewhere [41].
3.1.1. Design considerations
Key considerations that must be addressed for this approach to be successful include the
required mass transport properties, membrane stability, and cellular distribution with the
encapsulation material.
3.1.1.1. Mass transport. Survival of the entrapped cells as well as efficacy of the
therapeutic agent will be greatly affected by the mass transport properties of the
encapsulation membrane. Ideally, the pore size in the membrane should be carefully
controlled to allow bi-directional diffusion of the bioactive factor and molecules
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Biomaterials for cell immobilization
essential for cell survival (e.g., oxygen and glucose), while staying impervious to larger
molecules (host complement and antibodies) and immunogeneic cells. One of the major
hurdles of this technology is the fact that it is difficult to make a polymer membrane
with uniform pore sizes [44]. A distribution of pore sizes leads to a gradient of
diffusional resistances to molecules of varying molecular weight, instead of an absolute
cut-off. Another limitation to transport can be the fibrous capsule that often forms
around the implant due to the foreign body response. For example, a fibrous capsule
thickness of 160 microns is sufficient to cause hypoxia for transplanted islets of
Langerhans [45]. Due to this issue, materials that don't elicit a significant foreign body
response are required for these applications. Other factors that influence the mass
transport properties of a nanoporous membrane are the size and shape of the device, as
the surface area:volume ratio is a key parameter for diffusional transport [39]. Since the
metabolic requirements of each cell type are different, optimal membrane permeability
will depend on choice of cells [46]. A variety of structures including, intravascular
devices, spherical microcapsules, cylindrical hollow-fibre macrocapsules, flat sheets
[47] and planar macrocapsules [48] have been utilized. The spherical shape provides the
most surface area to volume ratio, which provides for efficient transfer of nutrients,
bioactive factors and waste. However, micro spheres can be difficult to retrieve at a later
time due to their small size. If retrieval of the device is desired or large numbers of cells
are required, cylindrical and plate designs have been employed, in spite of their inferior
mass transport properties [31].
3.1.1.2. Membrane stability. The need to periodically replenish encapsulated cells, due
to limitations on cell longevity, leads to specific requirements for membrane
biostability. Following cell death, the implant may be retrieved, or the implant may be
left in place and allowed to degrade over time. When short term drug delivery or
temporary replacement of biochemical function is desired, it may be desirable for the
membrane to slowly erode and allow the body's defences to destroy the implant. For
example, a microcapsule system has been designed to degrade and allow clearance of
the encapsulated cells following their death or loss of function [49]. These hydrogel
based microcapsules, made from alginate and poly (L-lactide), can be made to degrade
over several weeks or months and can eliminate the need for surgery to remove old
capsules. If retrieval of the implant is necessary or the implant is to be replenished in
vivo, a non-biodegradable membrane would be preferable. Having a non-degradable
membrane is also particularly useful in applications where genetically engineered cells
and cell lines are encapsulated, as the breakdown of the membrane could lead to escape
ofthe cells and potential tumour development [50].
3.1.1.3. Cellular distribution. Nanoporous materials must be designed to optimally
organize the encapsulated cells, as a common observation is the appearance of dead or
necrotic cells within biomaterial membranes (e.g. hollow fibre devices) [46]. Cells
loaded as dilute suspensions in aqueous growth media into these materials presumably
settle to create high-density aggregates that hinder diffusional transport of essential
nutrients. To overcome this problem, a variety of cell immobilization matrices,
including collagen, chitosan and alginate have been examined to more uniformly
distribute the cells within the membrane [51].
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Kathryn W. Riddle and David J. Mooney
3.2. MICROPOROUS AND MACROPOROUS
Potential applications for micro and macroporous cell immobilization include the
replacement of tissues with structural and mechanical functions, including heart, blood
vessels, bone and cartilage [52,53,54,55]. This contrasts with nanoporous cell
encapsulation technologies, which are typically utilized to replace only biochemical
functions of tissues. Micro and macroporous matrices, or scaffolds, can be fabricated
from polymers or other materials, and act as a temporary ECM for the forming tissue.
Example polymers used in these applications include both natural materials (e.g.,
hyaluronate), and synthetic polymers such as the polyanhydrides and polylactides.
These scaffolds mechanically support the formation of tissue, and serve to either
transplant cells and/or allow for cells from the surrounding tissue to migrate into this
space.
3.2.1. Design considerations
Ideally, a micro or macroporous cell immobilization scaffold should have the following
characteristics:
• Adequate porosity and pore sizes to control mass transport properties
• Appropriate mechanical properties for the desired application
• Controllable degradation and resorption to match tissue replacement.
• Suitable cell-interactions to allow for appropriate tissue development.
3.2.1.1. Mass transport properties. The pore size in micro or macroporous scaffolds
modulates the ability of cells and biological molecules to pass into and out of the
scaffold. In general, very small pores (e.g., d < II-I.m), while allowing free diffusion of
molecules, will not allow cellular migration, while pores in the 10 - 100's of microns
readily allow transplanted or host cells to migrate through the scaffold volume. In
addition, the rate of tissue ingrowth is dependent on the pore size and overall porosity of
the scaffold [56]. Other characteristics that can control transport properties with an
implanted scaffold include surface area and volume. A large surface area allows
significant cell attachment and growth on the material, a high porosity allows significant
host tissue infiltration. In general, it is believed that a porosity of greater than 90% will
provide an appropriate surface area for cell-polymer interactions, sufficient space for
ECM regeneration, and minimal diffusion constraints [57].
3.2.1.2. Mechanical design issues. Whereas nanoporous cell encapsulation aims at only
replacing a biochemical function, micro and macroporous scaffolds often provide
temporary structural support during tissue repair, and therefore require significant
mechanical stability. In soft tissue and non-load bearing applications, the mechanical
properties (e.g. elastic modulus) of the scaffold are often not required to be extremely
high, and a variety of natural polymers (e.g., collagen) and synthetic materials (e.g.,
poly(lactide-co-glycolide)) have been employed. In contrast, in load bearing
applications high strength synthetic materials or reinforced natural polymers (e.g.
chitosanlcalcium phosphate composites) are likely required.
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Biomaterials for cell immobilization
Mechanical stimuli conveyed to cells via the scaffold can also regulate the development
of many tissues. For example, under cyclical mechanical strain of the scaffold, smooth
muscle cells (SMCs) show increased proliferation, and elastin and collagen synthesis
[58]. This phenotypic control over SMCs has also been demonstrated using collagen
gels [59,60] and polyglycolide scaffolds [61]. Similarly, the development of engineered
cartilage has also been found to depend on the mechanical environment [62]. These
results indicate scaffolds must be designed to appropriately convey mechanical signals
to interacting cells.
3.2.1.3. Scaffold degradation. For each application, the desired rate and method of
biodegradation will playa large role in the choice of material. Generally, there are two
methods by which biodegradable polymers erode in the body: non-specific hydrolysis
and enzymatic degradation. These methods of erosion lead to varying degrees of local
vs. pre-programmed control over resorption of the biopolymer. For example, hydrolysis
of the ester linkage in poly(lactide-co-glycolide) polymers leads to bulk degradation
following the cleavage of ester bonds at random sites along the polymer chains until
eventually, lactic acid and/or glycolic acid is produced [63]. This bulk degradation can
bring about a rapid loss in mechanical properties and mass loss after prolonged periods
of reactive hydrolysis. In contrast, polymers that degrade by surface erosion via
enzymatic activity provide a more gradual resorption rate. Enzymatic degradation relies
on certain classes of enzymes (e.g. collagenase), secreted by cells in the body, which
have the ability to cleave the biopolymer [64]. Since polymer degradation in this case is
proportional to the amount of enzyme present, the polymer may erode at a rate
controlled by the local cell activity. One interesting approach to controlling polymer
degradation is crosslinking synthetic polymers with enzymatically degradable molecules
[65]. This concept combines the benefits of the highly controllable properties of
synthetic materials with local cellular control over degradation provided by the
enzymatically labile cross-links. Regardless of the material, scaffolds should ideally
biodegrade at a rate that is comparable to the rate of tissue regeneration so that the
scaffold is completely replaced and integrated into the host tissue.
3.2.1.4. Cellular interaction. The surface chemistry of scaffolds can be modified to
affect specific cellular responses. These responses may include the rate of host cell
invasion, the ingrowth of specific cell types, and cell differentiation. In the past, cell
adhesion to biomaterials was frequently based on the cells binding to non-specifically
adsorbed proteins (e.g., vitronectin) present in body fluids [65]. A more controlled
approach is to covalently, or physiochemically incorporate adhesion-promoting
oligopeptides and oligosaccharides on the biomaterial surface. One of the most
extensively studied adhesion-promoting peptide sequences is the arginine-glycine
aspartic acid (RGD) sequence, which has been shown to promote the adhesion and
spreading of a variety of anchorage dependant cell types [66,67,68]. Specific cell types
may also be targeted by utilizing peptides, which only those cells can recognize and
bind [69]. In addition to cell adhesion peptides, growth factors [70] and plasmid DNA
[71] may also be immobilized on the surface of a biomaterial to modify cellular
behaviour within and surrounding the implant.
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Kathryn W. Riddle and David J. Mooney
4. Material chemistry
In order to provide the varying requirements for each specific application, a variety of
naturally derived and synthetic biomaterials are used for cell encapsulation. These
materials can be processed into many different physical forms and geometries. A
representative group of these materials, many of the most commonly used, are discussed
in this section.
4.1. NATURAL POLYMERS
Natural polymers are attractive for cell immobilization due to their abundance and
apparent biocompatibility. These polymers can provide a wide range of physical
properties that offer unique characteristics for cell encapsulation technologies.
4.1.1. Collagen
Collagen is the major component of mammalian connective tissue and has been used in
cell immobilization due to its biocompatibility, biodegradability, abundance in nature,
and natural ability to bind cells. It is found in high concentrations in tendon, skin, bone,
cartilage and, ligament, and these tissues are convenient and abundant sources for
isolation of this natural polymer. Collagen can be readily processed into porous
sponges, films and injectable cell immobilization carriers. Collagen may be gelled
utilizing changes in pH, allowing cell encapsulation in a minimally traumatic manner
[72,73]. It may also be processed into fibres and macroporous scaffolds [74,75]. Its
natural ability to bind cells makes it a promising material for controlling cellular
distribution within immunoisolated devices, and its enzymatic degradation can provide
appropriate degradation kinetics for tissue regeneration in micro and macroporous
scaffolds. Challenges to using collagen as a material for cell immobilization includes its
high cost to purify, the natural variability of isolated collagen, and the variation in
enzymatic degradation depending on the location and state of the implant site. [76].
Collagen has been used to engineer a variety of tissues, including skin [77,78], bone
[79,80], heart valves [81], and ligaments [82].
4.1.2. Alginate
Alginate is a polysaccharide extracted from seaweed, and has widely been used for cell
immobilization due to its biocompatibility and simple gelation with divalent cations
(e.g. calcium ions). The polymer is made of the two sugars: D-mannuronate (M) and L
guluronate (G) (Figure 3). The M to G ratio in alginate and their distribution will dictate
the gelling and mechanical properties ofthe resulting gel [83,84,85]. Alginate can also
be covalently crosslinked using bi-functional molecules such as adipic dihydrazide,
methyl ester L-lysine, and polyethylene glycol (PEG) [86]. A potential limitation to the
use of alginate is its often uncontrollable and unpredictable dissolution, which occurs by
a process involving the loss of divalent ions into surrounding fluids [87]. In addition,
alginate gels do not promote high levels of protein adsorption [88], and have therefore
been modified with lectin or RGD-containing cell adhesion ligands to control cell
adhesion [89]. When used as a biomaterial for nanoporous immunoisolated devices,
alginate beads are typically coated with a polyamino acid such as poly-L-lysine (PLL)
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Biomaterials for cell immobilization
or poly-L-ornithine (PLO) to establish selective permeability and maintain the capsule
durability and biocompatibility in vivo [46]. Alginate has also been examined as a
carrier for a variety of cell types, including chondrocytes [85,90], for engineering
structurally integrated tissues. Its ability to be inje.=cte d ipn a m:in:im:allty invasive manner
into tissues is a significant attraction in this latter application .
HO~\
HO~OH
0;;
M o
Figure 3. Structure of alginate.
4.1.3. Hyaluronic acid
Hyaluronic acid, which is the largest glycosaminoglycan (GAG) found in nature, has
many attractive features for cell encapsulation (Figure 4). These include ready isolation
from abundant natural sources and the minimal inflammatory or foreign body reaction it
elicits following implantation [91]. Hyaluronic acid gels can be formed by covalently
cross-linking with various hydrazide derivatives [92], and these gels are enzymatically
degraded by hyaluronidase [93]. Potential limitations to the use of hyaluronic acid are
its long residence time in the body, and the limited range of mechanical properties
available from its gels. Moreover, hyaluronic acid requires thorough purification prior
to use to remove impurities and endotoxins that may potentially transmit disease or
elicit an immune response [87]. Hyaluronic acid has been used in micro and
macroporous cell encapsulation as a delivery vehicle for bone-marrow-derived
mesenchymal progenitors [94], or as an injectable microporous cell carrier for soft
tissue augmentation [91]. It has also been combined with collagen to create an
osteoconductive scaffold for bone regeneration [95].
23
Kathryn W. Riddle and David J. Mooney
HO~\
o~o
0=6
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eli>
Figure 4. Structure of hyaluronic acid.
4.1.4. Chitosan
Chitosan is a deacetylated derivative of chitin, which is widely found in crustacean
shells, fungi, insects, and molluscs (Figure 5). Chitosan forms hydrogels by ionic [96]
or chemical cross-linking with glutaraldehyde [97], and degrades via enzymatic
hydrolysis [98]. Chitosan and some of its complexes have been employed in a number
of biological applications including wound-dressings [8], drug delivery systems [99]
and space-filling implants [100]. Due to its weak mechanical properties and lack of
bioactivity, chitosan is often combined with other materials to achieve more desirable
mechanical properties. Specifically, chitosan has been combined with calcium
phosphate to increase its mechanical strength for micro and macroporous scaffold
applications [100], and has been combined with collagen to provide a more biomimetic
microenvironment in nanoporous cell encapsulation applications [l01].
I;;:7i-
, ,",'<' ')
Figure 5. Structure of chitosan.
Figure 6. Structure of agarose.
4.1.5. Agarose
Agarose, similar to alginate, is a seaweed derived polysaccharide, but one that has the
ability to form thermally reversible gels [46] (Figure 6). Mainly used for
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