along theway. The technical advances togetherwith the ever increasing knowledge and experience in the elde realization of the full potential of cell encapsulation in the future.

. . . .s a toollation .tion. .d modi. . . .. . . .. . . .

4.1. Microcapsule permeability and MWCO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 717

Advanced Drug Delivery Reviews 62 (2010) 711730

Contents lists available at ScienceDirect

Advanced Drug Delivery Reviews

j ourna l homepage: www.e lsev ie r.com/ locate /addr4.2. Mechanical integrity/stability/durability. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7174.3. Microcapsule size and morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7174.4. Biocompatibility and low immunogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7174.5. Cell choice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7174.6. Other issues. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 718

5. Therapeutic applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7195.1. Diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7195.2. Bone and cartilage defects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 720

5.3. Neurological diseases . . . . . .5.4. Cancer . . . . . . . . . . . . .5.5. Heart diseases. . . . . . . . . .5.6. Other diseases. . . . . . . . . .

This review is part of the Advanced Drug Delivery Re Corresponding author. Laboratory of Pharmacy an

Vitoria-Gasteiz, Spain. Tel.: +34 945 013095; fax: +3E-mail address: joseluis.pedraz@ehu.es (J.L. Pedraz).

0169-409X/$ see front matter 2010 Elsevier B.V. Adoi:10.1016/j.addr.2010.02.004aterials for cell encapsulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 716icrocarriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7163.5. Other polymers and types of biom4. Critical properties for the elaboration of mContents

1. Introduction . . . . . . . . . .2. Microcapsules and microcarriers a3. Biomaterials in cell microencapsu

3.1. Alginates for cell encapsula3.1.1. Functionalizing an

3.2. Collagen . . . . . . . .3.3. Chitosan . . . . . . . .3.4. Agarose . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 712for regenerative medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 712. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 714. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 715fying alginate gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 716. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 716. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 716. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 716. . . . . . . . . . . . . . . . . . . . . . .

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views theme issue on Therapeutic Cell Delivery for in sd Pharmaceutical Technology, Faculty of Pharmacy, Uni4 945 013040.

ll rights reserved. 2010 Elsevier B.V. All rights reserved.

will undoubtedly lead to thEngineered cellsStem cells

overview the current situatiMicrocapsules and microcarriers for in situ cell delivery

Rosa Ma Hernndez, Gorka Orive, Ainhoa Murua, Jos Luis Pedraz Laboratory of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of the Basque Country, 01006, Vitoria-Gasteiz, SpainNetworking Biomedical Research Center on Bioengineering, Biomaterials and Nanomedicine, CIBER-BBN, SLFPB-EHU, 01006, Vitoria-Gasteiz, Spain

a b s t r a c ta r t i c l e i n f o

Article history:Received 20 November 2009Accepted 3 February 2010Available online 11 February 2010

Keywords:Cell encapsulationAlginatePLLalginateCell therapyDrug delivery

In recent years, the use of transplanted living cells pumping out active factors directly at the site has proven tobe an emergent technology. However a recurring impediment to rapid development in the eld is the immunerejection of transplanted allo- or xenogeneic cells. Immunosuppression is used clinically to prevent rejection oforgan and cell transplants in humans, but prolonged usage canmake the recipient vulnerable to infections, andincrease the likelihood of tumorigenesis of the transplanted cells. Cell microencapsulation is a promising toolto overcome these drawbacks. It consists of surrounding cells with a semipermeable polymericmembrane. Thelatter permits the entry of nutrients and the exit of therapeutic protein products, obtaining in this way asustained delivery of the desirable molecule. The membrane isolates the enclosed cells from the host immunesystem, preventing the recognition of the immobilization cells as foreign. This review paper intends to

on in the cell encapsulation eld and discusses themain events that have occurred. . . . . . . . . . . . . . . . . . . . . . . . . 722

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itu Regenerative Medicine.versity of the Basque Country, Paseo de la Universidad 7, 01006

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action in the human body [1215]. host's genome [3337].

712 R.M. Hernndez et al. / Advanced Drug Delivery Reviews 62 (2010) 711730Since the pioneering study by TMS Chang in the early 1950s [16],when it was originally introduced as a basic research tool, theentrapment of cells has since been developed based on the promise ofits therapeutic usefulness in tissue transplantation and nowadaysrepresents an evolving branch of biotechnology and regenerative

Immunoprotection of transplanted cells and tissues by size-basedsemipermeable membranes allows the in situ delivery of secretedproteins to treat different pathological conditions such as CNSdiseases, diabetes mellitus, hepatic diseases, amyotrophic lateralsclerosis, hemophilia, hypothyroidism and cardiovascular diseases6. Concluding remarks. . . . . . . . . . . . . . . . . . . . . . . .Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . .References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction

Over the last decades various cell types including primary cells [1],stem cells [2] or bioengineered cells [3] have been consideredpotentially therapeutic for the treatment of many diseases includingthose with decient hormone production, such as insulin in diabetes[4] erythropoietin in anemia [5] and factors VIII and IX in hemophilia[6]. Moreover, delivering therapeutic products from nonautologousengineered cell lines has also been assayed in cancer therapy [7] andbone repair [8].

In general, the exciting developments in the eld of drug deliveryhave already had an enormous impact on medical technology,facilitating the administration of many drugs and improving thepharmacokinetics of many others. The past few years have also seenseveral rsts, including the design of novel tissue engineeredapproaches, intriguing advances in the elds of biomaterials and celltherapy and the improvements in the fabrication of more rened andtailored micro and nanocarriers for protein and drug delivery.

The synergyof someof thesepromisingeldshave fueled theprogressof cell encapsulation technology, a relatively old concept pioneered60 years ago. The ability to combine cells and polymer scaffolds to createliving cell medicines that provide long-term drug delivery has openednew doors in the use of allografts. In fact, transplanted cells may beisolated from the host's immune system by embedding them in apermeable device that controls the outward and inward diffusion ofmolecules and cells. As a result of this, the requirement for immunosup-pressant drugs can be eliminated or at least reduced [9,10].

At present, the burgeoning number of cutting edge discoveries isleading to the design of biomimetic and biodegradable microcarriersthat can easily combine with stem cells. These devices will improvethe protection and transport of the cells to the target injured tissueand then promote cell integration and consequently tissue repair orregeneration.

In the present review, we discussed the state of the art in the eldof cell encapsulation technology. The key elements in the design anddevelopment of cell-loaded microcarriers are summarized. Some ofthe most interesting therapeutic applications of this technology arepresented as are some of the limitations, future challenges anddirections in the eld.

2. Microcapsules and microcarriers as a tool forregenerative medicine

Cell therapy is one of the most exciting elds in translationalmedicine. It stands at the intersection of a variety of rapidly evolvingscientic disciplines: biomaterials, immunology, molecular biology,stem cell biology, tissue engineering, transplantation biology, regen-erative medicine, and clinical research. The aim of cell therapy is toreplace, repair, or enhance the function of damaged tissues or organs[11]. However, the success of anymedical treatment depends not onlyupon the pharmacokinetic/pharmacodynamic activity of the thera-peutic agent, but to a large extent, on its bioavailability at the site ofmedicine with numerous applications [17].. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 725

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Cell encapsulation is a strategy that aims to physically isolate a cellmass from an outside environment within the connes of asemipermeable membrane barrier without the use of long-termtherapies of modulating and/or immunosuppressive agents, whichhave potentially severe side-effects [1821]. Microcapsules are almostexclusively produced from hydrogels since they hold a number ofappealing features. They provide a highly hydrated microenviron-ment for embedded cells that can present biochemical, cellular, andphysical stimuli that guide cellular processes such as differentiation,proliferation, and migration [22]. Additionally, the frictional ormechanical irritation to the surrounding tissue is reduced by thesoft and pliable features of the hydrogel. Moreover, some authorsmention that due to the hydrophilic properties of thematerial, there isvirtually no interfacial tension with surrounding tissues and uidswhich minimizes cell adhesion and protein adsorption. Combinationof these two factors results in high biocompatibility [10]. Moreover,hydrogels provide a high degree of permeability for low-molecular-mass (Mr) nutrients and metabolites.

In addition to using natural biomaterials, synthetic polymers aswell as inorganic compounds have also been used [23]. Althoughsynthetic materials provide researchers with large exibility inmaterial design, they do not have an intrinsic mechanism forinteracting with cells, and cell adhesion is typically mediated bynon-specic cell adhesion [24,25]. This limits their use in applicationsthat require dened control over cellmatrix interactions, but this canbe achieved by functionalizing these matrices with bioactivemolecules, as it will be discussed later.

Microcapsule surrounding membranes are expected to be amena-ble to nutrient diffusion and molecules such as oxygen and growthfactors essential for cell survival [10]. Furthermore, the elimination ofcell secretions and catabolic products must be possible while keepingout all high molecular weight immune system components such asimmunoglobulins and immune cells. The permselective capsuleenvironment has been shown to support cellular metabolism,proliferation, differentiation and cellular morphogenesis [10,26,27].

The primary impetus behind the development of cell encapsulationtechnologies has been the aim to transplant cells across an immuno-logical barrier without the administration of immunosuppressantdrugs, an important issue to be considered in organ transplantationdue to their important adverse effects. Non-specic suppression of theimmune system may lead to a variety of undesired complications inpatients (e.g., opportunistic infections, failure of tumor surveillance)[2830]. By surrounding a transplant with a membrane barrier, theaccess of the host's immune system to the transplant can be physicallyprevented, acting as an articial immunoprivileged site shielding thegraft from destruction, which has initiated a urry of research intobioarticial organs and tissue engineering [31,32].

The encapsulation of cells has therefore two major potentialbenets: 1) transplantation without the need for immunosuppressivedrugs, and 2) use of cells from a variety of sources such as primary orstem cells, or genetically engineered cells which can be modied toexpress any desired protein in vivo without the modication of theamong others [3842]. Such cell-based devices are thought to hold

biotechnology, gene or cell therapy, etc.) [10,54,55]. This is a gentle,cell compatible method which has seen adaptation of the initialtechnique by independent laboratories in the last decades, naturallyleading to process improvements and development of superiorencapsulation materials. Many attempts have been made to optimizethe performance of the capsules, and numerous encapsulationtechniques have been developed over the years. Table 1 summarizesmain production methods of microbeads.

Antosiak-Iwaska et al. recently proposed the use of alginateprotamineheparine (APH) capsules as a more resistant alternative tothe conventional alginatepoly-L-lysinealginate (APA) microcapsules.However, long-term experiments indicate that immunoisolation withAPAmicrocapsules is more effective thanwith APHmicrocapsules [32].

Few cell immobilization technologies have allowed to obtain verysmall micrometric biocompatible microcapsules (3060 m) withhigh mechanical stability, of controlled size and uniformity. On thebasis of this new technology of producing very small microcapsuleswith a high mechanical stability, Herrero et al. succeeded in employ-

713R.M. Hernndez et al. / Advanced Drug Delivery Reviews 62 (2010) 711730great promise in applications requiring site-specic and sustainabledrug delivery of cell-synthesized molecules.

Cell immobilization shows an important advantage comparedwithencapsulation of proteins, allowing a sustained delivery of de novoproduced therapeutic products giving rise to more physiologicalconcentrations.

Furthermore, if the encapsulation device is broken, the toxicitycaused by a quick delivery of high concentrations of the drug could beavoided. However if cells manage to exit the encapsulation device thehost's immune system might attack them compromising theirsurvival. Moreover, the use of an inducible genetic system to avoidexcess expression of the therapeutic protein (which in many casesmight become hazardous) is an important challenge in the develop-ment of these delivery systems.

Numerous immunoisolation procedures have been developed overthe years. These techniques are generally classied as macroencapsula-tion (large usually at-sheet and hollow-core bers) and microencapsu-lation (involving small spherical vehicles andconformally coated tissues).

Regarding microcapsules, their spherical shape is consideredadvantageous from a mass transport perspective, offering optimalsurface-to-volume ratio for protein and nutrient diffusion, and thuscell viability compared to other immobilization scaffolds, whichimproves oxygen and nutrients' permeability [43]. The small size ofthe capsules (from 100 m to 500 m) allows their implantation inclose contact to the blood stream, which could be benecial in specicapplications later discussed for the long-term functionality of theenclosed cells due to an enhanced oxygen transfer into the capsules.Moreover, microcapsules are typically more durable than macro-capsules and difcult to mechanically disrupt [10].

Microcapsules can be classied in 3 categories: matrix-core/shellmicrocapsules manufactured by gelling alginate droplets in a solutioncontaining a bivalent ion followed by a surface treatment with apolycation (multi-step technique) [4449], liquid-core/shell microcap-sules produced by dropping a cell suspension containing bivalent ionsinto an alginate solution (one-step technique), and cells-core/shellmicrocapsules (or conformal coating). Matrix-core/shell microcapsulesinwhichcells are hydrogel-embedded, exempliedbyalginates capsule,are by far themost studiedmethod. Many renements of the techniquehave been attempted over the years such as correct biomaterialcharacterization and purication, improvements in microbead produc-tion procedures, and new microbead coating techniques.

All techniques typically start with a scheme to generate acontrolled-size droplet, followed by an interfacial process to stabilizethe droplet and to obtain a solid microcapsule membrane around thedroplet. However, aside those more traditional techniques (eithermatrix-core or liquid-core shells), new techniques are emerging inresponse to shortcomings of existing methods. More recently,conformal coating, where the surface of a cell mass is surroundedwith a membrane, has also been attempted to minimize membranethickness, internal mass transfer resistance and implant size [50,51].

Microcapsules and hollow spheres can be developed efcientlyusing many techniques well described for drug delivery and othernon-pharmacological applications [52,53]. However, in cell encapsu-lation applications, complex and conicting requirements have to bemet. Reproducible methods using very precise parameters (perme-ability, size, and surface) are of outstanding importance, but theseprocedures should also support cell viability and integrity during theencapsulation process and after implantation. Last but not least, thepreparation method must ensure adequate ux across the capsulemembrane for cell survival and functions.

The polyelectrolyte complexation of alginate with polycationicpoly(L-lysine) (PLL), initially developed by Lim and Sun [45], has beenthe most widely employed system for a variety of applications (in vivoand in vitro three-dimensional (3D) cell cultures, clonal selection ofdesired cell phenotypes, bioengineering, large-scale production of

cell-derived molecules in the biotechnology industry, reproductiveing a spraying technique (using atomization nozzles) to encapsulatemesenchymal stem cells and monocytes [11]. This method isadvantageous in terms of ease to set-up and scale up for the proposedindustrial, automatic dropwise of the polymer solution and obtainedspherical and uniform particles. This spraying technique and alginatemicroparticle formulations can further be optimized for oral deliveryof several pharmaceutical peptides and proteins [56].

Haeberle et al. [57] presented a novel technique which can processhighly viscous biopolymer solutions (up to 50,000 times the viscosityof water) while being sufciently gentle to maintain the viability ofthe cells. In this scheme, a commercially available polymer micro-nozzle [58] was spun on a centrifuge to dispense alginate dropletsthrough an air gap into a standard Eppendorf tube (Eppi) mountedon the ying bucket rotor. The tube contained an aqueous CaCl2solution to perform diffusion-based hardening to Ca-alginate beads.

A novel encapsulation system (ve-component/three-membranehybrid capsule) of sodium alginate (SA), CaCl2, polymethylene-co-guanidine (PMCG), cellulose sulfate (CS), and poly L-lysine (PLL) hasrecently been proven efcient in pancreatectomized canine allotrans-plantation experiments [33]. To improve the performance, a thininterwoven PMCGCS/PLLSA membrane was fused onto the PMCGCS/CaCl2SA capsule, forming permanent bonds. This union improvesthe immunoprotection function without jeopardizing the inux ofnutrients and oxygen and efux of therapeutic products and waste.To shield the PMCG and PLL on the surface of the capsule, a third(outer) membrane of CaCl2SA [59] was added to encase the system.

Conformal coating may be thought of as a special case ofmicroencapsulation where the term is used to describe a method offorming a barrier directly on a small cell mass or a small piece of

Table 1Main production methods of microbeads.

Methods Core polymer References

Solid-coreExtrusion methods Alginates/Ca2+ or Ba2+ [44,45,163]Emulsion (thermal gelation) Agarose [161]Microuidics Alginates/Ca2+ [47]Microlithography Alginates/Ca2+ [48]

Hollow-coreInterfacial precipitation HEMA-MMA, PLGA [159]Complex coacervation Cellulose sulfate/pDADMAC [160]

Conformal coatingPolyelectrolyte cell coating Alginates, PDADMAC/PSS [33]Interfacial precipitation coating Methacrylates, alginates/Ba2+ [50]Interfacial polymerization PEG-dyacrilates [51,52]

HEMAMAA, hydroxyethyl methacrylatemethacrylic acid; PLGA, poly(lactic glycolic)acid; pDADMAC, poly-diallyl-dimethyl-ammonium chloride; PSS, poly(styrene

sulfonate); PEG, poly(ethylene glycol).

tissue. The method eliminates unutilized space in a microcapsule coreby surrounding the cell mass with the encapsulation membrane. Thistheoretically provides an improved mass transport between thecapsule exterior and the cell mass, and increases the effectiveness ofcell packing (hence, minimizes implant size). Despite its potential, invivo performance of conformally coated islets remains to be reportedin the literature [50,51].

In addition to incorporating the living material, some approachesemploying microcapsules as microcarriers have also been de-scribed in the literature, where cells are attached to the surface ofthe biomaterial employed (Fig. 1). Tatard et al. developed pharma-cologically active biodegradable microcarriers (PAM) made with poly(D,L-lactic-coglycolic acid) (PLGA) and coated with adhesion mole-cules to serve as a support for cell culture [60]. Stover et al. haveproposed the use of Spheramine, an active component of culturedhuman retinal pigment epithelial (hRPE) cells, attached to an excipientpart of cross-linked porcine gelatinmicrocarrierswhichwas in Phase IIclinical trial [61] but had to be stopped recently due to adverse effectsencountered [62]. Gelatinmicrocarriers are also under study to beusedin three-dimensional cartilage- and bone like tissue engineering [63].

Integrated biodegradable devices have also been proposedrecently based on the integration of two techniques: microcapsulesand surface-coated poly (3-caprolactone) PCL capsules (diffusionchambers) [38]. Microcapsules provide a 3D microenvironment forspatial cell growth with good viability and proliferation. Coatingbiocompatible and hydrophilic PEG gelatin on the PCL surface couldmediate the inammatory response, prevent brosis formation, and

maintain controllable performance. Most importantly, the dualnanoporous construct provides a unique way to allow superior cellgrowth, immunoprotection, brosis prevention and controllablerelease of secreted products in a biodegradable device.

Cell immobilization has long been suggested as an efcientdelivery method for cell transplantation, but it was recently reportedthat cell immobilization can lead to modication of cell wall and cellmembrane compositions [64]. An increased understanding of thechemical signals that direct cell differentiation, migration andproliferation, advances in scaffold design and peptide engineeringthat allow this signaling to be recapitulated and the development ofnew materials, such as DNA-based and stimuli-sensitive polymers,have recently given engineers enhanced control over the chemicalproperties of a material and cell fate. Additionally, the immunesystem, which is often overlooked, has been shown to play a benecialrole in tissue repair, and future endeavors in material design willpotentially expand to include immunomodulation.

It is apparent that cell fate in growing tissues relies heavily on theadhesion ligands presented by the matrix, and the development ofmethods to functionalize materials with these molecules is central inrecapitulating these matrix effects and supporting the growth offunctional tissue.

3. Biomaterials in cell microencapsulation

Biomaterials are increasingly important in the development ofdrug delivery systems and tissue engineering approaches and play key

714 R.M. Hernndez et al. / Advanced Drug Delivery Reviews 62 (2010) 711730Fig. 1. Comparison between microcapsules and microcarriers.

715R.M. Hernndez et al. / Advanced Drug Delivery Reviews 62 (2010) 711730roles in overcoming the inherent insufciency of tailored therapies.Polymers of many types are used to create drug vehicles providingsustained delivery of potentially therapeutic agents, includingproteins, genes, cells and oligonucleotides. Biomaterials also makeexcellent scaffolds suitable for delivering cells to the host orimmobilizing them for long-term delivery of molecules to thesurrounding tissue. Scaffolds can be loaded with proteins and/orhave a surface morphology or extracellular matrix (ECM) capable ofcontrolling cell attachment, growth, and differentiation. In the lastfew decades, the eld of cell microencapsulation has also raised muchinterest in part due to the advancement and optimization of thebiomaterials used to elaborate the capsules [65]. These living cell-containing particles can be modied with surface characteristics thatallow them to control the proliferation and differentiation of theenclosed cells [6668].

It was recently acknowledged that the success of this therapeuticapproach requires a detailed analysis, at the atomic and molecularlevels, of the types of biomaterials employed and especially of themechanisms driving cellmaterial interactions. One of the rst issuesin this endeavor is the immunogenicity of the biomaterials used tofabricate the microcapsules and the biocompatibility of the micro-capsule system in its nal form. One critical limitation has been thepersistent lack of reproducibility of the different biomaterials and therequirements to achieve a better understanding of the chemistry andbiofunctionality of the biomaterials and microcapsule system. Moredetailed and in-depth knowledge will lead to the production ofstandardized transplantation-grade biomaterials and biocompatiblemicrocapsules.

3.1. Alginates for cell encapsulation

Alginates are certainly the most frequently employed biomaterialsfor cell immobilization due to their abundance, easy gelling propertiesand apparent biocompatibility. Although the suitability of other naturaland synthetic polymers is under investigation [69,70], nonehas reachedthe same level of performance as alginates. As natural polymers,alginates exist in brown seaweeds and bacterium [71] and theircompositions vary depending upon the source from which they areisolated [72]. The production of alginates with specic structures canalso be made by enzymatic modication using mannuronan C-5epimerases [73]. Alginates are a family of unbranched binary copoly-mers of 14 linked -D-mannuronic acid (M) and -L-guluronic acid(G), of widely varying compositions and sequential structures.Determining and standardizing these differences is of paramountimportance since they have a signicant impact on some of the alginategel properties including biocompatibility, stability, mechanical resis-tance, permeability, biodegradability and swelling behavior.

One particular critical issue is the biocompatibility of the alginatesand alginate microcapsules. A very high level of biocompatibility isessential assuming that the nal aim of the encapsulation device is toprotect the enclosed cellular tissue from the host's immune response.It is necessary to improve our knowledge about the biomaterial anddevice properties, and to optimize and characterize each of the stepsrelated to the cell encapsulation technology, from the alginateextraction and purication to the elaboration and administration ofthe microcapsule.

As a natural polymer, alginate's performance as a biomaterial islimited by its tendency to be largely contaminated. In addition, theindustrial processes used for extracting alginates from seaweed couldintroduce further contaminants into the raw alginates. Some of theseimpurities include endotoxins, certain proteins and polyphenols. Thelatter can be dangerous for humans as reported by the World HealthOrganization [74] and can possibly accumulate in the body [75].Moreover, endotoxins and proteins have been associated with areduced biocompatibility of the alginate. Therefore, a key element in

the validation of the alginate for implantation purposes is an efcientpurication process to monitor and remove all its contaminants.In the last few years, several research groups have developed theirown in-house protocols for alginate purication [7681]. The rstpublished method described by Zimmermann et al. used a free-owelectrophoresis technique [76] but, since it was difcult andexpensive, it was abandoned in favor of chemical extractionprocedures. Even, the rst comparative evaluation of some of thesein-house alginate purication protocols was published [82]. Resultsfrom this study showed that in general all of the studied puricationmethods reduced the amounts of endotoxins and polyphenols butwere less effective in eliminating proteins. A commercially puriedalginate was also analyzed in order to provide a comparison betweenthe in-house and commercial purication processes. Interestingly, thecommercially puried alginate also presented residual proteins inamounts that may be enough to compromise microcapsule biocom-patibility [82]. Overall, the results of this study reected that currentlyemployed methods to purify alginates may not be efcient enough tocompletely remove contaminating and potentially immunogenicspecies. It has been demonstrated that purifying the alginate inducesa number of changes in the polymer's characteristics. Alginatehydrophilicity was shown to increase by 10 to 40% followingpurication by different methods, in correlation with a decrease inprotein and polyphenol content. This increased hydrophilicitycorrelated with lower immunogenicity of the alginate gel. In thisstudy, reducing the contamination level of the alginate also correlatedwith an increased solution viscosity, a property that will inuence themorphology of the nal microcapsule.

The composition of the alginate is another critical issue to beconsidered. In fact, alginate composition regulates somemainpropertiesof the alginate gels including stability, biocompatibility and permeabil-ity. In the last decade, biocompatibilityof thealginates in relation to theircomposition has been a matter of much debate and controversy. Somegroups have reported that alginates with a high content in M evoke aninammatory response by stimulatingmonocytes to produce cytokinessuch as interleukin (IL)-1, IL-6 and tumor necrosis factor (TNF). Thismechanism may be driven via binding to CD14 [83,84]. Furthermore,antibodies to alginates were found when high-M alginates weretransplanted but not in the case of high-G alginates [85]. Soon-Shionget al. also observed a cellular overgrowth of 90% of the capsules whenhigh-Malginatewas used [86]. In contrast, Clayton et al. foundguluronicacids to be associatedwithmore severe cell overgrowth [87]. DeVos andco-workers have also reported that after transplantation in rats,the majority of high-G alginate capsules are overgrowth by inamma-tory cells and are adherent to the abdominal organs whereasintermediate-G capsules (with higher M content) are free of anyadhesion and areoating freely in theperitoneal cavity [88,89].With theaim of shedding light on this discussion, we evaluated the in vitro and invivo biocompatibility properties of microcapsules elaborated withalginates of different composition and purity. Our results suggestedthat the purity of individual alginate preparation, rather than theirchemical composition, was probably of greater importance in deter-mining microcapsule biocompatibility [90]. All this controversy mightbe caused in part by the lack of a standard denition for high-G alginatesand high-M alginates as well as for the different purity levels of bothmonomeric units and the different geometry of the capsules employedin the experimental studies [27]. However, further efforts are needed todevelop standardized assays that facilitate the evaluation of thebiocompatibility of alginates and other hydrogels. Recently, a highlysensitive cell assay based on the induction of apoptosis in Jurkat cells,capable of detecting low levels of immunogenic impurities present inalginate samples has been reported [91]. This in vitro test, as well asother similar assays, is certainly a useful tool to evaluate, select andimprove alginate preparations. Nevertheless, it should always be kept inmind that only the results of in vivo implantations can provide denitiveinformation on the immunogenicity of alginates. In general, further

research is still needed to precisely identify the alginate properties that

Challenges to using collagen as a material for cell immobilizationinclude its high cost to purify, the natural variability of isolated

implants [117]. Due to its weak mechanical properties and lack ofbioactivity, chitosan is often combinedwith othermaterials to achieve

but one that has the ability to form thermally reversible gels. Mainlyused for nanoencapsulation of cells, agarose/cell suspensions can be

formed by the presence of two or more polymerizable moieties,which is also known as radical cross-linking, have also been studied

716 R.M. Hernndez et al. / Advanced Drug Delivery Reviews 62 (2010) 711730can reliably predict its in vivoperformance. This information is necessaryto establish strictly outlined criteria for alginate selection andpurication and obtain results that are reproducible between researchgroups.

3.1.1. Functionalizing and modifying alginate gelsIn general, biomaterials have been considered as simple inert

scaffolds in which cells were merely entrapped. One current excitingapproach consists in modifying the biomaterials with differentpeptides and proteins that provide control over cell fate. By tailoringthe polymers with sequences that mimic the extracellular matrix(ECM) it is feasible to control cell proliferation and even celldifferentiation. Some examples of molecules that have been used todecorate the biomaterials include RGD, IKLLI, IKVAV, LRE, PDSGR andYIGSR [9294]. These moieties trigger a cascade of intracellularsignaling events through the focal contacts providing tight controlover cellmatrix interactions [24].

The most widely employed peptide sequence is arginineglycineaspartic acid (RGD) derived from bronectin, a natural proteinpresent in ECM [95,96]. The coupling of RGD sequences to alginatehydrogels has been extensively studied by Mooney et al. This groupshowed how it was possible to direct cell fate by controlling RGDdensity on alginate gels [97,98]. In addition, the inuence of differentnanopatterned islands of RGD on cell behavior has been extensivelyevaluated [99]. Recently, they reported the development of noveltools that allow for quantifying the interactions between cells andpresenting ligands [100,101]. Such advances make a step forward inthe understanding of cellECM interactions and conrm how integrinexpression varies depending on the stage of cell differentiation.

The elaboration of biomimetic scaffolds has also been applied tocell encapsulation technology by our research group [67]. Bydesigning biomimetic cellhydrogel capsules we were able topromote the in vivo long-term functionality of the encapsulatedmyoblast cells and improve the mechanical stability of the capsules.Biomimetic capsules were fabricated by coupling the adhesionpeptide arginineglycine aspartic acid (RGD) to alginate polymerchains and by using an alginate mixture providing a bimodalmolecular weight distribution. The biomimetic capsules providedcell adhesion for the enclosed cells, potentially also leading tomechanical stabilization of the cellpolymer system. Strikingly, thenovel cellhydrogel system signicantly prolonged the in vivo long-term functionality and drug release, providing a sustained erythro-poietin delivery during 300 days without immunosuppressive proto-cols. Additionally, regulating the cell-dose within the biomimeticcapsules enabled a controlled in vitro and in vivo drug delivery [67].

Another modication under evaluation is that focused on thecontrol over the biodegradation rate of the alginates. The easilybiodegradable alginates result in interesting tissue engineeringapproaches, especially when the repair, remodelling or regenerationof tissues is intended. In such an approach, the alginate is designed todegrade once the biomaterial has met its biological function. Thedegradation rate should be adjusted to the time required by graftedand host cells to replace the scaffold and provide new tissue. Oneinteresting example is the oxidation of alginate chains by generatingfunctional groups that are more susceptible to hydrolysis [102,103].

3.2. Collagen

Collagen is the major component of mammalian connective tissueand has been used in cell immobilization due to its biocompatibility,biodegradability, abundance in nature, and natural ability to bindcells. It is found in high concentrations in tendon, skin, bone, cartilageand, ligament, and these tissues are convenient and abundant sourcesfor isolation of this natural polymer. Collagen can be readily processedinto porous sponges, lms and injectable cell immobilization carriers.

Collagen may be gelled utilizing changes in pH, allowing cellfor cell encapsulation. Hyaluronic acid (HA) and poly(ethylene glycol)(PEG), functionalized with vinyl end groups, such as methacrylatesand acrylates, are the most used polymers for this polymerizationmechanism [121,122].

4. Critical properties for the elaboration of microcarriers

Although advances of outstanding importance have already beenachieved in the eld of cell microencapsulation, there are some criticalaspects that should be carefully taken into consideration if the clinicalsuccess of the technology is aimed. A compilation of importanttransformed into microbeads by utilizing a reduction in temperature[119]. A possible drawback to its use in this application is cellularprotrusion through the membrane after gelation. Other uses ofagarose in cell immobilization include the fabrication of microporousgels seeded with chondrocytes for the repair of cartilage defects [120].

3.5. Other polymers and types of biomaterials for cell encapsulation

Other biomaterials have been investigated in the eld of cellmicroencapsulation, although none of them is as much characterizedand studied as alginates. On the way to obtain alternative cell-basedtherapeutic strategies, we could benet from the advantages thatother biomaterials could offer. In addition to hydrogels created byionic interaction, biomaterials based on a cross-linked networkmore desirable mechanical properties. Specically, chitosan has beencombined with calcium phosphate to increase its mechanical strengthfor micro and macroporous scaffold applications [117], and has beencombined with collagen to provide a more biomimetic microenviron-ment in nanoporous cell encapsulation applications [118].

3.4. Agarose

Agarose, similar to alginate, is a seaweed derived polysaccharide,collagen, and the variation in enzymatic degradation depending onthe location and state of the implant site [108]. Collagen has beenused to engineer a variety of tissues, including skin [109,110], bone[111,112], heart valves [113], and ligaments [114].

3.3. Chitosan

Chitosan is a deacetylated derivative of chitin, which is widelyfound in crustacean shells, fungi, insects, andmolluscs. Chitosan formshydrogels by ionic or chemical cross-linking with glutaraldehyde, anddegrades via enzymatic hydrolysis. Chitosan and someof its complexeshave been employed in a number of biological applications includingwound dressings [115], drug delivery systems [116] and space-llingencapsulation in a minimally traumatic manner [104,105]. It mayalso be processed into bers and macroporous scaffolds [106,107]. Itsnatural ability to bind cells makes it a promising material forcontrolling cellular distribution within immunoisolated devices, andits enzymatic degradation can provide appropriate degradationkinetics for tissue regeneration in micro and macroporous scaffolds.capsule properties is provided in recent reviews [123,124].

717R.M. Hernndez et al. / Advanced Drug Delivery Reviews 62 (2010) 7117304.1. Microcapsule permeability and MWCO

The mass transport properties of an encapsulation membrane arecritical since the inux rate of molecules essential for cell survival andthe efux rate of therapeutic products will ultimately determine theextent of entrapped cell viability. Moreover, membrane pore sizemust be carefully controlled to avoid the undesired entrance ofimmune system components from the host that might destroy theinner cells. The metabolic requirements of different cell types arediverse and, hence, in principle optimal membrane permeabilitydepends on the choice of cells [10]. Although the role of permeabilityfor particular elements essential for cell survival has been explored(for example, oxygen) [125], no systematic approach has been takento determine the permeability requirements of each cell type. As aconsequence, an empirical approach has been typically taken to tailorcapsule permeability for cell survival. The upper limit of capsulepermeability, i.e., molecular weight cut-off (MWCO; size of the largestmolecule that is not substantially blocked by the semipermeablemembrane), will be application dependent. In the case of transplan-tation, the MWCO is expected to be different whether xenogeneic orallogeneic tissues are destined for engraftment [10].

4.2. Mechanical integrity/stability/durability

The mechanical role performed by the semipermeable barrierensures that no direct cellcell contact occurs between transplantedand host cells, while allowing for paracrine interaction between thebiological environment (host) and the transplant graft.

The assessment of capsule mechanical properties is important, notonly to determine the durability of capsules during production andhandling, but also as an indication of the capsule membrane integrity.The latter is most informative when long-term studies are carried out.

4.3. Microcapsule size and morphology

Another important issue that should be taken into account is thediameter of the capsule as it could inuence the immune responseagainst capsules. Sakai et al. observed that cellular reaction was muchlower when employing smaller microcapsules in comparison tobigger size microcapsules [126].

Rough surfaces of capsulesmust also be avoided due to the fact thatthey may elicit immunological reactions when implanted. In additionto a biomaterial's chemical properties, researchers have realized thatstructural aspects of themembranes can also have profound inuenceson cell function, fate and tissue formation [126129].

A smooth and clean device surface, controlled geometry anddimension, and polyethylene glycol (PEG) or gelatin modication onthe capsule surface could mediate the acute inammatory responseand minimize brosis formation [38].

Moreover, toguaranteea sufcientdiffusivemass transport, inoverall,the diameter of the microcapsules should not exceed 300400 m[130,131].

4.4. Biocompatibility and low immunogenicity

Biocompatibility is dened as the ability of a biomaterial toperform with an appropriate host response in a specic application[132]. Biocompatibility of microcapsules and their biomaterials'components is a critical issue if the long-term efcacy of thistechnology is aimed. Usually, a fully biocompatible system isconsidered to be a system manufactured of membranes which elicitno or not more than a minimal foreign body reaction. The hostresponse is a potentially serious and deleterious problem to theclinical implementation of the technology.

A key element in the validation of alginate for implantation

purposes is the efcient purication process to monitor and removeall its contaminants (inammatory components) which includeendotoxins, polyphenols and certain proteins. Not surprisingly, thepurity of the alginate has been found to be a pertinent factor in thebiocompatibility of alginatePLL capsules. Most purication methodshave been found to succeed in reducing endotoxins and polyphenols,these methods have not achieved a correct elimination of the proteincontent [82]. In addition, the purication process might induce anumber of changes in the polymers' features which should becarefully controlled [133].

The surgical implantation method is believed to be an additionalparameter that inuences the host reaction or biocompatibility tosuch implanted devices.

Several experiments have demonstrated that the surgical implan-tation method can inuence and activate a non-specic responseagainst implanted devices. Moreover, although it has been describedas a transient response, it is difcult to avoid as it cannot be solved bychemical modication of the capsule. In order to overcome thisobstacle, the use of transient immunosuppressive protocols has beenproposed [134,135].

Upon transplantation of encapsulated alien cells, the host responseis initiated by an acute inammatory reaction caused by thedisruption of host vasculature (associated with the release ofbioactive proteins from the host such as brinogen, thrombin,histamine and bronectin) (Fig. 2). Activated platelets, polymorpho-nuclear leukocytes, humoral components of serum, clot constituents,cell debris, and extracellular matrix are initially present at the hostmaterial interface. Tissue macrophages are recruited to the site andmediate the process of clean-up and initial wound healing. Mast cellsandmacrophages produce bioactive factors such as IL-1, TNF-, TGF- and histamine, which stimulate cells in the capsules. Finally,mesenchymally-derived cells mediate matrix production and con-tracture coupled with a neovascularization response which roundsout the process. Within two weeks, basophils and granulocytesgradually disappear from the graft site while macrophages and somemigratory cells that are primarily broblastic remain attached to anaverage of 210% of the capsules. These attachedmacrophages remainactivated and contribute to the deleterious circle of activation. As aconsequence, although the loss of 210% of capsules might not becrucial for the functionality of the remaining 9098%, different studiesshow that it is mandatory to completely delete overgrowthsurrounding microcapsules [43,122,136] due to the fact that it mayinterfere with diffusive transport of molecules and oxygenated bloodsupply [76,86,137].

In addition to the interactionbetween thebiomaterials and thehosttissue, a signicant interaction is the one between the biomaterial andthe encapsulated donor tissue. The response varies in degree and in thespecic cell types involved depending upon the site of implantation.

Neovascularization is another critical process which may deter-mine the success of encapsulation therapy. A number of studies haveshowed that the outer microarchitecture of the encapsulationmembrane exerts a profound inuence on the neovascularizationresponse, and not necessarily the membrane surface chemistry [138141]. Membranes with surface pores that allow host cell colonizationwithout inducing signicant cell spreading, in general, have resultedin the formation of vascular structures very near the hostmaterialinterface [10].

De Vos et al. have reported an interesting advance to predictbiocompatibility where the measurement of the electrical charge ofthe surface by means of zeta potential was found to predict theinterfacial reactions between the biomaterial and the surroundingtissue [142].

4.5. Cell choice

The choice of cells depends upon the intended application, such as

the secretion of a particular naturally occurring bioactive substance

718 R.M. Hernndez et al. / Advanced Drug Delivery Reviews 62 (2010) 711730like neurotransmitter, cytokine, chemokine, growth factor, growthfactor inhibitor, angiogenic factor; or the metabolism of a toxic agent,or the release of an immunizing agent; or based on a sense and releasefunction such as oxygen partial pressure and Epo or glucose andinsulin.

Cell encapsulation technology has in part failed to reach clinicalapproval so far mainly due to the high immunogenicity of theencapsulated cells (seed cells for therapeutic function), whicheventually evoke an inammatory reaction in the microenvironmentsurrounding the microdevices that leads to suffocation and death ofthe encapsulated cells [9,65,143]. The key issue to overcoming thisproblem could be to use cells that can downregulate or reduce thisimmune response [144].

The encapsulated nonautologous cells secrete cytokines and shedantigens, which eventually initiate a host immune response and leadto inammatory tissue surrounding the microcapsules. This inam-matory reaction leads to cell suffocation and decreased encapsulatedcell viability [65,143]. One promising solution to reduce host immunereaction is by administering anti-inammatory drugs along with thetherapeutic system [135,145]. Another approach under study toreduce host immune reaction is to replace the cell lines commonlyused for cell encapsulation with naive cells, such as stem cells. Humanmesenchymal stem cells (hMSCs) show promising properties as a cellof choice for cell microencapsulation and cell-based therapy. MSCsimprove the biocompatibility of the microcapsules in vivo, and can

Fig. 2. Diagram of the process of acute and chronic inammatory responses in the termed foRef. [122] 2009 Landes Bioscience.serve as a platform for continuous long-term delivery of therapeuticfactors, including potent cancer therapies [144].

4.6. Other issues

As previously mentioned, a gentle encapsulation technique isrequired if viability of the entrapped cells is aimed. In addition, animportant issue that involves the use of spherical-shaped micro-capsules mainly, is the formation of local domains of necrotic spotsdue to inadequate internal oxygen mass transfer. Various alternativeshave been proposed to overcome this obstacle. On the one hand, aspreviously mentioned, Sakai et al. developed alginateagarosesubsieve-size capsules of less than 100 m in diameter to improveoxygen transfer into the capsule where cell viability was observed notto be affected by the small size of the capsules [146]. Alternatively,Khattak et al. included synthetic oxygen carriers (peruorocarbons)in alginate gels to improve oxygen supply. An enhancement inmetabolic activity and cell viability was detected due to a reduction inanaerobic glycolysis which resulted in an increase in glucoseconsumption/lactate production efciency [147].

Another challenge in the eld of cell microencapsulation is theability to monitor the implanted devices. Once microcapsules aretransplanted, the only way until recently was to assess theirfunctional state is through invasive recovery surgery. Fortunately,imaging technologies have made possible an accurate non-invasive

reign body reaction against implanted biomaterials. Reproduced, with permission, from

719R.M. Hernndez et al. / Advanced Drug Delivery Reviews 62 (2010) 711730follow-up of engrafted tissues [148,149]. Non-invasive imagingtechniques using various reporter genes are complementary toex vivo molecularbiological assays and include additional spatialand temporal dimensions.

An alternative interesting approach to overcome this situation hasalso been recently proposed by Barnett et al. using alginate-basedradiopaque microcapsules containing either barium sulfate orbismuth sulfate which could be monitored by X-ray [150]. However,although cell viability and capsule permeability were not affected byradiopaque agents it should be mentioned that the metals employedin this work are toxic both for the encapsulated cells and the recipient.In a recent study by Fu et al., the group demonstrated that incor-poration of peruorooctylbromide into alginatePLL microcapsulesmay allow easy X-ray tracking, potentially providing scientists in theeld with a further tool to understand and improve cellular distribu-tion following implantation [151]. Additionally, magnetic resonance-guided imaging of magnetocapsules (alginate microcapsules elabo-rated using Feridex) has also been proposed and could be consideredan interesting non-invasive approach which might ease the in vivodetection of implanted devices [152].

Regarding the use of polymers for cell encapsulation, while bothnatural and synthetic polymers can be used for the preparation,natural polymers are more cell compatible, react under milderconditions and allow for the encapsulation of fragile cells, but thechallenge in producing such uniform capsules is to ensure excellentrepeatability and reproducibility both within and between batches[31]. A great deal of research work is still needed in order to obtain anincreased number of commercially available and clinically successfulnatural-based systems. Undoubtedly, natural-origin polymers ornature-inspired materials appear as the natural and desired choicefor the referred applications [153].

Despite many advances, researchers in the eld of cell microen-capsulation still face signicant challenges regarding the optimizationof scaffolds for each specic application. Scaffolds play an essentialrole as the extracellular matrix but they are often unable to mimic theexact microenvironment to promote the correct and accurateresponse. The emerging and promising next generation of engineeredbiomaterials is directed to producing scaffolds with an informationalfunction, e.g., biomaterials containing sequences of growth factorswhich ease cell attachment, proliferation and differentiation; farbetter than non-informational polymers. The use of growth factorshas been considered as an alternative to modify not only the hosthealing response at the site of injury to facilitate tissue repair, but alsoto manipulate and enhance the in vitro tissue growth in order toproduce more biofunctional tissues. Hence, the strategy is to modelthe extracellular matrix and provide the necessary information orsignaling for cell attachment, proliferation and differentiation to meetthe requirement of dynamic reciprocity for tissue engineering anddrug delivery.

5. Therapeutic applications

In this part of the article, the effect of microencapsulated-celltherapies on different disorders will be presented in addition tocommenting on available scientic data in this area.

5.1. Diabetes

Diabetes mellitus is a metabolic disorder characterized byhyperglycemia resulting from defects in insulin secretion, insulinaction or both. Current research efforts towards therapy of type 1diabetes are aimed at developing approaches for restoration ofregulated insulin supply. Transplantation of islets of Langerhans hasbeen proposed as a safe and effective method for treating patientswith insulin-dependent diabetes mellitus, although it is still, an

experimental procedure. In fact, the exciting improvements inoutcomes following clinical islet transplantation using the Edmontonprotocol, have renewed hope for patients with type 1 diabetes [154].The protocol is based on the use of human islets from cadavericdonors, which are implanted in the liver of carefully selected diabeticrecipients via portal vein injection. However, the limited availabilityof human tissue and the need for lifelong immunosuppression whichresults in long-term side-effects, makes the widespread application ofthis therapy difcult.

Using islets of Langerhans from other species is an obvious way ofproviding the large amounts of functional tissue required fortransplantation therapy. In 1980 Lim and Sun implanted micro-encapsulated xenograft islet cells into rats and the microencapsulatedislets corrected the diabetic state for several weeks [45]. Since then,there has been considerable progress toward understanding thebiological and technological requirements for successful transplanta-tion of encapsulated cells in experimental animal models, includingrodents and non-human primates. Bioarticial pancreatic constructsbased on islet microencapsulation could eliminate or reduce the needfor immunosuppressive drugs and offer a possible solution to theshortage of donors, as it may allow for the use of animal islets orinsulin-producing cells engineered from stem cells [4,155,156].

Different polymers have been used for islet encapsulation andimmunoprotection, photopolymerized poly(ethylene glycol) (PEG)[157], water insoluble polyacrylates [158,159], sodium cellulosesulfate [160], agarose [161], chitosan [162] and alginate [163].Among others, alginate-based microcapsules are widely used vehiclesfor introducing islets into the body. Several experiments havedemonstrated that these polymeric microcapsules could be useful inthe treatment of diabetes. Elliot et al. [164] have tested somemicroencapsulated piglet islet formulations into mice and monkeysand noted amelioration of disease. In another study with a placebo-controlled design [165], researchers assessed the safety and clinicalactivity of alginate-encapsulated porcine islets in a non-humanprimate model of streptozotocin-induced diabetes. They notedworsening of the disease in control animals: six out of eight controlmonkeys required increased doses of daily insulin; in contrast, six ofthe eight islet-transplanted monkeys had reduced insulin require-ments. After islet transplantation, individual blood glucose valuesvaried and one monkey was weaned off insulin for 36 weeks. In arecent study which reports the use of intraperitoneally implantedencapsulated allografts, type 1 diabetic patients remained nonimmu-nosuppressed but were unable to withdraw exogenous insulin[166,167].

In the last few years, the renewed interest in porcine isletxenotransplantation has generated some controversy about thehuman clinical trials carried out. The study by Living Cell TechnologiesLtd with the Diabecell device (neonatal porcine islets encapsulatedin alginate microcapsules) provided evidence of improvement inglycemic control individuals and showed no evidence of porcine viralo retroviral infection. Moreover, they reported evidence of residual,viable, encapsulated porcine islets being retrieved from a patient9.5 years after transplantation [168]. However, this approach has beencriticized by the International Xenotransplantation Association asbeing premature and potentially risky [169]. Recent progress in thisarea, like the use of closed, porcine endogenous retroviruses free(PERV-free) herds or advances in immunoisolation may help toimprove the formulations. In fact, a new open-label investigationabout the safety and effectiveness of Diabecell in patients withdiabetes type 1 is currently recruiting patients (NCT00940173) [170].

Besides alginate, polyethylene glycol (PEG) is widely used for isletencapsulation. The immobilization of PEG chains to the cell or tissuesurface creates a molecular barrier preventing molecular recognitionbetween cell surface receptors and soluble ligands. Therefore, surfacePEGylation has been used to improve the biocompatibility of islets[171,172]. Islet surfaces have been isolated either with a conformal

PEG coating, a technique in which a polyethylene glycol pre-polymer

720 R.M. Hernndez et al. / Advanced Drug Delivery Reviews 62 (2010) 711730is photopolymerized around an islet [173], or by direct covalentmodication of the protein surfaces of islets [174]. In an in vitro studyperformed with PEG-grafted islets cultured with peritoneal macro-phages and splenic lymphocytes, it was concluded that the graftedPEG molecules onto the islets could efciently prevent the activationof immune cells and secretion of cytokines. However, grafted PEGmolecules do not completely prevent the inltration of the cytotoxicmolecules into the islets [175]. Subsequently, these authors [176]have evaluated the clinical potential of a new combinatorial therapybased on PEGylation and immunosuppressant therapy with low dosesof cyclosporine A (CsA). For 1 year after transplantation, PEGylatedislets rmly controlled blood glucose levels, and enabled normalblood glucose responsiveness, hormone synthesis, and the existenceof PEG molecules at transplanted islets, suggesting that a PEGylation/CsA combinatorial therapy could semipermanently protect trans-planted islets from immune reactions at least in the rodent model.This technology is currently the basis for Phase I/II clinical trials byNovocell for encapsulated human islet allografts implanted into thesubcutaneous site. The trials began in 2005 (NCT00260234) [170].

Even though alginate and PEGylated microcapsules are beingtested in clinical trials, biocompatibility, immunoprotection andhypoxia [143,177] are main issues that need to be improved. Anumber of different strategies have been proposed as potentialsolutions to overcome these problems. The use of growth factors maybe useful for therapeutic stimulation of neovascularization, whichmay improve the survival and function of microencapsulated islets atthe transplantation site by allowing for adequate oxygen and nutrientexchange, as well as removal of waste products between encapsulatedislets and the systemic circulation [178,179]. Besides avoiding islethypoxia, the improvement of the biocompatibility of islets aftertransplantation is essential. On this respect, in a recent paper,Teramura and Iwata [180] have proposed a novel method using alayer of HEK293 living cells for islet encapsulation (Fig. 3). In thiscontext the use of bioactive peptides like the glucagon-like peptide 1(GLP-1) analog features an innovative strategy to modify PEGhydrogels which can signicantly enhance the efcacy of isletencapsulation [181]. Finally, in order to avoid acute inammationand its harmful effects on transplanted islets, different approacheshave been developed. For example co-administration with anti-inammatory drugs [134,182] or modulation of macrophage activa-tion [183,184] among others, are being studied to generate bioactivebarriers that locally modulate host response to microencapsulatedcells.

Much work is clearly needed before microencapsulated-celltherapy for diabetes can be advanced to the clinic. The challengescenter on generation of an abundant source of regulated insulin-producing cells and some aspects of the cell-based encapsulationmethods should be improved in order to increase the transplantlongevity and functional performance of the capsules in vivo [4].

5.2. Bone and cartilage defects

Bone defects resulting from trauma and tumor resection arecommon clinical problems. Bone tissue usually has the ability toregenerate, but when a defect of critical size needs to be bridged, therepair attempt fails in most cases.

The current standard tissue used is autologous tissue, which isusually harvested from the iliac crest of the patient. Althoughautografting has been a major treatment, it has several limitationsincluding patient pain, cost, and limited supply. As an alternative,allografting has been studied due to its abundant source. However, itsdrawbacks, including the uncertainty of biocompatibility and diseasetransmission, have limited its use [185]. On the other hand, articularcartilage has a limited capability for healing after trauma and only fewlong-tem treatments are available today, including mosaicplasty,

periosteal transplantation and autologous chondrocyte implantation.To overcome these drawbacks, investigators are considering alterna-tive therapies in which mesenchymal stem cells (MSCs) are involved.MSCs are multipotent progenitor cells that can be isolated from bonemarrow, adipose tissue, muscle tissue, umbilical cord blood, periph-eral blood, and other tissues [186188] and have the capability todifferentiate into multiple tissue-forming cell lineages, such asosteoblasts and chondrocytes, which contribute to the regenerationof bone and cartilage.

Lately, someworks have showed that microcapsules could create a3Dmicroenvironment that would provide a niche for stem cell growthand differentiation [189]. In this respect, Endres et al. [190] haveconrmed that, in vitro, these hMSC were able to differentiate alongthe chondrogenic lineage when encapsulated in Ca-alginate micro-capsules and stimulated with TGF-3. The size of these microcapsulesis in the injectable range (mean diameter of 600700 m)making thisadministration easier. Furthermore, Ca-alginatemight also protect thecells against shear forces during the injection process and overloaduntil they form their own functional extracellular matrix in thedefective site. In another study, Abbah et al. [191] investigated theeffect of connement within calcium cross-linked alginate micro-capsules on the survival of murine adipose-tissue stromal cells (ATSC)with osteogenic potential and their subsequent ability to elicitosteogenic response. It is important to emphasize that thesemicrocapsules were superior for murine ATSC proliferation andosteogenic differentiation when compared to the 2D monolayerplastic tissue culture surface. Similar results have been obtained bythese authors using rabbit bone marrow cells [192].

Moreover, MSCs can be genetically engineered to over-express therequired protein. Thus, using this ex vivo gene therapy approach, themicrocapsule containing the cells can be both a growth factor deliverycarrier and a 3Dmatrix for cellular activities in repair. Previous studieshave demonstrated that bone morphogenetic proteins (BMPs),especially BMP-2, are the most effective in inducing complete bonemorphogenesis [193,194]. In the MSCs expressing BMP-2 an impor-tant advantage accrues to the MSCs, since the genetically engineeredcells feature not only a paracrine effect on the host, but also anautocrine effect on the MSCs themselves [195]. Zilberman et al. [8]immobilized adult MSCs expressing rhBMP-2 within APA microcap-sules and studied the effect on mice. After subcutaneous administra-tion of capsules a physiological responsewas elicited and formation ofectopic cartilage and bone in the host was observed. The authorsconcluded that the angiogenic and osteogenic activities observedoutside the capsules are consequences of the paracrine effect of theengineered MSCs. In a parallel experiment, when encapsulated cellswere transplanted into local segmental bone defect, they were alsoable to form massive bone tissue in the defect. The bone in this casecomprised the host cells' response to the paracrine effect of thesecreted rhBMP-2. However, either during subcutaneous or bonedefect administration, encapsulated MSCs differentiated inside thecapsules mainly to cartilage cells. Therefore, microencapsulation ofgenetically engineered MSCs can be a useful tool to study anddistinguish between autocrine and paracrine mechanisms andintercellular interactions.

This approach based on genetically engineered cells to releasegrowth factors has also been assayed for cartilage regeneration. In aninteresting study, Paek et al. [196] examined the survival and themaintenance of funcitonality of microencapsulated genetically mod-ied broblasts in allogenic and xenogenic models. The growth factorreleased from Ca2+-alginate immobilized cells was human transform-ing growth factor-1 (hTGF-1). This substance is of particularimportance in intraoperative procedures for cartilage regenerationbecause it can induce chondrogenic differentiation or synovial cells.Both allogenic and xenogenic transplants could survive and maintainthe hTGF-1 secretory function in mice during 3 weeks. This period oftime is long enough since therapy format of intraoperative cartilage

repair envisions only 1-week in situ delivery of hTGF-1.

721R.M. Hernndez et al. / Advanced Drug Delivery Reviews 62 (2010) 711730A new design to obtain better cell-based therapies for boneregeneration involves co-immobilization of human osteoprogenitorsand endothelial cells within alginate microspheres. Together withosteoprogenitor cells, endothelial cells can regulate their osteogenicpotential in bone defects. Recent studies have already shown thatosteoblastic differentiation is improved by endothelial cells in 2Dculture systems [197,198]. In an elegant study, Grellier et al. [199] co-immobilized these two types of cells within RGD-modied alginatemicrocapsules. Both in vitro and in vivo studies revealed thatosteoprogenitor cells enhanced their mineralization potential whenthey were co-encapsulated with endothelial cells, thus setting up apromising new injectable strategy for bone tissue engineering.

Other approaches currently under investigation for their potentialto repair bone and cartilage include the use of cell-seeded micro-carriers [200]. Polymeric microcarriers made up of PLGA have been

Fig. 3. (A, B) Confocal laser-scanning and differential interference microscope images ofimmobilized with streptavidin-immobilized HEK293 cells. The HEK293 cells were labeled wculture at 0 and 1 days. HEK293 cells were immobilized on the surface of the islets and culturcells. (E, F) Histochemical analysis of HEK293 cell-immobilized islets cultured for 3 and 5 day488-labeled anti-insulin antibody and Hoechst 33342 dye for nuclear staining. The pictupermission, from Ref. [180] 2009 Elsevier Ltd.shown to be suitable as an injectable delivery system for chondro-cytes. In a recent report [201] PLGA/gelatin microspheres modiedwith RGD peptides were used to culture chondrocytes in vitro. Theresults observed were found to be particularly interesting due the factthat RGD sequences signicantly improved attachment, proliferationand viability in addition to glycosaminoglycan secretion fromchondrocytes. Bioceramics [202], calcium titanium phosphate [203]and hydroxyapatite [204], among others, have been proposed as celldelivery systems. These materials have osteoconductive nature andare extensively used in bone reconstruction.

In a recent paper Wang et al. [205] have proposed a novel gellangel-based microcarrier for anchorage-dependent cell delivery. In thisstudy, the gellan microspheres were covalently coated with gelatinlayers to create the cell binding ligands on which human cells,including broblasts and osteoblasts, were cultivated for appraisal of

surface-modied cells and islets. Hamster islets modied with biotinPEGlipid andith CellTracker. (C, D) Phase-contrast microscopy of HEK293 cell-immobilized islets ined on a non-treated dish in Medium 199 at 37 C. Arrows indicate immobilized HEK293s in medium. Frozen sections of HEK293 cell-immobilized islets were stained with Alexares are merged images from insulin and Hoechst 33342 staining. Reproduced, with

722 R.M. Hernndez et al. / Advanced Drug Delivery Reviews 62 (2010) 711730cell delivery and developmental efcacy. The in vivo results [206]suggest that these microcarriers, combined with a hydrogel, facilitateboth survival and differentiation of osteoprogenitor cells, whilemaintaining their favorable spread morphology in hydrogel matrices.This novel composite system could be benecial to clinical regener-ative medicine in the eld of bone engineering. Moreover, specicgrowth factors or extracellular matrix proteins can be included in themicrocarrier to further aid proliferation and differentiation [207].Hence, microcarriers can play a dual role as both delivery systems ofbioactive factors and scaffolds for proliferation and differentiation ofcells.

In conclusion, microcarriers made from awide range of establishedand novel biomaterials are being evaluated for their ability to facilitategrowth and differentiation, in addition to their capability to meet thecriteria for resorption post tissue-implantation. This is the drivingforce for emerging opportunities and the further application ofthe microcarrier culture in the eld of bone and cartilage tissueengineering [200].

5.3. Neurological diseases

Human neurological disorders such as Pakinson's disease (PD),Huntington's disease (HD), amyotrophic lateral sclerosis (ALS),Alzheimer's disease, stroke, and spinal cord injury (SCI) are causedby a loss of neurons and glial cells in the brain or spinal cord. In recentyears, the transplantation of cells to the brain and/or spinal cord hasbeen pursued as a potential curative treatment for a broad spectrumof human neurological diseases [208,209]. Promising results havealready been achieved in clinical trials, but much remains to be donebefore cell-based therapies can be practiced extensively.

One source of transplantable cells is the choroid plexus (CP). Inaddition to its well-dened role in cerebrospinal uid (CSF)production and maintenance of extracellular uid concentrationsthroughout the brain, the CP secretes numerous endogenousneurotrophic factors with therapeutic potential [210]. These primarycells are currently under study for the treatment of neurodegenerativedisorders. Emerich et al. developed an alginate-based encapsulationsystem where CP were immobilized in an effort to achieve anappropriate delivery of neurotrophic factors to the brain in rodent[211] and primate models of Huntington's disease (HD). In this latterstudy [212], cynomolgus primates received stereotaxic transplants ofeither empty capsules or porcine CP-loaded capsules directly into thecaudate and putamen. One week later, they received unilateralinjections of the excitotoxin quinolinic acid (QA). Researchersreported that QA administration produced a large lesion in both thecaudate and putamen in monkeys receiving implants of emptymicrocapsules. In contrast, the size of the lesion was signicantlyreduced (5-fold relative to control implanted monkeys) in animalsreceiving identical QA lesion but implants of encapsulated CP. It seemsthen that implants of alginate-encapsulated porcine CP prevent thedegeneration of striatal neurons typically occurring after QA intraes-triatal injections. Similar results have been obtained in a rodent modelof stroke [213]. The in vivo studies in a well-established middlecerebral artery occlusion model demonstrated that encapsulated CPsignicantly reduced the extent of cerebral infarction and associatedbehavioral decits.

Another important neurodegenerative disorder to take intoconsideration is Parkinson's disease (PD), characterized by anextensive loss of dopamine neurons in the substantia nigra parscompacta and their terminals in the striatum. An interestingapproach, using gelatin microcarriers (Spheramine), has been testedas a new drug delivery system for the treatment of this disease. Itconsists of an active component of cultured human retinal pigmentepithelial cells (hRPE), attached to a cross-linked porcine gelatinmicrocarrier. In this case immunosuppression was not required

because RPE cells are isolated from postmortem human eye tissue.Currently, it is postulated that the ability of hRPE cells to producelevodopa in the biosynthetic pathway for melanogenesis may serve asthe rationale for a therapeutic effect, but a role of trophic factorscannot be excluded. The studies carried out in PD animal models ofunilateral 6-hydroxydopamine (6-OHDA) striatal-lesioned rats andMPTP-induced hemiparkinsonian Macaca mulatta monkeys deter-mined the preclinical efcacy of hRPE cells [214,215]. Afterwards, in aPhase I clinical trial in humans the safety and efcacy of Spheramineimplanted in the putamen of patients with advanced PD wasevaluated [61]. However, the therapeutic potential of this platformhas been questioned due to the recently announced failure of thecommercial sponsored Phase II clinical trial [216]. While an in-depthanalysis of this disappointing result must await publication of thedetails of this trial, Falk et al. postulated that the failure to account forthe role of neurotrophic factors could be an important aspect and theverication of high expression of these factors, and not only forlevodopa should be considered [62].

Another interesting therapeutic application employing geneticallyengineered cells, instead of primary cells includes the immobilizationof growth factor-producing broblast for delivery of therapeuticproducts to the injured spinal cord (SCI). This pathology results in thedisruption of ascending and descending axons that produce adevastating loss of motor and sensory function. Several strategieshave been used to provide trophic and antiapoptotic molecules thatcan alter the environment of the injured central nervous system. Inthis sense, various studies [3,217] have pointed out that primarybroblasts genetically modied to produce brain-derived neuro-trophic factor (BDNF) survived in the injured spinal cord of adultSprague-Dawley rats rescuing axotomized neurons, promoting re-generation and contributing to recovery of locomotor function.However, immunosuppression was needed to prevent rejection ofgrafts. As an alternative, cell microencapsulation could replaceimmune suppression by protecting the cells after grafting. In 2005Tobias et al. [218] reported that alginatepoly-L-ornithine micro-capsules containing BDNF-producing broblasts grafted into a non-immunosuppressed SCI murine model resulted in partial recovery offorelimb usage in a test of vertical exploration and of hindlimbfunction while crossing an horizontal rope. Moreover, compared withthe animals that received unencapsulated BDNF-producing cellswithout immunosuppression, the recovery was signicantly higher.However, results were similar to those of immunosuppressed animalsthat had received unencapsulated cells. The study also showed noevidence of regeneration of rubrospinal axons in mice implanted withencapsulated cells presumably because the amounts of BDNF availablefrom the encapsulated graft were substantially less than thoseprovided by the much larger numbers of cells grafted in the non-encapsulated formulation in the presence of immunosuppression.Taken together, these results suggest that improvements in cellencapsulation systems to release higher concentrations of neurotro-phin are required to stimulate regeneration of axotomized brainstemneurons. Thus, after optimization of the drug delivery system,nonautologous engineered cells immobilized in microcapsules couldbe a feasible approach for recovery of lost function in the injuredspinal cord.

Future efforts will need to systematically approach each potentialclinical indication with emphasis on optimizing the cell source,determining which cells are most benecial, identifying the optimalpost injury timing, transplant location and dosage of cells to begrafted. In addition, reducing the diameter of cell-enclosing capsulesdesigning subsieve-size capsules [219], could be an interestingalternative for drug delivery in the CNS.

5.4. Cancer

One attractive approach to the treatment of disseminated cancer is

to enhance the immunogenicity of tumor cells, thus allowing systemic

723R.M. Hernndez et al. / Advanced Drug Delivery Reviews 62 (2010) 711730immune-mediated tumor cell death. Several different immune-mediating products, such as cytokines, assayed in the clinic, resultin enhanced immunity and decreased tumorigenicity. For example, IL-12 and TNF- encapsulated in poly-lactic acid microspheres havebeen implanted intratumorally into an experimental brosarcomamodel (MCA205 cell line) generating a systemic anti-tumor immuneresponse capable of eradicating distant disease [220]. However, themajor disadvantage of these polymeric biodegradable microsphereslies in the fact that multiple capsules would be required to maintaincontinuous delivery. To overcome this problem the use of micro-capsules containing cells secreting cytokines has been proposed.Cirone et al. [7] described a sustained release of IL-2, into tumor-bearing mice, after a single implantation of nonautologous mousemyoblasts (C2C12), genetically modied to secrete the cytokine,immobilized in microcapsules. The treatment with these encapsulat-ed cells led to a delay in tumor progression and prolonged survival ofthe animals. However, the long-term efcacy was limited by aninammatory reaction against the implanted microcapsules probablybecause of the secreted cytokine and the antigenic response againstthe xenogeneic fusion protein itself. Hence, although efcacious insuppressing tumor growth initially, this immune therapy protocolonly produced a transient effect, with the tumors resuming growthafter a delay of 15 days.

Another focus of interest in the treatment of cancer is angiogenesisinhibition. Tumors are dependent for their growthon the developmentof a blood vessel network and may trigger angiogenesis by release ofspecic growth factors such as the vascular endothelial growth factor(VEGF). Thus, the growth of tumors can be suppressed by inhibitingangiogenesis. In 2001 two independent groups [20,221] treated gliomamodels of cancer with encapsulated xenogenically derived cell linesgenetically modied to secrete endostatin, one of the most potentantiangiogenic drugs that can directly induce apoptosis in tumor cells.Both groups reported that local delivery of endostatin signicantlyinhibited tumor growth. Nevertheless, local delivery of drugs mightnot be feasible in the treatment of many tumors, especially formetastasis. In this respect, in a recent study by Teng et al. [222],Chinese hamster ovary (CHO) cells engineered to secrete humanendostatin encapsulated in APAmicrocapsules were transplanted intoperitoneal cavities of mice bearing subcutaneous B16 melanoma. Theresults proved that the intraperitoneally implanted microencapsu-lated cells could signicantly inhibit the subcutaneous growth ofmelanoma in mice. Using a similar approach Cirone et al. [223] testedangiostatin to treat amurinemodel ofmelanoma/breast cancer.Whenangiostatin was delivered systemically by implanting microencapsu-lated cells genetically modied to express angiostatin, tumor growthin the recipient animals was suppressed by N90% 3 weeks post-tumorinduction, while survival at this date was 100%, compared to 100%mortality in the untreated or mock-treated controls.

Currently, simultaneous delivery of cytokines and antiangiogenicdrugs is being explored for cancer therapy. Cirone et al. examined a two-pronged strategy by delivering interleukin 2 fusion protein (immu-notherapy) and angiostatin (antiangiogenic therapy) concurrently viamicroencapsulated cells, to evaluate their potential synergism in tumorsuppression. Twodifferentmethodshave beenused, capsules fabricatedto contain a mixture of both cell types, each type delivering the desiredtherapeutic product [224] or a mixture of different capsules, each onecontaining a single specic cell type [225]. The best results wereobtained with IL-2-secreting cells and angiostatin-secreting cellsencapsulated in separate microcapsules and implanted at differenttimes post-tumor induction. Thus, the use of a combined strategy withmicrocapsules containing cells engineered to release different mole-cules, with anti-tumor properties targeting multiple pathways, opensup new possibilities in the treatment of cancer.

The use of encapsulated cells over-expressing enzymes that canactivate chemotherapeutic agents or prodrugs, offers a promising

mean to treat tumors [226]. The rst demonstration that this methodto prove the use of this method in treating solid tumors was providedin 1998, in a murine model of pancreatic cancer [227]. In this study,feline kidney epithelial cells genetically modied to over-express acytochrome P450 enzyme (CYP) were encapsulated in polymers ofcellulose sulfate. In this study, encapsulated CYP over-expressing cellswere implanted into xenograft tumors and this was then followed bymultiple administrations of the prodrug ifosfamide, a well known andwidely used chemotherapeutic which is activated by CYP. Thiscombined cell therapy product plus chemotherapeutic treatmentwas shown to result in tumor reductions and, in some mice, evencomplete loss of the tumor. The data could be reproduced usingencapsulatedHEK293 cells over-expressing the sameCYP [228]. Basedon these results NovaCaps, an encapsulated cell therapy product, wasdeveloped and tested in a Phase I/II clinical trial. The results of thePhase I/II clinical trial, which involved the treatment of 14 patientssuffering from pancreatic cancer with encapsulated cells, were quitepromising. [229,230]. Therapeutic benet includes a 100% improve-ment inmedian survival over a control group and 1 year survival rates,whichwere almost twice as high as those documented after treatmentwith the current gold standard treatment, gemcitabine. Recently [231],NovaCaps has been designated an orphan drug in Europe by theEuropeanmedicines agency (EMEA)aswell as therst of a newclass oftherapeutics created by the EMEA called advanced therapymedicinalproducts (particularly somatic cell therapy medicinal products).

The ndings reviewed demonstrate the advantages of this deliverysystem, which allow continuous release of biologically active productsfor the treatment of cancer. However, the encapsulated nonautolo-gous cells secrete cytokines and shed antigens, which evoke a hostimmune response and lead to inammatory tissue surrounding thecapsules that may cause damage to the graft. In order to overcome thisshortcoming, stem cells could replace the cell lines commonly used incell microencapsulation. Moreover, as previously mentioned, mesen-chymal stem cells are hypoimmunogenic and can be geneticallymodied to express a variety of therapeutic agents. In a recent work,Goren et al. [144] encapsulated genetically engineered hMSCs toexpress hemopexin like protein (PEX), an angiogenesis inhibitor, inalginatePLL microcapsules and tested the efcacy of the microen-capsulation system in a model of human glioblastoma. The resultsrevealed a signicant reduction in the tumor volume 22 days post-treatment (89%) when compared with the tumor sizes of the controlgroups. On the other hand, immunohistological studies demonstrateda decrease in blood vessel formation and tumor cell proliferation andan increase in tumor cell apoptosis. These ndings indicate thepotential of microencapsulated engineered hMSCs to serve as aplatform for therapeutic applications in several pathologies likecancer.

Finally, immunoisolation of cells to deliver vaccines is anotherconcept in cancer therapy. The administration of recombinant puriedantibodies is an expensive process. Hence, delivery of antibodies byencapsulated cells could be an alternative and more cost-effectivemethod [232].

In conclusion, the use of encapsulated cells has great potential asthe basis for the treatment of various forms of cancer. This section hasattempted to summarize preclinical and clinical data from some of themore promising strategies involving encapsulated cells to treattumors. The authors believe that these results represent a glimmerof hope particularly for those tumors representing an unmet medicalneed.

5.5. Heart diseases

Heart failure is a leading cause of morbidity and mortalityworldwide. The critical cause of heart failure is myocardial ischemia,resulting in dysfunction and death of cardiomyocytes. The maincardiac response to myocardial infarction can be seen as cardiomyo-

cyte hypertrophy, apoptotic myocyte loss, progressive collagen

replacement, and enlargement of the left ventricle [233]. Progenitoror stem cells constitute a new prospect in the confrontation of cardiacinsufciency [234]. The benecial action of transplanted cells is underinvestigation and numerous trials examine their potential activecontribution to myocardial contractility, indirect improvement ofcardiac mechanisms, induced neovascularization, and other likelyactions in the biological operation of the heart [235237]. Themechanism by which stem cell-based therapy causes benecialeffects on cardiac function remains unclear and it may be possiblethat the benecial effects observed in cell therapy are due to multiplemechanisms; one system alone may not account for all observedimprovements in cardiac function. However heart regeneration is stillconfronted with great controversy. Moreover, several delivery routeshave been investigated for cell administration [238], but cell retentionfollowing implantation remains a major challenge in such cases. Theheart constantly contracts which contributes to the mechanical lossby