A Perspective on Bioactive Cell Microencapsulation

A Perspective on Bioactive Cell MicroencapsulationArgia Acarregui,1,2 Ainhoa Murua,1,2 Jose L. Pedraz,1,2 Gorka Orive1,2 and Rosa M. Hernandez1,2

1 NanoBioCel Group, Laboratory of Pharmaceutics, School of Pharmacy, University of the Basque Country (UPV/EHU),

Vitoria-Gasteiz, Spain

2 Networking Biomedical Research Center on Bioengineering, Biomaterials and Nanomedicine, CIBER-BBN, Vitoria-Gasteiz, Spain

Contents

Abstract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284

2. Cell Encapsulation Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284

2.1 Biomaterials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285

2.1.1 Alginate and Functionalized Alginate Gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285

2.1.2 Collagen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286

2.1.3 Chitosan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286

2.1.4 Agarose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286

2.1.5 Other Polymers: Poly(N-isopropylacrylamide), Gelatin, Poly(ethylene glycol), Hyaluronic Acid and Cellulose Sulphate 287

2.2 The Requirements of the Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287

2.2.1 Permeability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287

2.2.2 Mechanical Integrity, Stability, and Durability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288

2.2.3 The Size, Morphology, and Surface Properties of the Capsules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289

2.2.4 Biocompatibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289

2.2.5 Other Issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289

3. Therapeutic Applications and Clinical Trials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290

3.1 Diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290

3.2 Central Nervous System Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290

3.3 Cardiovascular Diseases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293

3.4 Liver Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293

3.5 Other Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 294

4. Concluding Remarks and Future Directions and Challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295

Abstract Bioactive cell encapsulation has emerged as a promising tool for the treatment of patients with various

disorders including diabetes mellitus, central nervous system diseases, and cardiovascular diseases. The

implantation of encapsulated cells that secrete a therapeutic product (protein, peptide, or antibody) within a

semipermeable membrane provides a physical barrier to mask the implant from immune surveillance at a

local level without the need for systemic immunosuppression; this serves to achieve a successful therapeutic

function following in vivo implantation. The aim of this review article is to provide an update on the progress

in this field. The current state of cell encapsulation technology as a controlled drug delivery system will be

covered in detail, and the essential requirements of the technology, the challenges, and the future directions

under investigation will be highlighted. The technical and biological advances, together with the increasing

experience in the field, may lead to the realization of the full potential of bioactive cell encapsulation in the

coming years.

REVIEWARTICLEBiodrugs 2012; 26 (5): 283-301

1173-8804/12/0005-0283/$49.95/0

Adis ª 2012 Springer International Publishing AG. All rights reserved.

1. Introduction

New molecules with high therapeutic effect are currently

being discovered; in some cases, traditional routes of adminis-

tration may not allow suitable delivery or guarantee maximum

efficacy. Many of these therapeutic molecules are peptides that

degrade easily and proteins that have limited half-lives in vivo;

they are especially difficult to administer at therapeutic con-

centrations and for extended periods of time. The purpose of

drug delivery systems is to maintain sustained therapeutic

concentrations with minimal side effects at specific locations in

the body, modifying the pharmacokinetics of the drug. Nu-

merous technologies using biomaterials in the form of fibers,

capsules, particles, three-dimensional porous scaffolds and in-

jectable gels have been investigated to better control the deliv-

ery of these drugs.[1]

The transplantation of encapsulated cells is a potent strategy

to enable the local and controlled delivery of therapeutic mol-

ecules to specific physiological sites as a means to recover a loss

of function caused by disease or degeneration.[2] Delivery could

be performed by cell encapsulation or by immobilization within

a semipermeable membrane.[1] The semipermeable membrane

may isolate the entrapped cells from the host immune system

and control the outward and inward diffusion of molecules.

Consequently, the long-term therapies of immunosuppressant

drugs could be eliminated or at least reduced. In this applica-

tion, cells may be considered ‘biological factories’ or ‘living cell

medicines’ that can continuously produce and release ther-

apeutic molecules in a biosafe manner.[3]

In the present review, drug delivery system strategies relying

on microcapsules, especially approaches based on cell micro-

encapsulation technology, are highlighted to provide a general

overview of the current research. The review focuses on some of

the most frequently used biomaterials and the most important

requirements of the technology. Special attention is paid to

some of the most encouraging therapeutic applications and

current clinical trials. Unresolved issues in the field and future

directions for investigation are discussed.

2. Cell Encapsulation Technology

Tissue and organ transplantation, as well as drug delivery

technologies, have significantly improved during the last few

years. Specifically, the immobilization of cells from an outside

environment within a permselective (semipermeable) mem-

brane barrier protects the inner cells from both mechanical

stress and from the components of the host immune system

(such as immunoglobulins, complement and immune cells); at

the same time, the membrane allows the inward diffusion of

nutrients and oxygen as well as the outward delivery of waste

products and therapeutic agents (figure 1). Moreover, this

membrane permits the transplantation of cells without the use

of long-term modulating and/or immunosuppressive therapies,

which have potentially severe side effects.[4,5] Cell encapsulation

technology can be used for the treatment of a large variety of

diseases, including hypoparathyroidism,[6] hemophilia B,[7,8]

anemia,[9] dwarfism,[10,11] liver failure,[12] central nervous sys-

tem (CNS) diseases,[13,14] diabetes mellitus,[15] cancer,[16] and

cardiovascular diseases.[17]

Cell encapsulation technology displays important advan-

tages over other delivery systems because it permits a sustained

and controlled release of de novo-produced therapeutic mole-

cules; this ensures the bioactivity of the therapeutic. Additional

advantages include the enhancement of biosafety, because the

toxicity caused by a rapid delivery of high concentrations of the

drug does not occur, and host immune system response might

NutrientsO2

Therapeutic moleculesWaste products

Hostimmune

mediators

Fig. 1. A schematic illustration of the encapsulated cells within the semipermeable membrane (dashed line) that allows the bidirectional diffusion of nutrients,

oxygen, therapeutic products and waste while preventing the entrance of immune cells and antibodies.

284 Acarregui et al.

Adis ª 2012 Springer International Publishing AG. All rights reserved. Biodrugs 2012; 26 (5)

attack the cells (compromising their survival) if they manage to

leave the microcapsule environment. Moreover, the genetically

modified cells can express any protein desired for in vivo ther-

apy without the modification of the host genome.[18] It is

possible to create small capsules (from 100 mm to 500 mm) and

implant them in close contact with the blood stream, improving

oxygen transfer into the cells, whichmay be beneficial in certain

applications. Furthermore, in comparison with other immobi-

lization systems, the high surface/volume ratio of the micro-

capsules increasesmembrane permeability, ensuring an optimal

product exchange to ensure adequate cell viability. Finally,

the microcapsules can be injected directly or transplanted

during minimally invasive surgery into implantation sites

such as the peritoneal cavity[19,20] and subcutaneous tissue,[21]

reducing the frequency of administration and thus improving

patient comfort.

The most widely employed cell microencapsulation method

was originally developed by Lim and Sun[22] and is based

on the polyelectrolyte complexation of alginate with a

polycation, normally poly-L-lysine (PLL) or poly-L-ornithine

(PLO).[20] These types of microcapsules are commonly referred

to as alginate-PLL-alginate (APA) or alginate-PLO-alginate

(APO) microcapsules, respectively. Apart from microcapsules

formed with these polycations, other polymers, such as chito-

san,[23] lactose-modified chitosan,[24,25] oligochitosans,[26,27]

poly(methylene-co-guanidine),[28] sodium silicate,[29] andmulti-

layers,[30-32] have also been employed to cover the alginate

cores. The drawback of these materials is that they are asso-

ciated with cytotoxicity and cell necrosis, which has generated

much controversy among research groups. Comparative stud-

ies using different polycations have alleviated some of these

concerns, but it is still unclear which of these strategies is the

best for cell encapsulation.[33,34] In recent decades, the initial

technique has undergone modifications, leading to several

methods to produce microcapsules, including extrusion meth-

ods,[35,36] emulsion (thermal gelation),[37] microfluidics,[38,39]

and microlithography.[40]

2.1 Biomaterials

Using an appropriate encapsulation biomaterial is one of the

most important requirements in the development of encapsu-

lated cells. These biomaterials play important roles, providing

the three-dimensional and synthetic extracellular environments

that mimic certain beneficial properties of the extracellular

matrix (ECM) and improving immune protection by isolating

entrapped cells from the host tissue.[41] These materials should

have the ability to form a gel in mild and physiological con-

ditions (in terms of temperature, pH, and ionic strength). In

addition, they must be biocompatible and should not interfere

with cellular function. Although the majority of the literature

has employed sodium alginate,[42] other types of natural and

synthetic polymers, including collagen[43] and hyaluronic acid

(HA)[44] or alginate in combination with chitosan,[45] agar-

ose,[46] gelatin,[47] and tyramine,[48] have been explored in the

field of cell encapsulation.

2.1.1 Alginate and Functionalized Alginate Gels

One of the reasons alginate has been so widely employed is

that it allows gel formation in minimally harmful conditions,

which is an attractive attribute for cell encapsulation.[49,50] The

most commonmethod to gel alginate is to combine the aqueous

alginate solution with ionic crosslinking agents such as divalent

cations (i.e. Ca2+). The divalent cations are believed to bind

solely to the G blocks of the alginate chains. Thus, the G blocks

of one polymer form junctions with the G blocks of adjacent

polymer chains in what is termed the egg-box model of cross-

linking[51] (figure 2). However, Donati et al.[52] have reported

similar characteristics of the mixed junctions formed between

alternating M and G blocks (MG blocks). Other studies have

shown that Ba2+ can also bind to M chains.[53] Several func-

tional and mechanical properties of the alginate microcapsules,

including their biocompatibility, biodegradability, mechanical

and chemical stability, and controllable swelling properties,

depend on the alginate composition and concentration, the type

and concentration of the cations added to induce gelling and

polycations.[34,54-56]

Recently, the functionalization and modification of algi-

nates with various peptides and proteins to provide control over

the cell fate is gaining significant attention. These functional

groups activate the intracellular signaling cascades through the

focal contacts that provide tight control over the cell-matrix

interactions.[57] The Arg-Gly-Asp (RGD) peptide sequence

derived from fibronectin is a natural protein sequence present

in the ECM.[58] Alginate itself does not support cell attach-

ment[59] but it is easily modified by the simple coupling of cell

Ca2+ ( )

Fig. 2. Alginate hydrogels prepared by ionic crosslinking (egg-box model).

Only guluronate blocks participate in the formation of a corrugated egg-box-

like structure with interstices in which calcium ions are placed. Reproduced

from Lee and Mooney (ª 2011),[42] with permission from Elsevier.

A Perspective on Bioactive Cell Microencapsulation 285


adhesion domains such as the RGD via carbodiimide chem-

istry. As a result of such a coupling, the interaction between

the alginate and the integrin receptors of the enclosed cells

is improved, enhancing cell survival and functionality.[60-64]

Alginate gels have also been functionalized with the Asp-Gly-

Glu-Ala (DGEA) and Tyr-Ile-Gly-Ser-Arg (YIGSR) sequences

derived from other ECM proteins to enhance the gels’ adhesive

interactions with various cell types.[65,66] In a recent study,

Mazzitelli et al.[67] have developed alginate-based micro-

capsules containing the ECM (isolated and purified from the

urinary bladder [UBM]) and Sertoli cells (SCs). The combina-

tion of alginate and the ECM resulted in a synergistic activity of

both materials. The microcapsules offer the mechanical and

material properties of the alginates, which can be controlled

with the consideration of some issues; at the same time, the

ECM increases the bioactivity of the microcapsules to modu-

late and improve the viability and function of the encapsulated

cells. Biomimetic microcapsules using RGD-alginate have also

been employed. These capsules performed the cell adhesion for

the enclosed cells and prolonged their long-term functionality

and drug release for more than 300 days,[68] suggesting that

these capsules promote the in vivo long-term functionality of

the enclosed cells and that the mechanical stability of the cap-

sules is improved.

Another attractive modification is focused on the control of

the biodegradation rate of the alginates under physiological

conditions. An example of such a modification is the partial

oxidation of the alginate chains so that they become sensitive to

hydrolysis.[69,70] The degree of this oxidation as well as the pH

and temperature of themedia influences the degradation rate of

the gels.[71] In a recent study, mesenchymal stem cells (MSC)

were entrapped in oxidized alginate-fibrin microbeads to ach-

ieve a rapid degradation profile and the release of the cells in an

effort to promote bone engineering.[72]

In addition, alginate may also be covalently crosslinked via

the addition of bi-functional molecules such as poly(ethylene

glycol) [PEG] or methacrylate moieties to the alginate chains.[73,74]

The selective modification of the alginate resulted in an en-

hanced covalent stabilization and an increased mechanical

strength of the alginate beads.

2.1.2 Collagen

Collagen is the main component of the ECM of the mam-

malian connective tissues. Some of the advantages it offers in-

clude its biocompatibility, abundance in nature, natural ability

to anchor cells anddegradabilitymediated bymetalloproteases. Its

gelation can be induced by changes in pH, allowing cells to be

encapsulated under mild conditions.[75,76] However, the chal-

lenges of using collagen as a material for cell immobilization

include the high cost of its purification, the natural variability

of the isolated collagen, its immunogenicity, and the variation

in its enzymatic degradation depending on the implant site.

A novel drug delivery system based on collagen-alginate com-

posite structures has been developed to prevent the cell leak-

age, maintain the cell growth, and control the protein secretion

of glial cell-derived neurotrophic factor (GDNF)-secreting

cells.[77] Collagen has been used to engineer a variety of tissues,

including skin,[78] bone,[79,80] heart valves,[81] and ligaments.[82]

2.1.3 Chitosan

Chitosan is a chitin isolated from the exoskeleton of shellfish,

and it has a structure similar to natural glycosaminoglycans. It

can be formed into hydrogels by ionic interaction[83] and

chemical crosslinking with glutaraldehyde.[84] Scaffolds made

of this material can be degraded by lysozymes. Zielinski and

Aebischer[85] encapsulated a genetically modified fibroblast cell

line in chitosan hydrogels to provide the sustained release of

nerve growth factor (NGF) and showed that the amount of

NGF secreted was sufficient to induce the differentiation of co-

cultured neural progenitor PC12 cells. Thermosensitive chito-

san has also been investigated as a material to create an aligned

cell sheet[86] and to encapsulate MSCs and disc cells.[87] In a

recent study, a bi-continuous morphology scaffold for the re-

pair of load-bearing soft tissues was designed and charac-

terized. A crosslinked chitosan hydrogel was used to distribute

the cells and enable cell growth, and an elastomer prepared

from a star-poly (e-caprolactone-co-D,L-lactide) triacrylate

was used to enhance the mechanical properties of the hydrogel.

The chondrocytes within the bi-continuous scaffolds prolif-

erated, exhibited increasedmetabolic activity, and accumulated

an ECM over a 14-day culture period.[88]

2.1.4 Agarose

Agarose is another algae-derived polysaccharide, but unlike

alginate, the formation of its gel microbeads is thermally re-

versible.[45] Its most appealing qualities include the ease of its

processing and a wide range of mechanical (e.g. matrix stiff-

ness) and structural (e.g. pore size) possibilities,[89] which are

influenced by its concentration in the gel and the cooling

rate.[90] There have been many studies involving the enclosure

of various cell types in agarose microparticles.[91-94] In a recent

study, porcine islets were encapsulated in agarose macrobeads

and transplanted into spontaneously diabetic biobreeding (BB)

rats. Normoglycemia was initially restored in all of the islet-

transplanted rats; moderate hyperglycemia developed approx-

imately 30 days after the transplantation and continued



throughout the study period. All the rats that received encap-

sulated porcine islets continued to gain weight and did not

require exogenous insulin therapy for the entire study.[94] In ad-

dition, agarose canbemodified to include cell-adhesionmoieties to

transform the polymer into a photolabile substrate that can be

controlled with biochemical cues.[95] A possible problem re-

ported in the use of this biomaterial is cellular protrusion.[96]

2.1.5 Other Polymers: Poly(N-isopropylacrylamide), Gelatin,

Poly(ethylene glycol), Hyaluronic Acid and Cellulose Sulphate

Poly(N-isopropylacrylamide) [pNIPAM] is a thermores-

ponsive and biocompatible polymer with a quick phase tran-

sition and a low critical solution temperature (approximately

32�C).[97,98] RGD-conjugated pNIPAM hydrogel microcarriers

produced using calcium alginate as a temporary mold have

been employed as cell culture substrates for enzyme-free chon-

drocyte detachment.[99] In addition to cell adhesion applica-

tions, cell encapsulation using pNIPAM particles has also been

reported. In this case, pancreatic cells were encapsulated into

pNIPAM-based microcapsules that exhibited a reversible sol-

to-gel transition at 30�C.[100] In another approach, pNIPAM

thermoresponsive hydrogels have been used to immobilize a

chondrogenic cell line, revealing that the biomaterial did not

affect the cell viability or proliferation. The results also dem-

onstrated an increase in the synthesis of glycosaminoglycans

during the culture period. This study supported the hypothesis

that thermally reversible pNIPAM hydrogel may be suitable as

an injectable cell carrier and has great potential for cartilage

repair.[101]

Gelatin is obtained by the partial hydrolysis of collagen

derived from the skin, white connective tissue, and bones of

animals. As a natural macromolecule, its major limitation is the

variability caused by its derivation from animal tissue, making

batch-to-batch reproducibility difficult. This problem could be

solved by the use of recombinant gelatins with defined molec-

ular weights.[102] The formation of gelatin cell microparticles is

based on crosslinking reactions.[103,104] Examples of gelatin

microcarriers include Spheramine�[105] and Cultispher�.

Cultispher� gelatin microcarriers (Percell Biolytica AB, Sweden)

are commercially available products and have been widely

evaluated as gelatin cell carriers. They are composed of type-A

porcine gelatin particles crosslinked to form a matrix. These

microcarriers supported the in vitro growth of variousmammalian

cells and have also been used as biodegradable scaffolds for guided

tissue regeneration and cell-based therapies in vivo.[106-108]

Other biomaterials employed for cell encapsulation in the

form of hydrogels include PEG combined with acrylates and

methacrylates, HA, and cellulose sulphate.[109-115] Despite the

fact that none have been characterized and studied as much as

alginate, we could benefit from the advantages that these bio-

materials might offer in the development of alternative cell-

based therapeutic strategies.

2.2 The Requirements of the Technology

During the past few years, important advances have been

achieved in the field of cell microencapsulation. These im-

provements in the technological properties have provided high

levels of permselectivity and structural stability that lasts

through the desired lifetime of the graft. To transition cell en-

capsulation to large-scale clinical applications, several re-

quirements must be considered.[116,117] In this section, we will

discuss the most important requirements of the cell encapsu-

lation technology with regard to alginate microcapsules.

2.2.1 Permeability

The permeability of the membrane is one of the most im-

portant requirements in the development of cell microcapsules.

As previously mentioned, this membrane must be able to con-

trol the influx rate of molecules essential for cell survival

(oxygen and nutrients) and the efflux rate of therapeutic fac-

tors, metabolites, and waste products, while completely ex-

cluding the harmful components of the host immune system

(antibodies and immune cells). Thus, the viability of the im-

mobilized cells will depend on the semipermeablemembrane.[96]

One of the factors that influences the diffusion and perme-

ability of the capsules is the rate of solute diffusion, which is

usually controlled by size-exclusion phenomena and diffusion

rate. The other factor is the membrane exclusion properties

reflected by the exclusion limit or the molecular weight cut-off

(MWCO). In APA microcapsules, using a PLL with a molec-

ular weight of 12–22 kDa, the MWCO (the size of the largest

molecule that is not essentially blocked by the semipermeable

membrane) is approximately 70 kDa. With this MWCO, the

membrane will exclude leukocytes and various immunoglobulins

such as IgM (950 kDa) and IgG (150 kDa) but will allow the

diffusion of the low molecular weight substances necessary for

cell survival (e.g. glucose [180Da], carbon dioxide [44Da] and

the secretory proteins from the cells including albumin

[66 kDa], growth factors [6–50 kDa] and insulin [6 kDa]).[116]

Table I shows the molecular weights of various enzymes, anti-

bodies, complement components, and common metabolites.

A successful method for the optimization of the permeability

of alginate beads is to modify the G and M blocks. The per-

meability of the capsules increases with increasing ratios of G

residues. This effect is directly connected to the character of the



long, stiff G blocks and the short, flexible MG blocks in the

high G ratio alginates.[118] In contrast, it was reported that

the reduction of the membranes’ pore size by increasing of the

alginate concentration could ultimately elicit a reduction in

the rate of diffusion.[119] Another approach to modulating

the permeability of themicrocapsule membrane is to change the

contact time with the polycation solution. Increasing the con-

tact time of the alginate beads with PLL solution to more

than 5 minutes significantly decreased the membrane perme-

ability.[120] In this case, the increased number of ionic bonds

between alginate and PLL results in an increased membrane

crosslinking density. Nevertheless, increasing the gelling time

did not change the pore size distribution and, consequently,

permeability was not altered.[15] Multilayered membranes, in

which the properties of each layer can be controlled inde-

pendently, have been developed to achieve the requirements of

permeability, biocompatibility, and suitable mechanical strength

of the capsules. Various approaches have achieved these re-

quirements through the use of alternating layers of alginate and

various combinations of polycations and polyanions[121] or the

use of three-layered agarose microcapsules.[122] The multi-

layered technique has also been used to ensure the complete

immunoisolation of the encapsulated islets through local-

ization of their biological functionalities within the PEG

capsules.[123]

It is essential to include the diffusion rate of the chosen

solute, the pore size, the pore size distribution, the chemistry of

the membrane and solutes, and the thickness of the membrane

in describing the permeability properties of a microcapsule.

However, these features are only rarely documented, making it

difficult to compare the permeability results between different

laboratories. Thus, the authors believe this topic requires fur-

ther investigation.

2.2.2 Mechanical Integrity, Stability, and Durability

The mechanical resistance of the microcapsule is usually

related to the configuration and chemical structure of the

semipermeable membrane. To bear the compression forces and

shear stresses at the implantation site, the semipermeable

membrane should provide an optimum mechanical strength as

for stiffness (resistance to deformation) and toughness (resis-

tance to fracture).[124]

The type of biomaterials employed for the formation of the

matrix and membrane, the type of gelling ion and the encap-

sulation technology are some of the main parameters that

control the mechanical resistance of the microcapsules.

There are various assays to quantify the mechanical resis-

tance, including osmotic pressure tests,[125,126] surface texture

analysis,[127] optical tweezers,[128] micropipette aspiration,[129]

atomic force microscopy (AFM),[130] magnetic bead measure-

ment techniques,[131] and microelectromechanical (MEMS)

microgrippers.[132] Nevertheless, one of the greatest difficulties

is the lack of standardization in the technology to measure the

mechanical integrity, stability, and durability of themicrocapsules.

Table I. The molecular weights of various enzymes, antibodies, complement components and common metabolites presumed to be excluded from or

permeate through alginate poly-L-lysine alginate microcapsules

Molecules presumed to be excluded

from APA microcapsules

Molecular weight (Da) Molecules presumed to permeate

through APA microcapsules

Molecular weight (Da)

Immunoglobulin M 950000 Albumin 66248

Complement C4 210000 Hemoglobin 64000

Complement C5 195000 Tumor necrosis factor 51 000

Immunoglobulin E 190000 Nerve growth factor 13 000

Complement C3 185000 Insulin 9 000

Immunoglobulin A 170000 Complement 3a 5 733

Complement C2 170000 Complement 5a 4 000

Complement C8 163000 Glucose 180

Immunoglobulin D 160000 Tyrosine 163

Immunoglobulin G 150000 Phenylalanine 147

Complement C6 110000 Glutamine 128

Complement C7 100000 Asparagine 114

Complement C9 79000 Creatinine 113

Oxygen (O2) 32

APA= alginate-poly-L-lysine-alginate.



The challenge is to determine the optimum mechanical resis-

tance for a specific application without modifying other rele-

vant capsule properties.

2.2.3 The Size, Morphology, and Surface Properties of the Capsules

The uniformity of the size and the shape ofmicrocapsules are

far-reaching characteristics because they could affect the in vivo

immune response to the microcapsules. Thus, to warrant a

correct diffusive mass transport, the overall diameter of the

microcapsules should not exceed 300–400 mm.[133] Beneficial

effects, including a better molecular exchange between the en-

closed cells and the surrounding environment as well as a higher

mechanical resistance and improved biocompatibility, could be

amended by reducing the diameter of the cell-enclosing micro-

capsules.[134-137] In fact, when the microcapsule diameter is too

large, it is difficult for the cells in the center of the core to survive

because the diffusion of oxygen and nutrients is limited.[138,139]

Other advantages of these small capsules are the ease of

their implantation into reduced sites such as the CNS[140] and

the eye.[141]

During the last few years, various technologies to produce

small particles (100–200 mm) have arisen, such as coaxial flow

techniques,[134,141,142] microfluidic technologies,[143] and a flow-

focusing system.[141] In a recent study, Santos et al.[141] devel-

oped a promising strategy to form monodisperse 100 mm APA

microcapsules using the flow-focusing system, and implanted

them successfully in the intravitreous space in vivo. After opti-

mizing all of the technical parameters required to produce

the small APA microcapsules, they showed that the cell via-

bility, cell proliferation, and vascular endothelial growth factor

soluble receptor (KDR) secretion were not affected by the

physicochemical changes arising from such a dramatic size re-

duction. Moreover, a strong signal was obtained from the

monitored encapsulated cells during the 3-week in vivo assay,

confirming the viability of the enclosed cells. The challenges

related to these small microcapsules include determining the

lowest cell load required to obtain a 100% encapsulation yield,

evaluating the changes in cell viability, and optimizing the ad-

ministration protocol to achieve the maximal dose within the

smallest administration volume.

2.2.4 Biocompatibility

Another important property that should be considered is the

ability of a biomaterial to result in an appropriate host response

in a specific application. This requirement is related to both the

interaction between the biomaterials and the host system and

the interaction between the biomaterial and the encapsulated

cells.[144] The biocompatibility of microcapsules is a complex

property that involves many characteristics, including material

composition, structure, morphology, degradation, and me-

chanical properties.[50,145,146]

In recent years, various assays have been proposed to

evaluate biocompatibility.[147-150] In a recent study, de Haan

et al.[150] studied the physicochemical changes caused by the

exposure of alginate beads and alginate-PLL capsules prepared

from various types of alginate to human peritoneal fluid by

applying micro-Fourier transform infrared spectroscopy. They

observed the adsorption of components from human peritoneal

fluid and physicochemical changes to the surface that are de-

pendent on the chemical composition of the capsules. Other

studies have reported that the use of intermediate-G alginate in

combination with calcium for the gelation process and PLO as

the polycation resulted in less immune cell adhesion. The period

of membrane formation, during which the polycation diffuses

and binds the alginate gel, governs the biocompatibility of

microcapsules and their physicochemical properties.[34,151] To

prevent and reduce an inflammatory response, some studies

have co-administered anti-inflammatory drugs along with the

encapsulated cells.[20,152-155] In a recent study, Murua et al.[153]

subcutaneously implanted a composite delivery system con-

sisting of erythropoietin (Epo)-secreting myoblasts immobi-

lized in APA microcapsules and dexamethasone-releasing

poly(lactic-co-glycolic acid) [PLGA] microspheres (figure 3).

This composite system enhanced the biocompatibility and

generated an immunoprivileged local environment to limit the

fibrotic layer caused by inflammation. Nevertheless, the major

challenge is to design standardized protocols that facilitate the

identification of alginate properties and the evaluation of bio-

compatibility; this will establish the criteria for alginate se-

lection and purification and thus result in reproducible results

between research groups.

2.2.5 Other Issues

The protection from the immune response that APA mi-

crocapsules provide to the encapsulated cells has allowed the

use of xenogeneic cells from non-human sources. The choice of

cells depends on the intended therapeutic application, such as

the secretion of a naturally bioactive substance (e.g. neuro-

transmitter, cytokine, growth factor, growth factor inhibitor,

or angiogenic factor), the metabolism of a toxic agent, or the

release of an immunizing agent.[3]

Tracking and monitoring the cell-containing microcapsules

in vivo is another requirement that needs to be resolved before

these products can reach large-scale clinical trials. Towards this

aim, various research groups have used non-invasive imaging

techniques, including X-ray,[156] magnetic resonance,[156,157]



and bioluminescent imaging.[158] In a recent study, Catena

et al.[158] incorporated the triple reporter gene TGL into

microencapsulated cells to control the location of the cells

and their viability by luminometry. They were able to interrupt

the therapy by the administration of ganciclovir. This system

may represent an appropriate tool to tightly control micro-

encapsulated cells and, moreover, to improve the biosafety of

this type of drug delivery system.

3. Therapeutic Applications and Clinical Trials

Finally, we will describe the most recent therapeutic appli-

cations and clinical trials to provide the readers with a general

overview of the current research area.

3.1 Diabetes

Diabetes mellitus is a metabolic disorder characterized by

hyperglycemia resulting from defects in insulin secretion, in-

sulin action, or both. A possible approach for the safe and

effective cure of these patients is the replacement of the dam-

aged islets of Langerhans.[159] However, the restricted avail-

ability of cadaveric human donor pancreases, coupled with the

need for life-long, general pharmacological immunosuppression,

makes the application of this therapy difficult. The many

contributions of these studies are shedding light on the main

challenges of the encapsulation technology.[160-166] For exam-

ple, an analysis of the global gene (mRNA) and selected

microRNA expression profiles was performed on the islets

derived from a human cadaver after decapsulation of the

cells. The study demonstrated that the tissue culture and

encapsulation process did not affect the viability of the cells.

Although some genes involved in the inflammatory response

were up-regulated, protein expression was not altered by the

encapsulation.[167]

In a recent study, SCs entrapped in alginate-based micro-

capsules induced the reversal of spontaneous diabetes by cre-

ating newly formed functional islets b-cells in the treated

non-obese diabetic mice.[168] Other researchers demonstrated

that the survival of the islet cell aggregates within the micro-

capsules in low-oxygen culture and during transplantation was

superior to that of the intact islets.[169] Opara et al.[170] have

described the new design of a bioartificial pancreas comprising

islets co-encapsulated with angiogenic proteins in permselec-

tive multilayer alginate microcapsules. In this study, the inner

alginate layer encapsulates the islets and the outer layer en-

capsulates the angiogenic proteins, which would otherwise in-

duce neovascularization around the graft within the omentum

pouch. Currently, stem cells from various sources and differ-

entiated into insulin-producing cells (IPCs) are also employed

to treat diabetes.[163,171,172]

3.2 Central Nervous System Diseases

The use of cell-loaded capsules implanted into the damaged

brain area is an interesting approach to the treatment of

CNS diseases requiring chronic administration of therapeutic

products.

Parkinson’s disease is a neurodegenerative disorder charac-

terized by the extensive loss of the dopamine neurons of

the substantia nigra pars compacta and their terminals in the

striatum. Several studies have been carried out using various

types of cells encapsulated in alginate microcapsules in animal

models of Parkinson’s disease; the most widely employed growth

factors in the treatment of this disease are GDNF[173-176] and

vascular endothelial growth factor (VEGF).[177-179] In one of

these studies, GDNF-secreting fibroblasts were immobilized in

Subcutaneous space: immunopriviledged environment

APA microcapsules embedding Epo-secreting C2C12 myoblasts

Dexamethasone-loaded PLGA microspheres

Fat tissue

Fig. 3. Schematic illustration of the immunomodulatory environment

created in the subcutaneous space of implanted mice. Reproduced from

Murua et al. (ª 2011),[153] with permission from Elsevier. APA =alginate-poly-L-lysine-alginate; PLGA =poly(lactic-co-glycolic acid).



APA microcapsules and implanted into the striatum of

6-hydroxydopamine (6-OHDA) lesioned rats. These micro-

capsules were able to deliver continuous and reliable levels of

GDNF for at least 6months, resulting in substantial behavioral

improvement and good biotolerance.[174] In general, most of

these studies conclude that GDNF and VEGF are more effec-

tive when administered before the induction of the lesion,

thereby exhibiting more of a neuroprotective than a neuro-

rescue effect. In a recent study by Skinner et al.,[13] neonatal

porcine choroid plexus (CP) cells were encapsulated within

alginate-PLO capsules and implanted into the striatum of

6-OHDA lesioned rats. They have shown that the neuro-

restorative proteins delivered by the encapsulated cells improve

the motor behavior and the nigrostriatal dopaminergic activity

of the lesioned rats.

The co-implantation of cells that induced functional recov-

ery and regeneration of the lesioned area combined with en-

capsulated cells that released various types of growth factors

with the aim of improving and increasing the survival of

transplanted, unencapsulated cells after transplantation has

also been studied.[180-184] Shanbhag et al.[184] developed a novel

alginate construct that acts as a multifunctional tissue scaffold

for CNS repair and as a localized growth factor delivery system.

In this alginate construct, they encapsulated genetically modi-

fied brain-derived neurotrophic factor (BDNF) or neuro-

trophin-3 (NT-3)-secreting fibroblasts, and neural progenitor

cell (NPCs) were seeded on the surface of the system. They

showed that the surface of the alginate construct acted as an

optimal growth environment for theNPC attachment, survival,

migration, and differentiation, and the continuous delivery of

BDNF or NT-3 directly influenced the lineage fates of the

NPCs.

Another interesting therapeutic application employing the

immobilization of genetically engineered cells to produce

growth factors is Alzheimer’s disease (AD).[140,185,186] AD is a

progressive neurodegenerative disorder of the elderly, charac-

terized by elevated levels of amyloid b-peptide (Ab) in the brain

and associated with neuronal and vascular toxicity; it is the

most prevalent form of dementia. Recently, Spuch et al.[140]

implanted microencapsulated fibroblast cells releasing VEGF

into a transgenic (APP/PS1) mouse model of AD with degen-

erative changes in the microvasculature. The study reported

an improvement in brain Ab removal and protection of the

cognitive behavior in the APP/PS1 mice after implantation

of themicrocapsules. In addition, they confirmed the promising

therapeutic advantages of VEGF therapy for brain angiogen-

esis, neuroprotection, and the cerebromicrovascular exchange

of substrates and nutrients; the implantation of VEGF-secreting

cells entrapped in microcapsules was presented as a therapeutic

tool for the treatment and prevention of brain amyloidosis.

Other similar approaches include the use of recombinant cells

secreting ciliary neurotrophic factor (CNTF) encapsulated

in alginate microcapsules that were implanted into the brain

of two different AD mouse models: the Ab oligomer-infused

and Tg2576 models. It was reported that the CNTF stimulated

a Janus kinase/signal transducer and activated a transcription-

mediated survival pathway that prevented synaptic and neu-

ronal degeneration; an improvement in both animal models

was observed after the implantation of these alginate micro-

capsules. In the Ab oligomer-infused murine model, a robust

improvement in the cognitive behavior caused by a continu-

ous secretion of the recombinant CNTF was demonstrated.

The results were more successful in the Tg2576 AD mouse

model, where a full recovery of the cognitive functions asso-

ciated with the stabilization of the synaptic protein levels was

observed.[185]

Huntington’s disease (HD) is another important neuro-

degenerative disorder to take into consideration. This disease

is characterized by the preferential loss of the striatal GABA-

ergic medium spiny neurons and includes an array of different

psychiatric manifestations, cognitive decline, and choreiform

movements. The CP epithelial cells secrete several neurotrophic

factors affecting the production of the cerebrospinal fluid

and the maintenance of the extracellular fluid concentra-

tions in the brain.[187] There have been various studies that

employ immobilized CP cells in alginate microcapsules to

achieve an appropriate release of neurotrophic factors into

the brain in rodent[13,14,188,189] and primate[190] models of HD.

These studies reported that the CP cells encapsulated in the

alginate microcapsules prevented the degeneration of the

striatal neurons. Baby hamster kidney (BHK) cells engineered

to produce human CNTF and immobilized into semipermeable

membranes have also been employed in the treatment of

HD with positive results, including protection of the neu-

rons from degeneration and restoration of the neostriatal

functions.[191]

Spinal cord injury (SCI) results in the disruption of the as-

cending and descending axons and subsequently produces a

devastating loss of motor and sensory function. In this regard,

BDNF-producing fibroblasts entrapped in alginate micro-

capsules were implanted in a murine model of SCI.[192,193] En-

capsulated MSCs have also been employed in the treatment of

the secondary inflammatory responses in the SCI animal

models.[194] The in vitro and in vivo results obtained in this study

demonstrated that the MSC-loaded microcapsules may over-

come many of the disadvantages that occur with the administra-



tion of the unencapsulated MSC (distribution to non-targeted

tissues after transplantation and retention at a low rate). At the

same time, encapsulation may serve to augment the tissue-

protective behavior of theMSC caused by the elimination of the

proinflammatory macrophages that is stimulated by the cell-

loaded microcapsules at the site of injury.[194]

The clinical trials performed for the previously mentioned

diseases and some others are summarized in table II.

Table II. Clinical trials already carried out or ongoing in the field of cell encapsulation

Therapeutic application Therapeutic agent Type of capsule or material Results References

Type 1 diabetes Encapsulated islets, low

dose immunosuppression

Alginate-PLL 9 months of insulin independence 195

Encapsulated islets, no

immunosuppression

Alginate-PLO-alginate Insulin consumption declined at 6–12 months

(2 patients)

196,197

Diabecell� device (living cell

technologies) encapsulated

neonatal porcine islets

Alginate 9.5 years’ improvement in glycemic control

(1 patient)

198

Little improvement in hypoglycemia

unawareness and a reduction in the exogenous

insulin schedule (12 patients)

199

Encapsulated islets, low

dose immunosuppression

Alginate Neither insulin requirements nor glycemic

control was affected (4 patients)

200

Monolayer islet device Alginate-monolayer

(1–3 cm2)

Recruiting patients, started in 2008

(NCT00790257)

201

Encapsulated beta cells Alginate Recruiting patients, started in 2011

(NCT01379729)

201

Viacyte (Novocell�) Conformal PEG coating Phase I/II study, concluded in 2011

(NCT00260234)

201

Parkinson’s disease Spheramine�. hRPE cells

implants

Gelatin microcarriers Safe and well tolerated implants and

improvement in motor symptoms

202

Ongoing phase II study, started in 2000

(NCT00761436)

201

Ongoing phase II study, started in 2003

(NCT00206687)

201

Alzheimer’s disease NsG0202 implants

(EC delivery/NsGene),

encapsulated NGF-

secreting cells

Hollow fiber Ongoing phase I study, started in 2008

(NCT01163825)

201

Huntington’s disease BHK cells that secrete CNTF Hollow fiber 2-year study (device changed every 6 months);

variability in CNTF levels (6 patients)

203

Amyotrophic lateral sclerosis BHK cells that secrete CNTF Hollow fiber 17 weeks post-implantation CNTF detected

in the CSF (6 patients)

204

Pancreatic carcinoma Encapsulated cells that

secrete the enzyme

cytochrome P450

Cellulose sulphate Tumor regression or stabilization,

no tumor growth

205,206

Hypoparathyroidism Encapsulated parathyroid

tissue

Alginate Calcium and vitamin D replacement reduced

(2 patients)

207

Retinitis pigmentosa Neurotech (NT-501) Hollow fibers Phase II study, no visual benefit 208,209

Intracerebral hemorrhage GLP-1 CellBeads,�.

encapsulated mesenchymal

cells that secrete GLP-1

Alginate Ongoing phase I/II study, started in 2008

(NCT01298830)

201

BHK cells= baby hamster kidney cells; CNTF= ciliary neurotrophic factor;CSF= cerebrospinal fluid; EC = encapsulated cell;GLP-1= glucagon-like peptide 1;

hRPE cells= human retinal pigment epithelial cells; NGF= nerve growth factor; PEG=poly(ethylene glycol); PLL= poly-L-lysine; PLO=poly-L-ornithine.



3.3 Cardiovascular Diseases

Cardiovascular diseases (CVDs) are the major causes

of morbidity and mortality worldwide. CVDs include myo-

cardial ischemia with the subsequent extended dysfunction

and death of the cardiomyocytes. After a myocardial infarc-

tion (MI), the heart attempts to restore its damaged tissue by

implementing various physiological and compensatory pro-

cesses, including cardiomyocyte hypertrophy, apoptotic myocyte

loss, progressive collagen replacement, and an enlargement of

the left ventricle.[210] Conventional medical management can im-

prove post-MI mortality, relieving symptoms and slowing de-

terioration, but it cannot restore cardiac function. This

treatment merely accommodates the damaged myocardium

rather than effecting direct repair to restore its normal structure

and function.[211] Thus, new therapeutic approaches for MI are

required.

Cell therapy is a novel and promising therapeutic approach

to sustain the endogenous regenerative mechanisms in ischemic

heart disease and heart failure; it is based on the transplantation

of human or animal cells to replace or repair damaged tissues or

cells.[212] Cell transplantation provides an effective and renew-

able source of proliferating and functional cardiomyocytes and

supports the development of a blood vessel network that sus-

tains and nourishes these newly formed cardiomyocytes and

their surrounding ischemic myocardium.[213] Toward this

aim, stem cell transplantation is being investigated as a novel

means to regenerate the heart tissue and enhance cardiac

function.[212,214] Several animal studies have demonstrated that

the administration of various types of adult stem cells using

various delivery routes can have therapeutic benefits, decreas-

ing cardiac infarction size by promoting angiogenesis and

myogenesis and effectively improving the left ventricular and

cardiac function.[215-222] However, there is an important draw-

back to myocardial cell therapy that is related to the low rate of

engraftment and survival of these transplanted cells. The heart

is constantly contracting, and this contributes to the mechan-

ical loss of the injected cells because they are squeezed out of the

myocardium. Large proportions of the injected cells are lost

from the myocardium within the first few minutes after their

injection.[223,224] Not more than 0.1–15% of all the injected cells

are retained within the myocardium.[211,225] To overcome the

massive loss of the cells and improve cell retention and survival,

diverse approaches are being studied.

One outstanding strategy could be the immobilization of the

cells within microcapsules. Microcapsules are larger than the

blood vessel diameter; thus, the heart’s contractile forces are

unable to eliminate the microcapsules into the bloodstream,

significantly increasing the amount of retained microcapsules

and the amount of cells.[225] In a recent study, Al Kindi et al.[17]

reasserted that the size of the microcapsules is an important

factor in the early loss of cells because encapsulated micro-

spheres in APAmicrocapsules (200–400 mm) exhibited a higher

retention rate than small microspheres (100 mm). In addition,

they showed that myocardial function did not improve with

escalating cell doses, showing that the optimal functional im-

provement began at 1.5 · 106 cells.The microencapsulated cells may exert a beneficial influence

on myocardial regeneration by means of a paracrine growth

factor effect. VEGF-secreting xenogeneic Chinese hamster

ovary (CHO) cells were encapsulated in APA microcapsules

and implanted into the infarcted myocardium of rats. This

study reported an improvement in global heart function and

enhanced angiogenesis.[226] In a similar approach, Yu et al.[227]

encapsulatedMSCs in RGD-modified alginate and covered the

droplets with PLL. They reported an improvement in cell at-

tachment, cell growth, and an increase in the expression of

angiogenic growth factor as well as the successful maintenance

of the shape of the left ventricle, the induction of angiogenesis,

and the prevention of the left ventricle remodeling, including

infarct wall thinning and chamber dilation after an MI. These

results suggest that these microencapsulated cells may have a

much greater potential for heart regeneration compared with

the unencapsulated stem cells. Another interesting and prom-

ising approach is the production of heart tissue grafts, in which

alginate and collagen have been combined to encapsulate neo-

natal rat cardiomyocytes. Cardiac cell proliferation and the

formation of interconnected multilayer heart-like tissue, as well

as the spontaneous synchronized contractility of the engineered

heart tissue grafts, were demonstrated.[228]

3.4 Liver Diseases

The liver is a central and vital organ. It performs a wide

range of fundamental metabolic functions, including the de-

toxification and elimination of endogenous wastes (such as

ammonia or bilirubin), drugs, and exogenous toxins, the syn-

thesis of amino acids and proteins, the metabolism and ab-

sorption of carbohydrates and lipids, and the storage and the

delivery of nutrients according to the requirements of the body.

Acute liver failure and chronic liver failure are diseases related

to the inability of the liver to perform its normal synthetic and

metabolic functions. Although liver dialysis can be used in the

short term, the only way to compensate for the absence of the

liver function in the long term is with an organ transplantation.

Several research groups have employed cell microencapsulation



technology to create a living cell-based replacement system or a

bioartificial liver to compensate for essential functions. There

have been various in vivo studies of this technology performed

inmany animalmodels, includingGunn rats,[229,230] mice,[12,231-233]

and pigs.[234] These studies generally reported enhanced re-

covery and support and improvement in hepatic metabolic

functions; the technology was presented as a potential alter-

native to transplantation for liver failure. In one of these

studies, immortalized human hepatocytes encapsulated in APA

microcapsules were transplanted intraperitoneally into a mice

model of acute liver failure to evaluate the effects on liver

metabolism and regeneration. This group reported an im-

provement in the survival and biochemical profile, including

lower levels of the cytokines (tumor necrosis factor [TNF]-a,interleukin [IL]-6, hepatocyte growth factor [HGF] and trans-

forming growth factor [TGF]-b1) in the serum of mice trans-

planted with hepatocytes compared with the mice receiving

empty capsules. These lower levels of cytokines could be ex-

plained by an increase in the clearance of these cytokines by

the encapsulated hepatocytes. However, the encapsulated

hepatocytes transplanted into the intraperitoneum did not

stimulate native liver regeneration.[231] Another possible ther-

apy involves the co-encapsulation of hepatocytes with other

types of cells that may maintain the specific function and phe-

notype of the encapsulated hepatocytes.[235,236] Shi et al.[237]

reported that rat hepatocytes and MSCs co-encapsulated in

APA microcapsules improved hepatocyte-specific functions

(albumin secretion and urea synthesis). In vivo studies in a

rat model of acute liver failure demonstrated an increase in

the liver’s survival rate and an improvement in its functions.

In addition, some MSCs transdifferentiated into hepatocyte-

like cells in vivo and expressed albumin, a typical marker for

hepatocytes.

An important issue in the transplantation of cells into the

liver is the evaluation of the mass transfer velocity between the

fluid and the encapsulated hepatocytes as well as the biological

functions of cells. Toward this aim, fluidized bed systems are

being investigated.[234,238-241] These devices provide a suitable

and valuable option to improve nutrient and oxygen transfers,

facilitating the inflow and transformation of lethal toxins as

well as the release of essential proteins from the cell-containing

beads (figure 4).

3.5 Other Diseases

Epo has been widely used in the treatment of the anemia

associated with various chronic conditions, including end-stage

renal disease, malignancy, and human immunodeficiency virus

(HIV) infection.[242] With consideration of that fact, our re-

search group has carried out a careful selection of biomate-

rials and cell lines to produce uniform and biocompatible

microcapsules with long-term functionality. C2C12 myoblasts

genetically engineered to secrete Epo have been successfully en-

trapped in APAmicrocapsules and implanted in allogeneic and

C3A

entrapped into alginate bead

Nutrients

Oxygen

Metabolites

Bioreactor

Red blood cells

Plasma

Plasmadetoxificated

Peristaltic pump

Patientwith

hepaticfailure

Plasmapheresis

a

b

Fig. 4. Principle of the fluidized-bed bioartificial liver for liver support (a). The patient’s blood is withdrawn and separated into plasma and blood cells. Plasma is

perfused through a fluidized-bed bioreactor hosting hepatocytes in order to be detoxified. Plasma and blood cells are mixed again and returned to the patient.

Transfer of nutrients, oxygen and metabolites occurs through the alginate bead structure (b). Reproduced from Gautier et al. (ª 2011),[238] with permission.



syngeneic mice[35,243,244] as well as in xenogeneic rats using

transient immunosuppression.[9] In a recent in vivo study carried

out in our laboratory, with the aim of generating an im-

munoprivileged local environment to avoid or at least diminish

the fibrotic layer caused by inflammation, we have developed a

composite delivery system that consists of Epo-secreting myo-

blasts immobilized in APAmicrocapsules and dexamethasone -

releasing PLGAmicrospheres.[153] All of these studies included

a single dose of microcapsules, which achieved high and con-

stant hematocrit levels.

The use of microcapsules containing cells that secrete vari-

ous therapeutic products has also been proposed for the treat-

ment of cancer. Some of these therapeutic products are cytokines

that enhance immunity and decrease tumorigenicity.[245] Others

are antiangiogenic proteins (endostatin or hemopexin) that

inhibit the release of VEGF and, thus, the growth and devel-

opment of blood vessels characteristic of and necessary to tu-

mors.[246-250] Others have employed a combined delivery of

the cytokines and antiangiogenic proteins.[251] In a recent

study, Dubrot et al.[252] encapsulated hybridoma cells that se-

crete immunostimulatory antibodies (anti-CD137 and anti-

OX40 mAb) and implanted them in mice with induced tumors.

The immunostimulatory monoclonal antibodies secreted by

the implanted encapsulated hybridoma cells enhance tumor-

specific cellular immunity.

Pelegrin et al.[253] employed cell encapsulation technology

for the treatment of viral diseases. Toward this aim, they im-

mobilized cells secreting an ectopic monoclonal antibody that

neutralizes the FrCasE retrovirus; neutralization prevents the

lethal neurodegeneration caused by the virus in infected new-

born mice. The treated mice showed reduced or undetectable

viremia as well as a lack of the histopathological lesions char-

acteristic of this disease.

Other approaches have recently been proposed by Piller

et al.[254] for the treatment of mucopolysaccharidosis type I

(MPSI). A murine myoblast cell line engineered to overexpress

the enzyme a-L-iduronidase (IDUA) was immobilized in algi-

nate microcapsules and implanted in a MPSI mouse model.

This resulted in a significant induction of enzyme activity,

a reduction in the accumulation of glycosaminoglycan and

a complete normalization of the deposits.

Finally, the treatment of deafness is being studied using

encapsulation technology. The effects of encapsulated BDNF-

expressing Schwann cells on the survival of the auditory neu-

rons in the deaf guinea pig were assessed. The study reported

that the delivery of the neurotrophic factors reduced or pre-

vented the auditory neuron degeneration that causes sensor-

ineural hearing loss.[255]

4. Concluding Remarks and Future Directions

and Challenges

Although several advances have been observed in cell micro-

encapsulation technology for the treatment of many significant

diseases, there are still major challenges to the technology.

These challenges include determining the effects of the encap-

sulation process, determining the appropriate deployment lo-

cation, achieving biocompatibility, increasing immunoprotection,

eliminating hypoxia, and preventing pericapsular fibrotic

overgrowth and post-transplant inflammation. These obstacles

must be resolved for the use of immunoprotective capsules in

clinical applications. Modifications to the microcapsule surface to

prevent fibrotic overgrowth, the application of genetic engineering,

co-encapsulation, an improvement in the oxygen supply or the

establishment of hypoxia resistance as well as other methods are

novel approaches to overcoming the obstacles mentioned above.

A continuing challenge in drug delivery applications is

achieving precise control over the delivery of single or multiple

drugs and sustained or sequential release in response to external

environmental changes, such as mechanical signals. Dynamic

control over the delivery can improve the safety and effective-

ness of the drugs and may provide new therapies.

Thus, the standardization and definition of the protocols for

the assay of the specific requirements of the technology must be

mandated to understand lab-to-lab variations and achieve ad-

equate reproducibility. This ambitious goal requires a multi-

disciplinary approach between scientists and researchers from the

biomedical, physical, and chemical fields as well as other fields.

Acknowledgments

The authors gratefully acknowledge the support to research cell micro-

encapsulation from the ‘‘Ministerio de Ciencia e Innovacion’’, University of

the Basque Country UPV/EHU (UFI11/32) and FEDER funds (SAF2008-

03157). A. Acarregui thanks the ‘‘Gobierno Vasco (Departamento de Edu-

cacion, Universidades e Investigacion)’’ for the Ph.D. fellowship.

In Memoriam

We would like to thank the contribution of Ainhoa Murua for her

extraordinary knowledge and capacity to foster scientific research. The

greatest tribute to her will be our continued promotion of the highest

scientific and educational work. She, who has touched the life of many

colleagues and PhD students, will remain forever alive.

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Correspondence: Professor Rosa Ma Hernandez, NanoBioCel Group, Labor-

atory of Pharmaceutics, School of Pharmacy, University of the Basque

Country (UPV/EHU), Paseo de la Universidad 7, 01006 Vitoria-Gasteiz,

Spain.

E-mail: [email protected]



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A Perspective on Bioactive Cell Microencapsulation