Cellular shellization: Surface engineering gives cells an exterior

Prospects & Overviews

Cellular shellization: Surfaceengineering gives cells an exterior

Ben Wang1)y, Peng Liu1)y and Ruikang Tang1)2)�

Unlike eggs and diatoms, most single cells in nature do

not have structured shells to provide extensive protec-

tion. It is a challenge to artificially confer shell structures

on living cells to improve their inherent properties and

functions. We discuss four different types of cellular

shellizations: man-made hydrogels, sol-gels, polyelectro-

lytes, and mineral shells. We also explore potential appli-

cations, such as cell storage, protection, delivery, and

therapy. We suggest that shellization could provide

another means to regulate and functionalize cells.

Specifically, the integration of living cells and non-living

functional shells may be developed as a novel strategy

to create ‘‘super’’ or intelligent cells. Unlike biological

approaches, this material-based bio-interface regulation

is inexpensive, effective, and convenient, opening up a

novel avenue for cell-based technologies and practices.

Keywords:.biointerface; biomimetic mineralization; cellular shell;

functional materials; polyelectrolyte

Introduction

Progress in biological science and materials engineering hasbegun to blur the boundary between living and non-livingsystems. Synthetic biologists can now engineer complex, arti-ficial biological systems to help understand natural biologicalphenomena and use in a variety of biomedical applications [1].Advances in directed evolution and membrane biophysicsmake the synthesis of simple living cells an imaginablegoal [2].

Cellular life is the basic unit of a living organism anddefines the presence of a stable information reservoir con-nected to the external world by a well-defined boundary [3].The cell membrane separates the interior of a cell from theoutside environment and must meet specific requirementssuch as semi-permeability to permit communication andmolecular transport across the border [4]. Using naturalmembranes as a model, it is possible to build an artificialshell from natural constituents or from synthetic soft or hardmaterials, introducing robustness to the capsule – the cell-shell combination. Furthermore, we can also design the shellto alter the inherent biological qualities of the cell.

In the evolution of natural systems, living organisms havedeveloped various mineralized structures, such as teeth,bones, shells, carapaces, and spicules. Those compositebiomaterials often exhibit complex hierarchical structuresand possess important functions such as mechanical

DOI 10.1002/bies.200900120

1) Center for Biomaterials and Biopathways and Department of Chemistry,Zhejiang University, Hangzhou, Zhejiang 310027, China

2) State Key Laboratory of Silicon Materials, Zhejiang University, Hangzhou,Zhejiang 310027, China

*Corresponding author:Ruikang TangE-mail: [email protected]

Abbreviations:ACT, adoptive cell therapy; ALP, alkaline phosphatase; bioMEMS, bio-Micro-Electro-Mechanical Systems; BMSC, bone marrow mesenchymal stem cells;BSA, bovine serum albumin; DMSO, dimethyl sulfoxide; EB, embryoid bodies;ECM, extracellular matrix; EDTA, ethylenediaminetetraacetic acid; HA,hyaluronic acid; hESC, human embryonic stem cell; HMS, hydrogel matrixshell; IMS, induced mineral shell; LbL, Layer by layer; MELN cells, MCF-7cells transfected with a construct expressing the luciferase gene under thecontrol of an estrogen-regulated promoter; mESCs, murine embryonic stemcells; MRI, Magnetic Resonance Imaging; MSC, mesenchymal stem cells;PAA, poly(acrylic sodium); PDADMAC, poly(diallyldimethylammoniumchloride); PE, polyelectrolyte; PES, polyelectrolyte shell; PLL, poly (L-lysine); PSS, polystyrene sulfonate; SGS, sol-gel shell; SLeX, SialylLewis(x); TMOS, tetramethyl orthosilicate; VNPs, viral nanoparticles.yThese authors contributed equally to this work.

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support, protection, motility, and sensing of signals [5].Marine organisms such as mollusks [6] and arthropods [7]use biominerals as their exterior coats to protect their softbodies from external stresses or aggression. A cocoon isanother example of an exterior coat that fulfills multiple rolesincluding protection against physical stress, toxic substancesand natural enemies, and even mediation of messages con-trolling the life cycle. Besides these examples, a number ofunicellular organisms have biogenic coverings as well.Chicken eggs are perhaps the most familiar example(Fig. 1A). Eggshells provide mechanical support for theembryo and are also extremely important for maintainingthe egg’s viability. It is well-known that an egg with an intactshell can be stored for several weeks in ambient conditions;however, the egg will decay quickly if the shell is broken. Theshell forms a shield to prevent the enclosed cells from con-tamination. Eggshells also maintain the balance of oxygen,carbon dioxide, water, and nutrients required for proper celldevelopment [8]. In addition, shells provide the embryo withthe minerals needed for the generation of organs high incalcium, such as the skeleton, muscles, and brain [9].

Diatoms, another example of a unicellular organismwith amineral coat, have unique cell walls made of silica (hydratedsilicon dioxide) called frustules. The ornately patternedsilicified shell has evolved as a biological protection fordiatoms [10].

However, most cells cannot make their own shells.Many attempts have been made to fabricate artificial shellsdirectly onto cells or to confer to cells an ability to formtheir own shells. This paper gives an overview of the

different approaches that have been undertaken toconstruct artificial shells, such as hydrogel matrix shell(HMS), sol-gel shell (SGS), polyelectrolyte shell (PES), andinduced mineral shell (IMS). Applications of cellular shelliza-tion – cell storage, protection, delivery, and therapy – are thendiscussed (Fig. 1C). Finally, conclusions and perspectives areoffered as to where these fascinating avenues of researchmight lead.

Hydrogel matrix shell (HMS)

The extracellular matrix (ECM) is the portion of animal tissuethat provides the essential microenvironment for cells. Due toits diverse nature and composition, the ECM can serve manyfunctions, such as support and anchorage for cells, segregat-ing tissues from one another, and regulating intercellularcommunication [11]. Synthetic and natural hydrogels havebecome popular as three-dimensional in vitro cell cultureplatforms that mimic ECM. Networks of hydrophilic polymersthat can retain large quantities of water, hydrogels can besynthesized by multiple methods. Hydrogels are biocompat-ible, supportive of cell maintenance and growth, since theyfacilitate the exchange of gases and nutrients in a way similarto the environment in vivo [12]. Hydrogel encapsulation mayprovide cells with structural support, chemical stability or ameasure of protection from immune attack. Both syntheticand naturally derived hydrogels have been explored for encap-sulation of a variety of cell types [13]. The use of hydrogels toencapsulate stem cell populations indicates the ability of this

Figure 1. Scheme of an egg and overview of cellular shellization.A: A chicken egg is a typical cell with a shell (its main inorganiccomposition is calcite). B: The unique structure of eggshells (theorganic matrix) provides crystallization sites for the deposition ofcalcite layer onto the cells. C: Different approaches for cellularshellization and its potential applications: HMS, SGS, PES, andIMS.

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technique to manipulate cell fate. For example, murine embry-onic stem cells (mESC) were encapsulated into 1.6% w/valginate microbeads. Differentiation was inhibited at the mor-ula-like stage, with no cystic embryoid bodies (EB) formedwithin the beads. Using the same strategy to coat humanembryonic stem cells (hESC), stem cell colonies could bemaintained for up to 260 days in an undifferentiated state.Upon release from the alginate microbeads, hESC were able toresume differentiation, as shown by the formation of intocystic EB containing beating cardiomyocytes. Retention of cellpluripotency by encapsulation occurred even under differen-tiation promoting conditions [14]. Recently, a newly developedintegrated bioprocess utilized alginate hydrogel encapsulationof mESC to form 3D mineralized constructs without the needfor passaging or handling of the cells [15]. Chan et al. alsofabricated injectable collagen–human mesenchymal stem cell(hMSC) microspheres using microencapsulation. Apart fromproviding a protective matrix for cell delivery, the collagenmicrospheres also acted as a bio-mimetic matrix facilitatingthe functional remodeling of hMSC [16]. The 3D hydrogelmicroenvironment offers a means to control cell-matrix inter-actions and thereby manipulate cell behavior. These studiesdemonstrate that hydrogel encapsulation can be considered ameans to not only provide structure and protection to a trans-plantable cell product, but also as a way to more efficientlyattain the desired cell population.

Hydrogel embedding has several advantages over othershellization techniques, including keeping the cells in anaqueous environment, in contact with soft and biocompatiblematerials, while protecting them from the stress of encapsu-lation. Their material properties can be engineered forbiocompatibility, selective permeability, mechanical, andchemical stability, as well as other requirements as specifiedby the application. Embedding cells in hydrogels requires theproduction of microbeads by an economical, gentle, andreproducible method. Methods of hydrogel bead generationinclude emulsification, extrusion, co-extrusion, hollowparticle formation [17], via microfluidic devices [18], micro-lithography [19], and micromolding [20]. Control over cell-laden bead size is important, because it can affect the numberof cells per bead, thereby varying the ratio of cells to matrix[16].

Sol-gel shell (SGS)

The sol-gel process is a new method of synthesizing silicaglasses at room temperature [21, 22]. Silica is present in allliving organisms and, after carbonates, it is the second mostabundant mineral formed by organisms. Silica structures withprecisely controlled morphologies are extensively producedby single-cell organisms such as diatoms [23, 24]. The recentextension of this process to enable the entrapment of func-tionally active biomolecules demonstrated the introductionand retention of biological activity within silica gels. The firstreport of the sol-gel encapsulation of enzymes was publishedin the early 1970s [25], but this process was actually developedby the Jerusalem group in 1990 [26]. Since then, enzymes,antibodies, and even whole cells have been encapsulatedwithin sol-gel glasses [27–29].

The silicate matrix is usually formed by hydrolysis of analkoxide precursor followed by condensation to yield a poly-meric oxo-bridged SiO2 network. In the process, molecules ofthe corresponding alcohol are liberated. The ability to formhybrid silica glasses under aqueous conditions and roomtemperature (at which proteins and cells are active) openedup the possibility of extending sol-gel processing to the encap-sulation of cells. However, while sol-gel conditions are mildenough for organic molecules, they are still too harsh for livingorganisms to retain viability. For example, alcohol andacidic pH lead to the denaturation of most proteins.Therefore, the sol-gel process had to be adapted to bioencap-sulation, which is currently achieved in two steps. The firststep is the hydrolysis of tetramethyl orthosilicate (TMOS) inthe presence of an acid to hydrolyze all alkoxy groups [30].Cells are then added to the Si(OH)4 hydrolyzed aqueoussolution in the presence of a buffer with a pH around 7.Condensation progresses form the silica network rapidlyaround the cells entrapped within the porous gel. The wholeprocess occurs at room temperature within a few minutes andwithout denaturation of most cells. A novel class of bioactivematerials is thus obtained, comprised of cells physicallytrapped within silica matrices.

The Biosil method is a versatile technique for combiningfunctional cells and cell aggregates within the uniquestructure provided by silica [31]. Biosil technology is basedon the encapsulation of whole cells by a sol-gel silica layerdeposited on the cell surface using silica precursors inthe gas phase (Fig. 2). It has been demonstrated that theBiosil process provides mechanical stability, porosity controlfor immunological protection, and maintenance of cellviability with sustained cellular functions. Extension ofthe Biosil process to alginate microencapsulation couldenhance biocompatibility for purposes of cell grafts andtherapy [32].

Figure 2. Schematic diagram of the Biosil process. A: Cellsuspension mixed with collagen fibers (red line) as a scaffold. Awater-layer forms around the cells (white layer) because of theirhydrophilic surface. B: Using alkoxides in the gas phase,a sol-gel membrane (yellow layer) can be built up directly onthe cell surface: the gaseous precursors react withsurface-adsorbed H2O and exposed ��OH, allowing removal ofbyproducts from the solid sol-gel in the gas phase. Si��OR,alkoxide precursors; ROH, reaction byproducts.

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An important property of the sol-gel process is the favorabledispersion of a variety of chemicals directly into the gellingsolution, including biomass, which gives a feasibleapproach for SGS modification. The intimate mixing oforganic molecules and alkoxides in the precursor solutionallows organic and inorganic components to be associatedat the molecular level. Organic molecules can simply beembedded within the silica matrix or chemically linkedvia Si��C bonds [33]. Reactions involving silica precursorslead to a sol (a nanometric dispersion of silica particles)which collapses to a solid network holding the liquid (thesol-gel state). Macromolecules, metal particles, and avariety of chemical compounds can all be combined inthe silica sol solution before gelling [34]. The extension ofthis approach to sol-gel silica loaded with bioactive mol-ecules, particularly enzymes, may create novel and valuablematerials for cellular shellization. Comprehensive reviews ofcell encapsulation within sol-gel materials have beenrecently published by Avnir and coworkers and Meunierand coworkers [35, 36].

Polyelectrolyte shells (PES)

Based on the electrostatic properties of the cell, polyelectrolyte(PE) shells may be directly grown or built onto negativelycharged cells. Layer by layer (LbL) fabrication has beenapplied as a general technique for the fabrication of multi-component films on solid supports: several alternating cyclesof absorption and deposition of opposite-charged PE [37, 38].

Living yeast cells have been singly encapsulated by thealternate adsorption of oppositely charged PEs. Fluorescentimaging revealed that the cells preserved their integrity andmetabolic activity after the coating procedure, and remainedcapable of dividing within the shell [39]. Shellization of mouseMSC with hyaluronic acid (HA) and poly (L-lysine) (5) main-tained morphology and viability for up to one week [40]. Cellscoated with PE layers could provide an inexpensive modelsystem for a wide range of biophysical and bioengineeringapplications, due to the tunable properties of the PES.

To create functional cell-based biosensors, it would beadvantageous to add protection to mammalian cells, whichwould be analogous to the cell wall protecting yeast or plantcells. As an example, MELN cells (MCF-7 cells transfected witha construct expressing the luciferase gene under the control ofan estrogen-regulated promoter) have been isolated with aPES using the LbL technique. Among several PE-couples,optimal cell survival (>80%) was obtained by alternatingpoly-cation poly-diallyldimethyl ammonium chloride layerswith the negatively charged poly-styrene sulfonate. Thecomposition of the buffer used for layer deposition wascrucial to preserving cell viability, e.g., potassium ions werepreferred to sodium ions during the coating. Furthermore,viability was increased when cells were allowed to recoverfor 2 hour between each bilayer deposition. Successful encap-sulation of MELN cells demonstrated that coating not onlypermits mammalian cell survival, but also allows essentialmetabolic functions such as RNA and protein synthesis to takeplace [41].

Besides the proposed shellization of cells, application of suchprocedures to viruses is also becoming both interesting andimportant in biological research. Viral nanoparticles (VNPs)have received great attention in recent nanotechnologyresearch, but their virulence must be eliminated before theirpractical application. Several biological approaches such asgene reassortment have been tried, but incompletely modifiedVNP cannot be avoided. They would likely stimulate undesir-able infections and immune responses during material fabri-cation. Another bottleneck in VNP applications is the difficultyof processing viruses on a large scale, mainly because of theineffective concentration and recovery of virus particles. Thegeneral approaches to concentrating viruses such as ultra-centrifugation and density gradient methods are too tediousand time-consuming to separate and purify the viruses insufficient amounts. Our experiments recently demonstratedthat shellization is a feasible strategy to manipulate the bio-logical security of viruses; the infectivity of the enclosed virusis effectively suppressed by the shell structure [42]. Thus virus-based PE composites could make large-scale application ofVNPs possible. In addition, the shell structure could reducethe surface repulsive force of virus particles, leading to newflocculation and aggregation behaviors. Therefore, the separ-ation and concentration of the virus-shell composites can evenbe achieved readily by normal centrifugation. Most virusshortages in VNPs applications could be addressed byshellization.

Maintaining viability of cells is the most important factorduring shellization. Unlike yeast cells, delicate mammaliancells are extremely sensitive to PEs. The toxicity of PEs duringthe LbL procedure is a serious threat to mammalian cellviability during biomimetic modification. Previous studieshave also demonstrated that positive PEs damage the celllayer even more than negative ones [43]. The toxicity of com-monly used positive PEs are ranked as poly(ethylenimine) ¼poly(l-lysine) > poly(diallyl dimethyl ammonium chloride) >diethylaminoethyl dextran>poly(vinyl pyridinium bromide) >starburst dendrimer > cationized albumin > native albumin.In addition, the toxic effects of all polymers can depend upontime and concentration [44]. Although a quick treatment (lessthan 10 minutes) can reduce the unfavorable influences of thePEs on living cells, it must be acknowledged that the toxicity ofPEs limits the usage of LbL on most mammalian cells. Thedesign and synthesis of the suitable positive PEwith acceptablelevels of toxicity for mammalian cells is necessary for cellularshellization in future applications.

Induced mineral shell (IMS)

Biomineralization is a biological process by which livingorganisms make use of organic matrices to control the for-mation of functional minerals [45]. Shellization is a specialexample, since the created crystals can enclose the wholeorganism via formed uniform shell structures. Previous stud-ies of biomineralization have demonstrated that cells withmineral shells always have outer proteinaceous membranes,which act as mineralization templates to induce hetero-geneous crystallizations [46]. Again using chicken eggs as



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an example (Fig. 2B), the foundation for cellular mineraliz-ation is the proteinaceous eggshell membrane (mineraliz-ation-induced layer). The mineralization of the calcite phasestarts with the nucleation of calcite crystals on the keratinsulfate-rich organic aggregations on the eggshell membrane[47]. Keratin sulfate is a calcium-binding polyanionic moleculereported to be closely associated with calcite crystals in thechicken ear statoconial membrane [48]. The shell membraneof eggshells, rich in calcium-binding polyanionic molecules,acts as a mineralization template to induce calcium carbonatecrystallization and control calcite growth.

Unlike conventional crystallization in laboratories, theorganic matrix can mediate the formation of inorganic crystalssuch as calcite in biomineralization. Inorganic mineralizationin the presence of organic additives is universally considered aperfect model for biomimetic mineralization [49]. Althoughhundreds of proteins have been identified during biomineral-ization, it is generally agreed that most proteins active in themediation of biologically directed mineral growth containacidic amino acid residues; specifically, regions rich in carbox-ylates that interact with mineral surfaces to influence bothcrystal morphology and rates of formation [50]. Negativelycharged groups such as carboxylate, sulfate, and phosphateare effective in binding Ca2þ ions to control nucleation andgrowth of calcium minerals by lowering the interfacial energybetween crystal and organic substrate and accumulatingsoluble calcium ions at suitable nucleation sites [51–53]. Inpractice, researchers have successfully used carboxylate-richcompounds to modify the mineralization ability of solid sub-strates. For example ethylenediaminetetraacetic acid (EDTA), arelatively smallmoleculewith four carboxylate groups, is oftenused to pretreat metal before implantation. It causes a layer ofuniform and continuous calcium coating to be precipitatedreadily on the metal surface, rendering it ‘‘living’’ for hardtissue formation [54]. This example demonstrates the regulat-ory effect of carboxylate on the mineralization of substrates. Italso offers a possible approach to changing the mineralizationcapability of living cells: introduction of mineralization factorssuch as carboxylate groups onto cell surfaces may turn non-mineralization cells into mineralization-preferred ones. Thus,it becomes possible to preparemineral shells for living cells viaa uniform enhancement of surface mineralization.

We must also emphasize that such regulation of cellularmineralization is also important in understanding pathologi-cal mineralization. In the human body, calcification ormineralization can occur on some calcification-free organsunder certain conditions, producing serious diseases suchas arteriosclerosis and kidney stones [55]. Pathological calci-fication is an important genesis of human diseases [56]. From aview of biomineralization, pathological calcification can beconsidered an unexpected promotion of biomineralizationability of cell or organism’s surfaces. It has also been notedthat the accumulation of mineralization factors (e.g., choles-terols and calcium-binding proteins) at cell-milieu interfacesalso causes pathological mineralization. Therefore, control-ling the mineralization factor density on cell surfaces is a keyto both artificial shellization and understanding pathologicalcalcification.

In order to improve the mineralization capability ofliving cells, mineralization factors could be inserted into or

adsorbed onto cell surfaces; they must be locally clusteredto adsorb calcium minerals at the specific sites to inducenucleation. Cells can also be modified using LbL to add therequired molecules or groups onto their surfaces [57, 58].Two PEs are used for cell membrane modification: polycationpoly(diallyldimethylammonium chloride) (PDADMAC) andpoly(acrylic sodium) (PAA). PAA has many polar carboxylategroups to bind dissolved Ca2þ, and the reorganized surfacescan significantly increase the electron density on the surface toinduce heterogeneous nucleation of calciumminerals [59, 60].After LbL treatment, the carboxylate groups can be integratedon the cell surfaces. When the treated yeast cells are in contactwith the supersaturated calcium phosphate solution, themineral phases are preferentially crystallized in situ at thecell-solution interfaces. Thus, an egg-liked structure can result(Fig. 3). Yeast cells have also been shellized with silica byInsung S. Choi and colleagues. In their experiment, PDADMACand sodium polystyrene sulfonate (PSS) were employedto modify cells by LbL treatment. The difference betweentheir experiment and ours is their use of PDADMAC asthe outermost layer, since synthetic polymers containingquaternary amines were found to be chemically catalytic forbiomimetic silica formation under physiologically mild con-ditions. This strategy could be universally used in the modi-fication of various cells and the preparation of differentmineral shells.

Besides calcium, nanoparticle shells can also be formed onthe cell surface using gold, silver, or silicon oxide. Yeast cellswere shellized using alternating deposition of PE and chargednanoparticles. The yeast cells were first shellized by usingalternating deposition of either PAH/PSS or bovine serumalbumin (BSA)/DNA onto the surface of cells as mentionedabove. Second the shellized cells were introduced into a sus-pension of either gold or silver nanoparticles, and then twoadditional PE layers were deposited onto them. Gold and silvernanoparticles were successfully immobilized on the surface ofthe cells, effectively altering their color and surface topogra-phy. Recently we also gave yeast cells silicon oxide nanopar-ticle shells. We used PDADMAC and PSS first; then the siliconoxide nanoparticles and PDADMACwere deposited in alternat-ing layers onto the surface of cells by the samemethod, formingsilicon oxide shells. The shellized cells remained viable andhave some special properties. Of course, it is important to retaincell viability during the treatment; live-dead viability probesshow that cell viability can remain almost unchanged if themineralization conditions are well designed.

In addition to LbL treatment, amphiphilic phospholipidscan also be used to direct the formation of biocompatible,uniform silica nanoshells on yeast, and bacterial cell surfaces[62]. Besidesmodifying the cell surfacewith the above chemicalengineering methods, we can also program membrane glyco-proteins using genetic approaches to introduce mineralizationfactors such as carboxylate groups onto cell surfaces and trans-form the non-mineralized cells into mineralization-preferredcells [63]. These shell structures maintain cell viability in theabsence of buffers, and cell surfaces are accessible to locallyadded proteins, plasmids, and nanocrystals (such as quantumdots) for cell tracking and imaging. Prolonged cell viabilitycombinedwith reporterprotein expression enables stand-alonecell-based sensing.



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It should be emphasized that IMS and PES are both flexible tosingle-cell shellization; however,HMSandSGSmake it difficultto achieve this goal. Because of good biocompatibility andbiodegradation of calcium phosphate, calcium shells also haveadvantages over SGS. IMS possesses many advantages overHMSandPES in cellular shellization, suchas bettermechanicaldurability and higher chemical and biological stability. Cellshape iswell-known to affect cell function [64, 65], and recentlyit has been shown that differentiation of hMSC is regulated bycell shape [66]. Inparticular, itwas shown thathMSCs thatwereallowed to spread on a 2D substrate underwent osteogenesis,while cells that were kept rounded became adipocytes. Controlover cell shape by IMS can therefore be an important tool inregulating stem cell differentiation.

Potential applications of cellularshellization

Cell storage

The storage of cells currently involves freezing cells insolutions containing dimethyl sulfoxide (DMSO) and sub-sequent transfer to liquid nitrogen. However, researchers have

noted that cryopreservation can induce permanent damage tothe stored cells, such as physical cell rupture caused by thedetrimental effects of cellular volumetric fluctuations andintracellular ice crystal formation. Apoptosis has also beenidentified as a major cause of cryopreservation-induced celldeath [67].

With the protection of shells, cells can be kept without theneed for any special facilities. Enclosed cells can be stored atroom temperature or in air without any rigorous requirements.The stored cells do not need to undergo rapid temperaturechange, so their natural properties can be maintained duringtreatment. Cells can be released by removal of the shell struc-ture, without the need for harsh treatments.

Shellized cells and bare cells have different biologicalproperties. Eukaryotic cell proliferation is controlled byspecific growth factors and the availability of essentialnutrients. Since these signals are blocked by themineral shells,cells captured within enter a specialized non-dividing restingstate, known as the stationary phase or G0, and become inert[61]. It is interesting that these inert cells can be reactivatedreadily by simple removal of the mineral shells, allowing themto grow and spontaneously return to the cycling mode, ame-nable to culture exactly as if theywere untreated cells (Fig. 4A).It appears that this ‘‘pause’’ function is not invoked in non-mineral shell: as mentioned above, LbL-treated yeast cells canstill undergo eukaryotic cell proliferation [39].

The encapsulation of enzymes within silica gels has beenextensively studied during the past decade for the design ofbiosensors and bioreactors [22, 32, 68]. Yeast spores and bac-teria have been recently immobilized within silica gels, wherethey retain their enzymatic activity [69–73]. Nassif et al.devised a method for the entrapment of bacteria(Escherichia coli) in silica gels, and demonstrated that theresulting mineral environment was more advantageous forcell survival than aqueous suspension. The metabolic activityof the bacteria decreased slowly, but half of the bacteria werestill viable after one month [74], . Yeast cells coated in a

Figure 3. Scheme of artificial shellization by PES. In most cases, thesurfaces of living cells are negatively charged. Positivepolyelectrolytes are adsorbed onto the surface by electrostaticinteraction so that the mineralization factors (always negativelycharged) can be subsequently linked onto the cell surfaces by LbL,forming the PES. The uniformity and density of the mineralizationfactors can be enhanced by repeating the LbL cycles. Finally,mineralization can be induced on the cell surfaces to form therequired shell structure. The SEM images show a bare yeast celland a yeast cell with artificial calcium phosphate shells aftershellization [61].



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mineralized shell were able to survive one month in water atfar higher levels than untreated cells in the same environment(Fig. 4B) 61]. However, the problem of the long-term viabilityof whole cells in an inorganic matrix has not been fullyaddressed. This is a serious challenge to the developmentof novel approaches for cell storage.

LbL treatment could be used to provide shells to livingcells. However, it is doubtful that the loose and flexible struc-ture of the pure PE covering would provide sufficient supportand protection for the enclosed cells. Hard mineral shells havethe comparative advantages of finely controlled structure andexcellent stability. We propose that IMS, as yet unexplored forthis application, would be an ideal technique for generatinghard mineralized shells to aid cell storage.

Cell protection

Containment of a cell within a porous shell permits onlymolecules smaller than the shell pore channel dimension topass through the shell and reach the cell inside. Shells essen-tially serve as an exoskeleton for cells and can protect againstforeign aggression. For example, enclosed yeast cells cansurvive under hostile conditions; a lytic enzyme mixture thatcould normally digest the yeast wall cannot digest the IMS andreach the enclosed cell. Therefore, enclosed yeast cells cansurvive in hypotonic solution while receiving sufficient smallnutrient transport [61]. The degree of protection provided bythe shell can be tuned by changing the porous structuralproperties of the shells. In a finely defined experiment, thepore size of shells was adjusted so that the cells receiveddifferent protection effects [75]. The shell thickness is anotherimportant factor that can be exploited to control transportacross the shell.

It is also possible to assign intelligent ‘‘guards’’ to the porechannel ‘‘gates,’’ achieved by using specific molecules andsubstrates that affect the passage of transport molecules(Fig. 5). For example, functional proteins or antibodies canact as the guards if they are introduced into the shell channels.Shells can also be associated with analogs of specific receptorson the cells surfaces and used to recognize the 3D structure ofspecific cytokines. These biologically modified shells can rec-ognize and monitor cytokines and molecules that associate

with a cell. Thus, the shell can maintain favorable cellularconditions and signals with the surrounding environment.Another protective advantage offered by shells is the abilityto inhibit contaminants and immunogens to associate withcells. Appropriate semi-permeable shells can prevent celldeath by permitting nutrients, waste, insulin, and similarmolecules to pass, but prevent passage of the larger moleculesassociated with immune rejection [75].

Although shells may be highly biocompatible, theirphysico-chemical functions are relatively limited, oftenrestricting their protective effect. However, artificial shelliza-tion can be engineered to provide protective barriers that arenot found in nature, for example, a UV coat. Rare earthmaterials adsorb ultraviolet light effectively and can be usedas shells to block mid-ultraviolet radiation penetration. In onestudy, zebrafish embryos enclosed with UV-blocking func-tional shells could develop normally when exposed to radi-ation where unprotected died [76]. Larvae could grow properlyand break the ultra-thin mineral shell. This technique could bedeveloped as a model of embryonic protection under an ozonehole. Shell engineering models such as these can help cellscounteract negative environments.

Figure 4. Scheme of pause function ofshellization during cell life cycle. A: Cellsare forced into G0 by shellization butthey can be reactivated by de-shellization; B: the yeast cells with shellhave longer life than the bare ones inwater, which demonstrates that theenclosed cells have become morerobust [61].

Figure 5. Gate role of shells. A: Environmental substances canaffect bare cells, and they may include hazardous components.B: Shells can control the substance transport by their micron- ornano-sized channels so that larger species (pentagons) cannotpenetrate the shell structure; C: the channel can be furtherfunctionalized by the use of recognition molecules such as proteins,which can choose the preferred species (small circles) to contactthe cells while blocking the others, which may have the similardimensions (squares). Thus, the shell acts as a smart gate-guard forthe cells.



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Cell delivery

Shellization is emerging as a platform for isolating, sorting,moving, and manipulating cells [77]. For example, Fe3O4

particles can be driven by magnetic fields and can be usedto control the movement of cells when incorporated into shelllayers. Fe3O4 nanocrystals can be co-precipitated with calciumphosphate and readily added into calcium shells. Thisapproach can be used as an effective method to separate,identify and concentrate different cell types. Since the func-tional shells can be moved under external control, the shellscan be considered as cell carriers for in vivo delivery. Thismethod is an inexpensive and relatively uncomplicated way tocontrol cell delivery (Fig. 6) [61].

Cell rolling is an important physiological and pathologicalprocess that is used to recruit specific cells in the bloodstreamto a target tissue. This process may be also exploited inengineering cell shells to capture and separate specific celltypes. One of the most commonly studied proteins thatregulate cell rolling is P-selectin. Adhesion bonds betweenP-selectin and Sialyl Lewis(x) (SLeX) dissociate readily undershear forces leading to cell rolling [78, 79]. Encapsulating cellsin hydrogel that has been covalently conjugated with struc-tural molecules of SLeX, could be control cell homing withoutmodifying cells directly. HMS coating over cells could functionas a chemical scaffold to mediate homing from vascular totissue compartments. Hydrogel shells conjugated with struc-tural molecules could be used to direct the homing of cells forregenerative therapy. The challenge of using cells like bonemarrow mesenchymal stem cells (MSC) for regenerativetherapy, inflammation treatment, and angiogenesis pro-motion is the targeting of these cells to the requisite treatmentsites with minimum morbidity and maximum efficiency[80, 81].

Magnetic nanoparticles [82] and quantum dot cell labeling[83] may be incorporated into mineral shell layers to facilitatein vivo tracking. Magnetic resonance tracking of magnetically

labeled stem and progenitor cells is an emerging technology,and there is an urgent need for appropriate probes that canmake cells highly magnetic. Enclosing cells with magneticallyvisible contrast agents is another possible approach, providedthat labeled cells maintain their viability and proliferationcapacity [84]. Therapeutic cells that are coated to permit celltracking by magnetic resonance imaging (MRI) techniqueshold great promise for answering various questions aboutthe optimum timing of administration, cell location, and cellviability over time.

Cell surface engineering and microfluidic systems can beintegrated to achieve effective cell manipulation on a chip [85,86]. Miltenyi et al. developed a flexible, fast, and simplemagnetic cell sorting system for separation of a large numberof cells according to specific surface markers. Cells stainedsequentially with biotinylated antibodies, fluorochrome-con-jugated avidin, and superparamagnetic biotinylated-micro-particles (about 100 nm diameter) were separated on high-gradient magnetic columns. Unlabeled cells pass freelythrough the column, while labeled cells are retained andcan be easily eluted. The simultaneous tagging of cells withfluorochromes and invisible magnetic beads makes this sys-tem an ideal complement to flow cytometry, as light scatterand fluorescent parameters of the cells are not changed by thebound particles, nor is cell viability or proliferation [87].

Cell therapy

Stem cells have great potential as therapeutic agents, sincethey can be induced to differentiate into specific tissue typesfor repair or regeneration [88]. Several studies [89, 90] havereported that nanoparticles of calcium minerals can promoteosteoblastic differentiation from stem cells. Differentiationand proliferation of stem cells can be effected significantlyby the presence of calcium phosphate nano-crystallites bysignificantly increasing the alkaline phosphatase (ALP)activity of BMSC [91]. In the presence of the mineral nano-phase, differentiation is directed specifically toward the osteo-blastic phenotype. These results indicate that an artificial shellof nanocalcium phosphate would not only provide protectionfor cell storage but would also act as an osteogenic promoter,making calcium phosphate-coated stem cells an ideal bonerepair material. Stem cells can be easily stored in shells so thatthey can be readily available for implantation, then degradedgradually, so that the cells can adapt appropriately to theirnew environment.

It is critical that the released cells maintain the advantageof differentiated osteoblasts. Generally, newly differentiatedosteoblasts have better mineralization ability and the mineralshells can provide an additional source of calcium and phos-phate ions for the osteoblasts to generate new bone tissue [92].The complex of BMSC and calcium phosphate shells can beconsidered a living bone repair material.

Adoptive cell therapy (ACT) is a treatment that uses acancer patient’s own T lymphocytes which have endogenousanti-tumor activity. These cells are expanded in vitro andreinfused into the patient. This approach involves the identi-fication ex vivo of autologous or allogeneic lymphocyteswith anti-tumor activity, often along with appropriate growthfactors, to stimulate their survival and expansion when

Figure 6. Magnetic particles (red dots) cannot be integrated intoliving cells (green circles) directly. However, they can be integratedinto shell structures (gray layers) readily so that magnetic cells canbe created using shell engineering. The optical images demonstratethat bare yeast cells are insensitive to magnetic fields so that theyare randomly placed, but the cells with calcium phosphate-Fe3O4

can be driven and concentrated by a magnet [61].



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implanted in the patient [93]. Tumor-killing cells may beinduced from a patient’s own T-cells by integrating growthfactors or cytokines within the outer-surface of shells as aself-support system to enhance their activities and to providean optimal environment for the transplanted cells. Such ashellized T-cell vaccine system can be a promising and highlyeffective immunotherapeutic approach for future cancertreatment.

Viruses with a mineral shell structure can also be used as atargeted cancer treatment. If a virus is wrapped by a mineralcoating, it cannot be detected by the immune system and isfree to reach host cells. Virus-mineral core-shell nanocompo-sites have the biological features of the mineral surfacematerials, but do not lose their virulence. Once they reachtarget cells, the mineral shells can be dissolved to expose thevirus. Interestingly, the cellular microenvironment for mostcancers is slightly acidic, which can dissolve calciummineralsto breakmineral shells, allowing for an innate cancer targetingsystem. In contrast, the pH environment of normal cells isslightly basic, maintaining stability of the shell and thuspreventing virus delivery to normal cells. It is expected thatthe viruses coated by calcium shells can be selectively acti-vated by cancer cells without affecting the normal cells [94].

Conclusions and perspectives

The combination of inorganic materials with biological sys-tems remains largely unexplored. Nature has been a source ofinspiration for similar technical developments. Inspired by theouter surface of eggshells and diatoms, cell surface engineer-ing – or shellization – is emerging as a new research focuswithin chemical biology. Four major methods for cellularshellization have been described in this paper. HMS is themost biocompatible method for cellular shellization and isnow widely used as mimic ECM for tissue engineering andregenerative medicine. However, its application to single cell,rather than bulk, encapsulation needs to be developed.Similar drawbacks apply to SGS, though some relevant trialshave been carried out as part of cell therapy development. PESis a relatively new method for cellular shellization. Its poten-tial has been shown in giving cells new properties, but thetoxicity of PEs tomammalian cells is the major limitation on itsuse. Compared to PES, IMS has many advantages, such ashigher stability, mechanical resistance and blocking of the cellcycle, ideal for cell storage, and cell delivery applications.With bio-Micro-Electro-Mechanical Systems (bioMEMS) inno-vation, microfluidic platforms will provide further oppor-tunities to exploit single-cell shellization. Integration ofmicrofluidic chips with cell surface engineering is expectedto yield novel cell-based microsystems devices and microflui-dic-based cell technologies.

Structure determines function, and designing novel shellsfrom either natural constituents or synthetic materials canprovide cells with new and unique properties. Shells maymimic natural features, such as semi-permeability, or canbe custom-designed to perform such functions as blockingUV radiation. Progress in cell surface engineering, especiallyin the design of shell materials with specific biological

functions, will find application throughout the fields of bio-technology and medicine.

AcknowledgmentsWe thank Jason Nichol, Stephanie Piecewicz, and Yanan Dufor revising the paper. This study was supported by theNational Natural Science Foundation of China (20871102),Zhejiang Provincial Natural Science Foundation (R407087),the Fundamental Research Funds for the Central Universities,and Daming Biomineralization Foundation.

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Cellular shellization: Surface engineering gives cells an exterior