Prospects & Overviews

Cellular shellization: Surface engineering gives cells an exterior Review essays Ben Wang1)y, Peng Liu1)y and Ruikang Tang1)2)

Unlike eggs and diatoms, most single cells in nature do Introduction not have structured shells to provide extensive protec- tion. It is a challenge to artificially confer shell structures Progress in biological science and materials engineering has begun to blur the boundary between living and non-living on living cells to improve their inherent properties and systems. Synthetic biologists can now engineer complex, arti- functions. We discuss four different types of cellular ficial biological systems to help understand natural biological shellizations: man-made hydrogels, sol-gels, polyelectro- phenomena and use in a variety of biomedical applications [1]. lytes, and mineral shells. We also explore potential appli- Advances in directed evolution and membrane biophysics cations, such as storage, protection, delivery, and make the synthesis of simple living cells an imaginable therapy. We suggest that shellization could provide goal [2]. Cellular life is the basic unit of a living organism and another means to regulate and functionalize cells. defines the presence of a stable information reservoir con- Specifically, the integration of living cells and non-living nected to the external world by a well-defined boundary [3]. functional shells may be developed as a novel strategy The cell membrane separates the interior of a cell from the to create ‘‘super’’ or intelligent cells. Unlike biological outside environment and must meet specific requirements approaches, this material-based bio-interface regulation such as semi-permeability to permit communication and molecular transport across the border [4]. Using natural is inexpensive, effective, and convenient, opening up a membranes as a model, it is possible to build an artificial novel avenue for cell-based technologies and practices. shell from natural constituents or from synthetic soft or hard materials, introducing robustness to the capsule – the cell- Keywords: shell combination. Furthermore, we can also design the shell .biointerface; biomimetic mineralization; cellular shell; to alter the inherent biological qualities of the cell. functional materials; polyelectrolyte In the evolution of natural systems, living organisms have developed various mineralized structures, such as teeth, bones, shells, carapaces, and spicules. Those composite often exhibit complex hierarchical structures and possess important functions such as mechanical

DOI 10.1002/bies.200900120

1) Center for Biomaterials and Biopathways and Department of Chemistry, Abbreviations: Zhejiang University, Hangzhou, Zhejiang 310027, China ACT, adoptive cell therapy; ALP, alkaline phosphatase; bioMEMS, bio-Micro- 2) State Key Laboratory of Materials, Zhejiang University, Hangzhou, Electro-Mechanical Systems; BMSC, bone marrow mesenchymal stem cells; Zhejiang 310027, China BSA, bovine serum albumin; DMSO, dimethyl sulfoxide; EB, embryoid bodies; *Corresponding author: ECM, ; EDTA, ethylenediaminetetraacetic acid; HA, Ruikang Tang hyaluronic acid; hESC, human embryonic stem cell; HMS, hydrogel matrix E-mail: [email protected] shell; IMS, induced mineral shell; LbL, Layer by layer; MELN cells, MCF-7 cells transfected with a construct expressing the luciferase gene under the control of an estrogen-regulated promoter; mESCs, murine embryonic stem cells; MRI, Magnetic Resonance Imaging; MSC, mesenchymal stem cells; PAA, poly(acrylic sodium); PDADMAC, poly(diallyldimethylammonium chloride); PE, polyelectrolyte; PES, polyelectrolyte shell; PLL, poly (L- lysine); PSS, polystyrene sulfonate; SGS, sol-gel shell; SLeX, Sialyl Lewis(x); TMOS, tetramethyl orthosilicate; VNPs, viral . yThese authors contributed equally to this work.

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

support, protection, motility, and sensing of signals [5]. different approaches that have been undertaken to Marine organisms such as mollusks [6] and arthropods [7] construct artificial shells, such as hydrogel matrix shell use biominerals as their exterior coats to protect their soft (HMS), sol-gel shell (SGS), polyelectrolyte shell (PES), and bodies from external stresses or aggression. A cocoon is induced mineral shell (IMS). Applications of cellular shelliza- another example of an exterior coat that fulfills multiple roles tion – cell storage, protection, delivery, and therapy – are then including protection against physical stress, toxic substances discussed (Fig. 1C). Finally, conclusions and perspectives are and natural enemies, and even mediation of messages con- offered as to where these fascinating avenues of research trolling the life cycle. Besides these examples, a number of might lead. unicellular organisms have biogenic coverings as well. Chicken eggs are perhaps the most familiar example (Fig. 1A). Eggshells provide mechanical support for the Hydrogel matrix shell (HMS) embryo and are also extremely important for maintaining the egg’s viability. It is well-known that an egg with an intact The extracellular matrix (ECM) is the portion of animal tissue shell can be stored for several weeks in ambient conditions; that provides the essential microenvironment for cells. Due to however, the egg will decay quickly if the shell is broken. The its diverse nature and composition, the ECM can serve many shell forms a shield to prevent the enclosed cells from con- functions, such as support and anchorage for cells, segregat- tamination. Eggshells also maintain the balance of oxygen, ing tissues from one another, and regulating intercellular carbon dioxide, water, and nutrients required for proper cell communication [11]. Synthetic and natural hydrogels have development [8]. In addition, shells provide the embryo with become popular as three-dimensional in vitro cell culture the minerals needed for the generation of organs high in platforms that mimic ECM. Networks of hydrophilic polymers calcium, such as the skeleton, muscles, and brain [9]. that can retain large quantities of water, hydrogels can be Diatoms, another example of a unicellular organism with a synthesized by multiple methods. Hydrogels are biocompat- mineral coat, have unique cell walls made of silica (hydrated ible, supportive of cell maintenance and growth, since they silicon dioxide) called frustules. The ornately patterned facilitate the exchange of gases and nutrients in a way similar silicified shell has evolved as a biological protection for to the environment in vivo [12]. Hydrogel encapsulation may diatoms [10]. provide cells with structural support, chemical stability or a However, most cells cannot make their own shells. measure of protection from immune attack. Both synthetic Many attempts have been made to fabricate artificial shells and naturally derived hydrogels have been explored for encap- directly onto cells or to confer to cells an ability to form sulation of a variety of cell types [13]. The use of hydrogels to their own shells. This paper gives an overview of the encapsulate stem cell populations indicates the ability of this

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technique to manipulate cell fate. For example, murine embry- The silicate matrix is usually formed by hydrolysis of an onic stem cells (mESC) were encapsulated into 1.6% w/v alkoxide precursor followed by condensation to yield a poly- alginate microbeads. Differentiation was inhibited at the mor- meric oxo-bridged SiO2 network. In the process, molecules of ula-like stage, with no cystic embryoid bodies (EB) formed the corresponding alcohol are liberated. The ability to form within the beads. Using the same strategy to coat human hybrid silica glasses under aqueous conditions and room embryonic stem cells (hESC), stem cell colonies could be temperature (at which and cells are active) opened maintained for up to 260 days in an undifferentiated state. up the possibility of extending sol-gel processing to the encap- Upon release from the alginate microbeads, hESC were able to sulation of cells. However, while sol-gel conditions are mild resume differentiation, as shown by the formation of into enough for organic molecules, they are still too harsh for living cystic EB containing beating cardiomyocytes. Retention of cell organisms to retain viability. For example, alcohol and pluripotency by encapsulation occurred even under differen- acidic pH lead to the denaturation of most proteins. tiation promoting conditions [14]. Recently, a newly developed Therefore, the sol-gel process had to be adapted to bioencap- integrated bioprocess utilized alginate hydrogel encapsulation sulation, which is currently achieved in two steps. The first Review essays of mESC to form 3D mineralized constructs without the need step is the hydrolysis of tetramethyl orthosilicate (TMOS) in for passaging or handling of the cells [15]. Chan et al. also the presence of an acid to hydrolyze all alkoxy groups [30]. fabricated injectable collagen–human mesenchymal stem cell Cells are then added to the Si(OH)4 hydrolyzed aqueous (hMSC) microspheres using microencapsulation. Apart from solution in the presence of a buffer with a pH around 7. providing a protective matrix for cell delivery, the collagen Condensation progresses form the silica network rapidly microspheres also acted as a bio-mimetic matrix facilitating around the cells entrapped within the porous gel. The whole the functional remodeling of hMSC [16]. The 3D hydrogel process occurs at room temperature within a few minutes and microenvironment offers a means to control cell-matrix inter- without denaturation of most cells. A novel class of bioactive actions and thereby manipulate cell behavior. These studies materials is thus obtained, comprised of cells physically demonstrate that hydrogel encapsulation can be considered a trapped within silica matrices. means to not only provide structure and protection to a trans- The Biosil method is a versatile technique for combining plantable cell product, but also as a way to more efficiently functional cells and cell aggregates within the unique attain the desired cell population. structure provided by silica [31]. Biosil technology is based Hydrogel embedding has several advantages over other on the encapsulation of whole cells by a sol-gel silica layer shellization techniques, including keeping the cells in an deposited on the cell surface using silica precursors in aqueous environment, in contact with soft and biocompatible the gas phase (Fig. 2). It has been demonstrated that the materials, while protecting them from the stress of encapsu- Biosil process provides mechanical stability, porosity control lation. Their material properties can be engineered for for immunological protection, and maintenance of cell biocompatibility, selective permeability, mechanical, and viability with sustained cellular functions. Extension of chemical stability, as well as other requirements as specified the Biosil process to alginate microencapsulation could by the application. Embedding cells in hydrogels requires the enhance biocompatibility for purposes of cell grafts and production of microbeads by an economical, gentle, and therapy [32]. reproducible method. Methods of hydrogel bead generation include emulsification, extrusion, co-extrusion, hollow particle 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 number of 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 silica glasses at room temperature [21, 22]. Silica is present in all living organisms and, after carbonates, it is the second most abundant mineral formed by organisms. Silica structures with precisely controlled morphologies are extensively produced by single-cell organisms such as diatoms [23, 24]. The recent extension of this process to enable the entrapment of func- Figure 2. Schematic diagram of the Biosil process. A: Cell tionally active biomolecules demonstrated the introduction suspension mixed with collagen fibers (red line) as a scaffold. A and retention of biological activity within silica gels. The first water-layer forms around the cells (white layer) because of their report of the sol-gel encapsulation of enzymes was published hydrophilic surface. B: Using alkoxides in the gas phase, a sol-gel membrane (yellow layer) can be built up directly on in the early 1970s [25], but this process was actually developed the cell surface: the gaseous precursors react with by the Jerusalem group in 1990 [26]. Since then, enzymes, surface-adsorbed H2O and exposed OH, allowing removal of antibodies, and even whole cells have been encapsulated byproducts from the solid sol-gel in the gas phase. SiOR, within sol-gel glasses [27–29]. alkoxide precursors; ROH, reaction byproducts.

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An important property of the sol-gel process is the favorable Besides the proposed shellization of cells, application of such dispersion of a variety of chemicals directly into the gelling procedures to viruses is also becoming both interesting and solution, including biomass, which gives a feasible important in biological research. Viral nanoparticles (VNPs) eiwessays Review approach for SGS modification. The intimate mixing of have received great attention in recent organic molecules and alkoxides in the precursor solution research, but their virulence must be eliminated before their allows organic and inorganic components to be associated practical application. Several biological approaches such as at the molecular level. Organic molecules can simply be gene reassortment have been tried, but incompletely modified embedded within the silica matrix or chemically linked VNP cannot be avoided. They would likely stimulate undesir- via SiC bonds [33]. Reactions involving silica precursors able infections and immune responses during material fabri- lead to a sol (a nanometric dispersion of silica particles) cation. Another bottleneck in VNP applications is the difficulty which collapses to a solid network holding the liquid (the of processing viruses on a large scale, mainly because of the sol-gel state). Macromolecules, metal particles, and a ineffective concentration and recovery of virus particles. The variety of chemical compounds can all be combined in general approaches to concentrating viruses such as ultra- the silica sol solution before gelling [34]. The extension of centrifugation and density gradient methods are too tedious this approach to sol-gel silica loaded with bioactive mol- and time-consuming to separate and purify the viruses in ecules, particularly enzymes, may create novel and valuable sufficient amounts. Our experiments recently demonstrated materials for cellular shellization. Comprehensive reviews of that shellization is a feasible strategy to manipulate the bio- cell encapsulation within sol-gel materials have been logical security of viruses; the infectivity of the enclosed virus recently published by Avnir and coworkers and Meunier is effectively suppressed by the shell structure [42]. Thus virus- and coworkers [35, 36]. based PE composites could make large-scale application of VNPs possible. In addition, the shell structure could reduce the surface repulsive force of virus particles, leading to new flocculation and aggregation behaviors. Therefore, the separ- Polyelectrolyte shells (PES) ation and concentration of the virus-shell composites can even be achieved readily by normal centrifugation. Most virus Based on the electrostatic properties of the cell, polyelectrolyte shortages in VNPs applications could be addressed by (PE) shells may be directly grown or built onto negatively shellization. charged cells. Layer by layer (LbL) fabrication has been Maintaining viability of cells is the most important factor applied as a general technique for the fabrication of multi- during shellization. Unlike yeast cells, delicate mammalian component films on solid supports: several alternating cycles cells are extremely sensitive to PEs. The toxicity of PEs during of absorption and deposition of opposite-charged PE [37, 38]. the LbL procedure is a serious threat to mammalian cell Living yeast cells have been singly encapsulated by the viability during biomimetic modification. Previous studies alternate adsorption of oppositely charged PEs. Fluorescent have also demonstrated that positive PEs damage the cell imaging revealed that the cells preserved their integrity and layer even more than negative ones [43]. The toxicity of com- metabolic activity after the coating procedure, and remained monly used positive PEs are ranked as poly(ethylenimine) ¼ capable of dividing within the shell [39]. Shellization of mouse poly(l-lysine) > poly(diallyl dimethyl ammonium chloride) > MSC with hyaluronic acid (HA) and poly (L-lysine) (5) main- diethylaminoethyl dextran>poly(vinyl pyridinium bromide) > tained morphology and viability for up to one week [40]. Cells starburst dendrimer > cationized albumin > native albumin. coated with PE layers could provide an inexpensive model In addition, the toxic effects of all polymers can depend upon system for a wide range of biophysical and bioengineering time and concentration [44]. Although a quick treatment (less applications, due to the tunable properties of the PES. than 10 minutes) can reduce the unfavorable influences of the To create functional cell-based , it would be PEs on living cells, it must be acknowledged that the toxicity of advantageous to add protection to mammalian cells, which PEs limits the usage of LbL on most mammalian cells. The would be analogous to the cell wall protecting yeast or plant design and synthesis of the suitable positive PE with acceptable cells. As an example, MELN cells (MCF-7 cells transfected with levels of toxicity for mammalian cells is necessary for cellular a construct expressing the luciferase gene under the control of shellization in future applications. an estrogen-regulated promoter) have been isolated with a PES using the LbL technique. Among several PE-couples, optimal cell survival (>80%) was obtained by alternating poly-cation poly-diallyldimethyl ammonium chloride layers Induced mineral shell (IMS) with the negatively charged poly-styrene sulfonate. The composition of the buffer used for layer deposition was Biomineralization is a biological process by which living crucial to preserving cell viability, e.g., potassium ions were organisms make use of organic matrices to control the for- preferred to sodium ions during the coating. Furthermore, mation of functional minerals [45]. Shellization is a special viability was increased when cells were allowed to recover example, since the created crystals can enclose the whole for 2 hour between each bilayer deposition. Successful encap- organism via formed uniform shell structures. Previous stud- sulation of MELN cells demonstrated that coating not only ies of biomineralization have demonstrated that cells with permits mammalian cell survival, but also allows essential mineral shells always have outer proteinaceous membranes, metabolic functions such as RNA and synthesis to take which act as mineralization templates to induce hetero- place [41]. geneous crystallizations [46]. Again using chicken eggs as

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an example (Fig. 2B), the foundation for cellular mineraliz- adsorbed onto cell surfaces; they must be locally clustered ation is the proteinaceous eggshell membrane (mineraliz- to adsorb calcium minerals at the specific sites to induce ation-induced layer). The mineralization of the calcite phase nucleation. Cells can also be modified using LbL to add the starts with the nucleation of calcite crystals on the keratin required molecules or groups onto their surfaces [57, 58]. sulfate-rich organic aggregations on the eggshell membrane Two PEs are used for cell membrane modification: polycation [47]. Keratin sulfate is a calcium-binding polyanionic molecule poly(diallyldimethylammonium chloride) (PDADMAC) and reported to be closely associated with calcite crystals in the poly(acrylic sodium) (PAA). PAA has many polar carboxylate chicken ear statoconial membrane [48]. The shell membrane groups to bind dissolved Ca2þ, and the reorganized surfaces of eggshells, rich in calcium-binding polyanionic molecules, can significantly increase the electron density on the surface to acts as a mineralization template to induce calcium carbonate induce heterogeneous nucleation of calcium minerals [59, 60]. crystallization and control calcite growth. After LbL treatment, the carboxylate groups can be integrated Unlike conventional crystallization in laboratories, the on the cell surfaces. When the treated yeast cells are in contact organic matrix can mediate the formation of inorganic crystals with the supersaturated calcium phosphate solution, the Review essays such as calcite in biomineralization. Inorganic mineralization mineral phases are preferentially crystallized in situ at the in the presence of organic additives is universally considered a cell-solution interfaces. Thus, an egg-liked structure can result perfect model for biomimetic mineralization [49]. Although (Fig. 3). Yeast cells have also been shellized with silica by hundreds of proteins have been identified during biomineral- Insung S. Choi and colleagues. In their experiment, PDADMAC ization, it is generally agreed that most proteins active in the and sodium polystyrene sulfonate (PSS) were employed mediation of biologically directed mineral growth contain to modify cells by LbL treatment. The difference between acidic amino acid residues; specifically, regions rich in carbox- their experiment and ours is their use of PDADMAC as ylates that interact with mineral surfaces to influence both the outermost layer, since synthetic polymers containing crystal morphology and rates of formation [50]. Negatively quaternary amines were found to be chemically catalytic for charged groups such as carboxylate, sulfate, and phosphate biomimetic silica formation under physiologically mild con- are effective in binding Ca2þ ions to control nucleation and ditions. This strategy could be universally used in the modi- growth of calcium minerals by lowering the interfacial energy fication of various cells and the preparation of different between crystal and organic substrate and accumulating mineral shells. soluble calcium ions at suitable nucleation sites [51–53]. In Besides calcium, shells can also be formed on practice, researchers have successfully used carboxylate-rich the cell surface using gold, silver, or silicon oxide. Yeast cells compounds to modify the mineralization ability of solid sub- were shellized using alternating deposition of PE and charged strates. For example ethylenediaminetetraacetic acid (EDTA), a nanoparticles. The yeast cells were first shellized by using relatively small molecule with four carboxylate groups, is often alternating deposition of either PAH/PSS or bovine serum used to pretreat metal before implantation. It causes a layer of albumin (BSA)/DNA onto the surface of cells as mentioned uniform and continuous calcium coating to be precipitated above. Second the shellized cells were introduced into a sus- readily on the metal surface, rendering it ‘‘living’’ for hard pension of either gold or silver nanoparticles, and then two tissue formation [54]. This example demonstrates the regulat- additional PE layers were deposited onto them. Gold and silver ory effect of carboxylate on the mineralization of substrates. It nanoparticles were successfully immobilized on the surface of also offers a possible approach to changing the mineralization the cells, effectively altering their color and surface topogra- capability of living cells: introduction of mineralization factors phy. Recently we also gave yeast cells silicon oxide nanopar- such as carboxylate groups onto cell surfaces may turn non- ticle shells. We used PDADMAC and PSS first; then the silicon mineralization cells into mineralization-preferred ones. Thus, oxide nanoparticles and PDADMAC were deposited in alternat- it becomes possible to prepare mineral shells for living cells via ing layers onto the surface of cells by the same method, forming a uniform enhancement of surface mineralization. silicon oxide shells. The shellized cells remained viable and We must also emphasize that such regulation of cellular have some special properties. Of course, it is important to retain mineralization is also important in understanding pathologi- cell viability during the treatment; live-dead viability probes cal mineralization. In the human body, calcification or show that cell viability can remain almost unchanged if the mineralization can occur on some calcification-free organs mineralization conditions are well designed. under certain conditions, producing serious diseases such In addition to LbL treatment, amphiphilic phospholipids as arteriosclerosis and kidney stones [55]. Pathological calci- can also be used to direct the formation of biocompatible, fication is an important genesis of human diseases [56]. From a uniform silica nanoshells on yeast, and bacterial cell surfaces view of biomineralization, pathological calcification can be [62]. Besides modifying the cell surface with the above chemical considered an unexpected promotion of biomineralization engineering methods, we can also program membrane glyco- ability of cell or organism’s surfaces. It has also been noted proteins using genetic approaches to introduce mineralization that the accumulation of mineralization factors (e.g., choles- factors such as carboxylate groups onto cell surfaces and trans- terols and calcium-binding proteins) at cell-milieu interfaces form the non-mineralized cells into mineralization-preferred also causes pathological mineralization. Therefore, control- cells [63]. These shell structures maintain cell viability in the ling the mineralization factor density on cell surfaces is a key absence of buffers, and cell surfaces are accessible to locally to both artificial shellization and understanding pathological added proteins, plasmids, and nanocrystals (such as quantum calcification. dots) for cell tracking and imaging. Prolonged cell viability In order to improve the mineralization capability of combined with reporter protein expression enables stand-alone living cells, mineralization factors could be inserted into or cell-based sensing.

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Figure 3. Scheme of artificial shellization by PES. In most cases, the noted that cryopreservation can induce permanent damage to surfaces of living cells are negatively charged. Positive the stored cells, such as physical cell rupture caused by the polyelectrolytes are adsorbed onto the surface by electrostatic detrimental effects of cellular volumetric fluctuations and interaction so that the mineralization factors (always negatively intracellular ice crystal formation. Apoptosis has also been charged) can be subsequently linked onto the cell surfaces by LbL, forming the PES. The uniformity and density of the mineralization identified as a major cause of cryopreservation-induced cell factors can be enhanced by repeating the LbL cycles. Finally, death [67]. mineralization can be induced on the cell surfaces to form the With the protection of shells, cells can be kept without the required shell structure. The SEM images show a bare yeast cell need for any special facilities. Enclosed cells can be stored at and a yeast cell with artificial calcium phosphate shells after room temperature or in air without any rigorous requirements. shellization [61]. The stored cells do not need to undergo rapid temperature change, so their natural properties can be maintained during treatment. Cells can be released by removal of the shell struc- It should be emphasized that IMS and PES are both flexible to ture, without the need for harsh treatments. single-cell shellization; however, HMS and SGS make it difficult Shellized cells and bare cells have different biological to achieve this goal. Because of good biocompatibility and properties. Eukaryotic cell proliferation is controlled by biodegradation of calcium phosphate, calcium shells also have specific growth factors and the availability of essential advantages over SGS. IMS possesses many advantages over nutrients. Since these signals are blocked by the mineral shells, HMS and PES in cellular shellization, such as better mechanical cells captured within enter a specialized non-dividing resting durability and higher chemical and biological stability. Cell state, known as the stationary phase or G0, and become inert shape is well-known to affect cell function [64, 65], and recently [61]. It is interesting that these inert cells can be reactivated it has been shown that differentiation of hMSC is regulated by readily by simple removal of the mineral shells, allowing them cell shape [66]. In particular, it was shown that hMSCs that were to grow and spontaneously return to the cycling mode, ame- allowed to spread on a 2D substrate underwent osteogenesis, nable to culture exactly as if they were untreated cells (Fig. 4A). while cells that were kept rounded became adipocytes. Control It appears that this ‘‘pause’’ function is not invoked in non- over cell shape by IMS can therefore be an important tool in mineral shell: as mentioned above, LbL-treated yeast cells can regulating stem cell differentiation. still undergo eukaryotic cell proliferation [39]. The encapsulation of enzymes within silica gels has been extensively studied during the past decade for the design of biosensors and bioreactors [22, 32, 68]. Yeast spores and bac- Potential applications of cellular teria have been recently immobilized within silica gels, where shellization they retain their enzymatic activity [69–73]. Nassif et al. devised a method for the entrapment of bacteria Cell storage (Escherichia coli) in silica gels, and demonstrated that the resulting mineral environment was more advantageous for The storage of cells currently involves freezing cells in cell survival than aqueous suspension. The metabolic activity solutions containing dimethyl sulfoxide (DMSO) and sub- of the bacteria decreased slowly, but half of the bacteria were sequent transfer to liquid nitrogen. However, researchers have still viable after one month [74], . Yeast cells coated in a

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Figure 4. Scheme of pause function of shellization during cell life cycle. A: Cells are forced into G0 by shellization but they can be reactivated by de- shellization; B: the yeast cells with shell have longer life than the bare ones in water, which demonstrates that the enclosed cells have become more robust [61]. Review essays

mineralized shell were able to survive one month in water at with a cell. Thus, the shell can maintain favorable cellular far higher levels than untreated cells in the same environment conditions and signals with the surrounding environment. (Fig. 4B) 61]. However, the problem of the long-term viability Another protective advantage offered by shells is the ability of whole cells in an inorganic matrix has not been fully to inhibit contaminants and immunogens to associate with addressed. This is a serious challenge to the development cells. Appropriate semi-permeable shells can prevent cell of novel approaches for cell storage. death by permitting nutrients, waste, insulin, and similar LbL treatment could be used to provide shells to living molecules to pass, but prevent passage of the larger molecules cells. However, it is doubtful that the loose and flexible struc- associated with immune rejection [75]. ture of the pure PE covering would provide sufficient support Although shells may be highly biocompatible, their and protection for the enclosed cells. Hard mineral shells have physico-chemical functions are relatively limited, often the comparative advantages of finely controlled structure and restricting their protective effect. However, artificial shelliza- excellent stability. We propose that IMS, as yet unexplored for tion can be engineered to provide protective barriers that are this application, would be an ideal technique for generating not found in nature, for example, a UV coat. Rare earth hard mineralized shells to aid cell storage. materials adsorb ultraviolet light effectively and can be used as shells to block mid-ultraviolet radiation penetration. In one Cell protection study, zebrafish embryos enclosed with UV-blocking func- tional shells could develop normally when exposed to radi- Containment of a cell within a porous shell permits only ation where unprotected died [76]. Larvae could grow properly molecules smaller than the shell pore channel dimension to and break the ultra-thin mineral shell. This technique could be pass through the shell and reach the cell inside. Shells essen- developed as a model of embryonic protection under an ozone tially serve as an exoskeleton for cells and can protect against hole. Shell engineering models such as these can help cells foreign aggression. For example, enclosed yeast cells can counteract negative environments. survive under hostile conditions; a lytic enzyme mixture that could normally digest the yeast wall cannot digest the IMS and reach the enclosed cell. Therefore, enclosed yeast cells can survive in hypotonic solution while receiving sufficient small nutrient transport [61]. The degree of protection provided by the shell can be tuned by changing the porous structural properties of the shells. In a finely defined experiment, the pore size of shells was adjusted so that the cells received different protection effects [75]. The shell thickness is another important factor that can be exploited to control transport across the shell. It is also possible to assign intelligent ‘‘guards’’ to the pore Figure 5. Gate role of shells. A: Environmental substances can channel ‘‘gates,’’ achieved by using specific molecules and affect bare cells, and they may include hazardous components. substrates that affect the passage of transport molecules B: Shells can control the substance transport by their micron- or (Fig. 5). For example, functional proteins or antibodies can nano-sized channels so that larger species (pentagons) cannot act as the guards if they are introduced into the shell channels. penetrate the shell structure; C: the channel can be further functionalized by the use of recognition molecules such as proteins, Shells can also be associated with analogs of specific receptors which can choose the preferred species (small circles) to contact on the cells surfaces and used to recognize the 3D structure of the cells while blocking the others, which may have the similar specific cytokines. These biologically modified shells can rec- dimensions (squares). Thus, the shell acts as a smart gate-guard for ognize and monitor cytokines and molecules that associate the cells.

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Cell delivery labeled stem and progenitor cells is an emerging technology, and there is an urgent need for appropriate probes that can Shellization is emerging as a platform for isolating, sorting, make cells highly magnetic. Enclosing cells with magnetically eiwessays Review moving, and manipulating cells [77]. For example, Fe3O4 visible contrast agents is another possible approach, provided particles can be driven by magnetic fields and can be used that labeled cells maintain their viability and proliferation to control the movement of cells when incorporated into shell capacity [84]. Therapeutic cells that are coated to permit cell layers. Fe3O4 nanocrystals can be co-precipitated with calcium tracking by magnetic resonance imaging (MRI) techniques phosphate and readily added into calcium shells. This hold great promise for answering various questions about approach can be used as an effective method to separate, the optimum timing of administration, cell location, and cell identify and concentrate different cell types. Since the func- viability over time. tional shells can be moved under external control, the shells Cell surface engineering and microfluidic systems can be can be considered as cell carriers for in vivo delivery. This integrated to achieve effective cell manipulation on a chip [85, method is an inexpensive and relatively uncomplicated way to 86]. Miltenyi et al. developed a flexible, fast, and simple control cell delivery (Fig. 6) [61]. magnetic cell sorting system for separation of a large number Cell rolling is an important physiological and pathological of cells according to specific surface markers. Cells stained process that is used to recruit specific cells in the bloodstream sequentially with biotinylated antibodies, fluorochrome-con- to a target tissue. This process may be also exploited in jugated avidin, and superparamagnetic biotinylated-micro- engineering cell shells to capture and separate specific cell particles (about 100 nm diameter) were separated on high- types. One of the most commonly studied proteins that gradient magnetic columns. Unlabeled cells pass freely regulate cell rolling is P-selectin. Adhesion bonds between through the column, while labeled cells are retained and P-selectin and Sialyl Lewis(x) (SLeX) dissociate readily under can be easily eluted. The simultaneous tagging of cells with shear forces leading to cell rolling [78, 79]. Encapsulating cells fluorochromes and invisible magnetic beads makes this sys- in hydrogel that has been covalently conjugated with struc- tem an ideal complement to flow cytometry, as light scatter tural molecules of SLeX, could be control cell homing without and fluorescent parameters of the cells are not changed by the modifying cells directly. HMS coating over cells could function bound particles, nor is cell viability or proliferation [87]. as a chemical scaffold to mediate homing from vascular to tissue compartments. Hydrogel shells conjugated with struc- Cell therapy tural molecules could be used to direct the homing of cells for regenerative therapy. The challenge of using cells like bone Stem cells have great potential as therapeutic agents, since marrow mesenchymal stem cells (MSC) for regenerative they can be induced to differentiate into specific tissue types therapy, inflammation treatment, and angiogenesis pro- for repair or regeneration [88]. Several studies [89, 90] have motion is the targeting of these cells to the requisite treatment reported that nanoparticles of calcium minerals can promote sites with minimum morbidity and maximum efficiency osteoblastic differentiation from stem cells. Differentiation [80, 81]. and proliferation of stem cells can be effected significantly Magnetic nanoparticles [82] and quantum dot cell labeling by the presence of calcium phosphate nano-crystallites by [83] may be incorporated into mineral shell layers to facilitate significantly increasing the alkaline phosphatase (ALP) in vivo tracking. Magnetic resonance tracking of magnetically 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 shell of nanocalcium phosphate would not only provide protection for cell storage but would also act as an osteogenic promoter, making calcium phosphate-coated stem cells an ideal bone repair material. Stem cells can be easily stored in shells so that they can be readily available for implantation, then degraded gradually, so that the cells can adapt appropriately to their new environment. It is critical that the released cells maintain the advantage of differentiated osteoblasts. Generally, newly differentiated osteoblasts have better mineralization ability and the mineral shells 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 be considered a living bone repair material. Adoptive cell therapy (ACT) is a treatment that uses a Figure 6. Magnetic particles (red dots) cannot be integrated into cancer patient’s own T lymphocytes which have endogenous living cells (green circles) directly. However, they can be integrated anti-tumor activity. These cells are expanded in vitro and into shell structures (gray layers) readily so that magnetic cells can reinfused into the patient. This approach involves the identi- be created using shell engineering. The optical images demonstrate that bare yeast cells are insensitive to magnetic fields so that they fication ex vivo of autologous or allogeneic lymphocytes are randomly placed, but the cells with calcium phosphate-Fe3O4 with anti-tumor activity, often along with appropriate growth can be driven and concentrated by a magnet [61]. factors, to stimulate their survival and expansion when

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implanted in the patient [93]. Tumor-killing cells may be functions, will find application throughout the fields of bio- induced from a patient’s own T-cells by integrating growth technology and medicine. factors or cytokines within the outer-surface of shells as a self-support system to enhance their activities and to provide an optimal environment for the transplanted cells. Such a Acknowledgments shellized T-cell vaccine system can be a promising and highly We thank Jason Nichol, Stephanie Piecewicz, and Yanan Du effective immunotherapeutic approach for future cancer for revising the paper. This study was supported by the treatment. National Natural Science Foundation of China (20871102), Viruses with a mineral shell structure can also be used as a Zhejiang Provincial Natural Science Foundation (R407087), targeted cancer treatment. If a virus is wrapped by a mineral the Fundamental Research Funds for the Central Universities, coating, it cannot be detected by the immune system and is and Daming Biomineralization Foundation. free to reach host cells. Virus-mineral core-shell nanocompo- sites have the biological features of the mineral surface Review essays materials, but do not lose their virulence. Once they reach References target cells, the mineral shells can be dissolved to expose the virus. Interestingly, the cellular microenvironment for most 1. Andrianantoandro E, Basu S, Karig DK, et al. 2006. Synthetic : cancers is slightly acidic, which can dissolve calcium minerals new engineering rules for an emerging discipline. Mol Syst Biol 2: 1–14. 2. Szostak JW, Bartel DP, Luisi PL. 2001. Synthesizing life. Nature 409: to break mineral shells, allowing for an innate cancer targeting 387–90. system. In contrast, the pH environment of normal cells is 3. Sole RV, Rasmussen S, Bedau M. 2007. Introduction. 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