Cell Compatibility of Three-Dimensional Porous Barium ... · 1Department of Biology, School of...

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*Corresponding author: E-mail: [email protected];

Journal of Scientific Research & Reports3(20): 2611-2621, 2014; Article no. JSRR.2014.20.002

SCIENCEDOMAIN internationalwww.sciencedomain.org

Cell Compatibility of Three-Dimensional PorousBarium-Cross-Linked Alginate Hydrogels

Ikuko Machida-Sano1*, Makoto Hirakawa1 and Hideo Namiki1

1Department of Biology, School of Education, Waseda University, 2-2 Wakamatsu-cho,Shinjuku-ku, Tokyo 162-8480, Japan.

Authors’ contributions

This work was carried out in collaboration between all authors. Authors IMS and MHcontributed equally to this work. Author IMS designed the study, carried out the laboratorywork, wrote the first draft of the manuscript and managed literature searches. Author MH

performed majority of the laboratory work. Author HN supervised and managed the analysesof the study. All authors read and approved the final manuscript.

Received 30th June 2014Accepted 5th August 2014

Published 19th August 2014

ABSTRACT

We attempted to clarify whether the suitability of three-dimensional porous barium-ion-crosslinked alginate (Ba-alginate) gels as a scaffold for cells, in comparison with calcium-ion-crosslinked alginate (Ca-alginate) gels, differed according the type of method used toinduce cross-linkage. We fabricated two types of three-dimensional porous Ba- and Ca-alginate gels in which the alginates were cross-linked under freeze-dried conditions or inaqueous solution, and evaluated their affinity for cells. Only Ba-alginate, cross-linked byfreeze-drying, exhibited a rough surface and high protein adsorption ability, in comparisonwith Ba-alginate that had been cross-linked in aqueous solution, and any type of Ca-alginate gel. Cells formed multicellular spheroids whenever alginate was used as ascaffold, but only on Ba-alginate porous gels cross-linked by freeze-drying did the numberof cells increase with culture time. These findings indicate that the properties of Ba-alginate influencing its suitability as a scaffold for cells change according to the methodused to induce cross-linkage. Our findings may be useful for extending the application ofBa-alginate, and may have significance in diverse biomedical fields.

Original Research Article

Machida-Sano et al.; JSRR, Article no. JSRR.2014.20.002

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Keywords: Barium-ion-crosslinked alginate (Ba-alginate); three-dimensional scaffold; proteinadsorption; cell proliferation; surface roughness.

1. INTRODUCTION

Culture substrates capable of supporting effective cell growth in vitro are required for variousapplications in a range of biomedical fields. In order for a material to function as a scaffold, itis necessary for it to adsorb cell-adhesive proteins, such as serum vitronectin andfibronectin, onto its surface [1]. Cells on the material surface adhere to the adsorbedextracellular proteins via integrin [2], and integrin-mediated cellular adhesion promotes cellgrowth [3]. It has been shown that the properties of material surfaces influence theirsuitability as culture substrates for cells. Surface properties of materials such as wettability[2,4-9], charge [10,11], and roughness [12-15] are reported to be major factors determiningtheir ability to adsorb protein. Therefore, an understanding of the characteristic features ofmaterial surfaces may aid formulation of better scaffolds for cell culture.

In a previous study, we demonstrated that two-dimensional barium-ion-crosslinked alginate(Ba-alginate) film was an efficient culture substrate, allowing good cell adhesion andproliferation [16]. Alginates are composed of 1,4-linked β-D-mannuronic acid (M) and α-L-guluronic acid (G) residues, forming gels with certain multivalent metal ions [17,18], and arewidely used in various biomedical fields. Calcium ions are used most frequently as a cross-linking agent for alginates, but it is known that calcium-ion-crosslinked alginate (Ca-alginate)is not an efficient culture substrate for cells because of its highly hydrophilic [7] andnegatively charged [19,20] surface, which prevents it from adsorbing protein. We havepreviously demonstrated that Ba-alginate film has a rough surface, even though its surfacewettability and charge are equivalent to those of Ca-alginate, and that this surfaceroughness is likely a key factor determining protein adsorption [16].

It is known that cells lose some of their functions when cultured as a monolayer under two-dimensional conditions in vitro [21-25]. Therefore, three-dimensional cell culture systems aredesirable for both research and therapeutic purposes. Many researchers have devisedporous scaffolds to provide three-dimensional cell culture environments [26-28]. Forfabrication of ionically cross-linked porous alginate gels (mainly Ca-alginate), two methodshave been reported: Cross-linkage with metal ions under freeze-dried conditions [29-32], orin aqueous solution [33-35].

In the present study, we prepared two types of three-dimensional porous Ba-alginate usingdifferent procedures, and investigated their suitability as a scaffold for cell culture, incomparison with Ca-alginate. We focused on protein adsorption ability and surfaceroughness as salient properties of Ba-alginate porous gels, and observed the responses ofcell to the gels. Here we describe how the properties of porous Ba-alginate gels, determiningtheir suitability as a culture substrate for cells, were altered according to the method used forgel preparation.

2. MATERIALS AND METHODS

2.1 Materials

A high G-content alginate (I-1G) was purchased from KIMICA Corporation (Tokyo, Japan).Eagle’s minimum essential medium (E-MEM) was obtained from Nissui Pharmaceutical


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(Tokyo, Japan), fetal bovine serum (FBS) was from Invitrogen (Carlsbad, CA, USA), andtrypsin was from Becton Dickinson (Sparks, MD, USA). A rat skin fibroblast cell line (FRcells) was obtained from DS Pharma Biomedical Co., Ltd. (Osaka, Japan). All otherchemicals were purchased from Wako Pure Chemical Co. (Tokyo, Japan).

2.2 Preparation of a Three-dimensional Porous Alginate Scaffold

Three-dimensional porous alginate gels were fabricated using two methods: alginates werecross-linked by metal ions under freeze-dried conditions (1) or in aqueous solution (2). (1)For preparation of alginate porous gels under freeze-dried conditions, 500 μl of 1% (w/v)alginate solution was poured into each well of a 24-well tissue culture plate, and frozen at -20ºC for 24 h, and then at -80ºC for 24 h. The frozen gels were then lyophilized, andsterilized by exposure to ultraviolet (UV) light for 1 h. Then 500 μl of 100 mM BaCl2 or CaCl2was added to each well to induce cross-linkage of the alginate at 25ºC for 30 min. Theresulting gel films were washed with deionized water to remove unreacted ions. (2) Forpreparation of alginate porous gels in aqueous solution, 1 ml of 2% (w/v) alginate solutionwas poured into each well of a 24-well tissue culture plate, and 1 ml of 20 mM BaCl2 orCaCl2 was then added to each well to induce cross-linkage of the alginate while stirringvigorously using a homogenizer at 25ºC. The final concentrations of polymer and cross-linking agents in the cross-linked solutions were 1% and 10 mM, respectively. The resultinggels were frozen at -20ºC for 24 h, and then at -80ºC for 24 h. The frozen gels were thenlyophilized, and cut into slices 2 mm thick. The gels were then sterilized by exposure to UVlight for 1 h. Before seeding of cells on the gels, both gel types were stabilized by immersionin E-MEM at 37ºC under a 5% CO2 atmosphere for 72 h, and the medium was replacedevery 24 h.

2.3 Measurement of the Structure and Surface Roughness of Alginate Gels

The structure and surface morphology of alginate gels were observed using a scanningelectron microscope (SEM) (VE-9800, KEYENCE, Osaka, Japan). Alginate gels, preparedas described above, were washed with deionized water and air-dried. The gels were thengold-coated and observed by SEM.

2.4 Evaluation of Protein Adsorption Capacity of the Alginate Gels

The protein adsorption ability of alginate gels was analyzed by measurement of the amountsof serum proteins adsorbed on them, as described previously [36]. Briefly, 0.2% FBS inCa2+-, Mg2+-free phosphate-buffered saline (PBS) or PBS (2 ml/well) was prepared in a 24-well tissue culture plate, and alginate gels were immersed in them at 37ºC for 2 h. Afterincubation, the protein concentrations of the solutions were measured using a bicinchoninicacid (BCA) protein assay reagent kit (Thermo Fisher Scientific Pierce Biotechnology, IL,USA). Alginate gels, immersed in PBS, were used as controls. The amount of adsorbedprotein was equivalent to the difference in the protein concentrations of 0.2% FBS beforeand after immersion of the alginate gels. Eventually, the adsorbed weight per 1 mg ofalginate scaffold (gel dry weight) was calculated.

2.5 Cell Culture

FR cells were cultured in E-MEM with 10% (v/v) FBS at 37ºC under a 5% CO2 atmosphere.To detach the cells by trypsinization, they were incubated with 0.25% (w/v) trypsin and


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0.02% (w/v) EDTA in PBS at 37ºC for 10 min. E-MEM containing 10% (v/v) FBS wassubsequently added to terminate the enzyme reaction. The cell suspension was centrifugedat 1000 x g for 5 min and resuspended in E-MEM containing 10% (v/v) FBS. The cells werecounted with a Coulter Counter (Beckman Coulter Corporation, FL, USA) and seeded ontoalginate gels in 24-well tissue culture plates at a density of 10 × 104 cells/well in 20 μl ofculture medium. Samples were incubated at 37ºC under a 5% CO2 atmosphere, and 1 ml ofculture medium was added to each well at 2 h after cell seeding. The medium was replacedevery day. After 1, 7, and 14 days, cell counting was performed to quantify the degree of cellproliferation. The numbers of cells on the scaffolds were assessed by 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8) assay using a Cell Counting Kit-8 (Dojindo Lab., Tokyo, Japan). The WST-8 assaymeasures the amount of formazan dye that is produced in the presence of an electroncarrier when WST-8 is reduced by dehydrogenases in cells, and the amount of the formazandye is directly proportional to the number of living cells. To count the cells on the scaffold,each alginate gel was transferred to another plate, and washed with PBS. Then 10 μl of CellCounting Kit solution and 1 ml of culture medium were added to each well and incubated at37ºC under a 5% CO2 atmosphere for 2 h. After incubation, the absorbance of culturesolutions at 450 nm with 650 nm as a reference was measured using a microplate reader(Maltiskan Spectrum, Thermo, MA, USA).

2.6 Examination of Cell Morphology on Alginate Gels

The morphology of cultured cells on the alginate scaffolds was observed using SEM asreported previously [36]. The cells on the gels were fixed in 2.5% glutaraldehyde in PBS for2 h at 4ºC, and dehydrated using four-step concentrations (50%, 70%, 90% and 99.5%) ofethanol. Then, the alginate gels were freeze-dried after placing them in tert-butyl alcohol.The alginate scaffolds, which were gold-coated, were observed using SEM.

2.7 Statistics

Statistical analysis was performed using KaleidaGraph version 4.0 software. The data weresubjected to analysis of variance (ANOVA). Scheffe test was used for post hoc evaluation ofdifferences between the respective groups. For all statistical evaluations, significance wasassigned at P<0.05.

3. RESULTS

3.1 Properties of the Gels

The morphology of Ba- and Ca-alginate scaffolds, cross-linked by freeze-drying or inaqueous solution, was observed by SEM (Fig. 1). In all cases, the alginate gels exhibited acomparable highly porous structure with interconnecting pores. However, the structures ofthe gels differed according to the method used for cross-linkage: those cross-linked byfreeze-drying exhibited a denser porous structure than those cross-linked in aqueoussolution. There were slight differences in gel morphology among the gels cross-linked withdifferent ions. To evaluate the surface properties of the gels, the three-dimensional surfacemorphology of Ba- and Ca-alginate gels was analyzed. This revealed that only Ba-alginategels cross-linked under freeze-dried conditions had a rough surface, whereas all the otheralginate gels had smooth surfaces (Fig. 2). Examination of the protein adsorption ability ofboth types of Ba- and Ca-alginate porous gels revealed that the amounts of adsorbed serum


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proteins were about 1.7-fold higher on the surface of Ba-alginate gel, cross-linked underfreeze-dried conditions, than on the other alginate scaffolds (Fig. 3).

Fig. 1. SEM micrographs of Ba- (a, b) and Ca- (c, d) alginate scaffoldsAlginates were cross-linked with metal ions under freeze-dried conditions (a, c) or in aqueous solution

(b, d). Bar equals 500 μm.

Fig. 2. SEM analysis of the three-dimensional surface morphology of Ba- (a, b) andCa- (c, d) alginate gels

Alginates were cross-linked with metal ions under freeze-dried conditions (a, c) or in aqueous solution(b, d). Images in lateral view are shown in the lower portion of each frame.

Bar represents 2 μm


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Fig. 3. Protein adsorption on Ba- and Ca-alginate gelsAlginates were cross-linked with metal ions under freeze-dried conditions (white columns) or in

aqueous solution (grey columns). The total amount of adsorbed serum proteins was quantified using aBCA protein assay kit. The amount of adsorbed protein was indicated as the weight of protein per 1 mg

of alginate gel. Error bars indicate standard deviation for n=4. *P<0.05

3.2 Cell Growth

The cell compatibility of three-dimensional porous alginate gels was analyzed by cellcultivation using FR cells. To evaluate cell growth on the gels, cell counting was performedafter 1, 7, 14 days. Increase in number of cells was confirmed only on the Ba-alginate gel,cross-linked under freeze-dried conditions, although almost no cell proliferation wasobserved on the other alginate gels. Between days 1 and 14, the number of attached cellson the Ba-alginate gel that had been cross-linked by freeze-drying increased about 2.6-fold(Fig. 4).

Fig. 4. Proliferation of cells on alginate scaffoldsCells were seeded onto Ba- and Ca-alginate gels, and the numbers of attached cells were counted

at 1, 7 and 14 days after the start of culture. Alginates were cross-linked with metal ions underfreeze-dried conditions (white columns) or in aqueous solution (grey columns). The relative cell

number was calculated by comparison with the number of cells at 1 day of cultivation on Ba-alginate gels that had been cross-linked under freeze-dried conditions.

Error bars indicate standard deviation for n=5. *P<0.05


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3.2 Cell Growth







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3.2 Cell Growth







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3.3 Cell Morphology

Fig. 5 shows SEM micrographs of cells cultured on the alginate gels. This indicated that thecells aggregated to form multicellular spheroids in all cases. Only on Ba-alginate gels thathad been cross-linked under freeze-dried conditions did the size of the spheroids increaseas the culture period was prolonged (Figs. 5(a) and 5(b)). These findings correlated well withthe quantitative cell growth data.

Fig. 5. SEM micrographs of cells seeded onto Ba- (a-d) and Ca- (e-h) alginate scaffoldsAlginates were cross-linked with metal ions under freeze-dried conditions (a, b, e, f) or in aqueous

solution (c, d, g, h). Cells were cultured on each type of gel, and observed after 1 day (a, c, e, g) and14 days (b, d, f, h) of culture. Bar equals 10 μm

4. DISCUSSION

Understanding the properties of materials is important for assessing their suitability asculture substrates for cells. In the present study, we evaluated two types of three-dimensional porous Ba-alginate gels, fabricated using two different cross-linking techniques.The results demonstrated that the properties of Ba-alginate gel that influenced its suitabilityas a cell scaffold changed according to the method used for cross-linkage.

Adsorption of proteins onto the surface of a material is required in order to support cellularadhesion and growth [2]. Surface roughness is known to be one of the critical factorsinfluencing protein adsorption ability [12-15]. In this study, we found that two types of Ba-alginate porous gel prepared using different methods had different surface properties.Surface roughness of the gels was recognized only when alginates had been cross-linkedwith barium ions under freeze-dried conditions (Fig. 2), and these gels had higher proteinadsorption ability than Ca-alginate (Fig. 3). SEM observation of the morphology of porousalginate gels indicated that those cross-linked under freeze-dried conditions had a smallerpore size than gels cross-linked in aqueous solution (Fig. 1). It is inferred that the amount ofproteins adsorbed increases when the pore size is smaller, due to an increase in surfacearea. However, as shown in Fig. 3, the amount of proteins adsorbed on the two types of Ca-alginate porous gels showed little difference, despite their difference in pore size, as was thecase for Ba-alginate gels. These observations suggested that the differences in the amountsof adsorbed proteins on the two types of Ba-alginate gels were attributable to the propertiesof the gel, rather than pore size.


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In a previous study where alginates were cross-linked with barium ions after drying of thealginate solution to fabricate Ba-alginate films, these films exhibited a rough surface, whichwas considered to enhance protein adsorption ability [16]. It has also been reported that Ba-alginate, cross-linked under liquid conditions, was able to act as a cell culture substrate, inview of its artificially created rough surface [37]. These observations suggest that the state ofa Ba-alginate gel affects its surface properties, and that formation of such a gel under dryconditions leads to the creation of a rough surface. It is considered that the radius of aspecific ion leads to differences in the surface properties of alginate gels. The larger ionicradius of the barium ion may reduce ionic diffusion in a viscous alginate solution, but mayfacilitate easier diffusion on a dried alginate surface. For this reason, the surface propertiesof Ba-alginate might vary according to the method used for cross-linkage. In fact, a study ofionically cross-linked multi-membrane spherical aerogels found that the properties of Ba-alginate varied when the alginate concentration was increased from 0.75% in the case ofmembranes to 1.5% in the case of spherical cores: the average pore diameter and overallpore volume were higher in the Ba-alginate core sample than in the membrane sample,being opposite to the results obtained for Ca-derived aerogels [38].

The interaction of alginate with barium ions was thought to differ from that with other cross-linking ions. Our previous investigation of two-dimensional alginate gel films cross-linkedwith five different metal ions (Ca2+, Ba2+, Sr2+, Fe3+ and Al3+) indicated that surfaceroughness, considered to enhance cell compatibility, was evident only when barium ionswere used [16]. For ferric and aluminum ion-cross-linked alginates, which – like Ba-alginate– support good cell proliferation, protein adsorption ability was considered to be related totheir surface wettability [16]. It has been reported that the properties of ionically cross-linkedalginate are affected by the types of cross-linking ions [39]. Various divalent ions differ intheir strength of affinity for alginates, and barium ions have much more marked affinity foralginate than calcium ions [40,41]. This higher affinity enables barium ions to form a denseralginate gel network than is the case for calcium ions [38]. It has also been reported thatbarium ions are able to form an egg box structure with alginate more efficiently throughstronger ionic bonds in comparison to calcium ions [39]. Moreover, the ion binding blockstructures in the alginate differ according to the type of cross-linker ion [39]. These areconsidered to be the main reasons for the specific properties of Ba-alginate.

The present study demonstrated that cells formed multicellular spheroids on both types ofBa-alginate gel (Fig. 5), and that the protein adsorption capacity of alginate gels wascorrelated with their ability to support cell growth (Figs. 3 and 4). Many studies havedemonstrated that cells cultured in a three-dimensional environment form multicellularspheroids, and are able to maintain their original functions [7,24,25,42-45]. Under suchthree-dimensional culture conditions – as with monolayer culture conditions – it is known thatcell-matrix interactions mediated by integrin are also critical for cell growth [46], and thatinhibition of β1 integrin function causes loss of spheroid-forming ability [47]. Therefore, it issuggested that cell growth on Ba-alginate, cross-linked under freeze-dried conditions, iscontrolled by integrin-mediated interactions between cells and adsorbed proteins on thescaffold. In case of HeLa cells that exhibit anchorage-independent growth, cell growth wasobserved on all types of alginate gels (data not shown). The present findings indicate thatBa-alginate gels fabricated using appropriate methods provide a favorable environment forthe growth of cells.

5. CONCLUSIONIn this study, we have revealed for the first time that the properties of Ba-alginate gels thatdetermine their suitability as a scaffold for cultured cells vary according to the method used


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for creating cross-linkage. In comparison with cross-linkage in aqueous solution, Ba-alginateporous gels exhibited a rough surface when the alginates were cross-linked with barium ionsunder freeze-dried conditions. It was considered that surface roughness enhanced thesuitability of gels as a culture substrate for cells by conferring a high capacity for proteinadsorption. These results confirm the effectiveness of Ba-alginate as a scaffold for culturedcells, and demonstrate that the properties of gels vary according to the method used forfabricating them. Therefore, it is anticipated that our findings will be of considerablesignificance in diverse biomedical fields.

COMPETING INTERESTS

Authors have declared that no competing interests exist.

REFERENCES

1. Underwood PA, Bennett FA. A comparison of the biological activities of the cell-adhesive proteins vitronectin and fibronectin. J Cell Sci. 1989;93(4):641-9.

2. Wilson CJ, Clegg RE, Leavesley DI, Pearcy MJ. Mediation of biomaterial-cellinteractions by adsorbed proteins: A review. Tissue Eng. 2005;1(1-2):1 1-18.

3. Mizushima H, Wang X, Miyamoto S, Mekada E. Integrin signal masks growth-promotion activity of HB-EGF in monolayer cell cultures. J Cell Sci.2009;122(23):4277-86.

4. Wei J, Igarashi T, Okumori N, Igarashi T, Maetani T, Liu B et al. Influence of surfacewettability on competitive protein adsorption and initial attachment of osteoblasts.Biomed Mater. 2009;4(4):045002.

5. Llopis-Hernández V, Rico P, Ballester-Beltrán J, Moratal D, Salmerón-Sánchez M.Role of surface chemistry in protein remodeling at the cell-material interface. PLoSOne. 2011;6(5):e19610.

6. Keselowsky BG, Collard DM, García AJ. Surface chemistry modulates fibronectinconformation and directs integrin binding and specificity to control cell adhesion. JBiomed Mater Res A. 2003;66(2):247-59.

7. Glicklis R, Shapiro L, Agbaria R, Merchuk JC, Cohen S. Hepatocyte behavior withinthree-dimensional porous alginate scaffolds. Biotechnol Bioeng. 2000;67(3):344-53.

8. Grinnell F, Feld MK. Fibronectin adsorption on hydrophilic and hydrophobic surfacesdetected by antibody binding and analyzed during cell adhesion in serum-containingmedium. J Biol Chem. 1982;257(9):4888-93.

9. Lee JH, Khang G, Lee JW, Lee HB. Interaction of different types of cells on polymersurfaces with wettability gradient. J Colloid Interface Sci. 1998;205(2):323-30.

10. Qiu Q, Sayer M, Kawaja M, Shen X, Davies JE. Attachment, morphology, and proteinexpression of rat marrow stromal cells cultured on charged substrate surfaces. JBiomed Mater Res. 1998;42(1):117-27.

11. Cai K, Frant M, Bossert J, Hildebrand G, Liefeith K, Jandt KD. Surface functionalizedtitanium thin films: Zeta-potential, protein adsorption and cell proliferation. Colloids andSurfaces B Biointerfaces. 2006;50(1):1-8.

12. Blanco EM, Horton MA, Mesquida P. Simultaneous investigation of the influence oftopography and charge on protein adsorption using artificial nanopatterns. Langmuir.2008;24(6):2284-7.

13. Xie HG, Zheng JN, Li XX, Liu XD, Zhu J, Wang F, et al. Effect of surface morphologyand charge on the amount and conformation of fibrinogen adsorbed ontoalginate/chitosan microcapsules. Langmuir. 2010;26(8):5587-94.


2620

14. Dufrêne YF, Marchal TG, Rouxhet PG. Influence of substratum surface properties onthe organization of adsorbed collagen films: In situ characterization by atomic forcemicroscopy. Langmuir. 1999;15(8):2871-8.

15. Boyan BD, Hummert TW, Dean DD, Schwartz Z. Role of material surfaces inregulating bone and cartilage cell response. Biomaterials. 1996;17(2):137-46.

16. Machida-Sano I, Hirakawa M, Matsumoto H, Kamada M, Ogawa S, Satoh N et al.Surface characteristics determining the cell compatibility of ionically cross-linkedalginate gels. Biomed Mater. 2014;9(2):25007.

17. Smidsrød O, Skjåk-Bræk G. Alginate as immobilization matrix for cells. TrendsBiotechnol. 1990;8(3):71-8.

18. Sriamornsak P, Kennedy RA. Development of polysaccharide gel-coated pellets fororal administration. 2. Calcium alginate. Eur J Pharm Sci. 2006;29(2):139-47.

19. Smetana K JR. Cell biology of hydrogels. Biomaterials. 1993;14(14):1046-50.20. Rowley JA, Madlambayan G, Mooney DJ. Alginate hydrogels as synthetic extracellular

matrix materials. Biomaterials. 1999;20(1):45-53.21. Du Y, Chia SM, Han R, Chang S, Tang H, Yu H. 3D hepatocyte monolayer on hybrid

RGD/galactose substratum. Biomaterials. 2006;27(33):5669–80.22. Fukui N, Ikeda Y, Tanaka N, Wake M, Yamaguchi T, Mitomi H, et al. αvβ5 Integrin

promotes dedifferentiation of monolayer-cultured articular chondrocytes. ArthritisRheum. 2011;63(7):1938–49.

23. Von Der Mark K, Gauss V, Von Der Mark H, Müller P. Relationship between cellshape and type of collagen synthesised as chondrocytes lose their cartilagephenotype in culture. Nature. 1997;267(5611):531–2.

24. Birgersdotter A, Sandberg R, Ernberg I. Gene expression perturbation in vitro--agrowing case for three-dimensional (3D) culture systems. Semin Cancer Biol.2005;15(5):405–12.

25. Hirschhaeuser F, Menne H, Dittfeld C, West J, Mueller-Klieser W, Kunz-Schughart LA.2010 Multicellular tumor spheroids: An underestimated tool is catching up again. JBiotechnol. 2010;148(1):3–15.

26. Cao H, Kuboyama N. A biodegradable porous composite scaffold of PGA/beta-TCPfor bone tissue engineering. Bone. 2010;46(2):386-95.

27. Laschke MW, Schank TE, Scheuer C, Kleer S, Schuler S, Metzger W, et al. Three-dimensional spheroids of adipose-derived mesenchymal stem cells are potentinitiators of blood vessel formation in porous polyurethane scaffolds. Acta Biomater.2013;9(6):6876-84.

28. Florczyk SJ, Wang K, Jana S, Wood DL, Sytsma SK, Sham JG, et al. Porouschitosan-hyaluronic acid scaffolds as a mimic of glioblastoma microenvironment ECM.Biomaterials. 2013;34(38):10143-50.

29. Li Z, Leung M, Hopper R, Ellenbogen R, Zhang M. Feeder-free self-renewal of humanembryonic stem cells in 3D porous natural polymer scaffolds. Biomaterials.2010;31(3):404-12.

30. Han J, Zhou Z, Yin R, Yang D, Nie J. Alginate-chitosan/hydroxyapatite polyelectrolytecomplex porous scaffolds: preparation and characterization. Int J Biol Macromol.2010;46(2):199-205.

31. Qi J, Chen A, You H, Li K, Zhang D, Guo F. Proliferation and chondrogenicdifferentiation of CD105-positive enriched rat synovium-derived mesenchymal stemcells in three-dimensional porous scaffolds. Biomed Mater. 2011;6(1):015006.

32. Luckanagul J, Lee LA, Nguyen QL, Sitasuwan P, Yang X, Shazly T, et al. Porousalginate hydrogel functionalized with virus as three-dimensional scaffolds for bonedifferentiation. Biomacromolecules. 2012;13(12):3949-58.


2621

33. Shapiro L, Cohen S. Novel alginate sponges for cell culture and transplantation.Biomaterials. 1997;18(8):583-90.

34. Turco G, Marsich E, Bellomo F, Semeraro S, Donati I, Brun F, et al.Alginate/Hydroxyapatite biocomposite for bone ingrowth: A trabecular structure withhigh and isotropic connectivity. Biomacromolecules. 2009;10(6):1575-83.

35. Re'em T, Tsur-Gang O, Cohen S. The effect of immobilized RGD peptide inmacroporous alginate scaffolds on TGFbeta1-induced chondrogenesis of humanmesenchymal stem cells. Biomaterials. 2010;31(26):6746-55.

36. Machida-Sano I, Ogawa S, Hirakawa M, Namiki H. Evaluation of three-dimensionalporous iron-cross-linked alginate as a scaffold for cell culture. ISRN Biomaterials.2014;2014:375-758.

37. Seo SJ, Kim IY, Choi YJ, Akaike T, Cho CS. Enhanced liver functions of hepatocytescocultured with NIH 3T3 in the alginate/galactosylated chitosan scaffold. Biomaterials.2006;27(8):1487-95.

38. Veronovski A, Knez Z, Novak Z. Comparison of ionic and non-ionic drug release frommulti-membrane spherical aerogels. Int J Pharm. 2013;454(1):58-66.

39. Mørch YA, Donati I, Strand BL, Skjåk-Braek G. Effect of Ca2+, Ba2+, and Sr2+ onalginate microbeads. Biomacromolecules. 2006;7(5):1471-80.

40. Haug A, Smidsrød O. Selectivity of some anionic polymers for divalent metal ions.Acta Chem Scand. 1970;24:843-54.

41. Haug A. The affinity of some divalent metals for different types of alginates. ActaChem Scand. 1961;15:1794-5.

42. Kim JB. Three-dimensional tissue culture models in cancer biology. Semin CancerBiol. 2005;15(5):365–77.

43. Sutherland RM. Cell and environment interactions in tumor microregions: The multicellspheroid model. Science. 1988;240(4849):177-84.

44. Becker JL, Blanchard DK. Characterization of primary breast carcinomas grown inthree-dimensional cultures. J Surg Res. 2007;142(2):256-62.

45. Karlsson H, Fryknäs M, Larsson R, Nygren P. Loss of cancer drug activity in coloncancer HCT-116 cells during spheroid formation in a new 3-D spheroid cell culturesystem. Exp Cell Res. 2012;318(13):1577-85.

46. Yoshii Y, Waki A, Yoshida K, Kakezuka A, Kobayashi M, Namiki H, et al. The use ofnanoimprinted scaffolds as 3D culture models to facilitate spontaneous tumor cellmigration and well-regulated spheroid formation. Biomaterials. 2011;32(26):6052-8.

47. Yoshida Y, Kurokawa T, Nishikawa Y, Orisa M, Kleinman HK, Kotsuji F. Laminin-1-derived scrambled peptide AG73T disaggregates laminin-1-induced ovarian cancercell spheroids and improves the efficacy of cisplatin. Int J Oncology. 2008;32(3):673-81.

_________________________________________________________________________© 2014 Machida-Sano et al.; This is an Open Access article distributed under the terms of the Creative CommonsAttribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.

Peer-review history:The peer review history for this paper can be accessed here:

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