Tissue Engineering www.biotechnology-journal.com

REVIEW

Biomaterial Substrate-Mediated Multicellular SpheroidFormation and Their Applications in Tissue Engineering

Ting-Chen Tseng, Chui-Wei Wong, Fu-Yu Hsieh, and Shan-hui Hsu*

Three-dimentional (3D) multicellular aggregates (spheroids), compared to thetraditional 2D monolayer cultured cells, are physiologically more similar tothe cells in vivo. So far there are various techniques to generate 3Dspheroids. Spheroids obtained from different methods have already beenapplied to regenerative medicine or cancer research. Among the cellspheroids created by different methods, the substrate-derived spheroids andtheir forming mechanism are unique. This review focuses on the formationof biomaterial substrate-mediated multicellular spheroids and their applica-tions in tissue engineering and tumor models. First, the authors will describethe special chitosan substrate-derived mesenchymal stem cell (MSC) sphe-roids and their greater regenerative capacities in various tissues. Second, theauthors will describe tumor spheroids derived on chitosan and hyaluronansubstrates, which serve as a simple in vitro platform to study 3D tumormodels or to perform cancer drug screening. Finally, the authors will mentionthe self-assembly process for substrate-derived multiple cell spheroids (co-spheroids), which may recapitulate the heterotypic cell–cell interaction for co-cultured cells or crosstalk between different types of cells. These uniquemulticellular mono-spheroids or co-spheroids represent a category of 3D cellculture with advantages of biomimetic cell–cell interaction, better functionali-ties, and imaging possibilities.

1. Introduction

Stem cells are a popular and attractive cell source forregenerative medicine because of their self-renewal andmultilineage differentiation capacities.[1] Conventional two-dimensional (2D) culture systems have been used as a standardtechnique for in vitro expansion of stem cells.[2] There are a fewlimitations for the 2D culture systems. First, cells grown on flatsolid surface (e.g., tissue culture polystyrene plates, TCPS) arestretched and lack of appropriate cell–cell and cell–matrixinteractions that exist in the native microenvironment. Addi-tionally, 2D-cultured cells undergo cytoskeletal rearrangementsacquiring artificial polarity, which in turn causes alternation innormal physiological behavior.[3] As a result, stem cells may lose

Dr. T.-C. Tseng, Dr. C.-W. Won, Dr. F.-Y. Hsieh, Prof. S.-h. HsuInstitute of Polymer Science and EngineeringNational Taiwan University, Taipei, TaiwanE-mail: shhsu@ntu.edu.tw

Prof. S.-h. HsuInstitute of Cellular and System MedicineNational Health Research Institutes, Miaoli, Taiwan

DOI: 10.1002/biot.201700064

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the capabilities to differentiate or self-renew after several passages in 2D cultiva-tion.[4] Therefore, sophisticated in vitro cellculture systems are highly wanted in orderfor the stem cells to possess their normalphysiological behavior.

Various three-dimensional (3D) in vitrocell culture systems have been developed tomimic the nativemicroenvironment of cellsin vivo.[5] In one category, cells cultured bythe 3D systems could become multicellularaggregates, also known as multicellularspheroids or spheroids. Spheroids gener-ated by differentmethods have already beenutilized in the field of stem cell biology[6,7]

and tissue engineering.[8–10] In this review,we will provide an overview of biomaterialsubstrate-derived spheroids and their appli-cations in tissue engineering.

2. Spheroids Formed byCulture Substrates VersusOther Methods

Stem cells in the form of 3D multicellularspheroids could reside in an environment

better mimicking the real in vivo situation, compared with thecells in traditional monolayer culture. Literatures have demon-strated that spheroid formation of MSCs could maintain theirstemness properties (Sox2, Oct4, and Nanog) and enhance theirmultilineage differentiation capacities.[7,11,12] Therefore, spher-oid formation of mesenchymal stem cells (MSCs) is animportant technique for stem cell-based research and clinicalstudies. Several 3D culture methods to obtain multicellualrspheroids have been developed, including the use of hangingdrop, non-adherent surfaces, micropatterned surfaces, suspen-sion culture, and microfluidic systems.[13–17] A list of themethods to generate multicellular spheroids and a comparisonof these different spheroid forming approaches are shown inTable 1. Although the above methods can generate 3Dmulticellular spheroids, they still have some limitations. Forinstance, using the hanging drop technique may harvestspheroids with uniform sizes but the average sizes of spheroidsare relatively small (50–250 μm) because of the limitation ofvolume accommodation.[13] Besides, the hanging drop techniquehas low throughput. The suspension culture method couldproduce a lot of spheroids. Particularly, the size of spheroids maybe manipulated by rotary suspension culture under thecontrolled revolution per minute (rpm).[16] However, the

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Shan-hui Hsu is a distinguishedprofessor at the Institute of PolymerScience and Engineering, NationalTaiwan University, Taiwan. Shereceived her Ph.D. in BiomedicalEngineering from Case WesternReserve University in 1992. Herresearch interest coversbiomaterials, polyurethane, tissueengineering, and 3D bioprinting. She

has published over 230 SCI papers and received anumber of academic awards. She also serves as editor/associate editor/editorial board for three SCI-indexedjournals.

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suspension culture requires special equipment and the shearflow from mixing may damage the cells. The non-adherentsurfaces may produce spheroids in high throughput, but the sizeof spheroids cannot be controlled. The spheroids generated frommicrofluidic systems are hard to collect and further analyze.[18]

Other than the above 3D culture methods, our earlier studieshave demonstrated thatMSCsgrownon chitosan substrates couldform self-assembled 3D cellular spheroids.[19] The chemicalstructure of chitosan is shown in Figure 1. Chitosan is a derivativeof the alkaline deacetylation of chitin and generally considered as anontoxic and biodegradable polymer. Chitosan hasmany physicaland chemical properties dominated by its functional groups(amino and hydroxyl groups) and related to its degree ofdeacetylation. Various functional groups could be introducedalong chitosan backbone to further extend its applications.Besides, chitosan has some unique and favorable properties,such as antibacterial[20] and mucoadhesive activities,[21] as well ashaemostatic properties.[22] Therefore, chitosan has become anoutstanding candidate for biomedical applications. Anotherbenefit of chitosan for use in tissue repair or regeneration istheir easy processing and manufacturing into a variety of forms,

Table 1. Methods to generate multicellular aggregates (spheroids) and com

Methods

Hanging drop

Non-adherent surfaces (e.g., polyvinyl alcohol, PVA)

Micropatterned surfaces

Suspension culture

Microfluidic system

Substrate-mediated spheroid formation (e.g., chitosan or chitosan-hyaluronan)

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includingfilms, sponges, andhydrogels. Its structural similarity tothe polysaccharide component of extracellular matrix (ECM) alsoendows it with high biocompatibility.

parison of different spheroid forming approaches.

Advantages Disadvantages

Simple

Uniform spheroid size

Low throughput

Time consuming

Simple

High-throughput

Not efficient

Non-uniform spheroid size

Simple

High-throughput

Complicated device fabrication

Simple

Mass production of spheroids

Special equipment required

Cell damaged by the shear flow from mixing

High-throughput

Uniform spheroid size

Complicated device fabrication

Hard to collect and further analyze

Simple

High-throughput

Self-assembled spheroids

Non-uniform spheroid size

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Figure 1. The chemical structure of chitosan and hyaluronan.

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The self-assembly process of stem cells on chitosan substrateswas different from that occurred in hanging drop, suspensionculture, or observed on non-adherent surfaces or micropatternedsubstrates. The expression of calcium signaling-associated genesparticipating in the process of spheroid formation on chitosanwas higher than that on non-adherent surfaces.[23] Besides, manygenes associated with the multilineage differentiation capacitiesand those associated with the anti-inflammatory properties ofMSCs were also up-regulated. On the other hand, the self-assembly process for different types of co-cultured cells on thesesubstrates could create various cellular patterns.[24] The co-spheroids with different morphologies were influenced by thecell–cell and cell–substrate interactions. This phenomenon wasonly observed in cells grown on chitosan-based substrates. Thesecharacteristics suggest that the substrate-derived spheroids maybe superior to other spheroids in their functions. However, the

Figure 2. Spheroids for tissue engineering. Spheroid formation of MSCs sengrafting potential, and regenerative properties. Spheroids as building blockbeen summarized.

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size of biomaterial substrate-derived spheroids may not beuniform (50–500 μm).

Spheroids with intensive cell–cell contacts exhibit betterdifferentiation capacity and excellent regenerative properties.These spheroids may be considered as building blocks for tissueengineering, as shown in Figure 2. Cellular spheroids could fuseto form a tissue due to the cell migration, cell–cell interactions,and cell–matrix interactions. The formation of a network of ECMthroughout the neighboring spheroids provides cues for thefusion process. Afterward the fused tissue becomes mature byculturing in a bioreactor and can be further used inimplantation. A literature has mentioned that spheroidsgenerated from suspension culture or hanging drop fusedrather slowly and that may affect the subsequent tissuematuration.[25] Thus, it requires more time for cell fusion andmaturation. On the contrary, cells on chitosan substrates migratevery fast which may accelerate the fusion into cellularspheroids.[19] Therefore, we suggest that these unique sub-strate-derived spheroids may be more easily fused for matura-tion and more suitable in tissue engineering.

In the following sections, the applications of substrate-derivedspheroids in tissue regeneration (e.g., cartilage, nerve, angio-genesis) and cancer research will be described, respectively.

3. Applications of Substrate-Derived Spheroidsin Tissue Regeneration

3.1. Spheroids and Cartilage Regeneration

The damaged articular cartilage was considered in earlier time tobe incapable of regeneration because of its avascular nature.Until in the 1990s, Brittberg et al. successfully repaired thecartilage defect by autologous chondrocyte transplantation.[26]

However, the autologous chondrocytes as a proper cell source forcartilage repair may be limited by the donor cartilage tissueavailability. Besides, the expansion of autologous chondrocytesrequires a period of time to achieve. As a result, MSCs with

hows better stemness properties, multilineage differentiation capacities,s or spheroids combined with scaffolds in current tissue engineering have

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self-renewal and multilineage differentiation capacities havebecome a promising cell source for cartilage tissueengineering.[27,28]

Many literatures have demonstrated that transplantation ofdispersed MSCs could repair the damaged cartilage.[29,30] MSCspheroids mimic the process of mesenchymal condensationduring chondrogenesis and show higher chondrogenic differ-entiation potential than dispersed MSCs.[19,31] Based on the invitro observation, transplantation of MSC spheroids was alsofound to enhance the therapeutic effect of MSCs in cartilagerepair.[32] However, different MSC spheroids obtained fromvarious methods has shown different repairing efficacy. Aprevious study has compared three kinds of MSC spheroidsgenerated by suspension culture, non-adherent substrates, andchitosan substrates in repairing rabbit articular cartilage defectmodel.[33] Results showed that transplantation of spheroidsderived on chitosan-based substrates could significantly improvethe cartilage regeneration efficiency, particularly for thosederived on chitosan-hyaluronan substrates. The finding maybe associated with higher expression level of N-cadherin (thecapacity for self-renewal and multilineage differentiation) andchemokine/receptor (the migration capacity) on chitosansubstrate-derived spheroids. Although transplantation of MSCspheroids could facilitate cartilage repair, there was a problemwith cell loss during the process of repairing. Therefore, thecombination of spheroids and scaffolds has emerged as apromising therapeutic strategy for cartilage tissue engineering.

Various scaffold-based culture systems such as hydrogels andporous scaffolds have been employed for cartilage regenera-tion.[34–36] Hydrogels with highly hydrated polymer networksmimic the native ECM while porous scaffolds provide forsuperior mechanical properties and mass transport of nutrientsand wastes. Many studies have indicated that MSC-ladenhydrogels improved cartilage repair.[37,38] However, those cell-laden constructs were lack of mechanical strength. On the otherhand, the mechanical properties of porous scaffolds could bematched to those of articular cartilage and offer the properstimulus for chondrogenesis. There are a variety of methods tofabricate porous scaffolds, including solvent casting/particulateleaching, freeze-drying, and etc. However, the pore size andporosity of these scaffolds are difficult to control. Theseconventional scaffolds may not be suitable for accommodatinglarge MSC spheroids. Biodegradable scaffolds with macro-porosity have been fabricated by 3D printing and used foraccommodation of the spheroids.[39] Their internal surfaceshould be further modified by chitosan coating for preservationof the morphology of spheroids. Results showed that MSCspheroids in macroporous scaffolds generated a larger amountof cartilage-associated extracellular matrix (�2.5-fold) comparedto that of MSC single cells. Implantation of MSC spheroid-ladenscaffolds into the chondral defects of rabbit knees revealedsuperior cartilage regeneration after only 1 week.

Regarding the different properties of various substrate-derivedMSC spheroids, it has been reported that MSC spheroidsobtained from different substrates displayed different cartilageregenerative capacity.[32] More specifically, MSC spheroidsderived on chitosan-hyaluronan substrates and combined witha 3D printed scaffold gave rise to the best regeneration, followedby those on chitosan substrates, and finally those on polyvinyl

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alcohol (PVA) substrates. To further overcome the seedingproblem of the spheroids, water-based polyurethane 3D printedscaffolds with the ability to promote the self-aggregation ofMSCs were developed.[40] The spheroids formed from the seededsingle cells in situ within the polyurethane scaffolds. Moreover,the polyurethane scaffolds were printed from a water-basedprocess, so the bioactive ingredient for cartilage tissueengineering such as growth factors or drugs could beincorporated and slowly released in 8 days without losingthe activity. Results showed that the polyurethane scaffolds withthe spontaneous self-aggregation of MSCs underwent chondro-genesis effectively. Transplantation of the MSC-laden 3D printedpolyurethane scaffolds with drug in rabbit chondral defectssignificantly improved the cartilage regeneration, based on theenhanced expression levels of glycosaminoglycan (GAG)contents (5-fold higher than polylactic-co-glycolic acid (PLGA)scaffolds) and type II collagen (1.5-fold higher than PLGAscaffolds). These specially designed scaffolds not only shortenedthe culturing time because of in situ spheroid formation, but alsohad the potential application in customized tissue engineeringbecause of the 3D printing capabilities. The polyurethaneactually activated calcium signaling[41] in a way similar tochitosan.[42]

3.2. Neuroregeneration and Stem Cell Spheroids

With the rapid increase of the aging population, the number ofpatients with neurological diseases has also raised. The diseaseshave caused a great burden for the community, family members,and the patient. However, there is no effective way to treatneurodegenerative diseases until now. For the past decades,tissue engineering and regenerative medicine based approacheshave been proposed as alternatives for neural repair/regenera-tion. Cell-based therapies have been highlighted for neuralregeneration,[43] as well as engineering approaches usingbiomaterials. The combination of biomaterials with celltransplantation has been widely explored for treating neurologi-cal diseases.

As a treatment option for repairing neurological diseases,stem cell replacement has attracted much attention re-cently.[43,44] However, transplantation of stem cells into theinjured sites often displayed extremely poor cell survival rate andengraftment (<20%).[45,46] Compared to traditional stem cellreplacement, dispersed neural stem cells (NSCs) embedded in achitosan-based self-healing hydrogel did not significantly rescuethe neural function when transplanted into the neural injurymodel.[47] Similarly, dispersed NSCs embedded into a two-component biomaterial composed of an outer PLGA scaffoldand an inner poly(ethylene glycol)/poly-L-lysine (PEG/PLL)macroporous hydrogel did not improve the efficacy oftransplantation or the survival rate of NSCs in the neural injurymodel compared to NSCs alone.[48] Based on these two studies, itseemed that the use of hydrogels or scaffolds combined withdispersed NSCs to treat the neural injury did not increase theefficacy of transplantation or cell survival rate, compared to thetraditional stem cell replacement.

Aggregation of MSCs may increase cell–cell contact andpromote cytokine secretion, thereby promoting the stem cell

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proliferation and differentiation. Consistent with these previousfindings, NSC spheroids and neurospheres also showed betterproliferation ability and differentiation capacities into neurons,oligodendrocytes, and astrocytes.[49,50] For NSC spheroidsencapsulated in type I collagen hydrogel, the hydrogel mimickedthe microenvironment of the central nervous system (CNS) andprovided a niche for NSC spheroid proliferation and differentia-tion into neurons.[51] It was thus reasonable to assume that thecombination of materials and stem cell spheroidsmay effectivelyincrease the efficacy of transplantation, the survival rate, andtissue repair capacity of cells. With respect to the use ofspheroids in peripheral nervous system (PNS), the sciatic nerveinjured rats receiving the polymeric nerve conduit with MSCspheroids (derived on chitosan-based substrates) had greaternerve regeneration and functional recovery at 31 days aftertransplantation compared to those receiving the polymeric nerveconduit with dispersedMSCs.[52] In the damaged CNS, the use ofNSC spheroids derived on chitosan-based substrates andincorporated into chitosan-based self-healing hydrogels alsopromoted NSC proliferation and differentiation, and increasedthe restoration of function.[47] In addition, NSCs incorporatedinto the thermoresponsive biodegradable polyurethane (PU)hydrogel with a stiffness of�600Pa could rapidly proliferate andaggregate into NSC spheroids, increase the secretion ofneurotrophic factors such as brain-derived neurotrophic factor(BDNF) and glial cell-derived neurotrophic factor (GDNF),promote NSC differentiation, as well as repair the injurednervous system in vivo.[53] Meanwhile, NSCs embedded in PUhydrogels with a stiffness of �1200 Pa could proliferate but thecells did not form neurosphere-like spheroids, which may resultin lower repairing capacities compared to those in PU hydrogelswith a stiffness of �600 Pa.[53] These studies demonstrated thatthe combination of biomaterials and NSC/MSC spheroids orusing biomaterials to stimulate NSC/MSC spheroid formationmay have potential applications in treating neurodegenerativediseases or neural injuries for both PNS and CNS.

3.3. New Blood Vessel Formation and Cell Spheroids

Growth and formation of the capillary network is a rate-limitingstep in pathological or physiological processes such as tissuegrowth, wound healing, compensation of ischemia, andinflammation. Capillary network formation (angiogenesis orneovascularization) is also a huge challenge in tissue engineer-ing. Because host-capillary invasion upon implantation ofartificial organs requires several weeks for neovascularization,the insufficient nutrient and waste metabolism may induce cellapoptosis in the core of the implant, which causes problems intissue integration. Therefore, neovascularization within theimplanted tissue/organ constructs is a very important issue.

The vascular endothelium is composed of a monolayer ofendothelial cells (ECs). Therefore, the standard method forculturing ECs is 2D cell culture, but ECs maintained in 2D cellculture often show a low expression of differentiation markerssuch as CD34, and thus lose the ability of vascular tubeformation.[54] Literature also demonstrated that the traditional2D cell culture could induce EC apoptosis. The culture of ECs isthus more challenging and complicated.[55] Plating ECs on

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non-adherent dishes could induce cell aggregation and spheroidformation. The EC spheroids expressed high levels of basicfibroblast growth factor 2 (FGF2), vascular endothelial growthfactor (VEGF), interleukin-1 (IL-1), tumor necrosis factor alpha(TNF-α), and the EC adhesion molecules. Besides, the ECspheroids had the ability to decrease cell apoptosis as comparedto the monolayer culture.[54] The EC spheroids seeded onelectrospun scaffolds composed of blended ethylene-co-acrylicacid and polycaprolactone could form capillary-like networks.Meanwhile, no capillary-like network was found for singlydispersed ECs seeded on the electrospun scaffolds.[56] Theseresults suggested that the aggregated ECs may induceangiogenesis through the increase of cell–cell contact andcytokine secretion.

Other than ECs, MSC spheroids could express high levels ofgrowth factors and cytokines such as angiogenin (ANG),angiopoietin-2 (ANGPT2), FGF2, hepatocyte growth factor(HGF), and vascular endothelial growth factor A (VEGFA).Among them, FGF2 and HGF are angiogenic molecules.ANGPT2 could also induce EC differentiation and promoteangiogenesis.[57,58] Therefore, culturing MSC spheroids or co-culturing MSCs with ECs or endothelial progenitor cells (EPCs)may enhance the ability of angiogenesis as compared toculturing ECs alone. Injection of MSC spheroids to the ischemicregion in mice significantly enhanced EC growth compared withinjection of dispersed ECs.[59] In addition, injection of MSCspheroids attracted ECs to induce angiogenesis and improvecardiac functions in a syngeneic rat model with infarctedmyocardium.[60] In literature, co-culturing MSCs and EPCs at aratio of 2:2 on chitosan-hyaluronan substrate could result inMSC-EPC core-shell spheroids. These MSC-EPC co-spheroids,when embedded into the Matrigel, had better angiogenesisability as compared to EPC spheroids. The above resultssuggested that MSCs when aggregated into spheroids maysecrete a larger amount of growth factors and cytokines, whichpromoted EPC differentiation to form tubes.[24] Based onprevious literature, both EC (orMSC) homo-spheroids andMSC-EPC (or EC) co-spheroids could be used for angiogenesis intissue engineering. For example, they could be transplanted withbiomaterials into patients with limb ischemia or coronary arterydisease to repair the damaged blood circulation system. A veryrecent study has explored that single dispersed ECs seeded in thechitosan-fibrin-based (CF) self-healing hydrogel could grow intospheroids and form capillary-like structures.[61] In addition, theinjection of the CF hydrogel alone could rescue the bloodcirculation in limb ischemic mice. Thus, the hydrogel whichpromotes the spheroid formation of ECs and tube formationmay offer new possibilities for future applications to vascularrepair.

Cellular spheroids are suitable for in vitro tests which do notalways require tissue function. On the other hand, theexpression of specific biomarkers in spheroids indeed did notmean functional tissue. There is a big step between markers andtissue function. However, the specific protein expressionexpressed in spheroids may exhibit a potential in functionaliza-tion after appropriate induction. Besides, the cellular environ-ment in vivo is more complicated than in vitro. The previousstudy has demonstrated that transplantation of pristine MSCspheroids could regenerate the injured sciatic nerve and recover

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the nerve function.[52] The transplanted MSC spheroids maydifferentiate into Schwann cells, a component of sciatic nerve.The findings suggested that spheroids after transplantation maybecome a functional tissue in vivo.

4. Spheroids in Cancer Research

2D cell culture has been employed as a tool for screeninganticancer therapeutics. However, the 2D cellular models do notrepresent the architecture of native tumors and the cell–cell andcell–ECM interactions that occur in real tumors. This discrep-ancy may lead to the wrong prediction of cancer cell response todrugs. 3D cell culture models have shown the potential torecapitulate the natural microenvironment of tumors and thusemerged as an alternative method for drug screening. Currently,there are several in vitro tumor models. They can be broadlydivided into transwell-based, spheroid-based, hybrid platforms,and tumor-microvessel models.[62] A spheroid-based model, thatis, tumor spheroids derived on chitosan and hyaluronansubstrates, will be described below as a simple in vitro platformto perform cancer drug screening.

Spheroid-based cancer models in general are one of thenumerous in vitro 3D tumor models. The sphere-like structuresare generated by a group of cancer cells aggregated in a free-floating culture or embedded in a matrix.[63–65] In the early1970s, the spherical cancer models were also known asmulticellular tumor spheroid (MCTS) model, which wasintroduced by the group of Sutherland.[66,67] Tumor size is animportant variable in MCTS, because it is correlated with cellfunction and drug penetration. The size of MCTS could rangefrom <100 to 3mm in diameter,[68,69] depending on differentcancer cell lines and different methods to generate thesespheroids.[70] Besides, it was observed that the size of spheroidsover 400mm in diamater usually required four days to grow

Figure 3. Alternation of cancer cell responses after forming the tumor sphinclude NSCLC spheroids, NSCLC/MSC co-spheroids, HCC spheroids, and caccording to the published literatures. CS-HA, chitosan-hyaluronan; CS, ch

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(from a cell density of 500 cells per 96-well) but the actual culturetime was highly dependent on the cancer cell types. Mostspheroids were roughly between 200 and 500mm in diameter,which were large enough to create an environment withgradients of nutrients, oxygen, and catabolites.[64] For MCTSlarger than 400–600mm in diameter, the cells in the core werenecrotic or apoptotic and there was a viable rim around thenecrotic core. The morphology and condition mimicked those inthe microregion of tumor in vivo.[64,71,72] Friedrich et al.indicated that spheroids less than 200mm used for drugscreening may be sufficient to develop the cell–cell and cell–matrix interactions but were not large enough to develop oxygengradients with hypoxic regions or proliferation gradients.[72] Inour previous study, we found that non-small cell lung cancer(NSCLC) could form tumor spheroids rapidly on the surface ofchitosan-hyaluronan. The average size of the tumor spheroidscould be regulated by the amount of hyaluronan grafting onchitosan substrates. The size of A549 tumor spheroids wasaround 70 μm (1.25� 105 cells density in 6-well plate, 3 days) inlow density hyaluronan (0.1mg cm�2) and �110 μm in the highhyaluronan density (1.8mg cm�2). These results suggested thata higher amount of hyaluronan on the surface could helpgenerate tumor spheroids of bigger sizes. In addition, formationof lung tumor spheroids on chitosan or chitosan-hyaluronansubstrates could alter the cancer cell responses, which is shownin Figure 3. The tumor spheroids showed cancer stem cell-likebehavior and up-regulated epithelial-mesenchymal transition(EMT)markers.[73] The cell motility and invasion ability of tumorspheroids also increased. Furthermore, cells in tumor spheroidswere more resistant to chemotherapeutic drugs (cisplatin andmethotrexate) compared to the conventional 2D cultures.

MSCs play a crucial role during cancer development. Thecrosstalk between MSCs and the tumor microenvironmentcould contribute to their tumor-promoting activities. To developan in vitro assay system that functionally mimics the tumor

eroids on chitosan or chitosan-hyaluronan substrates. Tumor spheroidsolon cancer cell line spheroids. The cellular responses were summarizeditosan; NA, not available; ", increased.

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microenvironment, MSCs, and A549 were co-cultured onchitosan-hyaluronan.[74] The two types of cells were self-organized into 3D tumor co-spheroids with core-shell structure(�75 μm, MSC core and A549 shell). The gene expression ofcancer stemness, EMTmarker, and cell mobility ability were up-regulated in the MSC-tumor co-spheroids. These resultsindicated that the co-spheroids derived from the chitosan-hyaluronan substrate promoted the expression of certain tumorenhancers and direct cell–cell interaction.[74] On the other hand,cancer stem cells (CSCs) are a small population of cancer cellswith the capability of self-renewal and driving tumorigenesis.Chang et al. recently demonstrated that colon and hepatocellularcarcinoma (HCC) cells on chitosan membranes increasedthe cell motility, drug resistance, quiescent population, self-renewal capacity, and the expression levels of stemness and CSCmarker genes. Meanwhile, they also indicated that chitosanmight activate the canonical Wnt/β-catenin-CD44 and nonca-nonical Wnt-STAT3 essential signaling pathways of CSC.[75]

Finally, all these evidences suggested that chitosan and chitosan-hyaluronan substrates may serve as a simple in vitro platform for3D culture of cancer cells. This platform may be convenientlyemployed as a tool for studying the cell–cell interaction in atumor-like microenvironment, for screening cancer drug, or fortherapeutic applications.[73–75]

5. Conclusions

Multicellular aggregates (stem cell spheroids or tumor spheroids)generated from the substrate-based 3D culture systemmay bettermimic the cells present in an in vivo microenvironment than in a2D monolayer culture system. Different types of spheroids havetheir own attractive properties. For instance, the substrate-derivedMSCspheroidsexhibitbetter self-renewalproperties,multilineagedifferentiation capacity, and engrafting potential. These spheroidsespecially for those derived from the chitosan substrates aresuitable for in vitro tests because of the imaging possibilities. Theyfurther own thepromises in thefield of regenerativemedicine.Onthe other hand, the substrate-mediated formation of tumorspheroids can enhance the aggressive characteristics, includingthe EMT phenotype of the tumor cells. Besides, the tumorspheroids exhibit higher malignancy and drug resistance. Thesecharacters make them suitable as an in vitro platform for drugscreening. Inadditiontospheroids for tissueengineeringanddrugscreening, the substrate-based 3D co-culture ofmultiple cell typesto form organoids deserves further investigations for basic andclinical applications in the future.

AcknowledgementsThis work was supported by the Program Project for RegenerativeMedicine, Ministry of Science and Technology, Taiwan, R.O.C. MOST106-3114-Y-043-021 and the Cutting-Edge Steering Research Project ofNational Taiwan University (NTU-106R7626).

Conflict of InterestThe authors declare no commercial or financial conflict of interest.

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Keywordscancer cells, chitosan, mesenchymal stem cells, spheroids, tissueengineering

Received: May 11, 2017Revised: September 1, 2017

Published online:

[1] L. G. van der Flier, H. Clevers, Annu. Rev. Physiol. 2009, 71, 241.[2] K. Tamama, V. H. Fan, L. G. Griffith, H. C. Blair, A. Wells, Stem Cells

2006, 24, 686.[3] E. Park, A. N. Patel, Cell Biol. Int. 2010, 34, 979.[4] A. W. Lund, B. Yener, J. P. Stegemann, G. E. Plopper, Tissue Eng. Part

B Rev. 2009, 15, 371.[5] W. Mueller-Klieser, Am. J. Physiol. 1997, 273, C1109.[6] Y. Sasai, Nature 2013, 493, 318.[7] S. Sart, A. C. Tsai, Y. Li, T. Ma, Tissue Eng. Part B Rev. 2014, 20, 365.[8] M. W. Laschke, M. D. Menger, Trends Biotechnol. 2017, 35, 133.[9] V. Mironov, R. P. Visconti, V. Kasyanov, G. Forgacs, C. J. Drake,

R. R. Markwald, Biomaterials 2009, 30, 2164.[10] B. Mattix, T. R. Olsen, Y. Gu, M. Casco, A. Herbst, D. T. Simionescu,

R. P. Visconti, K. G. Kornev, F. Alexis, Acta Biomater. 2014, 10, 623.[11] N. C. Cheng, S. Wang, T. H. Young, Biomaterials 2012, 33, 1748.[12] S. Mathews, P. K. Gupta, R. Bhonde, S. Totey, Cell Prolif. 2011,

44, 537.[13] J. M. Kelm, N. E. Timmins, C. J. Brown, M. Fussenegger,

L. K. Nielsen, Biotechnol. Bioeng. 2003, 83, 173.[14] G. Su, Y. Zhao, J. Wei, J. Han, L. Chen, Z. Xiao, B. Chen, J. Dai,

Biomaterials 2013, 34, 3215.[15] W. Wang, K. Itaka, S. Ohba, N. Nishiyama, U. I. Chung, Y. Yamasaki,

K. Kataoka, Biomaterials 2009, 30, 2705.[16] R. L. Carpenedo, C. Y. Sargent, T. C. McDevitt, Stem Cells 2007,

25, 2224.[17] X. J. Li, A. V. Valadez, P. Zuo, Z. Nie, Bioanalysis 2012, 4, 1509.[18] S. Halldorsson, E. Lucumi, R. G�omez-Sjöberg, R. M. Fleming,

Biosens. Bioelectron. 2015, 63, 218.[19] G. S. Huang, L. G. Dai, B. L. Yen, S. H. Hsu, Biomaterials 2011,

32, 6929.[20] G. J. Tsai, W. H. Su, J. Food Prot. 1999, 62, 239.[21] P. Hea, S. S. Davisa, L. Illumab, Int. J. Pharm. 1998, 166, 75.[22] A. E. Pusateri, S. J. McCarthy, K. W. Gregory, R. A. Harris, L. Cardenas,

A. T. McManus, C. W. Goodwin, Jr., J. Trauma 2003, 54, 177.[23] H. Y. Yeh, B. H. Liu, M. Sieber, S. H. Hsu, BMC Genomics 2014,

15, 10.[24] S. H. Hsu, T. T. Ho, N. C. Huang, C. L. Yao, L. H. Peng, N. T. Dai,

Biomaterials 2014, 35, 7295.[25] T. R. Olsen, F. Alexis, Bioprocess. Biotech. 2014, 4, 1000e112.[26] M. Brittberg, A. Lindahl, A. Nilsson, C. Ohlsson, O. Isaksson,

L. Peterson, N. Engl. J. Med. 1994, 331, 889.[27] R. S. Tuan, G. Boland, R. Tuli, Arthritis Res. Ther. 2003, 5, 32.[28] X. Wei, X. Yang, Z. Han, F. Qu, L. Shao, Y. F. Shi, Acta Pharmacol. Sin.

2013, 34, 747.[29] P. K. Gupta, A. K. Das, A. Chullikana, A. S. Majumdar, Stem Cell Res.

Ther. 2012, 3, 25.[30] Q. Li, J. Tang, R. Wang, C. Bei, L. Xin, Y. Zeng, X. Tang, Artif. Cells

Blood Substit. Immobil. Biotechnol. 2011, 39, 31.[31] S. H. Hsu, G. S. Huang, S. Y. Lin, F. Feng, T. T. Ho, Y. C. Liao, Tissue

Eng. Part A 2012, 18, 67.[32] J. I. Lee, M. Sato, H. W. Kim, J. Mochida, Eur. Cell Mater. 2011,

22, 275.[33] G. S. Huang, P. S. Hsieh, C. S. Tseng, S. H. Hsu, Biomater. Sci. 2014,

2, 1652.

© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim7 of 8)

www.advancedsciencenews.com www.biotechnology-journal.com

[34] C. H. Chen, V. B. Shyu, J. P. Chen, M. Y. Lee, Biofabrication 2014,6, 015004.

[35] S. H. Hsu, T. B. Huang, S. J. Cheng, S. Y. Weng, C. L. Tsai, C. S. Tseng,D. C. Chen, T. Y. Liu, K. Y. Fu, B. L. Yen, Tissue Eng. Part A 2011,17, 1549.

[36] S. W. Whu, K. C. Hung, K. H. Hsieh, C. H. Chen, C. L. Tsai, S. H. Hsu,Mater. Sci. Eng. C Mater. Biol. Appl. 2013, 33, 2855.

[37] I. E. Erickson, S. R. Kestle, K. H. Zellars, G. R. Dodge, J. A. Burdick,R. L. Mauck, Biomed. Mater. 2012, 7, 024110.

[38] Q. Meng, Z. Man, L. Dai, H. Huang, X. Zhang, X. Hu, Z. Shao, J. Zhu,J. Zhang, X. Fu, X. Duan, Y. Ao, Sci. Rep. 2015, 5, 17802.

[39] G. S. Huang, C. S. Tseng, L. B. Yen, L. G. Dai, P. S. Hsieh, S. H. Hsu,Eur. Cell Mater. 2013, 26, 179.

[40] K.C.Hung, C. S. Tseng, L.G.Dai, S.H.Hsu,Biomaterials 2016, 83, 156.[41] K. C. Hung, S. H. Hsu, Adv. Healthc. Mater. 2015, 4, 2186.[42] H. Y. Yeh, B. H. Liu, S. H. Hsu, Biomaterials 2012, 33, 8943.[43] J. Yoo, H. S. Kim, D. Y. Hwang, J. Cell Biochem. 2012, 114, 743.[44] M. S. Shoichet, C. C. Tate, M. Douglas Baumann, M. C. Laplaca,

Indwelling Neural Implants: Strategies for Contending with the In VivoEnvironment (Ed: W. M. Reichert), CRC Press/Taylor & Francis, BocaRaton (FL) 2008.

[45] K. H. Wu, X. M. Mo, Z. C. Han, B. Zhou, Ann. Thorac. Surg. 2011,92, 1917.

[46] A. Bakshi, C. A. Keck, V. S. Koshkin, D. G. LeBold, R. Siman,E. Y. Snyder, T. K. McIntosh, Brain Res. 2005, 14, 10658.

[47] T. C. Tseng, L. Tao, F. Y. Hsieh, Y. Wei, I. M. Chiu, S. H. Hsu, Adv.Mater. 2015, 27, 3518.

[48] R. C. AssunSc~ao-Silva, E. D. Gomes, N. Sousa, N. A. Silva,A. J. Salgado, Stem Cells Int. 2015, 2015, 948.

[49] K. Watanabe, M. Nakamura, H. Okano, Y. Toyama, Restor. Neurol.Neurosci. 2007, 25, 109.

[50] W. Ma, W. Fitzgerald, Q. Y. Liu, T. J. O’Shaughnessy, D. Maric,H. J. Lin, D. L. Alkon, J. L. Barker, Exp. Neurol. 2004, 190, 276.

[51] F. Huang, Q. Shen, J. Zhao, Neural Regen. Res. 2013, 8, 313.[52] T. C. Tseng, S. H. Hsu, Biomaterials 2014, 35, 2630.[53] F. Y. Hsieh, H. H. Lin, S. H. Hsu, Biomaterials 2015, 71, 48.

Biotechnol. J. 2017, 1700064 1700064 (

[54] D. Delia, M. G. Lampugnani, M. Resnati, E. Dejana, A. Aiello,E. Fontanella, D. Soligo, M. A. Pierotti, M. F. Greaves, Blood 1993,81, 1001.

[55] T. Korff, H. G. Augustin, J. Cell Biol. 1998, 143, 1341.[56] N. Nosoudi, W. Yin, D. A. Rubenstein, FASEB J. 2013, 27, 1.[57] I. A. Potapova, G. R. Gaudette, P. R. Brink, R. B. Robinson,

M. R. Rosen, I. S. Cohen, S. V. Doronin, Stem Cells. 2007, 25, 1761.[58] I. A. Potapova, P. R. Brink, I. S. Cohen, S. V. Doronin, J. Biol. Chem.

2008, 283, 13100.[59] C. C. Wang, C. H. Chen, S. M. Hwang, W. W. Lin, C. H. Huang,

W. Y. Lee, Y. Chang, H. W. Sung, Stem Cells 2009, 27, 724.[60] S. H. Bhang, S. Lee, J. Y. Shin, T. J. Lee, H. K. Jang, B. S. Kim, Mol.

Ther. 2014, 22, 862.[61] F. Y. Hsieh, L. Tao, Y. Wei, S. H. Hsu,NPG Asia Mater. 2017, 9, e363.[62] M. E. Katt, A. L. Placone, A. D. Wong, Z. S. Xu, P. C. Searson, Front.

Bioeng. Biotechnol. 2016, 4, 12. eCollection 2016.[63] W. Mueller-Klieser, J. Cancer Res. Clin. Oncol. 1987, 113, 101.[64] F. Hirschhaeuser, H. Menne, C. Dittfeld, J. West, W. Mueller-Klieser,

L. A. Kunz-Schughart, J. Biotechnol. 2010, 148, 3.[65] E. Fennema, N. Rivron, J. Rouwkema, C. van Blitterswijk, J. de Boer,

Trends Biotechnol. 2013, 31, 108.[66] W. R. Inch, J. A. McCredie, R. M. Sutherland, Growth 1970, 34, 271.[67] R. M. Sutherland, J. A. McCredie, W. R. Inch, J. Natl. Cancer Inst.

1971, 46, 113.[68] M. Haji-Karim, J. Carlsson, Cancer Res. 1978, 38, 1457.[69] M. T. Santini, G. Rainaldi, Pathobiology 1999, 67, 148.[70] B. Mayer, G. Klement, M. Kaneko, S. Man, S Jothy, J. Rak, R. S. Kerbel,

Gastroenterology 2001, 121, 839.[71] E. Gottfried, L. A. Kunz-Schughart, R. Andreesen, M. Kreutz, Cell

Cycle 2006, 5, 691.[72] J. Friedrich, C. Seidel, R. Ebner, L. A. Kunz-Schughart, Nat. Protoc.

2009, 4, 309.[73] Y. J. Huang, S. H. Hsu, Biomaterials 2014, 35, 10070.[74] H. W. Han, S. H. Hsu, Acta Biomater. 2016, 42, 157.[75] P. H. Chang, K. Sekine, H. M. Chao, S. H. Hsu, E. Chern, Sci. Rep.

2017, 8, 45751.

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