1072 Biochemical Society Transactions (2010) Volume 38, part 4

Developments in three-dimensional cell culturetechnology aimed at improving the accuracy ofin vitro analysesDaniel J. Maltman*† and Stefan A. Przyborski*†1

*School of Biological Sciences, Durham University, South Road, Durham DH1 3LE, U.K. and †Reinnervate Limited, NETPark Bioincubator, Thomas Wright Way,

Sedgefield, County Durham TS213FD, U.K.

AbstractDrug discovery programmes require accurate in vitro systems for drug screening and testing. Tradi-tional cell culture makes use of 2D (two-dimensional) surfaces for ex vivo cell growth. In such environments,cells are forced to adopt unnatural characteristics, including aberrant flattened morphologies. Thereforethere is a strong demand for new cell culture platforms which allow cells to grow and respond to theirenvironment in a more realistic manner. The development of 3D (three-dimensional) alternative substratesfor in vitro cell growth has received much attention, and it is widely acknowledged that 3D cell growth islikely to more accurately reflect the in vivo tissue environments from which cultured cells are derived. 3Dcell growth techniques promise numerous advantages over 2D culture, including enhanced proliferation anddifferentiation of stem cells. The present review focuses on the development of scaffold technologies for3D cell culture.

Demand for 3D (three-dimensional) cellculture technologyThe availability of accurate informative in vitro assays is anincreasingly important challenge facing the pharmaceuticalindustry. Rising cost-to-delivery ratios and the poor predict-ive value of existing in vitro tests places great emphasis on thedevelopment of more realistic cell culture models. Since costsmount as pharmaceuticals near market, efficient and realisticin vitro research is of huge potential value, enabling informedstrategic decisions to be made earlier rather than later.

Cell-based in vitro assays are a key component of drugdiscovery research. Cultured mammalian cells are importanttools for providing predictions of drug activity, metabolismand toxicity in vivo. Human stem cells are particularlyvaluable in this context since they promise to provide apotentially limitless supply of a wide range of human celltypes for use in drug testing. However, traditional cell cultureenvironments are far removed from real-life tissues. In vivo,cells are supported by a complex 3D ECM (extracellularmatrix), which facilitates cell–cell communication via directcontact and through the secretion of a plethora of cytokinesand trophic factors. In contrast, cells grown in culture aregenerally confined in 2D (two-dimensional) monolayerswithout many of the physical and chemical cues whichunderlie their identity and function in vivo. These factorsare particularly important when considering the potential fordirected differentiation of stem cells in vitro.

Key words: cell culture, drug discovery, in vitro model, polystyrene scaffold, three-dimensional

cell culture.

Abbreviations used: 2D, two-dimensional; 3D, three-dimensional; ECM, extracellular matrix;

HIPE, high internal phase emulsion.1To whom correspondence should be addressed (email stefan.przyborski@durham.ac.uk).

In vitro, cells can behave very differently depending onthe growth substrate employed. Conventional tissue cultureis carried out on 2D surfaces without scope for cells toadopt natural morphologies or to communicate efficientlywith their neighbours. This 2D confinement is far removedfrom the aforementioned 3D complexities of living tissue.Engineering the cell culture microenvironment to creategrowth conditions that more accurately mimic the in vivobehaviour of cells is an essential step for improving predictiveaccuracy during pharmaceutical development [1]. It has beenshown using alternative cell culture applications that thegrowth and function of cells as multicellular 3D structuresis significantly different to their growth as conventional 2Dmonolayer cultures [2]. The design of 3D culture systemsfor drug development is an important part of this process.Findings show that refinement of the in vitro environmentsignificantly influences the way in which cells respond tosmall molecules [3].

Considerable effort has therefore been made to engineermaterials for 3D in vitro cell culture in the belief that provisionof a 3D spatial environment will overcome some of therestrictions associated with 2D culture. Progress in this areais beginning to bridge the gap between traditional cell cultureand living tissue environments. The present article brieflyreviews some currently available technologies for in vitro 3Dcell culture, and we include reference to a recently developedtechnology designed to enable routine 3D cell culture.

Current technologies for in vitro 3D cellgrowthAlthough there are a variety of technologies available thatenable 3D cell growth, most of these have been developed

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for transplantation purposes and tissue engineering in vivo.Comparatively less attention has been devoted to the devel-opment of 3D culture systems for exclusive use in thelaboratory as an approach to improving the accuracy ofin vitro analyses. Recently, however, there have been severaladvances in this field, and this is partly in response to thedemand and interest in fabricating materials that facilitate3D growth and enhance cell function in ways that resembletissues in vivo.

A range of materials have been considered for applicationas 3D cell growth supports in vitro. These include naturallyoccurring materials as well as products fabricated fromnaturally derived and synthetic polymers. Natural substratessuch as alginate, which is a seaweed-derived material, havebeen used to support cell growth in a number of waysincluding cell encapsulation [4]. More recently, this materialhas been developed into a macroporous scaffold and hasbeen used, for example, to support the development of 3Dspheroids and the terminal differentiation of hepatocytesin vitro [5]. Similar such technology has been releasedcommercially, and AlgimatrixTM (Invitrogen) is marketed asan animal-free scaffold available for the development of high-fidelity cell culture models that are more predictive of diseasestates and drug responses. Granted that this technology doesenable a degree of 3D cell growth, however, growing cellsas individual spherical masses is not necessarily suitable forall requirements, as the distribution of cells throughout thematerial is not entirely even, and there are issues about masstransfer given the thickness of the scaffold under static growthconditions. Furthermore, it is not clear whether cells willrespond differently to the alginate substrate compared withconventional 2D plasticware, which is familiar and has beenused for many years.

An alternative and one of the early most successfulapproaches has been the culture of cells on biodegradablepolymers such as poly(glycolic acid), poly(lactic acid) andtheir copolymers poly(lactic-co-glycolic acid) [6]. Previouswork has shown that such materials benefit the repair ofarticular cartilage during implantation and encourage tissueregeneration [7]. Their degradation over time paves theway for replacement by viable functioning cells and aidsthe integration of co-transplanted cells with host tissues.Biodegradability, however, is not necessarily an attractivefeature for routine in vitro cell culture, where there are shelf-life issues, and improper storage of such biopolymers canrender such products useless due to degradation, leading tochanges in their properties, quality and variation of results.In addition, biodegradation during an in vitro experimentintroduces another variable that may influence how cellsbehave and is therefore not necessarily a desirable feature.

Hydrogels are a common form of material that hassuccessfully been used to support ex vivo 3D cell growthfor a variety of systems, such as bone, cartilage and nervoustissues [8–12]. Hydrogels comprise a cross-linked naturalbase material such as agarose, collagen, fibrin or hyaluronicacid with a high water content. They can be engineered tosupport preferential cell growth and function. Hydrogels in

essence trap cells in an artificial ECM environment and maybe modified to incorporate biologically active molecules suchas laminin [12] and altered physical parameters such as charge[13]. There are several commercially available hydrogel-basedproducts, including ExtracellTM (Glycosan Biosystems), achemically defined hyaluronan-based substrate. Injectablehydrogels have proven to be successful for tissue repair [8];however, their use for routine 3D cell culture is restrictedby various practical issues, including expense, shelf-life, gelpreparation, storage and consistency.

The development of inert non-degradable scaffolds madefrom synthetic polymers overcomes several of the limitationsthat other technologies experience. These structures consistof pores or voids into which cells can grow and whichare joined by interconnecting holes. Methods of fabricationfor porous materials include emulsion templating [14,15],leachable particles [16] and gas foaming technology [17].Gas-in-liquid foam templating has been used as a methodto create porous scaffolds for cell culture applications[18]. However, gas bubbles can coalesce, leading to abroad range of scaffold porosities and it is thereforedifficult to control the consistency of the material andconsequently the reproducibility of the cultures growntherein. Electrospinning is also a well-established techniquegiving rise to electrospun synthetic fibres woven intomats designed to support 3D cell growth [19]. However,the consistency and porosity of electrospun materials isdifficult to control. Ultra-WebTM (Corning) was developedas a commercial polyamide elecrospun nanofibre mat forcell culture. Cells grow as monolayers on the roughenedtopography created by the nanoscale Ultra-WebTM fibre mat,rather than within the physical lattice of the material.

Technology for routine 3D cell cultureA number of these fabrication technologies have been appliedto the manufacture of porous polystyrene-based scaffolds.Polystyrene is an attractive medium as a scaffold to support3D cell culture because it is chemically inert, stable and,most importantly, consistent and directly comparable withconventional 2D tissue culture plasticware. The vast majorityof in vitro cell culture experiments and data generated fromthem over the last few decades have been conducted onpolystyrene surfaces in one form or another. There is nodoubt that the transition to 3D cell culture models is a majorstep change; however, the development of polystyrene-basedscaffolds will ease the impact that this has, given that thepolystyrene substrate remains the same albeit it has changedfrom a 2D into a 3D geometry. Polystyrene scaffolds arealso advantageous given that they are generally simple andinexpensive to mass produce, and they are designed as aconsumable product with a long shelf-life. These attributesmake polystyrene-based substrates well suited for routine 3Dcell culture.

Emulsion templating enables the controlled manufactureof porous polystyrene that can subsequently be tailored tosupport 3D cell culture [14,15]. AlvetexTM (Reinnervate) is a

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1074 Biochemical Society Transactions (2010) Volume 38, part 4

Figure 1 Porous polystyrene scaffold developed for routine 3D

cell culture

(A) Scanning electron micrograph showing the structure of AlvetexTM, a

polystyrene scaffold consisting of voids and interconnecting pores. Scale

bar, 50 μm. (B) Polystyrene scaffold engineered into a 200-μm-thick

membrane and adapted to a conventional 12-well cell culture multi-well

plate.

new product that utilizes this technology and produces apolystyrene-based scaffold that has a relatively uniformstructure. The scaffold is formed by polymerization in abiphasic emulsion, consisting of an aqueous and a non-aqueous monomer/surfactant phase, termed HIPE (highinternal phase emulsion) [20]. The resulting polymer (poly-HIPE) product consists of a templated porous networkof voids, linked by interconnecting pores (Figure 1A).These structures in the final poly-HIPE material are highlycontrollable through manipulation of key manufacturingparameters [14,15]. AlvetexTM has been developed as asolution for routine 3D cell culture and is designed forincorporation into existing cell culture products, such aswelled plates or well inserts (Figure 1B). The scaffold hasbeen engineered into a thin 200-μm-thick membrane toaddress the issue of mass transfer, enabling cells to enter

Figure 2 Routine 3D cell culture using AlvetexTM porous

polystyrene scaffold

Human-derived cell lines representing (A) liver (HepG2) and (B)

pluripotent stem (TERA2.cl.SP12) cells cultured for 10 days and

subsequently prepared for histological analysis. Scale bar, 30 μm.

the material and allowing for sufficient mass exchange ofgases, nutrients and waste products during static culture.The polystyrene that forms the scaffold is cross-linked,which gives the material improved structural stability andstrength even when presented as a thin membrane. AlvetexTM

is compatible with standard cell culture plasma treatment,γ sterilization methods and, if required, can be coatedusing standard cell culture reagents such as collagen orfibronectin.

For routine 3D cell culture, the development of anynew technology must consider issues such as cost, easeof use, application and reproducibility, especially when theapplication is for drug discovery. A technology that isexpensive, difficult to use or is inconsistent in some mannerwill not satisfy these demands and will fail to be accepted bythe scientific community. Importantly, any such technologyrequires vigorous exemplification and validation as evidence

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of its ability to support true 3D cell culture over a range ofalternative cell types. Although AlvetexTM is developed as ageneric solution for 3D cell culture, it has also been promotedon the basis of demonstrating its application in several keyareas of research. For example, cultured liver cells are avaluable tool for the in vitro study of drug metabolism andtoxicity [21]. Liver-derived cell lines represent a convenientmodel for liver toxicology studies, although commonlyavailable lines often display poor metabolic responses whensubjected to drug treatments. However, responses by suchcells are significantly enhanced when cultured in 3D usingAlvetexTM [22,23]. Similarly, pilot data reveal that primaryhepatocytes have enhanced cell viability and superior capacityto metabolize hepatotoxic drugs when cultured in the poly-HIPE scaffold compared with 2D plasticware. Culturesystems able to contribute enhanced metabolic activity and/ormore realistic resistance/sensitivity in response to specificdrugs would be of potential value to the pharmaceuticalindustry, enabling more accurate toxicological assays.

Another essential element required is a reliable source ofcultured cells. Developments in stem cell technology arefocused on the differentiation of functional tissues fromhuman stem cells, and this potentially represents a valuableopportunity for a renewable source of functional hepatocytes[24,25]. Expansion of stem cell populations and control ofstem cell differentiation towards the formation of specificfunctional cell types will rely on the introduction of newmethods and technologies. This will no doubt require theneed for more sophisticated cell culture models includingsystems for 3D cell growth. Examples of human-derivedhepatocytes and pluripotent stem cells grown in 3D cultureare shown in Figure 2.

ConclusionsCulture technology has, in general, changed little over thelast few decades. The development of new technologiesin the physical sciences has, however, provided innovativesolutions to improving current practice, and the growth andperformance of cultured cells in 3D. The investment of timetowards developing and validating such in vitro models islikely to have a significant impact on the success and overallefficiency of pharmaceutical development in the future.

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Received 11 January 2010doi:10.1042/BST0381072

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