3d cell culture systems: advantages and applications · department of human genetics, faculty of...

3D Cell Culture Systems:Advantages and ApplicationsMADDALY RAVI,* V. PARAMESH, S.R. KAVIYA, E. ANURADHA, AND F.D. PAUL SOLOMONDepartment of Human Genetics, Faculty of Biomedical Sciences, Technology and Research, Sri Ramachandra University, Porur,

Chennai, India

Cell cultures are important material of study for the variety of advantages that they offer. Both established continuous cell lines and primarycell cultures continue to be invaluable for basic research and for direct applications. Technological advancements are necessary toaddress emerging complex challenges and the way cells are cultured in vitro is an area of intense activity. One important advancement incell culture techniques has been the introduction of three dimensional culture systems. This area is one of the fastest growing experimentalapproaches in life sciences. Augmented with advancements in cell imaging and analytical systems, as well as the applications of newscaffolds and matrices, cells have been increasingly grown as three dimensional models. Such cultures have proven to be closer to in vivonatural systems, thus proving to be useful material formany applications. Here, we review the three dimensional way of culturing cells, theiradvantages, the scaffolds and matrices currently available, and the applications of such cultures in major areas of life sciences.

J. Cell. Physiol. 230: 16–26, 2015. © 2014 Wiley Periodicals, Inc.

Cell cultures have been proven to be indispensable for avariety of applications, from research to industrialperspectives. Cell culture techniques and the number of celllines currently available have come a long way since the firstcell line, HeLa, was established. This is augmented by the factthat several tools of molecular biology, such as DNAfingerprinting/profiling and cytogenetic analysis are nowavailable to identify and characterize cell lines. With theincrease in the number of cell lines, parallel growth in cellculture techniques, imaging, data acquisition, and analysismethods are being witnessed.

Established cell lines are of either themonolayer types or thesuspension types. Cells in culture have a unique doubling timethat is determined by the cell cycle duration and otherphysiological events. All cells in culture exhibit the property ofgoing through distinct culture phases: the lag, log, plateau andthe decline phases. The log phase with an exponential increasein the number of cells provides us with the healthiest cells andbest cellular material for many studies.

Mammalian cell culture systems are probably some of themost exciting in vitro scientific models and have seen rapidadvancement in the past few decades. The advancements are inthe combined areas of types of culture methods and the mediasupplements. The promises that the in vitro cell culturesystems hold is due flexibility to experimental variations thatare possible in such systems. The advent of 3D cultureapproaches heralded a significant advancement, an indication asevident by the research publication trend in this area. Forexample a simple search in the PubMed site for the key words“3D cell cultures” gives us 2,546 results (as on this manuscriptpreparation date). It is interesting to note that the first paperswere published in 1968 and 1970, one each with a similar trenduntil the year 1992. The number of publications graduallyincreased from 7 in 1992 to 10 in 1997 and 11 in 1998. Theincrease in the publications showed a rapid increase from theyear 2000 onwards, touching a mark of 371 in the year 2012and 421 for the year 2013. As of this review preparation,40 papers have already been published even before the endof January, 2014. This exponential increase in the researchactivity conveys the importance and interest that the 3Dcell cultures warrant, one of the primary reasons for thisreview.

In this review we present the importance of the 3D cellculture systems and their advantages with focus on specificareas of applications. The areas of applications that arecomprehensively dealt with are differentiation studies, drug

discovery/pharmacological applications, cancer research, gene/protein expressions studies and for the understanding of cellphysiology. We have obtained information from all possibleprimary, secondary, and tertiary literature sources such aspublished papers, dedicated websites, commercial productcatalogues, etc. for this review on 3D cell culture systems.

3D Cell Culture Highlights and Advantages

Cells cultured as 3D models exhibit features that are closer tothe complex in vivo conditions (Vinci et al., 2012). The 3Dculture models have proven to be more realistic for translatingthe study findings for in vivo applications. While cell linesprovide us with excellent homogenous study material,culturing them as 3D models induces them to behave in amanner that is a step closer to the natural conditions. Till date,the 3D culture approach has been utilized to study more than380 cell lines. It is also known that the optimal 3D conditionrequirements vary between cell types and the characteristicfeatures of cells in 3D cultures differ in accordance to theirtypes. The differences exhibited by cells in 2D and 3D culturesystems and the areas where these differences can be ofadvantage are presented in Figure 1.

Matrices/Scaffolds Used for 3D Cultures

One important contribution for the “closer-to-in vivo”behavior of cells when grown as 3D cultures is thematrices andscaffolds that are used for obtaining such cultures. The mostcommonly used scaffolds are agarose, collagen, fibronectin,

[Correction added on 7October 2014, after first online publication29 September 2014: The citation year has been corrected to 2015.]

*Correspondence to: Maddaly Ravi, Department of HumanGenetics, Faculty of Biomedical Sciences, Technology andResearch, Sri Ramachandra University, Porur, Chennai 600116.India. E-mail: [email protected]

Manuscript Received: 3 May 2014Manuscript Accepted: 21 May 2014

Accepted manuscript online in Wiley Online Library(wileyonlinelibrary.com): 9 June 2014.DOI: 10.1002/jcp.24683

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gelatin, laminin, and vitronectin. From simple liquid overlaymethods to complex co-cultures, several matrices are utilized;from low-melting agarose to complex synthetic compounds.Of significance is to note that a specific type of matrix orscaffold can be the most suited to elicit a particular type ofmorphological and physiological behavior in cultured cells.More than 100 types of matrices and scaffolds of both organicand inorganic nature are being currently used. The choice ofsuch matrices and scaffolds is based on cell type and the natureof the study.

Type I collagen matrix is used commonly in 3D culturesystem. This is due to a number of reasons including the ease inprocessing, low-cost and the flexibility for live cellmanipulation. Also, the pore size, ligand density and stiffnesscan be varied by changing the concentration of collagen orintroducing chemical cross-linking compounds, thus making iteasy to change the structural properties of the gel (Baker et al.,2009, 2011; Harjanto and Zaman, 2011).

3D scaffolds are generated using various natural (collagen,gelatin, elastin, silk fibroin, chitosan, chitin, fibrin, fibrinogen,etc.) and synthetic polymers. The composite of both naturaland synthetic substances are also being used. These compositesmimic the native extracellular matrix by porosity, fibrous,permeability and mechanical stability. The micro architectureenhances the biophysical and biochemical interaction of theadhered cells to be better expressed in vitro. The 3D matrixprovides a biologically active environment for the cells toproliferate, differentiate and secreted cell specific extracellular

matrix which can be potentially used for a variety ofapplications. Agarose hydrogels are an example of simplematerials to obtain 3D aggregates for a variety of cell types.However, each cell type requires markedly different optimalconditions as defined by the composition, concentration andvolume of the agarose hydrogels. Also, different cell typesbehave differently when cultured using agarose hydrogels, thusproving to be simple, yet useful models. A few cell lines grownas 2D monolayers and as 3D aggregates using agarosehydrogels from our previous studies are presented in Figure 2.The salient points of various matrices and scaffolds used for 3Dcell cultures are presented in Table 1.

Applications of 3D Cell Cultures

The conventional 2D culture systems have helped usunderstand the complex cellular physiology; on how cellsfunction and respond to stimuli. The 3D culture approach hastaken us a step closer to the in vivo conditions. A majoradvantage of the 3D over the 2D approach is the decrease inthe gap between cell cultures system and the cellularphysiology (Cukierman et al., 2001). In conventional 2Dconditions, the extracellular matrix components, cell-to-celland cell-to-matrix interaction that are important fordifferentiation, proliferation and cellular functions in vivo arelost (Mazzoleni et al., 2009). Several studies show that 3Dorganization of cells reveals more novel and unanticipatedvisions into the tumorigenesis mechanism and could represent

Fig. 1. Some of the important areas for which 3D cell culture systems are excellent models include studies involving drug discovery,cytotoxicity, genotoxic, cell growth, apoptosis, survival, gene, and protein expressions, differentiations and developmental changes. Similarly,co-cultures in 3D systems give a better understanding of the cell interactions.

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3 D C E L L C U L T U R E S Y S T E M S 17

an integral missing component in the in vitro cancer studies(Muthuswamy, 2011). Also, when cells are allowed to grow inbasement membrane like gels, there is a mutual integration ofthe signaling pathways (Lee et al., 2007). A549 3D spheroidshave demonstrated constantly high levels of interleukin (IL)-6and IL-8 secretion when compared with their monolayercounterparts. Enhanced extracellular matrix deposition forbetter biomarker expression was reported using 3D culturesystems (Bazou, 2010). Cells in a 3D environment are goodmodels as “near-to-in vivo” systems and give us useful insightsfrom a variety of ways (Cukierman et al., 2001). They can serveas a cost effective screening platform for drug development andtesting. They provide us with a better and more realisticpredictive value for safety and risk assessment. In our ownstudies, when 3D aggregates were reverted to a 2Denvironment, the cells migrated from the aggregates andformed monolayers, a feature similar to that of an explantculture (bottom part of Fig. 2).

The 3D cell culture systems applications in differentiationstudies, drug discovery and pharmacological applications,cancer research, gene and protein expression studies, and tounderstand better, the complex cellular physiologicalmechanisms are reviewed and presented here.

For Differentiation Studies

3D culture systems have been used for many studies in theareas of stem cell research and differentiation studies. Stemcells are widely used as a cellular source for 3D models toengineer tissue constructs. This is largely due to theadvancements in the matrices and scaffolds that are bothflexible and complex, as suited for specific applications. The 3Dcell cultures for differentiation studies, apart from giving usefulinsights for basic research, are also making it possible to extendthe work to therapeutic applications.

Studies showed that 3D systems are useful to understandthe mechanisms of human osteoblast differentiation intoosteocytes, and for understanding the role of osteocytes inbone metastasis and tissue engineering applications. Efficientosteogenesis using mesenchymal rat stem cells was possibleusing expression of collagen type I (Farrell et al., 2007). Studiesshowed increased expression of the osteogenic marker CBFA-1, alkaline phosphatase, osteonectin, Collagen 1, osteopontin,and JNK2 during osteogenic differentiation of the bonemarrow stems cells in rat (Hosseinkhani et al., 2006). 3Dcultures showed increased differentiation of mouse embryonicstem cells into hematopoietic precursor cells (HPC) as showed

Fig. 2. Agarose hydrogels can induce the formation of floating aggregates or partially embedded aggregates (at the gel-liquid mediuminterphase), depending on the cell types. Finer differences also can be noticed among the floating and the partially embedded aggregates. Thedifferences can be noticed as the size of aggregates, the density of cells in the aggregates, the acellular zones within the aggregates, theextracellular matrix-like substance being formed within, around and outside the floating aggregates. With the capacity to induce suchfeatures, the agarose hydrogels prove to be excellent material for utilizing cancer cell lines much better than their utility as monolayers orsuspension cell cultures. The lower part shows the explant-like behaviors when 3D aggregates are reverted back into monolayers. However,their features/properties might differ from their original monolayer counterparts.


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by a twofold increase in the expression of surface-specificmarkers (CD34, Sca-1, etc.) and colony-forming cell (CFC)assays compared to their 2D counterparts (Liu and Roy, 2005).

Enhanced chondrogenesis as assessed by metachromasiaand expression of chondrocyte specific genes like COL II, COLX, etc. in stromal cells of human marrow was possible using 3Dculture systems. Smith et al. (2011) performed a comparativestudy between the 2D and nanofibrous architecture andbiochemical cues of 3D scaffold on osteogenesis using

embryonic stem cells. The osteogenic differentiation markerexpression is seen even after 3 weeks of culture in 3Dwithout supplements and growth factors which has a potentialfor tissue engineering. Li et al. (2002) studied the embryonicstem cells with fibrous matrix to derive hematopoietic celllineage.

In a study, the differentiation of mesenchymal stromal cellsto chondrocytes using hyaluronic acid hydrogels as 3D modelswas analyzed. It was found that the cell receptors could interact

TABLE 1. Scaffolds and matrices are of primary importance for culturing cells as 3D systems

Sl no. Matrix/scaffold classification Examples Properties Major applications

1. Extracellular matrix—natural •Decellularized tissue•Collagen•Basement membranes such as

•Maximum resemblance to thein vivo conditions

•Batch to batch variation

• Induce specific cell and tissueresponse, enhancing the nativetissue integration

laminin• Fibrin, alginates, chitosan, hyaluronic

•Risks associated with biologicalmaterial

• Impart cell differentiation capacity byproviding an environment that

acid, silk fibroin, cellulose acetate,casein, chitin, fibrinogen, gelatine,elastin, Poly-(hydroxylalkanoate)

• Favourite substrate for cellularattachment, proliferation, anddifferentiation

• Poor mechanical properties with

mimics the native ECM

variable physical properties withdifferent sources

• Immunogenic problems

2. Extracellular matrix—syntheticpolymers (bulkbiodegradable and surface

•Hyaluronic acid (HA) modified forms• Poly-ethylen glycol (PEG) modified

forms

• Possess predictable andreproducible mechanical andphysical properties

•Used as carriers for growth factors,drug delivery, gene transfection, celltherapy with hydrogels

biodegradable polymers) • Self-assembling protein hydrogels • It can be combined with • Surface modification induce the• Poly(lactic-co-glycolic acid) (PLGA) biomolecules such as growth bioactivity• Polycaprolactone (PCL) factors and antibiotics• Polyurethane• PGS

3. Biological and synthetic hybrids • Polycaprolactone-chitosan • Possess predictable andreproducible mechanical andphysical properties

• Stimulate healing injury and impartstability until the cells integrate withthe native tissue

• PLLA-Hydroxyapatite

• Enhance the biodegradability• Flexible mechanical property

• In vitro 3D models to study thedisease of organ specific

•Hydroxyapatite-bioglass-ceramic• Poly-(hydroxylalkanoate)-bioglass•Hydroxyapatite-collagen• PCL-gelatin• PCL-collagen

4. Metals •Tantalam • Lack of biological activity on thesurface materials

•Coated with collagen, RGD peptides,vibronectin, fibronectin leads toosseointegration and in vivo boneformation

•Magnesium and its alloys

•Used as implanting materials fordental and orthopedic defects

•Titanium and its alloys•Nitinol (nickel and titanium alloys)

5. Ceramics and bioactive glass •Titanium and tri calcium phosphate • Excellent osteoblast cell supportability

• Improved bioactivity with high boneingrowth observed•Hydroxyapatite and Tricalcium

phosphate •Cells infiltrate and proliferateeffectively in the porousstructures

•Bone mineralization induced•Bioactive silicate glass(SiO2–Na2O–

CaO–P2O5)•Cell delivery enhanced by surface

silanization•Hydroxyapatite and bioglass•Calcium phosphate glass• Phosphate glass

6. Carbon nanotubes areconstructed using graphiteranging from 0.4 to 2 nm

•CNT-polycaprolactone • Imparts scaffold electricallyconductive to transmits the cellto cell signals that regulatemuscle contractions

• In vitro cardiac tissue constructs•CNT-ceramic matrix

•CNT resembles native ECMproteins similar to collagen andlaminin

•Accelerate osteoblast cell growth• 45S5 bioglass-CNT

• Large surface area and highelectrical conductivity

• Enhance the restoration of lost nervefunction•CNT studded with gelatine hydrogel

•Mechanical property is foundsimilar to ECM structuralproteins

•Neural stem cell differentiation andtheir excitation

•CNT-TiO2

•Regulates the stimulating effectsbetween the scaffold andbiological cell membranes

•Generation of chondrocytes frommesenchymal stem cells from adultbone marrow

•CNT-laminin•CNT grafted with polyacrylic acid

• Embryonic stem cells to neural lineage

•CNT-TGF-b

The nature of cell behavior in the 3D systems can be influenced by the type of scaffold or matrix used and many types have been developed currently for 3D cell culture applications. The type ofscaffold or matrix will depend on the cell type being cultured and also as determined by the aim and nature of the study. The scaffolds and matrices as can be used for 3D cell culture can bebroadly classified as natural and synthetic material. These can be further grouped as natural and synthetic extracellular matrices, natural and synthetic hybrids, metals, ceramics, bioactive glass,and carbon nanotubes. Each of the scaffolds and matrices type has characteristic properties and can be used for a specific application.



with the HA better and influence the cell differentiation. Thevarious factors like biologically functional microenvironment,material chemistry, cellular interactions, and the mechanicalproperty enhanced chondrogenesis (Erickson et al., 2009).

Studies suggest that the stiffness of the 3Dmodels influencesthe differentiation of cell lineages (Rockwood et al., 2011). Theincrease in the mechanical stiffness of silk fibroin matrix filledwith a silk fibroin microparticle influences the humanembryonic mesenchymal stem cells (hEMCs) to differentiateinto osteogenic lineage. The same group also studiedchondrogenic lineage differentiation using softer matrices. 3Dmodels provide more flexibility in choosing the matrixproperty as required for certain studies. For example, the softmatrigels proved to be efficient material to obtainneuroepithelial cysts from hESCs which can differentiate intoretinal epithelium and neuronal cell lineages (Zhu et al., 2013).

Polyethylene glycol based photo polymerizing hydrogelencapsulation of embryonic stem cells lead to chondrogenicdifferentiation, a potential approach for cartilage tissueengineering. Poly-lactide-co-glycolide (PLGA) andnanohydroxyapatite together with high-aspect ratio vessel(HARV) bioreactor induced the proliferation anddifferentiation of human mesenchymal stem cells withincreased mineralization (Seong et al., 2010).

An optimal scaffold material, micro architecture and surfacenanotopography with HARV provided enhanced cellulardifferentiation to form alternative grafts for bone tissueregeneration. Microgravity rotating culture system withpolyglycolic acid scaffold is compared with 3D static culture ofmesenchymal stem cells. Chondrogenic induction was foundelevated in microgravity rotating culture as confirmed by theexpression of collagen type II and aggrecan. This approach haspotential applications in obtaining tissue engineered cartilageconstructs.

In one study, poly(L-lactic acid) and poly(glycolic acid) werefabricated to form a strong flexible porous 3D matrix andmicrogravity was stimulated using a rotating bioreactor.Embryonic stem cells when grown in this set-up along withgrowth factors and hormones differentiated into cells withtypical hepatocytes morphology, gene, and proteinexpressions. The 3D scaffolds thus obtained when transplantedinto the severe combined immunodeficient mice, remainedviable and further differentiated into matured hepatocytes invivo.

3D cell culture systems also are good sources forcontinuously obtaining a supply of specifically differentiatedcells. For example, induced pluripotent stem cells (iPSCs) andhuman embryonic stem cells (hESCs) provided an unlimitedsource of human hepatocytes due to their indefinite self-renewal and pluripotent properties. The prolonged survival ofdifferentiated cells in 3D models is a major advantage in tissueengineering which can be alternative for animal usage in drugdiscovery, gene therapy, cancer biology, therapeutics, andregenerative medicine. The collagen tissue constructs havebeen used for long term maintenance (more than 2 months) ofdifferentiated neuronal cells from umbilical cord blood (Bercuet al., 2013). The undifferentiated cells survival is also enhancedin 3D culture systems creating the best environment foroptimal differentiation. This can be useful to study the effect ofphysico-chemical reactions associated with the environmenton embryonic cells.

Drug Discovery and Pharmacological Applications

Until 1980s, animal models were widely used for drugdiscovery studies. Later, increase in drug compounds and therequired high throughput screening made the animal usagemore expensive and unethical. 3D cultures have potential togreatly improve cell-based drug screening and identify toxic and

ineffective substances at an earlier stage of the drug discoverypipeline than animal or clinical trials. Moreover, they canreduce ethically controversial animal testing (Pampaloni et al.,2009) and greatly diminish the cost and the experimentalcomplexities that involve animal models. The 3D approachwould be a simpler but an effective tool for drug genotoxicity,cytotoxicity, and for anti-cancer drug/agent discovery.

Drug testing in 2D provides very little information and oftenmisleading, whereas the 3D multicellular cell structure canbridge the gap between the conventional in vitro and the animaldrug testing models. The way cells are grown have animportant bearing on how they respond pharmacologically.Changes in the cellular response to the drugs Paclitaxel,KU174, Alimta, Zactima, Doxorubicin, etc. in 3D model wasreported (Nirmalanandhan et al., 2010). 3D cell culturesshowed differences in the efficacy on prediction of druginduced hepatotoxicity (Meng, 2010). Gel encapsulated HepG2cells were capable of metabolizing the pro-drug EFC (7-ethoxy-4-trifluromethyl) in a straight fashion over a time period,showing the optimal pharmacokinetic response of in vitrotissue model systems (Shih-Feng et al., 2010). 3D cultures ofhepatocytes or HepG2 cells were less susceptible tomethotrexate than their 2D counterparts proving their betterreliability for drug testing. Increase in drug resistance tomethotrexate was also observed in 3D cell culture of rathepatocytes (Yin et al., 2009).

The effects of anticancer drugs doxorubicin and paclitaxelwere markedly different when cells were exposed to them inthe 2D and 3D systems (Loessner et al., 2010; Millerot-Serrurot et al., 2010). An increased survival rate of HEP-G2 cellline further to anticancer drug treatment was observed whenthey were cultured as 3-D multicellular spheroids. Likewise, adecrease in the chemosensitivity of Lovo and MCF-7 3Dcultures was reported (Nakamura et al., 2003) and a reduceddrug-induced antiproliferative in 3-D models were alsoreported (Horning et al., 2008).

Apart from studies on the direct drug effects, the 3Dsystems have proved to be efficient models for studying thesynergistic effects of biologically important substances on cells.For example, it was shown that monolayers of hepatocytesshowed an increased drug resistance while the resistance didnot show any change with hormonal supplementation in themedium (Yin et al., 2009).

Tumor Models and Cancer Biology Applications

Many cell types grown as 3D tumor spheroids have threelayers, the central necrotic, inner quiescent and the outerproliferating ones that mimic the microenvironment of humansolid tumors. Several features of the in vivo solid tumors havebeen exhibited by the tumor spheroids in vitro. The similaritiesin the drug responsiveness among the tumor spheroids and theanimal models might largely be due to their similarities inenhanced cellular interactions via adhesion and secretion ofsoluble factors of tumors that lead to the low pH and hypoxia.The usefulness of 3D cell cultures for cancer research includeapproaches from signaling pathways, expression andinteractions with ECM components, cell communication,differences in cell proliferation rates, and patterns (Lee et al.,2007; Mazzoleni et al., 2009). Cancer research involving studieson biomarkers, invasion, metastasis, and tumor angiogenesishave been widely carried out (Mueller-Klieser, 1997; Bazou,2010; Liu et al., 2010).

There aremany peculiar features of cancerous cells grown in3D conditions and which are parallel to the in vivo tumors,particularly in the early events of tumor growth, before theappearance of the tumor vascularization. The hollow coresresembling the necrotic areas of the in vivo tumors can be seenin 3D cultures. Adding to this, the proliferative index of the



tumor cells are low in the three dimensional cultures. Alsothere are reports on the differential metabolism of the tumorcells between the monolayer culture and 3D culture. Whencompared with 2D cultures, the cancerous cells show adecreased sensitivity to apoptosis that is induced by radio-chemo therapy or by death receptor ligation. Likewise, thecytostatic sensitivity, interferon induced or chemotherapybased cytotoxicity are significantly lower in 3D cell cultures.

The multicellular spheroid drug resistance was found to besimilar to the in vivo tumors when exposed to alkylating agents.It was observed that mouse mammary carcinoma cell line(EMT-6) were resistant to various alkylating agents whentreated as spheroids cultures and not when treated asmonolayers(Kobayashi et al., 1993). IC50 increased 100-fold forthe anti-cancer drug paclitaxel for the spheroids of MCF-7 andDLD-1 cell lines when comparedwith their monolayer cultures(Nicholson et al., 1997).

Gene and Protein Expression Applications

Interesting facts emerge when we compare the genes and theirproducts as induced by 3D cultures. A similar observation ismade when we compare the gene and protein expressions ofcells in 3D cultures with those expressed in the natural in vivocancers. A comparison of gene and protein expressions by cellsgrown as 3D cultures and in vivo cancers is presented inTable 2.

Cell Physiology Applications

3D cell cultures help to understand better, various cellfunctions such as proliferation, adhesion, viability, morphology,microenvironment, and response to drugs.

Cell Proliferation and Cell-Cycle Studies

Investigations using three dimensional cultures of mammarycells, vascular cells, mouse fibroblasts and osteoblasts have ledto better understanding of mechanisms involved in cellproliferation. By controlling the compliance of hydrogels usingacrylamide, scaffolds differing in their elastic moduli wereestablished. Cell proliferation in these matrices was measuredand this data was used to study the mitogenesis regulation ofcell cycle in tissues. The results indicate that physiologicalstiffness is a well conserved inhibitor of mitogenesis at theelastic moduli range 600–4,300 Pa as shown by tissues isolatedfrom mammary gland, thoracic aortae, and femoral arteries ofmouse (Klein et al., 2009).

Electrospun nanostructured 3D grids made of Polyanilinewere synthesized and the morphology and viability of HeLa(Human Cervical epithelial cells) were studied at 24, 48, and72 h on the scaffolds. Viability of cells increased significantlyafter 48 h of incubation in the scaffolds (Wolun-Cholewa et al.,2013)

2D cell cultures and 3D models of Human Aortic SmoothMuscle Cells (HASMC)were studied after layering them on topand casting into 0.1% collagen gels, respectively (Li et al., 2002).TheDNA and RNA content of the cells in both the systemswasmeasured and cell growth was found to be slower in 3Dmatrix.This was supported by the flow-cytometric data whichconfirms cells in 3D matrix had more cells in G0/G1 phase andfewer cells in S phase. Thus, cells grown in matrices instead ofasmonolayers are able to survive for extended time and slowerproliferation rates. Similar results were reported for primaryembryonic rat hippocampal neurons grown in rat tail Type Icollagen (Xu et al., 2009). This study showed that 50% ofneurons entrapped in 3D collagen matrix survived while lessthan 30% survival was observed in 2D cultures on Day 21. Thiswas attributed to the property of collagen hydrogels to keep

cells hydrated and nourished for a longer time along withproviding attachment and development support. This studyhighlights the potential of 3D collagen scaffolds in studying theneuronal networks and in constructing tissue basedbiosensors. DNA microarray analyses showed that matrixgeometry alters the expression of genes involved in cell cycleregulation (Li et al., 2002).

TC-71 Ewing Sarcoma cells were cultured as 2Dmonolayersand in 3D PCL scaffolds. Cells within 3D scaffolds had asubstantially slower rate similar to cells that were grown asxenografts. Studies such as these highlight the advantages of 3Dfibers; due to their reduced pore and fiber diameter, the largesurface area allows for precise assessment of cell proliferation(Fong et al., 2013).

Matrix stiffness has been found to have an important effecton cell functions such as self renewal, proliferation, etc. In aparticular study, the stiffness of the methacrylated alginatematrix was varied and preadipocyte cells (3T3-1) werecultured on moderate, compliant, and stiff hydrogels. Cellproliferation was significantly higher in moderate and stiffhydrogels. Also, adipocytes seemed to differentiate in thematrix that has a modulus analogous to the adipose tissue(compliant type) whereas stiffer matrices promotedproliferation of cells (Chandler et al., 2011).

Cell Cytoskeleton Studies

The composition of cell cytoskeleton and the extracellularmatrix (ECM) varies between cells grown as monolayers andthree dimensional cultures. Oral squamous cell carcinomas(OSCC) cultured as monolayers and scaffold engineeredtumors expressed different levels of proteins that constitutethe cytoskeleton. A diminished expression of laminins andincreased fibronectin levels in 3D models was observed, whichis correlated to their enhanced malignancy (Fischbach et al.,2007). Fluorescence staining revealed that smooth muscle cellson 3D matrix had fewer actin stress fibers, focal adhesions(FA), and reduced cell surface area due to restriction by thesurrounding matrix.

Enhanced expression of mesenchymal characteristics thanepithelial traits was observed in the cell cytoskeleton of humanovarian cancer cell line SKOV3. In an innovative microchipcontaining cell supporting bioengineered membrane, tumorcells show increased expression of N-cadherin, vimentin, andfibronectin, which are well characterized mesenchymalmarkers. The expression of epithelial cell adhesion molecule,CD326, was lower when compared with conventional 2Dcultures (Kuo et al., 2012). Increased expression of a5b1integrin fibronectin receptor and N-cadherin was observed intransformed epithelial cells; only cells grown on 3D scaffoldswere seen to attain metastatic capability (Fischbach et al.,2007).

Apoptosis Studies

Interactions of the cell cytoskeleton and the ECM haveimportant effects on various activities, including inhibition oracceleration of apoptosis. Among the early studies, interfacebetween b1 integrins of cells and ECM receptors was shown torepress apoptosis of mammary epithelial cells (MEC) grown inexogenous basement ECM. Cells plated on tissue cultureplastic showed signs of apoptosis (DNA laddering, nuclearcondensation and expression of SGP-2, a gene related toapoptosis) after 4–5 days of seeding. But cells in ECM werehealthy even after 10 days since plating. On 12th day, whenantibodies were introduced against b1 integrins, MECs in ECMexhibited apoptotic characteristics similar to cells grown on 2Dconditions. Studies in transgenic mice showed that disruptingthe integrity of basement ECM using metalloproteinase



stromelysin 1 induced apoptosis in 10–15% of MECs, similar toresults obtained in 3D cultures.

Cancer therapy of malignant breast cancer cells was studiedin laminin rich ECM (lrECM) cultures with an aim to address theaberrant expression of b1 integrin.When b1 integrin inhibitoryantibody AIIB2 was administered to both malignant tumor likecolonies in vitro, and non-malignant cells, inhibition of b1integrins the malignant cells was seen. The cells showedincreased apoptosis, decreased proliferation and changes intissue organization. The non-malignant acinar structureshowever, did not show any significant effects to AIIB2.With the

authors also confirming these results in mouse models, b1integrin inhibition opens up a new therapeutic approach;establishing ECMbased 3D cell cultures as successful models tostudy tumor response to therapy (Park et al., 2006).

Cell Adhesion and Signaling Studies

In multi-cellular organisms, cells grow in a complexenvironment, surrounded by various other cells and a well-structured matrix. Communications and adhesion with thesediverse elements appear to control their behavior. Emerging

TABLE 2. A comparison of the gene and protein expression between 3D cultures of 30 different cell lines (27 cancerous and 3 non-cancerous) and their tissue specificin vivo cancers reveal shared gene and protein expressions

Cell lines

In vitro—3D cell cultures In vivo—cancerous

Genes and proteins lessexpressed

Genes and proteins overexpressed

Genes and proteinsless expressed

Genes and proteins overexpressed

Cancer cell linesOSCC3 NIL IL-8, bFGF, and VGFR NIL VGFR, IL-8, and bFGFT4-2 NIL b1-Intergrin and VGFR, ERK,

MAPK, PI3KNIL b1-Intergrin and VGFR, ERK,

MAPK, PI3KMCF-10A NIL BIM and BCL2 BIM and Rb BCL2, Cyclin D1, ERBB2,

CSF1R, SRC, and IGF1RCaco2 LKB1 LKB1 NILHCC1569, CAMA-1,BT-549, ZR-75-1, andMDA-MB-468

PTEN AKT PTEN AKT

1-LN NIL PAP NIL NILU4F NIL Urogential sinus dervived

growth inhibitory factorNIL NIL

PC3 NIL Cytokeratin, CD44, E-cadherin. PAP and PSA

NIL PSA

DU 145 NIL PAP, EGFR, PSA,transforming growth factorb, collagen IV, cytokeratin,

CD44 and E-cadherin

NIL EGFR and PSA

LnCaP NIL Cytokeratin, CD44, and E-cadherin

NIL NIL

MDA-MB-415 NIL pAkt NIL pAktSKBR-3 and AU565 E-Cadherin ErbB2 E-Cadherin NILBT-474, HCC1569,MDA-MB-361,MDA-MB-453, ZR-75-1,BT-483, and UACC-812

NIL ErbB2 NIL NIL

Neuroblastoma culture NIL HSP 90-b, HSC 71,transketolase, HSP 60,pyruvate kinase M1/M2,adenyl CAP-1, tubulin

b-2chain, a enolase, actin,septin 2, PSAT, PGAM1,TPI, TCTP, CRABP1,cofilin, pin-1, and

thioredoxin

NIL NIL

HepG2 b-Catenin CYP1A1, CYP2C9, CYP3A4,integrins, cadherins,

catenins, members of theCD44 family, b1-integrin,

EGFR, and VEGF

NIL EGFR and VEGF

A549 NIL Ki-67, MMP-9 NIL MMP-9Non-cancer cell linesPrimary fallopian tubesecretory epithelial cells(FTSECs)

NIL Mucin 9, PAPPA NIL NIL

Neonatal rat cardiacventricle cells (NRCVCs)

ANP and CARP BMP-2, ID3, and THBS1 NIL NIL

ImM10 (as induced bysupplementing mediumwith specific growthfactors)

FGFL5, SFRP2, RAX, Hey1,NTN1, EGF, FLNA, BDNF,

and ARNT2

Nestin, SOX2, PAX6, MSI1,GNL3, PAX6, CCNS1,

SIX, NPTX, chemokine (C-X-C motif) ligand 1, DLL1,

MEF2C , BMP2, PTN,OPN4, CABP5, RHOK,CRX, GRM6, OPN1MW,

MAP1, and CRX

NIL NIL

Non-specific CDH1 and E-cadherin CDH1 andE-cadherin

Cells in the comparative group (cancerous and non-cancerous) grown as 3D cultures over-expressed 77 genes and 14 genes were less-expressed by them. Cancerous cells in the comparativegroup over-expressed 49 genes of which 16 were also found to be expressed in in vivo cancers. The genes and proteins as commonly expressed by the 3D cell cultures and in vivo cancers arehighlighted.



studies highlight the usefulness of 3D cultures for studyingthese intricate details. While cell morphology differedsignificantly between conventional monolayers and laminin richECM (lrECM) cultures of A549 and UT-SCC15 cell lines, thedoubling time of the cells were similar for the first 4 days.During this time period, the resistance of cells to increasingsingle doses of X-rays and cisplatin was studied. Also, wholegenome gene expression analysis of cells as monolayers and 3Dcultures revealed differential expressions of many genesinvolved in cell adhesion, biological adhesion, immune responseand tissue development. An important contribution of thisstudy was the finding that inspite of no prominent difference ingene expression of DNA repair mechanisms (e.g., increasedexpression of TXNIP, Theoredoxin Interacting Protein) inboth 2D and 3D culture systems, 3D cultures exhibitedincreased resistance to the radiation and cytotoxic drug.Though a correlation was established between increasedresistance in 3D culture systems and the enhanced expressionof genes involved in the above mentioned processes (e.g.,increased CEACAM1 expression in 3D cultures), theexact mechanism of this is yet to be studied (Zschenker et al.,2012).

Primary cardiac cells grown as 2D and 3D cultures werecompared for expression profiles, tissue markers, multicellularorganization pathways, etc. It was reported that in 3D cultures,a multicellular organization was formed with up-regulation ofendothelial migratory pathways, decrease in Nppa and Ankrdproteins and an increase in sensitivity to tri-idothyronine. It wasshown that 3D aggregates of cardiac cells exhibitedphysiological alterations of cellular phenotype that consisted ofcardiac ventricular tissue formation and maturation (Akinset al., 2010).

Focal adhesion proteins are multi-protein assemblies andmediate cell signaling, force transduction, and adhesion tosubstrate. The role of FAs in 3D cell culture systems wereanalyzed and it was reported that FAs including vinculin, talin,FAK, etc. are found in the cytoplasm of 3D cultured cells. Inmatrix-embedded cells, these proteins regulate the cell speedand persistence there by affecting the protrusion activity anddeformation of matrix. The study also reveals that themembrane protrusion affects the cell motility in 3D matrix(Fraley et al., 2010). Various other studies have observed that3D cultures show distinct differences from their 2Dcounterparts in terms of cell signaling pathways. It has beenreported that 3D collagen matrix can reduce the expressionof PIK3/Akt pathway which was consistent with theirreduced migration speed and cell proliferation rates (Fallicaet al., 2012). When KRAS wild-type cell line CACO-2 is grownin laminin-rich-extracellular matrix, the EGFR inhibition wasfound to be lower when compared with 2D monolayers (Lucaet al., 2013).

Also, signaling pathways are of growing interest to cancerbiologists and clinicians due to their role in tumor developmentand influence on therapy modules. For example, the insulin-likegrowth factor-1 receptor (IGF-1R)/mammalian target ofrapamycin (mTOR) signaling cascade is an actively investigatedpathway in Ewing Sarcoma patients due to its role inchemotherapy resistance. When 2D EWS monolayers and 3Dpoly(capro lactone) engineered tumors were compared, cellsfrom 3D cultures showed high amount of phosphorylated IGF-1R, indicating abnormal expression of IGF-1R/mTOR genes(Fong et al., 2013).

Cell Motility Studies

The movement of cells into or within biomaterial scaffolds is aprerequisite which enables tissue repair and regeneration.There is a requirement for engineered biomaterials in whichthe three dimensional cell migration can be optimized.

Constructing such a scaffold will entail thorough understandingof the parameters that control cell migration in an artificiallysynthesized scaffold. Two different modes of cell migrationthrough the ECM, namely proteolytic (mesenchymal) migrationand non-proteolytic (amoeboid) migration has been studied(Ehrbar et al., 2010). The 3D scaffold constructed by theauthors is poly(ethylene glycol)(PEG)-based macromersformed via the transglutaminase (TG) factor XIII. The stiffnessof the gel seems to be the deciding factor for the proteolyticdegradation of murine preosteoblasts MC3T3-E1by MMPs. Onthe other hand, softer gels showed migration of cells withoutmatrix degradation, suggesting that amoeboid type of migrationoccurs through the pores of the scaffold. Further, the matrixcomposition had similar effect on the formation of cellularnetworks in vitro and tissue regeneration studies in rat modelswith calvarial defects.

Similar observations were made with collagen hydrogelswhose compliance was manipulated by the addition of agarose(Ulrich et al., 2010). Higher percentage of agarose in thecollagen matrix (higher stiffness) showed that U373 malignantglioma cells had a greater percentage of mesenchymal-amoeboid transition (MAT) when compared to cells in purecollagen matrix or matrix with low agarose. Thus, with theincrease in collagen stiffness, ECM remodeling and directionalmovement of cells was reduced. All these results highlightvarious parameters of the ECM as can be induced in 3D thatseems to control cell behavior.

Microenvironment Studies

The environment surrounding the cells plays a key function indeciding their differentiation fate and functions. This has beenextensively studied in the tumor microenvironment, whichalong with its many aberrations and abnormal conditions,stresses the role of a healthy milieu in shaping the cell behavior.

Important changes were reported when oral squamous cellcarcinoma (OSCC) cells were cultured on synthetic polylactide-co-glycide (PLG) scaffolds, in vitro and in vivo. 3D PLGcultures showed increased secretion of vascular endothelialgrowth factor (VEGF), basic fibroblast growth factor (FGF) andInterleukin 8 (IL-8) by 2-, 23-, and 98-fold, respectively. Sincethe literature reports that these factors are regulated throughhypoxia, the monolayers were exposed to 0–2%O2. It led to anoverall increased expression of VEGF and FGF which wassimilar to in vivo tumors. The results also indicated that, whilecertain factors of microenvironment (hypoxia) controlexpression of VEGF and FGF, certain other features found in3D cultures but lacking in 2Dmodels, are important for the IL-8expression. By calculating the percentage of cumulativesecretion scores of these pro angiogenic factors, they provethat IL-8, not VEGF, has a most important role in angiogenesis.This changed the previously held notion from 2D cell culturedata regarding the role of IL8 for its angiogenic potential(Fischbach et al., 2007).

Various 3D models have different characteristics which givethem a distinct edge. For example, PLG engineered tumorswere closer to in vivo cancers than standard 3DMatrigel growntumors, as the level of IL-8 expression was reduced in thesetumors. This shows that Matrigel grown tumors lack certainmicroenvironmental factors that the engineered tumors areprovided with (Fischbach et al., 2007). Research data seem tostress upon the importance of using appropriate 3D culturemethod depending on the focus of the study. For example,development of organotypic cultures and correlation studiesbetween morphology and genotypic expression can be doneefficiently using 3D “on-top” assay (Lee et al., 2007).

The role of matrix metalloproteinase in controlling the cell-to-cell and cell–ECM organization was analyzed (Aubin et al.,2010). The activity of MMP in the 3T3 fibroblast cells cultured



in micropatterned 3D GelMA hydrogels was inhibited usingdoxycycline. It was found that ECM remodeling was mediatedby the MMPs and they have an important role in alignment andelongation of cells.

Collagen I hydrogel scaffolds with varying degrees ofcompliance were chosen as 3Dmatrix to culture breast cancerMDA-MB-231 cells. HIF-1a protein was expressed on day 1itself, with an increase observed on day 5. As HIF-1a activatesVEGF-A, the later was found to accelerate progressively afterday 3. Both the levels reduced after day 7, indicating thehypoxic death of cells due to lack of oxygen and nutrientavailability. The authors indicate that this 3Dmodel promotes atumor microenvironment that is analogous to the pre-vascularized stages of in vivo tumor progression (Szot et al.,2011). Similarly, when pre-adipocytes (3T3-L1) were culturedon alginate beads, stiffer matrices promoted angiogenesis aswitnessed by upregulated VEGF levels (Chandler et al., 2011).

Cell Morphology and Tissue Architecture Studies

Distinct differences have been noted in the morphology of cellswhen grown as monolayers and as three dimensional cultures.A panel of breast cancer cell lines exhibited non-distinctmorphology while culturing them as monolayers. But as 3Dcultures on top of lrECM, they adopted morphologies whichcan be classified into four types. Phase contrast and confocalmicroscopy examination with phalloidin staining revealed thedifferences between round, mass, grape-like, and stellatemorphologies. Also, a correlation between the cellmorphology and factors like tissue invasiveness, a differentialability to metastasize were arrived at. For example, cell linesthat formed grape-like morphology (AU565, CAMA-1, MDA-MB-361, etc.) formed loosely associated colonies with reducedcell-to-cell adhesion which might explain their highly metastaticpotential as the tumor progresses (Kenny et al., 2007).

Studies have observed that geometry of scaffolds has animportant effect on cell behavior, which can be related to theimpact of tissue architecture on cell activities in vivo. Fourscaffolds with different orientation of syntheticpolycaprolactone (PCL) fibers, namely: basic (B), basic-offset(BO), crossed (C), and crossed-offset (CO) were constructed.Bone marrow mesenchymal stem cells (MSC) isolated fromrats were incubated with the synthesized scaffolds and checkedfor tissue engineering applications, cell proliferation, andalkaline phosphatase (ALP) activity after 7, 14, and 21 days. Theresults show that an offset in scaffolds gave more surface areafor cells to attach on the direct flow path of media thusaccounting for high cell proliferation rates in BO and COscaffolds. Further, Basic (B) scaffold gave more surface area forcells to attach, resulting in more cell proliferation. Highest rateof differentiation was supported by crossed (C) scaffolds(Yilgor et al., 2008; Klein et al., 2009).

When a renal adenocarcinoma cell line, RENCA wasencapsulated in agarose macrobeads, distinct changes incellular morphology were observed. While cells in monolayerswere epithelioid shaped, the surviving cells in macrobeadswereeither large and irregular with a high cytoplasm-to-nuclearratio, or small and round with a much lower ratio ofcytoplasmic-to-nuclear volume. These cells developed intolarger, metabolically active and viable tumor colonies (Smithet al., 2011). MDA-MB-231 human breast cancer cells werecultured on Collagen I hydrogels and it was observed that thecells took up a stellate, elongated morphology withdisorganized nuclei by day 3. These cells proliferatedthroughout the hydrogel, exhibiting invasive processes andaggregation into clusters depicting cell–cell and cell–matrixinteractions (Szot et al., 2011).

Cell and ECM alignment is an important factor in definingthe biological and mechanical role of cells. In micropatterned

cell-laden 3D gelatin methacrylate (GelMA) hydrogels createdby photolithographic techniques, the morphology of NIH 3T3were studied (Aubin et al., 2010). The authors observedthat cell alignment and elongation in patterned gels aredependent on various parameters such as cell and hydrogelconcentrations, culture time, micropattern width, inherentpotential of cells to align into organized tissues in vivo, etc.

Drug Response Studies

When malignant cells are exposed to drugs, they showenhanced resistance than normal cells. Drug screening studiesrequire an authentic in vitro model that can effectively mimicthe tumor tissues and help in measuring their in vivo drugresponse. In this context, researchers have often observed that2D cell cultures fall short of this requirement. Oral squamouscell carcinomas engineered in 3D poly lactide-co-glycide (PLG)and as 2D monolayers were treated with the cytotoxic drugPI3-kinase inhibitor LY294002. The monolayers were sensitiveto the drug, whereas 3D tumors with their microenvironmenthad significant resistance (Fischbach et al., 2007).

Apart from being screened for their therapeutic potentials,cytotoxic drugs are used in cancer research to learn howsignaling pathways and alternate survival mechanisms areadopted by tumors to evade regulatory checks (Pickl and Ries,2009; Weigelt et al., 2010). In one such approach, HumanEpidermal Growth factor Receptor type 2 (HER2) moleculeswhich is over expressed in breast cancers was studied. In spiteof various HER2 targeting agents available in the market, thetreatment has not been effective, due to resistance of tumorsto this drug. It requires more studies with models that canreplicate themolecularmechanisms as seen in in vivo tumors tounderstand their resistance, to plan a therapeutic strategy.

Breast cancer cell lines with similar genetic profiles (AU565,SKBR3, HCC1569, and BT549) were grown as conventional2D monolayers and as 3D cultures on lrECM and wereexposed to Trastuzumab, Pertuzumab (monoclonal antibodiestargeting HER2), Lapatinib (dual inhibitor of HER2 and EFGR),the b1 integrin inhibitory rat monoclonal IgG1 antibody AIIB2along with required controls. While all the cell lines possessingthe target molecules showed increased resistance in 3Dcultures when compared to their 2D counterparts, differencesbetween the responses of each cell line to the drugs in bothculture conditions were also observed. This finding suggeststhat each cell line may exhibit different signal transductionpathways when cultured on lrECM (Weigelt et al., 2010). Suchfindings provide us with insights into the specific requirementsfor each of the cancer types, as applicable for specific studies.

TC-71 EW cells responded differentially to doxorubicinwhen cultured as 2D monolayers and as PCL scaffoldengineered tumors. 3D tumors were highly resistant with anIC50 of 2.738mM whereas monolayers had an elevatedsusceptibility to the drug with an IC50 of 0.0122mM (Fong et al.,2013). Resistance to chemotherapeutic drugs such as Cisplatin,Gemcitabine, 5-Fluorouracil and Camptothecin in differentNSCLC cell lines varied between the 2D cultures and their 3Dcounterparts; the 3D spheroids showed higher IC50 values than2D cells. Anti-cancer drug induced apoptosis studies revealedhigher expression in 2D models of H1650 and H460 cell linesthan in the 3Dmodels. Anitapoptotic Bcl-2 mRNA expressionswere significantly lower in 2D systems than the 3D systems(Godugu et al., 2013).

3D models have another distinct edge over the 2Dmonolayers in drug toxicity studies; due to their lowproliferation rate, they can be effectively utilized for studyinglong term effects of drugs. This is not feasible in 2D models asconfluency limits the duration of culture. Consequences ofexposure to varied concentration of doxorubicin for differentduration were successfully estimated in 3D PCL engineered



EWS cells (Fong et al., 2013). This will be particularly useful toelucidate the antineoplastic effects of drugs in the time framesset for targeted therapies.

Behavior as Co-Cultures

Despite advancement in cell culture techniques, successfultransition of the results from research to the clinic is notefficient in all cases. Various studies with mammary epithelialcells in 3D culture systems stress the influence of themicroenvironment on the cell behavior (Boudreau and Bissell,1988; Howlett et al., 1995; Weaver et al., 1997; Gudjonssonet al., 2002). 3D cultures of homogenous cell types have notonly provided insights to tissue architecture and biochemicalcues on cell functioning, also have given us an importanttechnological edge to establish heterogeneous cell line co-cultures. This resulted in a means to better understanding thecomplexity prevailing in a complete organism (Schmeichel andBissell, 2003). In two such pioneering studies, humankeratinocytes on dermis and fibroblasts embedded withincollagen gels gave rise to satisfactory organotypic cultures ofthe skin. These models also successfully replicated skin in vivofunctioning. Such co-culture models have been utilized toexplore various factors governing cell behavior (Fusenig andBoukamp, 1998; Bagutti et al., 2001) as well as for applicationsthat include drug testing and grafting/transplantations. Oneother most studied organotypic culture is that of the mammarygland which has played a key role in understanding the role ofmyoepithelial–luminal epithelial interaction in the process ofmalignancy (Sternlicht et al., 1997; Man et al., 2002).

Another facade in organotypic models are the mousexenograft transplants of 3D cultured cells to study the effect ofmicroenvironment on tumor progression. Several authorsstress that promotion of epithelial cells into malignant stage isindeed supported by the conditions in stroma and themicroenvironment it provides. In a particular study, prostatederived fibroblasts (normal and tumor derived) were mixedwith prostatic epithelial cells (normal and immortalized by SV-40) in collagen gels and transplanted behind the renal capsule ofathymic mice. The fibroblasts and epithelial cells in normal orcancerous condition were not able to induce tumor formationindividually. Malignancy was induced only when immortalizedepithelial cells were grafted along with cancerous fibroblasts(Olumi et al., 1999). Similar co-culture studies using cell typessuch as mammary gland have highlighted the importance of thestromal cell ability to induce tumors (Parmar et al., 2002).

Conclusion

With the advent of 3D cell cultures, in vitro studies are nowcloser to animal models in many aspects. They offer biologicallysuperior structures which can be utilized to study complexinteractions that were not possible with 2D cultures. 3Dcultures can efficiently be used to understand complex studiesfor basic and applied research. The dynamic advancements inthe instrumentation technology and material sciences haveaugmented the parallel increase in complexity in cell culturetechniques. 3D cell cultures have a myriad of applications; fordrug discovery, pharmacological studies, understanding cellphysiology, gene and protein expressions, cancer research,tissue engineering and also for increasing the biotech industryproductivity.

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3d cell culture systems: advantages and applications · department of human genetics, faculty of...

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