State of the art paper2D and 3D cell cultures – a comparison of differenttypes of cancer cell culturesMarta Kapałczyńska1, Tomasz Kolenda1,2, Weronika Przybyła1, Maria Zajączkowska1, Anna Teresiak1,Violetta Filas3, Matthew Ibbs3, Renata Bliźniak1, Łukasz Łuczewski4, Katarzyna Lamperska1Laboratory of Cancer Genetics, Greater Poland Cancer Centre, Poznan, PolandPostgraduate School of Molecular Medicine, University of Warsaw, Warsaw, Poland3 Department of Tumour Pathology, Greater Poland Cancer Centre, Poznan, Poland4 Department of Head and Neck Surgery, Greater Poland Cancer Centre, PoznanUniversity of Medical Sciences, Poznan, Poland1 2 Submitted: 9 February 2016Accepted: 27 June 2016Arch Med Sci 2018; 14, 4: 910–919DOI: 2016 Termedia & BanachAbstractCell culture is a widely used in vitro tool for improving our understandingof cell biology, tissue morphology, and mechanisms of diseases, drug action, protein production and the development of tissue engineering. Mostresearch regarding cancer biology is based on experiments using two-dimensional (2D) cell cultures in vitro. However, 2D cultures have many limitations, such as the disturbance of interactions between the cellular andextracellular environments, changes in cell morphology, polarity, and method of division. These disadvantages led to the creation of models which aremore closely able to mimic conditions in vivo. One such method is three-dimensional culture (3D). Optimisation of the culture conditions may allow fora better understanding of cancer biology and facilitate the study of biomarkers and targeting therapies. In this review, we compare 2D and 3D culturesin vitro as well as different versions of 3D cultures.Key words: co-culture, cell culture methods, 3D culture, 2D culture, cancerresearch.Cell cultures as a research modelStudies on the mechanisms underlying the formation, function andpathology of tissues and organs are manageable largely due to the useof cell culture systems and animal models [1].Harrison carried out the first cell cultures in 1907 during researchinto the origin of nerve fibres [2]. Since then, the method has been improved and used to observe the growth and differentiation of cells outside the body [3, 4]. Nowadays, experiments can be conducted usingprimary cells isolated directly from the donors’ material or using established cultures deposited in cell banks [5]. Primary cultures are isolatedfrom living organisms and usually contain populations of different celltypes present in the source tissue. In this case, it is important to isolatethe correct cell type [5]. Characteristic features of primary cell lines are:i) difficulties with isolation and ii) short life span. On the other hand,they closely mimic the in vivo genetic features of tumours and thus makeit possible to perform some functional experiments. An alternative option is the use of an established cell line. Bioresource centres, such asCorresponding author:Tomasz Kolenda,Katarzyna LamperskaLaboratoryof Cancer GeneticsGreater PolandCancer Centre15 Garbary St61-866 Poznan, PolandPhone: 48 61 885 06 68E-mail: [email protected], [email protected]

2D and 3D cell cultures – a comparison of different types of cancer cell culturesIn adherent 2D cultures, cells grow as a monolayer in a culture flask or in a flat petri dish, attached to a plastic surface [10].The advantages of 2D cultures are associatedwith simple and low-cost maintenance of the cellculture and with the performance of functionaltests. Unfortunately, adherent cultures also havenumerous disadvantages. First, 2D cultured cellsdo not mimic the natural structures of tissues ortumours (Figure 2 A). In this culture method, cellcell and cell-extracellular environment interactionsare not represented as they would be in the tumourmass. These interactions are responsible for celldifferentiation, proliferation, vitality, expressionof genes and proteins, responsiveness to stimuli, drug metabolism and other cellular functions[9, 11–13]. After isolation from the tissue and transfer to the 2D conditions, the morphology of the cellsis altered, as is the mode of cell division. The lossof diverse phenotype is also a result of 2D culturing [14, 15]. The changed morphology of the cellscan affect their function [16, 17], the organizationof the structures inside the cell, secretion and cellsignalling [18, 19]. Due to disturbances in interactions with the external environment, cells growingadherently lose their polarity [20], which changesthe response of those cells to various phenomena,such as to apoptosis [21, 22]. Another drawback of2D culture is that the cells in the monolayer haveunlimited access to the ingredients of the mediumsuch as oxygen, nutrients, metabolites and signalmolecules. For cancer cells in vivo, the availabilityof nutrients, oxygen, and so forth, is more variablebecause of the natural architecture of the tumourmass [9]. Furthermore, it has been observed thatthe 2D system changes the gene expression andsplicing, topology and biochemistry of the cell[23–26]. In addition, adherent cultures are usuallymonocultures and allow for the study of only onecell type [27], which results in a lack of tumourABthe ATCC (American Type Culture Collection), offercharacterized models of various types of cancercell lines that are routinely used in research [6].Cell cultures make it possible to understand cellbiology, tissue morphology, mechanisms of diseases, drug action, protein production and the development of tissue engineering [7]. They are often usedin the preclinical research of many drugs, in cancerresearch, and in studies on gene function [5].The choice of the most appropriate cell culturemethods in the area of cancer research may allow us to better understand tumour biology, andhence to optimize radio- and chemotherapy, oreven to find new treatment strategies [8].The cultures can be carried out under adherent conditions wherein the cells are attached toa glass or plastic dish or in a suspension, which insome cases (e.g. cultures of lymphocytes) corresponds more faithfully to the natural environment[6]. The most commonly used type of cell cultureis the 2D model, but recently the 3D culture method has been gaining in popularity (Figure 1) [9].Depending on the type of culture chosen, cell behaviour differs in many aspects [7].2D culturesCell-to-cell contact surfaceCell andscaffoldcontactsurfaceCell and Petri dishcontact surfaceCell and medium contact surfaceScaffoldCCell and mediumcontact surfaceCell ell-to-cellcontact surfaceFigure 1. Types of cell culture methods commonly used in research studies. A – Cells flattened ina monolayer on the bottom of the culture vessel.They are in contact with the culture vessel, neighbouring cells, and the culture medium. B – Cellsattached to a scaffold are in contact with the scaffolding, neighbouring cells, and the culture medium. C – A group of cells suspended in the culturemedium or cultivated in gel-like substance; thecells are in contact with neighbouring cells andwith the culture mediumArch Med Sci 4, June / 2018 911

M. Kapałczyńska, T. Kolenda, W. Przybyła, M. Zajączkowska, A. Teresiak, V. Filas, M. Ibbs, R. Bliźniak, Ł. Łuczewski, K. LamperskaABCDEFFigure 2. FaDu cell line cultured under various conditions. The FaDu cells were maintained in adherent conditionswith standard medium (10% FBS) and next detached and placed as single cells in different (A–F) culture conditions in standard medium. A – flattened cells growing as a monolayer under 2D conditions (scale bar represents100 µm); B – 3D structures in soft agar, single cells suspended in a gel are visible (scale bar represents 200 µm);C – adherent colonies formed between layers of soft agar (scale bar represents 200 µm); D – 3D structure formedon non-adherent plate (scale bar represents 100 µm); E – tissue-like structures formed by attached single spherescultivated on ultra-low attachment plates (scale bar represents 200 µm); F – cells (red) cultured using 3D scaffoldsystem with visible membrane pores (scale bar represents 100 µm)microenvironment, or niches, which in vivo are required by cancer-initiating cells [28, 29].Owing to the many disadvantages of 2D systems, there was a need to find alternative models,better able to mimic a natural tumour mass, suchas 3D culture systems (Table I).3D culturesOne of the first three-dimensional cultureswas made in soft agar solution, and was carried912 out by Hamburg and Salmon in the 1970s [30].Since then, striking similarities between the morphology and behaviour of cells growing in a tumour mass and in cells cultured under 3D conditions have been well described and documented[9, 31].Due to the method of preparation, 3D models can be divided into: i) suspension cultures onnon-adherent plates (Figure 2 C); ii) cultures in concentrated medium or in gel-like substances (Fig-Arch Med Sci 4, June / 2018

2D and 3D cell cultures – a comparison of different types of cancer cell culturesTable I. Comparison of 2D and 3D cell culture methodsType of culture2D3DRef.Time of cultureformationWithin minutes to a few hoursFrom a few hours to a few days[11, 34, 57]Culture qualityHigh performance, reproducibility,long-term culture, easy to interpret,simplicity of cultureWorse performance and reproducibility,difficult to interpret, cultures moredifficult to carry out[12]In vivo imitationDo not mimic the natural structureof the tissue or tumour massIn vivo tissues and organs are in 3Dform[35]Deprived of cell-cell and cellextracellular environment interactions,no in vivo-like microenvironment andno “niches”Proper interactions of cell-cell andcell-extracellular environment,environmental “niches” are created[13, 28, 29,36, 37]Changed morphology and way ofdivisions; loss of diverse phenotypeand polarityPreserved morphology and way ofdivisions, diverse phenotype andpolarity[1, 14–17,20, 38]Access toessentialcompoundsUnlimited access to oxygen, nutrients,metabolites and signalling molecules(in contrast to in vivo)Variable access to oxygen, nutrients,metabolites and signalling molecules(same as in vivo)[10, 46]MolecularmechanismsChanges in gene expression, mRNAsplicing, topology and biochemistryof cellsExpression of genes, splicing, topologyand biochemistry of cells as in vivo[23–26,42–45]Cost ofmaintaininga cultureCheap, commercially available tests andthe mediaMore expensive, more time-consuming,fewer commercially available tests[8, 48, 58,75]CellsinteractionsCharacteristicsof cellsure 2 B) and iii) cultures on a scaffold (Figure 2 E).All of these models are characterized in Table II.The concept of 3D spheres is based on the creation of spheroid structures in which cells form various layers. This structure mimics the physical andbiochemical features of a solid tumour mass. Morphological analysis of 40 tumour cell lines (originating from: glioblastoma, astrocytoma, Wilms’tumour, neuroblastoma, head and neck squamouscell carcinoma, melanoma, lung, breast, colon,prostate, ovarian, hepatocellular and pancreaticcancers) cultured in 3D spheroid conditions led tothe identification of three distinct groups according to the architecture of spheroid shapes: i) tightspheroids, ii) compact aggregates and iii) loose aggregates [32, 33]. Some cells under non-adhesiveconditions display reduced cell-cell and cell-matrixinteractions, lose their anchorage, escape fromanoikis, divide and create spheres [34].Cells from the donor’s tissues are cultured inmulticellular, three-dimensional structures, imitating the architecture of the parental tissue moreaccurately than is possible in 2D models (Figure 3)[35]. This feature of 3D models is the result of theproper cell-cell and cell-environment interactions,created in order to obtain imitation of tissue structure. Cells can receive stimuli from the local environment, as happens in vivo [36, 37]. Moreover, in3D cultures, the morphology and polarity of thecells are maintained, and they can be restored tocells previously cultivated in 2D [1, 15, 38]. Furthermore, in some 3D systems, e.g. acinar-likespheroids, specific internal architecture with lumen formation is observed. This is the result ofcell apoptosis in the central part of the spheroids.Cell proliferation depends on cell location and ishigher in the peripheral part of the 3D structures[39–41]. Another important attribute of 3D cultureis its similarity to cells growing in vivo in terms ofcellular topology, gene expression, signalling andmetabolism [42–47].All these features create a specific platformwhich can be used for the study of the biologyof cancer-initiating cells, invasion and metastaticprocesses, as well as for drug testing or for testingthe response of cells to irradiation.3D suspension culture systems are widely usedas a model in studies, e.g. for increasing the population of cancer-initiating cells. This method allows for simple and low cost biological research[37, 48]. The spheroids obtained from oral cancercell lines show an increased proportion of cancer-initiating cells, probably due to the epithelial-mesenchymal transition process occurringunder 3D conditions. The spheres exhibit loss ofE-cadherin expression, and overexpression of fibronectin, Sox2, Oct4 and Nanog. Expression ofputative stem cell markers such as CD133 andALDH also occurs. Sphere-related enrichment ofthe cancer-initiating cell population is also whya lower number of cells, derived from the spheres,is needed to generate a tumour in xenograft mice,compared to parental cells [37]. It has been observed that the number of spheres is reduced withArch Med Sci 4, June / 2018 913

M. Kapałczyńska, T. Kolenda, W. Przybyła, M. Zajączkowska, A. Teresiak, V. Filas, M. Ibbs, R. Bliźniak, Ł. Łuczewski, K. LamperskaTable II. Characteristics of different 3D cell culture methodsType of 3DsystemDescription of cell es on nonadherent plates Single cells are seededon non-adherent plateswith medium 3D structures can beobserved after 3 days ofculture Simplicity, easiness andspeed of conductingculture Bacterial plates ornon-adherent cultureplates can be used butonly for some cell lines Cells can be easily extracted from the mediumand used for furtherexperiments Some cell lines needexpensive plates coatedwith specific materials,for example polystyreneor covalently boundhydrogel, because ofstrong adhesion abilitiesof cells Formation of aggregates of cells as a resultof cells’ movement inmedium[8, 48,58, 59]Cultures inconcentratedmedium or ingel-likesubstances Single cells grow inmedium containingsubstances with gellingproperties: i) dissolvedlow-melting agarose withcell medium is pouredon plate and incubateduntil solidifying to obtainthe first, lower layer;the top layer consisting of agarose and themedium with single cellsis added; ii) the cells areflooded in Matrigel (multiprotein hydrogel) 3D structures can beobserved after 7 days ofculture Soft agar allows to studyboth the growth ofa single cell regardlessof attachment and thephenomenon of escapefrom anoikis Cells cultured in Matrigelcan be easily recoveredfor further analysis Cells in Matrigel havethree-dimensional interactions with the localenvironment and formtissue-like structures Used to study the aggressiveness of the cellsand their potential formetastasis Difficulty in obtainingspheres for certainlines, inconvenient andtime-consuming preparation of the two layers ofagar and requirement oflong-term cultures Low repeatability of theresults The difficulty of extracting cells from the agarand immunofluorescencestaining of spheres, Materials constitutingthe Matrigel containendogenous bioactive ingredients that influencethe structure formation[7, 48,58, 59,75–81]Cultures onscaffold The cells can migrateamong fibres and attachto the scaffold, made ofbiodegradable materialsuch as silk, collagen,laminin, alginate, and fillthe space among fibres,grow and divide System is compatiblewith commercially available functional tests, aswell as with DNA/RNAand protein isolation kits Easy to prepare forimmunohistochemicalanalysis Cells attached to thescaffolds flatten andspread like the cellscultured under adherentconditions Scale of scaffolds and topography of cell distribution may cause variousbehaviour of the cell Materials used toconstruct the scaffoldmay affect the adhesion, growth and cellbehaviour Cell observation andcell extraction for someanalyses are restricted[7, 8, 37,82–90]the passage of time, and that the percentage ofALDH /CD44 cells decreases during the cultureperiod, which may indicate an ongoing differentiation process [48]. Campos et al. observed thatALDH /CD44 cells create more orospheres, havehigher proliferation rates and are more resistantto anoikis than ALDH-/CD44- cells growing in suspension conditions. This is probably caused by secreted endothelial factors which activate PI3k-Aktsignalling, which in turn influences cells obtainedfrom primary and metastatic cancers differently[49]. Sphere formation assays (in suspension or914 soft-agar cultures) are commonly used and inexpensive assays for evaluating the role of examined genes in self-renewal and maintenance oftumour stemness [50].It is not surprising that tumour cells are lesssensitive to drugs in 3D than in 2D cultures. Thiseffect may be caused by reduced access to compounds in the medium or by pathophysiologicaldifferences due to hypoxia, or by changes in thecell cycle [35, 36]. Hsieh and colleagues showedthat unstable culture conditions, which sometimes occur in vitro, and the type of culture meth-Arch Med Sci 4, June / 2018

2D and 3D cell cultures – a comparison of different types of cancer cell cultures2D3DSCC-040FaDuABLength 482.20 µmFigure 3. Structural architecture of 3D spheroids. The SCC-040 and FaDu cells were maintained in adherent condition with standard medium (10% FBS) and next detached and placed as single cells on non-adherent plates instandard medium. The created spheroids were taken to make the formalin-fixed paraffin-embedded tissue sections (FFPET) and H&E staining as well as DAPI staining. A – cross section through the cells growing in 2D and 3Dcultures of SCC-040 and FaDu cell lines, H&E staining (scale bars represent 20 µm and 50 µm, respectively); B – 3Dstructure stained with DAPI; blue – nuclei, pink – cells (scale bar represents 50 µm)od used can significantly influence cellular metabolic activity, cell proliferation and, ultimately,changes in cell sensitivity to tested drugs. Theyalso indicated that among 2D, 3D and spheroidmodels, only 3D cell culture, with the same celldensity as natural tissue, shows a drug responsecomparable to that of a solid tumour [51].Cell extracellular matrix (ECM) interactionsseem to play an important role in the drug resistance of tumours. Cells growing in a 3D silk scaffold system, which have been found to be similarin fibre orientation and dimensions to native tumour ECM, are more resistant to paclitaxel. Theuse of artificial ECM is a good way to mimic thenatural architecture of a tumour mass. It has beenfound that changes in ECM composition are associated with cancer progression and tumour features [52]. In this context, the use of 3D systemscould avoid over- or underestimation of a specificdrug in case of drug sensitivity and resistance, aswell as its dosage [35, 36].As mentioned above, the spheroids show different responses to drugs, but also the spatialstructure of spheroids influences the irradiationresponse. Increased radiation survival is broughtabout by 3D architecture, which influencesDNA heterochromatinization, characterized bydeacetylation of histone H3 and high expressionof heterochromatin protein 1α (HP1α). It shouldbe noted that higher levels of heterochromatinpartly protect DNA against radiation-dependentinduction of double strand breaks in 3D structures. This phenomenon could be overcome byknockdown of histone deacetylase (HDAC) 1/2/4or by application of the HDAC inhibitor LBH589.However, neither growth conditions nor HDACmodification affects ATM phosphorylation [53].It has been shown that in some cases, such ashead and neck cancers, integrins and their signalling cascades are critical for cell proliferation andsurvival. Use of the FAK/IGF-IR inhibitor TAE226demonstrates strong radiosensitizing potentialArch Med Sci 4, June / 2018 915

M. Kapałczyńska, T. Kolenda, W. Przybyła, M. Zajączkowska, A. Teresiak, V. Filas, M. Ibbs, R. Bliźniak, Ł. Łuczewski, K. Lamperskaunder in vitro 3D conditions, which strongly suggests that this inhibitor has potential in clinicalpractice [54]. Furthermore, the behaviour of cellscultured under 3D conditions shows that the combined targeting of FAK/IGF-IR by cetuximab andTAE226 induces cell death without the need forfurther irradiation [55].Spheroids can be used to study the processof cell migration on ECM proteins, invasion intoMatrigel, or simultaneously tissue invasion andangiogenesis. Characteristic features of migrationare visible flattened cells surrounding spheroids(dispersed or radial migration). In the case of invasion the cells extend in visible invadopodia [35,36]. Tissue invasion and angiogenesis assays canbe performed by means of co-cultures of spheroidsand embryoid bodies generated from mouse embryonic stem cells. This assay is designed to mimicxenograft tumour transplant systems [35, 36].Moreover, 3D tissue culture system allows forthe creation of imitation cancer tissue, with greenfluorescent protein (GFP) expression and with thefeatures of a solid tumour. The efficiency of transfection of anti-GFP oligonucleotides can be measured simply by fluorescence microscopy. Creationof systems with over- or down-regulated genesexamined for usage in new treatment strategiesis also possible. An example of application for 3Dcollagen matrix tissue structures could be in theestablishment of an intracellular delivery systemfor oligonucleotides using the microneedle technique [56].Apart from using 3D systems in the area ofcancer research, they can also be applied in tissueengineering. For example, primary human salivarygland cells may be encapsulated in a 3D hyaluronic acid-based hydrogel scaffold, in order to obtainorganized acinar-like spheroids with active protein secretion pathways. This approach might beused in the future to restore function to salivaryglands damaged by radiation treatment [39–41].A disadvantage of 3D cultures is that it requiresthe separation of single cells from spheroid structures by proteolytic degradation of single layers,which takes from several hours to a few days [57].In many 3D methods, the efficiency, life-span, repeatability, and comfort of work are poorer thanin the case of 2D systems [12]. It is often emphasized that a disadvantage of 3D structures is thefact that “spheres” can be formed, not from a single cell, but from a few cell clusters. However, evenstructures created as aggregates of several cellsstill have a three-dimensional form and seem tobe a better model than flat, adherent cultures [58].A tumour is not a homogeneous structure, but isbuilt from tumour cells of various phenotypes. Furthermore, 2D cultures are, in fact, also a compoundof various cell phenotypes. In spite of this, a homogeneous structure can be achieved from culture of916 a single cell, with only one genetic background inthe concentrated culture medium, as is the case,for example, in soft agar or Matrigel [59].The problem of low reproducibility in 3D culture was solved by Vinci et al., who describeda three-dimensional spheroid-based functionalassay for tumour target validation and drug evaluation. They used 96-well ultra-low attachmentplates to create just one spheroid per well. Thesize of the obtained spheroids was reproducibleand showed Gaussian distribution [32, 33].Owing to the large number of problems associated with 2D systems, 3D models would appearto be a good alternative, that could be an intermediate model between 2D and animal studies[1, 30]. The different technical approaches to obtaining 3D models possess their advantages andtheir limitations (Table II). The proper choice of 3Dsystem mostly depends on the nature of the research. It must be emphasized that choosing thewrong model may influence the results. Clearly,the ideal 3D model does not exist. In some casesthe use of a 2D culture system is enough, but 3Dwill be used more frequently in the future due toimprovements to automation and cost reductions.2D and 3D methods in co-culture systemsA tumour is a mass composed of multiple celltypes [60]. In co-cultures, different cell types aregrown together in the same environment [61].This type of culture was described in the 1970sas a system by which to examine communicationamong cells [62]. Such communication includesthree types of intercellular interactions: cell-cell,cell-microenvironment and paracrine signalling bydissolved factors [61]. This allows us to observeinteractions in functional structures that closelyresemble interactions in vivo [63].In co-culture we can distinguish between target cells and assistance cells that support theirgrowth and development [61]. Studies show thatboth types of cells gain through co-culture [64].Co-cultures can be divided into two types: direct and indirect [61]. In the first model, differenttypes of cells are mixed and cultured together. Inthe second, cells are separated by a physical barrier [61]. Both types of co-culture can be carriedout in 2D systems [65, 66], as well as in 3D models [67, 68]. In direct cultures, we can observe allthree types of interactions described above, whichwould appear to be of great importance in thestudy of cellular behaviour [69, 70]. In contrast tothe direct system, in the indirect model, the cellsare deprived of interactions between the types ofcells by the presence of a physical barrier [68, 71].Our experience has shown that cells of different phenotypes do not grow with each other,even in direct models. Figure 4 shows mesenchy-Arch Med Sci 4, June / 2018

2D and 3D cell cultures – a comparison of different types of cancer cell culturesABFigure 4. Co-culture of epithelial SCC-25 (red) and fibroblast MSU-1.1 (green) cell lines (scale bar represents 100µm). A – cells cultured under 2D conditions are flattened and attached to the plate surface. The epithelial SCC-25cells (red) have typical rhombus-like shape and MSU-1.1 cells (green) are spindle-like and surround SCC-25 cells;B – SCC-25 (red) and MSU-1.1 (green) cells cultured under 3D conditions changed their own morphology due tothe lack of attachment. Cells lose their typical shape and aggregate, creating more (SCC-25) and less (MSU-1.1)compact structuresmal fibroblast cells (green) cultured with epithelial cancer cells (red). It can be seen that the cellsgrow within the limits of their own line both in2D and 3D systems. Moreover, it was observedthat under 3D conditions, the SCC-25 line (red)formed a 3D structure, while the MSU-1.1 line(green) surrounded the established structures asassistance cells.The downside of co-cultures is the inclusion,by necessity, of many variables (i.e. the degree ofsimilarity and separation of the population, thecomponents of the medium, volume and durationof culture), which must all be optimized so thatall types of cultured cells might thrive. All thesefactors make co-cultures difficult to conduct [72].ConclusionsCell cultures are commonly used in geneticand biological cancer research [6, 48]. They mimic in vivo conditions, to varying degrees, and mayprovide an alternative to animal models [73].Currently, there are many forms of cell culture,which allows for the selection of a method wellsuited to the purpose of the study [74]. The mostcommon research model is still the 2D culturesystem. However, owing to its limitations, 2Dcultures are increasingly being seen as an inefficient model with which to study the processes associated with cellular responses to ionizingradiation or to exposure to chemotherapeutics.The 3D models are potentially a better approachin the search for new biomarkers and new treatment strategies, leading us closer to the goal ofpersonalized medicine.AcknowledgmentsThe experimental work to prepare figures wassupported by Greater Poland Cancer Centre – grantno. 3/2015 (95) and grant no. 17/2015 (109); thepreparation of the publication was supported bygrant no. 13/2016 (128).Marta Kapałczyńska and Tomasz Kolenda havecontributed equally to this work.Conflict of interestThe authors declare no conflict of interest.References1. Yamada K, Cukierman E. Modeling tissue morphogenesis and cancer in 3D. Cell 2007; 130: 601-10.2. Harrison R. Observations of the living developing nervefiber. Anat Rec 1907; 1: 116-28.3. Harrison RG. The outgrowth of the nerve fiber as a modeof protoplasmic movement. J Exp Zool 1910; 9: 787-846.4. Scudiero D, Shoemaker RH, Paull KD, et al. Evaluationof a soluble tetrazolium formazan assay for cell growthand drug sensitivity in culture using human and othertumor cell lines. Cancer Res 1988; 48: 4827-33.5. Jacoby W, Pasten I. Methods in Enzymology: Cell Culture. Vol. 58. Academic Press, New York 1979.6. Ryan J. Introduction to Animal Cell Culture. TechnicalBulletin Corning 2003; 1-8.7. Sanyal S. Culture and assay systems used for 3D cellculture. Corning 2014; 9: 1-18.8. Aggarwal B, Danda D, Gupta S, Gehlot P. Models for prevention and treatment of cancer: problems vs promises.Biochem Pharmacol 2009; 78: 1083-94.9. Pampaloni F, Reynaud EG, Stelzer EH. The third dimension bridges the gap between cell culture and live tissue. Nat Rev Mol Cell Biol 2007; 8: 839-45.10. Breslin S, O’Driscoll L. Three-dimensional cell culture:the missing link in drug discovery. Drug Discov Today2013; 18: 240-9.11. Baker B, Chen C. Deconstructing the third dimension –how 3D culture microenvironments alter cellular cues.J Cell Sci 2012; 125: 3015-24.12. Hickman JA, Graeser R, de Hoogt R, et al.

61-866 Poznan, Poland Phone: 48 61 885 06 68 E-mail: [email protected], [email protected] 1Laboratory of Cancer Genetics, Greater Poland Cancer Centre, Poznan, Poland 2Postgraduate School of Molecular Medicine . Greater Poland Cancer Centre, Poznan University of Medical Sciences, Poznan, Poland Submitted: 9 February 2016 Accepted: 27 .