a growing number of studies recognize the limitations of two - dimensional ( 2d ) monolayer cultures for in vitro cancer biology study [ 16 ] . the adhesion and organization of cancer cells growing in conventional 2d culture may differ from those in vivo in terms of proliferation , cellular signal transduction , and response to drugs and radiation [ 713 ] . in vivo cancer - associated stroma is a three - dimensional ( 3d ) structure consisting of neighboring cells , extracellular matrix ( ecm ) , blood vessels , immune cells , and cytokines . recent studies of 3d culture have demonstrated that cancer cells interact extensively with their microenvironment during proliferation , angiogenesis , metastasis , and chemo- or radiotherapy [ 1422 ] . it is therefore critical to mimic cancer cells and their surrounding environment during in vitro study . several strategies have been used to develop an in vitro cancer model that can accurately represent in vivo conditions . the most commonly used matrix gel 3d culture method involves the encapsulation of cancer cells within collagen , laminin - based matrix , or hydrogel . using matrix gel in multiwell or transwell plates , breast cancer and ovarian cancer cell lines have been studied for gene expression , cellular signal pathways , angiogenesis , and chemotherapy response [ 2225 ] . recently there has been great interest in using multicellular tumor spheroid models to study gene expression and antitumor treatment therapy [ 14 , 20 , 23 , 26 , 27 ] . spheroid culture was developed 40 years ago and gradually more advanced models have been created to accommodate the fast - growing field of cancer targeted therapy . in contrast to the matrix and spheroid technologies , 3d models using porous scaffolds are relatively new . in 2007 established a 3d model using oral squamous cell carcinoma and compared the 3d model both in vitro and in vivo conditions with very encouraging results . since then porous collagen scaffolds , silk porous scaffolds , and nanoparticle - incorporated scaffolds have been developed to mimic angiogenesis , tumorigenicity , cancer metastasis , cancer stem cell properties , and drug uptake and release [ 2839 ] . however , only a few solid tumors have been tested using these approaches [ 15 , 40 ] . there is a vast demand for 3d scaffold models for solid tumors directed at improved cellular attachment for cancer biology study . the aim of the present study was to establish a 3d cancer model using a synthetic composite scaffold ( variotis tissue scaffolds , biometic , sydney , australia ) . this scaffold comprises highly interconnected and porous polyester - based material with a pore size in excess of 100  m . it was initially developed for cartilage repair and has been used in chronic wound - healing studies . here we studied the growth pattern of a non - small - cell lung cancer cell line using this scaffold compared with standard monolayer culture . the nci - h460 cell line ( a human non - small - cell lung cancer cell derived from metastatic pleural fluid ) was obtained from american type culture collection ( manassas , usa ) . nci - h460 has been studied extensively in 2d culture [ 41 , 42 ] . this cell line was chosen because of its metastatic potential [ 41 , 43 , 44 ] . nci - h460 cells were cultured in rpmi1640 ( gibco , life technologies , victoria , australia ) supplemented with 2  mm glutamine and 5% fetal calf serum ( fcs , interpath services , melbourne , australia ) at 37c in 5% co2/humidified air . all chemicals were sourced from sigma ( sigma - aldrich , sydney , australia ) . variotis tissue scaffolds ( biometic , sydney , australia ) were cut into 100  mm , sterilized with uv radiation for 60 minutes , and then placed in 2  ml eppendorf tubes . briefly nci - h460 cells were grown in t75 flasks ; when the culture reached 80% confluence , cells were trypsinized , washed with phosphate buffered saline ( pbs ) , and counted . cell suspensions of 1  10 to 3  10 per ml were prepared in culture medium ( 10% fcs in advanced rpmi1640 ) and 100  l of the suspension was dispended onto the scaffold slowly to ensure the cells were captured by the scaffold . the seeded scaffolds were incubated for 1 to 2 hours at 37c without the addition of culture medium . the scaffolds were then transferred into 6-well plates ( corning incorporated life sciences , lowell , usa ) with 5  ml of medium and incubated for up to 2 weeks . the medium was changed every 2 to 3 days by transferring the scaffold culture carefully to a new plate with fresh culture medium containing 20% conditioned medium . at least three scaffolds were established for each testing condition , and each experiment was performed in duplicate . to examine the presence and distribution of nci - h460 cells on scaffolds , 1- and 3-day cultures were washed twice with pbs . the scaffolds were then fixed and stained in 0.3% methylene blue/50% ethanol solution for 30 minutes then rinsed in water . morphological features were examined by light and fluorescent microscopy . at 1 , 3 , and 7 days the scaffold cultures were gently washed and fixed , stained with hoechst 33528 for 15 minutes , and washed with pbs . the 7- and 14-day scaffold cultures were fixed in formalin acidic acid for 3 days , embedded in paraffin , cut into 5  m sections , and stained with hematoxylin and eosin ( h&e ) . the cells grown in monolayer were collected , made into cell blocks , and processed as for the 3d scaffolds . in addition , the attachment of cells on scaffold was monitored daily and quantified by cell counting after 1 and 3 days incubation , respectively . the cells were dissociated from the scaffold with 200  l of 0.25% trypsin , washed in pbs , and resuspended in 0.5  ml pbs . the cell proliferation rate was determined by a mtt ( 3 - 4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ) assay at 1 , 3 , and 7 days . briefly , the scaffold cultures were transferred into 96-well plates and incubated with 200  l solution of mtt at 1  mg / ml for 4 hours at 37c . then the scaffolds were transferred into 5  ml tubes with 2 to 3  ml dimehyl sulfoxide ( dmso ) to completely dissolve the formazan product converted by living cells . eight aliquots of 150  l dissolved mtt / dmso solution were pipetted to 96-well plates and the absorbance was read at 540  nm in a multiskan ex plate reader ( thermo , melbourne , australia ) . monolayer cultures of 5  10 to 1  10 cells were also seeded in 24-well plates to create a standard curve using mtt assay . the absorbance from 3d scaffold culture was then plotted on the standard curve to obtain the cell number in 3d culture . immunohistochemistry was performed using an autostainer ( dako , melbourne , australia ) following the manufacturer 's procedures . following treatment with antigen retrieval buffer ph6 , sections were incubated for 60 minutes with the primary antibodies : ki67 ( 1  :  200 rabbit monoclonal , ab16667 , abcam , cambridge , uk ) , cd44 ( 1  :  500 mouse mab , 156 - 3c11 , cell signaling , danvers , usa ) , or carbonic anhydrase ix ( ca ix 1  :  1000 rabbit anti - human nb100 - 417 , novus , littleton , usa ) . the envision dual link system peroxidase ( dako ) was used as secondary antibody , and sections were counter - stained with mayer 's hematoxylin . for negative controls staining was visualized with an olympus provis microscope and scored by the study pathologist ( csl ) . in 2d culture , the nci - h460 exhibited characteristics of an adherent epithelial cancer cell line ; the cells were large and predominantly polygonal with a relatively high nuclear / cytoplasmic ratio that was estimated to be 0.5 to 1 ( figure 1 ) . in 3d culture , using high - density low - volume seeding , nci - h460 readily attached onto the scaffold without surface pretreatment . using quantitative cell counting , we found that the highest attachment rate was 35% with 1.5  10 cells seeded for 2 hours . figure 2 demonstrates that increasing the initial seeding period improved cell attachment in a time and cell density dependent manner . however , increasing the initial seeding time to 24 hours did not enhance cell attachment but caused cell damage possibly due to the very low volume of culture medium . the distribution and presence of the cells on scaffolds can be seen clearly with methylene blue staining ( figure 3 ) . on day 1 , there were only a few stained cells on the surface of the scaffold seeded with 1  10 cells ; in comparison , more dark - blue staining was observed spreading randomly on the scaffold seeded with 1  10 . we noted that after incubating for 3 days the dark - blue staining covered the surface of the scaffold as well as infiltrated deep into the centre for both 1  10 and 1  10 scaffolds . examination of the scaffold cultures under light microscopy after overnight incubation ( figure 4(a ) ) showed that 20% of the scaffold surface was covered with cells . growth extended horizontally and reached 50% surface coverage at 2 weeks culture ( figure 4(b ) ) . h&e staining of a cross section of the scaffold culture revealed that most of the larger clumps were located towards the exterior of the scaffold ( figure 4(c ) ) . cancer cells growing from single to multiple layers could be seen on most fields ; there were also nonviable cell populations especially in the centre of the large clumps ( figure 4(d ) ) . three types of growth patterns were observed . besides expanding horizontally , the majority of cells grew vertically to form clumps along the surface or on the end of the scaffold fiber ( figures 5(a)5(c ) ) . the size of the cell clusters was variable and irregular with the largest clumps reaching diameters of 500600  m ( figure 4(b ) ) . as the cell clumps continued to develop , they frequently separated from the scaffold culture to form structures similar to cancer spheroids ( figure 5(d ) ) . often the detached clumps settled onto the bottom of the culture plate and continued to grow as a monolayer . as demonstrated in figure 6 , nci - h460 cells had a slower proliferation rate in 3d scaffold than in monolayer culture ; the cell doubling time in 3d culture was 2 to 3 days compared with less than 24 hours in monolayer . mtt assays confirmed 3040% initial attachment after overnight incubation for all seeding concentrations which is indicated in the dark grey bar in figure 6 . all of the 3d culture was grown continuously in a seeding - number dependent manner . when seeded with at lower density ( 1  10 ) , the 3d culture expanded rapidly and reached 2  10 cells on day 7 which exceeded cultures with higher initial seeding cell numbers . similar growth behavior was observed for the 3d culture seeded with 5  10 cells . in comparison , cells only doubled in number by day 3 for scaffolds with higher seeding numbers ( 1  10 and 2  10 ) ; after 3 days of incubation the 3d culture reached a plateau and the difference between 3 days and 7 days of incubation did not appear to be statistically significant . immunohistochemistry studies were performed on both 3d culture and monolayer cell pellets ( figure 7 ) . for ki67 , 3d culture showed positive cells at the periphery of the clumps and mostly negative cells in the centre ( figure 7(a ) ) , whereas monolayer cells showed a uniform pattern of staining ( figure 7(b ) ) . for the cell - surface glycoprotein cd44 , the 3d culture was characterized by upregulation of cd44 . there was positive staining of more than 50% of the cells , and dynamic changes were evident among the cells in each cell clump that represent cellular functional changes ( figure 7(c ) ) ; in contrast , less than 30% of monolayer cells were cd44 positive ( figure 7(d ) ) . . cells in 3d culture showed enhanced expression represented by very strong membrane staining ( figure 7(e ) ) ; in contrast , cells in 2d culture showed very low positivity ( figure 7(f ) ) . although there have been numerous investigations aimed at developing 3d cancer models , challenges still remain for initial attachment on porous scaffolds . in this study , we used the high - density random seeding technique to enhance the initial attachment of a non - small - cell lung cancer cell line . this represents an improvement over previous rates and allows controlled cell loading suitable for further evaluation with different surface coating and pore size . our study of this 3d cancer model has demonstrated that the variotis tissue scaffold is suitable for assessing cell adhesion , polarity , and morphology . the stiffness of variotis tissue scaffolds is similar to that of biological connective tissue thus the nci - h460 cells could readily attach and migrate along the scaffold surface . mechanical forces have been demonstrated to alter gene expression and regulation of cell signaling , providing appropriate substrate stiffness vital to living cell functions . recent studies have found that the rigidity of the matrix can influence stem cell differentiation , the migration of cells through cell membrane receptors , and activation of actin cytoskeleton [ 4853 ] . our results revealed that nci - h460 cells grown on scaffolds formed tumor clusters of various sizes . the morphology of the cells appeared to represent their tissue origin , with distinct cellular membrane connection and distribution of necrotic cells . the porous nature of the scaffold allowed oxygen and nutrient to circulate freely in the culture . , these specific growth patterns were not seen in the 2d monolayer culture which showed the typical pattern of cellular uniformity in size and shape and contact inhibition at confluence . the cells formed clusters between single layers of cells along the surface of scaffold to form multilayer clumps of sizes up to 500  m . both h&e and ki67 staining revealed the resting and nonviable cells in the center of cell clumps . this indicated that passive diffusion could no longer provide either sufficient nutrients or removal of waste products in the centre of the clumps . lack of sufficient detoxification of this static system resulted in growth arrest and development of central necrosis . this pattern of central necrosis mimics tumor mass in vivo and provides a more realistic in vitro cancer model . considerable research has been devoted to determining how the surface of living cells senses their physical environment and translates into a biological response of altered protein and gene expression [ 6 , 5458 ] . cd44 is a membrane receptor for hyaluronic acid ; it interacts with other cell - adhesion ligands such as collagens and matrix metalloproteinases . nci - h460 cells had increased expression of cd44 in 3d scaffold compared with monolayer culture , indicating extracellular matrix protein activation . furthermore , cd44 as a cell surface marker has been identified in some breast and prostate cancer stem cells . although the pathways of the stem cell differentiation remain unknown , the expression of cd44 in our 3d culture suggests that the complex structure of scaffolds and the close communication of neighboring cells encouraged the differentiation of cancer stem cells . it has been shown that ca ix regulates endothelial cell proliferation , and expression of ca ix has correlated with poor prognosis in various tumors [ 59 , 60 ] . as cells form a 3d structure , they rely on their microenvironment to maintain the ability to proliferate and communicate . in our 3d culture , there was necrosis in the centre of the cell cluster and detachment of cells from the scaffold . the overexpression of ca ix reflects these functional changes and demonstrates exchanges between cancer cells and their environment .   unlike the culture of cancer cells in matrix gel or cancer spheroid , our tumor 3d scaffold model proved very easy to handle . in addition to its use in immunohistochemistry this porous scaffold also provides a unique opportunity for studying soluble factors present in culture . for example , the hollow channel between the cell clusters allows soluble chemicals and secreted proteins to diffuse . introducing specific culture conditions such as additional growth factors and chemokines will provide new insight into the mechanisms that regulate tumor proliferation and metastasis . the released soluble cellular products from scaffold culture can also be collected and detected by elisa assays . in conclusion , the present work compared the attachment and growth patterns of a non - small - cell cancer cell line in monolayer with a 3d culture in scaffold . our experiments confirmed that the initial attachment to scaffold is enhanced by high - density seeding , and the behavior of cancer cell in our 3d model is similar to tumor growth in vivo .
background . human cancer is a three - dimensional ( 3d ) structure consisting of neighboring cells , extracellular matrix , and blood vessels . 
 it is therefore critical to mimic the cancer cells and their surrounding environment during in vitro study . 
 our aim was to establish a 3d cancer model using a synthetic composite scaffold . 
 methods . 
 high - density low - volume seeding was used to promote attachment of a non - small - cell lung cancer cell line ( nci - h460 ) to scaffolds . 
 growth patterns in 3d culture were compared with those of monolayers . 
 immunohistochemistry was conducted to compare the expression of ki67 , cd44 , and carbonic anhydrase ix 
 . results . 
 nci - h460 readily attached to the scaffold without surface pretreatment at a rate of 35% from a load of 1.5  106 cells . 
 most cells grew vertically to form clumps along the surface of the scaffold , and cell morphology resembled tissue origin ; 2d cultures exhibited characteristics of adherent epithelial cancer cell lines . 
 expression patterns of ki67 , cd44 , and ca ix varied markedly between 3d and monolayer cultures . 
 conclusions . 
 the behavior of cancer cells in our 3d model is similar to tumor growth in vivo . 
 this model will provide the basis for future study using 3d cancer culture .