Using Three-dimensional Polystyrene (PS) Insert™ to Produce Tumor Spheroids

Caicedo-Carvajal, CE., Zhang, A., Sridharan, A, and Q., Liu. 3D Biotek 675 US Highway One, North Brunswick, NJ, 08902

Abstract In vitro tumor spheroids are generated using scaffold-free technologies, e.g. hanging drop, agar gel sandwich, and low adhesive surfaces, etc, for drug screening applications. Some of these platforms are currently adapting towards reducing spheroid time formation and increasing highthroughput workflow. Here, we show the use of 3-dimensional (3D) polymer scaffolds to generate multicellular spheroids in an efficient manner. Using 3D Polystyrene (PS) Inserts™, a 3D scaffold with 100% pore interconnectivity, several cancer cell lines were able to produce spheroids as early as 2 days in culture. Further characterization of spheroid formation showed that the number and size of the spheroids appear to be dependent on cell type and scaffold geometry. For example, endometrial cancer cells generated a large pool of small spheroids and osteosarcoma cultured in the scaffold produced a fewer pool of larger spheroids. In conclusion, the 3D PS Insert™ represents a novel platform to generate spheroids for standard drug screening and potential in vitro cancer and stroma co-culture models.

and it may be the most important obstacle to cancer treatment [9].g. creating scaffolds with controlled fiber diameter and openpore geometry for facilitated exchange of waste/nutrients. For example. embryoid body (EB) formation. increasing aggressive and highly oncogenic cancer phenotypes [7]. e.Introduction Tumor spheroids are the closest organotypic structures mimicking in vivo cancer tumor morphology. cells at the surface of the spheroid aggregate are highly proliferative. they have been used to amplify liquid tumors in the presence of 3D stroma [12] and as a 3D platform for stem cell-based therapies [13. 3-dimensional (3D) tumor spheroids are intrinsically more resistant to most anticancer drugs than conventional monolayer in vitro models [8]. while in the middle of the spheroids. cells are quiescence. The resistance encountered in cells cultured as spheroids seems to be analogous to cancer drug resistance observed in patients. 14]. Tumor resistance to anticancer drugs is a real phenomenon. For example. so the use of three-dimensional cell culture may provide a model for studies on the development of anti-cancer drugs. Thus. 11]. and soft-agar [1. allowing cells to self-assemble in the absence of scaffolding. There are some reported methods to generate tumor spheroids. The 3D scaffolds have been successfully used in Tissue and Cancer Engineering. we found that 3D polymer scaffolds can be used to produce tumor spheroids in an efficient manner. Recent evidence has shown that angiogenic suppression and extracellular matrix deposition in adenocarcinomas causes hypoxic/oxidative microenvironments. Figure 1 is an illustration showing the 4-layer structure of the 3D polymer scaffold. In this study. partly because of the so-called multicellular resistance (MCR). tumor cells experience different metabolic patterns. It has been shown that solid tumors contain significant areas of chronic or transient hypoxia [3]. . In this study. reconstituted basement membrane. Direct comparison between cancer cells grown as 3D spheroid and 2D monolayer showed that 3D spheroids closely resemble the morphology and biology of primary tumors [1]. the same polymer that was used to produce tissue culture plates and flasks. tumor spheroids form through anchorage-independent mechanisms. Evidence suggests that hypoxia may have a profound impact on malignant progression and on responsiveness to therapy [4-6]. and cells are under hypoxic conditions and necrotic in the spheroid center [2]. In these methodologies. Depending on the position within the tumor spheroid. 10. Each layer was purposely colored for easy viewing of the 3D layering structure. we report a novel tumor spheroid producing method that uses 3D polymer scaffolds. The 3D polymer scaffolds (3D Inserts™ -PS) were made from polystyrene. The scaffolds are produced using microfabrication technology.

cells were grown on non-treated 12-well culture plates (2D Control) under the similar culture conditions. However. but ECC-1 tumor cells did not produce any spheroid. aggregate compaction was different for spheroids formed in 3D culture. we used 3D Inserts™ -PS as a tool to generate tumor spheroids.Figure 1. HepG2 (Hepatocacinoma). Spheroid formation was seen at day 2 for ECC-1 cells and at day 4 for both HepG2 and U2OS-β-actin-RFP when they were culture in 3D Inserts™ -PS scaffolds. cellular aggregation were seen on 2D cell culture controls. As control. Anchorage-Dependent Spheroid Formation In this study. middle . HepG2 and U2OS-β-actin-RFP formed some type of spheroid-like aggregates under shaking conditions. shows a qualitative comparison between aggregates formed in 2D plates and spheroids formed in 3D scaffolds. this points out to potential differences where the 3D scaffold geometry does play a role in the formation of cell spheroids.000 cells/cm2 following manufacturer’s recommended protocols. 2. As expected. CAD models of 3D scaffold. spheroids formed easily and quickly even for the ECC1 cells which did not even produce unbound aggregates in 2D control plates (Fig. After one day in culture. The scaffold is made of four layers (different color represent different layers from bottom to top) with each layer of fibers 90° perpendicular to its adjacent layers (ac). Figure 2. the scaffolds were transferred to 12-well non-treated culture plates and shaken for 6 days. and U2OS-actin-RFP (Osteosarcoma) were seeded and cultured on 12-Well 3D Inserts™ -PS scaffolds at a seeding density of 25. Several cancer cells lines such as ECC-1 (Endometrial Adenocarcinoma). Thus. when using 3D scaffolds. On the other aggregates probably due to strong cellular adhesion to non-treated 2D plates.

The U2OS-β-actin-RFP spheroids were live-imaged using fluorescence microscopy. Facin phalloidin) and peripheral DAPI staining (Blue). In general. Fixed stained spheroids for F-actin phalloidin (Green) and cell nucleus (Blue) showed pseudo-peripheral cellular organization (Right panel. 2.panel). and U2OS-β-actin-RFP cell lines (top to bottom) cultured using conventional 2D plate (left) and 3D Inserts™ -PS (1520) scaffold (middle). cells grown on 3D scaffold yielded spheroids s while conventional 2D culture only produces some types of loose cellular aggregate (Scale bar 100-micrometers) . HepG2. 2. bottom). showing evidence of cytoskeletal function (Fig. Phase contrast and fluorescent microscopic images of ECC1. These spheroids showed an even distribution of βactin-RFP fluorescent throughout the spheroid’s surface. right panel. Fig. A close inspection of the spheroid morphology using fluorescent staining on fixed spheroids (ECC-1 and HepG2) showed cortical localization of F-actin between cells (Green. top and mid images).

0. Characterization of spheroid shape factor and diameter in 3D PS Insert™ and 2D plates. ECC-1 had the smallest average diameter of 110.0. In addition. In table I all spheroids from 3D culture had shape factors close to 1.A). Although cells were seeded at comparable seeding density.09 +/.81 N.A 179. spheroid diameter variation was more prominent for HepG2 3D cell culture with an average diameter of 179.05 +/.81-µm. The shape factor of a perfect spheroid is 1 and for non-spherical aggregates the shape factor is greater than 1.08 N.12 +/.7 N.A .60 1.59 +/.0. spheroids from different cell lines showed different number of spheroid and average diameters.5. The shape factor data from ECC1 2D culture was not listed because 2D conditions did not yield detached tissue aggregates (N.12.71 +/. Table I.44 ± 12.Effect of 3D Scaffold on Spheroid Shape To further illustrate the differences in aggregation.54.93 N.0. we computed the shape factor (Length to Width Ratio) and spheroid diameter to show quantitative differences between 3D spheroids and 2D aggregates. Among the 3 cell lines.A 45 22 20 20 Shape Factor 1.A 162 +/. while the shape factor from 2D aggregates had mean values of 1.45 +/.9.0.09 1.05 1.35 Spheroid Diameter (um) 110.93-µm.A 1.49 ± 54.4 +/. Cell Type/Geometry ECC1 (3D) ECC1 (2D) HepG2 (3D) HepG2 (2D) U2OS (3D) U2OS (2D) *Error Bars ± SD Total Aggregates 92 N.

On the other hand. For scaffolds with 300-um fiber diameter. In smaller fiber and pore scaffolds PS(1520). On figure 3(a). After one day. we tested two different scaffold geometries. Figure 4. At day 6. larger number of spheroids with smaller diameters were generated. scaffolds with fiber diameter 150-um and fiber-to-fiber space 200-um (PS(1520)).000 cells/cm2 and grown for 1 day.Effect of Scaffold Fiber Diameter on Spheroid Size To understand the mechanism of spheroid formation in 3D scaffolds. ECC1 cells were seeded at 25. Figure 3 shows differences in distribution and spheroid diameter.e. Spheroid size distribution as a function of scaffold fiber diameter. i. there were a total of 46 single spheroids formed with an average diameter of 151 +/-20-um (STDEV). the spheroids pool was characterized using spheroid size distribution. and scaffolds with fiber size 300-um and fiber –to fiber space 400-um (PS(3040)). . ECC-1 cells seeded on different 3D Inserts™ configurations yielded different spheroid distributions. indicating that scaffold fiber diameter does have an effect on spheroid size and size distribution. the scaffolds were transferred into non-treated wells and placed under shaking conditions. PS(3040) had almost half the number of spheroids with larger size distributions (Right panels with blue (PS(1520)) and red (PS(4030)) tracing on the size distribution graphs. the number of spheroids from ECC1 cells on scaffolds with 150-um fiber diameter was 92 with an average diameter of 110 +/-10-um (STDEV).

Life Science for providing the Zinc-Finger Modified (ZFN™) modified osteosarcoma cell line. allow the formation and easy retrieval of these multicellular spheroids. . this is the first report that uses solid polymer scaffolds to produce tumor spheroids of varying size distribution. The mechanism of spheroid formation seemed to be related to the formation of 3D aggregates on the curved surface of polymer fiber and the amount of pore volume available. the small spheroids will grow to certain sizes and then detach from the curved surfaces. Since the surface seems to play a role. Acknowledgements We would like to thank Sigma Aldrich. Once the initial small spheroids formed on the surface of the polymer fiber. The novel geometry of the scaffold such as homogeneous fiber/pore sizes and 100% pore-to-pore connectivity. we show multicellular spheroids can be easily produced using 3D porous scaffolds. To our knowledge. The detachment of the spheroids from polymer fiber is very likely due to the dynamic momentum generated from the shaking culture conditions. This offers an integrative approach to more complex models in cancer drug screening.Discussion As opposed to scaffold-free technology. the scaffold can be coated with different extracellular matrix (ECM) molecules or stroma to study the rate of spheroid formation n the presence of other cells and under drug treatment conditions. U2OS-β-actin-RFP used in this study.

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