Agarose multi-wells for tumour spheroid formation and anti-cancer drug test
Graphical abstract
Introduction
Cancer metastasis is a leading cause of mortality in the world for which tremendous efforts are now devoted to anti-cancer drug development [1], [2], [3]. Before metastasis, a primary tumour was formed at the anatomical site where tumour progression started to yield a cancerous mass. Therefore, one strategy in drug discovery is against the tumour progression. However, the most previously used cell based assays for cancer drug studies are based on two-dimensional (2D) culture which does not take properly into account the three dimensional (3D) characteristics of tumour formation in vivo [4], [5]. Suspension culture is frequently used for multicellular spheroid formation but it is not easy to control the size consistency of the spheroids and to handle them for systematic studies [6], [7]. Suspension culture using spinning flask or rotating bioreactors can improve the size uniformity of the spheroids but undesired shear stress can be introduced during the spheroid formation. Culture with hanging drops can also improve the size uniformity and overcome the problem of mechanical disturbance. This technique is however time-consuming and not easy to perform drug tests during spheroid progression. More recently 2D and 3D patterned substrates are used to support spheroid formation [8], [9]. In particular, various micro-well arrays are proposed for 3D cell aggregation, depending on microfabrication techniques [10], [11]. This approach allows controlling cell population in each spheroid as well as high-throughput screening. However, the available systems are mostly appropriate for small size spheroid formation, which cannot efficiently recapture the spheroid complexity of solid tumours.
To facilitate the diffusion of nutrients, drugs and other factors into the spheroid area, it is also important to form the 3D assays with the materials of high permeability [12]. In this work, we developed a multi-well assay form by agarose moulding. We first describe our technique and then compare the formation of spheroid culture in agarose, PDMS and PEGDA wells. We then show the relevance of agarose wells for the formation of millimetre size tumour spheroids. Finally, the results of anticancer drug test performed on the same platform on the progressing tumours are reported and the potentials of the proposed assay form for other applications are also briefly discussed.
Section snippets
Fabrication of multi-well array
Fig. 1 shows the schematic process flow of multi-well array fabrication. A flat PDMS film of 2 mm thickness was prepared by casting a mixture of PDMS pre-polymer and cross-linker (GE RTV 615) at a ratio of 10:1 on a silicon wafer. After curing at 80 °C for 1 h, the PDMS layer was peeled-off and an array of holes was created on the PDMS layer using a computer numeric controlled (CNC) milling machine and a biopsy punch of 2 mm diameter. Afterward, the PDMS layer was exposed in trimethylchlorosilane
Material comparisons of multi-wells
Multi-wells made of PDMS, PEGDA and hydrogels could be easily obtained by casting or moulding and used for the formation of either tumour spheroids or embryoid bodies [13], [14], [15]. However, their performances for the spheroid formation should be different due to difference in cell compatibility and material permeability. Fig. 2 shows microphotographs of tumour spheroids formed in multi-wells made of PDMS, PEGDA and agarose. After 2 days, spheroid shaped cell aggregates could be found in all
Conclusion
We have demonstrated a simple but reliable technique for the fabrication of agarose multi-wells and their usefulness in anti-cancer drug tests. In 2 mm diameter agarose wells, 1.4 mm diameter U87-MG tumour spheroids can be formed and cultured for many days. Interestingly, the size of the tumour spheroids does not change with the further increase of seeding cell density. Anti-cancer drug CA4 added at different stages of tumour spheroid formation have different drug effects. More systematic studies
Acknowledgment
This work was supported by the European Commission under contract No. 604263 (Neuroscaffolds) and Agence de Recherche Nationale under contract No. ANR-13-NANO-0011-01 (Pillarcell). Y.T. is grateful to the Chinese Scholar Council for grant.
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