Controllable Fabrication Of Microwell Arrays And Their Application In Three-Dimensional Cell Culture

As one of a high fatality rate disease,malignant tumor threats public health in the world.A lot of efforts have been made in malignant tumor treating.However,only a few of medications can be turned into the clinical treatments from lab experiments.Currently,two-dimensional?2D?cancer cell monolayer platform is mainly used for the drugs screening and the drug efficacy evaluating in preclinical testing.However,the lack of physico-biochemical characteristics using the monolayer platform leads to overestimated drug efficiency.Compared with 2D cell culture,three-dimensional?3D?tumor spheroid model has been considered to better simulate the biological properties of actual tumors in vivo,such as the spatial complexity,complex interaction between cells and extracellular matrix?ECM?,chemical and phenotypical gradients related to nutrients,O₂,pH,metabolites and growth factors.Recently,3D tumor spheroid model has gained great attention and becomes one of the most representative models of actual tumor in vitro.Many 3D tumor spheroid culture tools based on spinner flask or rotating bioreactors,non-adherent surface,hanging drop and microfluidic have been used to fabricate in vivo-like 3D tumor spheroids.However,these methods are time-consuming,labor-intensive,complex and requiring expertise.Precisely fabricating of 3D tumor spheroid model remains challenging.There is an increasing concern of microwell array due to its excellent structures,which can promote the formation of 3D tumor spheroid with uniform size.Many methods such as micromilling,gas expansion,surface tension-mediated and photolithography have been applied to the fabrication of microwell array.However,it is still a challenge to realize simple and controllable fabrication of microwell arrays.In order to overcome the above-mentioned difficulties,the microspheres were arrayed with through-hole stainless steel meshes?TSSM?,and the array was served as template for the simple and efficient preparation of PDMS microwell array.A high-throughput 3D multicellular sphere culture platform based on microwell arrays was proposed.The preparation of microwell arrays with different structure and size,3D tumor spheroid cultivation,3D tumor spheroid recovery and drug resistance testing had been investigated.The obtained results from this study are as follows:1.Microsphere arrays were simply and efficiently fabricated based on resin microspheres and through-hole stainless steel meshes.Large-scale spherical cavity microwell arrays and through-hole arrays were successfully prepared by using microwell arrays as templates.The obtained results showed that the preparation of spherical cavity PDMS microwell arrays with different size in the range of 300-700?m can be realized by changing the size of microspheres.The entrance size and shape of the microwell are determined by the rotation speed of spin coating.The PDMS through-hole microwell arrays were obtained by fabricating microsphere arrays into sandwich structures.The 300,400,500,600,700?m spherical cavity microwell arrays and through-hole arrays prepared in this experiment were uniform and highly ordered.2.Multi-size HepG2 tumor spheroid culture platform was established based on microwell array.The result showed that simple,controllable and high-throughput generation and culture of HepG2 tumor spheroid of different sizes?224.2±10.8,320.0±10.8,349.8±11.1,380.1±24.5 and 410.4±15.4?m?can be achieved by changing the size of the microwell array.Cell viability was above 92%after 3 days of culture,meeting the needs of biomedical research.3.The through-hole microwell devices used for 3D tumor spheroid culture was successfully fabricated by PDMS through-hole array,and applied in the cultivation and recovery of 3D tumor spheroid.Simple and low-damage recovery of 3D tumor spheroid with high cell viability,complete cytoskeleton and close interconnection can be obtained by blowing.4.On-chip drug resistance evaluation for the antitumor drug Doxorubicin?Dox?was also achieved in microwell array.The lower cell viability of 3D tumor spheroid caused by a higher anticancer drug concentration.And the size of tumor spheroid was markedly inhibited when Dox concentration exceeded the lethal dose(LC50 8.0?g mL^-1).In conclusion,the simple and efficient method for the generation and culture of 3D tumor spheroid was proposed in this study.It is expected to be widely used in anti-tumor drug screening and efficacy evaluation.