Patterned substrates fabricated by a controlled freezing approach and biocompatibility evaluation by stem cells

• A directional freezing approach is developed to fabricate aligned surface patterns.
• Various colloidal suspensions and polymer solutions can be used to produce such patterns.
• Aligned patterns from silica colloids, PLGA and chitosan are non-cytotoxic.
• Guided growth of mouse mesenchymal stem cells is observed.

Patterned substrates have been widely used in the studies investigating how to regulate cell growth and alignment. Such substrates may be fabricated by various techniques such as photolithography, soft lithography and microcontact printing. We report here a facile approach to fabricate aligned and grid surface patterns by a controlled freezing approach and further investigate their biocompatibility. The fabrication has been demonstrated with polymers (hydrophilic & hydrophobic), nanoparticles (organic & inorganic), or mixtures of these components. For the aligned surface patterns, the spacings between the patterned ridges can be tuned by varying the freezing rates. The biocompatibility of the substrates is evaluated by WST-8 viability tests with cell counting kit-8 (CCK-8) and by culturing with mouse mesenchymal stem cells (mMSCs). Three surface-patterned substrates (PLGA, PLGA nanospheres with chitosan, and silica colloids) are evaluated in more details to show that the mMSCs can grow alongside the aligned ridges while the cells grow randomly when plain glass slides are used as control. Further observations show that PLGA substrates undergo degradation, and are thus unsuitable for cell culture over the longer term. On the other hand, the PLGA-chitosan substrate and silica substrate were stable and could maintain mMSC alignment throughout the culture period.

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