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We developed a simple and efficient method for thermo-responsive surface fabrication. The thermo-responsive properties, film thickness and retention of the fabricated surface were characterized. Cell studies were conducted to prove the effectiveness of the developed thermal-responsive substrate for cell culture and detachment.
For the fabrication, we used a common adhesion promoter, 3-aminopropyltriethoxysilane (APTES), to enhance the retention of spin-coated poly(N-isopropylacrylamide) (pNIPAAm) films onto the surface of a glass substrate to fabricate thermoresponsive substrate for cell culture and detachment. Briefly, polymer blend films were produced by spin-coating the pNIPAAm/APTES mixed solution onto pre-cleaned substrates for 30 s at 2000 rpm (p-6000 Spin Coater, Specialty Coating Systems Inc., Indianapolis, IN). The spin-coated samples were cured inside a vacuum (<100 mTorr) oven (VWR International, Radnor, PA) for 3 days at the temperature of 160°C. The thermo-response properties of the fabricated surface were confirmed by contact angel measurement, showing the surface became more hydrophobic with the increased temperature (see Fig. 1.).
Findings
Cell studies were conducted to prove the effectiveness of the fabricated thermal-responsive substrate for cell culture and detachment. Human mesenchymal stem cells (hMSCs) (Passage 3) were seeded onto different substrates: a clean glass substrate (as negative control), a pNIPAAm film (as experimental control), a thermo-responsive culture substrate, and a tissue culture polystyrene (TCPS) dish (as positive control) at a density of 1.5 × 104 cells cm–2. An MTT cell proliferation assay was performed to compare the proliferative potential of cells on different substrates. Our data showed that the fabricated substrates are biocompatible and support the maintenance of normal cellular functions. (Fig 2)
hMSCs were cultured on the developed thermo-responsive surface to assess individual cell and cell sheet detachment resulting from temperature change. Upon 80% confluence, cell detachment was achieved by replacing the cell culture medium with fresh cold medium (4 °C). The cell detachment behaviors are shown in Fig. 3. All cells detached within 2.5 min after adding cold medium. Under the same conditions, it took nearly 3 h for the cells to completely detach from the commercial thermoresponsive cell culture surface (UpCell®). Cell viability after detachment was also examined using Live/Dead assay.
The results demonstrate that thermo-responsive cell culture substrate can be generated by simple blending, spin-coating and thermal curing. These fabricated substrates exhibit enhanced cell adhesion and proliferation, and greatly accelerate detachment of the cells upon cooling
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