Development of a cell culture surface conversion technique using alginate thin film for evaluating effect upon cellular differentiation

Abstract

Here, we sought to develop a cell culture surface conversion technique that would not damage living cells. An alginate thin film, formed on a glass plate by spin coating of sodium alginate solution and dipping into calcium chloride solution, was used to inhibit adhesion of cells. The film could be removed by ethylenediaminetetraacetate (EDTA) at any time during cell culture, permitting observation of cellular responses to conversion of the culture surface in real time. Additionally, we demonstrated the validity of the alginate thin film coating method and the performance of the film. The thickness of the alginate thin film was controlled by varying the rotation speed during spin coating. Moreover, the alginate thin film completely inhibited the adhesion of cultured cells to the culture surface, irrespective of the thickness of the film. When the alginate thin film was removed from the culture surface by EDTA, the cultured cells adhered to the culture surface, and their morphology changed. Finally, we achieved effective differentiation of C2C12 myoblasts into myotube cells by cell culture on the convertible culture surface, demonstrating the utility of our novel technique.

Creation of cell micropatterns using a newly developed gel micromachining technique

“…Cells were disseminated by micropipette, and cell micropatterns were created along those in the alginate thin film because cells cannot adhere to the alginate-covered surfaces on the glass plate (figure 3(v)). Thereafter, the alginate thin-film micropattern was removed by 0.2 g l −1 EDTA during cultivation (figure 3(vi)), which is below the level of cytotoxicity [32], and a different cell type was disseminated (figure 3(vii)). Thus, the second type of cells could be patterned alongside the first within the same culture dish (figure 3…”

“…We first evaluated the influence of EDTA and alginate lyase on cell proliferation to determine whether they would affect cell viability during removal of the alginate thin-film micropattern. Figures 4 [32] and 5 show the cell viability of cells in culture media containing EDTA or alginate lyase, respectively. There were no significant differences in cell proliferation compared with normal medium for 0.2 g ml −1 EDTA or 200 μg ml -1 alginate lyase.…”

“…Figure 4. Relationship between the number of cultured cells and cultivation time in culture media containing various concentrations of EDTA[32]. At 0.2 g l −1 EDTA, cell numbers gradually increased over time similar to the normal culture medium (control).…”

“…The alginate gel was chosen from biocompatible hydrogel materials. The alginate gel is inexpensive, easy to prepare, and easily removable comparing with other biocompatible hydrogel materials such as photocrosslinkable gelatin (GelMA) [29,30] and polyethylene glycol diacrylate [31], and the thickness can be controlled [32]. In addition, cell adhesion or non-adhesion to alginate gel can be controlled by changing the crosslinking ions [33].…”