Preparation of EpH4 and 3T3L1 cells aggregates incorporating gelatin hydrogel microspheres for a cell condition improvement

The objective of this study is to prepare three dimensional (3D) of mouse mammary epithelial EpH4 and mouse preadipocyte 3T3L1 cells in the presence of gelatin hydrogel microspheres (GM) and evaluate the effect of GM presence on the survival and functions of cells in the 3D cell aggregates. Gelatin was dehydrothermally crosslinked at 140 °C for 48 h in a water-in-oil emulsion state to obtain the GM with average diameters of 50 and 200 μm, followed by treatment with fibronectin (FN). EpH4 and/or 3T3L1 cells were cultured with or without the FN-treated GM in round U-bottom wells of 96-multiwell culture plates which had been coated with poly (vinyl alcohol) (PVA) to allow the cells to form their aggregates. On the other hand, EpH4 cells were precultured with the FN-treated GM, and then continued to culture with 3T3L1 cells in the same condition described above. The EpH4 cells attached onto the GM in the cell number dependent manner, irrespective of their size. When 3T3L1 cells were incubated with the original and GM-preincubated EpH4 cells in the presence of both the FN-treated GM, the number of alive cells in the aggregates was significantly high compared with that for the absence of FN-treated GM. In addition, higher β-casein expression level of EpH4 cells in EpH4/3T3L1 cells aggregates in the presence of FN-treated GM was observed than that of cells in the absence of FN-treated GM. Laminin secretion was also promoted for the cells aggregates cultured with FN-treated GM. It is concluded that the presence of FN-treated GM in the EpH4/3T3L1 cells aggregates gave a better condition to cells, resulting in an enhanced generation of β-casein from EpH4 cells in the aggregates.