*2.6. Cell Preparation*

Three different cell types, including human adipose-derived stromal cells (hASCs), L929 mouse fibroblasts, and SaOS-2 human osteoblast-like cells, were chosen to investigate their activities while culturing on the developed SF:eADF4(C16) hydrogels. Primary hASCs were used as a model for tissue engineering applications. L929 and SaOS-2 were selected to represent normal and tumor cells, respectively.

Human ASCs were isolated from subcutaneous fat tissues collected from female participants enrolled to Chulalongkorn Memorial Hospital, Thailand for laparotomy with an approval from institutional ethic committee on human research, Faculty of Medicine, Chulalongkorn University (project no. 416/61). Isolation and culture procedures were conducted following established protocols [17,18]. Briefly, 10–15 g fat tissue was washed with PBS before enzymatically digested using 0.1% collagenase type II (Gibco, New York, NY, USA) supplemented with 1% bovine serum albumin at 37 ◦C with continuous shaking for 1 h. The digested specimen was then centrifuged, and the upper oil layer was removed. The bottom dark brown layer, known as stromal vascular fraction (SVF), was collected, resuspended with PBS and centrifuged. After that, SVF was resuspended with culture medium (DMEM/F12 + 10% FBS + 1% antibiotics) and transferred to a T-75 tissue culture flask. The culture was maintained at 37 ◦C with fed air supplemented with 5% CO2.

After initial plating for 48 h, cells were washed with PBS to remove unattached cells and refed with the new medium. Typically, hASCs reached 80–90% confluency within 2 weeks. Subculture was performed using TrypLE Express enzyme (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's advice with a subculture ratio of 1:2.

L929 and SaOS-2 were cultured in DMEM supplemented with 10% FBS and 1% antibiotics. The cells were maintained in a CO2-incubator, and the subculture was treated using trypsin (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's protocol.
