Preadipocyte basal medium 2

After printing or casting, ASC spheroid-laden constructs were cultured in an adipogenic differentiation medium consisting of Preadipocyte Basal Medium-2 (Lonza, Basel, Switzerland) supplemented with 10% FBS, 1% penicillin/streptomycin, and the hormonal inducers insulin (final concentration 1.7 µM; PromoCell, Heidelberg, Germany), dexamethasone (1 µM; Sigma-Aldrich, St. Louis, MO, USA), 3-isobutyl-1-methylxanthine (IBMX, 500 µM; Serva-Electrophoresis, Heidelberg, Germany) and indomethacin (200 µM; Sigma-Aldrich, St. Louis, MO, USA). Constructs were cultured for up to 21 days, with media exchange every other day.

The human pre-adipocyte and mouse fibroblast cell line (L929) were purchased from Lonza (Basel, Switzerland) and American Type Culture Collection (ATCC; VA, USA), respectively. Pre-adipocytes were cultured on tissue culture polystyrene (TCPS; SPL Life Science, Gyeonggi-do, Republic of Korea) dishes in Pre-adipocyte Basal Medium-2 (PBM-2, Lonza). L929 was cultured on TCPS in Dulbecco’s modified Eagle’s medium-low glucose (DMEM; Gibco, MA, USA) with supplementation of fetal bovine serum (10% v/v, Gibco) and penicillin–streptomycin (1% v/v, Gibco). When cells were propagated until they reached 80% confluence, they were passaged by detachment using 0.25% trypsin/ethylenediaminetetraacetic acid (Gibco).
Sprague-Dawley rats (8 weeks old and male) and BALB/c nude mice (6 weeks old and male) were purchased from Orient Inc. (Gyeonggi-do, Republic of Korea). The New Zealand white rabbits were purchased from DooYeol Biotech (Seoul, Republic of Korea). The Institutional Animal Care and Use Committee of the Yonsei Laboratory Animal Research Center (permit no. 2019-0205 and 2020-0071) and Korea Testing & Research Institute (permit no. IAC2022-1336) approved all animal protocols.