Hascs

hASCs were purchased from Lonza (Switzerland) and cultured in low-glucose Dulbecco's modified Eagle's medium (DMEM; Welgene, South Korea) supplemented with 10% fetal bovine serum (Biowest, France) and 1% penicillin/streptomycin (Gibco, USA). The medium was changed every two days. To compare the fabricated cell aggregates (experimental group), conventional hASCs spheroids (control group) were fabricated via the hanging drop method 6 (link). Briefly, 1 × 10⁵ hASCs suspended in 20 µL of medium were dropped onto the inside of the lid of a cell culture plate (SPL, South Korea). The lid of the plate was then gently inverted over a cell culture dish filled with phosphate-buffered saline (PBS) and cultured for three days to generate cell spheroids.

Human adipose-derived stem cells (hASCs) were purchased from Lonza (PT-5006, Lot.0000605220). hASCs were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco^®, Thermo Fisher Scientific, lnc., Walthan, MA, USA) with 10% Fetal Bovine Serum (FBS, Gibco^®, Thermo Fisher Scientific, lnc., Walthan, MA, USA) and 100 units/mL of penicillin–streptomycin (Gibco^®) in a humidified 37 °C incubator with 5% CO₂. hASCs (passage 5) were seeded at a density of 1 × 10⁵ cells/well with culture media onto a 12-well plate. After 24 h of culture, the culture medium was replaced with an extract medium which was eluted for 24 h by soaking each bone graft. The control groups were exposed only to standard culture medium. After 24 or 48 h of incubation, cell viability was evalutated using a CCK-8 assay (Dojindo Molecular Technologies, Inc., Rockville, MD, USA). CCK reagent was added per well and incubated at 37 °C for 3 h. The absorbance values were measured at 450 nm using a microplate reader (Epoch™ Microplate Spectrophotometer, BioTek Instruments, Inc., Winooski, VT, USA). All experiments were performed in triplicate wells for each group.

hASCs were purchased from Lonza Walkersville, Inc. (Walkersville, MD, USA); the cells were derived from a female donor (non-diabetic, BMI 26 kg/m², 36 years old) (PT5006 lot 0000410257). The cells (passage 4) were pre-cultured as described by Yamada et al. [33 (link)]. Briefly, 2 × 10⁴ cells/mL of the cells were cultured in 50% Dulbecco’s modified Eagle’s medium (DMEM)/50% α minimum essential medium supplemented with 1% fetal bovine serum (FBS), 1 × ITS, 10 ng mL^-1 bFGF (PeproTech Inc., NJ, USA), and 400 ng mL^-1 of hydrocortisone on a 24-well plate for 3 days, until cells became confluent. To induce adipocyte-differentiation, post-confluent preadipocytes were stimulated in MDI differentiation medium (DMEM containing 10% FBS, 1 μM DEX, 0.5 mM IBMX, 0.2 mM indomethacin, 10 μg mL^-1 insulin, and 33 μM biotin) for 7 days (Day 0–7). During cultivation, the culture was replaced with fresh MDI differentiation medium every 2 days with or without Euglena extract. Then, cells were maintained for a further 7 days (Day 7–14) in adipocyte nutrition medium (DMEM containing 10% FBS, 10 μg mL^-1 insulin, and 33 μM biotin), which was replaced with fresh culture every 2 days. All cells were cultured at 37°C under a 5% CO₂ atmosphere. All the above reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).