Stempro human adipose derived stem cells

Four cryovials of StemPro^™ Human Adipose-derived Stem Cells (#R7788155) from a single donor were purchased from Thermo Fisher Scientific (Cambridge, MA, USA). The cells were thawed rapidly at 37 °C, pooled, and divided equally to initiate the described serial cultures with different growth factor supplements.

StemPro® Human Adipose-Derived Stem Cells (#R7788-115, Gibco™, Thermo Fischer Scientific) derive from human adipose tissue collected during liposuction procedures and were cryopreserved at passage 1 from primary cultures. Each lot of hASCs originates from a single donor of human lipoaspirate tissue, and the two previously characterized lots used here [7] (link), are named hASCs-1 and hASCs-2. In some experiments, we used hASCs derived from 13 different donors, after a signed informed consent was obtained [19] (link).
These cells were generally grown in MesenPRO RS™ medium (Gibco™, Thermo Fischer Scientific), containing 5 mM glucose and 2% fetal bovine serum (FBS) complemented with MesenPRO RS™ growth supplement and 2 mM L-glutamine prior to use. To avoid undesired phenotypic effects, cells were grown in the absence of antibiotics [20] (link).
To induce adipogenic differentiation, confluent hASCs were incubated for 21 days with StemPro® Adipogenesis Differentiation Kit (Gibco™), as previously described [7] (link).

The human ADSCs used in the present study were purchased from StemPro (StemPro Human Adipose-Derived Stem Cells, Gibco BRL, Thermo Fisher Scientific, Waltham, MA, USA). Human ADSCs were cultured in K-NAC medium for ADSC expansion per the protocol presented in our previous papers [31 (link),32 (link)]. The K-NAC medium comprised a keratinocyte SFM basal medium (Gibco BRL, Rockville, MD, USA) supplemented with 25 mg of bovine pituitary extract (Gibco BRL), 2.5 µg of human recombinant epidermal growth factor (Gibco BRL), 2-mM N-acetyl-L-cysteine, 0.2-mM L-ascorbic acid 2-phosphate sesquimagnesium salt, and 5% fetal bovine serum (FBS) [31 (link),32 (link)]. The cells were cultured and expanded in a humidified atmosphere with 5% CO₂ at 37 °C until a sufficient number of cells was acquired.

Human adipose-derived stem cells (hASCs; STEMPRO® Human Adipose-Derived Stem Cells; Invitrogen, Carlsbad, CA, USA) were used in this experiment. The hASCs were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS, 10 ng/mL epidermal growth factor, 10 ng/mL basic fibroblast growth factor, 100 U/mL penicillin, and 100 μg/mL streptomycin, and were used at passage 6–8 in the experiments.

hADSCs (StemPro^® Human Adipose-Derived Stem Cells, R7788-115, Invitrogen, USA) were generously provided by Gwo Xi Stem Cell Applied Technology. The hADSCs were suspended in the medium and seeded into scaffolds at a density of 1 × 10⁶ cells/scaffold. The scaffolds with hADSCs were placed in a 24-well culture plate for 24 h at 37 °C under 5% CO₂ for cell adhesion and transferred to a functionally closed process bioreactor system. The medium was circulated at an initial pump setting of 1 mL/min using a peristaltic pump. The hADSCs spheres were obtained from Ca-Ag scaffolds by dissolving the scaffolds in 50 mM EDTA (324503, Sigma, USA) solution for 5 min at 37 °C.

STEMPRO™ Human Adipose-Derived Stem Cells (Invitrogen, Grand Island, NY) were used in this study. ADSCs were cultured in ADSC basal medium consisting of MesenPROTM RS (Gibco, Carlsbad, CA) with GlutaMAXTM-I (Gibco).