Chemically Defined Xeno- and Serum-Free Cell Culture Medium to Grow Human Adipose Stem Cells

Abstract

Adipose tissue is an abundant source of stem cells. However, liposuction cannot yield cell quantities sufficient for direct applications in regenerative medicine. Therefore, the development of GMP-compliant ex vivo expansion protocols is required to ensure the production of a “cell drug” that is safe, reproducible, and cost-effective. Thus, we developed our own basal defined xeno- and serum-free cell culture medium (UrSuppe), specifically formulated to grow human adipose stem cells (hASCs). With this medium, we can directly culture the stromal vascular fraction (SVF) cells in defined cell culture conditions to obtain hASCs. Cells proliferate while remaining undifferentiated, as shown by Flow Cytometry (FACS), Quantitative Reverse Transcription PCR (RT-qPCR) assays, and their secretion products. Using the UrSuppe cell culture medium, maximum cell densities between 0.51 and 0.80 × 105 cells/cm2 (=2.55–4.00 × 105 cells/mL) were obtained. As the expansion of hASCs represents only the first step in a cell therapeutic protocol or further basic research studies, we formulated two chemically defined media to differentiate the expanded hASCs in white or beige/brown adipocytes. These new media could help translate research projects into the clinical application of hASCs and study ex vivo the biology in healthy and dysfunctional states of adipocytes and their precursors. Following the cell culture system developers’ practice and obvious reasons related to the formulas’ patentability, the defined media’s composition will not be disclosed in this study.

Human Adipose Stem Cells (hASCs) Grown on Biodegradable Microcarriers in Serum- and Xeno-Free Medium Preserve Their Undifferentiated Status

“…The production of the microcarriers was extensively described in Muoio et al [ 19 ]. Briefly, the manufacturing process relies on a double emulsion solvent evaporation technique where the most critical components for the manufacture are 17 KDa Poly(D, L-lactide-co-glycolide) (PLGA) (lactide/glycolide ratio 50:50), Purasorb PDLG 5002 purchased from Corbion Purac (Amsterdam, Netherlands).…”

“…The donors of the subcutaneous adipose tissue used in this study provided written agreement as required from the local Ethics Committee of the Canton of Ticino, which approved the project and its procedures (project reference number: CE 2915). The surgical removal of adipose tissue was described by Panella et al [ 19 ]. Adipose tissue samples were stored at room temperature and processed at the latest 24 h after their collection [ 20 ].…”

“…Isolation of the cells of the SVF from human adipose tissue, in vitro culturing in defined xeno- and serum-free conditions, cell cryopreservation, followed the ethical principles traced in the Declaration of Helsinki and according to the instructions of the Ethics Committee of the Canton of Ticino (Switzerland). The protocol we routinely use to extract the SVF is described precisely by Panella et al [ 19 ]. We briefly summarize the essential parameters here: the adipose tissue was homogenized in a blender then digested for 45 min at 37 °C with 0.28 Wünsch Unit/mL of Collagenase AB (Worthington Biochemical Corp., Lakewood, NJ, USA).…”

“…Freshly isolated cells of the SVF were stained for analytical flow cytometry as described by Panella et al [ 19 ]. Briefly, the cells were incubated 20 min with the following monoclonal mouse anti-human antibodies and fluorescent dyes: CD34-BV650, CD45-PC7, CD73-FITC (BioLegend, San Diego, CA, USA), CD146-PE, CD36-APC (Miltenyi BioTech, Bergisch, Germany), 7-amino-actinomycin D (7-AAD) (Becton Dickinson, Franklin Lakes, NJ, USA), and Syto40 (Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA).…”

“…The medium was replaced every 2 days, keeping 50% of it, until the cells were between 80% and 90% confluent. In the Supplementary Information of Panella et al [19], we describe in detail the initial plating of the cells (Section 3), and we give some suggestions on how to work in defined serum-free conditions (Section 4). For passaging, the cells were detached with TrypLE TM Select (Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA) after 2 min of incubation at 37 • C. The hASCs were washed with DPBS at least once to remove the protease, counted, resuspended in UrSuppe medium, and finally seeded in new cell culture vessels (Corning PureCoat™ ECM Mimetic Fibronectin Peptide, Corning Inc., Corning, NY, USA) at a density of 10,000 hASCs/cm 2 .…”