[Frontiers in Bioscience E3, 256-263, January 1, 2011]

Isolation and expansion of adipose-derived stem cells for tissue engineering

Trine Fink1, Jeppe Grondahl Rasmussen1,2, Pia Lund1, Linda Pilgaard1, Kjeld Soballe3, Vladimir Zachar1

1Laboratory for Stem Cell Research, Department of Health Science and Technology, Aalborg University, Denmark, 2Department of Pharmacology, Aarhus University, Denmark, 3Orthopaedic Research Laboratory, Aarhus University Hospital, Denmark

FIGURES

Figure 1. Absolute yield of nucleated cells and colony forming units from 5 different donors. (A) The yield of nucleated cells pr mL of adipose tissue from the use of different enzyme preparations. (B) The yield of colony-forming units pr mL of adipose tissue using different enzymes. B: Blendzyme, L1: Liberase H1, CCM: crude collagenase mix, cfu: colony-forming units.

Figure 2. Effect of serum, serum replacements and media on growth and osteogenic differentiation of human ASCs. The cells were cultured in DMEM/F12 supplemented with different amounts of serum or serum replacements (Panel A) or in in different basal media supplemented with 10% FCS (Panel B). The numbers of cells on day 8 were normalized to those cultured in DMEM/F12 10% FCS (Black bars). After culture in the respective media/sera the cells were induced to undergo osteogenesis. Deposition of calcium deposits were quantified and normalized to values from cells cultured in DMEM/F12 10% FCS (open bars). The data are presented as a mean + standard error of mean from five different donors. Asterisks denote statistically significant difference (p < 0.05) when compared to DMEM/F12 10% FCS. FCS: Fetal calf serum; SR1: Serum replacement 1; SR3: Serum replacement 3; KOSR: Knockout serum replacement; DMEM/F12: Dulbecco's Modified Eagle Medium Nutrient Mixture F-12; DMEM HG: Dulbecco's Modified Eagle Medium with 4,500 mg/ml glucose; DMEM LG: Dulbecco's Modified Eagle Medium with 1,500 mg/ml glucose; F12: Nutrient Mixture F-12; A-MEM: Alpha Modified Eagle Medium; LP02: LP02 complete medium.

Figure 3. The effect of reduced oxygen tension on the growth rate of ASCs. (A) Cell density was determined for short term cultures. The cells were seeded in 24-well plates, and half the cultures incubated in 5% oxygen (open circles), the other half at ambient oxygen tensions (closed circles). Cells were counted using picogreen. The experiments were performed in duplicate with cells from 5 donors. Error bars represent standard error of mean and asterisks denote a significant difference (p<0.05) (B) Cumulative population doublings were calculated for long term cultures cells from three different donors (donor a: circles; donor b: squares; donor c: triangles). The cells were cultured in T25 flasks for 35 days at either 21% oxygen (filled symbols) or 5% oxygen (open symbols). Cultures were continuously monitored, subcultured and counted when 90% confluent.