Human adipose-derived stem cells (ADSCs)

ADSCs were isolated from human subcutaneous adipose tissue obtained during abdominal plastic surgery from healthy women or men aged 17 - 69 years as previously described in chapter 3 (Page 47).

For 2-D monolayer cultures, cells were seeded in basal medium (PBM-2 supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 µg/ml)) at a density of 30,000 cells/cm². At confluence, cells were induced to undergo adipogenesis by exchanging half of the medium with basal media supplemented with 1.7 µM insulin, 1 µM dexamethasone, 0.5 mM IBMX, and 200 µM indomethacin (final concentration). Different concentrations of EDHB (0-200 µM) were added to the medium. The time point of induction was referred to as day 0 throughout all experiments. Both inducers and EDHB were present during the entire

Chapter 5 Effect of EDHB on Adipogenesis in 2-D and 3-D Culture

adipogenic period. Medium was exchanged every other day up to day 14. Feeding was performed by exchanging only half of the medium volume in order to treat cells in the same way as in 3-D culture (see 3.2.). Cells were incubated in a humidified atmosphere at 37°C and 5% CO2.

3.2. 3-D cell culture

Spheroids were generated according to the liquid overlay technique (Figure 1) [13,14].

Figure 1: Generation of spheroids utilizing the liquid overlay technique (from Weiser B. [14]).

Initially, 3T3-L1 preadipocytes were expanded in conventional 2-D cell culture. Then, cells were seeded into agarose-coated 96-well plates. Cells cannot adhere to the agarose surface, but, supported by the meniscus of the agarose layer and the agitation on an orbital shaker, cells accumulated in the center of the well, attached to each other and formed one multicellular spheroid per well.

Tissue culture polystyrene (TCPS) 96-well plates were coated with 50 µl of 1.5% agarose in α-MEM per well [31]. Spheroid formation was initiated by seeding cells in basal medium (3.1.1 and 3.1.2) into the agarose-coated wells (Figure 1). The outermost rows and columns of the well plate, respectively, were not used for culture due to the increased evaporation of medium from the wells during prolonged culture; they were only filled with PBS supplemented with penicillin (100 U/ml) and streptomycin (100 µg/ml). Two days after seeding, cells were induced to undergo adipogenesis by exchanging half of the medium with induction medium (see 3.1.1 and 3.1.2). Feeding was performed by exchanging only half of the medium volume, otherwise spheroids would be aspirated with complete removal of the medium from the wells. The induction medium was applied either without or with EDHB (50, 100, 200 µM). For 3T3-L1 preadipocytes, adipogenic induction was performed for another two days. Then half of the medium was exchanged with differentiation medium (see 3.1.1).

This medium exchange was performed three times to further reduce the concentrations of inducers. Because ADSCs were cultured in the presence of inducers during the entire adipogenic phase, this washing step was not necessary. For either cell type, subsequently, cells were fed by exchanging half of the medium with differentiation medium every other day

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(see 3.1.1 and 3.1.2). Spheroid cultures were incubated in a humidified atmosphere at 37°C and 5% CO2 on an orbital shaker at 60 rpm (Heidolph, Schwabach, Germany).

During the present study, the 3-D spheroids of ADSCs consisted of 3000 cells per aggregate.

Notably, the size of the 3-D spheroids of 3T3-L1 was changed. The initial experiments for studying the effect of EDHB on adipogenesis as well as the cytotoxicity caused by EDHB were performed with spheroids consisting of 750 cells per aggregate (Figure 3, 4, 7, and 8, Page 116, 117, 120 and 121). Previous studies showed that this cell aggregate size ensured the generation of homogeneous adipocyte constructs due to a sufficient supply of nutrients, oxygen, and adipogenic inducers. However, this small spheroid size generates disadvantages especially concerning the practical handling. For certain analyses, a higher cell number is necessary for quantification. Hence, in the following experiments (Figure 9, Page 123) larger spheroids were generated by seeding 3000 cells per well. In Figure 2, spheroids consisting of 750 or 3000 cells per construct are compared. Spheroids grew significantly up to day 9 and the relative increase in spheroid size was comparable in both groups (Figure 2 A). Adipogenic maturation of the cells occurred homogeneously throughout both spheroids until day 9 after induction as detected by oil red O staining (Figure 2 B). A gradient in cellular TG accumulation from the outside to the center area of the spheroids was not observed. Small and large spheroids did not differ in the TG content per cell (Figure 2 B). For the spheroids consisting of 750 cells per aggregate, a reducing effect of EDHB on spheroid volume was described (Figure 3 A and C, Page 116). This effect was also observed with the spheroids composed of 3000 cells. In presence of 100 µM EDHB, the increase of the spheroid volume was suppressed (Figure 2 C).

In conclusion, these results revealed that the adipogenic development was independent from spheroid size. Furthermore, the larger spheroids did not alter the effect of EDHB and therefore, the change of the use of spheroids consisting of 3000 cells was uncritical.

Chapter 5 Effect of EDHB on Adipogenesis in 2-D and 3-D Culture

Figure 2: Comparison of different sizes of 3T3-L1 spheroids with regard to morphology, TG accumulation and effect of EDHB at day 9 after induction. 750 and 3000 cells were seeded per 96-well and induced to undergo adipogenesis. A) Phase contrast images of 3-D spheroids of differentiated 3T3-L1. On the right, kinetics of spheroid growth are displayed (n=10). B) Oil red O staining of intracellular lipid droplets in cryosections of 3-D spheroids. Sections from the center area are displayed. The intracellular TG contents were quantified (n=3) and normalized to cell numbers (on the right). C) Phase contrast images of 3-D spheroids treated without or with 100 µM EDHB.

Development of spheroid sizes is shown on the right. Equivalent spheroid volumes at different time points of differentiation in absence or presence of EDHB were quantified morphometrically from n=10 randomly selected spheroids. Statistically significant differences are denoted by * (p<0.05).

Three independent cell culture experiments were performed (A-C); representative photographs from one experiment are shown. Bar=100 µm.

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