9 days after hormonal induction of adipogenesis, 2-D and 3-D cells, respectively, were harvested under appropriate conditions according to the specific protocols described below in this section. To allow for separate investigation of the outer and inner areas of the cell-polymer constructs, entire constructs were aseptically divided by die-punching with a sterile stainless-steel dermal punch 3 mm in diameter (Aesculap; Tuttlingen, Germany). Thereby, a ring consisting of external tissue as well as a disc composed of internal areas was produced.
Unless otherwise stated, subsequent analysis was independently carried out for entire constructs, outer rings and inner discs.
Histology:
Complete generated fat constructs were fixed overnight in 10% buffered formalin, dehydrated and then embedded in paraffin. To assess cellularity of different areas, deparaffinized sections of 5 µm were stained with hematoxylin and eosin (H&E) (Sigma-Aldrich; Deisenhofen, Germany). Due to lipid extraction from the cells by organic solvents used in routine staining procedures, examination of lipid storage was carried out with oil red O pre-stained specimen. For oil red O staining of cytoplasmic triglyceride droplets, 2-D cells, as well as complete 3-2-D cell-polymer constructs, were washed with PBS.
Subsequently, they were subjected to an overnight fixation step in 10% buffered formalin.
The fixed 2-D and 3-D samples were stained as described [10], but only the 3-D specimen were mounted in Tissue Tek (Sakura Finetek; Torrane, CA, USA). Finally, they were rapidly frozen and cut with a 2800 Frigocut E cryostat (Cambridge Instruments GmbH; Nussloch, Germany) in 12 µm sections, which could directly be investigated.
Immunohistological detection of the ECM component laminin was performed by staining of deparaffinized sections, 5 µm thick, with a rabbit anti-mouse laminin antibody (Novus Biologicals, Inc.; Littleton, CO, USA). The staining procedure was conducted as follows: Endogenous peroxidase activity was blocked by incubation in 1% H2O2. After extensive rinsing, antigen retrieval was performed by pepsin digestion. To prevent non-specific antibody binding, sections were incubated with 5% normal horse serum (Vector
Laboratories Inc.; Burlingame, CA, USA) in PBS. Subsequently, the primary antibody was applied at a dilution of 1:600 (in PBS with 5% horse serum) for 60 min at room temperature (RT). In control sections, the primary antibody was replaced by PBS with 5% normal horse serum. After washing with PBS, the sections were incubated with biotinylated horse anti-rabbit IgG antibody (1:100) (Vector Laboratories Inc.; Burlingame, CA, USA) for 30 min at RT. To form the streptavidin-biotin-peroxidase complex, the Vectastain Elite ABC-Kit was used. Peroxidase localization was performed with the DAB Substrate Kit for Peroxidase. Both kits were obtained from Vector Laboratories Inc.; Burlingame, CA, USA. Finally, the sections were counter-stained with hematoxylin and mounted in DPX Mountant (Fluka, Biochemika;
Taufkirchen, Germany).
Scanning electron microscopy (SEM):
Entire cell-polymer constructs were washed with PBS. Afterwards, they were fixed for 15 min with 2.5% glutardialdehyde in PBS and for 30 min with 1% osmium tetroxide (Carl Roth GmbH & Co.; Karlsruhe, Germany). After extensive rinsing and freezing at –80°C, samples were subjected to lyophilization (Christ Beta 2-16, Martin Christ Gefriertrocknungsanlagen; GmbH, Osterode am Harz, Germany). For differential examination of internal and external areas, lyophilized samples were halved (Fig. 3) and mounted on aluminum stubs using conductive carbon tape and coated with gold - palladium (Polaron SC515, Fisons surface systems; Grinstead, UK). All micrographs were obtained at 10 kV on a DSM 950 (Zeiss; Oberkochen, Germany).
Glycerol-3-phosphate dehydrogenase (GPDH) measurement:
2-D cells and 3-D cell-polymer constructs (entire and die-punched) were washed with PBS, harvested in lysis buffer (50 mM Tris, 1 mM EDTA, 1 mM mercaptoethanol, pH 7.5), and sonicated afterwards with a digital sonifier. The GPDH assay was performed as described in the literature [11,12]. Cytosolic protein concentration was determined after precipitation with trichloracetic acid using the Lowry assay [13]. GPDH activity was normalized to protein content and is expressed as mU per mg protein.
Analysis of triglyceride (TG) content:
To facilitate the determination of TG content, 2-D cell cultures and die-punched samples of 3-D cell-polymer constructs were washed with PBS, harvested in 0.5% thesit, and sonicated with a digital sonifier (Branson Ultrasonic Corporation, Danburg, CT, USA).
Subsequently, spectroscopic quantification of TG was performed using the enzyme coupled glycerol assay kit 337 (Sigma Diagnostics; Deisenhofen, Germany). Cytosolic protein concentration was determined after precipitation with trichloracetic acid using the Lowry assay[13]. TG content was normalized to protein content and is expressed as mg TG per mg protein.
Reverse transcription polymerase chain reaction (RT-PCR):
Subsequent to washing in PBS, harvest of 2-D and 3-D derived cells (from entire and die-punched constructs) was performed with Trizol reagent (Invitrogen GmbH; Karlsruhe, Germany). Total RNA was isolated according to manufacturers instructions.
Table 1: Primer sequences and PCR conditions for the investigated genes
Oligonucleotide sequences of mouse forward (sense) and reverse (antisense) primers. Gene amplification was performed by PCR according to the specified annealing temperatures (AT) and number of cycles for each gene. The reaction conditions of one cycle were composed as follows: denaturation for 45 sec at 94°C, annealing for 45 sec at the indicated temperatures, and extension for 1 min at 72°C.
Gene Forward and reverse primers of examined genes AT (°C) / Cycles PPARg 5´-AAC CTG CAT CTC CAC CTT ATT ATT CTG A-3´
5´-GAT GGC CAC CTC TTT GCT CTG CTC CTG-3´ 60 / 35 GLUT-4 5´-CCC CGC TGG AAT GAG GTT TTT GAG GTG AT-3´
5´-CAG ACA GGG GCC GAA GAT TGG GAG ACA GT-3´ 61 / 35 laminin-b1 5´-GCT GGA TCC GCT TGC AGC AGA GTG CAG CTG A-3´
5´-CGC GAA TTC GCT AAG CAG GTG CTG TAA ACC G-3´ 60 / 30 angiotensinogen 5´-CTG GCC GCC GAG AAG CTA GAG GAT GAG GA-3´
5´-GAG AGC GTG GGA AGA GGG CAG GGG TAA AGA G-3´ 62 / 35 beta3-AR 5´-CAG TGG TGG CGT GTA GGG GCA GAT-3´
5´-CGG GTT GAA GGC GGA GTT GGC ATA G-3´ 63 / 36 18 S 5´-TCA AGA ACG AAA GTC GGA GGT TCG-3´
5´-TTA TTG CTC AAT CTC GGG TGG CTG-3´ 60 / 22
First-strand cDNA was synthesized from total RNA by using random hexamers (Roche Diagnostics; Mannheim, Germany) and Superscript II RNase H- Reverse Transcriptase (Invitrogen GmbH; Karlsruhe, Germany). For reverse transcription, samples were incubated at 42°C for 50 min and heated afterwards for 15 min at 70°C to inactivate the enzyme.
Subsequently, PCR was performed with Sawady Taq-DNA-Polymerase (PeqLab; Erlangen, Germany); initial denaturation occurred at 94°C for 120 sec, final extension at 72°C for 30 sec, and holding at 4°C. The amplification was carried out using specific primers and appropriate conditions for each gene (Tab. 1). 18 S rRNA served as internal control. Reverse transcription and PCR were performed using a Mastercycler Gradient (Eppendorf AG;
Hamburg, Germany). The PCR products were analyzed by electrophoresis on 2% agarose gels containing ethidium bromide, followed by imaging and densitometric scanning of the resulting bands using UV light (l = 312 nm) and a Kodak EDAS 290 (Fisher Scientific;
Schwerte, Germany).