Patients
Ten healthy patients (5 males and 5 females) between 27 and 59 years of age had elective liposuction (i.e., fat harvesting) in the Department of Plastic, Reconstructive and Hand Surgery – Burns Center at RWTH Aachen University Hospital. Each patient signed the consent form. The protocol and the use of human material were approved by the ethics committee of the Faculty of Medicine at RWTH University in Aachen, Germany (Name in German: Ethik-Kommission des Universitätsklinikums Aachen, Votum Number: EK163/07). The experiments were conducted in compliance with the Declaration of Helsinki Principles.
Liposuction and centrifugation of the obtained lipoaspirate
Fat was harvested via tumescent liposuction, as described previously [5, 7]. The harvesting cannula used in this study was the st’RIM cannula (Thiebaud Biomedical Devices, Margencel, France), which was developed by Guy Magalon for micro-lipografting. This cannula was 2 mm in diameter with a blunt tip and four 600-μm gauge orifices. After the liposuction procedure, the samples were centrifuged for 3 min at 3,000 rpm using a Sigma 2–16 K centrifuge (Osterode am Harz, Germany). After centrifugation, the purified lipoaspirate was immediately used in experiments (Figure 1).
Isolation of ASCs from lipoaspirate
Isolation of the cellular pellet was performed as described previously [5]. Briefly, the purified lipoaspirate was transferred into a sterile tube and normal saline was added to remove cell debris and blood. A second centrifugation process was then completed for 10 min at 300 × g. The extracellular matrix was digested with 0.075% collagenase I (Biochrom, Berlin, Germany) for 45 min at 37°C. The digested tissue solution was subsequently filtered using a 250 μm filter (Neolab, Heidelberg, Germany). The pellet was resuspended in 30 ml of a NaCl solution and centrifuged for another 10 min at 300 × g to obtain the SVF, which contained the ASCs. The pellet was then resuspended in DMEM/F12 supplemented with 100 U/ml of penicillin and 100 μg of streptomycin, without foetal calf serum or proliferation factors. The isolated cells were not cultured or passaged prior to direct transplantation onto the collagen and elastin matrix.
Incubation of ASCs on collagen and elastin matrices to assess cellular adherence
Circular 1 mm thick pieces of non–cross-linked native bovine collagen and elastin matrix containing type I, III, and V collagen derived from bovine skin were used (Matriderm® sheet; MedSkin Solutions Dr. Suwelack AG, Billerbeck, Germany). A circular punch biopsy device measuring 0.8 cm in diameter was used to cut the matrix into small pieces, which were placed into 48-well culture plates. The isolated cells were added to 48-well culture plates lined with collagen and elastin matrix at a density of 50,000 cells per well and incubated at 37°C with 5% CO2 for 1, 3 or 24 h. The collagen and elastin matrix that was incubated with the isolated cells was separated after a 1-, 3- or 24-h incubation period (Figure 1). The matrices were washed carefully with normal saline (0.9% NaCl) and transferred into a clean culture Plate. A 270 μl volume of DMEM/F12 supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin was added, and 30 μl of the alamarBlue® resazurin reagent (AbD Serotec, Oxford, UK), was subsequently delivered to each well. This assay can be used to detect the metabolic activity of cells using a fluorescence spectrometer. The medium/alamarBlue® mix was repeated and carefully removed from the well after 2 hours at 37°C and 5% CO2. The samples were then measured at room temperature using a Fluostar Optima fluorescence spectrometer (BMG Labtech, Offenburg, Germany), with an excitation wavelength of 540 nm and an emission wavelength 590 nm. We avoided measuring the matrix itself to avoid the influence of the matrix on fluorescence. The alamarBlue® reagent was added to the medium without cells as a negative control.
Analysis of cellular attachment and migration by histology
Cell-loaded pieces of collagen and elastin matrix were histologically investigated directly after cellular transplantation onto the matrix. The samples were fixed overnight in Lidi’s 4% formalin (Merck, Darmstadt, Germany). The formalin was then removed by extensive washing, and the samples were dehydrated using an increasing gradient of isopropanol, embedded into paraplast, and cut into 15 μm sections. Staining was then performed using hematoxylin and eosin. Microscopic analyses of all samples were performed via light microscopy in our laboratory.
Analysis of cellular distribution on collagen and elastin matrices using two-photon microscopy
Two-photon microscopy using an FV1000MPE microscope (Olympus Corp., Tokyo, Japan) attached to a pulsed Ti-Sapphire laser (MaiTai DeepSee, SpectraPhysics, Santa Clara, CA, USA) was performed to visualise the 3D-structure of the collagen and elastin matrices, including the organisation of the isolated cells within the matrix. Hoechst 33342 was added for vital staining of the nuclei. The enriched collagen and elastin matrices were also stained with fluorescein diacetate (FDA) to image the isolated cells in combination with Hoechst 33342 for nuclei staining. The enriched collagen and elastin matrix was then visualised using the non-linear optical effect of second harmonic generation (SHG). Hoechst was excited at 730 nm and detected at 418–468 nm. Series of subsequent 1024 × 1024 pixel xy-frames were then obtained in 1 mm z-steps for structural 3D reconstruction using Imaris Software (Bitplane, Zurich, Switzerland).
Statistical analysis
Data analysis was performed using Prism® software, version 5.01c (GraphPad, La Jolla, CA, USA). A Gaussian distribution of the values was assessed using the D’Agostino & Pearson omnibus normality test. One-way repeated measures ANOVA was performed followed by an appropriate post-hoc multiple comparison test (i.e., Tukey method). Differences for which p < 0.05 were considered statistically significant.
