Claudia Fischbach, Michael Hacker, Barbara Weiser, Torsten Blunk, Achim Göpferich
Department of Pharmaceutical Technology, University of Regensburg, 93040 Regensburg, Germany
To be submitted to Cells Tissues Organs
Introduction
Donor shortage problems have led to the emergence of the interdisciplinary field of tissue engineering (TE), which pursues the creation of biologically compatible substitutes to restore, maintain, or improve diseased tissue functions [1]. Originally intended to be used for transplantation and reconstructive purposes, TE approaches are increasingly recognized as a means of providing valuable three-dimensional (3-D) model systems applicable for basic research in vitro. Growing cells in a 3-D environment, e.g. in highly porous polymer scaffolds or gel systems, may lead to the recreation of the particular cell-cell and cell-extracellular matrix (ECM) interactions present within native tissues. Consequently, an environment resembling in vivo conditions can be imitated more thoroughly than in conventional two-dimensional (2-D) cell culture. Thereby, comprehensive investigations of cellular features characteristic for native tissues are enabled in vitro.
In recent years, a number of studies has been performed with the objective of demonstrating the advantages of 3-D cell culture techniques over conventional 2-D methodologies. For instance, it has been shown that dedifferentiated cartilage cells, yielded from monolayer proliferation, regained their differentiated phenotype when cultivated in a 3-D environment [2,3]. In these specific studies, the application of TE strategies proved to be beneficial to overcome the intrinsic drawbacks of conventional 2-D cell culture. Basic research of adipose tissue represents another scope potentially benefiting from a tissue-engineered 3-D culture system. It has been described that in some cases that adipose cell functions observed in animal studies are impaired in vitro; e.g. in vivo production of TNFa and leptin by far exceed protein secretion in 2-D cell culture [4,5]. Apparently, essential factors provided by the physiological environment are seemingly absent from conventional 2-D cell culture and thereby lead to the development of an incomplete adipose phenotype. In order to adequately address the potential reasons, it may, therefore, be helpful to conduct the particular investigations in a 3-D culture system, which is suitable for analyzing inherent tissue properties.
We recently reported the establishment of a 3-D in vitro model of adipogenesis consisting of 3T3-L1 preadipocytes, induced to undergo adipose differentiation, and highly porous polymer scaffolds made from polyglycolic acid (PGA) [6]. Generation of the model system under dynamic conditions resulted in a coherent fat-like construct, which exhibited the appropriate adipose characteristics and, thus, implied the promise for being of use for basic
research purposes. The first promising indications of improved adipose properties compared to conventional 2-D cell culture were obtained through microscopic examination of the 3-D model. Thereby, large lipid-filled, nearly unilocular adipocytes rarely detectable in monolayer cultures could be observed. However, microscopic investigation of cross-sections prepared from the constructs uncovered considerable differences of the outer areas relative to the inner ones. For other in vitro engineered tissues such as bone, this observation has been made before [7,8] and is also reported for human preadipocyte seeded collagen sponges [9].
Nevertheless, neither of those studies addressed the observed heterogeneity more in detail.
Therefore, the focus of this study was not only to thoroughly characterize the 3-D model, but to also address the non-uniform construct composition. Due to the preliminarily determined heterogeneity, a method was developed enabling separate investigation of the different construct parts. This approach allowed for more detailed evaluation as the results, hypothesized to depend on the particular construct part, could be related to a clearly defined area of the generated tissues. In detail, adipose tissue constructs were generated from 3T3-L1 preadipocytes and 3-D polyglycolic acid (PGA) fiber meshes. Subsequently, they were divided into outer and inner parts via a stainless-steel dermal punch and the resulting construct parts were characterized in terms of key features of adipose tissue. Afterwards, the gained results were compared to those obtained from adipocytes cultured under conventional 2-D conditions. At first, histological examination of entire adipose tissue constructs was performed in order to comprehensively assess spatial differences. Subsequently, intracellular lipid accumulation was quantitatively determined by measurement of triglyceride content and activity of glycerol-3-phosphate dehydrogenase (GPDH), a key enzyme involved in triglyceride biosynthesis. Expression analysis of a variety of fat cell genes, known to be characteristic for the adipose phenotype, was carried out on the mRNA level. Finally, lipolysis was determined under stimulating and inhibiting conditions in order to investigate the functionality of the adipocytes derived from the distinct construct parts.
Materials and Methods Materials:
3T3-L1 preadipocytes were obtained from ATCC (Manassas, VA, USA). DMEM with 1.0 g/l glucose, fetal bovine serum (FBS), and trypsin (1:250) were purchased from Biochrom KG Seromed (Berlin, Germany); phosphate buffered saline (PBS) and penicillin-streptomycin
solution were from Life Technologies (Karlsruhe, Germany). a-MEM, corticosterone, indomethacin, and oil red O were from Sigma-Aldrich (Deisenhofen, Germany). 3-isobutyl-1-methylxanthine (IBMX) was purchased from Serva Electrophoresis GmbH (Heidelberg, Germany). Insulin was kindly provided by Hoechst Marion Roussel (Frankfurt a. M., Germany). Cell culture materials were obtained from Sarstedt AG & Co. (Nuembrecht, Germany) and BD Biosciences Labware (Heidelberg, Germany).
Spinner flasks were self-made (250 ml volume, 6 cm bottom diameter, side arms for gas exchange). Silicon stoppers were obtained from Schuber & Weiss (München, Germany);
needles were from Unimed (Lausanne, Switzerland). Polyglycolic acid (PGA) non-woven fiber meshes (12-14 µm fiber diameter; 96% porosity; 62 mg/cm3 bulk density) were purchased from Albany Int. Research Co. (Mansfield, MA, USA) and die-punched into discs 5 mm in diameter and 2 mm thick.
Cell culture:
3T3-L1 preadipocytes were expanded during 4 passages and frozen in liquid nitrogen.
Subsequent to defrosting, the cells were further expanded; cells from the fourth passage (following cryo-storage) were used for experiments. Growth of stock cultures was performed in DMEM supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (0.1 mg/ml).
For 2-D cell culture preadipocytes were plated on tissue culture polystyrene (TCPS) at a density of 5,000 cells per cm². Development of the 3-D model system was performed as previously described [6]. Briefly, expanded 3T3-L1 preadipocytes were dynamically seeded onto PGA scaffolds in stirred spinner flasks. Thereby, each flask comprised 8 scaffolds and 100 ml of a suspension containing 16x106 cells, i.e., 2x106 cells per scaffold. Stirring for two days at 80 rpm allowed for cell attachment to the polymer fibers. Cell-polymer constructs were transferred into 6-well plates (one construct and 5 ml culture medium per well) and cultured in the incubator (37°C, 5% CO2) dynamically on an orbital shaker at 50 rpm (Dunn Labortechnik GmbH; Asbach, Germany). Cultivation of the cell-seeded constructs was performed in MEM (alpha-modification) supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (0.1 mg/ml). Four days after plating (2-D) and seeding (3-D), adipogenesis was induced by adding 0.1 µM corticosterone, 0.5 mM IBMX, and 60 µM indomethacin to differentiation medium (a-MEM, 5% FBS, 1 µM insulin, penicillin (100 U/ml), and streptomycin (0.1 mg/ml)) and referred to as day 0. After 2 days, the induction medium was
replaced by differentiation medium alone. Cultures were maintained under these conditions until cell harvest, whereby the culture medium was changed every other day.