Institut für Ernährungswissenschaften

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Der Einfluss verschiedener Ölemulsionen und der IL-6-Typ-Zytokine CNTF und LIF auf die Differenzierung von 3T3-L1-Präadipozyten zu Fettzellen
(2009) Stäbler, Antje; Graeve, Lutz
Type 2 diabetes is a disease of civilization spreading all over the world like an epidemic. Among other things it is characterized by increased glucose and triglyceride levels of the blood. Thus the differentiation of preadipocytes into adipocytes that are specialized in storing large fat depots is essential for the removal of excess nutrients from the circulation. This process can be simulated through the differentiation of 3T3-L1 cells by treatment with insulin, IBMX, and dexamethasone. In this study a compensative effect of olive oil as well as soy bean oil on the differentiation of 3T3-L1 cells was found. The differentiation triggered by insulin, IBMX, and dexamethasone was somewhat inhibited by incubating the cells with one of the two oils, whereas both olive and soy bean oil had a certain potential to act as differentiating agents them-selves when insulin, IBMX, and dexamethasone were not added. This points to benefits of certain oils contained in food compared to thiazolidinediones that are used as antidiabetics and lead to weight gain in the long term. Furthermore the inhibitory effect of the proinflammatory cytokine TNFα on the differentiation process could be compensated to some degree by either oil. The transcription factor PPARγ was used to measure the state of differentiation. Caveolin1 is involved in both buildup and degradation of intracellular fat stores and was induced by oil incubation in differentiated as well as in undifferentiated 3T3-L1 cells. Moreover the signal transduction of the IL-6-related cytokine CNTF in 3T3-L1 cells and its influence on the differentiation process of these cells was investigated. In recent years CNTF was spotlighted because of its leptin-like properties that persist even in leptin resistant states. In this study an increasing sensitivity to CNTF during the differentiation of 3T3-L1 cells was shown. Furthermore the positive effects of the cytokine on fatty acid metabolism and adipocyte development were verified. CNTF favored the emergence of many small adipocytes compared to fewer, but larger adi-pocytes under different conditions. Thereby the functionality of the cells is influenced in a positive way and the protection from the development of insulin resistance is increased. The effect of LIF - another IL-6-related cytokine - was not similar to that of CNTF but was more comparable to the effect of the oils.
Establishment of defined culture conditions for the differentiation, long-term maintenance and co-culture of adipose-derived stem cells for the setup of human vascularized adipose tissue
(2018) Volz, Ann-Cathrin; Kluger, Petra Juliane
Most current attempts in engineering adipose tissue are based on the supplementation with human or animal-derived sera. However, especially the use of animal-derived serum is linked to many disadvantages, like potential contaminations, ethical issues and in general the missing identification of many ingredients. Therefore, serum supplementation impedes the actual application of engineered adipose tissue constructs as implants, to substitute lost tissue after tumor resection, severe burnings or trauma. Equally, due to a potential cover up of the cellular response by unidentified components, it impairs the in vitro use of such models as test systems to elucidate mechanisms of disease development, screen for new drugs or generally assess pharmaceutical safety levels. To be capable for functional anastomosis with the host tissue after implantation and for the use in time- and maturation-dependent in vitro purposes, engineered constructs have to exhibit a minimum sustainability. So far, only few authors addressed the serum-free, defined differentiation of adipocytes. And there are hardly any trials available on the defined maintenance of adipocytes. In this study, the development of a defined culture medium for the adipogenic differentiation of primary human adipose-derived stem cells (ASCs) was aimed. Based on the addition of specific factors for the replacement of serum, ASCs were differentiated to viable and characteristic adipocytes for 14 days, which was proven through the accumulation of lipids, the expression of perilipin A and by the release of leptin and glycerol. Furthermore, a defined maintenance medium was developed, which supported the maturation and stability of cells for a long-term period of additional 42 days until day 56. In order to pursue the goal of a physiological tissue substitute of relevant size, the integration of a vascular component is of fundamental importance to allow sufficient nutrient supply of all peripheral tissue areas. For this purpose, a natural vascular system based on a cellular component would be ideal. Due to the lack of an adequate co-culture medium, a major challenge in adipose tissue vascularization is represented by the setup of an adipocyte/endothelial cell (EC) co-culture. In this study, the development of a tissue-tailored co-culture medium based on adipocyte- and EC-factors was developed. Thereby the critical role of epidermal growth factor (EGF) and hydrocortisone (HC) in adipocyte/microvascular (mv)EC co-culture was determined. Through the adjustment of their supplementation, a functional co-culture of adipocytes and mvECs was achieved. In there, mvECs maintained the cell-specific expression of von Willebrand factor (vWF) and cluster of differentiation 31 (CD31). Additionally, cells kept their ability to incorporate acetylated low density lipoprotein (acLDL). By combining the experiences from both mentioned attempts, a defined adipocyte/EC co-culture medium was developed. Next to the maintenance of functional and characteristic adipocytes, the medium facilitated the formation of vascular-like structures in the direct co-culture. To be able to establish tissue constructs of relevant size, current in vitro attempts have to be transferred to a three-dimensional (3D) environment. In this trial, a 3D adipose tissue model was set up based on the differentiation of ASCs in a collagen type I hydrogel in co-culture with mvECs for 21 days in total. The comparison of these models with native adipose tissue demonstrated high accordance in the gene expression levels related to differentiation and fatty acid metabolism. Some deviations were found mostly in maturation-dependent genes linked to tissue functionality and angiogenic mediation. Differentiation and the maintenance of a homeostatic tissue state highly rely on the physical and chemical characteristics of the applied scaffold. As another part, the influence of a novel cellulose-based material (CBM) on defined adipogenic differentiation of ASCs and the defined maintenance of mvECs was investigated in this thesis. An accelerating effect of CBM on the defined differentiation of ASCs was proven by enhanced release of leptin and the increased expression of perilipin A. CBM was further shown to facilitate the formation of vascular-like structures by mvECs under defined conditions in the absence of another supporting cell type. Additionally, the successful co-culture of adipocytes and mvECs was demonstrated on CBM under defined conditions. Summarized, defined culture media for the differentiation, maintenance and co-culture of primary ASC and mvECs were developed. The supporting effect of CBM on the defined establishment of cultures was proven. Further the successful setup of a 3D adipocyte/mvEC co-culture model with a high predictive power was shown. Combined these achievements can be used for the in vitro setup of a 3D vascularized adipose tissue under defined conditions.

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