Browsing by Person "Volz, Ann-Cathrin"

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Bioprinting of 3D adipose tissue models using a GelMA-bioink with human mature adipocytes or human adipose-derived stem cells
(2022) Albrecht, Franziska Brigitte; Schmidt, Freia F.; Volz, Ann-Cathrin; Kluger, Petra J.
Adipose tissue is related to the development and manifestation of multiple diseases, demonstrating the importance of suitable in vitro models for research purposes. In this study, adipose tissue lobuli were explanted, cultured, and used as an adipose tissue control to evaluate in vitro generated adipose tissue models. During culture, lobule exhibited a stable weight, lactate dehydrogenase, and glycerol release over 15 days. For building up in vitro adipose tissue models, we adapted the biomaterial gelatin methacryloyl (GelMA) composition and handling to homogeneously mix and bioprint human primary mature adipocytes (MA) and adipose-derived stem cells (ASCs), respectively. Accelerated cooling of the bioink turned out to be essential for the homogeneous distribution of lipid-filled MAs in the hydrogel. Last, we compared manual and bioprinted GelMA hydrogels with MA or ASCs and the explanted lobules to evaluate the impact of the printing process and rate the models concerning the physiological reference. The viability analyses demonstrated no significant difference between the groups due to additive manufacturing. The staining of intracellular lipids and perilipin A suggest that GelMA is well suited for ASCs and MA. Therefore, we successfully constructed physiological in vitro models by bioprinting MA-containing GelMA bioinks.
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.