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

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.Die meisten aktuellen Ansätze zum Aufbau eines künstlichen Fettgewebekonstruktes basieren auf dem Einsatz von Blutseren. Speziell die Verwendung von tierischem Serum ist jedoch mit vielen Nachteilen verbunden. Dazu zählen potentielle Kontaminationen, große Variationen zwischen den Chargen und die vielen, nicht identifizierten Komponenten. Die Supplementierung mit Serum behindert so den Einsatz von künstlichen Fettgewebekonstrukten als Implantat zum Ersatz von Gewebe nach einer Tumorentfernung, schweren Verbrennungen oder Traumata. In vitro Ansätze z.B. zur Aufklärung von Krankheitsentstehungen, der Entwicklung oder Sicherheitseinstufung von Medikamenten, basieren auf der zellulären Antwort, welche ebenfalls durch Serum-Komponenten verschleiert werden kann. Zudem sollten Fettgewebekonstrukte eine grundsätzliche Stabilität aufweisen um nach der Implantation eine ausreichende Anastomose mit dem Empfängergewebe und die Nutzung für zeit- und reifeabhängige in vitro Fragestellungen zu ermöglichen. Die definierte serumfreie Differenzierung von Adipozyten wurde bisher nur von wenigen Autoren adressiert. In dieser Arbeit wurde die Entwicklung definierter Kulturmedien für die adipogene Differenzierung humaner primärer Stammzellen aus dem Fettgewebe (adipose-derived stem cells, ASCs) angestrebt. Durch die Zugabe von spezifischen Faktoren als Alternative zu Serum, erfolgte eine 14-tägige Differenzierung zu viablen und charakteristischen Adipozyten, was sich durch die Einlagerung von Lipiden, der Expression von Perilipin A und der Freisetzung von Leptin und Glycerol bestätigen ließ. Weiterhin wurde ein definiertes Erhaltungsmedium entwickelt, welches die Reifung der Adipozyten über einen Kulturzeitraum von 42 Tagen bis zu Tag 56 unterstützte. Zur ausreichenden Versorgung, auch peripher gelegener Geweberegionen, ist die Integration einer vaskulären Komponente beim Aufbau eines physiologischen Gewebeersatzes in relevanter Größe fundamental. Idealerweise sollte das vaskuläre System aus einer natürlichen, zellulären Komponente bestehen. Durch den Mangel eines adäquaten Co-Kulturmediums, stellt der Aufbau einer Adipozyten/Endothelzell (endothelial cell, EC) Co-Kultur bei der Vaskularisierung von Fettgewebe eine zentrale Herausforderung dar. In dieser Arbeit wurde ein gewebespezifisches Co-Kulturmedium mit Adipozyten- und EC-Faktoren entwickelt. Hierbei zeigte sich die kritische Rolle vom epidermalen Wachstumsfaktor (epidermal growth factor, EGF) und Hydrocortison (HC) in der Co-Kultur von Adipozyten und mikrovaskulären (mv)EC. Durch die Supplementanpassung ließ sich eine funktionelle Adipozyten/mvEC Co-Kultur aufbauen. Darin erhielten die mvECs die zellspezifische Expression des von Willebrand Faktors (vWF) und des Oberflächenmarkers 31 (Cluster of differentiation, CD31). Außerdem behielten sie die Fähigkeit zur Aufnahme von acetyliertem Lipoprotein niedriger Dichte (acetylated low density liporotein, acLDL). Durch die Erkenntnisse aus beiden Ansätzen ließ sich ein definiertes Adipozyten/EC Co-Kultur-medium entwickeln. Neben dem Erhalt funktioneller und charakteristischer Adipozyten, unterstütze das Medium in direkter Co-Kultur die Ausbildung vaskulärer Strukturen. Zum Aufbau von Gewebekonstrukten relevanter Größe ist die Überführung der aktuellen Ansätze in eine dreidimensionale (3D) Umgebung notwendig. In dieser Thesis wurde ein 3D Fettgewebekonstrukt mit differenzierten ASCs in einem Kollagen Typ I Hydrogel in Co-Kultur mit mvECs über 21 Tage aufgebaut. Im Vergleich der Modelle mit nativem Gewebe zeigte sich eine größtenteils übereinstimmende Expression von Genen, die mit der Differenzierung und dem Fettstoffwechsel verbunden sind. Abweichungen wurden hingegen bei zumeist reifeabhängigen Genen, die im Zusammenhang mit der Gewebefunktionalität und angiogenen Prozessen stehen, festgestellt. Die Differenzierung und eine homeostatische Gewebeerhaltung hängen maßgeblich von den physikalischen und chemischen Eigenschaften des eingesetzten Biomaterials ab. In einem weiteren Teil dieser Thesis wurde der Einfluss eines neuartigen Cellulose-basierten Materials (CBM) auf die definierte adipogene Differenzierung und die definierte mvEC Erhaltung untersucht. Die erhöhte Ausschüttung von Leptin und die Expression von Perilipin A zeigte einen beschleunigenden Effekt von CBM auf die Differenzierung von ASCs. Weiterhin ermöglichte CBM die Ausbildung vaskulärer Strukturen in der definierten Kultur ohne die Unterstützung weiterer Zelltypen. Schließlich gelang die definierte Co-Kultur von Adipozyten und mvECs auf CBM. Zusammengefasst wurden in dieser Arbeit definierte Medien zur Differenzierung, Erhaltung und Co-Kultur von primären ASCs mit mvECs entwickelt. CBM zeigte einen unterstützenden Effekt im definierten Ansatz dieser Kulturen. Außerdem gelang der Aufbau einer 3D Adipozyten/mvEC Co-Kultur. In Kombination können diese Ergebnisse zum Aufbau eines vaskularisierten 3D Fettgewebekonstruktes unter definierten Bedingungen genutzt werden

Completely serum-free and chemically defined adipocyte development and maintenance

Background aims: In vitro engineered adipose tissue is in great demand to treat lost or damaged soft tissue or to screen for new drugs, among other applications. However, today most attempts depend on the use of animal-derived sera. To pave the way for the application of adipose tissue–engineered products in clinical trials or as reliable and robust in vitro test systems, sera should be completely excluded from the production process. In this study, we aimed to develop an in vitro adipose tissue model in the absence of sera and maintain its function long-term. Methods: Human adipose tissue–derived stem cells were expanded and characterized in a xeno- and serum-free environment. Adipogenic differentiation was induced using a completely defined medium. Developed adipocytes were maintained in a completely defined maturation medium for additional 28 days. In addition to cell viability and adherence, adipocyte-specific markers such as perilipin A expression or leptin release were evaluated. Results: The defined differentiation medium enhanced cell adherence and lipid accumulation at a significant level compared with the corresponding negative control. The defined maturation medium also significantly supported cell adherence and functional adipocyte maturation during the long-term culture period. Conclusions: The process described here enables functional adipocyte generation and maintenance without the addition of unknown or animal-derived constituents, achieving an important milestone in the introduction of adipose tissue–engineered products into clinical trials or in vitro screening

How do culture media influence in vitro perivascular cell behavior?

Perivascular cells are multilineage cells located around the vessel wall and important for wall stabilization. In this study, we evaluated a stem cell media and a perivascular cell-specific media for the culture of primary perivascular cells regarding their cell morphology, doubling time, stem cell properties, and expression of cell type-specific markers. When the two cell culture media were compared to each other, perivascular cells cultured in the stem cell medium had a more elongated morphology and a faster doubling rate and cells cultured in the pericyte medium had a more typical morphology, with several filopodia, and a slower doubling rate. To evaluate stem cell properties, perivascular cells, CD146 cells, and mesenchymal stem cells (MSCs) were differentiated into the adipogenic, osteogenic, and chondrogenic lineages. It was seen that perivascular cells, as well as CD146 cells and MSCs, cultured in stem cell medium showed greater differentiation than cells cultured in pericyte-specific medium. The expression of pericyte-specific markers CD146, neural/glial antigen 2 (NG2), platelet-derived growth factor receptor-β (PDGFR-β), myosin, and α-smooth muscle actin (α-SMA) could be found in both pericyte cultures, as well as to varying amounts in CD146 cells, MSCs, and endothelial cells. The here presented work shows that perivascular cells can adapt to their in vitro environment and cell culture conditions influence cell functionality, such as doubling rate or differentiation behavior. Pericyte-specific markers were shown to be expressed also from cells other than perivascular cells. We can further conclude that CD146⁺ perivascular cells are inhomogeneous cell population probably containing stem cell subpopulations, which are located perivascular around capillaries

Understanding the effects of mature adipocytes and endothelial cells on fatty acid metabolism and vascular tone in physiological fatty tissue for vascularized adipose tissue engineering

Engineering of large vascularized adipose tissue constructs is still a challenge for the treatment of extensive high-graded burns or the replacement of tissue after tumor removal. Communication between mature adipocytes and endothelial cells is important for homeostasis and the maintenance of adipose tissue mass but, to date, is mainly neglected in tissue engineering strategies. Thus, new coculture strategies are needed to integrate adipocytes and endothelial cells successfully into a functional construct. This review focuses on the cross-talk of mature adipocytes and endothelial cells and considers their influence on fatty acid metabolism and vascular tone. In addition, the properties and challenges with regard to these two cell types for vascularized tissue engineering are highlighted

Adipose-derived stem cell differentiation as a basic tool for vascularized adipose tissue engineering

The development of in vitro adipose tissue constructs is highly desired to cope with the increased demand for substitutes to replace damaged soft tissue after high graded burns, deformities or tumor removal. To achieve clinically relevant dimensions, vascularization of soft tissue constructs becomes inevitable but still poses a challenge. Adipose-derived stem cells (ASCs) represent a promising cell source for the setup of vascularized fatty tissue constructs as they can be differentiated into adipocytes and endothelial cells in vitro and are thereby available in sufficiently high cell numbers. This review summarizes the currently known characteristics of ASCs and achievements in adipogenic and endothelial differentiation in vitro. Further, the interdependency of adipogenesis and angiogenesis based on the crosstalk of endothelial cells, stem cells and adipocytes is addressed at the molecular level. Finally, achievements and limitations of current co-culture conditions for the construction of vascularized adipose tissue are evaluated

Defined adipocyte differentiation and long term stability through a cellulose-based culture matrix

White adipose tissue (WAT) comprises about one fourth of the human body, interacts with many other organs via paracrine and endocrine signals and sequestrates lipophilic substances. Thereby it impacts distribution and levels of drugs [1]. Additionally WAT is the origin of different diseases and thus target of several drugs addressing those [2]. Therefore an adipose tissue testsystem is highly needed to analyze fundamental biological issues in physiological or diseased state, screen for potential drugs or create affiliated safety profiles. Additionally adipose tissue substitutes are highly desired to treat lost, deformed or burned subcutaneous fatty tissue [3]. In several promising attempts substantial progress was achieved e.g. by the encapsulation of adipocytes in a 3D environment with native matrix components like gelatine [4] or the optimization of media composition [5]. However, regardless of its potential in vivo or in vitro application, an adipose tissue model has to fulfill two main requirements which could not be implemented so far. First, the model has to show in vitro stability for a period of several weeks to enable its maturation and time-dependent investigations. Second, culture conditions have to be free of animal derived components or preferably completely defined to exclude possible impairments through unknown constituents and allow for GMP-compliant production. In this approach a novel cellulose based matrix and a specific media supplementation are combined to achieve defined adipocyte differentiation and long term maintenance. Human primary adipose-derived stem cells (ASCs) were isolated, characterized and expanded xenofree. ASCs were next to tissue culture polystyrol applied to a collagen coated surface and the cellulose-based matrix. Adipogenic differentiation of ASCs was induced by addition of a composed defined differentiation medium and continued for 14 days. On day 14 medium was switched to a composed defined adipocyte maintenance medium. Obtained adipocytes were subsequently cultured for additional 28 days. Preservation of adipocyte characteristics were evaluated by immunofluorescence staining of specific markers like perilipin A and the quantitative analysis of stored lipids. Retained adipocyte metabolism was determined by released leptin, lipolysis by released glycerol levels. Our results classify the cellulose-based matrix as a useful tool to realize and optimize defined adipogenic differentiation of ASCs and the subsequent adipocyte maintenance. The matrix constitution enables strong cell matrix interaction and thereby strengthens cell adherence which is often diminished under defined culture conditions. Due to matrix-induced cell quiescence adipocytes’ long-term stability was enhanced. Based on matrix-derived support successful differentiation and maintenance were confirmed via the composed media under completely defined conditions. Therefore the cellulose-based matrix is a promising biomaterial for stable long-term culture of quiescent cells with preservation of cell specific functions and characteristics which could as well be applied in other setups. Consecutive defined adipose tissue models could be further expanded e.g. to co-culture models and used as testsystems or tissue substitutes

Generation of an azide-modified extracellular matrix by adipose-derived stem cells using metabolic glycoengineering

Natural extracellular matrix (ECM) represents an ideal biomaterial for tissue engineering and regenerative medicine approaches. For further functionalization, there is a need for specific addressable functional groups within this biomaterial. Metabolic glycoengineering (MGE) provides a technique to incorporate modified monosaccharide derivatives into the ECM during their assembly, which was shown by us earlier for the production of a modified fibroblast-derived dermal ECM

Completely defined co-culture of adipogenic differentiated ASCs and microvascular endothelial cells

Vascularized adipose tissue models are in high demand as alternatives to animal models to elucidate the mechanisms of widespread diseases, screen for new drugs or assess drug safety levels. Animal-derived sera such as fetal bovine serum (FBS), which are commonly used in these models, are associated with ethical concerns, risk of contaminations and inconsistencies of their composition and impact on cells. In this study, we developed a serum-free, defined co culture medium and implemented it in an adipocyte/endothelial cell (EC) co culture model. Human adipose-derived stem cells were differentiated under defined conditions (diffASCs) and, like human microvascular ECs (mvECs), cultured in a defined co culture medium in mono-, indirect or direct co-culture for 14 days. The defined co-culture medium was superior when compared to mono-culture media and facilitated the functional maintenance and maturation of diffASCs including perilipin A expression, lipid accumulation, and also glycerol and leptin release. The medium also allowed mvEC maintenance, confirmed by the expression of CD31 and von Willebrand factor (vWF), and by acetylated low density lipoprotein (acLDL) uptake. Thereby, mvECs showed strong dependence on EC-specific factors. Additionally, mvECs formed vascular structures in direct co-culture with diffASCs. The completely defined co-culture system allows for the serum-free culture of adipocyte/EC co-cultures and thereby represents a valuable and ethically acceptable tool for the culture and study of vascularized adipose tissue models

Comparing the use of differentiated adipose-derived stem cells and mature adipocytes to model adipose tissue in vitro

In vitro models of human adipose tissue may serve as beneficial alternatives to animal models to study basic biological processes, identify new drug targets, and as soft tissue implants. With this approach, we aimed to evaluate adipose-derived stem cells (ASC) and mature adipocytes (MA) comparatively for the application in the in vitro setup of adipose tissue constructs to imitate native adipose tissue physiology. We used human primary MAs and human ASCs, differentiated for 14 days, and encapsulated them in collagen type I hydrogels to build up a three-dimensional (3D) adipose tissue model. The maintenance of the models was analyzed after seven days based on a viability staining. Further, the expression of the adipocyte specific protein perilipin A and the release of leptin and glycerol were evaluated. Gene transcription profiles of models based on dASCs and MAs were analyzed with regard to native adipose tissue. Compared to MAs, dASCs showed an immature differentiation state. Further, gene transcription of MAs suggests a behavior closer to native tissue in terms of angiogenesis, which supports MAs as preferred cell type. In contrast to native adipose tissue, genes of de novo lipogenesis and tissue remodeling were upregulated in the in vitro attempts

Cell-derived extracellular matrix as maintaining biomaterial for adipogenic differentiation

The extracellular matrix (ECM) naturally surrounds cells in humans, and therefore represents the ideal biomaterial for tissue engineering. ECM from different tissues exhibit different composition and physical characteristics. Thus, ECM provides not only physical support but also contains crucial biochemical signals that influence cell adhesion, morphology, proliferation and differentiation. Next to native ECM from mature tissue, ECM can also be obtained from the in vitro culture of cells. In this study, we aimed to highlight the supporting effect of cell-derived- ECM (cdECM) on adipogenic differentiation. ASCs were seeded on top of cdECM from ASCs (scdECM) or pre-adipocytes (acdECM). The impact of ECM on cellular activity was determined by LDH assay, WST I assay and BrdU assay. A supporting effect of cdECM substrates on adipogenic differentiation was determined by oil red O staining and subsequent quantification. Results revealed no effect of cdECM substrates on cellular activity. Regarding adipogenic differentiation a supporting effect of cdECM substrates was obtained compared to control. With these results, we confirm cdECM as a promising biomaterial for adipose tissue engineering