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The world population is growing and alternative ways of satisfying the increasing demand for meat are being explored, such as using animal cells for the fabrication of cultured meat. Edible biomaterials are required as supporting structures. Hence, we chose agarose, gellan and a xanthan-locust bean gum blend (XLB) as support materials with pea and soy protein additives and analyzed them regarding material properties and biocompatibility. We successfully built stable hydrogels containing up to 1% pea or soy protein. Higher amounts of protein resulted in poor handling properties and unstable gels. The gelation temperature range for agarose and gellan blends is between 23–30 °C, but for XLB blends it is above 55 °C. A change in viscosity and a decrease in the swelling behavior was observed in the polysaccharide-protein gels compared to the pure polysaccharide gels. None of the leachates of the investigated materials had cytotoxic effects on the myoblast cell line C2C12. All polysaccharide-protein blends evaluated turned out as potential candidates for cultured meat. For cell-laden gels, the gellan blends were the most suitable in terms of processing and uniform distribution of cells, followed by agarose blends, whereas no stable cell-laden gels could be formed with XLB blends.
The coculture of osteogenic and angiogenic cells and the resulting paracrine signaling via soluble factors are supposed to be crucial for successfully engineering vascularized bone tissue equivalents. In this study, a coculture system combining primary human adiposederived stem cells (hASCs) and primary human dermal microvascular endothelial cells (HDMECs) within two types of hydrogels based on methacryloyl‐modified gelatin (GM) as three‐dimensional scaffolds was examined for its support of tissue specific cell functions. HDMECs, together with hASCs as supporting cells, were encapsulated in soft GM gels and were indirectly cocultured with hASCs encapsulated in stiffer GM hydrogels additionally containing methacrylate‐modified hyaluronic acid and hydroxyapatite particles. After 14 days, the hASC in the stiffer gels (constituting the “bone gels”) expressed matrix proteins like collagen type I and fibronectin, as well as bone‐specific proteins osteopontin and alkaline phosphatase. After 14 days of coculture with HDMEC‐laden hydrogels, the viscoelastic properties of the bone gels were significantly higher compared with the gels in monoculture. Within the soft vascularization gels, the formed capillary‐like networks were significantly longer after 14 days of coculture than the structures in the control gels. In addition, the stability as well as the complexity of the vascular networks was significantly increased by coculture. We discussed and concluded that osteogenic and angiogenic signals from the culture media as well as from cocultured cell types, and tissue‐specific hydrogel composition all contribute to stimulate the interplay between osteogenesis and angiogenesis in vitro and are a basis for engineering vascularized bone.
In bioprinting approaches, the choice of bioink plays an important role since it must be processable with the selected printing method, but also cytocompatible and biofunctional. Therefore, a crosslinkable gelatin-based ink was modified with hydroxyapatite (HAp) particles, representing the composite buildup of natural bone. The inks’ viscosity was significantly increased by the addition of HAp, making the material processable with extrusion-based methods. The storage moduli of the formed hydrogels rose significantly, depicting improved mechanical properties. A cytocompatibility assay revealed suitable ranges for photoinitiator and HAp concentrations. As a proof of concept, the modified ink was printed together with cells, yielding stable three-dimensional constructs containing a homogeneously distributed mineralization and viable cells.
Though bioprinting is a forward-looking approach in bone tissue engineering, the development of bioinks which are on the one hand processable with the chosen printing technique, and on the other hand possess the relevant mechanical as well as osteoconductive features remains a challenge. In the present study, polymer solutions based on methacrylated gelatin and methacrylated hyaluronic acid modified with hydroxyapatite (HAp) particles (5 wt%) were prepared. Encapsulation of primary human adipose derived stem cells in the HAp-containing gels and culture for 28 d resulted in a storage moduli significantly increased to 126% ± 9.6% compared to the value on day 1 by the sole influence of the HAp. Additional use of osteogenic media components resulted in an increase of storage module up to 199% ± 27.8%. Similarly, the loss moduli was increased to 370% ± 122.1% under the influence of osteogenic media components and HAp. Those changes in rheological material characteristics indicate a distinct change in elastic and viscous hydrogel properties, and are attributed to extensive matrix production in the hydrogels by the encapsulated cells, what could also be proven by staining of bone matrix components like collagen I, fibronectin, alkaline phosphatase and osteopontin. When using the cell-laden polymer solutions as bioinks to build up relevant geometries, the ink showed excellent printability and the printed grid structure's integrity remained intact over a culture time of 28 d. Again, an intense matrix formation as well as upregulation of osteogenic markers by the encapsulated cells could be shown. In conclusion, we demonstrated that our HAp-containing bioinks and hydrogels on basis of methacrylated gelatin and hyaluronic acid are on the one hand highly suitable for the build up of relevant three-dimensional geometries with microextrusion bioprinting, and on the other hand exhibit a significant positive effect on bone matrix development and remodeling in the hydrogels, as indicated by rheological measurements and staining of bone components. This makes the developed composite hydrogels an excellent material for bone bioprinting approaches.
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.
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 of 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 fo unknown or unimal-derived constituents, achieving an important milestone in the introduction of adipose tissue engineered products into clinical trials or in vitro screening.
In vitro composed vascularized adipose tissue is and will continue to be in great demand e.g. for the treatment of extensive high-graded burns or the replacement of tissue after tumor removal. Up to date, the lack of adequate culture conditions, mainly a culture medium, decelerates further achievements. In our study, we evaluated the influence of epidermal growth factor (EGF) and hydrocortisone (HC), often supplemented in endothelial cell (EC) specific media, on the co-culture of adipogenic differentiated adipose derived stem cells (ASCs) and microvascular endothelial cells (mvECs). In ASCs, EGF and HC are thought to inhibit adipogenic differentiation and have lipolytic activities. Our results showed that in indirect co-culture for 14 days, adipogenic differentiated ASCs further incorporated lipids and partly gained an univacuolar morphology when kept in media with low levels of EGF and HC. In media with high EGF and HC levels, cells did not incorporate further lipids, on the contrary, cells without lipid droplets appeared. Glycerol release, to measure lipolysis, also increased with elevated amounts of EGF and HC in the culture medium. Adipogenic differentiated ASCs were able to release leptin in all setups. MvECs were functional and expressed the cell specific markers, CD31 and von Willebrand factor (vWF), independent of the EGF and HC content as long as further EC specific factors were present. Taken together, our study demonstrates that adipogenic differentiated ASCs can be successfully co-cultured with mvECs in a culture medium containing low or no amounts of EGF and HC, as long as further endothelial cell and adipocyte specific factors are available.
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.
Artificial adipose tissue (AT) constructs are urgently needed to treat severe wounds, to replace removed tissue, or for the use as in vitro model to screen for potential drugs or study metabolic pathways. The clinical translation of products is mostly prevented by the absence of a vascular component that would allow a sustainable maintenance and an extension of the construct to a relevant size. With this study, we aimed to evaluate the suitability of a novel material based on bacterial cellulose (CBM) on the defined adipogenic differentiation of human adipose-derived stem cells (ASCs) and the maintenance of the received adipocytes (diffASCs) and human microvascular endothelial cells (mvECs) in mono- and coculture. A slight acceleration of adipogenic differentiation over regular tissue culture polystyrene (TCPS) was seen on CBM under defined conditions, whereas on the maintenance of the generated adipocytes, comparable effects were detected for both materials. CBM facilitated the formation of vascular like structures in monoculture of mvECs, which was not observed on TCPS. By contrast, vascular-like structures were detected in CBM and TCPS in coculture by the presence of diffASCs. Concluding, CBM represents a promising material in vascularized AT engineering with the potential to speed up and simplify the in vitro setup of engineered products.
Completely defined co-culture of adipogenic differentiated ASCs and microvascular endothelial cells
(2018)
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.