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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.
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.
New approaches to respiratory assist: bioengineering an ambulatory, miniaturized bioartificial lung
(2019)
Although state-of-the-art treatments of respiratory failure clearly have made some progress in terms of survival in patients suffering from severe respiratory system disorders, such as acute respiratory distress syndrome (ARDS), they failed to significantly improve the quality of life in patients with acute or chronic lung failure, including severe acute exacerbations of chronic obstructive pulmonary disease or ARDS as well. Limitations of standard treatment modalities, which largely rely on conventional mechanical ventilation, emphasize the urgent, unmet clinical need for developing novel(bio)artificial respiratory assist devices that provide extracorporeal gas exchange with a focus on direct extracorporeal CO2 removal from the blood. In this review, we discuss some of the novel concepts and critical prerequisites for such respiratory lung assist devices that can be used with an adequate safety profile, in the intensive care setting, as well as for long-term domiciliary therapy in patients with chronic ventilatory failure. Specifically, we describe some of the pivotal steps, such as device miniaturization, passivation of the blood-contacting surfaces by chemical surface modifications, or endothelial cell seeding, all of which are required for converting current lung assist devices into ambulatory lung assist device for long-term use in critically ill patients. Finally, we also discuss some of the risks and challenges for the long-term use of ambulatory miniaturized bioartificial lungs.
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.
Human adipose-derived stem cells (hASCs) have become an important cell source for the use in tissue engineering and other medical applications. Not every biomaterial is suitable for human cell culture and requires surface modifications to enable cell adhesion and proliferation. Our hypothesis is that chemical surface modifications introduced by low-discharge plasma enhance the adhesion and proliferation of hASCs. Polystyrene (PS) surfaces were modified either by ammonia (NH3), carbon dioxide (CO2) or acrylic acid (AAc) plasma. The results show that the initial cell adhesion is significantly higher on all modified surfaces than on unmodified material as evaluated by bright field microscopy, live/dead staining, total DNA amount and scanning electron microscopy. The formation of focal adhesions was well pronounced on the Tissue Culture PS, NH3-, and CO2 plasma modified samples. The number of matured fibrillar adhesions was significantly higher on NH3 plasmamodified surfaces than on all other surfaces. Our study validates the suitability of chemical plasma activation and represents a method to enhance hASCs adhesion and improved cell expansion. All chemical modification promoted hASCs adhesion and can therefore be used for the modification of different scaffold materials whereby NH3-plasma modified surfaces resulted in the best outcome concerning hASCs adhesion and proliferation.
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.
Size and function of bioartificial tissue models are still limited due to the lack of blood vessels and dynamic perfusion for nutrient supply. In this study, we evaluated the use of cytocompatible methacryl-modified gelatin for the fabrication of a hydrogel-based tube by dip-coating and subsequent photo-initiated cross-linking. The wall thickness of the tubes and the diameter were tuned by the degree of gelatin methacryl-modification and the number of dipping cycles. The dipping temperature of the gelatin solution was adjusted to achieve low viscous fluids of approximately 0.1 Pa s and was different for gelatin derivatives with different modification degrees. A versatile perfusion bioreactor for the supply of surrounding tissue models was developed, which can be adaped to several geometries and sizes of blood-vessel mimicking tubes. The manufactured bendable gelatin tubes were permeable for water and dissolved substances, like Nile Blue and serum albumin. As a proof of concept, human fibroblasts in a three-dimensional collagen tissue model were sucessfully supplied with nutrients via the central gelatin tube under dynamic conditions for 2 days. Moreover, the tubes could be used as scaffolds to build-up a functional and viable endothelial layer. Hence, the presented tools can contribute to solving current challenges in tissue engineering.
Due to its availability and minimal invasive harvesting human adipose tissue-derived extracellular matrix (dECM) is often used as a biomaterial in various tissue engineering and healthcare applications. Next to dECM, cell-derived ECM (cdECM) can be generated by and isolated from in vitro cultured cells. So far both types of ECM were investigated extensively toward their application as (bio)material in tissue engineering and healthcare. However, a systematic characterization and comparison of soft tissue dECM and cdECM is still missing. In this study, we characterized dECM from human adipose tissue, as well as cdECM from human adipose-derived stem cells, toward their molecular composition, structural characteristics, and biological purity. The dECM was found to exhibit higher levels of collagens and lower levels of sulfated glycosaminoglycans compared with cdECMs. Structural characteristics revealed an immature state of the fibrous part of cdECM samples. By the identified differences, we aim to support researchers in the selection of a suitable ECM-based biomaterial for their specific application and the interpretation of obtained results.
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.
Due to its wide-ranging endocrine functions, adipose tissue influences the whole body’s metabolism. Engineering long-term stable and functional human adipose tissue is still challenging due to the limited availability of suitable biomaterials and adequate cell maturation. We used gellan gum (GG) to create manual and bioprinted adipose tissue models because of its similarities to the native extracellular matrix and its easily tunable properties. Gellan gum itself was neither toxic nor monocyte activating. The resulting hydrogels exhibited suitable viscoelastic properties for soft tissues and were stable for 98 days in vitro. Encapsulated human primary adipose-derived stem cells (ASCs) were adipogenically differentiated for 14 days and matured for an additional 84 days. Live-dead staining showed that encapsulated cells stayed viable until day 98, while intracellular lipid staining showed an increase over time and a differentiation rate of 76% between days 28 and 56. After 4 weeks of culture, adipocytes had a univacuolar morphology, expressed perilipin A, and secreted up to 73% more leptin. After bioprinting establishment, we demonstrated that the cells in printed hydrogels had high cell viability and exhibited an adipogenic phenotype and function. In summary, GG-based adipose tissue models show long-term stability and allow ASCs maturation into functional, univacuolar adipocytes.