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Within this interdisciplinary study, we demonstrate the applicability of a 6D printer for soft tissue engineering models. For this purpose, a special plant was constructed, combining the technical requirements for 6D printing with the biological necessities, especially for soft tissue. Therefore, a commercial 6D robot arm was combined with a sterilizable housing (including a high-efficiency particulate air (HEPA) filter and ultraviolet radiation (UVC) lamps) and a custom-made printhead and printbed. Both components allow cooling and heating, which is desirable for working with viable cells. In addition, a spraying unit was installed that allows the distribution of fine droplets of a liquid. Advanced geometries on uneven or angled surfaces can be created with the use of all six axes. Based on often used bioinks in the field of soft tissue engineering (gellan gum, collagen, and gelatin methacryloyl) with very different material properties, we could demonstrate the flexibility of the printing system. Furthermore, cell-containing constructs using primary human adipose-derived stem cells (ASCs) could be produced in an automated manner. In addition to cell survival, the ability to differentiate along the adipogenic lineage could also be demonstrated as a representative of soft tissue engineering.
Skin wound healing involves many cell types in a stepwise process of tissue regeneration. Reepithelialization is an essential characteristic of successful healing. In tissue engineering, mimicking the complex process of injury repair in vitro is challenging and requires the development of advanced skin models. In this study, a simple and reproducible method for wounding three-layered skin models on membranes with different pore sizes (0.4, 1, 3 μm) was established. The model allows the investigation of reepithelialization processes in a more complex environment. Hemalaun-eosin (HE) and 3-[4,5-dimethylthiazole-2-yl]-2,5- diphenyltetrazolium bromide (MTT) staining proved sufficient removal of the epidermis directly after wounding. An increasing pore size of the culture membrane delayed the reepithelialization time. Transepithelial electrical resistance (TEER) measurements provided non-invasive monitoring of reepithelialization, showing increasing values 4 days after wounding for the skin models on 0.4 and 1 μm membranes but not for those on 3 μm membranes. Cytokine quantification of interleukin (IL)-6 and IL-8 complemented the TEER results with increasing levels directly after wounding for all skin models. This skin wounding model could be used to simulate different wounding scenarios and test wound matrix materials. Furthermore, it could be adapted by adding immune cells to resemble the in vivo setup more closely.
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.
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.
Improvement of a three-layered in vitro skin model for topical application of irritating substances
(2020)
In the field of skin tissue engineering, the development of physiologically relevant in vitro skin models comprising all skin layers, namely epidermis, dermis, and subcutis, is a great challenge. Increasing regulatory requirements and the ban on animal experiments for substance testing demand the development of reliable and in vivo-like test systems, which enable high-throughput screening of substances. However, the reproducibility and applicability of in vitro testing has so far been insufficient due to fibroblast-mediated contraction. To overcome this pitfall, an advanced 3-layered skin model was developed. While the epidermis of standard skin models showed an 80% contraction, the initial epidermal area of our advanced skin models was maintained. The improved barrier function of the advanced models was quantified by an indirect barrier function test and a permeability assay. Histochemical and immunofluorescence staining of the advanced model showed well-defined epidermal layers, a dermal part with distributed human dermal fibroblasts and a subcutis with round-shaped adipocytes. The successful response of these advanced 3-layered models for skin irritation testing demonstrated the suitability as an in vitro model for these clinical tests: only the advanced model classified irritative and non-irritative substances correctly. These results indicate that the advanced set up of the 3-layered in vitro skin model maintains skin barrier function and therefore makes them more suitable for irritation testing.
White adipose tissue (WAT) plays a crucial role in energy homeostasis and secretes numerous adipokines with far-reaching effects. WAT is linked to diseases such as diabetes, cardiovascular disease, and cancer. There is a high demand for suitable in vitro models to study diseases and tissue metabolism. Most of these models are covered by 2D-monolayer cultures. This study aims to evaluate the performance of different WAT models to better derive potential applications. The stability of adipocyte characteristics in spheroids and two 3D gellan gum hydrogels with ex situ lobules and 2D-monolayer culture is analyzed. First, the differentiation to achieve adipocyte-like characteristics is determined. Second, to evaluate the maintenance of differentiated ASC-based models, an adipocyte-based model, and explants over 3 weeks, viability, intracellular lipid content, perilipin A expression, adipokine, and gene expression are analyzed. Several advantages are supported using each of the models. Including, but not limited to, the strong differentiation in 2D-monolayers, the self-assembling within spheroids, the long-term stability of the stem cell-containing hydrogels, and the mature phenotype within adipocyte-containing hydrogels and the lobules. This study highlights the advantages of 3D models due to their more in vivo-like behavior and provides an overview of the different adipose cell models.
