Robust Generation of ASC Spheroids for Use as 3D Cultures and in Bioprinted Tissue Models.

Three-dimensional (3D) cell culture techniques have become a valuable tool to mimic the complex interactions of cells with each other and their surrounding extracellular matrix as they occur in vivo. In this respect, 3D spheroids are widely acknowledged as self-assembled cellular aggregates that can be generated from a variety of cell types without the need for exogenous material while being highly reproducible, easy to handle, and cost-effective. Furthermore, due to their capacity to be developed into microtissues, spheroids represent potential building blocks for various tissue engineering applications, including 3D bioprinting approaches for tissue model development. Adipose-derived stromal/stem cells (ASCs), due to their ease of isolation, multipotent nature, and secretory capacity, represent an attractive cell source employed in numerous tissue engineering studies and other cell-based therapy approaches. In this chapter, we describe two procedures for robust spheroid generation, namely the liquid overlay technique, either using agarose-coated 96-well plates or employing agarose-cast micromolds. Furthermore, we show, in principle, the generation of ASC spheroids with subsequent adipogenic differentiation and the spheroid generation using adipogenically differentiated ASCs, as well as the morphological characterization of generated spheroids.

Three-Dimensional Breast Cancer Model to Investigate CCL5/CCR1 Expression Mediated by Direct Contact between Breast Cancer Cells and Adipose-Derived Stromal Cells or Adipocytes.

The tumor microenvironment (TME) in breast cancer is determined by the complex crosstalk of cancer cells with adipose tissue-inherent cells such as adipose-derived stromal cells (ASCs) and adipocytes resulting from the local invasion of tumor cells in the mammary fat pad. This leads to heterotypic cellular contacts between these cell types. To adequately mimic the specific cell-to-cell interaction in an in vivo-like 3D environment, we developed a direct co-culture spheroid model using ASCs or differentiated adipocytes in combination with MDA-MB-231 or MCF-7 breast carcinoma cells. Co-spheroids were generated in a well-defined and reproducible manner in a high-throughput process. We compared the expression of the tumor-promoting chemokine CCL5 and its cognate receptors in these co-spheroids to indirect and direct standard 2D co-cultures. A marked up-regulation of CCL5 and in particular the receptor CCR1 with strict dependence on cell-cell contacts and culture dimensionality was evident. Furthermore, the impact of direct contacts between ASCs and tumor cells and the involvement of CCR1 in promoting tumor cell migration were demonstrated. Overall, these results show the importance of direct 3D co-culture models to better represent the complex tumor-stroma interaction in a tissue-like context. The unveiling of tumor-specific markers that are up-regulated upon direct cell-cell contact with neighboring stromal cells, as demonstrated in the 3D co-culture spheroids, may represent a promising strategy to find new targets for the diagnosis and treatment of invasive breast cancer.

Bioink Platform Utilizing Dual-Stage Crosslinking of Hyaluronic Acid Tailored for Chondrogenic Differentiation of Mesenchymal Stromal Cells.

3D bioprinting often involves application of highly concentrated polymeric bioinks to enable fabrication of stable cell-hydrogel constructs, although poor cell survival, compromised stem cell differentiation, and an inhomogeneous distribution of newly produced extracellular matrix (ECM) are frequently observed. Therefore, this study presents a bioink platform using a new versatile dual-stage crosslinking approach based on thiolated hyaluronic acid (HA-SH), which not only provides stand-alone 3D printability but also facilitates effective chondrogenic differentiation of mesenchymal stromal cells. A range of HA-SH with different molecular weights is synthesized and crosslinked with acrylated (PEG-diacryl) and allylated (PEG-diallyl) polyethylene glycol in a two-step reaction scheme. The initial Michael addition is used to achieve ink printability, followed by UV-mediated thiol-ene reaction to stabilize the printed bioink for long-term cell culture. Bioinks with high molecular weight HA-SH (>200 kDa) require comparably low polymer content to facilitate bioprinting. This leads to superior quality of cartilaginous constructs which possess a coherent ECM and a strongly increased stiffness of long-term cultured constructs. The dual-stage system may serve as an example to design platforms using two independent crosslinking reactions at one functional group, which allows adjusting printability as well as material and biological properties of bioinks.

Bioprinting and Differentiation of Adipose-Derived Stromal Cell Spheroids for a 3D Breast Cancer-Adipose Tissue Model.

Biofabrication, including printing technologies, has emerged as a powerful approach to the design of disease models, such as in cancer research. In breast cancer, adipose tissue has been acknowledged as an important part of the tumor microenvironment favoring tumor progression. Therefore, in this study, a 3D-printed breast cancer model for facilitating investigations into cancer cell-adipocyte interaction was developed. First, we focused on the printability of human adipose-derived stromal cell (ASC) spheroids in an extrusion-based bioprinting setup and the adipogenic differentiation within printed spheroids into adipose microtissues. The printing process was optimized in terms of spheroid viability and homogeneous spheroid distribution in a hyaluronic acid-based bioink. Adipogenic differentiation after printing was demonstrated by lipid accumulation, expression of adipogenic marker genes, and an adipogenic ECM profile. Subsequently, a breast cancer cell (MDA-MB-231) compartment was printed onto the adipose tissue constructs. After nine days of co-culture, we observed a cancer cell-induced reduction of the lipid content and a remodeling of the ECM within the adipose tissues, with increased fibronectin, collagen I and collagen VI expression. Together, our data demonstrate that 3D-printed breast cancer-adipose tissue models can recapitulate important aspects of the complex cell-cell and cell-matrix interplay within the tumor-stroma microenvironment.

Human Adipose-Derived Mesenchymal Stromal/Stem Cell Spheroids Possess High Adipogenic Capacity and Acquire an Adipose Tissue-like Extracellular Matrix Pattern.

Adipose-derived mesenchymal stromal/stem cells (ASCs) represent a commonly used cell source for adipose tissue engineering. In this context, ASCs have routinely been cultured in conventional 2D culture and applied as single cell suspension for seeding onto scaffold materials or direct injection. However, this approach is associated with the loss of their intrinsic 3D microenvironment and leads to impaired regenerative capacity of the cells. Thus, the application of ASCs as self-assembled 3D spheroids with cells residing in their own matrix is an attractive alternative. However, characterization of the structural features and differentiation capacity of the spheroids is necessary to effectively apply them as building blocks in adipose tissue engineering. In this study, we focus on extracellular matrix (ECM) development in ASC spheroids, as well as adipogenic differentiation in comparison to conventional 2D culture using different induction protocols. Reproducible assembly of ASCs into spheroids was achieved within 24 h using the liquid overlay technique. Undifferentiated spheroids displayed a stromal ECM pattern, with fibronectin, collagen V, and VI as the main components. In the course of adipogenesis, a dynamic shift in the ECM composition toward an adipogenic phenotype was observed, associated with enhanced expression of laminin, collagen I, IV, V, and VI, similar to native fat. Furthermore, adipogenic differentiation was enhanced in spheroids as compared with 2D cultured cells, with the spheroids needing a distinctly shorter adipogenic stimulus to sustain adipogenesis, which was demonstrated based on analysis of triglyceride content and adipogenic marker gene expression. In summary, culturing ASCs as spheroids can enhance their adipogenic capacity and generate adipose-like microtissues, which may be a promising cell delivery strategy for adipose tissue engineering approaches. Impact statement Adipose-derived mesenchymal stromal/stem cells (ASCs) as a widely used cell source for adipose tissue engineering have been shown to be limited in their regenerative capacity when applied as single cells. As an alternative approach, the delivery as spheroids, consisting of cells in a 3D context, may be favorable. However, insights into extracellular matrix (ECM) development and efficient adipogenic differentiation are required for their effective application. In this study, we show that differentiated ASC spheroids develop an ECM, resembling native adipose tissue. Furthermore, the ASC spheroids exhibited a superior differentiation capacity as compared with conventional 2D culture, and required only a short adipogenic induction stimulus. Our results identify ASC-derived spheroids as an attractive cell delivery method for adipose tissue engineering approaches.

Transgenic systems for unequivocal identification of cardiac myocyte nuclei and analysis of cardiomyocyte cell cycle status.

Even though the mammalian heart has been investigated for many years, there are still uncertainties in the fields of cardiac cell biology and regeneration with regard to exact fractions of cardiomyocytes (CMs) at different developmental stages, their plasticity after cardiac lesion and also their basal turnover rate. A main shortcoming is the accurate identification of CM and the demonstration of CM division. Therefore, an in vivo model taking advantage of a live reporter-based identification of CM nuclei and their cell cycle status is needed. In this technical report, we describe the generation and characterization of embryonic stem cells and transgenic mice expressing a fusion protein of human histone 2B and the red fluorescence protein mCherry under control of the CM specific αMHC promoter. This fluorescence label allows unequivocal identification and quantitation of CM nuclei and nuclearity in isolated cells and native tissue slices. In ventricles of adults, we determined a fraction of <20 % CMs and binucleation of 77-90 %, while in atria a CM fraction of 30 % and a binucleation index of 14 % were found. We combined this transgenic system with the CAG-eGFP-anillin transgene, which identifies cell division and established a novel screening assay for cell cycle-modifying substances in isolated, postnatal CMs. Our transgenic live reporter-based system enables reliable identification of CM nuclei and determination of CM fractions and nuclearity in heart tissue. In combination with CAG-eGFP-anillin-mice, the cell cycle status of CMs can be monitored in detail enabling screening for proliferation-inducing substances in vitro and in vivo.