Generation of Tissue Spheroids via a 3D Printed Stamp-Like Device.

Advances in 3D cell culture have developed more physiologically relevant in vitro models, such as tissue spheroids. Cells cultivated as spheroids have more realistic biological responses that resemble the in vivo environment. Due to their advantages, tissue spheroids represent an emerging trend toward superior, more reliable, and more predictive study models with a broad range of biotechnological applicability. However, reproducible platforms that can achieve large-scale production of tissue spheroids have become an unmet need in fully exploring and boosting their potential. Herein, the large-scale production of homogeneous tissue spheroids is reported using a low-cost and time-effective methodology. A 3D printed stamp-like device is developed to generate up to 4,716 spheroids per 6-well plate. The device is fabricated by the stereolithography method using a photocurable resin. The final device is composed of cylindrical micropins, with a height of 1.3 mm and a width of 650 µm. This approach allows the fast generation of homogeneous spheroids and co-cultured spheroids with uniform shape and size and >95% cell viability. Moreover, the stamp-like device is tunable for different sizes of well plates and Petri dishes. It is easily sterilized and can be reused for long periods. The efficient large-scale production of homogeneous tissue spheroids is essential to leverage their translation for multiple areas of industry, such as tissue engineering, drug development, disease modeling, and on-demand personalized medicine.

Fabrication, morphological, mechanical and biological performance of 3D printed poly(ϵ-caprolactone)/bioglass composite scaffolds for bone tissue engineering applications.

Several techniques, such as additive manufacturing, have been used for the manufacture of polymer-ceramic composite scaffolds for bone tissue engineering. A new extruder head recently developed for improving the manufacturing process is an experimental 3D printer Fab@CTI that enables the use of ceramic powders in the processing of composite materials or polymer blends. Still, the manufacturing process needs improvement to promote the dispersion of ceramic particles in the polymer matrix. This article addresses the manufacture of scaffolds by 3D printing from mixtures of poly(ϵ-caprolactone) (PCL) and a glass powder of same composition of 45S5Bioglass®, labeled as synthesized bioglass (SBG), according to two different methods that investigated the efficiency of the new extruder head. The first one is a single extrusion process in a Fab@CTI 3D printer, and the other consists in the pre-processing of the PCL-SBG mixture in a mono-screw extruder with a Maddock® element, followed by direct extrusion in the experimental Fab@CTI 3D printer. The morphological characterization of the extruded samples by scanning electron microscope showed an architecture of 0°/90° interconnected struts and suitable porosity for bone tissue engineering applications. Scaffolds fabricated by two methods shows compressive modulus ranging from 54.4 ± 14.2 to 155.9 ± 20.4 MPa, results that are compatible to use in bone tissue engineering. Cytotoxicity assays showed non-toxic effects and viability forin vitroMG-63 cell proliferation. Alizarin Red staining test showed calcium deposition in all scaffolds, which suggests PCL/SBG composites promising candidates for use in bone tissue engineering. Results of cell morphology suggest more cell growth and adhesion for scaffolds fabricated using the pre-processing in a mono-screw extruder.

The absence of the autoimmune regulator gene (AIRE) impairs the three-dimensional structure of medullary thymic epithelial cell spheroids.

BACKGROUND: Besides controlling the expression of peripheral tissue antigens, the autoimmune regulator (AIRE) gene also regulates the expression of adhesion genes in medullary thymic epithelial cells (mTECs), an essential process for mTEC-thymocyte interaction for triggering the negative selection in the thymus. For these processes to occur, it is necessary that the medulla compartment forms an adequate three-dimensional (3D) architecture, preserving the thymic medulla. Previous studies have shown that AIRE knockout (KO) mice have a small and disorganized thymic medulla; however, whether AIRE influences the mTEC-mTEC interaction in the maintenance of the 3D structure has been little explored. Considering that AIRE controls cell adhesion genes, we hypothesized that this gene affects 3D mTEC-mTEC interaction. To test this, we constructed an in vitro model system for mTEC spheroid formation, in which cells adhere to each other, establishing a 3D structure. RESULTS: The comparisons between AIRE wild type (AIREWT) and AIRE KO (AIRE-/-) 3D mTEC spheroid formation showed that the absence of AIRE: i) disorganizes the 3D structure of mTEC spheroids, ii) increases the proportion of cells at the G0/G1 phase of the cell cycle, iii) increases the rate of mTEC apoptosis, iv) decreases the strength of mTEC-mTEC adhesion, v) promotes a differential regulation of mTEC classical surface markers, and vi) modulates genes encoding adhesion and other molecules. CONCLUSIONS: Overall, the results show that AIRE influences the 3D structuring of mTECs when these cells begin the spheroid formation through controlling cell adhesion genes.

Predicted miRNA-mRNA-mediated posttranscriptional control associated with differences in cervical and thoracic thymus function.

A secondary cervical thymus (CT) is present in the neck region in about 50% of human and mice. CT in mice is an independent and functional organ, which can be colonized by T lymphocyte progenitors and generate thymocytes that are selected by the T cell receptor repertoire following the positive and negative selection. However, CT and the main thoracic thymus (TT) have been shown in mice to have significant functional differences. In this study, we use transcriptional profiling to compare mRNA or miRNAs expression patterns in murine CT and TT. We used these data to perform functional enrichment of the expression signatures and reconstruction of posttranscriptional miRNA-mRNA interaction networks. For this purpose, we compared the transcriptome profiling of paired RNA samples of whole CTs, TTs and parathyroid gland (PT), which was used as an external group, from Foxn1-GFP;Pth-Cre;R26dTomato transgenic mice that differentially label CT and TT. As expected, CT and TT featured comprehensive transcriptome similarity and this suggests that these organs are subjected to correlated transcriptional control. Nevertheless, significant differences were also observed between TT and CT, characterized by 107 differentially expressed (DE) mRNAs, and in 13 DE miRNAs, that in turn established interactions. These results suggest that functional similarity between TT and CT is reflected in their transcriptional activity and that CT functional uniqueness might be under posttranscriptional control.

Posttranscriptional Interaction Between miR-450a-5p and miR-28-5p and STAT1 mRNA Triggers Osteoblastic Differentiation of Human Mesenchymal Stem Cells.

We demonstrate that the interaction between miR-450a-5p and miR-28-5p and signal transducer and activator of transcription 1 (STAT1) mRNA correlates with the osteoblastic differentiation of mesenchymal stem cells from human exfoliated deciduous teeth (shed cells). STAT1 negatively regulates runx-related transcription factor 2 (RUNX2), which is an essential transcription factor in this process. However, the elements that trigger osteoblastic differentiation and therefore pause the inhibitory effect of STAT1 need investigation. Usually, STAT1 can be posttranscriptionally regulated by miRNAs. To test this, we used an in vitro model system in which shed cells were chemically induced toward osteoblastic differentiation and temporally analyzed, comparing undifferentiated cells with their counterparts in the early (2 days) or late (7 or 21 days) periods of induction. The definition of the entire functional genome expression signature demonstrated that the transcriptional activity of a large set of mRNAs and miRNAs changes during this process. Interestingly, STAT1 and RUNX2 mRNAs feature contrasting expression levels during the course of differentiation. While undifferentiated or early differentiating cells express high levels of STAT1 mRNA, which was gradually downregulated, RUNX2 mRNA was upregulated toward differentiation. The reconstruction of miRNA-mRNA interaction networks allowed the identification of six miRNAs (miR-17-3p, miR-28-5p, miR-29b, miR-29c-5p, miR-145-3p, and miR-450a-5p), and we predicted their respective targets, from which we focused on miR-450a-5p and miR-28-5p STAT1 mRNA interactions, whose intracellular occurrence was validated through the luciferase assay. Transfections of undifferentiated shed cells with miR-450a-5p or miR-28-5p mimics or with miR-450a-5p or miR-28-5p antagonists demonstrated that these miRNAs might play a role as posttranscriptional controllers of STAT1 mRNA during osteoblastic differentiation. J. Cell. Biochem. 118: 4045-4062, 2017. © 2017 Wiley Periodicals, Inc.

Oxidative Nanopatterning of Titanium Surface Influences mRNA and MicroRNA Expression in Human Alveolar Bone Osteoblastic Cells.

Titanium implants have been extensively used in orthopedic and dental applications. It is well known that micro- and nanoscale surface features of biomaterials affect cellular events that control implant-host tissue interactions. To improve our understanding of how multiscale surface features affect cell behavior, we used microarrays to evaluate the transcriptional profile of osteoblastic cells from human alveolar bone cultured on engineered titanium surfaces, exhibiting the following topographies: nanotexture (N), nano+submicrotexture (NS), and rough microtexture (MR), obtained by modulating experimental parameters (temperature and solution composition) of a simple yet efficient chemical treatment with a H2SO4/H2O2 solution. Biochemical assays showed that cell culture proliferation augmented after 10 days, and cell viability increased gradually over 14 days. Among the treated surfaces, we observed an increase of alkaline phosphatase activity as a function of the surface texture, with higher activity shown by cells adhering onto nanotextured surfaces. Nevertheless, the rough microtexture group showed higher amounts of calcium than nanotextured group. Microarray data showed differential expression of 716 mRNAs and 32 microRNAs with functions associated with osteogenesis. Results suggest that oxidative nanopatterning of titanium surfaces induces changes in the metabolism of osteoblastic cells and contribute to the explanation of the mechanisms that control cell responses to micro- and nanoengineered surfaces.