Scaffold-free 3D cell culture of skin cells
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ABSTRACT:
PROVIDER: PRJEB43316 | ENA |
REPOSITORIES: ENA
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Project description:We analyze the effect of magnetic 3D bioprinting using NanoShuttle-PL on the proteome of primary human skin fibroblasts and epidermal squamous cell carcinoma cells. NanoStuttle-PL employs magnetic nanoparticles that interact electrostatically with cells rendering them magnetic and allowing manipulation of their 3D assembly using external magnetic forces.
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Project description:Development of systems allowing the maintenance of native properties of mesenchymal stromal cells (MSC) is a critical challenge for studying physiological functions of skeletal progenitors, as well as towards cellular therapy and regenerative medicine applications. Conventional stem cell culture in monolayer on plastic dishes (2D) is associated with progressive loss of functionality, likely due to the absence of a biomimetic microenvironment and the selection of adherent populations. Here we demonstrate that 2D MSC expansion can be entirely bypassed by culturing freshly isolated bone marrow cells within the pores of 3D scaffolds in a perfusion-based bioreactor system, followed by enzymatic digestion for cell retrieval. The 3D-perfusion system supported MSC growth while maintaining cells of the hematopoietic lineage, and thus generated a cellular environment mimicking some features of the bone marrow stroma. As compared to 2D-expansion, sorted CD45- cells derived from 3D-perfusion culture after the same time (3 weeks) or a similar extent of proliferation (7-8 doublings) maintained a 4.3-fold higher clonogenicity and exhibited a superior differentiation capacity towards all typical mesenchymal lineages, with similar immunomodulatory function in vitro. Transcriptomic analysis performed on MSC from 5 donors validated the robustness of the process and indicated a reduced inter-donor variability as well as a significant upregulation of multipotency-related gene clusters following 3D-perfusion as compared to 2D expansion. The described system offers a model to study how factors of a 3D engineered niche may regulate MSC function and, by streamlining conventional labor-intensive processes, is prone to automation and scalability within closed bioreactor systems. Nucleated cells were isolated from 5 fresh human bone marrow aspirates by means of red blood cells lyses buffer and then were seeded into a 3D perfusion bioreactor system using a pure hydroxyapatite 3D scaffold and in conventional Petri dishes (2D). After culture for 19 days, cells from both systems were enzymatically retrieved and sorted using anti-CD45-coated magnetic beads. Total RNA was extracted from CD45- cells, QCed and hybridized to Affymetrix microarrays.
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Project description:Retinoblastoma Y79 cell line was grown on 3D scaffolds and compared its gene expression profile with Y79 cells grown without scaffold. Agilent one-color experiment, Organism: Human, Agilent-014850 Whole Human Genome Microarray 4x44K G4112F, Labeling kit: Agilent Quick-Amp labeling Kit (p/n5190-0442)
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Project description:The intestinal muscle layers execute various gut wall movements to achieve controlled propulsion and mixing of intestinal content. Engineering intestinal muscle layers with complex contractile function is critical for developing bioartificial intestinal tissue to treat patients with short bowel syndrome. A living intestinal muscle patch is created for the first time in vitro to generate three distinct motility patterns and display multiple digesta manipulations. Developing such intestinal muscle patches combines a differentiation medium and a 3D scaffold. Next-generation RNA sequencing was performed across primary cells isolated from the intestinal muscle layers that were further cultured with/without the differentiation medium or with/without the 3D scaffold to characterize the effect of the medium and the scaffold on the transcriptomic profile of the cells. We found that the successful generation of the contracting intestinal muscle patch relies on both biological factors in the medium and environmental cues from the scaffold. In cooperation, the medium and the scaffold both greatly enhance the differentiation and maturity of the enteric neurons. Separately, the medium profoundly impacts ion transport activities. The scaffold markedly reshapes cell communication and protein binding.
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Project description:Understanding the molecular underpinnings of adipogenesis is of fundamental importance for the development of novel therapeutics against metabolic disorders. However, translational approaches are hampered as current models do not accurately recapitulate adipogenesis. Here, a scaffold-free versatile 3D adipocyte culture platform with chemically defined conditions is presented in which primary human preadipocytes accurately recapitulate adipogenesis. Following differentiation, multi-omics profiling and functional tests demonstrate that 3D adipocyte cultures feature mature molecular and cellular phenotypes similar to freshly isolated mature adipocytes. Spheroids exhibit physiologically relevant gene expression signatures with 4704 differentially expressed genes compared to conventional 2D cultures (false discovery rate < 0.05), including the concerted expression of factors shaping the adipogenic niche. Furthermore, lipid profiles of >1000 lipid species closely resemble patterns of the corresponding isogenic mature adipocytes in vivo (R^2 = 0.97). Integration of multi-omics signatures with analyses of the activity profiles of 503 transcription factors using global promoter motif inference reveals a complex signaling network, involving YAP, Hedgehog, and TGFβ signaling, that links the organotypic microenvironment in 3D culture to the activation and reinforcement of PPARγ and CEBP activity resulting in improved adipogenesis.
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Project description:3D cell cultures are becoming more and more important in the field of regenerative medicine due to their ability to mimic the cellular physiological microenvironment. Among the different types of 3D scaffolds there is the Nichoid: an engineered 3D micro-scaffold with a structure inspired by the natural staminal niche. The Nichoid can activate cellular response simply by subjecting the cells to mechanical stimuli. This kind of influence results in different cellular morphology and organization, but the molecular bases of these changes remain largely unknown. Here, we show that the main cellular response to the Nichoid is an alteration of the mechanotransduction, which in turn affects the global cellular activity. Through RNA-seq approach on murine Neural Precursors Stem Cells (NPCs) expanded inside the Nichoid, we investigated the deregulated genes and pathways showing that the Nichoid causes alteration in genes strongly connected to mechanical properties. We found 1934 deregulated RNAs, 81% of which were coding genes. Moreover, we fully dissected this mechanism highlighting how the changes start at a membrane level, with subsequent alterations in the cytoskeleton, signalling pathways, and metabolism, all leading to a final alteration in gene expression. The results shown here demonstrate that the 3D scaffold Nichoid influences the biological and genetic response of stem cells thorough specific alterations of cellular signaling.
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