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Establishing a reproducible approach to study cellular functions of plants cells with 3D bioprinting

ABSTRACT: Capturing cell-to-cell and cell-to-environment signals in a defined 3 dimensional (3D) microenvironment is key to study cellular functions, including cellular reprogramming towards tissue regeneration. A major challenge in current culturing methods is that these methods cannot accurately capture this multicellular 3D microenvironment. In this study, we established the framework of 3D bioprinting with plant cells to study cell viability, cell division, and cell identity. To investigate the cellular reprogramming underlying the initial cell divisions prior to microcallus formation, we collected RNA from isolated differentiated root cells immediately, 1, and 3 days after bioprinting. We showed the cell cycle re-entry of the isolated Arabidopsis and soybean cells leading to the formation of microcalli and of which the timing coincides with the induction of core cell cycle genes and regeneration-related genes. Finally, we showed that the identity of isolated cells of Arabidopsis roots expressing endodermal markers maintained longer periods of time. The framework established in this study paves the way for a general use of 3D bioprinting for studying cellular reprogramming and cell cycle re-entry towards tissue regeneration.

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE208176 | GEO | 2022/08/15

REPOSITORIES: GEO

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Nanoengineered Osteoinductive Bioink for Three-Dimensional Bioprinting Bone Tissue

Project description:Bioprinting is an emerging additive manufacturing approach to the fabrication of patient-specific, implantable three-dimensional (3D) constructs for regenerative medicine. However, developing cell-compatible bioinks with high printability, structural stability, biodegradability, and bioactive characteristics is still a primary challenge for translating 3D bioprinting technology to preclinical and clinal models. To overcome this challenge, we develop a nanoengineered ionic covalent entanglement (NICE) bioink formulation for 3D bone bioprinting. The NICE bioinks allow precise control over printability, mechanical properties, and degradation characteristics, enabling custom 3D fabrication of mechanically resilient, cellularized structures. We demonstrate cell- induced remodeling of 3D bioprinted scaffolds over 60 days, demonstrating deposition of nascent extracellular matrix proteins. Interestingly, the bioprinted constructs induce endochondral differentiation of encapsulated human mesenchymal stem cells (hMSCs) in absence of osteoinducing agents such as dexamethasone or bone morphogenic protein-2 (BMP-2). Using next-generation transcriptome sequencing (RNA-seq) technology, we establish the role of nanosilicates, a bioactive component of NICE bioink, to stimulate endochondral differentiation at the transcriptome level. Overall, the osteoinductive bioink has the ability to induce formation of osteo-related mineralized extracellular matrix by encapsulatedhMSCsingrowthfactor-freeconditions.Furthermore,wedemonstratedtheabilityofNICEbioinktofabricatepatient-specific, implantable 3D scaffolds for repair of craniomaxillofacial bone defects. We envision transformation of this NICE bioink technology toward a realistic clinical process for 3D bioprinting patient-specific bone tissue for regenerative medicine.

2020-03-04 | GSE146309 | GEO

Scaffold-free 3D cell culture of primary skin fibroblasts induces profound changes of the matrisome

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.

2021-07-16 | PXD023701 | Pride

High-speed live cell interferometry for screening bioprinted organoids

Project description:We developed bioprinted tumor organoids linked to real-time growth pattern quantitation via high-speed live cell interferometry (HSLCI). We demonstrate that bioprinting gives rise to 3D organoid structures that preserve histology and gene expression.

2022-11-26 | GSE218693 | GEO

Rapid 3D Bioprinting of Multicellular Model Recapitulating Pterygium Microenvironment

Project description:Pterygium is an ocular surface disorder with high prevalence that can lead to vision impairment. As a pathological outgrowth of conjunctiva, pterygium involves neovascularization and chronic inflammation, but its pathogenesis remains largely unknown. Over the last decade, various types of disease models have been built to study pterygium. Here, we developed a 3D multicellular in vitro pterygium model using the digital light processing (DLP)-based 3D bioprinting of human conjunctival stem cells (hCjSCs). A novel feeder-free culture system was adopted and efficiently expanded the primary hCjSCs with homogeneity, stemness and differentiation potency. The DLP-based 3D bioprinting was able to fabricate hydrogel scaffolds that support the viability and biological integrity of the encapsulated hCjSCs. The bioprinted 3D pterygium model was fabricated with hCjSCs, immune cells and vascular cells to recapitulate the disease microenvironment. Transcriptomic analysis using RNA sequencing (RNA-seq) identified a distinct profile correlated to inflammation response, angiogenesis, and epithelial mesenchymal transition in the bioprinted 3D pterygium model. In addition, the pterygium signatures and disease relevance of the bioprinted model were validated with the public RNA-seq data from patient-derived pterygium tissues. By integrating the stem cell technology and 3D bioprinting, this is the first reported 3D in vitro disease model for pterygium that can be utilized by future studies towards the personalized medicine and the drug screening.

2022-06-01 | GSE180343 | GEO

Tuning the 3D microenvironment of reprogrammed tubule cells enhances biomimetic modeling of polycystic kidney disease

Project description:Embedding cells in 3D environments can enhance their physiological properties and tissue architecture can be controlled using bioprinting approaches. One cellular source for in vitro reconstituted kidney tubules are directly reprogrammed induced renal tubular epithelial cells (iRECs). Here, we investigated the compatibility of iRECs with different biomaterials as self assembly or bioprinting dependent guided growth approaches. We assess cell viability, morphology, their transcriptional changes and protein expression properties. iRECs showed high viability and biocompatibility with dispensing methods and bioinks. However, the morphology of multicellular aggregates was dramatically influenced by the microenvironment. Transcriptomic analyses revealed differentially expressed gene-signatures specific to each of the used biomaterials and to bioprinted tubule-like structures. In conclusion, we find that the 3D environment elicits a strong influence on the morphology and expression profiles of iRECs that unmasks disease phenotypes and can be harnessed for and tailored to experimental demands. Combining directly reprogrammed cells with the appropriate biomaterials will facilitate optimal construction of biomimetic kidney tubule and disease models at scale.

2022-11-22 | GSE184531 | GEO

Mechanical forces direct the cell fate of inner ear organoids in a stage-dependent manner

Project description:During the development, mechanical force plays an important role in shaping the inner ear and establishing regular cellular patterns, however, the effects of ubiquitous mechanical cues on cellular fate determination are not clear. Here we developed stiffness adjustable hydrogels for three-dimensional (3D) cochlear organoid culture, which could precisely simulate the mechanical force from the extracellular matrix (ECM) during the expansion of cochlear progenitor cells (CPCs) and organoid formation. Within a certain range, suitable stiffness can stimulate CPC proliferation through a mechanism requiring integrin/cytoskeleton/YAP signaling. Surprisingly, increasing stiffness could inhibit the proliferation of CPCs and induce the differentiation of organoids, which was mediated by the increased intracellular Ca2+ signaling in CPCs, and further activated Klf2 to promote the differentiation of HCs with bundles. Furthermore, this chemical-defined hydrogel is also suitable for 3D bioprinting, and we printed the spiral-like structure with CPCs and HCs, which provided a promising way for cochlear organoids to further improve our understanding of the organization of the inner ear and contribute to developing new therapeutic approaches for HCs regeneration.

2023-10-26 | GSE202640 | GEO

Inhibition of the mTOR pathway and reprogramming of protein synthesis by MDM4 reduce ovarian cancer metastatic properties.

Project description:Background: The expression of MDM4, a well-known p53-inhibitor, is positively associated with chemotherapy response and overall survival in epithelial ovarian cancer (EOC). The basis of this association remains elusive. Since the occurrence of metastasis is one of the factors responsible for the high death rate of this cancer, we analyzed MDM4 involvement in EOC metastatic process. Methods: In vivo and in vitro models, based on 2D and 3D assays, were employed to assess the activity of MDM4 in ovarian cancer progression. A 3D-bioprinting co-culture system was ad hoc developed for this study. Proteomic analysis was conducted on 3D multicellular tumour spheroids to assess pathways triggered by MDM4 overexpression. Results: In mouse models, increased MDM4 reduced intraperitoneal dissemination of human and murine EOC cells, independently of p53 and in a cell-autonomous way. Consistently, high MDM4 correlates with increased overall survival probability in large public data sets. 2D and 3D assays indicated that MDM4 impairs the early steps of the metastatic process. The 3D-bioprinting co-culture system showed reduced dissemination and intravasation into vessel-like structures of MDM4-expressing cells. Proteomic analysis of EOC spheroids revealed that MDM4 reduces protein synthesis and decreases mTOR signaling. Accordingly, MDM4 did not further inhibit EOC cell migration when its activity towards mTOR is blocked genetically or pharmacologically. Conversely, increased MDM4 reduced the efficacy of mTOR inhibitors in constraining EOC cell migration. Conclusions: Overall, these data clarify the antagonism of MDM4 towards EOC progression and suggest the usefulness of MDM4 assessment for tailored application of mTOR targeted therapy.

2021-05-27 | PXD019935 | Pride

3D-Bioprinting allows the establishment of long-term 3D culture model for Chronic Lymphocytic Leukemia cells

Project description:Studying primary Chronic Lymphocytic Leukemia cells in vitro is very challenging due to their short survival in culture, and also due to the fact that traditional two-dimensional in vitro models lack cellular and spatial complexity present in vivo. Based on these considerations, we exploited three-dimensional (3D) bioprinting to advance in vitro models for CLL. Through RNA sequencing (RNAseq) analysis, we observed a consistent difference in gene expression profile between 2D and 3D samples, indicating a different behavior of the cells in the two different culture settings.

2021-04-21 | GSE163977 | GEO

Bioprinted 3D Outer Retina Barrier Uncovers RPE-dependent Choroidal Phenotype in Advanced Macular Degeneration

Project description:We engineered a native-like 3D-oBRB tissue by bioprinting endothelial cells, pericytes, and fibroblasts on the basal side of a biodegradable scaffold and establishing an RPE monolayer on top. To investigate the communication between endothelial cells, fibroblasts, pericytes and retinal pigment epithelial cells, we grew them as either 2D cell cultures or as 3D bioprinted outer blood retinal barrier tissues and harvested cells for single-cell RNAseq after 6 weeks of maturation.

2022-10-21 | GSE214928 | GEO

Paxbp1 is indispensable for cell cycle re-entry of adult muscle stem cells

Project description:Adult muscle stem cells (MuSCs) transit from a quiescent state to a highly proliferative state to support muscle repair. Paxbp1 deletion in MuSCs blocked their cell cycle re-entry and caused subsequent muscle regeneration failures. To understand the underlying molecular mechanisms, we compared the transcriptomes of control and Paxbp1 mutant MuSCs by RNA-seq. Our data confirmed a marked repression of cell cycle machineries in Paxbp1 mutant MuSCs. We also uncovered down-regulation of mutliple metabolic pathways in mutant MuSCs. In summary, our study highlights an essential role of Paxbp1 in MuSCs growth and cell cycle re-entry.

2021-03-23 | GSE141881 | GEO

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