Scaffold-free 3D cell culture of primary skin fibroblasts induces profound changes of the matrisome
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Skin, Fibroblast
SUBMITTER: 
Joern Dengjel
LAB HEAD: Joern Dengjel
PROVIDER: PXD023701 | Pride | 2021-07-16
REPOSITORIES: Pride
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| 20201106_BV_NHF_1_1.raw | Raw | |||
| 20201106_BV_NHF_1_2.raw | Raw | |||
| 20201106_BV_NHF_1_3.raw | Raw | |||
| 20201106_BV_NHF_1_4.raw | Raw | |||
| 20201106_BV_NHF_1_5.raw | Raw |
Items per page: 5 1 - 5 of 37 |
Similar Datasets
Project description:The human skin has a highly developed extracellular matrix (ECM) that is vital for proper skin functioning, its 3D architecture playing a pivotal role in support and guidance of resident and invading cells. To establish relevant in vitro models mimicking the complex design observed in vivo, scaffold-based and scaffold-free 3D cell culture systems have been developed. Here we show that scaffold-free systems are well suited for the analysis of ECM protein regulation. Using quantitative mass spectrometry-based proteomics in combination with magnetic 3D bioprinting we characterize changes in the proteome of skin fibroblasts and squamous cell carcinoma cells. Transferring cells from 2D to 3D without any additional scaffold induces a profound upregulation of matrisome proteins indicating the generation of a complex, tissue-like ECM.
| S-EPMC8377039 | biostudies-literature
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.
2014-08-01 | E-GEOD-52896 | biostudies-arrayexpress
Project description:Classical methods of investigating protein-protein interactions (PPIs) are generally performed in non-living systems, yet in recent years new technologies utilizing proximity labeling (PL) have given researchers the tools to explore proximal PPIs in living systems. PL has distinct advantages over traditional protein interactome studies, such as the ability to identify weak and transient interactions in vitro and in vivo. Most PL studies are performed on targets within or on the cell membrane. We have adapted the original PL method to investigate PPIs within the extracellular compartment, using both BioID2 and TurboID, that we term extracellular PL (ePL). To demonstrate the utility of this modified technique, we investigate the interactome of the widely expressed matrisome protein Tissue inhibitors of metalloproteinases 2 (TIMP2). Tissue inhibitors of metalloproteinases (TIMPs) are a family of multi-functional proteins that were initially defined by their ability to inhibit the enzymatic activity of metalloproteinases (MPs), the major mediators of extracellular matrix (ECM) breakdown and turnover. TIMP2 exhibits a broad expression profile and is often abundant in both normal and diseased tissues. Understanding the functional transformation of matrisome regulators, like TIMP2, during the evolution of tissue microenvironments associated with disease progression is essential for the development of ECM targeted therapeutics. Using carboxyl- and amino-terminal fusion proteins of TIMP2 with BioID2 and TurboID, we describe the TIMP2 proximal interactome. We also illustrate how the TIMP2 interactome changes in the presence of different stimuli, in different cell types, in unique culture conditions (2D vs 3D), and with different reaction kinetics (BioID2 vs. TurboID); demonstrating the power of this technique versus classical PPI methods. We propose that the screening of matrisome targets in disease models using ePL will reveal new therapeutic targets for further comprehensive studies.
2024-01-09 | GSE252575 | GEO
Project description:Enzyme-catalyzed proximity labeling (PL) with biotin is a novel approach to map organelle compartmentalization and protein interaction networks in living cells. Here, we present a new method based on semi-permeabilized yeast cells and the enzyme APEX2, an engineered ascorbate peroxidase. Combined with stable isotope labeling by amino acids in cell culture (SILAC), we demonstrate proteomic mapping of a membrane-enclosed and a semi-open compartment, the mitochondrial matrix and the nucleus. APEX2 PL revealed nuclear proteins that were previously not identified by conventional techniques. One of these, the Yer156C protein, is highly conserved but of unknown function. In follow-up experiments using quantitative PL with Yer156C-APEX2 we discovered an array of Yer156C interactors. Thus, our approach establishes APEX2 PL as an effective and versatile tool that complements the powerful methods palette for the model system yeast.
2019-07-17 | PXD013215 | Pride
Project description:Regenerating critical-sized long bone defects poses substantial challenges due to limitations of autografts and processed allografts. Biomaterial scaffolds offer versatile alternatives, yet their effectiveness is often constrained by their limited innate osteoinductivity. While growth factors and cells can enhance osteoinduction, the inclusion of biologics in biomaterial scaffolds creates regulatory challenges for clinical translation. To address this, here we describe three-dimensional (3D) printed polycaprolactone (PCL) scaffolds for temporally controlled delivery of osteoimmunomodulatory amorphous calcium phosphate-chitosan nanoparticles (ACPC-NP). In vitro, the ACPC-NP exhibit concentration dependent effects on osteoblasts, monocytes, and osteoclasts. At increasing concentrations up to 500 μg/ml, these nanoparticles stimulate osteogenesis, modulate M2/M1 macrophage polarization, and inhibit osteoclast maturation and activity. Leveraging these concentration-dependent effects in vivo through temporally controlled release of ACPC-NP from 3D-printed PCL scaffolds, we observe the complete regeneration and the restoration of biomechanical strength of critically sized radial defects in rats. Such healing is absent in defects implanted with bare PCL scaffolds or those loaded with calcium-phosphate microparticles. The tunable osteoimmunomodulation by the NP underscore the translational potential of this technology to yield structurally sound and functionally robust bone regeneration outcomes.
2024-07-02 | GSE262295 | GEO
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.
2014-08-01 | GSE52896 | GEO
Project description:Analysis of transcriptome changes in rabbit cartilage tissue following surgical induction of critical size defects on knee joints with or without collagen-chondroitin sulphate (CLCS) 3D scaffolds.
2021-12-07 | GSE149098 | GEO
Project description:* To compare surgical and oncological outcomes in patients underwent to colorectal resection with 3D vs 2D laparoscopic technique.
* To evaluate the visual overload in surgeons using 3D laparoscopic technique.
| 2220465 | ecrin-mdr-crc
Project description:Here, we introduce a 3D cell culture system based on a mixture of collagen I and matrigelTM amenable to stable isotope labeling by amino acids in cell culture and quantitative mass spectrometry-based proteomics analyses. We study the extra- and intracellular proteomic response of skin fibroblast isolated from healthy volunteers in comparison to cancer-associated fibroblasts (CAFs) on 3D culture conditions
2018-09-10 | PXD009424 | Pride
Project description:Scaffold-free 3D cell culture of primary skin fibroblasts induces profound changes of the matrisome
| PRJEB43316 | ENA