Protein-engineered scaffolds for in vitro 3D culture of primary adult intestinal organoids

2015 ◽  
Vol 3 (10) ◽  
pp. 1376-1385 ◽  
Author(s):  
Rebecca L. DiMarco ◽  
Ruby E. Dewi ◽  
Gabriela Bernal ◽  
Calvin Kuo ◽  
Sarah C. Heilshorn
Keyword(s):  
Extracellular Matrix ◽  
Recombinant Protein ◽  
3D Culture ◽  
Intestinal Organoids ◽  
Microenvironmental Factors

A family of recombinant, protein-engineered mimics of the extracellular matrix were used to identify the microenvironmental factors that impact the 3D culture efficiency of primary adult intestinal organoids.

A rhabdomyosarcoma hydrogel model to unveil cell-extracellular matrix interactions

2021 ◽  
Author(s):  
Mattia Saggioro ◽  
Stefania D'Agostino ◽  
Anna Gallo ◽  
Sara Crotti ◽  
Sara D'Aronco ◽  
...  
Keyword(s):  
Cell Culture ◽  
Extracellular Matrix ◽  
Three Dimensional ◽  
3D Culture ◽  
3D Cell Culture ◽  
Culture Systems ◽  
Matrix Interactions ◽  
In Vitro Systems ◽  
Extracellular Matrix Interactions

Three-dimensional (3D) culture systems are progressively getting attention given their potential in overcoming limitations of the classical 2D in vitro systems. Among different supports for 3D cell culture, hydrogels (HGs)...

The regenerative capacity of the notochordal cell: tissue constructs generated in vitro under hypoxic conditions

2009 ◽  
Vol 10 (6) ◽  
pp. 513-521 ◽  
Author(s):  
W. Mark Erwin ◽  
Facundo Las Heras ◽  
Diana Islam ◽  
Michael G. Fehlings ◽  
Robert D. Inman
Keyword(s):  
Extracellular Matrix ◽  
Tissue Culture ◽  
Sodium Alginate ◽  
Cultured Cells ◽  
3D Culture ◽  
Culture Conditions ◽  
Notochordal Cells ◽  
Collagen Ii

Object The intervertebral disc (IVD) is a highly avascular structure that is occupied by highly specialized cells (nucleus pulposus [NP] cells) that have adapted to survive within an O2 concentration of 2–5%. The object of this study was to investigate the effects of long-term hypoxic and normoxic tissue cultures of nonchondrodystrophic canine notochordal cells—cells that appear to protect the disc NP from degenerative change. Methods The authors obtained notochordal cells from nonchondrodystrophic canines according to their established methods and placed them into monolayer and 3D culture using sodium alginate globules under either hypoxic (3.5% O2) or normoxic (21% O2) conditions. Histological, immunohistochemical, scanning electron microscopy, and histomorphometric methods were used to evaluate the cells within the globules after 5 months in culture. Results Notochordal cells under in vitro hypoxic tissue culture conditions produced a highly complex, organized, 3D cellular construct that was strikingly similar to that observed in vivo. In contrast, traditional normoxic tissue culture conditions resulted in notochordal cells that failed to produce an organized matrix. Hypoxia resulted in a matrix rich in aggrecan and collagen II, whereas normoxic cultured cells did not produce any observable aggrecan or collagen II after 5 months of culture. Conclusions Hypoxia induces notochordal cells to organize a complex 3D cellular/extracellular matrix without an external scaffold other than suspension within sodium alginate. These cells produce an extracellular matrix and large construct that shares exactly the same characteristics as the in vivo condition—robust aggrecan, and type II collagen production. Normoxic tissue culture conditions, however, lead to a failure of these cells to thrive and a lack of extracellular matrix production and significantly smaller cells. The authors suggest that future studies of NP cells and, in particular, notochordal cells should utilize hypoxic tissue culture conditions to derive meaningful, biologically relevant conclusions concerning possible biological/molecular interventions.

scholarly journals Bioprintable Lung Extracellular Matrix Hydrogel Scaffolds for 3D Culture of Mesenchymal Stromal Cells

Polymers ◽  
2021 ◽  
Vol 13 (14) ◽  
pp. 2350
Author(s):  
Bryan Falcones ◽  
Héctor Sanz-Fraile ◽  
Esther Marhuenda ◽  
Irene Mendizábal ◽  
Ignacio Cabrera-Aguilera ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Cultured Cells ◽  
3D Culture ◽  
Focal Adhesions ◽  
Fold Increase ◽  
Paracrine Factors ◽  
Hydrogel Scaffolds ◽  
Vitro Model ◽  
Cells Cultured

Mesenchymal stromal cell (MSC)-based cell therapy in acute respiratory diseases is based on MSC secretion of paracrine factors. Several strategies have proposed to improve this are being explored including pre-conditioning the MSCs prior to administration. We here propose a strategy for improving the therapeutic efficacy of MSCs based on cell preconditioning by growing them in native extracellular matrix (ECM) derived from the lung. To this end, a bioink with tunable stiffness based on decellularized porcine lung ECM hydrogels was developed and characterized. The bioink was suitable for 3D culturing of lung-resident MSCs without the need for additional chemical or physical crosslinking. MSCs showed good viability, and contraction assays showed the existence of cell–matrix interactions in the bioprinted scaffolds. Adhesion capacity and length of the focal adhesions formed were increased for the cells cultured within the lung hydrogel scaffolds. Also, there was more than a 20-fold increase of the expression of the CXCR4 receptor in the 3D-cultured cells compared to the cells cultured in plastic. Secretion of cytokines when cultured in an in vitro model of lung injury showed a decreased secretion of pro-inflammatory mediators for the cells cultured in the 3D scaffolds. Moreover, the morphology of the harvested cells was markedly different with respect to conventionally (2D) cultured MSCs. In conclusion, the developed bioink can be used to bioprint structures aimed to improve preconditioning MSCs for therapeutic purposes.

scholarly journals Crosstalk between invadopodia and the extracellular matrix

2020 ◽  
Author(s):  
Shinji Iizuka ◽  
Ronald P. Leon ◽  
Kyle P. Gribbin ◽  
Ying Zhang ◽  
Jose Navarro ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Type I Collagen ◽  
Complex Structure ◽  
Three Dimensional ◽  
3D Culture ◽  
Model Systems ◽  
Type I ◽  
Extracellular Matrix Components

ABSTRACTThe scaffold protein Tks5α is required for invadopodia-mediated cancer invasion both in vitro and in vivo. We have previously also revealed a role for Tks5 in tumor cell growth using three-dimensional (3D) culture model systems and mouse transplantation experiments. Here we use both 3D and high-density fibrillar collagen (HDFC) culture to demonstrate that native type I collagen, but not a form lacking the telopeptides, stimulated Tks5-dependent growth, which was dependent on the DDR collagen receptors. We used microenvironmental microarray (MEMA) technology to determine that laminin, collagen I, fibronectin and tropoelastin also stimulated invadopodia formation. A Tks5α-specific monoclonal antibody revealed its expression both on microtubules and at invadopodia. High- and super-resolution microscopy of cells in and on collagen was then used to place Tks5α at the base of invadopodia, separated from much of the actin and cortactin, but coincident with both matrix metalloprotease and cathepsin proteolytic activity. Inhibition of the Src family kinases, cathepsins or metalloproteases all reduced invadopodia length but each had distinct effects on Tks5α localization. These studies highlight the crosstalk between invadopodia and extracellular matrix components, and reveal the invadopodium to be a spatially complex structure.

scholarly journals The Impact of the Extracellular Matrix Environment on Sost Expression by the MLO-Y4 Osteocyte Cell Line

2022 ◽  
Vol 9 (1) ◽  
pp. 35
Author(s):  
Robert T. Brady ◽  
Fergal J. O’Brien ◽  
David A. Hoey
Keyword(s):  
Extracellular Matrix ◽  
Cell Line ◽  
Three Dimensional ◽  
3D Culture ◽  
Specific Gene ◽  
Collagen Scaffolds ◽  
Sost Gene ◽  
The Impact

Bone is a dynamic organ that can adapt its structure to meet the demands of its biochemical and biophysical environment. Osteocytes form a sensory network throughout the tissue and orchestrate tissue adaptation via the release of soluble factors such as a sclerostin. Osteocyte physiology has traditionally been challenging to investigate due to the uniquely mineralized extracellular matrix (ECM) of bone leading to the development of osteocyte cell lines. Importantly, the most widely researched and utilized osteocyte cell line: the MLO-Y4, is limited by its inability to express sclerostin (Sost gene) in typical in-vitro culture. We theorised that culture in an environment closer to the in vivo osteocyte environment could impact on Sost expression. Therefore, this study investigated the role of composition and dimensionality in directing Sost expression in MLO-Y4 cells using collagen-based ECM analogues. A significant outcome of this study is that MLO-Y4 cells, when cultured on a hydroxyapatite (HA)-containing two-dimensional (2D) film analogue, expressed Sost. Moreover, three-dimensional (3D) culture within HA-containing collagen scaffolds significantly enhanced Sost expression, demonstrating the impact of ECM composition and dimensionality on MLO-Y4 behaviour. Importantly, in this bone mimetic ECM environment, Sost expression was found to be comparable to physiological levels. Lastly, MLO-Y4 cells cultured in these novel conditions responded accordingly to fluid flow stimulation with a decrease in expression. This study therefore presents a novel culture system for the MLO-Y4 osteocyte cell line, ensuring the expression of an important osteocyte specific gene, Sost, overcoming a major limitation of this model.

scholarly journals Distinct niches within the extracellular matrix dictate fibroblast function in (cell free) 3D lung tissue cultures

2018 ◽  
Vol 314 (5) ◽  
pp. L708-L723 ◽  
Author(s):  
Gerald Burgstaller ◽  
Arunima Sengupta ◽  
Sarah Vierkotten ◽  
Gerhard Preissler ◽  
Michael Lindner ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Lung Tissue ◽  
Ex Vivo ◽  
3D Culture ◽  
Focal Adhesions ◽  
3D Cell Culture ◽  
Small Gtpases ◽  
Cellular Migration

Cues from the extracellular matrix (ECM) and their functional interplay with cells play pivotal roles for development, tissue repair, and disease. However, the precise nature of this interplay remains elusive. We used an innovative 3D cell culture ECM model by decellularizing 300-µm-thick ex vivo lung tissue scaffolds (d3D-LTCs) derived from diseased and healthy mouse lungs, which widely mimics the native (patho)physiological in vivo ECM microenvironment. We successfully repopulated all d3D-LTCs with primary human and murine fibroblasts, and moreover, we demonstrated that the cells also populated the innermost core regions of the d3D-LTCs in a real 3D fashion. The engrafted fibroblasts revealed a striking functional plasticity, depending on their localization in distinct ECM niches of the d3D-LTCs, affecting the cells’ tissue engraftment, cellular migration rates, cell morphologies, and protein expression and phosphorylation levels. Surprisingly, we also observed fibroblasts that were homing to the lung scaffold’s interstitium as well as fibroblasts that were invading fibrotic areas. To date, the functional nature and even the existence of 3D cell matrix adhesions in vivo as well as in 3D culture models is still unclear and controversial. Here, we show that attachment of fibroblasts to the d3D-LTCs evidently occurred via focal adhesions, thus advocating for a relevant functional role in vivo. Furthermore, we found that protein levels of talin, paxillin, and zyxin and phosphorylation levels of paxillin Y118, as well as the migration-relevant small GTPases RhoA, Rac, and CDC42, were significantly reduced compared with their attachment to 2D plastic dishes. In summary, our results strikingly indicate that inherent physical or compositional characteristics of the ECM act as instructive cues altering the functional behavior of engrafted cells. Thus, d3D-LTCs might aid to obtain more realistic data in vitro, with a high relevance for drug discovery and mechanistic studies alike.

scholarly journals Vascular lung triculture organoid via soluble extracellular matrix suspension

2021 ◽  
Author(s):  
Jonard Corpuz Valdoz ◽  
Nicholas A Franks ◽  
Collin G Cribbs ◽  
Dallin J Jacobs ◽  
Ethan L Dodson ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Cell Biology ◽  
Disease Modeling ◽  
3D Culture ◽  
Culture Method ◽  
Matrix Proteins ◽  
Dependent Relationship ◽  
Shaped Cell ◽  
And Function

Scaffold-free tissue engineering is desired in creating consistently sized and shaped cell aggregates but has been limited to spheroid-like structure and function, thus restricting its use in accurate disease modeling. Here, we show formation of a viable lung organoid from epithelial, endothelial, and fibroblast stable cell lines in suspension culture supplemented with soluble concentrations of extracellular matrix proteins (ECM). We demonstrate the importance of soluble ECM in organotypic patterning with the emergence of air space-like gas exchange units, formation of branching, perfusable vasculature, and increased 3D growth. Our results show a dependent relationship between enhanced fibronectin fibril assembly and the incorporation of ECM in the organoid. Endothelial branching was found to depend on both soluble ECM and fibroblast. We successfully applied this technology in modeling lung fibrosis via bleomycin induction and test a potential antifibrotic drug in vitro while maintaining fundamental cell-cell interactions in lung tissue. Our human fluorescent lung organoid (hFLO) model accurately represents features of pulmonary fibrosis which were ameliorated by fasudil treatment. We demonstrate a 3D culture method with potential of creating organoids from mature cells, thus opening avenues for disease modeling and regenerative medicine, enhancing understanding of lung cell biology in health and lung disease.

scholarly journals Robust Three-Dimensional (3D) Expansion of Bovine Intestinal Organoids: An In Vitro Model as a Potential Alternative to an In Vivo System

Animals ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 2115
Author(s):  
Bo-Ram Lee ◽  
Hyeon Yang ◽  
Sang-In Lee ◽  
Inamul Haq ◽  
Sun-A Ock ◽  
...  
Keyword(s):  
Stem Cells ◽  
Small Intestine ◽  
Adult Stem Cells ◽  
Three Dimensional ◽  
3D Culture ◽  
Great Promise ◽  
Specific Expression ◽  
Intestinal Organoids

Intestinal organoids offer great promise for disease-modelling-based host–pathogen interactions and nutritional research for feed efficiency measurement in livestock and regenerative medicine for therapeutic purposes. However, very limited studies are available on the functional characterisation and three-dimensional (3D) expansion of adult stem cells in livestock species compared to other species. Intestinal crypts derived from intestinal organoids under a 3D culture system from the small intestine in adult bovine were successfully established and characterised for functionality testing, including the cellular potentials and genetic properties based on immunohistochemistry, immunocytochemistry, epithelial barrier permeability assay, QuantSeq 3′ mRNA-Seq. data and quantitative reverse transcription-polymerase chain reaction. Intestinal organoids were long-term cultivated over several passages of culture without loss of the recapitulating capacity of crypts, and they had the specific expression of several specific markers involved in intestinal stem cells, intestinal epithelium, and nutrient absorption. In addition, they showed the key functionality with regard to a high permeability for compounds of up to FITC-dextran 4 kDa, while FITC-dextran 40 kDa failed to enter the organoid lumen and revealed that the genetic properties of bovine intestinal organoids were highly similar to those of in vivo. Collectively, these results provide a reliable method for efficient isolation of intestinal crypts from the small intestine and robust 3D expansion of intestinal organoids in adult bovine and demonstrate the in vitro 3D organoids mimics the in vivo tissue topology and functionality. Finally, intestinal organoids are potential alternatives to in vivo systems and will be facilitated as the practical model to replace animal experiments for various purposes in the fields of animal biotechnology.

scholarly journals Decellularized extracellular matrix mediates tissue construction and regeneration

2021 ◽  
Author(s):  
Chuanqi Liu ◽  
Ming Pei ◽  
Qingfeng Li ◽  
Yuanyuan Zhang
Keyword(s):  
Extracellular Matrix ◽  
Wound Healing ◽  
Tissue Regeneration ◽  
Healing Process ◽  
3D Culture ◽  
Tissue Remodeling ◽  
Specific Cell ◽  
Culture Development

AbstractContributing to organ formation and tissue regeneration, extracellular matrix (ECM) constituents provide tissue with three-dimensional (3D) structural integrity and cellular-function regulation. Containing the crucial traits of the cellular microenvironment, ECM substitutes mediate cell—matrix interactions to prompt stem-cell proliferation and differentiation for 3D organoid construction in vitro or tissue regeneration in vivo. However, these ECMs are often applied generically and have yet to be extensively developed for specific cell types in 3D cultures. Cultured cells also produce rich ECM, particularly stromal cells. Cellular ECM improves 3D culture development in vitro and tissue remodeling during wound healing after implantation into the host as well. Gaining better insight into ECM derived from either tissue or cells that regulate 3D tissue reconstruction or organ regeneration helps us to select, produce, and implant the most suitable ECM and thus promote 3D organoid culture and tissue remodeling for in vivo regeneration. Overall, the decellularization methodologies and tissue/cell-derived ECM as scaffolds or cellular-growth supplements used in cell propagation and differentiation for 3D tissue culture in vitro are discussed. Moreover, current preclinical applications by which ECM components modulate the wound-healing process are reviewed.

Practical Applications of 3D Cell Culture Biotechnologies for Translational Oncology and Personalized Therapy

2020 ◽  
Vol 36 (3) ◽  
pp. 3-15
Author(s):  
D.A. Chudakova ◽  
E.Yu. Skorova ◽  
I.V. Reshetov
Keyword(s):  
Cell Culture ◽  
Extracellular Matrix ◽  
3D Culture ◽  
3D Cell Culture ◽  
Personalized Therapy ◽  
3D Bioprinting ◽  
Culture Systems ◽  
Culture Models ◽  
2D And 3D

The creation of in vitro three-dimensional cellular model systems (in vitro 3D cultures) is a fast-growing leading-edge segment of the biotechnological industry. We have examined in this work the key 80 articles published after 2008, and focused on applications of in vitro 3D culture in translational oncology. We described a broad range of 3D culture systems, including models with and without extracellular matrix (ECM). 3D culture models based on decellularized ECM were discussed in more detail. The role of ECM in pathogeneis of malignant neoplasms, in particular, in the phenomenon of the tumor resistance to chemotherapy, was evaluated. 2D and 3D culture systems were compared, and natural and synthetic ECM were described, as well as the model creation based on 3D bioprinting. Particular attention was paid to in vitro models of various cancers, including those at the metastatic stage, based on 3D cell cultures, which maximally mimic the in vivo tumor behavior. The prospects of the practical application of 3D cell culture models in preclinical drag screening and in personalized therapy were discussed. We also presented our data on in vitro 2D and 3D culturing of human cells on various substrates. 3D cellular models, 3D bioprinting, biotechnology, extracellular matrix, cancer, translational medicine, personalized medicine, drag development, in vitro, ex vivo, oncology The authors are grateful to Dr. E. Shabalina for providing part of the experimental data and to OKA-Biotech Company for the samples of recombinant Funding-The work was supported by a Grant from the Russian Science Foundation (no. 18-15-00391). doi: 10.21519/0234-2758-2020-36-3-3-15

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