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 Elastin synthesis by ligamentum nuchae fibroblasts: effects of culture conditions and extracellular matrix on elastin production.

1981 ◽  
Vol 90 (2) ◽  
pp. 332-338 ◽  
Author(s):  
R P Mecham ◽  
G Lange ◽  
J Madaras ◽  
B Starcher
Keyword(s):  
Extracellular Matrix ◽  
Cultured Cells ◽  
Elastic Fiber ◽  
Calf Serum ◽  
Phenotypic Expression ◽  
Fetal Tissue ◽  
Culture Conditions ◽  
Population Doubling

Fetal bovine ligamentum nuchae fibroblasts maintained in culture synthesized soluble elastin but were unable to form the insoluble elastic fiber. Secreted elastin precursors accumulated in culture medium and were measured using a radioimmunoassay for elastin. When elastin production was examined in ligament tissue from fetal calves of various gestational ages, cells from tissue taken during the last trimester of development produced significantly more elastin than did cells from younger fetal tissue, with maximal elastin synthesis occurring shortly before birth. Soluble elastin was detected in ligament cells plated at low density until proliferation began to be density inhibited and the cells became quiescent. Also, soluble elastin production per cell declined with increasing population doubling or with age in culture. Cells grown in the presence of 5% fetal calf serum produced approximately four times as much soluble elastin as cells grown in serum-free medium. The addition of dexamethasone (0.1 microM) and bleomycin (1 microgram/ml) increased soluble elastin production by cultured cells 180% and 50%, respectively, whereas theophylline (5 micrograms/ml) depressed production 50% and antagonized stimulation by dexamethasone. Ascorbate (50 micrograms/ml), soybean trypsin inhibitor (1 mg/ml), insulin (100 microunits/ml), and aminoacetonitrile (50 micrograms/ml) had no effect, but cycloheximide at 10(-4) M completely inhibited soluble elastin production. In contrast to cells in culture, ligament tissue minces (ligament cells surrounded by in vivo extracellular matrix) efficiently incorporated soluble elastin precursors into insoluble, cross-linked elastin. In addition, soluble elastin production per cell (per microgram of DNA) was higher in tissue minces than elastin production by cells maintained on plastic. These results suggest a role for extracellular matrix in formation of the elastic fiber and in stabilizing elastin phenotypic expression by ligament fibroblasts. Fibroblasts from the bovine ligamentum nuchae present an excellent model for in vitro studies of elastin biosynthesis.

scholarly journals A macrophage and theca cell-enriched stromal cell population influences growth and survival of immature murine follicles in vitro

Reproduction ◽  
2011 ◽  
Vol 141 (6) ◽  
pp. 809-820 ◽  
Author(s):  
Candace M Tingen ◽  
Sarah E Kiesewetter ◽  
Jennifer Jozefik ◽  
Cristina Thomas ◽  
David Tagler ◽  
...  
Keyword(s):  
Stromal Cells ◽  
Culture System ◽  
Stromal Cell ◽  
Ovarian Follicle ◽  
3D Culture ◽  
Culture Conditions ◽  
Growth And Survival ◽  
3D Culture System

Innovations in in vitro ovarian follicle culture have revolutionized the field of fertility preservation, but the successful culturing of isolated primary and small secondary follicles remains difficult. Herein, we describe a revised 3D culture system that uses a feeder layer of ovarian stromal cells to support early follicle development. This culture system allows significantly improved primary and early secondary follicle growth and survival. The stromal cells, consisting mostly of thecal cells and ovarian macrophages, recapitulate the in vivo conditions of these small follicles and increase the production of androgens and cytokines missing from stromal cell-free culture conditions. These results demonstrate that small follicles have a stage-specific reliance on the ovarian environment, and that growth and survival can be improved in vitro through a milieu created by pre-pubertal ovarian stromal cell co-culture.

scholarly journals 3D Hepatic Organoid-Based Advancements in LIVER Tissue Engineering

2021 ◽  
Vol 8 (11) ◽  
pp. 185
Author(s):  
Amit Panwar ◽  
Prativa Das ◽  
Lay Poh Tan
Keyword(s):  
Tissue Engineering ◽  
Self Assembly ◽  
Liver Tissue ◽  
3D Culture ◽  
Culture Method ◽  
Culture Conditions ◽  
Phenotypic Properties ◽  
Liver Tissue Engineering

Liver-associated diseases and tissue engineering approaches based on in vitro culture of functional Primary human hepatocytes (PHH) had been restricted by the rapid de-differentiation in 2D culture conditions which restricted their usability. It was proven that cells growing in 3D format can better mimic the in vivo microenvironment, and thus help in maintaining metabolic activity, phenotypic properties, and longevity of the in vitro cultures. Again, the culture method and type of cell population are also recognized as important parameters for functional maintenance of primary hepatocytes. Hepatic organoids formed by self-assembly of hepatic cells are microtissues, and were able to show long-term in vitro maintenance of hepato-specific characteristics. Thus, hepatic organoids were recognized as an effective tool for screening potential cures and modeling liver diseases effectively. The current review summarizes the importance of 3D hepatic organoid culture over other conventional 2D and 3D culture models and its applicability in Liver tissue engineering.

scholarly journals Current Status of the Instructional Cues Provided by Notochordal Cells in Novel Disc Repair Strategies

2021 ◽  
Vol 23 (1) ◽  
pp. 427
Author(s):  
Ajay Matta ◽  
William Mark Erwin
Keyword(s):  
Extracellular Matrix ◽  
Intervertebral Disc ◽  
Current Status ◽  
Scientific Publications ◽  
Seminal Paper ◽  
Notochordal Cell ◽  
Notochordal Cells ◽  
Disc Repair

Numerous publications over the past 22 years, beginning with a seminal paper by Aguiar et al., have demonstrated the ability of notochordal cell-secreted factors to confer anabolic effects upon intervertebral disc (IVD) cells. Since this seminal paper, other scientific publications have demonstrated that notochordal cells secrete soluble factors that can induce anti-inflammatory, pro-anabolic and anti-cell death effects upon IVD nucleus pulposus (NP) cells in vitro and in vivo, direct human bone marrow-derived mesenchymal stem cells toward an IVD NP-like phenotype and repel neurite ingrowth. More recently these factors have been characterized, identified, and used therapeutically to induce repair upon injured IVDs in small and large pre-clinical animal models. Further, notochordal cell-rich IVD NPs maintain a stable, healthy extracellular matrix whereas notochordal cell-deficient IVDs result in a biomechanically and extracellular matrix defective phenotype. Collectively this accumulating body of evidence indicates that the notochordal cell, the cellular originator of the intervertebral disc holds vital instructional cues to establish, maintain and possibly regenerate the intervertebral disc.

scholarly journals In vivo engineered extracellular matrix scaffolds with instructive niches for oriented tissue regeneration

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Meifeng Zhu ◽  
Wen Li ◽  
Xianhao Dong ◽  
Xingyu Yuan ◽  
Adam C. Midgley ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Tissue Regeneration ◽  
Spatial Organization ◽  
Cultured Cells ◽  
Functional Integration ◽  
Host Cells ◽  
Hierarchical Porous ◽  
Parallel Microchannels

Abstract Implanted scaffolds with inductive niches can facilitate the recruitment and differentiation of host cells, thereby enhancing endogenous tissue regeneration. Extracellular matrix (ECM) scaffolds derived from cultured cells or natural tissues exhibit superior biocompatibility and trigger favourable immune responses. However, the lack of hierarchical porous structure fails to provide cells with guidance cues for directional migration and spatial organization, and consequently limit the morpho-functional integration for oriented tissues. Here, we engineer ECM scaffolds with parallel microchannels (ECM-C) by subcutaneous implantation of sacrificial templates, followed by template removal and decellularization. The advantages of such ECM-C scaffolds are evidenced by close regulation of in vitro cell activities, and enhanced cell infiltration and vascularization upon in vivo implantation. We demonstrate the versatility and flexibility of these scaffolds by regenerating vascularized and innervated neo-muscle, vascularized neo-nerve and pulsatile neo-artery with functional integration. This strategy has potential to yield inducible biomaterials with applications across tissue engineering and regenerative medicine.

scholarly journals Simulated Cholinergic Reinnervation ofβ(INS-1) Cells: Antidiabetic Utility of Heterotypic Pseudoislets ContainingβCell and Cholinergic Cell

2018 ◽  
Vol 2018 ◽  
pp. 1-13 ◽  
Author(s):  
Ao Jiao ◽  
Feng Li ◽  
Chengshuo Zhang ◽  
Wu Lv ◽  
Baomin Chen ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Pancreatic Islets ◽  
3D Culture ◽  
Cholinergic Neurons ◽  
Fold Increase ◽  
Culture Conditions ◽  
Glucose Stimulated Insulin Secretion ◽  
E Cadherin

Cholinergic neurons can functionally support pancreatic islets in controlling blood sugar levels. However, in islet transplantation, the level of cholinergic reinnervation is significantly lower compared to orthotopic pancreatic islets. This abnormal reinnervation affects the survival and function of islet grafts. In this study, the cholinergic reinnervation of beta cells was simulated by 2D and 3D coculture of INS-1 and NG108-15 cells. In 2D culture conditions, 20 mM glucose induced a 1.24-fold increase (p<0.0001) in insulin secretion from the coculture group, while in the 3D culture condition, a 1.78-fold increase (p<0.0001) in insulin secretion from heterotypic pseudoislet group was observed. Glucose-stimulated insulin secretion (GSIS) from 2D INS-1 cells showed minimal changes when compared to 3D structures. E-cadherin expressed in INS-1 and NG108-15 cells was the key adhesion molecule for the formation of heterotypic pseudoislets. NG108-15 cells hardly affected the proliferation of INS-1 cells in vitro. Heterotypic pseudoislet transplantation recipient mice reverted to normoglycemic levels faster and had a greater blood glucose clearance compared to INS-1 pseudoislet recipient mice. In conclusion, cholinergic cells can promote insulin-secreting cells to function better in vitro and in vivo and E-cadherin plays an important role in the formation of heterotypic pseudoislets.

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 Bacterial Infection-Mimicking Three-Dimensional Phagocytosis and Chemotaxis in Electrospun Poly(ε-caprolactone) Nanofibrous Membrane

Membranes ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 569
Author(s):  
Seung-Jun Lee ◽  
Perry Ayn Mayson A Maza ◽  
Gyu-Min Sun ◽  
Petr Slama ◽  
In-Jeong Lee ◽  
...  
Keyword(s):  
Culture System ◽  
Three Dimensional ◽  
3D Culture ◽  
Culture Conditions ◽  
Lung Epithelial Cells ◽  
Nanofibrous Membrane ◽  
Infection Model ◽  
Layer System

In this study, we developed a three-dimensional (3D) in vitro infection model to investigate the crosstalk between phagocytes and microbes in inflammation using a nanofibrous membrane (NM). Poly(ε-caprolactone) (PCL)-NMs (PCL-NMs) were generated via electrospinning of PCL in chloroform. Staphylococcus aureus and phagocytes were able to adhere to the nanofibers and phagocytes engulfed S. aureus in the PCL-NM. The migration of phagocytes to S. aureus was evaluated in a two-layer co-culture system using PCL-NM. Neutrophils, macrophages and dendritic cells (DCs) cultured in the upper PCL-NM layer migrated to the lower PCL-NM layer containing bacteria. DCs migrated to neutrophils that cultured with bacteria and then engulfed neutrophils in two-layer system. In addition, phagocytes in the upper PCL-NM layer migrated to bacteria-infected MLE-12 lung epithelial cells in the lower PCL-NM layer. S. aureus-infected MLE-12 cells stimulated the secretion of tumor necrosis factor-α and IL-1α in 3D culture conditions, but not in 2D culture conditions. Therefore, the PCL-NM-based 3D culture system with phagocytes and bacteria mimics the inflammatory response to microbes in vivo and is applicable to the biomimetic study of various microbe infections.

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.

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