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.

Gonadotrophic regulation of prolactin mediated progesterone secretion in vitro

1985 ◽  
Vol 110 (2) ◽  
pp. 251-256 ◽  
Author(s):  
J. Steven Alexander ◽  
Thomas M. Crisp
Keyword(s):  
Granulosa Cells ◽  
In Vitro Model ◽  
Fold Increase ◽  
Model System ◽  
Regulatory Mechanisms ◽  
Progesterone Secretion ◽  
Vitro Model ◽  
Dose Dependent ◽  
Cells Cultured

Abstract. The effects of preincubating rat granulosa cells with FSH, LH, and Prl on subsequent Prl mediated progesterone secretion were investigated. Granulosa cells were isolated from ovarian follicles 50 h after injection of 5 IU PMSG and were then plated on poly-l-lysine coated coverslips in serum supplemented medium. Cells were preincubated for 24 h in the absence of hormones (control) or with the addition of either 0.25, 2.5, 25 ng/ml rat FSH or rat LH, or 1 μg/ml rat Prl. Following the preincubation period, cells were maintained for an additional 6 or 8 days in the presence or absence of 1 μg/ml Prl. When cells were preincubated with FSH or LH, only the two higher concentrations (2.5 and 25 ng/ml) stimulated significantly more progesterone secretion than control cultures during the 24 h preincubation period. For each series of preincubations, cells cultured for 6 or 8 days in the presence of Prl secreted significantly more progesterone at each day of culture than cells cultured without Prl. Cells preincubated and cultured with Prl secreted only 3–7-fold more progesterone than cells preincubated in control medium and then cultured with Prl. Preincubation with FSH or LH promoted a 20–45-fold increase in Prl mediated progesterone secretion compared to control preincubation cultures that also subsequently were cultured with Prl. The magnitude of Prl mediated progesterone secretion observed through 6 days of culturing was dose dependent on the preincubation concentration of FSH or LH. The establishment of an in vitro model system in which gonadotrophins enhance the responsiveness of granulosa cells to Prl in serum supplemented medium provides the opportunity for study of the regulatory mechanisms involved with the induction and maintenance of such responsiveness.

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 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.

Development of an in vitro Model for the Study of the Response of Equine Tendon Fibroblasts to Injury and Medication

1997 ◽  
Vol 10 (01) ◽  
pp. 6-11 ◽  
Author(s):  
R. F. Rosenbusch ◽  
L. C. Booth ◽  
L. A. Dahlgren
Keyword(s):  
Electron Microscopy ◽  
Extracellular Matrix ◽  
Light Microscopy ◽  
Light And Electron Microscopy ◽  
Tendon Healing ◽  
Matrix Proteins ◽  
Isolated Cells ◽  
Superficial Digital Flexor Tendon ◽  
Vitro Model

SummaryEquine tendon fibroblasts were isolated from explants of superficial digital flexor tendon, subcultured and maintained in monolayers. The cells were characterized by light microscopy, electron microscopy and radiolabel studies for proteoglycan production. Two predominant cell morphologies were identified. The cells dedifferentiated toward a more spindle shape with repeated subcultures. Equine tendon fibroblasts were successfully cryopreserved and subsequently subcultured. The ability to produce proteoglycan was preserved.The isolated cells were identified as fibroblasts, based on their characteristic shape by light microscopy and ultrastructure and the active production of extracellular matrix proteins. Abundant rough endoplasmic reticulum and the production of extracellular matrix products demonstrated active protein production and export. Proteoglycans were measurable via liquid scintillation counting in both the cell-associated fraction and free in the supernatant. This model is currently being utilized to study the effects of polysulfated glycosaminoglycan on tendon healing. Future uses include studying the effects of other pharmaceuticals, such as hyaluronic acid, on tendon healing.A model was developed for in vitro investigations into tendon healing. Fibroblasts were isolated from equine superficial digital flexor tendons and maintained in monolayer culture. The tenocytes were characterized via light and electron microscopy. Proteoglycan production was measured, using radio-label techniques. The fibroblasts were cryopreserved and subsequently subcultured. The cells maintained their capacity for proteoglycan production, following repeated subculturing and cryopreservation.

A New In Vitro Model for Predicting the Toxicity of Cosmetic Products

1992 ◽  
Vol 20 (1) ◽  
pp. 138-143
Author(s):  
Maria Carrara ◽  
Lorenzo Cima ◽  
Roberto Cerini ◽  
Maurizio Dalle Carbonare
Keyword(s):  
Cell Culture ◽  
Cultured Cells ◽  
Neutral Red ◽  
Total Protein Content ◽  
Cosmetic Products ◽  
Cell Culture Insert ◽  
A Cell ◽  
Vitro Model ◽  
Cosmetic Emulsions

A method has been developed whereby cosmetic products which are not soluble in water or in alcohol can be brought into contact with cell cultures by being placed in a cell culture insert, which is then placed in the cell culture well. Preliminary experiments were carried out with L929 cells, and cytotoxicity was evaluated by measuring neutral red uptake and the total protein content of treated cultured cells. Encouraging results were obtained in comparisons of three cosmetic emulsions and of one emulsion containing a range of concentrations of two preservatives, Kathon CG and Bronopol.

scholarly journals Ionic Dissolution Products of NovaBone® Promote Osteoblastic Proliferation via Influences on the Cell Cycle

2009 ◽  
Vol 37 (3) ◽  
pp. 737-745 ◽  
Author(s):  
Z Qiu ◽  
H Yang ◽  
J Wu ◽  
L Wei ◽  
J Li
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Regulation ◽  
Cultured Cells ◽  
Bone Morphogenic Protein ◽  
Mg63 Cells ◽  
Silicon Ions ◽  
Cycle Regulation ◽  
Bone Morphogenic Protein 2 ◽  
Cells Cultured

This study investigated the effects of the ionic dissolution products of NovaBone® on osteoblastic proliferation and cell cycle regulation. MG63 osteoblast-like cells were cultured in NovaBone®-conditioned Dulbecco's Modified Eagle's Medium (DMEM) or control DMEM for 10 days. The concentration of silicon ions was significantly higher in NovaBone®-conditioned DMEM than control DMEM. MG63 cells cultured in NovaBone®-conditioned DMEM exhibited greater proliferation on days 1 and 4 than control cells. There were increased proportions of Novabone®-conditioned DMEM-cultured cells in the S and G2/M phases, and decreased proportions in the G0/G1 phase on days 1 and 4 versus control cells, while no differences were observed on days 7 and 10 between the two groups. Bone morphogenic protein 2 production increased in both groups, but was significantly higher for the NovaBone®-conditioned DMEM-cultured cells on day 10 compared with the controls. In conclusion, the NovaBone® ionic dissolution products, particularly the silicon ions, promoted proliferation of MG63 osteoblast-like cells in vitro via influences on the cell cycle.

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)...

Abstract 9: Temperature-responsive Cell Delivery Biopolymers for Cardiac Tissue Engineering

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Brisa Pena ◽  
Valentina Martinelli ◽  
Susanna Bosi ◽  
Carmen Sucharov ◽  
Mark Jeong ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Body Temperature ◽  
Cell Viability ◽  
Cultured Cells ◽  
Ventricular Myocytes ◽  
3D Culture ◽  
Polymeric Matrix ◽  
Neonatal Rat ◽  
Cell Delivery

Background: Advances in cell therapy and material science have made tissue engineering a promising strategy for heart regeneration. We developed an injectable biomimetic reverse thermal gel (RTG) that is liquid at room temperature but gel-like at body temperature, with the ultimate goal of being able to serve as a vehicle for cell-based delivery (liquid) to targeted tissue areas (gel-phase at 37°C). In this study we tested the suitability of this biomimetic RTG on cell viability. Methods and results: We tested different biomimetic RTG systems with and without the chemical incorporation of lysine. In vitro 3D culture experiments were performed with neonatal rat ventricular myocytes (NRVM) by mixing 3x104 cells with 50 μl of polymeric solution and allowing gel formation at 37°C. The cultured cells were incubated for 21 days. For controls we used NRVMs plated on 2D traditional gelatin coated dishes. We found that the 3D polymeric matrix induces rapid coordinated contraction with improved functionality when compared with standard 2D-cultured NRVM. By immunostaining for the morphology of the sarcomere (alpha-actinin) and DAPI, we also observed that the 3D polymeric matrix stimulates cells to spread and form 3D syncytia. Conclusion: These proof-of-concept results demonstrate long-term cell viability in this unique biomimetic system and therefore provide feasibility of a polymeric cell delivery system that permits reversible liquid-to-gel transition at body temperature. These results offer potential for a tissue engineering approach to cardiac regeneration.

Activation of human keratinocyte migration on type I collagen and fibronectin

1990 ◽  
Vol 96 (2) ◽  
pp. 197-205
Author(s):  
M. Guo ◽  
K. Toda ◽  
F. Grinnell
Keyword(s):  
Type I Collagen ◽  
Cultured Cells ◽  
Focal Adhesions ◽  
Human Keratinocytes ◽  
Type I ◽  
Integrin Subunit ◽  
Basal Cells ◽  
Keratinocyte Migration ◽  
Beta 1 Integrin ◽  
Cells Cultured

The purpose of our studies was to learn more about the regulation of keratinocyte migration. Human keratinocytes freshly harvested from skin were relatively immotile cells, whereas keratinocytes harvested from cell culture migrated on type I collagen or fibronectin as measured in a phagokinesis assay. Development of migratory competence by keratinocytes varied depending on the culture substratum. Cells cultured on plastic were activated more quickly and to a greater extent than cells cultured on dermis. The effect of the culture substratum on migratory competence was reversible. That is, cells cultured on plastic showed reduced activity after subculture on dermis. Cells cultured on dermis showed increased activity after subculture on plastic. Freshly isolated as well as cultured keratinocytes contained beta 1 integrin subunits, but only cultured cells were able to organize the subunits into focal adhesions. These adhesion sites also contained vinculin. In epidermal explants, beta 1 integrin subunits were mostly in basal cells, often more prominent between lateral cell borders than at the epidermal-dermal interface. In keratinocytes that migrated out of skin explants, there appeared to be an increase in the intensity of beta 1 integrin subunit immunostaining, possibly because of the change in shape of migrating cells. Also, beta 1 integrin subunits were found around and beneath migrating keratinocytes. These results show that changes in the distribution of beta 1 integrin subunits accompany development of migratory competence.

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