Generating an in vitro 3D cell culture model from zebrafish larvae for heart research.
Ontology highlight
ABSTRACT:
SUBMITTER: Grunow B
PROVIDER: S-EPMC4436572 | biostudies-literature | 2015 Apr
REPOSITORIES: biostudies-literature
Publications
Generating an in vitro 3D cell culture model from zebrafish larvae for heart research.
Grunow Bianka B Mohamet Lisa L Shiels Holly A HA
The Journal of experimental biology 20150224 Pt 8
We describe here a novel, fast and inexpensive method for producing a 3D 'heart' structure that forms spontaneously, in vitro, from larval zebrafish (ZF). We have named these 3D 'heart' structures 'zebrafish heart aggregate(s)' (ZFHAs) and have characterised their basic morphology and structural composition using histology, immunohistochemistry, electron microscopy and mass spectrometry. After 2 days in culture, the ZFHA spontaneously form and become a stable contractile syncytium consisting of ...[more]
Similar Datasets
Project description:This protocol describes a culture system in which inner-ear sensory tissue is produced from mouse embryonic stem (ES) cells under chemically defined conditions. This model is amenable to basic and translational investigations into inner ear biology and regeneration. In this protocol, mouse ES cells are aggregated in 96-well plates in medium containing extracellular matrix proteins to promote epithelialization. During the first 14 d, a series of precisely timed protein and small-molecule treatments sequentially induce epithelia that represent the mouse embryonic non-neural ectoderm, preplacodal ectoderm and otic vesicle epithelia. Ultimately, these tissues develop into cysts with a pseudostratified epithelium containing inner ear hair cells and supporting cells after 16-20 d. Concurrently, sensory-like neurons generate synapse-like structures with the derived hair cells. We have designated the stem cell-derived epithelia harboring hair cells, supporting cells and sensory-like neurons as inner ear organoids. This method provides a reproducible and scalable means to generate inner ear sensory tissue in vitro.
| S-EPMC6453571 | biostudies-literature
Project description:BackgroundThree-dimensional (3D) in-vitro cultures are recognized for recapitulating the physiological microenvironment and exhibiting high concordance with in-vivo conditions. Taking the advantages of 3D culture, we have developed the in-vitro tumor model for anticancer drug screening.MethodsCancer cells grown in 6 and 96 well AlgiMatrix™ scaffolds resulted in the formation of multicellular spheroids in the size range of 100-300 µm. Spheroids were grown in two weeks in cultures without compromising the growth characteristics. Different marketed anticancer drugs were screened by incubating them for 24 h at 7, 9 and 11 days in 3D cultures and cytotoxicity was measured by AlamarBlue® assay. Effectiveness of anticancer drug treatments were measured based on spheroid number and size distribution. Evaluation of apoptotic and anti-apoptotic markers was done by immunohistochemistry and RT-PCR. The 3D results were compared with the conventional 2D monolayer cultures. Cellular uptake studies for drug (Doxorubicin) and nanoparticle (NLC) were done using spheroids.ResultsIC(50) values for anticancer drugs were significantly higher in AlgiMatrix™ systems compared to 2D culture models. The cleaved caspase-3 expression was significantly decreased (2.09 and 2.47 folds respectively for 5-Fluorouracil and Camptothecin) in H460 spheroid cultures compared to 2D culture system. The cytotoxicity, spheroid size distribution, immunohistochemistry, RT-PCR and nanoparticle penetration data suggested that in vitro tumor models show higher resistance to anticancer drugs and supporting the fact that 3D culture is a better model for the cytotoxic evaluation of anticancer drugs in vitro.ConclusionThe results from our studies are useful to develop a high throughput in vitro tumor model to study the effect of various anticancer agents and various molecular pathways affected by the anticancer drugs and formulations.
| S-EPMC3548811 | biostudies-literature
Project description:We describe here a protocol for culturing epicardial cells from adult zebrafish hearts, which have a unique regenerative capacity after injury. Briefly, zebrafish hearts first undergo ventricular amputation or sham operation. Next, the hearts are excised and explanted onto fibrin gels prepared in advance in a multiwell tissue culture plate. The procedure allows the epicardial cells to outgrow from the ventricle onto a fibrin matrix in vitro. This protocol differs from those used in other organisms by using a fibrin gel to mimic blood clots that normally form after injury and that are essential for proper cell migration. The culture procedure can be accomplished within 5 h; epicardial cells can be obtained within 24-48 h and can be maintained in culture for 5-6 d. This protocol can be used to investigate the mechanisms underlying epicardial cell migration, proliferation and epithelial-to-mesenchymal transition during heart regeneration, homeostatic cardiac growth or other physiological processes.
| S-EPMC3541820 | biostudies-literature
Project description:Microfluidic devices that combine an extracellular matrix environment, cells, and physiologically relevant perfusion, are advantageous as cell culture platforms. We developed a hydrogel-based, microfluidic cell culture platform by loading polyethylene glycol (PEG) hydrogel-encapsulated U87 glioblastoma cells into membrane-capped wells in polydimethyl siloxane (PDMS). The multilayer microfluidic cell culture system combines previously reported design features in a configuration that loads and biomimetically perfuses a 2D array of cell culture chambers. One dimension of the array is fed by a microfluidic concentration gradient generator (MCGG) while the orthogonal dimension provides loading channels that fill rows of cell culture chambers in a separate layer. In contrast to typical tree-like MCGG mixers, a fractional serial dilution of 1, ½, ¼, and 0 of the initial solute concentration is achieved by tailoring the input microchannel widths. Hydrogels are efficiently and reproducibly loaded in all wells and cells are evenly distributed throughout the hydrogel, maintaining > 90% viability for up to 4 days. In a drug screening assay, diffusion of temozolomide and carmustine to hydrogel-encapsulated U87 cells from the perfusion solution is measured, and dose-response curves are generated, demonstrating utility as an in vitro mimic of the glioblastoma microenvironment.
| S-EPMC9588086 | biostudies-literature
Project description:The in vitro study of the pathogenesis of inflammatory bowel disease (IBD) requires a cell model which closely reflects the characteristics of the in vivo intestinal epithelium. This study aimed to investigate the application of L-pNIPAM hydrogel as a scaffold to develop a long-term 3D co-culture model of Caco-2 and HT29-MTX cells under conditions analogous to inflammation, to determine its potential use in studying IBD. Monocultures and co-cultures were layered on L-pNIPAM hydrogel scaffolds and maintained under dynamic culture conditions for up to 12 weeks. Treatments with IL-1β, TNFα, and hypoxia for 1 week were used to create an inflammatory environment. Following prolonged culture, the metabolic activity of Caco-2 monoculture and 90% Caco-2/10% HT29-MTX co-cultures on L-pNIPAM hydrogels were increased, and finger-like structures, similar in appearance to villi were observed. Following treatment with IL-1β, TNFα and hypoxia, ALP and ZO-1 were decreased, MUC2 increased, and MUC5AC remained unchanged. ADAMTS1 was increased in response to hypoxia. Caspase 3 expression was increased in response to TNFα and hypoxic conditions. In conclusion, L-pNIPAM hydrogel supported long-term co-culture within a 3D model. Furthermore, stimulation with factors seen during inflammation recapitulated features seen during IBD.
| S-EPMC6372635 | biostudies-literature
Project description:BackgroundIn vitro cystic fibrosis (CF) models are crucial for understanding the mechanisms and consequences of the disease. They are also the gold standard for pre-clinical efficacy studies of current and novel CF drugs. However, few studies have investigated expansion and differentiation of primary CF human bronchial epithelial (CF-HBE) cells. Here we describe culture conditions to expand primary CF airway cells while preserving their ability to differentiate into 3D epithelial cultures expressing functional cystic fibrosis transmembrane conductance regulator (CFTR) ion channels that responds to CFTR modulators.MethodsPrimary CF airway cells were expanded using PneumaCultTM-Ex Plus (StemCell Technologies) medium with no feeder cells or added Rho kinase (ROCK) inhibitor. Differentially passaged CF-HBE cells at the air-liquid interface (ALI) were characterized phenotypically and functionally in response to the CFTR corrector drug VX-661 (Tezacaftor).ResultsCF-HBE primary cells, expanded up to six passages (~25 population doublings), differentiated into 3D epithelial cultures as evidenced by trans-epithelial electrical resistance (TEER) of >400 Ohms∙cm2 and presence of pseudostratified columnar ciliated epithelium with goblet cells. However, up to passage five cells from most donors showed increased CFTR-mediated short-circuit currents when treated with the corrector drug, VX-661. Ciliary beat frequency (CBF) also increased with the corrector VX-661.ConclusionsCF donor-derived airway cells can be expanded without the use of feeder cells or additional ROCK inhibitor, and still achieve optimal 3D epithelial cultures that respond to CFTR modulators. The study of rare CF mutations could benefit from cell expansion and could lead to the design of personalized medicine/treatments.
| S-EPMC7796805 | biostudies-literature
Project description:The development of in vitro 3D models to get insights into the mechanisms of bone regeneration could accelerate the translation of experimental findings to the clinic, reducing costs and duration of experiments. This work explores the design and manufacturing of multi-compartments structures in poly(ε-caprolactone) (PCL) 3D-printed by Fused Filament Fabrication technique. The construct was designed with interconnected stalls to host stem cells and endothelial cells. Cells were encapsulated within an optimised gellan gum (GG)-based hydrogel matrix, crosslinked using strontium (Sr2+) ions to exploit its bioactivity and finally, assembled within compartments with different sizes. Calcium (Ca2+)-crosslinked gels were also used as control for comparison of Sr2+ osteogenic effect. The results obtained demonstrated that Sr2+ ions were successfully diffused within the hydrogel matrix and increased the hydrogel matrix strength properties under compressive load. The in vitro co-culture of human-TERT mesenchymal stem cells (TERT- hMSCs) and human umbilical vein endothelial cells (HUVECs), encapsulated within Sr2+ ions containing GG-hydrogels and inter-connected by compartmentalised scaffolds under osteogenic conditions, enhanced cell viability and supported osteogenesis, with a significant increase of alkaline phosphatase activity, osteopontin and osteocalcin respect with the Ca2+-crosslinked GG-PCL scaffolds. These outcomes demonstrate that the design and manufacturing of compartmentalised co-culture of TERT-hMSCs and HUVEC populations enables an effective system to study and promote osteogenesis.
| S-EPMC6181937 | biostudies-literature
Project description:BackgroundIntradiscal transplantation of mesenchymal stromal cells (MSCs) has emerged as a promising therapy for intervertebral disc degeneration (IDD). However, the hostile microenvironment of the intervertebral disc (IVD) may compromise the survival of implanted cells. Interestingly, studies reported that paracrine factors, such as extracellular vesicles (EVs) released by MSCs, may regenerate the IVD. The aim of this study was to investigate the therapeutic effects of Wharton's Jelly MSC (WJ-MSC)-derived EVs on human nucleus pulposus cells (hNPCs) using an in vitro 3D alginate-bead culture model.MethodsAfter EV isolation and characterization, hNPCs isolated from surgical specimens were encapsulated in alginate beads and treated with 10, 50, and 100 μg/mL WJ-MSC-EVs. Cell proliferation and viability were assessed by flow cytometry and live/dead staining. Nitrite and glycosaminoglycan (GAG) content was evaluated through Griess and 1,9-dimethylmethylene blue assays. hNPCs in alginate beads were paraffin-embedded and stained for histological analysis (hematoxylin-eosin and Alcian blue) to assess extracellular matrix (ECM) composition. Gene expression levels of catabolic (MMP1, MMP13, ADAMTS5, IL6, NOS2), anabolic (ACAN), and hNPC marker (SOX9, KRT19) genes were analyzed through qPCR. Collagen type I and type II content was assessed with Western blot analysis.ResultsTreatment with WJ-MSC-EVs resulted in an increase in cell content and a decrease in cell death in degenerated hNPCs. Nitrite production was drastically reduced by EV treatment compared to the control. Furthermore, proteoglycan content was enhanced and confirmed by Alcian blue histological staining. EV stimulation attenuated ECM degradation and inflammation by suppressing catabolic and inflammatory gene expression levels. Additionally, NPC phenotypic marker genes were also maintained by the EV treatment.ConclusionsWJ-MSC-derived EVs ameliorated hNPC growth and viability, and attenuated ECM degradation and oxidative stress, offering new opportunities for IVD regeneration as an attractive alternative strategy to cell therapy, which may be jeopardized by the harsh microenvironment of the IVD.
| S-EPMC10782051 | biostudies-literature
Project description:Current conventional cancer drug screening models based on two-dimensional (2D) cell culture have several flaws and there is a large need of more in vivo mimicking preclinical drug screening platforms. The microenvironment is crucial for the cells to adapt relevant in vivo characteristics and here we introduce a new cell culture system based on three-dimensional (3D) printed scaffolds using cellulose nanofibrils (CNF) pre-treated with 2,2,6,6-tetramethylpyperidine-1-oxyl (TEMPO) as the structural material component. Breast cancer cell lines, MCF7 and MDA-MB-231, were cultured in 3D TEMPO-CNF scaffolds and were shown by scanning electron microscopy (SEM) and histochemistry to grow in multiple layers as a heterogenous cell population with different morphologies, contrasting 2D cultured mono-layered cells with a morphologically homogenous cell population. Gene expression analysis demonstrated that 3D TEMPO-CNF scaffolds induced elevation of the stemness marker CD44 and the migration markers VIM and SNAI1 in MCF7 cells relative to 2D control. T47D cells confirmed the increased level of the stemness marker CD44 and migration marker VIM which was further supported by increased capacity of holoclone formation for 3D cultured cells. Therefore, TEMPO-CNF was shown to represent a promising material for 3D cell culture model systems for cancer cell applications such as drug screening.
| S-EPMC8301137 | biostudies-literature
Project description:Three-dimensional cell culture has the potential to revolutionize toxicology studies by allowing human-based reproduction of essential elements of organs. Beyond the study of toxicants on the most susceptible organs such as liver, kidney, skin, lung, gastrointestinal tract, testis, heart and brain, carcinogenesis research will also greatly benefit from 3D cell culture models representing any normal tissue. No tissue function can be suitably reproduced without the appropriate tissue architecture whether mimicking acini, ducts or tubes, sheets of cells or more complex cellular organizations like hepatic cords. In this review, we illustrate the fundamental characteristics of polarity that is an essential architectural feature of organs for which different 3D cell culture models are available for toxicology studies in vitro. The value of tissue polarity for the development of more accurate carcinogenesis studies is also exemplified, and the concept of using extracellular gradients of gaseous or chemical substances produced with microfluidics in 3D cell culture is discussed. Indeed such gradients-on-a-chip might bring unprecedented information to better determine permissible exposure levels. Finally, the impact of tissue architecture, established via cell-matrix interactions, on the cell nucleus is emphasized in light of the importance in toxicology of morphological and epigenetic alterations of this organelle.
| S-EPMC5623165 | biostudies-other