Selection and validation of reference gene for RT-qPCR studies in co-culture system of mouse cementoblasts and periodontal ligament cells

  1. RT-qPCR is a reliable method for gene expression analysis, but the accuracy of the quantitative data depends on the appropriate selection of reference genes.
  2. A Co-culture system consisting of periodontal ligament cells (SV-PDL) and cementoblasts (OCCM-30) to investigate the crosstalk between these two cell lines under orthodontic condition is essential for experimental orthodontic setups in-vitro. Therefore, we aimed to identify a set of reliable reference genes suitable for RT-qPCR studies for prospective co-culture systems of OCCM-30 and SV-PDL cells.
  3. The results demonstrated that PPIB, GUSB and RPLP0 turned out to be the three most stable reference genes for OCCM-30 in the co-culture system, while PPIB, POLR2A and RPLP0 have the three highest rankings for SV-PDL cells in the co-culture system. The most stable gene combination were PPIB and POLR2A in the co-culture system.
  4. In conclusion, PPIB is overall the most stably expressed reference gene for OCCM-30 or SV-PDL cell line in the system. The combination of PPIB and POLR2A as reference genes are indicated to be the potential and mandatory to obtain accurate quantification results for normalizing RT-qPCR data in genes of interest expression in these two cell lines co-culture systems.
  5. Assessment of Collagen-Based Nanostructured Biomimetic Systems with a CoCulture of Human Bone-Derived Cells.

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Osteoporosis is a worldwide disease resulting in the increase of bone fragility and enhanced fracture risk in adults. In the context of osteoporotic fractures, bone tissue engineering (BTE), i.e., the use of bone substitutes combining biomaterials, cells, and other factors, is considered a potential alternative to conventional treatments.
Innovative scaffolds need to be tested in in vitro systems where the simultaneous presence of osteoblasts (OBs) and osteoclasts (OCs), the two main players of bone remodeling, is required to mimic their crosstalk and molecular cooperation.
To this aim, two composite materials were developed, based on type I collagen, and containing either strontium-enriched mesoporous bioactive glasses or rod-like hydroxyapatite nanoparticles. The developed nanostructured systems underwent genipin chemical crosslinking and were then tested with an indirect co-culture of human trabecular bone-derived OBs and buffy coat-derived OC precursors, for 2-3 weeks.
The favorable structural and biological properties of the materials proved to successfully support the viability, adhesion, and differentiation of cells, encouraging a further investigation of the developed bioactive systems as biomaterial inks for the 3D printing of more complex scaffolds for BTE.

Stem Cells from Human Exfoliated Deciduous Teeth (SHED) Have Mitochondrial Transfer Ability in Stromal-Derived Inducing Activity (SDIA) Coculture System

  • Stem cells from human exfoliated deciduous teeth (SHED) have stromal-derived inducing activity (SDIA): which means these stromal cells induce neural differentiation where they are used as a substratum for embryonic stem cell (ESCs) culture.
  • Recent studies show that mitochondria or mitochondrial products, as paracrine factors, can be released and transferred from one cell to another.
  • With this information, we were curious to know whether in the SDIA co-culture system, SHED release or donate their mitochondria to ESCs. For this purpose, before co-culture, SHED s’ mitochondria and ESCs s’ cell membranes were separately labeled with specific fluorescent probes.
  • After co-culture, SHED s’ mitochondria were tracked by fluorescent microscope and flow cytometry analysis. Co-culture also performed in the presence of inhibitors that block probable transfer pathways suchlike tunneling nanotubes, gap junctions or vesicles.
  • Results showed that mitochondrial transfer takes place from SHED to ESCs. This transfer partly occurs by tunneling nanotubes and not through gap junctions or vesicles; also was not dependent on intracellular calcium level.
  • This kind of horizontal gene transfer may open a new prospect for further research on probable role of mitochondria on fate choice and neural induction processes.

Lymphangion-chip: a microphysiological system that supports coculture and bidirectional signaling of lymphatic endothelial and muscle cells.

The pathophysiology of several lymphatic diseases, such as lymphedema, depends on the function of lymphangions that drive lymph flow.
Even though the signaling between the two main cellular components of a lymphangion, endothelial cells (LECs) and muscle cells (LMCs), is responsible for crucial lymphatic functions, there are no in vitro models that have included both cell types.
Here, a fabrication technique (gravitational lumen patterning or GLP) is developed to create a lymphangion-chip. This organ-on-chip consists of co-culture of a monolayer of endothelial lumen surrounded by multiple and uniformly thick layers of muscle cells.
The platform allows construction of a wide range of luminal diameters and muscular layer thicknesses, thus providing a toolbox to create variable anatomy.
In this device, lymphatic muscle cells align circumferentially while endothelial cells aligned axially under flow, as only observed in vivo in the past.
This system successfully characterizes the dynamics of cell size, density, growth, alignment, and intercellular gap due to co-culture and shear.
Finally, exposure to pro-inflammatory cytokines reveals that the device could facilitate the regulation of endothelial barrier function through the lymphatic muscle cells. Therefore, this bioengineered platform is suitable for use in preclinical research of lymphatic and blood mechanobiology, inflammation, and translational outcomes.

Cytotoxicity of mancozeb on Sertoli-germ cell coculture system: Role of MAPK signaling pathway

Mancozeb (MZB) is a worldwide fungicide for the management of fungal diseases in agriculture and industrial contexts.
Human exposure occurs by consuming contaminated plants, drinking water, and occupational exposure.
There are reports on MZB’s reprotoxicity such as testicular structure damage, sperm abnormalities, and decrease in sperm parameters (number, viability, and motility), but its molecular mechanism on apoptosis in testis remains limited.
To investigate the molecular mechanisms involved in male reprotoxicity induced by MZB, we used primary cultures of mouse Sertoli-germ cells. Cells were exposed to MZB (1.5, 2.5, and 3.5 μM) for 3 h to evaluate viability by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay, reactive oxygen species (ROS) generation, and oxidative stress parameters (lipid peroxidation).

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CytoSelect 24-Well Cell Co-Culture System

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Cell Culture Grade Water, Cell Culture Tested, Sterile

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ViraSafe Lentiviral Packaging System, Ecotropic

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ViraSafe Lentiviral Packaging System, Pantropic

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Platinum Retroviral Expression System, Ecotropic

VPK-300 Cell Biolabs 1 kit 1169 EUR
Cell death and mitogen-activated protein kinase (MAPK) signaling were measured in these cells using flow cytometry and western blotting. In addition, some groups were exposed to N-acetylcysteine (NAC, 5 mM) in the form of co-treatment with MZB.
Mancozeb reduced viability and increased the level of intracellular ROS, p38 and c-Jun N-terminal kinases (JNK) MAPK proteins phosphorylation, and apoptotic cell death, which could be blocked by NAC as an inhibitor of oxidative stress.
The present study indicated for the first time the toxic manifestations of MZB on the Sertoli-germ cell co-culture. Redox imbalance and p38 and JNK signaling pathway activation might play critical roles in MZB-induced apoptosis in the male reproductive system.