Enhanced growth and myogenic differentiation of spheroid-derived C2C12 cells

2021 ◽  
Author(s):  
Guang-Zhen Jin
Keyword(s):  
Myogenic Differentiation ◽  
Three Dimensional ◽  
3D Culture ◽  
Stem Cell Differentiation ◽  
Culture Conditions ◽  
C2c12 Cells ◽  
Dependent Manner ◽  
Physical Force ◽  
Myod Gene ◽  
2D And 3D

Abstract Among many factors of controlling stem cell differentiation, the key transcription factor upregulation via physical force is a good strategy on the lineage-specific differentiation of stem cells. The study aimed to compare growth and myogenic potentials between the parental cells (PCs) and the 1-day-old C2C12 spheroid-derived cells (SDCs) in two-dimensional (2D) and three-dimensional (3D) culture conditions through examination of the cell proliferation and the expression of myogenic genes. The data showed that 1-day-old spheroids had more intense expression of MyoD gene with respect to the PCs. The proliferation of the SDCs significantly higher than the PCs in a time dependent manner. The SDCs had also significantly higher myogenic potential than the PCs in 2D and 3D culture conditions. The results suggest that MyoD gene upregulation through cell-cell contacts is the good approach for preparation of seed cells in muscle tissue engineering.

Extracellular vesicles from three dimensional culture of human placental mesenchymal stem cells ameliorated renal ischemia/reperfusion injury

2021 ◽  
pp. 039139882098680
Author(s):  
Xuefeng Zhang ◽  
Nan Wang ◽  
Yuhua Huang ◽  
Yan Li ◽  
Gang Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Extracellular Vesicles ◽  
Therapeutic Potential ◽  
Expression Profiles ◽  
Ischemia Reperfusion ◽  
Three Dimensional ◽  
3D Culture ◽  
Placental Mesenchymal Stem Cells ◽  
2D And 3D

Background: Three-dimensional (3D) culture has been reported to increase the therapeutic potential of mesenchymal stem cells (MSCs). The present study assessed the therapeutic efficacy of extracellular vesicles (EVs) from 3D cultures of human placental MSCs (hPMSCs) for acute kidney injury (AKI). Methods: The supernatants from monolayer culture (2D) and 3D culture of hPMSCs were ultra-centrifuged for EVs isolation. C57BL/6 male mice were submitted to 45 min bilateral ischemia of kidney, followed by renal intra-capsular administration of EVs within a 72 h reperfusion period. Histological, immunohistochemical, and ELISA analyses of kidney samples were performed to evaluate cell death and inflammation. Kidney function was evaluated by measuring serum creatinine and urea nitrogen. The miRNA expression profiles of EVs from 2D and 3D culture of hPMSCs were evaluated using miRNA microarray analysis. Results: The 3D culture of hPMSCs formed spheroids with different diameters depending on the cell density seeded. The hPMSCs produced significantly more EVs in 3D culture than in 2D culture. More importantly, injection of EVs from 3D culture of hPMSCs into mouse kidney with ischemia-reperfusion (I/R)-AKI was more beneficial in protecting from progression of I/R than those from 2D culture. The EVs from 3D culture of hPMSCs were more efficient against apoptosis and inflammation than those from 2D culture, which resulted in a reduction in tissue damage and amelioration of renal function. MicroRNA profiling analysis revealed that a set of microRNAs were significantly changed in EVs from 3D culture of hPMSCs, especially miR-93-5p. Conclusion: The EVs from 3D culture of hPMSCs have therapeutic potential for I/R-AKI.

scholarly journals Extending In-Plane Impedance Measurements from 2D to 3D Cultures: Design Considerations

2021 ◽  
Vol 8 (1) ◽  
pp. 11
Author(s):  
Sorel E. De Leon ◽  
Lana Cleuren ◽  
Zay Yar Oo ◽  
Paul R. Stoddart ◽  
Sally L. McArthur
Keyword(s):  
Three Dimensional ◽  
3D Culture ◽  
Collagen Gel ◽  
Impedance Measurement ◽  
Impedance Spectrum ◽  
3D Models ◽  
Low Frequencies ◽  
3D Cell Cultures ◽  
3D Collagen ◽  
2D And 3D

Three-dimensional (3D) cell cultures have recently emerged as tools for biologically modelling the human body. As 3D models make their way into laboratories there is a need to develop characterisation techniques that are sensitive enough to monitor the cells in real time and without the need for chemical labels. Impedance spectroscopy has been shown to address both of these challenges, but there has been little research into the full impedance spectrum and how the different components of the system affect the impedance signal. Here we investigate the impedance of human fibroblast cells in 2D and 3D collagen gel cultures across a broad range of frequencies (10 Hz to 5 MHz) using a commercial well with in-plane electrodes. At low frequencies in both 2D and 3D models it was observed that protein adsorption influences the magnitude of the impedance for the cell-free samples. This effect was eliminated once cells were introduced to the systems. Cell proliferation could be monitored in 2D at intermediate frequencies (30 kHz). However, the in-plane electrodes were unable to detect any changes in the impedance at any frequency when the cells were cultured in the 3D collagen gel. The results suggest that in designing impedance measurement devices, both the nature and distribution of the cells within the 3D culture as well as the architecture of the electrodes are key variables.

Thermally Induced Apoptosis, Necrosis, and Heat Shock Protein Expression in Three-Dimensional Culture

2014 ◽  
Vol 136 (7) ◽  
Author(s):  
Alfred S. Song ◽  
Amer M. Najjar ◽  
Kenneth R. Diller
Keyword(s):  
Prostate Cancer ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Fluorescent Protein ◽  
Three Dimensional ◽  
3D Culture ◽  
Hsp70 Expression ◽  
Three Dimensional Culture ◽  
Apoptosis And Necrosis ◽  
2D And 3D

This study was conducted to compare the heat shock responses of cells grown in 2D and 3D culture environments as indicated by the level of heat shock protein 70 expression and the incidence of apoptosis and necrosis of prostate cancer cell lines in response to graded hyperthermia. PC3 cells were stably transduced with a dual reporter system composed of two tandem expression cassettes—a conditional heat shock protein promoter driving the expression of green fluorescent protein (HSPp-GFP) and a cytomegalovirus (CMV) promoter controlling the constitutive expression of a “beacon” red fluorescent protein (CMVp-RFP). Two-dimensional and three-dimensional cultures of PC3 prostate cancer cells were grown in 96-well plates for evaluation of their time-dependent response to supraphysiological temperature. To induce controlled hyperthermia, culture plates were placed on a flat copper surface of a circulating water manifold that maintained the specimens within ±0.1 °C of a target temperature. Hyperthermia protocols included various combinations of temperature, ranging from 37 °C to 57 °C, and exposure times of up to 2 h. The majority of protocols were focused on temperature and time permutations, where the response gradient was greatest. Post-treatment analysis by flow cytometry analysis was used to measure the incidences of apoptosis (annexin V-FITC stain), necrosis (propidium iodide (PI) stain), and HSP70 transcription (GFP expression). Cells grown in 3D compared with 2D culture showed reduced incidence of apoptosis and necrosis and a higher level of HSP70 expression in response to heat shock at the temperatures tested. Cells responded differently to hyperthermia when grown in 2D and 3D cultures. Three-dimensional culture appears to enhance survival plausibly by activating protective processes related to enhanced-HSP70 expression. These differences highlight the importance of selecting physiologically relevant 3D models in assessing cellular responses to hyperthermia in experimental settings.

Comparison of gene expression and activation of transcription factors in organoid-derived monolayer intestinal epithelial cells and organoids

2021 ◽  
Author(s):  
Yu Takahashi ◽  
Yu Inoue ◽  
Keitaro Kuze ◽  
Shintaro Sato ◽  
Makoto Shimizu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Gene Expression Regulation ◽  
Intestinal Epithelial Cells ◽  
Three Dimensional ◽  
Culture Conditions ◽  
Dependent Manner ◽  
Intestinal Epithelial

Abstract Intestinal organoids better represent in vivo intestinal properties than conventionally used established cell lines in vitro. However, they are maintained in three-dimensional culture conditions that may be accompanied by handling complexities. We characterized the properties of human organoid-derived two-dimensionally cultured intestinal epithelial cells (IECs) compared with those of their parental organoids. We found that the expression of several intestinal markers and functional genes were indistinguishable between monolayer IECs and organoids. We further confirmed that their specific ligands equally activate intestinal ligand-activated transcriptional regulators in a dose-dependent manner. The results suggest that culture conditions do not significantly influence the fundamental properties of monolayer IECs originating from organoids, at least from the perspective of gene expression regulation. This will enable their use as novel biological tools to investigate the physiological functions of the human intestine.

scholarly journals Characterization of Phenotypic and Transcriptional Differences in Human Pluripotent Stem Cells under 2D and 3D Culture Conditions

2016 ◽  
Vol 5 (22) ◽  
pp. 2951-2958 ◽  
Author(s):  
Ken-ichiro Kamei ◽  
Yoshie Koyama ◽  
Yumie Tokunaga ◽  
Yasumasa Mashimo ◽  
Momoko Yoshioka ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
3D Culture ◽  
Culture Conditions ◽  
Human Pluripotent Stem Cells ◽  
2D And 3D

scholarly journals Regulation of podocalyxin trafficking by Rab small GTPases in 2D and 3D epithelial cell cultures

2016 ◽  
Vol 213 (3) ◽  
pp. 355-369 ◽  
Author(s):  
Paulina S. Mrozowska ◽  
Mitsunori Fukuda
Keyword(s):  
Cell Cultures ◽  
Three Dimensional ◽  
Culture Conditions ◽  
Small Gtpases ◽  
Cell Polarization ◽  
Rab Gtpases ◽  
3D Cell Cultures ◽  
Polarized Epithelia ◽  
3D Environments ◽  
2D And 3D

MDCK II cells, a widely used model of polarized epithelia, develop into different structures depending on culture conditions: two-dimensional (2D) monolayers when grown on synthetic supports or three-dimensional (3D) cysts when surrounded by an extracellular matrix. The establishment of epithelial polarity is accompanied by transcytosis of the apical marker podocalyxin from the outer plasma membrane to the newly formed apical domain, but its exact route and regulation remain poorly understood. Here, through comprehensive colocalization and knockdown screenings, we identified the Rab GTPases mediating podocalyxin transcytosis and showed that different sets of Rabs coordinate its transport during cell polarization in 2D and 3D structures. Moreover, we demonstrated that different Rab35 effectors regulate podocalyxin trafficking in 2D and 3D environments; trafficking is mediated by OCRL in 2D monolayers and ACAP2 in 3D cysts. Our results give substantial insight into regulation of the transcytosis of this apical marker and highlight differences between trafficking mechanisms in 2D and 3D cell cultures.

Inhibition of Drp1-dependent mitochondrial division impairs myogenic differentiation

2013 ◽  
Vol 305 (8) ◽  
pp. R927-R938 ◽  
Author(s):  
Boa Kim ◽  
Ji-Seok Kim ◽  
Yisang Yoon ◽  
Mayra C. Santiago ◽  
Michael D. Brown ◽  
...  
Keyword(s):  
Muscle Development ◽  
Myogenic Differentiation ◽  
Mitochondrial Dynamics ◽  
C2c12 Cells ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Dominant Negative Mutant ◽  
Serum Starvation ◽  
Dependent Manner ◽  
Mitochondrial Division

Mitochondria are dynamic organelles forming a tubular network that is continuously fusing and dividing to control their morphology and functions. Recent literature has shed new light on a potential link between the dynamic behavior of mitochondria and muscle development. In this study, we investigate the role of mitochondrial fission factor dynamin-related protein 1 (Drp1) in myogenic differentiation. We found that differentiation of C2C12 myoblasts induced by serum starvation was accompanied by a gradual increase in Drp1 protein expression (to ∼350% up to 3 days) and a fast reduction of Drp1 phosphorylation at Ser-637 (to ∼30%) resulting in translocation of Drp1 protein from the cytosol to mitochondria. During differentiation, treatment of myoblasts with mitochondrial division inhibitor ( mdivi-1), a specific inhibitor of Drp1 GTPase activity, caused extensive formation of elongated mitochondria, which coincided with increased apoptosis evidenced by both enhanced caspase-3 activity and increased number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells. Furthermore, the mdivi-1-treated myotubes ( day 3 in differentiation media) showed a reduction in mitochondrial DNA content, mitochondrial mass, and membrane potential in a dose-dependent manner indicating defects in mitochondrial biogenesis during myogenic differentiation. Most interestingly, mdivi-1 treatment significantly suppressed myotube formation in both C2C12 cells and primary myoblasts. Likewise, stable overexpression of a dominant negative mutant Drp1 (K38A) dramatically reduced myogenic differentiation. These data suggest that Drp-1-dependent mitochondrial division is a necessary step for successful myogenic differentiation, and perturbation of mitochondrial dynamics hinders normal mitochondrial adaptations during muscle development. Therefore, in the present study, we report a novel physiological role of mitochondrial dynamics in myogenic differentiation.

scholarly journals Comparative Microsomal Proteomics of A Model Lung Cancer Cell Line NCI-H23 Reveals Distinct Differences Between Molecular Profiles of 3D and 2D Cultured Cells

2020 ◽  
Author(s):  
Josip Blonder ◽  
Jan A. Kaczmarczyk ◽  
Rhonda R. Roberts ◽  
Brian T. Luke ◽  
King C. Chan ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cancer Cells ◽  
Cell Line ◽  
3D Culture ◽  
Culture Conditions ◽  
Attrition Rate ◽  
Dependent Manner ◽  
Drug Candidates ◽  
High Attrition Rate ◽  
Culture Dependent

Abstract Background: Lung cancer is the leading cause of cancer-related deaths in the USA and worldwide. Yet, about 95% of new drug candidates validated in preclinical phase eventually fail in clinical trials. Such a high attrition rate is attributed mostly to the inability of conventional two-dimensionally (2D) cultured cancer cells to mimic native three-dimensional (3D) growth of malignant cells in human tumors. Thus, it is expected that 3D cell culture systems would more accurately represent the phenotype of cancer cells growing in tumors. To ascertain phenotypical differences between these two distinct culture conditions, we carried out a comparative proteomic analysis of membrane fraction obtained from 3D- and 2D-cultured NSCLC model cell line NCI-H23. Methods: Global shotgun membrane (SGM) proteomics that relies on strong cation exchange (SCX)-based peptide fractionation and accurate-mass, liquid chromatography mass spectrometry (HR/AM LC-MS) was employed to analyze microsomal fractions obtained from the NCI-H23 cells grown in both 2D and 3D culture conditions. Results: Comparative proteomics revealed a map of 1,166 (24%) nonredundant protein species regulated in culture dependent manner in NCI-H23 cell line. Of these, a subset of 234 (i.e., 21 %) proteins were found significantly dysregulated (p-value ≤ 0.05) under both culture conditions whereas a total of 334 (27.8%) and 598 (51,2%) proteins were uniquely identified in 3D and 2D culture, respectively. The Ingenuity Pathway Analysis revealed extensive metabolic changes and differential regulation of a subset of CD molecules in culture-dependent manner. Using western blotting we verified exclusive 3D-culture expression of CD99, CD146 and CD239, involved in development of malignant stroma extracellular matrix, neo-angiogenesis and metastasis. Furthermore, using label-free quantitation we unambiguously confirmed upregulation of wild type and monoallelic KRas4B G12C mutant in 3D cultured NCI-H23 cells, targeting exclusively proteoform-specific tryptic peptides. Conclusions: In this study we generated a large-scale proteomic resource/atlas of a preclinical testing model NCI-H23 cell line grown in 3D- and 2D-culture, providing insight into phenotypical/proteomic changes unique to each culture type, that would not have been discovered using only conventional 2D-culture. To reduce high attrition rate of new drug candidates it is critical to profile a large number of patient-derived NSCLC cell lines.

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 MUNC, an Enhancer RNA Upstream from the MYOD Gene, Induces a Subgroup of Myogenic Transcripts in trans Independently of MyoD

2018 ◽  
Vol 38 (20) ◽  
Author(s):  
Magdalena A. Cichewicz ◽  
Manjari Kiran ◽  
Róża K. Przanowska ◽  
Ewelina Sobierajska ◽  
Yoshiyuki Shibata ◽  
...  
Keyword(s):  
Noncoding Rna ◽  
Myogenic Differentiation ◽  
Regulatory Region ◽  
C2c12 Cells ◽  
Dependent Manner ◽  
Exon 2 ◽  
Functional Sites ◽  
Enhancer Rna ◽  
Distal Regulatory Region ◽  
Exon 1

ABSTRACT MyoD upstream noncoding RNA (MUNC) initiates in the distal regulatory region (DRR) enhancer of MYOD and is formally classified as an enhancer RNA (DRReRNA). MUNC is required for optimal myogenic differentiation, induces specific myogenic transcripts in trans (MYOD, MYOGENIN, and MYH3), and has a functional human homolog. The vast majority of eRNAs are believed to act in cis primarily on their neighboring genes (1, 2), making it likely that MUNC action is dependent on the induction of MYOD RNA. Surprisingly, MUNC overexpression in MYOD−/− C2C12 cells induces many myogenic transcripts in the complete absence of MyoD protein. Genomewide analysis showed that, while many genes are regulated by MUNC in a MyoD-dependent manner, there is a set of genes that are regulated by MUNC, both upward and downward, independently of MyoD. MUNC and MyoD even appear to act antagonistically on certain transcripts. Deletion mutagenesis showed that there are at least two independent functional sites on the MUNC long noncoding RNA (lncRNA), with exon 1 more active than exon 2 and with very little activity from the intron. Thus, although MUNC is an eRNA of MYOD, it is also a trans-acting lncRNA whose sequence, structure, and cooperating factors, which include but are not limited to MyoD, determine the regulation of many myogenic genes.

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