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.

Abstract 141: Influence of the Collagen Processing Heat Shock Protein 47 on Cardiomyocyte Homeostasis and Maturation

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Sumon Sur ◽  
Peter Christalla ◽  
Angelica Roa ◽  
Wolfram H Zimmermann
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Cardiac Tissue ◽  
Isometric Force ◽  
Embryonic Stem ◽  
3D Culture ◽  
Heat Shock Protein 47 ◽  
Culture Models ◽  
2D And 3D

PURPOSE: Heat shock protein 47 (Hsp47) is a collagen-specific molecular chaperone required for maturation of collagen type 1. Little is known about the role of Hsp47 in the heart. This study aims to investigate whether Hsp47 is important for cardiomyocyte maturation and assembly into functional syncytia. METHODS: We made use of recently developed 2D and 3D culture platforms to scrutinize the role of Hsp47 for cardiomyocyte maturation and function, i.e., (1) 2D model: culture of embryonic stem cell-derived cardiomyocytes (ESC-CM) on ECMs derived from both mouse embryonic wild type (WT)- and Hsp47 (-/-) fibroblasts and (2) 3D model: construction of engineered heart muscle (EHM) consisting of purified ESC-CMs with WT- or Hsp47 (-/-) fibroblasts. Cultures were investigated using confocal microscopy and isometric force measurements. RESULTS: ESC-CMs showed mainly immature polygonal shaped morphology and disorganized sarcomeric structures when cultured on ECM secreted by Hsp47 (-/-) fibroblasts; this morphology was in clear contrast to the mostly anisotropic cardiomyocyte appearance in control conditions. EHM with Hsp47 (-/-) fibroblasts did not develop measurable contractile forces. In contrast, WT fibroblast-supplemented EHMs contracted regularly (WT: 0.14±0.1 mN at 2.4 mM Ca2+; n=8/group). Histological analysis of Hsp47 (-/-) EHMs showed mainly rounded and underdeveloped cardiomyocytes comparable to the cardiomyocyte phenotype observed in EHMs containing only ESC-CMs, without fibroblasts. CONCLUSIONS: Hsp47 influences the deposition of ECM-collagen and affects cardiomyocyte morphology and functionality in our 2D and 3D culture models. Our data collectively suggest that Hsp47 may be an attractive target for the regulation of cardiac tissue homeostasis.

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.

scholarly journals Effects ofHelicobacter pyloriand Heat Shock Protein 70 on the Proliferation of Human Gastric Epithelial Cells

2014 ◽  
Vol 2014 ◽  
pp. 1-5 ◽  
Author(s):  
Liping Tao ◽  
Hai Zou ◽  
Zhimin Huang
Keyword(s):  
Helicobacter Pylori ◽  
Heat Shock ◽  
Epithelial Cells ◽  
Heat Shock Protein ◽  
Mtt Assay ◽  
Heat Shock Protein 70 ◽  
Hsp70 Expression ◽  
Gastric Epithelial Cells ◽  
Ags Cells ◽  
H Pylori

Infection ofHelicobacter pylori (H. pylori)changed the proliferation of gastric epithelial cells and decreased the expression of heat shock protein 70 (HSP70). However, the effects ofH. pylorion the proliferation of gastric epithelial cells and the roles of HSP70 during the progress need further investigation.Objective.To investigate the effects ofHelicobacter pylori (H. pylori)and heat shock protein 70 (HSP70) on the proliferation of human gastric epithelial cells.Methods. H. pyloriand a human gastric epithelial cell line (AGS) were cocultured. The proliferation of AGS cells was quantitated by an MTT assay, and the expression of HSP70 in AGS cells was detected by Western blotting. HSP70 expression in AGS cells was silenced by small interfering RNA (siRNA) to investigate the role of HSP70. ThesiRNA-treated AGS cells were cocultured withH. pyloriand cell proliferation was measured by an MTT assay.Results.The proliferation of AGS cells was accelerated by coculturing withH. pylorifor 4 and 8 h, but was suppressed at 24 and 48 h. HSP70 expression was decreased in AGS cells infected byH. pylorifor 48 h. The proliferation in HSP70-silenced AGS cells was inhibited after coculturing withH. pylorifor 24 and 48 h compared with the control group.Conclusions.Coculture ofH. pylorialtered the proliferation of gastric epithelial cells and decreased HSP70 expression. HSP70 knockdown supplemented the inhibitory effect ofH. pylorion proliferation of epithelial cells. These results indicate that the effects ofH. pylorion the proliferation of gastric epithelial cells at least partially depend on the decreased expression of HSP70 induced by the bacterium.

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.

Effects of ferulic acid on oxidative stress, heat shock protein 70, connexin 43, and monoamines in the hippocampus of pentylenetetrazole-kindled rats

2017 ◽  
Vol 95 (6) ◽  
pp. 732-742 ◽  
Author(s):  
Abdelaziz M. Hussein ◽  
Khaled M. Abbas ◽  
Osama A. Abulseoud ◽  
El-Hussainy M.A. El-Hussainy
Keyword(s):  
Oxidative Stress ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Ferulic Acid ◽  
Group Treatment ◽  
Heat Shock Protein 70 ◽  
Connexin 43 ◽  
Hsp70 Expression ◽  
Neuroprotective Effects ◽  
Cx43 Expression

The present study investigated the effects of ferulic acid (FA) on pentylenetetrazole (PTZ)-induced seizures, oxidative stress markers (malondialdehyde (MDA), catalase, and reduced glutathione (GSH)), connexin (Cx) 43, heat shock protein 70 (Hsp 70), and monoamines (serotonin (5-HT) and norepinephrine (NE)) levels in a rat model of PTZ-induced kindling. Sixty Sprague Dawley rats were divided into 5 equal groups: (a) normal group; (b) FA group: normal rats received FA at a dose of 40 mg/kg daily; (c) PTZ group: normal rats received PTZ at a dose of 50 mg/kg i.p. on alternate days for 15 days; (d) FA-before group: treatment was the same as for the PTZ group, except rats received FA; and (e) FA-after group: rats received FA from sixth dose of PTZ. PTZ caused a significant increase in MDA, Cx43, and Hsp70 along with a significant decrease in GSH, 5-HT, and NE levels and CAT activity in the hippocampus (p < 0.05). Pre- and post-treatment with FA caused significant improvement in behavioral parameters, MDA, CAT, GSH, 5-HT, NE, Cx43 expression, and Hsp70 expression in the hippocampal region (p < 0.05). We conclude that FA has neuroprotective effects in PTZ-induced epilepsy, which might be due to attenuation of oxidative stress and Cx43 expression and upregulation of neuroprotective Hsp70 and neurotransmitters (5-HT and NE).

scholarly journals The effect of red-fleshed pitaya (Hylocereus polyrhizus) on heat shock protein 70 and cortisol expression in strenuous exercise induced rats

F1000Research ◽  
2019 ◽  
Vol 8 ◽  
pp. 130
Author(s):  
Novita Sari Harahap ◽  
Aznan Lelo ◽  
Ambrosius Purba ◽  
Awaluddin Sibuea ◽  
Rina Amelia ◽  
...  
Keyword(s):  
Body Weight ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Heat Shock Protein 70 ◽  
The Body ◽  
Male Rats ◽  
Strenuous Exercise ◽  
Hsp70 Expression ◽  
Average Weight ◽  
Significant Difference

Background: Oxidative stress from exercise can contribute to damaging cells, increasing heat shock protein 70 (HSP70) and suppressing the immune system in the body. This research aimed to determine the antioxidant potential of red-fleshed pitaya extract on HSP70 and cortisol expression in rats which were subjected to strenuous exercise. Methods: The subjects of this research were 32 Sprague Dawley male rats, aged 3 months, with an average weight of 200 g. Red-fleshed pitaya extract was obtained from methanol extraction process; a maceration technique was performed and the extract was concentrated using an air-drying method. Rats were randomly divided into four groups. Group 1 were subjected to strenuous exercise and treated with distilled water only; while Groups 2, 3 and 4 were subjected to strenuous exercise and treated with 100 mg/kg body weight, 200 mg/kg body weight and 300 mg/kg body weight of red-fleshed pitaya extract, respectively. Strenuous exercises in rats was performed by intense swimming of 20 min/day, 3 days a week for 3 weeks. HSP70 expression and cortisol were measured with Enzyme-Linked Immune Sorbent Assay (ELISA) method. Results: There was a significant reduction of HSP70 (p=0.000) and cortisol expression (p=0.000) between the groups. Also, there was a significant difference in the average decreasing of HSP70 expression between group 4 and either groups 1 or 2 (p=0.000). However, a significant difference between groups 4 and 3 was not observed (p=0.813). Lastly, a significant difference was found in the average decrease of cortisol expression between groups 4 and 1 (p=0.000), 2 (p=0.000), and 3 (p=0.000) respectively. Conclusion: Red-fleshed pitaya is potential to be utilized as antioxidant to decrease the HSP70 and cortisol expression.

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