scholarly journals Expression of Otx Genes in Müller Cells Using an In Vitro Experimental Model of Retinal Hypoxia

2021 ◽  
Vol 2021 ◽  
pp. 1-10
Author(s):  
Claudio Azzolini ◽  
Simone Donati ◽  
Giovanni Micheloni ◽  
Vittoria Moretti ◽  
Roberto Valli ◽  
...  
Keyword(s):  
Recovery Time ◽  
Culture Medium ◽  
Cobalt Chloride ◽  
Retinal Diseases ◽  
Cellular Events ◽  
In Vitro Response ◽  
Retinal Pathologies ◽  
Eye Morphogenesis

Introduction. Müller glial cells typically activate to react to hypoxic tissue damage in several retinal diseases. We evaluated the in vitro response to a hypoxia-mimicking stimulus on the expression of a set of genes, known to contribute to eye morphogenesis and cell differentiation. Materials and Methods. A MIO-M1 Müller cell line was cultured in a hypoxia-mimicking environment by the addition of cobalt chloride to the culture medium, followed by a recovery time in which we mimic restoration from the hypoxic insult. The HIF-1α protein and VEGF-A gene expression were quantified to verify the induction of a hypoxia-like state. Results. Among the genes under study, we did not observe any difference in the expression levels of Otx1 and Otx2 during treatment; conversely, Otx1 was overexpressed during recovery steps. The VEGF-A gene was strongly upregulated at both the CoCl2 and recovery time points. The transactivated isoform (TA) of the TP73 gene showed an overexpression in long-term exposure to the hypoxic stimulus with a further increase after recovery. Discussion. Our molecular analysis is able to describe the activation of a set of genes, never before described, that can drive the response to a hypoxia-like status. The improved comprehension of these cellular events will be useful for designing new therapeutical approaches for retinal pathologies.

scholarly journals Temperature-induced reorganisation of Schistocephalus solidus (Cestoda) proteome during the transition to the warm-blooded host

Biology Open ◽  
2021 ◽  
Vol 10 (11) ◽  
Author(s):  
Ekaterina V. Borvinskaya ◽  
Albina A. Kochneva ◽  
Polina B. Drozdova ◽  
Olga V. Balan ◽  
Victor G. Zgoda
Keyword(s):  
Model Experiment ◽  
Protein Production ◽  
Culture Medium ◽  
Gasterosteus Aculeatus ◽  
Protein Composition ◽  
Tegument Protein ◽  
Schistocephalus Solidus ◽  
Induced Genes

ABSTRACT The protein composition of the cestode Schistocephalus solidus was measured in an experiment simulating the trophic transmission of the parasite from a cold-blooded to a warm-blooded host. The first hour of host colonisation was studied in a model experiment, in which sticklebacks Gasterosteus aculeatus infected with S. solidus were heated at 40°C for 1 h. As a result, a decrease in the content of one tegument protein was detected in the plerocercoids of S. solidus. Sexual maturation of the parasites was initiated in an experiment where S. solidus larvae were taken from fish and cultured in vitro at 40°C for 48 h. Temperature-independent changes in the parasite proteome were investigated by incubating plerocercoids at 22°C for 48 h in culture medium. Analysis of the proteome allowed us to distinguish the temperature-induced genes of S. solidus, as well as to specify the molecular markers of the plerocercoid and adult worms. The main conclusion of the study is that the key enzymes of long-term metabolic changes (glycogen consumption, protein production, etc.) in parasites during colonisation of a warm-blooded host are induced by temperature.

scholarly journals Zeaxanthin Inhibits Hypoxia-Induced VEGF Secretion by RPE Cells through Decreased Protein Levels of Hypoxia-Inducible Factors-1α

2015 ◽  
Vol 2015 ◽  
pp. 1-11 ◽  
Author(s):  
Richard Rosen ◽  
Tommaso Vagaggini ◽  
Yueqin Chen ◽  
Dan-Ning Hu
Keyword(s):  
Culture Medium ◽  
Retinal Pigment ◽  
Retinal Diseases ◽  
Hypoxia Inducible Factors ◽  
Rpe Cells ◽  
Protein Levels ◽  
Hypoxic Conditions ◽  
Cell Hypoxia ◽  
Important Stimulus

Hypoxia is the most important stimulus leading to upregulation of VEGF in the retina and this is caused by accumulation of hypoxia-inducible factors-1α(HIF-1α) protein. The effects of zeaxanthin, a natural phytochemical, on the VEGF and HIF-1αexpression in the primary culture of human retinal pigment epithelial (RPE) cells were studied. An in vitro RPE cell hypoxia model was established by placing cells under 1% oxygen pressure or by adding cobalt chloride (CoCl2) to the culture medium. RPE cells and conditioned media were collected from cultures treated with and without zeaxanthin under normoxic and hypoxic conditions. VEGF and HIF-1αprotein and RNA levels were measured by ELISA kits and RT-PCR, respectively. Hypoxia caused a significant increase of VEGF expression and accumulation of HIF-1αin RPE cells. Zeaxanthin at 50–150 μM significantly inhibited the expression of VEGF and accumulation of HIF-1αprotein caused by hypoxia but did not affect expression of VEGF and HIF-1αunder normoxic conditions. This is the first report on the effect of zeaxanthin on VEGF and HIF-1αlevels in cultured RPE cells and suggests that zeaxanthin may have potential value in the prevention and treatment of various retinal diseases associated with vascular leakage and neovascularization.

scholarly journals Long-term in Vitro Regeneration of Hamelia patens

HortScience ◽  
1995 ◽  
Vol 30 (4) ◽  
pp. 873G-874
Author(s):  
D. Sankhla ◽  
T.D. Davis ◽  
N. Sankhla ◽  
A. Upadhyaya
Keyword(s):  
Culture Medium ◽  
In Vitro Regeneration ◽  
Shoot Formation ◽  
Shoot Tips ◽  
Shoot Cultures ◽  
Ms Medium ◽  
Prolific Shoot ◽  
Ornamental Shrub

This report describes an efficient in vitro regeneration protocol for H. patens (firebush), a heat-tolerant ornamental shrub native to tropical and subtropical America. Shoot cultures were initially established using shoot tips placed on MS-revised medium containing 2.3 μM 2,4-D, 2.3 μM kinetin, and 0.25% polyvinylpyrrolidone. Other types of explants (nodal and internodal segments, leaf pieces, floral buds) did not regenerate shoots when placed on this medium. Two-month-old plantlets derived from the shoot tips were subcultured on MS medium supplemented with 0.5 μM thidiazuron (TDZ), and within 3 to 4 weeks, some callus was produced at the root–shoot junction. When this callus, with a small portion of the root and shoots, was placed on MS medium with 0.05 μM TDZ and 0.01 μM ABA, prolific shoot formation occurred within 3 to 4 weeks followed by root formation. By regular subculturing every 5 to 6 weeks, hundreds of plantlets have been obtained over the past 3 years with no apparent decline in regeneration potential. Addition of activated charcoal (0.5%) to the culture medium has greatly improved growth of the plantlets.

scholarly journals The Growth Response of Several Potato Genotypes (Solanum tuberosum L.) to Induced Water Stress Using Sorbitol and Polyethylene Glycol

2016 ◽  
Vol 8 (4) ◽  
pp. 511-519 ◽  
Author(s):  
Mihaela A. CIOLOCA ◽  
Andreea M. TICAN ◽  
Maria IANOŞI ◽  
Carmen L. BĂDĂRĂU
Keyword(s):  
Polyethylene Glycol ◽  
Water Stress ◽  
Drought Tolerance ◽  
Growth Response ◽  
Culture Medium ◽  
Growth Parameters ◽  
The Other ◽  
Addition Lines ◽  
In Vitro Response

The current paper aimed to study the in vitro response of potato genotypes to water stress induced by adding sorbitol and polyethylene glycol in the culture medium. The biological material analysed in the experiment was represented by a Romanian line ‘LP 11-1525/1’ and two isogenic lines ‘LI 101’ and ‘LI 102’. For cultures initiation, the line ‘LP 11-1525/1’ was started from meristems and for the other two genotypes true potato seeds were used. The studied potato genotypes behaved differently depending on the analysed parameters and on the treatment applied for drought tolerance. It was noted that the line ‘LP 11-1525/1’ achieved good results for most of the growth parameters studied, and also the lines derived from true potato seeds behaved well, in some cases even exceeding the line derived from meristems. Of the lines derived from true potato seeds, the best performance was noted for line ‘LI 101-6’ in all the analysed parameters, both on sorbitol and PEG medium. In addition, lines ‘LI 101-7’ and ‘LI 102-4’ achieved good results on both variants of medium used to mediate water stress. Therefore, establishing drought tolerance individuals within populations derived from true potato seeds using sorbitol and polyethylene glycol might be applied.

scholarly journals Extract of Amburana cearensis maintains the survival of ovine preantral follicles during long-term ovarian tissue transport and promotes primordial follicle activation after in vitro culture

2018 ◽  
Vol 39 (5) ◽  
pp. 2001
Author(s):  
Vanúzia Gonçalves Menezes ◽  
Ricássio De Sousa Barberino ◽  
Bruna Bortoloni Gouveia ◽  
Rodrigo José de Souza Gonçalves ◽  
Jackson Roberto Guedes da Silva Almeida ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Culture Medium ◽  
Ovarian Tissue ◽  
Primordial Follicle ◽  
Chi Square ◽  
Preantral Follicles ◽  
Primordial Follicle Activation ◽  
Chi Square Test

This study evaluated the effect of Amburana cearensis extract as a preservation or culture medium for ovine ovarian tissue. Ovarian fragments were fixed in 4% buffered formaldehyde for 18 h (fresh control), stored in Minimal Essential Medium (MEM) or in A. cearensis extract (0.1; 0.2 or 0.4 mg/mL) at a temperature of 4ºC for 6, 12 or 24 h (preservation - experiment 1) or cultured for 7 days in ?-MEM+ or in A. cearensis extract without (0.1; 0.2 or 0.4 mg/mL) or with supplements (0.1+ ; 0.2+ or 0.4+ mg/ mL; experiment 2). The percentages of morphologically normal follicles and follicular activation were submitted to analysis of variance (ANOVA) and Tukey´s test. The values of TUNEL-positive cells were submitted to Chi-square test (P < 0.05). The storage of fragments for 6 h in MEM showed higher percentages of normal follicles (62%) and a lower rate of TUNEL positive cells (36.17%) compared to other treatments (normal follicles: 46%; 43% and 52%; TUNEL positive cells: 58.57%; 55.30% and 55.63% for Amb 0.1; Amb 0.2 and Amb 0.4 mg/mL, respectively). However, after 12 or 24 h, MEM (12 h: 48%; 24 h: 45%) and Amb 0.2 mg/mL (12 h: 37%; 24 h: 39%) showed similar percentages of normal follicles and TUNEL positive cells (MEM - 12 h: 43.26%; 24 h: 58%; Amb 0.2 mg/mL - 12 h: 50%; 24 h: 61%). After culture, ?-MEM+ recorded a higher percentage of normal follicles (58.25%) than A. cearensis treatments (32.8%; 25.4% and 34.2% for Amb 0.1; Amb 0.2 and Amb 0.4 mg/mL, and 22.25%; 20.0% and 36.6% for Amb 0.1+ ; Amb 0.2+ and Amb 0.4+ mg/mL, respectively) (P < 0.05). Follicular activation increased in all treatments (52.5%; 36.73%; 54.05%; 47.5% and 58.19% for ?-MEM+ ; Amb 0.1; Amb 0.1+ ; Amb 0.2+ and Amb 0.4+ mg/mL, respectively) compared to the fresh control (11.65%), except for Amb 0.2 mg/mL (23.69%) and Amb 0.4 mg/mL (28.85%) (P > 0.05). Moreover, after in vitro culture, A. cearensis at a concentration of 0.1 mg/mL maintained the percentage of TUNEL positive cells (30.0%) in a way that is similar to that observed in the fresh control (22%) (P > 0.05). In conclusion, ovine preantral follicles can be preserved at 4°C in MEM for 6 h. For longer periods of preservation (24 h), MEM and 0.2 mg/mL A. cearensis are recommended. Moreover, after in vitro culture, A. cearensis extract (0.1 mg/mL) showed higher activation and lower DNA fragmentation in ovine preantral follicles.

scholarly journals Improving In Vitro Culture of Human Male Fetal Germ Cells

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 2033
Author(s):  
Myriam Martin-Inaraja ◽  
Monica Ferreira ◽  
Jasin Taelman ◽  
Cristina Eguizabal ◽  
Susana M. Chuva De Sousa Lopes
Keyword(s):  
Germ Cells ◽  
Culture Medium ◽  
Culture Media ◽  
Culture Conditions ◽  
Pronounced Difference ◽  
Coated Substrate ◽  
Male Gametes ◽  
The Media

Male human fetal germ cells (hFGCs) give rise to spermatogonial stem cells (SSCs), which are the adult precursors of the male gametes. Human SSCs are a promising (autologous) source of cells for male fertility preservation; however, in contrast to mouse SSCs, we are still unable to culture them in the long term. Here, we investigated the effect of two different culture media and four substrates (laminin, gelatin, vitronectin and matrigel) in the culture of dissociated second trimester testes, enriched for hFGCs. After 6 days in culture, we quantified the presence of POU5F1 and DDX4 expressing hFGCs. We observed a pronounced difference in hFGC number in different substrates. The combination of gelatin-coated substrate and medium containing GDNF, LIF, FGF2 and EGF resulted in the highest percentage of hFGCs (10% of the total gonadal cells) after 6 days of culture. However, the vitronectin-coated substrate resulted in a comparable percentage of hFGCs regardless of the media used (3.3% of total cells in Zhou-medium and 4.8% of total cells in Shinohara-medium). We provide evidence that not only the choices of culture medium but also choices of the adequate substrate are crucial for optimizing culture protocols for male hFGCs. Optimizing culture conditions in order to improve the expansion of hFGCs will benefit the development of gametogenesis assays in vitro.

scholarly journals Effects of Exposure to Tobacco Cigarette, Electronic Cigarette and Heated Tobacco Product on Adipocyte Survival and Differentiation In Vitro

Toxics ◽  
2020 ◽  
Vol 8 (1) ◽  
pp. 9 ◽  
Author(s):  
Zoi Zagoriti ◽  
Mohamed A. El Mubarak ◽  
Konstantinos Farsalinos ◽  
Stavros Topouzis
Keyword(s):  
Culture Medium ◽  
Electronic Cigarettes ◽  
Body Weight Loss ◽  
Tobacco Product ◽  
Dependent Manner ◽  
Ppar Γ ◽  
Beige Adipocytes ◽  
Thermogenic Adipocytes

Cigarette smoking (CS) causes significant morbidity worldwide, attributed to the numerous toxicants generated by tobacco combustion. Electronic cigarettes (ECIG) and heated tobacco products (HTP) are considered alternative smoking/vaping products that deliver nicotine through an inhaled aerosol and emit fewer harmful constituents than CS. However, their long-term impacts on human health are not well established. Nicotine exposure has been linked to lipolysis and body weight loss, while smoking has been associated with insulin resistance and hyperinsulinemia. Enhanced function of beige (thermogenic) adipocytes has been proposed as a means to reduce obesity and metabolic disorders. In this study, we compared the effect of extract-enriched media via exposure of culture medium to CS, HTP aerosol, and ECIG aerosol on the viability and the differentiation of 3T3-L1 pre-adipocytes to beige adipocytes. Only CS extract caused a decrease in cell viability in a dose- and time-dependent manner. Furthermore, relative lipid accumulation and expression levels of the adipocyte markers Pgc-1α, Ppar-γ and Resistin were significantly decreased in cells exposed to CS extract. Our results demonstrate that CS extract, in contrast to HTP and ECIG extracts, significantly impairs differentiation of pre-adipocytes to beige adipocytes and may therefore impact significantly adipose tissue metabolic function.

Limitations to oxygen diffusion and equilibration in in vitro cell exposure systems in hyperoxia and hypoxia

2001 ◽  
Vol 281 (4) ◽  
pp. L1021-L1027 ◽  
Author(s):  
Corrie B. Allen ◽  
B. Kelly Schneider ◽  
Carl W. White
Keyword(s):  
Tissue Culture ◽  
Time Course ◽  
Culture Medium ◽  
Cultured Cells ◽  
Long Term Effects ◽  
Temporal Precision ◽  
Cellular Events ◽  
Solubility Of Oxygen

Exposure of cultured cells to changing gaseous environments is used as a model for understanding both the immediate and long-term effects of such exposures on lung cells in vivo. We conducted experiments with polystyrene tissue culture flasks and plates to determine the time course of changes in oxygen concentration occurring under in vitro conditions. Only a few minutes were required for the concentration of oxygen in the environmental chamber to reach equilibrium with that of the flushing gas. However, >3 h were required for the oxygen content in the medium in the tissue culture flasks and plates to achieve equilibrium. The low solubility of oxygen in aqueous solutions and the limited diffusion of oxygen through a boundary layer of gas above the medium are the major barriers to rapid oxygen transport into the culture medium. The delay in achieving the desired Po 2 within the culture medium limits the temporal precision of any assessment of the correlation of cellular events with the concentration of oxygen to which those cells are exposed.

Long-term survival and aldosterone secretion pattern of aldosteronoma in culture

1983 ◽  
Vol 104 (4) ◽  
pp. 495-501 ◽  
Author(s):  
Tetsuro Okabe ◽  
Hiroshi Hidaka ◽  
Nakaaki Ohsawa ◽  
Toshio Tsushima
Keyword(s):  
Angiotensin Ii ◽  
Primary Aldosteronism ◽  
Culture Medium ◽  
Cultured Cells ◽  
Aldosterone Secretion ◽  
Term Survival ◽  
Eosinophilic Cytoplasm

Abstract. In an attempt to obtain an in vitro experimental model for aldosteronoma, primary culture was initiated with adenomas from 3 patients with primary aldosteronism. The cells grown in culture retained the morphology and functional properties characteristic of aldosteronoma cells well for periods of up to 200 days. The cells formed monolayer cell colonies and showed an epithelioid morphology with small nuclei containing prominent nucleoli. The cells possessed a clear, eosinophilic cytoplasm resembling that of aldosteronoma cells in vivo. The cultured cells continued to secrete large amounts of aldosterone throughout the culture period. The cells responded to angiotensin II and III by increased release of aldosterone into the culture medium. They also responded to Db-cAMP and ACTH by increased secretion of the hormone.

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