scholarly journals Modulation of the morphology and glycosaminoglycan biosynthesis of human monocytes, induced by culture substrates

1984 ◽  
Vol 219 (3) ◽  
pp. 793-799 ◽  
Author(s):  
S O Kolset ◽  
R Seljelid ◽  
U Lindahl
Keyword(s):  
Foreign Body ◽  
Culture Media ◽  
Chondroitin Sulphate ◽  
Morphological Changes ◽  
Artificial Substrates ◽  
Plastic Substrate ◽  
Incubation Periods ◽  
Cells Cultured

Monocytes were isolated from human blood and cultured in vitro on plastic culture dishes or on fibronectin-coated dishes. After 5 days in vitro, the cells on plastic dishes displayed marked morphological changes compared with day 1, with an epithelioid appearance resembling that of foreign-body cells. This transition was inhibited in cells cultured on fibronectin-coated dishes. 35S-labelled polysaccharides were isolated from the culture media after 24h incubation periods with inorganic [35S]sulphate. The cells cultured for 5 days on a plastic substrate synthesized, and secreted into the medium, an oversulphated galactosaminoglycan previously shown to contain 4,6-di-O-sulphated N-acetylgalactosamine units [Kolset, Kjellén, Seljelid & Lindahl (1983) Biochem. J. 210, 661-667]. In contrast, 35S-labelled polysaccharide produced by cells cultured on plastic for 1 day only, or on fibronectin for either 1 or 5 days, contained only minor amounts of such disulphated sugar units. These findings indicate that the formation of oversulphated chondroitin sulphate is coupled to the conversion of monocytes into epithelioid cells. Furthermore, they suggest that the overall process is induced by contact with artificial substrates, and that it may be regarded as the equivalent of a foreign-body reaction in vivo.

99 DIFFERENTIAL GENE REGULATION OF STEROIDOGENIC TRANSCRIPTS AND ESTRADIOL PRODUCTION FOLLOWING IN VITRO PIG EMBRYO ELONGATION IN ALGINATE HYDROGEL THREE-DIMENSIONAL MATRIX

2012 ◽  
Vol 24 (1) ◽  
pp. 162
Author(s):  
J. R. Miles ◽  
C. N. Sargus ◽  
S. A. Plautz ◽  
J. L. Vallet ◽  
A. K. Pannier
Keyword(s):  
Equal Opportunity ◽  
Culture Media ◽  
Morphological Changes ◽  
Three Dimensional ◽  
Differential Gene Regulation ◽  
Alginate Hydrogels ◽  
The Media ◽  
And Control

Between Day 10 and 12 of gestation, the pig embryo elongates from a sphere to a long thin, filament. During this time, the embryo increases the production of oestrogen via an increase in steroidogenic transcripts, which is critical for maternal recognition of pregnancy. To date, attempts to elongate porcine embryos in vitro have been unsuccessful. Therefore, the objective of this study was to utilise alginate hydrogels to establish a culture system that promotes in vitro embryo elongation with a corresponding increase in steroidogenic transcripts and oestradiol production. In 3 replicate collections, White crossbred gilts (n = 15) were bred at Day 0 of the oestrous cycle. At Day 9 of gestation, reproductive tracts were collected and flushed with RPMI-1640 containing antibiotics. Embryos were recovered, grouped according to size and washed with RPMI-1640 containing antibiotics and 10% fetal bovine serum (FBS). Embryos were randomly assigned to be encapsulated using a double encapsulation technique (0.7% sodium alginate and 1.5% calcium chloride solution) or used as controls. Encapsulated and control embryos were cultured for 96 h in CO2 -pretreated RPMI-1640 containing antibiotics and 10% FBS at 38°C, 5% CO2 in air and 100% humidity. Every 24 h, the embryos were imaged and half of the media was replaced. The removed media was stored at –20°C and used to assess oestradiol levels by radioimmunoassay. At the end of culture, a subset of encapsulated and control embryos were snap frozen and used to assess the expression level of steroidogenic transcripts (STAR, CYP11 and CYP19) using quantitative PCR. All data were analysed using general linear model (GLM) procedures for ANOVA. Cell survival, assessed by blastocyst fragmentation and confirmed by live/dead staining in representative embryos, was greater (P = 0.01) for encapsulated embryos (60.1 ± 4.8%) compared with controls (33.3 ± 4.8%). Of encapsulated embryos, 27% had some morphological change (minor flattening and tubal formation) and 14% had significant morphological changes (considerable flattening and tubal formation elongating through the gel), consistent with in vivo embryo elongation. In contrast, the control embryos had no morphological changes observed and remained spherical during culture. The expression levels of STAR, CYP11 and CYP19 were significantly (P < 0.05) greater in encapsulated embryos compared with control embryos. Furthermore, a significant (P < 0.01) time-dependent increase in oestradiol levels in the culture media of encapsulated embryos was identified compared with controls and culture media alone. These results illustrate that cultured pig embryos encapsulated in alginate hydrogels undergo limited morphological changes with increased expression of steroidogenic transcripts and oestrogen production. †USDA is an equal opportunity provider and employer.

scholarly journals Morphological Changes in Astrocytes by Self-Oxidation of Dopamine to Polydopamine and Quantification of Dopamine through Multivariate Regression Analysis of Polydopamine Images

Polymers ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 2483
Author(s):  
Anik Karan ◽  
Elnaz Khezerlou ◽  
Farnaz Rezaei ◽  
Leon Iasemidis ◽  
Mark A. DeCoster
Keyword(s):  
Cell Culture ◽  
Regression Analysis ◽  
Multivariate Regression ◽  
Culture Media ◽  
Morphological Changes ◽  
Multivariate Regression Analysis ◽  
Cell Number ◽  
Polymerization Process

Astrocytes, also known as astroglia, are important cells for the structural support of neurons as well as for biochemical balance in the central nervous system (CNS). In this study, the polymerization of dopamine (DA) to polydopamine (PDA) and its effect on astrocytes was investigated. The polymerization of DA, being directly proportional to the DA concentration, raises the prospect of detecting DA concentration from PDA optically using image-processing techniques. It was found here that DA, a naturally occurring neurotransmitter, significantly altered astrocyte cell number, morphology, and metabolism, compared to astrocytes in the absence of DA. Along with these effects on astrocytes, the polymerization of DA to PDA was tracked optically in the same cell culture wells. This polymerization process led to a unique methodology based on multivariate regression analysis that quantified the concentration of DA from optical images of astrocyte cell culture media. Therefore, this developed methodology, combined with conventional imaging equipment, could be used in place of high-end and expensive analytical chemistry instruments, such as spectrophotometry, mass spectrometry, and fluorescence techniques, for quantification of the concentration of DA after polymerization to PDA under in vitro and potentially in vivo conditions.

scholarly journals Fluid Menisci and In Vitro Particle Dosimetry of Submerged Cells

2021 ◽  
Author(s):  
Sandor Balog ◽  
Barbara Rothen-Rutishauser ◽  
Alke Fink
Keyword(s):  
Culture Media ◽  
In Vivo Studies ◽  
Cell Culture Media ◽  
Air Interface ◽  
Inner Surface ◽  
Particle Dosimetry ◽  
Meta Analyses ◽  
Cells Cultured

Understanding the mechanisms of interaction between cells and particulate nanomaterials lies in the heart of assessing the hazard associated with nanoparticles. The paradigm of toxicology requires quantifying and interpreting dose-response relationships, and cells cultured in vitro and exposed to particle dispersions rely on mathematical models that estimate the received nanoparticle dose. Yet, none of these models acknowledges the fact that aqueous cell-culture media wet the inner surface of hydrophilic open wells, which results in curved fluid-air interface called meniscus. We show that omitting this phenomenon leads to a nontrivial but systematic error and twists the fundamental concept of nanotoxicology. Given that reproducibility and harmonization between meta analyses, in vitro, in silico, and in vivo studies must be improved, we present an adequate mathematical model that greatly advances such efforts.

Mechanical stretching increases the number of epithelial cells synthesizing DNA in culture

1984 ◽  
Vol 69 (1) ◽  
pp. 35-45
Author(s):  
D.M. Brunette
Keyword(s):  
Volume Fraction ◽  
Tritiated Thymidine ◽  
Unit Length ◽  
Plastic Substrate ◽  
Mechanical Stretching ◽  
Foetal Bovine Serum ◽  
Flexible Plastic Substrate ◽  
Cells Cultured

The influence of mechanical stretching on epithelial (E) cells was examined by culturing E cells derived from the epithelial cell rests of Malassez on a flexible plastic substrate and stretching the substrate by means of an orthodontic screw. A significant increase in the number of E cells synthesizing DNA was observed after just 30 min of stretching. In 17 experiments the ratio of cells labelled with tritiated thymidine in cultures stretched for 2 h to the number of labelled cells in unstretched controls was 1.92 +/− 0.34. An increase in labelling as a result of stretching was found for E cells cultured at either high or low cell-population densities but the effect was most pronounced for E cells cultured at higher concentrations of foetal bovine serum. Morphometric analysis of electron micrographs of stretched and unstretched cultures indicated that the stretched cultures had a higher volume fraction of filamentous structures and more desmosomes per unit length of cell membrane than unstretched cultures. The behaviour of E cells in response to stretching in vitro appears to be similar to the response of the epithelial rests in vivo when the latter are exposed to tension as a result of forces produced by orthodontic techniques.

scholarly journals In vitro development of preimplantation porcine embryos using alginate hydrogels as a three-dimensional extracellular matrix

2014 ◽  
Vol 26 (7) ◽  
pp. 943 ◽  
Author(s):  
Catherine N. Sargus-Patino ◽  
Elane C. Wright ◽  
Sarah A. Plautz ◽  
Jeremy R. Miles ◽  
Jeff L. Vallet ◽  
...  
Keyword(s):  
Cell Survival ◽  
Culture System ◽  
Culture Media ◽  
Morphological Changes ◽  
Three Dimensional ◽  
In Vitro Development ◽  
Alginate Hydrogels ◽  
Vitro Development

Between Days 10 and 12 of gestation, porcine embryos undergo a dramatic morphological change, known as elongation, with a corresponding increase in oestrogen production that triggers maternal recognition of pregnancy. Elongation deficiencies contribute to embryonic loss, but exact mechanisms of elongation are poorly understood due to the lack of an effective in vitro culture system. Our objective was to use alginate hydrogels as three-dimensional scaffolds that can mechanically support the in vitro development of preimplantation porcine embryos. White cross-bred gilts were bred at oestrus (Day 0) to Duroc boars and embryos were recovered on Days 9, 10 or 11 of gestation. Spherical embryos were randomly assigned to be encapsulated within double-layered 0.7% alginate beads or remain as non-encapsulated controls (ENC and CONT treatment groups, respectively) and were cultured for 96 h. Every 24 h, half the medium was replaced with fresh medium and an image of each embryo was recorded. At the termination of culture, embryo images were used to assess morphological changes and cell survival. 17β-Oestradiol levels were measured in the removed media by radioimmunoassay. Real-time polymerase chain reaction was used to analyse steroidogenic transcript expression at 96 h in ENC and CONT embryos, as well as in vivo-developed control embryos (i.e. spherical, ovoid and tubular). Although no differences in cell survival were observed, 32% (P < 0.001) of the surviving ENC embryos underwent morphological changes characterised by tubal formation with subsequent flattening, whereas none of the CONT embryos exhibited morphological changes. Expression of steroidogenic transcripts STAR, CYP11A1 and CYP19A1 was greater (P < 0.07) in ENC embryos with morphological changes (ENC+) compared with CONT embryos and ENC embryos with no morphological changes (ENC–), and was more similar to expression of later-stage in vivo-developed controls. Furthermore, a time-dependent increase (P < 0.001) in 17β-oestradiol was observed in culture media from ENC+ compared with ENC– and CONT embryos. These results illustrate that preimplantation pig embryos encapsulated in alginate hydrogels can undergo morphological changes with increased expression of steroidogenic transcripts and oestrogen production, consistent with in vivo-developed embryos. This alginate culture system can serve as a tool for evaluating specific mechanisms of embryo elongation that could be targeted to improve pregnancy outcomes.

207 Female Reproductive Fluids and Epigenetics

2021 ◽  
Vol 99 (Supplement_1) ◽  
pp. 114-114
Author(s):  
Sebastian Canovas ◽  
Raquel Romar ◽  
Pilar Coy
Keyword(s):  
Assisted Reproductive Technologies ◽  
Culture Media ◽  
Morphological Changes ◽  
Adult Life ◽  
Specific Effect ◽  
Reproductive Technologies ◽  
Early Embryo ◽  
Vitro Fertilization

Abstract Physiological fertilization, and early embryo development, involves dramatic transcriptomic, epigenetic and morphological changes in a short temporal window. During this period gametes and early embryos are surrounded by reproductive fluids (oviductal and uterine), which contain nutrients, growth factors, hormones and extracellular vesicles acting as carriers of DNA, RNA, proteins and other factors with putative roles in intercellular communication. Under in vitro conditions, and in the absence of these fluids, embryos derived from Assisted Reproductive Technologies (ART) reveal transcriptional and epigenetic differences compared with in vivo embryos, which could result in long-term phenotypic consequences in adult life. Therefore, reproductive fluids supplementation in the culture medium offers an alternative to imitate physiological conditions and decrease these consequences. In vitro, oviductal fluid (OF) can modulate capacitation-associated events and sperm-zona pellucida interactions and contribute to the control of polyspermy in pigs. The use of in vitro fertilization media supplemented with reproductive fluids (Natur-IVF) improves embryo quality and blastocysts hatching ability. Moreover, Natur-IVF embryos show expression and methylation patterns closer to in vivo blastocysts. In cows, supplementation of culture media with reproductive fluids, or some isolated factors, improves blastocyst rate and survival after embryo transfer, and reverses the expression of some altered genes. However, considering the complexity of the oviductal and uterine fluids, it seems difficult that the use of just a few factors in isolation can reverse all undesired consequences of the IVP. On the other hand, sex-specific embryonic plasticity, as a consequence of the oviductal regulatory signals, have been proposed. Thus, we have analysed the sex-specific effect of supplementation with reproductive fluids in bovine embryos and data reveal sex-dependent impact in DNA methylation. All these results confirm that developmental programme can be modulated by reproductive fluids and it shows sex-specific effects. This strategy allows the possibility of minimizing undesired in vitro derived consequences.

The Anti-tumor Activity and Mechanisms of rLj-RGD3 on Human Laryngeal Squamous Carcinoma Hep2 Cells

2020 ◽  
Vol 19 (17) ◽  
pp. 2108-2119
Author(s):  
Yang Jin ◽  
Li Lv ◽  
Shu-Xiang Ning ◽  
Ji-Hong Wang ◽  
Rong Xiao
Keyword(s):  
Wound Healing ◽  
Western Blot ◽  
Morphological Changes ◽  
In Vivo Studies ◽  
Migration And Invasion ◽  
Tunel Staining ◽  
Dna Ladder ◽  
Western Blot Assay

Background: Laryngeal Squamous Cell Carcinoma (LSCC) is a malignant epithelial tumor with poor prognosis and its incidence rate increased recently. rLj-RGD3, a recombinant protein cloned from the buccal gland of Lampetra japonica, contains three RGD motifs that could bind to integrins on the tumor cells. Methods: MTT assay was used to detect the inhibitory rate of viability. Giemsa’s staining assay was used to observe the morphological changes of cells. Hoechst 33258 and TUNEL staining assay, DNA ladder assay were used to examine the apoptotic. Western blot assay was applied to detect the change of the integrin signal pathway. Wound-healing assay, migration, and invasion assay were used to detect the mobility of Hep2 cells. H&E staining assay was used to show the arrangement of the Hep2 cells in the solid tumor tissues. Results: In the present study, rLj-RGD3 was shown to inhibit the viability of LSCC Hep2 cells in vitro by inducing apoptosis with an IC50 of 1.23µM. Western blot showed that the apoptosis of Hep2 cells induced by rLj- RGD3 was dependent on the integrin-FAK-Akt pathway. Wound healing, transwells, and western blot assays in vitro showed that rLj-RGD3 suppressed the migration and invasion of Hep2 cells by integrin-FAKpaxillin/ PLC pathway which could also affect the cytoskeleton arrangement in Hep2 cells. In in vivo studies, rLj-RGD3 inhibited the growth, tumor volume, and weight, as well as disturbed the tissue structure of the solid tumors in xenograft models of BALB/c nude mice without reducing their body weights. Conclusion: hese results suggested that rLj-RGD3 is an effective and safe suppressor on the growth and metastasis of LSCC Hep2 cells from both in vitro and in vivo experiments. rLj-RGD3 might be expected to become a novel anti-tumor drug to treat LSCC patients in the near future.

scholarly journals Influence of Light Conditions and Medium Composition on Morphophysiological Characteristics of Stevia rebaudiana Bertoni In Vitro and In Vivo

Horticulturae ◽  
2021 ◽  
Vol 7 (7) ◽  
pp. 195
Author(s):  
Alla A. Shulgina ◽  
Elena A. Kalashnikova ◽  
Ivan G. Tarakanov ◽  
Rima N. Kirakosyan ◽  
Mikhail Yu. Cherednichenko ◽  
...  
Keyword(s):  
Culture Media ◽  
Stevia Rebaudiana ◽  
Adventitious Shoot ◽  
Optimal Growth ◽  
Medium Composition ◽  
Light Spectra ◽  
Stevia Rebaudiana Bertoni ◽  
Multiplication Coefficient

We investigated the influence of different conditions (light composition and plant growth regulators (PGRs) in culture media) on the morphophysiological parameters of Stevia rebaudiana Bertoni in vitro and in vivo. Both PGRs and the light spectra applied were found to significantly affect plant morphogenesis. During the micropropagation stage of S. rebaudiana, optimal growth, with a multiplication coefficient of 15, was obtained in an MS culture medium containing 2,4-epibrassinolide (Epin) and indole-3-acetic acid (IAA) at concentrations of 0.1 and 0.5 mg L−1, respectively. During the rooting stage, we found that the addition of 0.5 mg L−1 hydroxycinnamic acid (Zircon) to the MS medium led to an optimal root formation frequency of 85% and resulted in the formation of strong plants with well-developed leaf blades. Cultivation on media containing 0.1 mg L−1 Epin and 0.5 mg L−1 IAA and receiving coherent light irradiation on a weekly basis resulted in a 100% increase in the multiplication coefficient, better adventitious shoot growth, and a 33% increase in the number of leaves. S. rebaudiana microshoots, cultured on MS media containing 1.0 mg L−1 6-benzylaminopurine (BAP) and 0.5 mg L−1 IAA with red monochrome light treatments, increased the multiplication coefficient by 30% compared with controls (white light, media without PGRs).

scholarly journals Protein kinase D promotes plasticity-induced F-actin stabilization in dendritic spines and regulates memory formation

2015 ◽  
Vol 210 (5) ◽  
pp. 771-783 ◽  
Author(s):  
Norbert Bencsik ◽  
Zsófia Szíber ◽  
Hanna Liliom ◽  
Krisztián Tárnok ◽  
Sándor Borbély ◽  
...  
Keyword(s):  
Synaptic Plasticity ◽  
Protein Kinase ◽  
Dendritic Spines ◽  
Morphological Changes ◽  
Long Term Potentiation ◽  
Memory Formation ◽  
Protein Kinase D

Actin turnover in dendritic spines influences spine development, morphology, and plasticity, with functional consequences on learning and memory formation. In nonneuronal cells, protein kinase D (PKD) has an important role in stabilizing F-actin via multiple molecular pathways. Using in vitro models of neuronal plasticity, such as glycine-induced chemical long-term potentiation (LTP), known to evoke synaptic plasticity, or long-term depolarization block by KCl, leading to homeostatic morphological changes, we show that actin stabilization needed for the enlargement of dendritic spines is dependent on PKD activity. Consequently, impaired PKD functions attenuate activity-dependent changes in hippocampal dendritic spines, including LTP formation, cause morphological alterations in vivo, and have deleterious consequences on spatial memory formation. We thus provide compelling evidence that PKD controls synaptic plasticity and learning by regulating actin stability in dendritic spines.

scholarly journals Astrocyte-Derived CCL2 is Associated with M1 Activation and Recruitment of Cultured Microglial Cells

2016 ◽  
Vol 38 (3) ◽  
pp. 859-870 ◽  
Author(s):  
Mingfeng He ◽  
Hongquan Dong ◽  
Yahui Huang ◽  
Shunmei Lu ◽  
Shu Zhang ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Microglial Activation ◽  
Culture Media ◽  
Microglial Cells ◽  
Migration Ability ◽  
Migration Assay ◽  
Tnf Α

Background/Aims: Microglia are an essential player in central nervous system inflammation. Recent studies have demonstrated that the astrocytic chemokine, CCL2, is associated with microglial activation in vivo. However, CCL2-induced microglial activation has not yet been studied in vitro. The purpose of the current study was to understand the role of astrocyte-derived CCL2 in microglial activation and to elucidate the underlying mechanism(s). Methods: Primary astrocytes were pre-treated with CCL2 siRNA and stimulated with TNF-α. The culture medium (CM) was collected and added to cultures of microglia, which were incubated with and without CCR2 inhibitor. Microglial cells were analyzed by quantitative RT-PCR to determine whether they polarized to the M1 or M2 state. Microglial migratory ability was assessed by transwell migration assay. Results: TNF-α stimulated the release of CCL2 from astrocytes, even if the culture media containing TNF-α was replaced with fresh media after 3 h. CM from TNF-α-stimulated astrocytes successfully induced microglial activation, which was ascertained by increased activation of M1 and enhanced migration ability. In contrast, CM from astrocytes pretreated with CCL2 siRNA showed no effect on microglial activation, compared to controls. Additionally, microglia pre-treated with RS102895, a CCR2 inhibitor, were resistant to activation by CM from TNF-α-stimulated astrocytes. Conclusion: This study demonstrates that the CCL2/CCR2 pathway of astrocyte-induced microglial activation is associated with M1 polarization and enhanced migration ability, indicating that this pathway could be a useful target to ameliorate inflammation in the central nervous system.

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