Trifolium pratense: Friable calli, cell culture protocol and isoflavones content in wild plants, in vitro and cell cultures analyzed by UPLC

The physiological characteristics of the callus cell cultures of Alhagi persarum Boiss et Buhse, a member of the legume family, widely used in folk medicine, have been studied. It was shown that the source of the explant was an important factor in the initiation of callusogenesis: more intense callusogenesis (almost 100%) was observed for explants from various organs of sterile seedlings, rather than intact plants (less than 30%). As a result, more than 20 lines of morphologically different callus cell cultures were obtained, and the growth parameters for the 5 most intensively growing lines were determined. The composition of fatty acids (FA) of total lipids and secondary metabolites in the most physiologically stable callus line Aр-207 was analyzed. Using capillary gas-liquid chromatography with mass spectrometric detection (GLC-MS), 19 individual C12--C24 FAs were identified, the main fraction of which were palmitic (~ 23%), stearic (~ 22%), linoleic (~ 14%) and α-linolenic (~ 33%) acids. The established atypical ratio of FAs (a simultaneous high content of both saturated FAs and polyunsaturated α-linolenic acid) is possibly due to the adaptation of cells to in vitro growth conditions. Phytochemical analysis of the secondary metabolites was carried out using ultra-performance liquid chromatography with electrospray ionization mass spectrometric detection (UPLC MS). Compounds belonging to different structural groups of isoflavones were found. Aglycones (calycosin, formononetin and afrormosin isomer), glucosides (formononetin glucoside), as well as esters of glucosides (malonylglycosides of calicosin, formononetin, afrormosin isomers, glycitein and genistein) were detected. These secondary metabolites are widespread in plants of the Fabaceae family; however, isoflavones are rare in representatives of the Alhagi genus. The presence of malonylated isoflavone glycosides in Alhagi spp. was shown for the first time. endemic plant species, Alhagi, in vitro cell culture, callus cell culture, isoflavones, fatty acids All studies were carried out using the equipment of the "Experimental Biotechnological Facility" and the "All-Russian Collection of Cell Cultures of Higher Plants" of IРР RAS. This work was supported by the Russian Foundation for Basic Research (RFBR), contract no.18-54-06021 (Az_a), and the Government of the Russian Federation, Megagrant Project no. 075-15-2019-1882.

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Feeder-Dependent/Independent Mouse Embryonic Stem Cell Culture Protocol

10.1007/7651_2021_402 ◽

2021 ◽

Author(s):

Hatice Burcu Şişli ◽

Selinay Şenkal ◽

Derya Sağraç ◽

Taha Bartu Hayal ◽

Ayşegül Doğan

Keyword(s):

Cell Culture ◽

Stem Cell ◽

Embryonic Stem Cell ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cell ◽

Stem Cell Culture ◽

Cell Culture Protocol ◽

Culture Protocol

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THE PROLONGED GROWTH AND SURVIVAL OF OVARIAN TISSUE OF THE PROMETHEA MOTH (CALLOSAMIA PROMETHEA) IN VITRO

The Journal of General Physiology ◽

10.1085/jgp.41.5.1027 ◽

1958 ◽

Vol 41 (5) ◽

pp. 1027-1034 ◽

Cited By ~ 30

Author(s):

T. D. C. Grace

Keyword(s):

Cell Culture ◽

Cell Migration ◽

Cell Cultures ◽

Ovarian Tissue ◽

Explant Culture ◽

Growth And Survival ◽

Live Tissue ◽

Cell Divisions ◽

Callosamia Promethea

1. The ovarian tissues from diapausing pupae of the promethea moth (Callosamia promethea) have survived and grown for 186 days under in vitro conditions. There was continual cell migration and multiplication for a period of 53 days, followed by a period of 47 days during which no cells migrated from the tissues. Between the 100th and 105th days after setting up the cultures, cell migration was resumed, and by the 111th day 250 cells were present in the medium. A few cell divisions were observed between the 126th and 136th days. After the tissues were subcultured on the 140th day, the explant culture continued to survive, but the cell culture died 3 days later. 2. The tissues were subcultured a total of 6 times during the 186 days. By the introduction of a piece of live tissue into the cell cultures, the growth and survival of the cells were increased from 8 days to about 20 days. 3. It is possible that the tissues had become adapted to the medium during their long survival, as the cells which migrated from them after 100 days showed considerably longer survival than those in earlier cultures.

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Three-Dimensional Cell Cultures as an In Vitro Tool for Prostate Cancer Modeling and Drug Discovery

International Journal of Molecular Sciences ◽

10.3390/ijms21186806 ◽

2020 ◽

Vol 21 (18) ◽

pp. 6806 ◽

Cited By ~ 2

Author(s):

Fabrizio Fontana ◽

Michela Raimondi ◽

Monica Marzagalli ◽

Michele Sommariva ◽

Nicoletta Gagliano ◽

...

Keyword(s):

Prostate Cancer ◽

Cell Culture ◽

Drug Discovery ◽

Cancer Cells ◽

Cell Cultures ◽

Antitumor Agents ◽

Three Dimensional ◽

3D Cell Culture ◽

Cancer Modeling

In the last decade, three-dimensional (3D) cell culture technology has gained a lot of interest due to its ability to better recapitulate the in vivo organization and microenvironment of in vitro cultured cancer cells. In particular, 3D tumor models have demonstrated several different characteristics compared with traditional two-dimensional (2D) cultures and have provided an interesting link between the latter and animal experiments. Indeed, 3D cell cultures represent a useful platform for the identification of the biological features of cancer cells as well as for the screening of novel antitumor agents. The present review is aimed at summarizing the most common 3D cell culture methods and applications, with a focus on prostate cancer modeling and drug discovery.

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Ratiometric Nanoviscometers: Applications for Measuring Cellular Physical Properties in 3D Cultures

SLAS TECHNOLOGY Translating Life Sciences Innovation ◽

10.1177/2472630319901262 ◽

2020 ◽

Vol 25 (3) ◽

pp. 234-246

Author(s):

Charles McRae White ◽

Mark A. Haidekker ◽

William S. Kisaalita

Keyword(s):

Cell Culture ◽

Cell Cultures ◽

Cell Biology ◽

Low Cost ◽

3D Cell Culture ◽

Biomechanical Properties ◽

Practical Applications ◽

3D Cell Cultures

New insights into the biomechanical properties of cells are revealing the importance of these properties and how they relate to underlying molecular, architectural, and behavioral changes associated with cell state and disease processes. However, the current understanding of how these in vitro biomechanical properties are associated with in vivo processes has been developed based on the traditional monolayer (two-dimensional [2D]) cell culture, which traditionally has not translated well to the three-dimensional (3D) cell culture and in vivo function. Many gold standard methods and tools used to observe the biomechanical properties of 2D cell cultures cannot be used with 3D cell cultures. Fluorescent molecules can respond to external factors almost instantaneously and require relatively low-cost instrumentation. In this review, we provide the background on fluorescent molecular rotors, which are attractive tools due to the relationship of their emission quantum yield with environmental microviscosity. We make the case for their use in both 2D and 3D cell cultures and speculate on their fundamental and practical applications in cell biology.

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BeO-OSL detectors for dose measurements in cell cultures

Nuklearmedizin ◽

10.3413/nukmed-0261 ◽

2009 ◽

Vol 48 (06) ◽

pp. 227-232

Author(s):

D. Sommer ◽

R. Freudenberg ◽

U. Reichelt ◽

J. Henniger ◽

J. Kotzerke ◽

...

Keyword(s):

Cell Culture ◽

Monte Carlo Simulation ◽

Monte Carlo ◽

Cell Cultures ◽

Rate Sensitivity ◽

Culture Vessel ◽

Qualitative Properties ◽

Dose Profile ◽

Ambient Lighting

Summary Aim: The absorbed dose is an important parameter in experiments involving irradiation of cells in vitro with unsealed radionuclides. Typically, this is estimated with a model calculation, although the results thus obtained cannot be verified. Generally used real-time measurement methods are not applicable in this setting. A new detector material with in vitro suitability is the subject of this work. Methods: Optically-stimulated luminescence (OSL) dosimeters based on beryllium oxide (BeO) were used for dose measurement in cell cultures exposed to unsealed radionuclides. Their qualitative properties (e. g. energy-dependent count rate sensitivity, fading, contamination by radioactive liquids) were determined and compared to the results of a Monte Carlo simulation (using AMOS software). OSL dosimeters were tested in common cell culture setups with a known geometry. Results: Dose reproducibility of the OSL dosimeters was ± 1.5%. Fading at room temperature was 0.07% per day. Dose loss (optically-stimulated deletion) under ambient lighting conditions was 0.5% per minute. The Monte Carlo simulation for the relative sensitivity at different beta energies provided corresponding results to those obtained with the OSL dosimeters. Dose profile measurements using a 6 well plate and 14 ml PP tube showed that the geometry of the cell culture vessel has a marked influence on dose distribution with 188Re. Conclusion: A new dosimeter system was calibrated with β-emitters of different energy. It turned out as suitable for measuring dose in liquids. The dose profile measurements obtained are suitably precise to be used as a check against theoretical dose calculations.

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A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Journal of Visualized Experiments ◽

10.3791/2720 ◽

2011 ◽

Cited By ~ 108

Author(s):

Ramsey Foty

Keyword(s):

Cell Culture ◽

Hanging Drop ◽

Cell Culture Protocol ◽

Culture Protocol ◽

3D Spheroids

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Cytogenetic analysis of rat pancreatic cell cultures at early passages

Cell and Organ Transplantology ◽

10.22494/cot.v4i1.8 ◽

2016 ◽

Vol 4 (1) ◽

pp. 66-69

Author(s):

V. Kovpak ◽

O. Kovpak

Keyword(s):

Cell Culture ◽

Mitotic Index ◽

Cell Cultures ◽

Cytogenetic Analysis ◽

Pancreatic Cell ◽

Binucleated Cells ◽

Treatment Of Diabetes ◽

Cultivation In Vitro ◽

Number Of Cells

Cell culture obtained from the pancreas can serve as a source of physiologically competent substitute for primary islets of Langerhans in the treatment of diabetes. However, it is possible to obtain the required number of cells only at long-term cultivation in vitro. Therefore, it is necessary to investigate the risks of neoplastic transformation of cells in vitro before transplantation.Materials and methods. Cell culture was obtained by explant method from pancreas of 12-day-old rats. Cell cultures of the first to sixth passages were used for the cytogenetic analysis. In this study the number of cells with altered karyotype, cells with micronuclei, binucleated cells and the cells in a state of apoptosis were considered, mitotic index was calculated.Results. Aneuploid cells were noted at all passages in an amount of 2.2 % (1st) to 16.6 % (4th). Polyploidy manifested in a population of cells from the second (1.1 %) to the sixth passage (4.4 %) with a maximum at passage four (7.8 %). A significant increase in their number was observed since the second passage (0.3 %). We have seen a significant increase in the number of binucleated cells from the first (0.1 %) to the sixth passage (0.8 %). During the study there was a decrease in mitotic index from the first (2.7 %), to the third passage (1.5 %) and its gradual increase in fourth (1.7 %) and sixth (2.0 %) passages. In addition, there was discovered a small percentage of cells in apoptosis, their number gradually increased to the 4th passage (0.5 %). The 5th-6th passages showed decrease in the number of apoptotic cells to 0.1 %.Conclusion. There have been revealed changes in the rat pancreatic cells culture as aneuploidies, polyploidies and micronuclei, the intensity of which varied depending on the passage. However, karyotype variability of mentioned cell did not exceed the level of spontaneous mutations characteristic of mammalians.

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Development of an In Vitro Exposure System for Live Visualization of the Health Impacts of Oily Marine Aerosol on the Human Respiratory System

International Oil Spill Conference Proceedings ◽

10.7901/2169-3358-2017.1.000349 ◽

2017 ◽

Vol 2017 (1) ◽

pp. 2017349

Author(s):

David Murphy ◽

Nima Afshar-Mohajer ◽

Kristine Nishida ◽

Yury Ronzhes ◽

Ramana Sidhaye ◽

...

Keyword(s):

Cell Culture ◽

Cell Cultures ◽

Human Lung ◽

Major Advance ◽

Marine Aerosol ◽

Long Distance ◽

Exposure System ◽

Optical Access ◽

Cleanup Workers

Aerosolization of oily water droplets has recently been recognized as a potential respiratory health threat to oil spill cleanup workers, communities near spills, and marine mammals in oil-polluted waters. These sub-micron to millimeter scale droplets may be aerosolized by bursting bubbles, breaking waves, and splashing raindrops. Furthermore, dispersant applied to oil slicks also may become aerosolized as oil-water-dispersant emulsion droplets and subsequently inhaled, with unknown health consequences. With the goal of investigating the effects of inhaled oily marine aerosol on human lung health, we present the design of a novel in vitro bioreactor which mimics the conditions and exposures that human lungs might experience in the field. The bioreactor provides the ability to expose human lung cell cultures to laboratory-produced, well-characterized and chemically analyzed oily marine aerosols. A major advance over similar systems currently used to study the effects of smoking is the incorporation of optical access to allow visualization of the cells throughout exposure. In the bioreactor, differentiated, primary human bronchial epithelial cell cultures reside on membranes at the air-liquid interface between the flow-through test atmosphere and a temperature-controlled bath of culture media, thereby simulating the human lung. Oily marine aerosol is produced by a 1-Jet Collison Nebulizer (Mesa Labs Inc.) to match realistic concentrations produced and measured in a wave tank and is sampled via scanning mobility particle sizer (TSI Inc) to characterize its size distribution. The aerosol-laden air is humidified and injected at a controlled flow rate of ~1 ml/s into a module containing the cell culture, allowing particles to deposit on the cells. The module has sealed glass windows to allow optical access. An optical setup incorporating a 20× long-distance objective, 1× tube lens, and camera is used to visualize the cells over time. Preliminary testing involves determining the effectiveness of deposition of oily marine aerosol droplets at various concentrations onto the cell culture surface. Phase contrast microscopy is used to examine contact between cells as a determinant of monolayer integrity. Immunofluorescence of live cells is used with FITC- or mCherry-labelled cytoskeletal and cell-cell adhesion proteins, such as actin and E-cadherin, to determine underlying mechanisms disrupting the monolayer. A system such as this allowing for live visualization of cells during the exposure currently does not exist and will provide significant understanding of how changes within the epithelium may disrupt tissue integrity in response to inhalation of oily marine aerosol.

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Effects of Lapatinib on HER2-Positive and HER2-Negative Canine Mammary Carcinoma Cells Cultured In Vitro

Pharmaceutics ◽

10.3390/pharmaceutics13060897 ◽

2021 ◽

Vol 13 (6) ◽

pp. 897

Author(s):

Antonio Fernando Leis-Filho ◽

Patrícia de Faria Lainetti ◽

Priscila Emiko Kobayashi ◽

Carlos Eduardo Fonseca-Alves ◽

Renée Laufer-Amorim

Keyword(s):

Breast Cancer ◽

Cell Culture ◽

Mammary Gland ◽

Cell Cultures ◽

Kinase Inhibitor ◽

Predictive Marker ◽

Her2 Positive ◽

Her2 Breast Cancer ◽

Mammary Gland Cancer

HER2 is a prognostic and predictive marker widely used in breast cancer. Lapatinib is a tyrosine kinase inhibitor that works by blocking the phosphorylation of the receptor HER2. Its use is related to relatively good results in the treatment of women with HER2+ breast cancer. Thus, this study aimed to verify the effects of lapatinib on four canine primary mammary gland carcinoma cell cultures and two paired metastatic cell cultures. Cultures were treated with lapatinib at concentrations of 100, 500, 1000 and 3000 nM for 24 h and the 50% inhibitory concentration (IC50) for each cell culture was determined. In addition, a transwell assay was performed to assess the ability of lapatinib to inhibit cell migration. Furthermore, we verified HER2 expression by RT-qPCR analysis of cell cultures and formalin-fixed paraffin-embedded tissues from samples corresponding to those used in cell culture. Lapatinib was able to inhibit cell proliferation in all cell cultures, but it was not able to inhibit migration in all cell cultures. The higher the expression of HER2 in a culture, the more sensitive the culture was to treatment. This relationship may be an indication that the expression of HER2 may be a predictive factor and opens a new perspective for the treatment of primary and metastatic mammary gland cancer.

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