Long-term videomicroscopy of living cells in vitro: Opportunities and prospects

Aim. Intravital video microscopy of cells is a highly informative approach to the study of cell cultures. Often, this method allows refining and complementing the data obtained by researchers at the visual study of living cultures or fixed preparations. The main problem of the long intravital video microscopy is the maintenance of cell activity. To solve this problem, video-computer "Tsitomir" has been developed. Methods. During cultivation the images of the cell culture areas (from one to several hundred) specified by researcher are captured at regular intervals (time-lapse method of photography). A motorized sample stage allows moving the culture vessel with the joystick, as well as to scan the specified cell culture sites automatically. Results. In our investigations, we studied such processes as cell division, death, differentiation, motility and massive changes of cell cultures associated with cancerous transformation, including abnormal morphological changes and cell aggregation. The effectiveness of the intravital cell microscopy use to test the anti-cancer drugs is shown as well. Conclusions. Opportunities of video-complex enable its use in biomedical research, in the development of cell technologies, the study of the action of pharmacological agents and sanitary-hygienic regulation of chemicals in the cell assay systems. Obtained through "Tsitomir" photos and videos can also be used as educational material for students of biological, medical and agricultural universities.Keywords: cell culture, intravital videomicroscopy, differentiation, proliferation, anti-cancer protection.

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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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Detection of Cell Aggregation and Altered Cell Viability by Automated Label-Free Video Microscopy

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057114562158 ◽

2014 ◽

Vol 20 (3) ◽

pp. 372-381 ◽

Cited By ~ 4

Author(s):

Obaid Aftab ◽

Mårten Fryknäs ◽

Ulf Hammerling ◽

Rolf Larsson ◽

Mats G. Gustafsson

Keyword(s):

Cell Viability ◽

Phase Contrast ◽

Growth Factor Receptor ◽

Cell Aggregation ◽

Well Being ◽

Time Lapse ◽

Video Microscopy ◽

Label Free ◽

Monitoring Method

Automated phase-contrast video microscopy now makes it feasible to monitor a high-throughput (HT) screening experiment in a 384-well microtiter plate format by collecting one time-lapse video per well. Being a very cost-effective and label-free monitoring method, its potential as an alternative to cell viability assays was evaluated. Three simple morphology feature extraction and comparison algorithms were developed and implemented for analysis of differentially time-evolving morphologies (DTEMs) monitored in phase-contrast microscopy videos. The most promising layout, pixel histogram hierarchy comparison (PHHC), was able to detect several compounds that did not induce any significant change in cell viability, but made the cell population appear as spheroidal cell aggregates. According to recent reports, all these compounds seem to be involved in inhibition of platelet-derived growth factor receptor (PDGFR) signaling. Thus, automated quantification of DTEM (AQDTEM) holds strong promise as an alternative or complement to viability assays in HT in vitro screening of chemical compounds.

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Obtaining and Characterization of Alhagi persarum Boiss. et Buhse Callus Cell Cultures, Isoflavonoid Producers

Biotekhnologiya ◽

10.21519/0234-2758-2020-36-6-35-48 ◽

2020 ◽

Vol 36 (6) ◽

pp. 35-48

Author(s):

D.V. Коchkin ◽

G.I. Sobolkovа ◽

А.А. Fоmеnkov ◽

R.А. Sidorov ◽

А.М. Nоsоv

Keyword(s):

Fatty Acids ◽

Cell Culture ◽

Liquid Chromatography ◽

Secondary Metabolites ◽

Cell Cultures ◽

Mass Spectrometric ◽

Mass Spectrometric Detection ◽

Gas Liquid Chromatography ◽

Callus Cell

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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Chitosan Elicitation for Enhancing of Vincristine and Vinblastine Accumulation in Cell Culture of Catharanthus roseus (L.) G. Don

Journal of Agricultural Science ◽

10.5539/jas.v10n12p287 ◽

2018 ◽

Vol 10 (12) ◽

pp. 287

Author(s):

Pawnpirun Pliankong ◽

Padungsak Suksa-Ard ◽

Surawit Wannakrairoj

Keyword(s):

Molecular Weight ◽

Cell Culture ◽

Stationary Phase ◽

Liquid Medium ◽

Cell Cultures ◽

Catharanthus Roseus ◽

Dry Weight ◽

Shrimp Shell ◽

Anti Cancer ◽

Herbal Plant

Catharanthus roseus (L.) G. Don is an important herbal plant. There are two important alkaloids, vinblastine and vincristine, use in anti-cancer drugs. In this study production of the two alkaloids was enhanced in C. roseus cell cultures, in a Murashige and Skoog (MS) liquid medium supplemented with 1.5 mg/L 2,4-D, 0.5 mg/L kinetin and 30 g/L sucrose, by adding 0, 50, 100, 250 or 500 mg/L medium molecular weight chitosan or chitosan derived from shrimp shell. After 14 days of culture, the cell suspension at stationary phase in the 100 mg/L medium molecular weight chitosan could produce the highest amounts of vinblastine and vincristine at 4.15 and 5.48 µg/mg cell dry weight, respectively. At the same time, the controls (0 mg/L chitosan) produced the two alkaloids at only 2.43 and 2.49 µg/mg cell dry weight, respectively. For chitosan from shrimp shell, it was found that 100 mg/L chitosan could lead to the highest quantity of 4.09 µg vinblastine/mg cell dry weight. The highest amount of 5.47 µg vincristine/mg cell dry weight was obtained when 250 mg/L chitosan was added.

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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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Inhibition of Nanobacteria by Antimicrobial Drugs as Measured by a Modified Microdilution Method

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.46.7.2077-2086.2002 ◽

2002 ◽

Vol 46 (7) ◽

pp. 2077-2086 ◽

Cited By ~ 48

Author(s):

N. Çíftçíoglu ◽

M. A. Miller-Hjelle ◽

J. T. Hjelle ◽

E. O. Kajander

Keyword(s):

Cell Culture ◽

Kidney Stones ◽

Morphological Changes ◽

Human Kidney ◽

Test System ◽

Antimicrobial Drugs ◽

Microdilution Method ◽

Kidney Cyst ◽

Cyst Fluids

ABSTRACT Compounds from 16 classes of antimicrobial drugs were tested for their abilities to inhibit the in vitro multiplication of nanobacteria (NB), a newly discovered infectious agent found in human kidney stones and kidney cyst fluids from patients with polycystic kidney disease (PKD). Because NB form surface calcifications at physiologic levels of calcium and phosphate, they have been hypothesized to mediate the formation of tissue calcifications. We describe a modified microdilution inhibitory test that accommodates the unique growth conditions and long multiplication times of NB. This modified microdilution method included inoculation of 96-well plates and determination of inhibition by periodic measurement of the absorbance for 14 days in cell culture medium under cell culture conditions. Bactericidal or bacteriostatic drug effects were distinguished by subsequent subculture in drug-free media and monitoring for increasing absorbance. NB isolated from fetal bovine serum (FBS) were inhibited by tetracycline HCl, nitrofurantoin, trimethoprim, trimethoprim-sulfamethoxazole, and ampicillin at levels achievable in serum and urine; all drugs except ampicillin were cidal. Tetracycline also inhibited multiplication of isolates of NB from human kidney stones and kidney cyst fluids from patients with PKD. The other antibiotics tested against FBS-derived NB either had no effect or exhibited an inhibitory concentration above clinically achievable levels; the aminoglycosides and vancomycin were bacteriostatic. Antibiotic-induced morphological changes to NB were observed by electron microscopy. Bisphosphonates, aminocaproic acid, potassium citrate-citric acid solutions, and 5-fluorouracil also inhibited the multiplication of NB in a cidal manner. Insights into the nature of NB, the action(s) of these drugs, and the role of NB in calcifying diseases may be gained by exploiting this in vitro inhibition test system.

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Proliferation and Migration of Human Osteoblasts on Porous Three Dimensional Scaffolds

Materials Science Forum ◽

10.4028/www.scientific.net/msf.638-642.506 ◽

2010 ◽

Vol 638-642 ◽

pp. 506-511 ◽

Cited By ~ 5

Author(s):

Claudia Bergemann ◽

Ernst Dieter Klinkenberg ◽

Frank Lüthen ◽

Arne Weidmann ◽

Regina Lange ◽

...

Keyword(s):

Cell Culture ◽

Morphological Changes ◽

Three Dimensional ◽

3D Cell Culture ◽

Porous Tantalum ◽

Human Osteoblasts ◽

A Cell ◽

And Migration ◽

Migration Of Cells

Porous tantalum (Ta) biomaterial is designed to function as a scaffold for osseous ingrowths and has found applications in orthopedics. Integration of this Ta foam into the neighboring bone requires that osteoprogenitor cells attach to the implant, grow into the scaffold, proliferate and differentiate to osteoblasts. The aim of the present study was to create an in vitro 3D model system to investigate the interaction of human osteoblasts with porous Ta in the depth of the corpus. To explore active migration of osteoblasts into the Ta scaffold two porous Ta discs (Zimmer, Poland) were horizontally fixed within a clamping ring. Thereby a 3D Ta module with 4 levels is generated, which is placed into a cell culture well with the appropriate medium. Osteoblast-like cells were seeded apical onto the Ta module and cultured for 7 days in humidified atmosphere. Active migration of cells into the scaffold was monitored by field emission scanning electron microscopy (FESEM) imaging of the apical, medial and basal layers. A problem in 3D cell culture is the nutrition of cells inside of the scaffold. Therefore morphological changes and differentiation of the cells in distinct layers were analyzed.

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