scholarly journals Cell culture-based shark karyotyping as a resource for chromosome-scale genome analysis

2020 ◽  
Author(s):  
Yoshinobu Uno ◽  
Ryo Nozu ◽  
Itsuki Kiyatake ◽  
Nobuyuki Higashiguchi ◽  
Shuji Sodeyama ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cultured Cells ◽  
Culture Conditions ◽  
Technical Solution ◽  
Shark Species ◽  
Bamboo Shark ◽  
Genome Assemblies ◽  
Osmoregulatory Mechanism

AbstractKaryotyping is indispensable for validating genome assemblies whose sequence lengths can be scaled up to chromosome sizes using modern methods and is traditionally performed using cytogenetic techniques. Karyotype reports of chondrichthyans are scarce, mainly because of their unique osmoregulatory mechanism, which hinders cell culture. Here, we focused on carpet shark species and the culture conditions for fibroblasts and lymphocytes. Using this method, we performed high-fidelity characterization of their karyotypes, namely 2n = 102 for the whale shark (Rhincodon typus) and zebra shark (Stegostoma fasciatum), and 2n = 106 for the brownbanded bamboo shark (Chiloscyllium punctatum) and whitespotted bamboo shark (C. plagiosum). We identified heteromorphic XX/XY sex chromosomes for the two latter species and demonstrated the first-ever fluorescence in situ hybridization of shark chromosomes prepared from cultured cells. Our technical solution is applicable to diverse chondrichthyan species and will deepen the understanding of early vertebrate evolution at the molecular level.

scholarly journals Cell culture-based karyotyping of orectolobiform sharks for chromosome-scale genome analysis

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Yoshinobu Uno ◽  
Ryo Nozu ◽  
Itsuki Kiyatake ◽  
Nobuyuki Higashiguchi ◽  
Shuji Sodeyama ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cultured Cells ◽  
Molecular Level ◽  
Culture Conditions ◽  
Shark Species ◽  
Rhincodon Typus ◽  
Bamboo Shark ◽  
Genome Assemblies

Abstract Karyotyping, traditionally performed using cytogenetic techniques, is indispensable for validating genome assemblies whose sequence lengths can be scaled up to chromosome sizes using modern methods. Karyotype reports of chondrichthyans are scarce because of the difficulty in cell culture. Here, we focused on carpet shark species and the culture conditions for fibroblasts and lymphocytes. The utility of the cultured cells enabled the high-fidelity characterization of their karyotypes, namely 2n = 102 for the whale shark (Rhincodon typus) and zebra shark (Stegostoma fasciatum), and 2n = 106 for the brownbanded bamboo shark (Chiloscyllium punctatum) and whitespotted bamboo shark (C. plagiosum). We identified heteromorphic XX/XY sex chromosomes for the two latter species and demonstrated the first-ever fluorescence in situ hybridization of shark chromosomes prepared from cultured cells. Our protocols are applicable to diverse chondrichthyan species and will deepen the understanding of early vertebrate evolution at the molecular level.

A Microfluidic Sensing System with a Multichannel Surface Plasmon Resonance Chip: Damage-free Characterization of Cells by Pattern Recognition

2020 ◽  
Author(s):  
Hiroka Sugai ◽  
Shunsuke Tomita ◽  
Sayaka Ishihara ◽  
Kyoko Yoshioka ◽  
Ryoji Kurita
Keyword(s):  
Cell Culture ◽  
Pattern Recognition ◽  
Surface Plasmon Resonance ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Cultured Cells ◽  
Culture Media ◽  
Biological Research ◽  
Cell Culture Media

<p>The development of a versatile sensing strategy for the damage-free characterization of cultured cells is of great importance for both fundamental biological research and industrial applications. Here, we present a pattern-recognition-based cell-sensing approach using a multichannel surface plasmon resonance (SPR) chip. The chip, in which five cysteine derivatives with different structures are immobilized on Au films, is capable of generating five unique SPR sensorgrams for the cell-secreted molecules that are contained in cell culture media. An automatic statistical program was built to acquire kinetic parameters from the SPR sensorgrams and to select optimal parameters as “pattern information” for subsequent multivariate analysis. Our system rapidly (~ 10 min) provides the complex information by merely depositing a small amount of cell culture media (~ 25 µL) onto the chip, and the amount of information obtained is comparable to that furnished by a combination of conventional laborious biochemical assays. This non-invasive pattern-recognition-based cell-sensing approach could potentially be employed as a versatile tool for characterizing cells. </p>

Enhancing cell culture in magnetic vesicle gels

2010 ◽  
Vol 1272 ◽  
Author(s):  
Felicity Leng ◽  
Julie E Gough ◽  
Simon J Webb
Keyword(s):  
Cell Culture ◽  
Magnetic Nanoparticles ◽  
Cultured Cells ◽  
Culture Conditions ◽  
Chondrocyte Viability ◽  
Smart Biomaterials ◽  
Cell Culture Conditions

AbstractSeveral different hydrogel compositions have been incorporated into magnetic vesicle gels and the resulting “smart” biomaterials assessed as cell culture scaffolds. The compatibility of these hydrogels with the “smart” component of these biomaterials, thermally sensitive vesicles (TSVs) crosslinked by magnetic nanoparticles, was assessed by the leakage of fluorescent 5/6-carboxyfluorescein from the TSVs under cell culture conditions. Subsequently the ability of the hydrogels to support 3T3 fibroblast and chondrocyte viability was assessed. These studies revealed that alginate-based gels were the most compatible with both the TSVs and the cultured cells, with an alginate:fibronectin mix proving to be the most versatile. Nonetheless these studies also suggest that TSV composition needs to be modified to improve the performance of these “smart” cell culture scaffolds in future applications.

An embryonic pineal body as a multipotent system in cell differentiation

Development ◽  
1988 ◽  
Vol 103 (1) ◽  
pp. 17-26
Author(s):  
K. Watanabe ◽  
H. Aoyama ◽  
N. Tamamaki ◽  
T. Sonomura ◽  
T.S. Okada ◽  
...  
Keyword(s):  
Cell Culture ◽  
Striated Muscle ◽  
Neural Retina ◽  
Culture Conditions ◽  
Muscle Fibres ◽  
Muscle Type ◽  
Mesodermal Origin ◽  
Skeletal Muscle Fibres ◽  
Cells Cultured

The differentiating potency of pineal cells from 8-day quail embryos was studied with cell culture. It was found that the differentiation of striated muscle fibres occurred abundantly in the pineal cells cultured in hypertonic culture conditions. Muscle nature of these fibres was confirmed by utilizing the antiserum against the striated muscle type creatine kinase (MM-CK). When CO2, NAHCO3, NaCl, KCl and MgCl2 were added in hypertonic concentrations, extensive myogenesis occurred in cultured pineal cells. Myogenesis in pineal cultures began as early as 2 days and, after 3 days in the medium with 75 mM additional NaCl, reached 100-fold when compared with that in the isotonic medium. Muscle fibres from pineal cells in culture were similar in morphology to the skeletal muscle fibres of mesodermal origin in situ. Myogenesis of pineal cells under hypertonic conditions was accompanied by the synthesis of a unique 56 × 10(3) Mr protein, which was not found in the intrinsic muscle cells. Clonal cell culture revealed that about 80% of clonable pineal cells were myogenic precursors. Pineal cells of 8-day quail embryos were not only myogenic but oculopotent (melanogenic and lentoidogenic) in cultures. This study examined whether multipotential progenitor cells with both potentials are present in the pineal or not. The results showed that at least 16% of all clonable pineal cells were multipotent precursors. The embryonic pineal is considered to be a typical multipotent system in parallel with the pigmented and neural retina, the neural crest and the teratocarcinoma.

A Microfluidic Sensing System with a Multichannel Surface Plasmon Resonance Chip: Damage-free Characterization of Cells by Pattern Recognition

2020 ◽  
Author(s):  
Hiroka Sugai ◽  
Shunsuke Tomita ◽  
Sayaka Ishihara ◽  
Kyoko Yoshioka ◽  
Ryoji Kurita
Keyword(s):  
Cell Culture ◽  
Pattern Recognition ◽  
Surface Plasmon Resonance ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Cultured Cells ◽  
Culture Media ◽  
Biological Research ◽  
Cell Culture Media

<p>The development of a versatile sensing strategy for the damage-free characterization of cultured cells is of great importance for both fundamental biological research and industrial applications. Here, we present a pattern-recognition-based cell-sensing approach using a multichannel surface plasmon resonance (SPR) chip. The chip, in which five cysteine derivatives with different structures are immobilized on Au films, is capable of generating five unique SPR sensorgrams for the cell-secreted molecules that are contained in cell culture media. An automatic statistical program was built to acquire kinetic parameters from the SPR sensorgrams and to select optimal parameters as “pattern information” for subsequent multivariate analysis. Our system rapidly (~ 10 min) provides the complex information by merely depositing a small amount of cell culture media (~ 25 µL) onto the chip, and the amount of information obtained is comparable to that furnished by a combination of conventional laborious biochemical assays. This non-invasive pattern-recognition-based cell-sensing approach could potentially be employed as a versatile tool for characterizing cells. </p>

In Situ and In Vitro Identification and Characterization of Cardiac Ganglion Neurons in the Crab, Carcinus maenas

1999 ◽  
Vol 81 (6) ◽  
pp. 2964-2976 ◽  
Author(s):  
Michelle A. Saver ◽  
Jerrel L. Wilkens ◽  
Naweed I. Syed
Keyword(s):  
Cell Culture ◽  
Carcinus Maenas ◽  
Membrane Properties ◽  
Cardiac Ganglion ◽  
Shore Crabs ◽  
Ganglion Neurons ◽  
Identification And Characterization

In situ and in vitro identification and characterization of cardiac ganglion neurons in the crab, Carcinus maenas. The aim of this study was to investigate the intrinsic membrane properties and hormonal responses of individual central pattern generating neurons in the cardiac ganglion of the shore crab Carcinus maenas. Because the cardiac ganglion in this crustacean species is buried within the heart musculature and is therefore inaccessible for direct morphological and electrophysiological analysis, we developed two novel in vitro preparations. First, to make the ganglion accessible, we established a brief enzymatic treatment procedure that enabled us to isolate the entire cardiac ganglion, in the absence of muscle tissue. Second, a cell culture procedure was developed to isolate individual neurons in vitro. With the use of both isolated ganglionic and neuronal cell culture techniques, this study provides the first direct account of the neuroanatomy of the cardiac ganglion in shore crabs. We demonstrate that cultured neurons not only survived the isolation procedures, but that they also maintained their intrinsic membrane and transmitter response properties, similar to those seen in the intact ganglion. Specifically, we tested the peptides proctolin, crustacean cardioactive peptide, the FLRFamide-related peptide F2, and an amine (serotonin) on both isolated ganglion and in vitro culture neurons. We measured changes in neuronal burst rate, burst amplitude, pacemaker slope, and membrane potential oscillation amplitude in response to the above four hormones. Each hormone either increased neuronal activity in spontaneously bursting neurons, or induced a bursting pattern in quiescent cells. The in vitro cell culture system developed here now provides us with an excellent opportunity to elucidate cellular, synaptic and hormonal mechanisms by which cardiac activity is generated in shore crabs.

scholarly journals Degradation of Extracellular NAD+ Intermediates in Cultures of Human HEK293 Cells

Metabolites ◽  
2019 ◽  
Vol 9 (12) ◽  
pp. 293 ◽  
Author(s):  
Veronika Kulikova ◽  
Konstantin Shabalin ◽  
Kirill Nerinovski ◽  
Alexander Yakimov ◽  
Maria Svetlova ◽  
...  
Keyword(s):  
Cell Culture ◽  
Nicotinic Acid ◽  
Culture Medium ◽  
Cultured Cells ◽  
Culture Conditions ◽  
Hek293 Cells ◽  
Vitamin B3 ◽  
Serum Free ◽  
Nicotinamide Riboside ◽  
Nicotinamide Mononucleotide

Nicotinamide adenine dinucleotide (NAD) is an essential redox carrier, whereas its degradation is a key element of important signaling pathways. Human cells replenish their NAD contents through NAD biosynthesis from extracellular precursors. These precursors encompass bases nicotinamide (Nam) and nicotinic acid and their corresponding nucleosides nicotinamide riboside (NR) and nicotinic acid riboside (NAR), now collectively referred to as vitamin B3. In addition, extracellular NAD+ and nicotinamide mononucleotide (NMN), and potentially their deamidated counterparts, nicotinic acid adenine dinucleotide (NAAD) and nicotinic acid mononucleotide (NAMN), may serve as precursors of intracellular NAD. However, it is still debated whether nucleotides enter cells directly or whether they are converted to nucleosides and bases prior to uptake into cells. Here, we studied the metabolism of extracellular NAD+ and its derivatives in human HEK293 cells using normal and serum-free culture medium. Using medium containing 10% fetal bovine serum (FBS), mono- and dinucleotides were degraded to the corresponding nucleosides. In turn, the nucleosides were cleaved to their corresponding bases. Degradation was also observed in culture medium alone, in the absence of cells, indicating that FBS contains enzymatic activities which degrade NAD+ intermediates. Surprisingly, NR was also rather efficiently hydrolyzed to Nam in the absence of FBS. When cultivated in serum-free medium, HEK293 cells efficiently cleaved NAD+ and NAAD to NMN and NAMN. NMN exhibited rather high stability in cell culture, but was partially metabolized to NR. Using pharmacological inhibitors of plasma membrane transporters, we also showed that extracellular cleavage of NAD+ and NMN to NR is a prerequisite for using these nucleotides to maintain intracellular NAD contents. We also present evidence that, besides spontaneous hydrolysis, NR is intensively metabolized in cell culture by intracellular conversion to Nam. Our results demonstrate that both the cultured cells and the culture medium mediate a rather active conversion of NAD+ intermediates. Consequently, in studies of precursor supplementation and uptake, the culture conditions need to be carefully defined.

scholarly journals A simple method for the preparation of cell cultures for ultrastructural investigation.

1981 ◽  
Vol 29 (1) ◽  
pp. 84-86 ◽  
Author(s):  
H Kuhn
Keyword(s):  
Cell Culture ◽  
Cell Cultures ◽  
Cultured Cells ◽  
Microscopical Level ◽  
Simple Method ◽  
Electron Microscopical Level ◽  
Cell Groups ◽  
Electron Microscopical ◽  
Culturing Conditions

A method is described that allows investigation of cultured cells at the light or electron microscopical level without changing the culturing conditions or the in situ situation during dehydration or embedding. Fixed and dehydrated cell culture layers were cut into small pieces and separated from the plastic dish by adding propylene oxide. With this simple method we could obtain orientated sections of selected cell groups.

Sign in / Sign up

Export Citation Format

Share Document