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Journal of Ophthalmology 2020To develop a method for the rapid isolation of rat RPE cells with high yield and maintain its epithelial state in modified culture system.
OBJECTIVE
To develop a method for the rapid isolation of rat RPE cells with high yield and maintain its epithelial state in modified culture system.
METHODS
The eyeballs were incubated with dispase. The retina was isolated with RPE attached and cut into several pieces. Following a brief incubation in growth medium, large RPE sheets can be harvested rapidly. RPE cells were divided into four groups and cultured for several weeks, that is, (1) in cell culture dishes with 10% FBS containing medium (CC dish-FBS), (2) in petri dishes with 10% FBS containing medium (Petri dish-FBS), (3) in cell culture dishes with N2 and B27 containing medium (CC dish-N2B27), and (4) in petri dishes with N2 and B27 containing medium (Petri dish-N2B27). Morphological and biological characteristics were investigated using light microscopy, Q-PCR, and western blot.
RESULTS
The retina would curl inwardly during the growth medium incubation period, releasing RPE sheets in the medium. Compared with low density group (5,000 cells/cm), RPE cells plated at high density (15,000 cells/cm) can maintain RPE morphology for a more extended period. Meanwhile, plating RPE cells at low density significantly reduced the expression of RPE cell type-specific genes (RPE65, CRALBP, and bestrophin) and increased the expression of EMT-related genes (N-cadherin, fibronectin, and -SMA), in comparison with the samples from the high density group. The petri dish culture condition reduced cell adhesion and thus inhibited RPE cell proliferation. As compared with other culture conditions, RPE cells in the petri dish-N2B27 condition could maintain RPE phenotype with increased expression of RPE-specific genes and decreased expression of EMT-related genes. The AKT/mTOR pathway was also decreased in petri dish-N2B27 condition.
CONCLUSION
The current study provided an alternative method for easy isolation of RPE cells with high yield and maintenance of its epithelial morphology in the petri dish-N2B27 condition.
PubMed: 32855817
DOI: 10.1155/2020/4892978 -
World Journal of Stem Cells Oct 2019Mesenchymal stem cells (MSCs) are stromal multipotent stem cells that can differentiate into multiple cell types, including fibroblasts, osteoblasts, chondrocytes,... (Review)
Review
Mesenchymal stem cells (MSCs) are stromal multipotent stem cells that can differentiate into multiple cell types, including fibroblasts, osteoblasts, chondrocytes, adipocytes, and myoblasts, thus allowing them to contribute to the regeneration of various tissues, especially bone tissue. MSCs are now considered one of the most promising cell types in the field of tissue engineering. Traditional petri dish-based culture of MSCs generate heterogeneity, which leads to inconsistent efficacy of MSC applications. Biodegradable and biocompatible polymers, poly(3-hydroxyalkanoates) (PHAs), are actively used for the manufacture of scaffolds that serve as carriers for MSC growth. The growth and differentiation of MSCs grown on PHA scaffolds depend on the physicochemical properties of the polymers, the 3D and surface microstructure of the scaffolds, and the biological activity of PHAs, which was discovered in a series of investigations. The mechanisms of the biological activity of PHAs in relation to MSCs remain insufficiently studied. We suggest that this effect on MSCs could be associated with the natural properties of bacteria-derived PHAs, especially the most widespread representative poly(3-hydroxybutyrate) (PHB). This biopolymer is present in the bacteria of mammalian microbiota, whereas endogenous poly(3-hydroxybutyrate) is found in mammalian tissues. The possible association of PHA effects on MSCs with various biological functions of poly(3-hydroxybutyrate) in bacteria and eukaryotes, including in humans, is discussed in this paper.
PubMed: 31692924
DOI: 10.4252/wjsc.v11.i10.764 -
EMBO Reports Mar 2018Interfering with mitosis for cancer treatment is an old concept that has proven highly successful in the clinics. Microtubule poisons are used to treat patients with... (Review)
Review
Interfering with mitosis for cancer treatment is an old concept that has proven highly successful in the clinics. Microtubule poisons are used to treat patients with different types of blood or solid cancer since more than 20 years, but how these drugs achieve clinical response is still unclear. Arresting cells in mitosis can promote their demise, at least in a petri dish. Yet, at the molecular level, this type of cell death is poorly defined and cancer cells often find ways to escape. The signaling pathways activated can lead to mitotic slippage, cell death, or senescence. Therefore, any attempt to unravel the mechanistic action of microtubule poisons will have to investigate aspects of cell cycle control, cell death initiation in mitosis and after slippage, at single-cell resolution. Here, we discuss possible mechanisms and signaling pathways controlling cell death in mitosis or after escape from mitotic arrest, as well as secondary consequences of mitotic errors, particularly sterile inflammation, and finally address the question how clinical efficacy of anti-mitotic drugs may come about and could be improved.
Topics: Apoptosis; Cell Cycle Checkpoints; Cell Death; Humans; Microtubules; Mitosis; Neoplasms
PubMed: 29459486
DOI: 10.15252/embr.201745440 -
Current Microbiology May 2021Conventional cultivation methods, including petri dish plating, are selective and biased to enrich specific microorganisms, such as big population and fast-growing...
Conventional cultivation methods, including petri dish plating, are selective and biased to enrich specific microorganisms, such as big population and fast-growing bacteria. In this study, we evaluated the ability of isolation chip (ichip) to reduce cultivation bias. We used the ichip and petri dish plating methods to cultivate bacteria from soil contaminated with (contaminated soil) or without (natural soil) crude oil. Ichip improved the richness and evenness of bacterial isolates in both the natural and contaminated soil samples. Using the petri dish plating method, Pseudomonas and Lysinibacillus isolates were found to be the most abundant, with over 50% of the relative abundance in the natural and oil-polluted soil-cultured communities, respectively. In comparison, using the ichip method, the isolates with the highest relative abundances were from Bacillus and Aeromonas in natural and contaminated soil-cultured communities, which only accounted for 20% and 28% of the total isolates, respectively. Interestingly, the evenness and richness of the bacteria varied slightly between the natural and oil-polluted soil samples, indicating that ichip had the ability to reduce the cultivation bias. In addition, oil selective pressure enriched the functional bacteria isolated using the petri dish plating method. In summary, ichip allows bacteria to grow evenly, as well as allowing for substance exchange between the environment and single cells. As such, it is a very good method for increasing culturable bacterial diversity and reducing cultivation bias.
Topics: Bacteria; Petroleum; Soil; Soil Microbiology; Soil Pollutants
PubMed: 33821359
DOI: 10.1007/s00284-021-02474-0 -
Scientific Reports Sep 2021Nanoneedle structures on dragonfly and cicada wing surfaces or black silicon nanoneedles demonstrate antibacterial phenomena, namely mechano-bactericidal action. These...
Nanoneedle structures on dragonfly and cicada wing surfaces or black silicon nanoneedles demonstrate antibacterial phenomena, namely mechano-bactericidal action. These air-exposed, mechano-bactericidal surfaces serve to destroy adherent bacteria, but their bactericidal action in the water is no precedent to report. Calcium carbonate easily accumulates on solid surfaces during long-term exposure to hard water. We expect that aragonite nanoneedles, in particular, which grow on TiO during the photocatalytic treatment of calcium-rich groundwater, exhibit mechano-bactericidal action against bacteria in water. Here, we showed that acicular aragonite modified on TiO ceramics prepared from calcium bicarbonate in mineral water by photocatalysis exhibits mechanical bactericidal activity against E. coli in water. Unmodified, calcite-modified and aragonite-modified TiO ceramics were exposed to water containing E. coli (in a petri dish), and their bactericidal action over time was investigated under static and agitated conditions. The surfaces of the materials were observed by scanning electron microscopy, and the live/dead bacterial cells were observed by confocal laser scanning microscopy. As a result, the synergistic bactericidal performance achieved by mechano-bactericidal action and photocatalysis was demonstrated. Aragonite itself has a high biological affinity for the human body different from the other whisker-sharpen nanomaterials, therefore, the mechano-bactericidal action of acicular aragonite in water is expected to inform the development of safe water purification systems for use in developing countries.
PubMed: 34584148
DOI: 10.1038/s41598-021-98797-w -
Advances in Applied Microbiology 2021Petri dish contaminations are commonplace and personally witnessed by every microbiologist. The vast majority of such contaminations result in nothing more than...
Petri dish contaminations are commonplace and personally witnessed by every microbiologist. The vast majority of such contaminations result in nothing more than annoyance following which the Petri dishes are discarded. However, a handful of incidents of contaminations have led to momentous outcomes, the most renowned of which being that perceived by Alexander Fleming on the basis of the immense number of lives saved by penicillin. Petri dish contaminations as reported upon in the literature fall broadly into two categories; those in which the contaminant caused antagonism toward the species being cultured, and those in which the contaminant was established to be a species novum. Accounts of both of these categories of contaminations are set out here.
Topics: Culture Media
PubMed: 34353504
DOI: 10.1016/bs.aambs.2021.04.002 -
Analytical Chemistry Jun 2018In the last few decades, new types of cell cultures have been introduced to provide better cell survival and development, with micro- and nanoenvironmental...
In the last few decades, new types of cell cultures have been introduced to provide better cell survival and development, with micro- and nanoenvironmental physicochemical conditions aimed at mimicking those present in vivo. However, despite the efforts made, the systems available to date are often difficult to replicate and use. Here, an easy-to-use surface-acoustic-wave (SAW)-based platform is presented for realizing dynamic cell cultures that is compatible with standard optical microscopes, incubators, and cell-culture dishes. The SAW chip is coupled to a standard Petri dish via a polydimethylsiloxane (PDMS) disc and consists of a lithium niobate (LN) substrate on which gold interdigital transducers (IDTs) are patterned to generate the SAWs and induce acoustic streaming in the dish. SAW excitation is verified and characterized by laser Doppler vibrometry, and the fluid dynamics is studied by microparticle image velocimetry (μPIV). Heating is measured by an infrared (IR) thermal camera. We finally tested this device with the U-937 monocyte cell line for viability and proliferation and cell-morphological analysis. The data demonstrate that it is possible to induce significant fluid recirculation within the Petri dish while maintaining negligible heating. Remarkably, cell proliferation in this condition was enhanced by 36 ± 12% with respect to those of standard static cultures. Finally, we show that cell death does not increase and that cell morphology is not altered in the presence of SAWs. This device is the first demonstration that SAW-induced streaming can mechanically improve cell proliferation and further supports the great versatility and biocompatibility of the SAW technology for cell manipulation.
Topics: Biotechnology; Cell Culture Techniques; Cell Proliferation; Hot Temperature; Humans; Hydrodynamics; Sound; U937 Cells
PubMed: 29791795
DOI: 10.1021/acs.analchem.8b00972 -
Biophysics Reviews Mar 2022With a kind of magnetism, the human retina draws the eye of neuroscientist and physicist alike. It is attractive as a self-organizing system, which forms as a part of... (Review)
Review
With a kind of magnetism, the human retina draws the eye of neuroscientist and physicist alike. It is attractive as a self-organizing system, which forms as a part of the central nervous system via biochemical and mechanical cues. The retina is also intriguing as an electro-optical device, converting photons into voltages to perform on-the-fly filtering before the signals are sent to our brain. Here, we consider how the advent of stem cell derived analogs of the retina, termed retina organoids, opens up an exploration of the interplay between optics, electrics, and mechanics in a complex neuronal network, all in a Petri dish. This review presents state-of-the-art retina organoid protocols by emphasizing links to the biochemical and mechanical signals of retinogenesis. Electrophysiological recording of active signal processing becomes possible as retina organoids generate light sensitive and synaptically connected photoreceptors. Experimental biophysical tools provide data to steer the development of mathematical models operating at different levels of coarse-graining. In concert, they provide a means to study how mechanical factors guide retina self-assembly. In turn, this understanding informs the engineering of mechanical signals required to tailor the growth of neuronal network morphology. Tackling the complex developmental and computational processes in the retina requires an interdisciplinary endeavor combining experiment and theory, physics, and biology. The reward is enticing: in the next few years, retina organoids could offer a glimpse inside the machinery of simultaneous cellular self-assembly and signal processing, all in an setting.
PubMed: 38505227
DOI: 10.1063/5.0077014 -
PloS One 2022The purpose of this study was to investigate the abundance and distribution of psychrophilic microorganisms associated with spoilage in beef slaughterhouse environments...
The purpose of this study was to investigate the abundance and distribution of psychrophilic microorganisms associated with spoilage in beef slaughterhouse environments after cleaning. The processing lines and equipment used in slaughtering and boning were swabbed, and the microbial count was determined using a TSA and MRS medium and Chromocult® Coliform agar incubated at 15ºC and 37ºC, respectively. As a result, the brisket saw (handle side) and trolley hook were the most heavily contaminated with microorganisms, with each having a microbial adhesion rate of 66.7%. The microbial adhesion rates of the apron and milling cutter (edge side) were 50%, respectively, and those of the foot cutter (edge and handle side), splitting saw (edge side), and knife (handle side) were 33.3%, respectively. Next, four colonies were randomly isolated from the petri dish used for the bacterial count measurement to identify the predominant microbial species of the microorganisms attached to each equipment. As a result of Sanger sequencing analysis, yeasts such as Candida zeylanoides and Rhodotorula sp. and bacteria including Pseudomonas sp. and Rhodococcus sp. were identified from the equipment used in the slaughtering line, and it was assumed that these microorganisms were of environmental origin. In contrast, only Pseudomonas sp. and Candida zeylanoides were isolated from the boning line. Despite the use of cleaning operations, this study identified some equipment was contaminated with microorganisms. Since this equipment frequently comes into direct contact with the carcass, it is critical to thoroughly remove the microorganisms through accurate cleaning to prevent the spread of microbial contamination on the carcasses.
Topics: Abattoirs; Animals; Cattle; Colony Count, Microbial; Food Handling; Food Microbiology; Japan; Meat; Saccharomycetales; Yeasts
PubMed: 35921278
DOI: 10.1371/journal.pone.0268411 -
Frontiers in Microbiology 2019The new era of multidrug resistance of pathogens against frontline antibiotics has compromised the immense therapeutic gains of the 'golden age,' stimulating a... (Review)
Review
The new era of multidrug resistance of pathogens against frontline antibiotics has compromised the immense therapeutic gains of the 'golden age,' stimulating a resurgence in antimicrobial research focused on antimicrobial and immunomodulatory components of botanical, fungal or microbial origin. While much valuable information has been amassed on the potency of crude extracts and, indeed, purified compounds there are too many reports that uncritically extrapolate observed activity to presumed ingestive and/or topical therapeutic value, particularly in the discipline of ethnopharmacology. Thus, natural product researchers would benefit from a basic pharmacokinetic and pharmacodynamic understanding. Furthermore, therapeutic success of complex mixtures or single components derived therefrom is not always proportionate to their MIC values, since immunomodulation can be the dominant mechanism of action. Researchers often fail to acknowledge this, particularly when 'null' activity is observed. In this review we introduce the most up to date theories of oral and topical bioavailability including the metabolic processes affecting xenobiotic biotransformation before and after drugs reach the site of their action in the body. We briefly examine the common methodologies employed in antimicrobial, immunomodulatory and pharmacokinetic research. Importantly, we emphasize the contribution of synergies and/or antagonisms in complex mixtures as they affect absorptive processes in the body and sometimes potentiate activity. Strictly in the context of natural product research, it is important to acknowledge the potential for chemotypic variation within important medicinal plants. Furthermore, polar head space and rotatable bonds give indications of the likelihood of bioavailability of active metabolites. Considering this and other relatively simple chemical insights, we hope to provide the basis for a more rigorous scientific assessment, enabling researchers to predict the likelihood that observed anti-infective activity will translate to outcomes in a therapeutic context. We give worked examples of tentative pharmacokinetic assessment of some well-known medicinal plants.
PubMed: 31736910
DOI: 10.3389/fmicb.2019.02470