Tissue Culture Preparations Research Articles

DETERMINATION OF NUTRITIONAL VALUE OF PLEUROTUS OSTREATUS JACQ. EXFR. P.KUMN (OYSTER MUSHROOM) CULTIVATED ON SOME PLANT SUBSTRATES

Oyster mushroom plays a vital role in human nutrition as it supplies the necessary growth factors such as protein, minerals, vitamins in the body. Fresh samples of Oyster mushroom (Pleurotus ostreatus) were collected from the agricultural research farm of Ibrahim Badamasi Babagida. University Lapai, Niger State, Nigeria located at latitude 9.0674oN, longitude 6.5698o E. The aim of this study is to determine the nutritional value of Oyster Mushroom cultivated on some plant substrates. Sawdust, Maize (Zea mays) husk Rice (Oryza sativa) straw and Banana (Musa acuminata) leaves were collected from the Botanical garden of the Department of Biological Sciences, Ibrahim Badamasi Babagida. University Lapai, Niger State. Tissue culture techniques, spawn production, preparation and pasteurization of the plant substrates, inoculation of the mushroom spawn were conducted on the mushroom, nutritional analysis of the cultivated mushroom were determined and conducted using standard methods. The results indicated that among the selected substrates used, mushroom cultivated on banana leaves was found to be high in ash and moisture contents with 7.00% and 16.00 % respectively, rice straw was found the to be high in carbohydrate content with 51.70%. Maize husk was found to be high in crude fat and protein contents with 28.60% and 7.31% respectively, crude fibre content of 7.69% was found to be high in sawdust when compared with the other selected substrates .The present work demonstrated almost all the substrates used have certain levels of nutritive values. Mushroom cultivation under different plant substrates which serve as a bio-remedy of plant waste...

Quality criteria for in vitro human pluripotent stem cell-derived models of tissue-based cells

The advent of the technology to isolate or generate human pluripotent stem cells provided the potential to develop a wide range of human models that could enhance understanding of mechanisms underlying human development and disease. These systems are now beginning to mature and provide the basis for the development of in vitro assays suitable to understand the biological processes involved in the multi-organ systems of the human body, and will improve strategies for diagnosis, prevention, therapies and precision medicine. Induced pluripotent stem cell lines are prone to phenotypic and genotypic changes and donor/clone dependent variability, which means that it is important to identify the most appropriate characterization markers and quality control measures when sourcing new cell lines and assessing differentiated cell and tissue culture preparations for experimental work. This paper considers those core quality control measures for human pluripotent stem cell lines and evaluates the state of play in the development of key functional markers for their differentiated cell derivatives to promote assurance of reproducibility of scientific data derived from pluripotent stem cell-based systems.

Cooperation between the department of ophthalmology at the University of Düsseldorf (Germany) and the Fundación Visión in Asunción (Paraguay) as an example of the German hospital partnerships programm

HintergrundSeit 2014 besteht auf der Basis eines Memorandum of Understanding eine Partnerschaft zwischen der Universitätsaugenklinik Düsseldorf (UAK Düsseldorf) und der Fundación Visión (FV) in Asunción, die eine führende Rolle in der augenärztlichen Behandlung sowie der Prävention von Erblindung in Paraguay einnimmt. Seit 2016 existiert ein Programm zur Förderung internationaler Klinikpartnerschaften der Bundesregierung.Material und MethodenDie Klinikpartnerschaft Düsseldorf–Asunción wird nach schriftlichem Antrag im Jahr 2016 durch das Ministerium für wirtschaftliche Zusammenarbeit gefördert. Im Rahmen der Projektlaufzeit sollen mithilfe mehrerer Aktivitäten in einem definierten Zeitplan moderne, minimal-invasive Hornhauttransplantationstechniken, die eine schnellere postoperative Rehabilitation und weniger Nachsorge erfordern, in Paraguay etabliert werden. Zusätzlich sollen erste Prävalenzdaten von Augenerkrankungen erhoben und die Basis für moderne Hornhautbanktechniken geschaffen werden.ErgebnisseZunächst erfolgte die Ausbildung eines paraguayischen Operateurs in der Durchführung von DMEK-Transplantationen in Deutschland. Zeitgleich wurden die instrumentellen Voraussetzungen für die Etablierung der DMEK-Technik in der FV geschaffen. Im September 2018 wurden unter Supervision eines erfahrenen OP-Teams aus Düsseldorf die ersten lamellären Hornhauttransplantationen durch das Team der FV erfolgreich in Paraguay durchgeführt. In der Folge wurden in Asunción auch die Verwendung einer Spenderhornhaut für je ein Transplantat für eine hintere und eine vordere lamelläre Keratoplastik (DMEK und DALK) umgesetzt. Im November 2019 erfolgte eine epidemiologische Datenerhebung zu Augenerkrankungen im Rahmen einer Feldkampagne der Fundación in einer ländlichen Region Paraguays. In deren Zug erlernte der datenerhebende Mitarbeiter der Universitätsaugenklinik Düsseldorf die Technik der manuellen Kleinschnittkataraktchirurgie. Im März 2019 wurde ein Mitarbeiter der FV in Düsseldorf in den Techniken der Hornhautgewebekultur und der Herstellung von Amnionmembrantransplantaten in der Lions-Hornhautbank NRW geschult.SchlussfolgerungMithilfe einer geförderten Klinikpartnerschaft können moderne minimal-invasive Hornhauttransplantationstechnik erfolgreich in die Augenklinik eines Schwellenlandes transferiert und im Gegenzug chirurgische Fertigkeiten für in Ausbildung befindliche Operateure aus Deutschland vermittelt werden. Damit stellt eine effiziente Klinikpartnerschaft die Grundlage für einen wechselseitigen Austausch dar und ist keine reine Einbahnstraße.

Viral Packaging and Cell Culture for CRISPR-Based Screens.

This protocol describes how to perform the tissue culture and high-throughput sequencing library preparation for a CRISPR-based screen. First, pantropic lentivirus is prepared from a single guide RNA (sgRNA) plasmid pool and applied to the target cells. Following antibiotic selection and a harvest of the initial population, cells are then cultured under the desired screening condition(s) for 14 population doublings. The sgRNA barcode sequences integrated in the genomic DNA of each cell population are amplified and subject to high-throughput sequencing. Guidelines for downstream analysis of the sequencing data are also provided.

Use of synthetic polymer hydrogels to prepare scaffoldless 3D tissue constructs

The use of 3-dimensional tissue cultures is gaining popularity in many fields including drug discovery, toxicity testing and tissue engineering. Currently most of the techniques used to prepare these 3D tissues are time consuming and cannot be reproduced easily. There is an urgent need to optimize the preparation of these 3D tissue cultures. This study evaluated the use of synthetic hydrogel polymers used to manufacture soft contact lenses to guide cells to form multicellular tissue-like structures. It was found that bovine chondrocytes and porcine dental pulp stem cells were able to form 3D tissue structures when placed inside a soft contact lens. Commercially available microarrays, 96 or 384 well plates manufactured using synthetic hydrogel polymers may help overcome many reproducibility issues and simplify the 3D tissue culture process.

Use of synthetic polymer hydrogels to prepare scaffoldless 3D tissue constructs

The use of 3-dimensional tissue cultures is gaining popularity in many fields including drug discovery, toxicity testing and tissue engineering. Currently most of the techniques used to prepare these 3D tissues are time consuming and cannot be reproduced easily. There is an urgent need to optimize the preparation of these 3D tissue cultures. This study evaluated the use of synthetic hydrogel polymers used to manufacture soft contact lenses to guide cells to form multicellular tissue-like structures. It was found that bovine chondrocytes and porcine dental pulp stem cells were able to form 3D tissue structures when placed inside a soft contact lens. Commercially available microarrays, 96 or 384 well plates manufactured using synthetic hydrogel polymers may help overcome many reproducibility issues and simplify the 3D tissue culture process.

Primary Cultures of Astrocytes: Their Value in Understanding Astrocytes in Health and Disease

During the past few decades of astrocyte research it has become increasingly clear that astrocytes have taken a central position in all central nervous system activities. Much of our new understanding of astrocytes has been derived from studies conducted with primary cultures of astrocytes. Such cultures have been an invaluable tool for studying roles of astrocytes in physiological and pathological states. Many central astrocytic functions in metabolism, amino acid neurotransmission and calcium signaling were discovered using this tissue culture preparation and most of these observations were subsequently found in vivo. Nevertheless, primary cultures of astrocytes are an in vitro model that does not fully mimic the complex events occurring in vivo. Here we present an overview of the numerous contributions generated by the use of primary astrocyte cultures to uncover the diverse functions of astrocytes. Many of these discoveries would not have been possible to achieve without the use of astrocyte cultures. Additionally, we address and discuss the concerns that have been raised regarding the use of primary cultures of astrocytes as an experimental model system.

Metadynamics-Based Mechanistic Interpretation of Functional Crosstalk Between Serotonin 2A and Metabotropic Glutamate 2 receptors

Serotonin 2A (2AR) and metabotropic glutamate 2 (mGluR2) receptors have been shown to form a functional and specific heteromeric complex in mammalian brain and in tissue culture preparations with possible implications in the psychotic symptoms of schizophrenia. Unlike non-antipsychotic drugs, clinically effective atypical antipsychotics (e.g., clozapine) that bind to 2AR increase the glutamate-mediated Gi signaling through the 2AR/mGluR2 heterodimer. The molecular mechanisms underlying these allosteric effects and functional crosstalk are unknown. Using molecular dynamics (MD) simulations enhanced with metadynamics, we investigated at the molecular level the conformational changes induced by atypical antipsychotic or non-antipsychotic 2AR ligands in atomistic representations of interacting 2AR and mGluR2 embedded in an explicit lipid-water environment. First, we sampled the conformational transitions from inactive to activated (opsin-like) models of the ligand-free transmembrane regions of 2AR or mGluR2 with adiabatic biased MD simulations. We then reconstructed the free-energy landscape of the 2AR/mGluR2 heterodimer along the pre-determined transition trajectories in the presence of ligands, using a path collective variable approach based on metadynamics. The CHARMM force-field with the CMAP backbone energy correction was used to describe the full systems. All calculations were performed using NAMD enhanced with the Plumed plug-in. Our results suggest that the conformational transitions of 2AR and mGluR2 are populated by different inactive and active metastable states of the receptors, which are differentially stabilized by antipsychotic and non-antipsychotic ligands.

Role of the supraoptic nucleus in regulation of parturition and milk ejection revisited.

This review will focus on the activity of oxytocin neurons in the supraoptic nucleus (SON) and some factors that regulate their function during parturition and milk ejection in the rat. The level of oxytocin increases in the blood during parturition following a regression of the corpus luteum. The increase in oxytocin secretion is presumably a consequence of releasing the oxytocin neurons from restraining inhibitory influences of endogenous opioids-, nitric oxide-, and GABA-containing neurons following declining blood levels of progesterone on the one hand and increasing levels of estrogen on the other during late pregnancy. However, the principal stimulus for the increased oxytocin release is believed to originate, at least in part, from mechanical stimulation to the uterine cervix by fetuses near term, the resultant uterine contractile activity, and the fetal expulsion reflex. Hence, the contractile activity of the uterus acts through positive feedback mechanisms during parturition to stimulate oxytocin neurons as well, and this further increases the secretion of oxytocin. During suckling in lactating rats, somatosensory stimuli from the pups induce intermittent synchronized burst firing of oxytocin neurons, resulting in pulsatile increases in blood oxytocin concentrations to cause milk ejection. The oxytocin neurons appear to have an intrinsic capability to fire in a bursting fashion as determined by observation of this phenomenon in brain slice or tissue culture preparations. The release of oxytocin within the microenvironment of the SON and paraventricular nucleus coupled with morphological reorganization in these nuclei play important roles in the bursting activity of each oxytocin neuron and synchronization in vivo. However, the mechanism responsible for the synchronization of electrical activity in oxytocin neurons in the four discrete hypothalamic nuclei remains an interesting unanswered question.

Tissue culture of the enteric nervous system from equine ileum.

Ileal samples were harvested fresh from euthanized adult horses. The tissues were microdissected to prepare wholemount preparations for immunohistochemistry and for either explant or dissociated culture systems of the enteric nervous system. Explant culture systems were established using whole-mounts of either the submucous plexus or the muscularis externa (including the myenteric plexus). Dissociated cell cultures could only be obtained from the submucous plexus. Culture systems were maintained for up to 5 days. Immunoreactivity for a neuronal marker (Pan-N) and for glial cell markers (GFAP and S100) indicated the presence of both neurons and enteric glia in the tissue culture preparations. This is the first report of equine enteric neurons being grown in tissue culture Further refinements to the techniques will be required before this in vitro model can be used for quantitative analysis.

A novel fluorescent GSH-adduct binds to the NMDA receptor

In an attempt to develop various fluorescent probes to label glutathione (GSH) receptors, we have serendipitously synthesized a probe that binds to and antagonizes the NMDA receptor. Probe 1, a GSH adduct, displaces the competitive NMDA antagonist [ 3H]-CGP 39653 with a higher affinity than NMDA or cysteine in rat synaptic membranes. In recording experiments from a rat cortical ‘wedge’ preparation, Probe 1 reversibly blocks both NMDA- and cysteine-induced depolarization. In mixed astrocyte-neuron tissue culture preparations, Probe 1 labels parts of both cell bodies as well as processes. The present data suggest that Probe 1 binds to the NMDA receptor and antagonizes channel function.

Development of chemosensitivity of rat medullary raphe neurons

In many neonatal mammals, including humans and rats, there is a developmental increase in the ventilatory response to elevated pCO 2. This maturation of central respiratory chemoreception may result from maturation of intrinsic chemosensitivity of brainstem neurons. We have examined age-related changes in chemosensitivity of neurons from the rat medullary raphe, a putative site for central chemoreception, using perforated patch-clamp recordings in vitro. In brain slices from rats younger than 12 days old, firing rate increased in 3% of neurons and decreased in 17% of neurons in response to respiratory acidosis ( n=36). In contrast, in slices from rats 12 days and older, firing rate increased in 18% of neurons and decreased in 15% of neurons in response to the same stimulus ( n=40). A tissue culture preparation of medullary raphe neurons was used to examine changes in chemosensitivity with age from three to 74 days in vitro. In cultured neurons younger than 12 days in vitro, firing rate increased in 4% of neurons and decreased in 44% of neurons in response to respiratory acidosis ( n=54). In contrast, in neurons 12 days in vitro and older, firing rate increased in 30% of neurons and decreased in 24% of neurons in response to respiratory acidosis ( n=105). In both types of chemosensitive neuron (“stimulated” and “inhibited”), the magnitudes of the changes in firing rate were greater in older neurons than in young neurons. These results indicate that the incidence and the degree of chemosensitivity of medullary raphe neurons increase with age in brain slices and in culture. This age-related increase in cellular chemosensitivity may underlie the development of respiratory chemoreception in vivo. Delays in this maturation process may contribute to developmental abnormalities of breathing, such as sudden infant death syndrome.

Myocardial architecture and ventricular arrhythmogenesis.

Conduction of the cardiac impulse is dependent on both the active membrane properties of cardiac cells (generating the action potential) and the passive properties determined by architectural features of the myocardium. The influence of tissue architecture on conduction is determined principally by the size, shape, and packing of individual myocytes and by the quantity, three-dimensional distribution, and physiological behavior of the specialized intercellular junctions responsible for impulse propagation from cell to cell, the gap junctions.1 It has long been recognized that abnormalities in conduction of the cardiac impulse are an important cause of arrhythmias2 in that they alter the relationships between conduction velocity, path length, and recovery of excitability (the determinants of reentrant excitation). However, it was not until more recently that myocardial architecture was considered important in determining patterns of activation and conduction velocity3 4 and was therefore thought to have a central role in arrhythmogenesis,5 and some of the evidence for this forms the basis of this review. ### Myocardial Syncytium: Anisotropy of Structure and Function The early observation that conduction properties in myocardium were different in different directions,6 with more rapid conduction in the direction parallel to the myocardial fiber axis than in the transverse direction (the definition of the anisotropic conduction characteristic of heart muscle [Figure 1⇓]), is attributable principally to the lower resistivity of myocardium in the longitudinal than the transverse direction. Gap-junctional channels create continuity between the cytoplasmic compartments of abutting myocytes but act as resistive discontinuities to the cytoplasmic current flow between the intracellular compartments of the cells (the “intracellular” conduction pathway). Longitudinal resistivity is lower than transverse because this intracellular pathway encounters fewer cell boundaries per unit distance in the longitudinal than the transverse direction. In normal ventricular myocardium, conduction in the direction parallel to the long axis of the myocardial fiber bundles …

Advances in the microscopy of osteoarthritis

This review describes recent contributions made by microscopy to the understanding of osteoarthritis, a clinical syndrome the pathological features of which are well defined by classical white light microscopy. The fluorescence and reflected light, conventional and scanning optical microscopy of excised osteoarthritic tissue preparations, from human and animal sources, has enabled the identification of cell proteins such as S100, of matrix components such as the proteoglycans and collagens, and of adhesion molecules including fibronectin, the integrins and tenascin. Comparable microscopic studies have been made of cell and tissue culture preparations of osteoarthritic cartilage and synovium. Scanning optical microscopy also allows the rapid measurement, in hydrated osteoarthritic tissues, of cell density, cell size, surface roughness and other parameters. The importance of water in sustaining the physical attributes of cartilage is accepted and new forms of electron microscopy can play important parts in the study of unfixed osteoarthritic cartilage. These methods include the low temperature scanning electron microscopy and electron probe x-ray microanalysis of hydrated bulk material and the high resolution transmission electron microscopy of low temperature replicas of cartilage surfaces. Understanding of osteoarthritis has been facilitated by these advances and will continue to be enhanced as new techniques of microscopy evolve. Microsc. Res. Tech. 37:245–270, 1997. © 1997 Wiley-Liss, Inc.

Advances in the microscopy of osteoarthritis.

This review describes recent contributions made by microscopy to the understanding of osteoarthritis, a clinical syndrome the pathological features of which are well defined by classical white light microscopy. The fluorescence and reflected light, conventional and scanning optical microscopy of excised osteoarthritic tissue preparations, from human and animal sources, has enabled the identification of cell proteins such as S100, of matrix components such as the proteoglycans and collagens, and of adhesion molecules including fibronectin, the integrins and tenascin. Comparable microscopic studies have been made of cell and tissue culture preparations of osteoarthritic cartilage and synovium. Scanning optical microscopy also allows the rapid measurement, in hydrated osteoarthritic tissues, of cell density, cell size, surface roughness and other parameters. The importance of water in sustaining the physical attributes of cartilage is accepted and new forms of electron microscopy can play important parts in the study of unfixed osteoarthritic cartilage. These methods include the low temperature scanning electron microscopy and electron probe x-ray microanalysis of hydrated bulk material and the high resolution transmission electron microscopy of low temperature replicas of cartilage surfaces. Understanding of osteoarthritis has been facilitated by these advances and will continue to be enhanced as new techniques of microscopy evolve.

Detection and differentiation of epizootic haemorrhagic disease of deer and bluetongue viruses by serogroup-specific sandwich ELISA.

A serogroup specific sandwich ELISA was developed for the detection of epizootic haemorrhagic disease of deer viruses (EHDV) in infected insects and tissue culture preparations. Polyclonal rabbit antiserum against purified EHDV core particles was used to capture viral antigen and specific binding detected using guinea pig antisera against EHDV core particles followed by anti-guinea pig immunoglobulin enzyme-labelled conjugate. The assay is EHDV specific and detects all 8 serotypes. No cross-reactions were found with related viruses such as bluetongue (BTV), Palyam, Tilligery or African horse sickness virus (AHSV). A similar serogroup specific sandwich ELISA was also developed for BTV. The assays showed a similar sensitivity in detecting the respective EHDV or BTV antigens in a pool of 500 midges where only 2 were infected. These assays allow a simple and rapid means of detecting and differentiating members of these closely related serogroups. The sensitivity of the tests will allow more extensive studies on vector competence and virus/vector distribution.

Cryptococcal cellulitis in a diabetic, kidney transplant patient

The cutaneous manifestations of disseminated cryptococcosis are variable and include papules, plaques, nodules, draining sinuses, acneiform lesions, tumors, abscesses, ulcers, pustules, bullae, molluscum-like lesions, and cellulitis.8-1 0 Cryptococcal cellulitis has only been seen in patients with severe underlying diseases such as leukemiaf myeloma,8.11 systemic lupus erythematosusj chronic active hepatitis,'? intestinal lymphangiectasia and congenital lymphedema.P as well as in kidney transplant recipients. 1-7 These patients had received immunosuppressive therapy, which in all cases included prednisone. Only a few cases of cryptococcal cellulitis in kidney transplant patients have been reported. 1-7 Most lesions occurred on an extremity and appeared abruptly as a single, tender, indurated, erythematous plaque that was initially mistaken for bacterial cellulitis and erroneously treated with antibiotics. Ours is only the third reported case of cryptococcal cellulitis with multiple sites of involvement.': 7 Methods of directly diagnosing cryptococcal cellulitis include Gram's stain or india ink preparation of tissue aspirates and cultures of skin specimens. Cryptococcal cellulitis may histologically demonstrate a variable lymphohistiocytic infiltrate in the Hugh M. Gloster, Jr., MD, Robert A. Swerlick, MD, and Alvin R. Solomon, MD Atlanta, Georgia

Common Sequence Elements in Structurally Unrelated Genomes of Defective Interfering Semliki Forest Virus

The polymerase chain reaction was used to amplify defective interfering (DI) Semliki Forest virus (SFV) genomes from tissue culture preparations which had in vitro interfering and in vivo mouse-protecting activity. The products were molecularly cloned and sequenced (pSFVDI-6, 2146 nt and pSFVDI-19, 1244 nt). Comparison with the sequence of the SFV genome showed that both were derived from three noncontiguous regions: the 5′ terminus including the start of the nsP1 coding region, part of the nsP2 coding region, and the 3′ untranslated region. Both clones possessed open reading frames, including one with the potential of encoding truncated versions of nsP1. Transcribed RNAs from pSFVDI-6 and pSFVDI-19 were translated in an in vitro system into polypeptides of M r 27,000 and 25,000, respectively. Although pSFVDI-6 and pSFVDI-19 did not possess the rearranged sequences or extensive repeat regions of previously cloned DI SFV genomes (Lehtovaara et al., Proc. Natl. Acad. Sci. USA 78, 5353-5357, 1981; Lehtovaara et al., J. Mol. Biol. 156, 731-748, 1982), they were derived from similar regions of the SFV genome, suggesting a common sequence requirement for DI SFV particles.

Unique method of tissue culture preparation for EM

A simplified technique of growing monolayer cells for electron microscopic examination has been developed. Our procedure has eliminated many difficult steps and therefore is easier than those reported by others.Regular Beem capsules for routine embedding for electron microscopy were used(Fig. 1). Prior to tissue inoculation capsules were washed with 5% HCl and gas sterilized. A 0.5 ml cell suspension of MCF-7 cells (10,000/ml) was placed in the capsules and cells settled in the pyramid portion (Fig. 2). Culture medium was alpha-MEM with 10% FCS. Capsules were incubated at 37°C for 3 days. They were then fixed in 70% ethanol for 20 minutes and stained with crystal violet. The pyramid portion of the capsule was cut off and monolayer cell growth was confirmed by examination under a microscope (Fig. 3).

Herpes simplex virus antigen direct detection in standard virus transport medium by Du Pont Herpchek enzyme-linked immunosorbent assay

A commercial 5-h direct herpes simplex virus (HSV) antigen detection enzyme immunoassay kit (Du Pont Herpchek) was compared with a cell culture isolation system by using primary rabbit kidney and MRC-5 cells with 779 clinical specimens received in virus transport medium and with stock tissue culture preparations of HSV types 1 and 2. In the first study of 422 specimens from symptomatic patients, Herpchek detected 110 of 111 HSV-positive specimens (26.3% of all specimens), with a sensitivity of 99% and a specificity of 100%. In the second study of 357 specimens primarily from asymptomatic pregnant women, however, Herpchek detected 70 of 119 HSV-positive specimens (33% of all specimens), with a sensitivity of 58.8% and a specificity of 99.5%. Stock virus dilution experiments showed that Herpchek was 10 to 100 times less sensitive than culture. Herpchek was found to be an acceptable test for symptomatic patients, but for asymptomatic patients shedding a low titer of HSV it was not as sensitive and cell culture of Herpchek-negative specimens is recommended for such cases.