Localization of protein secretion in fungal colonies using a novel culturing technique; the ring-plate system

A novel culturing technique, called the ring-plate system, is described. Fungal colonies are grown on a polycarbonate membrane placed on a plate with 6 ring-shaped wells that are filled with liquid medium. This culturing technique enables simultaneous monitoring of environmental conditions and secretion in different parts of the fungal colony.     

Does ex vivo labelling of proliferating cells in colonic and vaginal mucosa reflect the S-phase fraction in vivo?

The ex vivo labelling of DNA-synthesizing epithelial cells in colonic and vaginal mucosa was compared with in vivo labelling. For this purpose, in vivo S-phase cells were labelled with [3H]thymidine (Tdr) and ex vivo labelling was continued by culturing tissue specimens in bromodeoxyuridine (BrdU). Various methods of tissue culture were employed in order to improve diffusion of medium (and BrdU) in the tissue. BrdU and 3H-TdR labelling were evaluated by immunohistochemistry and autoradiography respectively. Ex vivo labelling resulted in a patchy distribution of labelled cells, which did not correspond with the 3H-TdR labelling pattern obtained in vivo. Under the described conditions ex vivo labelling does not appear to be a reliable for estimation of the proliferative activities in vivo.     

Characteristics of human embryonic neuronal cells procured by non-enzyme method

Isolation and culturing of human neuronal progenitor cells is of significant value for both fundamental research and therapeutic purposes. In this work, human embryonic neuronal cells were characterized as a heterogeneous population of progenitor cells with various differentiation potentials. During in vitro culturing the cells are capable of re-inoculating, proliferating, differentiating and migrating. While differentiating, these cells form neurons and glial cells. The present research demonstrates that depending upon the culturing conditions the embryonic neuronal cells may either form floating aggregates (incubation with embryonic serum), attach (incubation without serum), proliferate, or form neurospheres. Besides, peculiarities of aggregate differentiation during their incubation under various media are described.     

Does ex vivo labelling of proliferating cells in colonic and vaginal mucosa reflect the S-phase fraction in vivo?

Summary The ex vivo labelling of DNA-synthesizing epithelial cells in colonic and vaginal mucosa was compared with in vivo labelling. For this purpose, in vivo S-phase cells were labelled with [³H]thymidine (Tdr) and ex vivo labelling was continued by culturing tissue specimens in bromodeoxyuridine (BrdU). Various methods of tissue culture were employed in order to improve diffusion of medium (and BrdU) in the tissue. BrdU and ³H-TdR labelling were evaluated by immunohistochemistry and autoradiography respectively. Ex vivo labelling resulted in a patchy distribution of labelled cells, which did not correspond with the ³H-TdR labelling pattern obtained in vivo. Under the described conditions ex vivo labelling does not appear to be a reliable for estimation of the proliferative activities in vivo.     

Rapid sampling culture chamber.

An all-glass chamber for culturing anaerobic and aerobic bacteria in liquid medium is described. The system permits both rapid sampling and turbidimetric measurements under controlled atmospheric conditions.     

Rapid sampling culture chamber.

An all-glass chamber for culturing anaerobic and aerobic bacteria in liquid medium is described. The system permits both rapid sampling and turbidimetric measurements under controlled atmospheric conditions.     

Culturing chick embryos—a simplification of New's method

A simplified version of New's method for culturing early chick embryos is described. The technique allows continuous observation of the critical first three days of development. The conditions for setting up successful cultures are not at all stringent and the method could become a very useful teaching aid.     

Initiation of dedifferentiation and structural changes in in vitro cultured petiole of Arabidopsis thaliana

Although the method of tissue culturing has been used widely in practice for a long time, and there are numerous hypotheses to explain the dedifferentiation phenomenon in the tissue culturing, many details of mechanism of dedifferentiation remain unclear. In the study, dedifferentiation process is initiated in the residual procambium, followed by the procambium-derived cells and finally xylem parenchyma cells under the culturing of Arabidopsis thaliana petiole explants. The procambium may induce its derivative cells to undergo dedifferentiation, which in turn induce the xylem parenchyma cells to dedifferentiate. This phenomenon is very similar to the activity of interfascicular cambium induced by intrafascicular cambium in secondary growth of plant stems. In the present study, only the paired procambium-derived cells and xylem parenchyma truly underwent dedifferentiation, whereas the initial changes in the procambium simply recovered the inherent meristematic capacity of those cells. In transverse section of petiole of A. thaliana, parenchyma cells outside the vascular bundle did not participate in dedifferentiation and gradually disintegrated under the culture conditions. Obviously, the time for initiation and difficulty underlain for undergoing dedifferentiation are dependent on the differential degree and location of parenchyma cells in the petiole.     

Electrical stimulation of cultured lepidopteran dorsal vessel tissue: an experiment for development of bioactuators

An insect dorsal vessel (DV) is well suited for a bioactuator since it is capable of contracting autonomously, and its tissue and cells are more environmentally robust under culturing conditions compared with mammalian tissue. In this study, electrical pulse stimulation was examined so as to regulate a bioactuator using the DV tissue. The DV tissue of a larva of Ctenoplusia agnate was assembled on a micropillar array, which was stimulated after culturing for about 3 wk. The contraction of the DV tissue was evaluated by image analysis to measure lateral displacements at the micropillar top. As a result, suitable stimulation conditions in a 35-mm petri dish were determined as: applied voltage of 10 V with 20-ms duration. Next, the time lag between the onset of electrical stimulus and the onset of mechanical contraction (electromechanical delay (EMD)) was estimated. A light-emitting diode (LED) was connected serially with the petri dish, and the LED flashed when electrical pulses were given. Movie images were analyzed in which electrical pulses made the DV tissue contract and the LED flashed virtually simultaneously; from these, the EMD was estimated as approximately 50 ms. These results suggest that the electrical pulse stimulation is capable of regulating the DV tissue, and the micropillar array is a useful biological tool to investigate physiological properties of muscle tissue.     

Epithelial–mesenchymal transition in rhesus monkey embryonic stem cell colonies: the role of culturing conditions

Colonies of rhesus monkey embryonic stem cells (rhESC; cell line R366.4) have been described before to show a spatially ordered process of epithelial–mesenchymal transition in vitro. In the present investigations, we have studied variables of culturing conditions which influence the reproducibility of the formation of crater-like ingression centers in the colonies. Critical parameters are found to be age and density of mouse embryonic fibroblast (MEF) feeder cell layers, the mode of mitotic inactivation of the MEFs (mitomycin C, or irradiation), and the mode of rhESC isolation during subculturing (enzymatic/mechanical cell cluster isolation; type of enzyme). The described culturing system appears to offer a reproducible in vitro model potentially useful for studies on cellular processes involved in gastrulation in the primate.     

Adaptation of mammalian cells to growth in serum-free media

A three-step protocol is described for adapting an anchorage-dependent, serum-dependent recombinant mammalian cell lineage to high density serum-free suspension culture. The objective is a cell lineage that is well-suited for the manufacture of a recombinant protein. The first step of the protocol generates an anchorage-independent cell lineage by culturing trypsin-treated cells in spinner flasks using serum-containing medium. The second step adapts the lineage to serum-free medium through a series of serum reduction steps in the presence of defined growth-promoting additives. The third step adapts the lineage to high-cell-density conditions by culturing the cells in a bioreactor in a manner that allows development of tolerance to growth-inhibiting substances released by the cells. Examples are presented for the use of this protocol for recombinant CHO cells.     

Cockroach tissueIn Vitro: A system for the study of insect cell biology

Summary Anin vitro technique of culturing insect tissue is described in which isolated tissues and cells of the cockroach,Leucophaea maderae (F.), are maintained in a chemically defined nutrient medium. The system can be used for three types of cultures: organ cultures of nymphal leg regenerates for endocrine studies; drop cultures of embryo leg tissue for screening biologically active chemicals; and dispersed embryo cell cultures for physiological studies at the cellular and subcellular levels. The dispersed cultures show measurable mitotic rates and can be maintained as long as 170 days without adding undefined substances to the medium.     

Effect of adhesion on the status of corneal tissue during in vitro culturing

Newt and rat cornea with and without the corneal limbus was cultured under conditions of adhesion to a substrate (stationary culturing) and in the absence of adhesion (roller culturing). It was found that the contact of the tissue with the substrate play the key role in the transduction of the regulatory signal that ensures the maintenance of cornea viability and affects proliferation and migration of cornea cells     

Morphological characteristics of cultured fresh and thawed pericardium cells

The need for selection of the optimal material for the manufacturing of cardio-patches can be resolved by the use of cryostored autologous pericardial tissue. This short communication is a concise fragment of a large-scale research and demonstrates only the efficiency of cell culturing before and after pericardial preservation in the low temperature conditions.     

Traditional and modern approaches to culture of preimplantation mammalian embryos in vitro

This review covers the basic principles and methods of in vitro culture of preimplantation mammalian embryos. The features of in vitro development of embryos of various species of animals with allowance for the composition of nutrient media are described, with special attention paid to those species that have traditionally been considered as laboratory (i.e., mice, rats, and hamsters). The effects of suboptimal culturing conditions of preimplantation embryos on the formation of the phenotype of individuals developed from these embryos are discussed. New approaches to optimize the conditions of the development of preimplantation mammalian embryos in vitro are analyzed.     

Karelian birch (Betula pendula Roth. var. carelica Merkl.) as a model for studying genetic and epigenetic variation related to the formation of patterned wood

The results of long-term pioneering studies on in vitro micropropagation of Karelian birch patterned forms and simultaneous cytological analysis of plants multiplied using different periods of in vitro culturing are published for the first time. The patterned wood character has been shown to be correlated with the degree of mixoploidy of its somatic tissue, which is higher in the plants obtained from callus cultures during the first years of culturing. Subsequent intracellular selection leads to a decrease in mixoploidy and, hence, in a later expression and lower expressivity of the patterned wood character in regenerant plants. It is also known that extreme growth conditions stimulate the formation of patterned wood. Thus, Karelian birch may serve as a model object for studying the forms of variability (both genetic and epigenetic) that result in patterned wood. The genetic variability is expressed in the variation of the degree of mixoploidy of somatic tissue as a result of various mitotic aberrations. The epigenetic variability is not related to changes in the DNA structure; it is caused by different phenotypic effects of genes located in cells with different ploidy/aneuploidy levels, the ratio between which varies depending on the environmental conditions. The expression of genes, in particular, rRNA genes, is affected by extreme conditions. The appearance of a residual nucleolus at the mitotic metaphase-telophase stages is a cytological expression of this phenomenon.     

Karelian birch (<Emphasis Type="Italic">Betula pendula</Emphasis> Roth. var. <Emphasis Type="Italic">carelica</Emphasis> Merkl.) as a model for studying genetic and epigenetic variation related to the formation of patterned wood

The results of long-term pioneering studies on in vitro micropropagation of Karelian birch patterned forms and simultaneous cytological analysis of plants multiplied using different periods of in vitro culturing are published for the first time. The patterned wood character has been shown to be correlated with the degree of mixoploidy of its somatic tissue, which is higher in the plants obtained from callus cultures during the first years of culturing. Subsequent intracellular selection leads to a decrease in mixoploidy and, hence, in a later expression and lower expressivity of the patterned wood character in regenerant plants. It is also known that extreme growth conditions stimulate the formation of patterned wood. Thus, Karelian birch may serve as a model object for studying the forms of variability (both genetic and epigenetic) that result in patterned wood. The genetic variability is expressed in the variation of the degree of mixoploidy of somatic tissue as a result of various mitotic aberrations. The epigenetic variability is not related to changes in the DNA structure; it is caused by different phenotypic effects of genes located in cells with different ploidy/aneuploidy levels, the ratio between which varies depending on the environmental conditions. The expression of genes, in particular, rRNA genes, is affected by extreme conditions. The appearance of a residual nucleolus at the mitotic metaphase-telophase stages is a cytological expression of this phenomenon.     

Cardosins improve neuronal regeneration after cell disruption: a comparative expression study

The establishment of primary cell cultures is invaluable for studying cell and molecular biological questions. Although primary cell cultures more closely resemble and function like in the native environment, during the culture establishment the cells undergo several changes including the damage sustained during their removal from original tissue. The resultant cells have to rebalance the expression of their processing molecules to ascertain matrix signalling that ensure cell adaptation and consequent proliferation. Hence, we used cardosin, a novel plant enzyme for tissue disaggregation, for isolating and culturing neuronal cells from embryonic rats. The present investigation reports the molecular events, mainly related with matrix metalloproteinases (MMPs)/tissue inhibitor of metalloproteinase (TIMPs) expression, which could substantiate the superior neurite outgrowth and dendritic extension previously described. It was observed that 24 h after primary culture establishment, MMP-2 and MMP-9 messenger RNA (mRNA) are significantly upregulated, while the expression of TIMP-1 and TIMP-2 is unaltered. Regarding the role of laminin in neuronal pathfinding, it was found that the use of anti-laminin antibody and arginine–glycine–aspartate (RGD) peptide exerted inhibitory effects on neurite outgrowth after mechanical lesion where the expression of MMP-9 and TIMP-1 is upregulated under non-permissive conditions in response to mechanical injury.     

A method for the harvest,culture, and characterization of human adult atrial myocardial cells: Correlation with age of donor

Summary Myocardial cell culture methods are now well established for animal and fetal human tissue. We present here a method for harvesting and culturing adult human atrial myocardiocytes. Cells are obtained from fresh atrial tissue normally discarded after being removed to cannulate the right atrium during open heart surgery. The atrial tissue is minced and then digested using collagenase. The single cell suspension is initially cultured in serum-containing growth medium, then transferred to defined medium, selective for myocardial cell growth. The cells are characterized by immunoperoxidase stains and transmission electron microscopy. The cultured cells stain positive for myoglobin, whereas control cultured fibroblasts and endothelial cells do not. Electron microscopy shows the presence of numerous myofibrils, Z-bodies, pleomorphic mitochondria, and secretory granules. The chronological age of the donor was an important factor in culturing the adult tissue, the younger tissue correlated with a higher success rate. This method provides a means for in vitro study of human adult myocardial cells and provides guidelines for appropriate atrial tissue to use.     

Chromosome preparations of human blood lymphocytes—Evaluation of techniques

Several parameters of human lymphocyte culturing techniques and metaphase chromosome preparation procedures were studied and quantitatively evaluated in regard to their influence on the results of Q and G banding procedures. The culturing conditions were studied using³H thymidine incorporation as a parameter. A whole blood culturing technique using Ham's F12 medium was found to give optimal and consistent results. Colcemid concentration proved to be of no influence on chromosome contraction or on the number of metaphases obtained over the concentration range investigated. Prolonged exposure to colcemid was found to cause a decrease in the mean chromosome length but the absolute number of metaphases with a low degree of chromosomal contraction hardly decreased. Different spreading techniques were quantitatively analysed and factors important for the spreading of chromosomes were evaluated. Based on the results obtained, an optimal procedure is described which over a period of one year has given consistent results.