Use of glycine as a non-enzymic procedure for separation of mouse embryonic tissues and dissociation of cells

A non-enzymic procedure for separation and cell dissociation of mouse embryonic tissues is described, utilizing solutions of either 1 or 0.5 M glycine with EDTA. Incubation of blastocysts in glycine resulted in decompaction of cells without complete dissociation. Post-implantation egg cylinders were readily separated into component tissue layers, with subsequent dissociation into single cells. Cell viability was high after such treatment, and all cell types adhered rapidly and spread out on plastic culture dishes. Parietal endoderm, visceral yolk sac and amnion tissues from 10th and 14th day embryos were incubated in glycine, with resulting cell dissociation and/or tissue separation. Changes in cell adhesion properties appear to take place during development of these tissues. PSA1 embryoid bodies readily separated into the outer ‘endoderm’ layer and inner embryonal carcinoma cell core after a brief incubation in glycine. However, this procedure was unsuccessful for dissociation of cells from a plastic or gelatin substrate in vitro, suggesting that cell-substrate adhesion in vitro may differ from that in vivo.     

The movement of single cells within solid tissue masses

Individual heart and liver cells isolated from chick embryos labelled in ovo with ³H-thymidine were seeded, in culture, onto the surfaces of unlabelled, embryonic heart and liver tissue masses (both tissue fragments and cellular reaggregates). Single, labelled cells, as observed in autoradiographs, infiltrated the interiors of the tissue masses in most cases. These results might be unexpected in light of previous experiments and current notions of ‘contact inhibition of cell movement’.     

Actin polymerization and synthesis in cultured neurones

The protein content of sympathetic neurones explanted from 10–11-day old chick embryos into culture medium containing nerve growth factor (NGF) increases steadily from about 100 to about 400 pg/cell in 7 days. Actin remains at close to 5% of the total protein during this period, but the proportion of unpolymerized actin falls. As measured by the inhibition of DNase I activity, rounded neurones without neurites contain 70 ± 7% of their total actin in monomeric form, whereas cells in mature, neurite-bearing cultures contain 39 ± 7%. When allowance is made for the increase in size of the neuronal cell bodies, the actin present in the neurites (‘axons’) alone is found to be almost entirely in filamentous form.Cultures exposed to radioactive leucine rapidly incorporate radioactivity into both sedimentable and non-sedimentable forms of actin. Actin-specific activities in the two fractions—estimated after isolation of the actin on small DNase I—Sepharose affinity columns—are similar after labelling for less than 1 h. Direct incorporation of newly-synthesized actin into filaments is suggested from these results.Pulse-chase experiments show that non-sedimentable protein in cultured sympathetic neurones turns over more rapidly than sedimentable protein. However, this is not true for actin, which shows a similar specific activity in sedimentable and non-sedimentable forms—even after 6 days of cold chase. This anomalous behaviour is simply explained by an exchange of actin molecules between filamentous and non-filamentous forms. Control experiments indicate that exchange does not occur to this degree during preparation of subcellular fractions. It is consequently attributed to exchange processes in the living cell.     

"Culture shock" from the bone cell's perspective: emulating physiological conditions for mechanobiological investigations

Bone physiology can be examined on multiple length scales. Results of cell-level studies, typically carried out in vitro, are often extrapolated to attempt to understand tissue and organ physiology. Results of organ- or organism-level studies are often analyzed to deduce the state(s) of the cells within the larger system(s). Although phenomena on all of these scales—cell, tissue, organ, system, organism—are interlinked and contribute to the overall health and function of bone tissue, it is difficult to relate research among these scales. For example, groups of cells in an exogenous, in vitro environment that is well defined by the researcher would not be expected to function similarly to those in a dynamic, endogenous environment, dictated by systemic as well as organismal physiology. This review of the literature on bone cell culture describes potential causes and components of cell "culture shock," i.e., behavioral variations associated with the transition from in vivo to in vitro environment, focusing on investigations of mechanotransduction and experimental approaches to mimic aspects of bone tissue on a macroscopic scale. The state of the art is reviewed, and new paradigms are suggested to begin bridging the gap between two-dimensional cell cultures in petri dishes and the three-dimensional environment of living bone tissue. osteoblast; osteocyte; tissue engineering; mechanobiology; mechanochemical transduction; fluid flow     

Long term ‘marine diet’ in Eskimos is not associated with altered urinary excretion of total tetranor prostaglandin metabolites

The total urinary excretion of tetranor prostaglandin metabolites, measured as tetranorprostanedioic acid (TPD), was quantified in traditionally living Greenland Eskimos (E) and compared with that in Caucasian Danes (D). TPD excretion (μg/24h) was not significantly different between both groups, neither for males (331 ± 62.4 (E) vs. 331 ± 25.7 (D), mean ± SEM, n = 9 and 10) nor for females (190 ± 31.7 (E) vs. 264 ± 27.4 (D), n = 11 and 10, P₂ > 0.05). Since urinary prostaglandin metabolites are thought to reflect the total prostaglandin turnover in vivo, these results suggest that a long-term intake of relatively large amounts of polyunsaturated fatty acids of the (n-3) family does not alter total prostaglandin turnover in vivo. This is in contrast to stimulated prostanoid formation in vitro, and thus suggests a different regulatory role of dietary and tissue fatty acids for ‘stimulated’ and ‘basal’ prostaglandin production.     

Growth and Development of Romanomermis culicivorax In Vitro

Various combinations of vertebrate and invertebrate tissue culture and microbiological media were utilized in an attempt to culture Romanomermis culicivorax (Mermithidae: Nematoda) in vitro. Most media were unsuitable and caused nematodes to become lumpy, vacuolated, and granular. Slow and limited growth and development of internal structures of the nematodes were obtained with variously supplemented Grace''s tissue culture and Schneider''s Drosophila media. In an enriched Grace''s medium, development attained by the nematodes after 3-4 wk was comparable to 4-5-day-old parasites grown in vivo in the mosquito host, Culex pipiens. Two molts were observed in vitro. Maximum dimensions in vitro were 7.0-mm length and 87-μm width at the widest point. The stichosome, stichocytes, and trophosome developed prominently. A filiform tail and highly cuticularized tube persisted throughout the culture period in vitro.     

Experimental evidence for negative turgor pressure in small leaf cells of Robinia pseudoacacia L versus large cells of Metasequoia glyptostroboides Hu et W.C.Cheng. 1. Evidence from pressure‐volume curve analysis of dead tissue.

This paper provides a mini‐review of evidence for negative turgor pressure in leaf cells starting with experimental evidence in the late 1950s and ending with biomechanical models published in 2014. In the present study, biomechanical models were used to predict how negative turgor pressure might be manifested in dead tissue, and experiments were conducted to test the predictions. The main findings were as follows: (i) Tissues killed by heating to 60 or 80 °C or by freezing in liquid nitrogen all became equally leaky to cell sap solutes and all seemed to pass freely through the cell walls. (ii) Once cell sap solutes could freely pass the cell walls, the shape of pressure‐volume curves was dramatically altered between living and dead cells. (iii) Pressure‐volume curves of dead tissue seem to measure negative turgor defined as negative when inside minus outside pressure is negative. (iv) Robinia pseudoacacia leaves with small palisade cells had more negative turgor than Metasequoia glyptostroboides with large cells. (v) The absolute difference in negative turgor between R. pseudoacacia and M. glyptostroboides approached as much as 1.0 MPa in some cases. The differences in the manifestation of negative turgor in living versus dead tissue are discussed.     

Organotypic collagen I assay: a malleable platform to assess cell behaviour in a 3-dimensional context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic. Furthermore, interaction with other cell types, including stromal fibroblasts and immune cells, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo. Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo. As such, they can serve an important role in reducing the need for experiments on living animals.     

Insect epidermis: disturbance of supracellular tissue polarity does not prevent the expression of cell polarity

Summary The insect integument displays planar tissue polarity in the uniform orientation of polarized cuticular structures. In a body segment, for example, the denticles and bristles produced by the constituent epidermal cells point posteriorly. Colchicine can abolish this uniform orientation while still allowing individual cells to form orientated cuticular structures and thereby to express cell polarity. This suggests that an individual cell in a sheet can establish planar polarity without reference to some kind of covert supracellular cue (such as a morphogen gradient) in the epidermis as a whole. The results also indicate that colchicine interferes — directly or indirectly — with the mechanisms involved in aligning the polarity axes of individual cells into a common orientation, thereby generating supracellular or tissue polarity.     

Hydrogels as extracellular matrix mimics for 3D cell culture

Methods for culturing mammalian cells ex vivo are increasingly needed to study cell and tissue physiology and to grow replacement tissue for regenerative medicine. Two‐dimensional culture has been the paradigm for typical in vitro cell culture; however, it has been demonstrated that cells behave more natively when cultured in three‐dimensional environments. Permissive, synthetic hydrogels and promoting, natural hydrogels have become popular as three‐dimensional cell culture platforms; yet, both of these systems possess limitations. In this perspective, we discuss the use of both synthetic and natural hydrogels as scaffolds for three‐dimensional cell culture as well as synthetic hydrogels that incorporate sophisticated biochemical and mechanical cues as mimics of the native extracellular matrix. Ultimately, advances in synthetic–biologic hydrogel hybrids are needed to provide robust platforms for investigating cell physiology and fabricating tissue outside of the organism. Biotechnol. Bioeng. 2009;103: 655–663. © 2009 Wiley Periodicals, Inc.     

Interactions between living and zinc-fixed cells in culture

When chick heart fibroblasts collide with zinc-fixed fibroblasts in culture, they show a typical ‘contact inhibition of locomotion’ reaction both an inhibition of overlapping and contact paralysis occurring. Previous reports in the literature have suggested that contact inhibition of locomotion only occurs when the collision is between living cells.     

From axenic spore germination to molecular farming

The first bryophyte tissue culture techniques were established almost a century ago. All of the techniques that have been developed for tissue culture of seed plants have also been adapted for bryophytes, and these range from mere axenic culture to molecular farming. However, specific characteristics of bryophyte biology—for example, a unique regeneration capacity—have also resulted in the development of methodologies and techniques different than those used for seed plants. In this review we provide an overview of the application of in vitro techniques to bryophytes, emphasising the differences as well as the similarities between bryophytes and seed plants. These are discussed within the framework of physiological and developmental processes as well as with respect to potential applications in plant biotechnology.     

Cell cultivation under different gravitational loads using a novel random positioning incubator

Important in biotechnology is the establishment of cell culture methods that reflect the in vivo situation accurately. One approach for reaching this goal is through 3D cell cultivation that mimics tissue or organ structures and functions. We present here a newly designed and constructed random positioning incubator (RPI) that enables 3D cell culture in simulated microgravity (0 g). In addition to growing cells in a weightlessness‐like environment, our RPI enables long‐duration cell cultivation under various gravitational loads, ranging from close to 0 g to almost 1 g. This allows the study of the mechanotransductional process of cells involved in the conversion of physical forces to an appropriate biochemical response. Gravity is a type of physical force with profound developmental implications in cellular systems as it modulates the resulting signaling cascades as a consequence of mechanical loading. The experiments presented here were conducted on mouse skeletal myoblasts and human lymphocytes, two types of cells that have been shown in the past to be particularly sensitive to changes in gravity. Our novel RPI will expand the horizon at which mechanobiological experiments are conducted. The scientific data gathered may not only improve the sustainment of human life in space, but also lead to the design of alternative countermeasures against diseases related to impaired mechanosensation and downstream signaling processes on earth. Biotechnol. Bioeng. 2014;111: 1180–1190. © 2013 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

猪十二指肠组织灌流培养与静态培养比较

组织灌流培养和静态培养是肠道组织体外培养最常见的两种方法,但目前尚不清楚这两种培养方法哪种更适合用于体外胃肠激素分泌的研究.以猪十二指肠组织为研究对象,首先比较L-精氨酸下两种体外培养方法对组织胆囊收缩素(Cholecystokinin,CCK)分泌和组织活性乳酸脱氢酶(Lactate dehydrogenase,LD...     

Expression of high molecular weight (67K) keratin in human keratinocytes cultured on dead de-epidermized dermis

The influence of living dermal tissue upon epidermal differentiation during embryonic development as well as in vitro culture has been documented. Living dermal tissue contains both cellular and matricial elements. In the present study, third-passage subcultured adult human keratinocytes were either seeded on plastic dishes or recombined with dead de-epidermized dermis and further cultured for 3 weeks. After this time, keratins were extracted and analysed by one- and two-dimensional gel electrophoresis. The 67K keratin subunit, which is thought to be involved in the process of in vivo type skin differentiation, was absent in ordinary cultures; however, it was expressed in air-exposed cultures on dead de-epidermized dermis. Quantitatively, however, it did not reach the in vivo level. This suggests that in principle, the induction of the expression of this protein does not require the presence of living dermal cells.     

Update on the Search for Miss Waldron’s Red Colobus Monkey

Miss Waldron’s red colobus (Procolobus badius waldroni) has a restricted distribution in eastern Ivory Coast and western Ghana. There have been no confirmed sightings of them since 1978 and surveys carried out from 1993 to the present have yet to reveal any living individual. Since the announcement of the monkey’s probable extinction (Oates et al., 2000), new evidence from forest in the extreme southeast of Ivory Coast suggests that a handful of individuals have remained undetected to this point. I discuss the evidence—a tail, a skin and a photograph—and results of accompanying surveys. Additional surveys of the Ehy Forest are required to confirm the presence of Procolobus badius waldroni and an Action Plan for the conservation of all red colobus across the continent is needed to prevent the disappearance of other taxa.     

Utilization of H-5-uridine for RNA synthesis in ehrlich ascites tumour cells

Incorporation of [³H]uridine into the total RNA of a hyperdiploid line of Ehrlich ascites tumour cells reaches a plateau after 10–11 min, both in vivo and in vitro. Such patterns of incorporation —characteristic of a pulse-labelling situation—were achieved without the use of antibiotics or a ‘cold’ chase and suggest that these cells could be a very useful model for detailed kinetic studies of RNA synthesis and migration. With the addition of glucose to the incubation medium in vitro the cessation of incorporation after 10 min is removed and a continuous pattern observed. These results are discussed in relation to the transport and utilization of uridine by these cells.