In vitro micropropagation of Dracaena sanderiana Sander ex Mast: An important indoor ornamental plant

A protocol has been developed for in vitro plant regeneration from a nodal explant of Dracaena sanderiana Sander ex Mast. Nodal explant showed high callus induction potentiality on MS medium supplemented with 6.78 μM 2,4-dichlorophenoxyacetic acid (2,4-D) followed by 46.5 μM chlorophenoxy acetic acid (CPA). The highest frequency of shoot regeneration (85%) and number of shoots per explant (5.6) were obtained on medium supplemented with 7.84 μM N⁶-benzylaminopurine (BA). Rooting was high on MS solid compared to liquid medium when added with 7.38 μM indole-3-butyric acid (IBA). Fifty percent of the roots were also directly rooted as microcuttings on soil rite, sand and peat mixture (1:1:1). In vitro and ex vitro raised plantlets were used for acclimatization. More than 90% of the plantlets was successfully acclimatized and established in plastic pots. Ex vitro transferred plantlets were normal without any phenotypic aberrations.     

Cryo‐Immunoelectron Microscopy of Adherent Cells Improved by the Use of Electrospun Cell Culture Substrates

Electrospun nanofibres are an excellent cell culture substrate, enabling the fast and non‐disruptive harvest and transfer of adherent cells for microscopical and biochemical analyses. Metabolic activity and cellular structures are maintained during the only half a minute‐long harvest and transfer process. We show here that such samples can be optimally processed by means of cryofixation combined either with freeze‐substitution, sample rehydration and cryosection‐immunolabelling or with freeze‐fracture replica‐immunolabelling. Moreover, electrospun fibre substrates are equally suitable for complementary approaches, such as biochemistry, fluorescence microscopy and cytochemistry.     

Opinion survey of the Hong Kong general public regarding genomic science and technology and their ethical and social implications

The study's objective is to survey the attitudes of Chinese people living in Hong Kong toward genomic science and technology (GST) and their ethical and social implications. Using a 24-item questionnaire, 877 Cantonese-speaking residents between age 18 and 64 with minimum high school education are interviewed by telephone. Multiple regression analysis identifies education level as the most important demographic variable. Overall, respondents have mild agreement with genetic determinism and the use of GST for disease prevention but not for non-therapeutic genetic enhancement and production of “genetically modified” crops or meat. Respondents strongly believe that GST tampers with nature and resources should be used to solve other healthcare problems first. Respondents also show little concern that personal genetic information may be abused by their employers or schools and have only a minimal willingness to share personal genetic information with their family members.     

Chronic exposure to a 2 mT static magnetic field affects the morphology,the metabolism and the function of in vitro cultured swine granulosa cells

In recent years, the exposure of organisms to static magnetic fields (SMFs) is continuously increasing. Thus, we investigated the effect of chronic exposure to a 2 mT SMF on in vitro cultured swine granulosa cells (GCs). In particular, the culture expansion (cell viability and doubling time), the cell phenotype (cell morphology and orientation, actin and α-tubulin cytoskeleton), the cell metabolism (intracellular Ca²⁺ concentration [Ca²⁺]_i and mitochondrial activity) and the cell function (endocrine activity) were assessed. It has been found that the exposure to the field did not affect the cell viability, but the doubling time was significantly reduced (p < 0.05) in exposed samples after 72 h of culture. At the same time, the cell length and thickness significantly changed (p < 0.05), while the cell orientation was unaffected. Evident modifications were induced on actin and α-tubulin cytoskeleton after 3 days of exposure and, simultaneously, a change in [Ca²⁺]_i and mitochondrial activity started to become evident. Finally, the SMF exposure of GCs longer than 72 h determined a significant alteration of progesterone and estrogen production (p < 0.05). In conclusion, our results demonstrate that the chronic exposure of swine GCs to a 2 mT SMF exerts a negative effect on cell proliferation, morphology, biochemistry and endocrine function in an in vitro model.     

Prolongation of Life in Anephric Rats following de novo Renal Organogenesis

One solution to the shortage of human organs available for transplantation envisions growing new organs in situ. This can be accomplished by transplantation of developing organ anlagen/primordia. Allotransplantation of embryonic day 15 metanephroi into the omentum of adult hosts is followed by differentiation, growth, vascularization and function of the implants. Here we show that survival of rats with all native renal mass removed can be increased by prior metanephros transplantation and ureteroureterostomy. Excretion of urine formed by metanephroi is prerequisite for enhanced survival. This is the first demonstration that life can be extended following de novo renal organogenesis.     

A simple method for using silicone elastomer masks for quantitative analysis of cell migration without cellular damage or substrate disruption

Cell migration is fundamental to many biological processes, including development, normal tissue remodeling, wound healing, and many pathologies. However, cell migration is a complex process, and understanding its regulation in health and disease requires the ability to manipulate and measure this process quantitatively under controlled conditions. This report describes a simple in vitro assay for quantitative analysis of cell migration in two-dimensional cultures that is an inexpensive alternative to the classic “scratch” assay. The method described utilizes flexible silicone masks fabricated in the lab according to the research demands of the specific experiment to create a cell-free area for cells to invade, followed by quantitative analysis based on widely available microscopic imaging tools. This experimental approach has the important advantage of visualizing cell migration in the absence of the cellular damage and disruption of the substrate that occurs when the “wound” is created in the scratch assay. This approach allows the researcher to study the intrinsic migratory characteristics of cells in the absence of potentially confounding contributions from cellular responses to injury and disruption of cell–substrate interactions. This assay has been used with vascular smooth muscle cells, fibroblasts, and epithelial cell types, but should be applicable to the study of practically any type of cultured cell. Furthermore, this method can be easily adapted for use with fluorescence microscopy, molecular biological, or pharmacological manipulations to explore the molecular mechanisms of cell migration, live cell imaging, fluorescence microscopy, and correlative immunolabeling.     

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.     

Density Gradient Multilayered Polymerization (DGMP): A Novel Technique for Creating Multi-compartment,Customizable Scaffolds for Tissue Engineering

Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix structure (e.g. composition, concentration, or stiffness of the matrix) vary over space, would enable a wide range of investigations concerning how these variables affect cell differentiation, migration, and other phenomena. The major challenge in creating layered matrices is maintaining the structural integrity of layer interfaces without diffusion of individual components from each layer¹. Current methodologies to achieve this include photopatterning^2-3, lithography⁴, sequential functionalization5, freeze drying⁶, microfluidics⁷, or centrifugation⁸, many of which require sophisticated instrumentation and technical skills. Others rely on sequential attachment of individual layers, which may lead to delamination of layers⁹. DGMP overcomes these issues by using an inert density modifier such as iodixanol to create layers of varying densities¹⁰. Since the density modifier can be mixed with any prepolymer or bioactive molecule, DGMP allows each scaffold layer to be customized. Simply varying the concentration of the density modifier prevents mixing of adjacent layers while they remain aqueous. Subsequent single step polymerization gives rise to a structurally continuous multilayered scaffold, in which each layer has distinct chemical and mechanical properties. The density modifier can be easily removed with sufficient rinsing without perturbation of the individual layers or their components. This technique is therefore well suited for creating hydrogels of various sizes, shapes, and materials.A protocol for fabricating a 2D-polyethylene glycol (PEG) gel, in which alternating layers incorporate RGDS-350, is outlined below. We use PEG because it is biocompatible and inert. RGDS, a cell adhesion peptide¹¹, is used to demonstrate spatial restriction of a biological cue, and the conjugation of a fluorophore (Alexa Fluor 350) enables us to visually distinguish various layers. This procedure can be adapted for other materials (e.g. collagen, hyaluronan, etc.) and can be extended to fabricate 3D gels with some modifications¹⁰.