Plasma-derived serum as a selective agent to obtain endothelial cultures from swine aorta

Summary Endothelial cell and smooth muscle cell cultures from artery wall provide a potential model system for studying cellular processes involved in atherogenesis. To prepare serial subcultures of swine arterial endothelial cells that are free of smooth muscle cells without either selecting a small population or subjecting the cells to cytotoxic conditions, we used swine plasma-derived serum (SPDS) to establish conditions in which endothelial cells have a growth advantage. Endothelial cells were collected by collagenase digestion and smooth muscle cell cultures were prepared by outgrowth from explants of arterial medial segments. Growth rates were compared when each cell type was maintained on SPDS, or fetal bovine serum (FBS), or swine whole serum (SWS). When 20% FBS or SWS were used the doubling times were <30 h for both endothelial cells and smooth muscle cells. On 20% SPDS the doubling time for endothelial cells was 32 h, but for smooth muscle cells it was at least 168 h. Using SPDS, we prepare endothelial subcultures from swine aorta that express principally polygonal morphology at confluence. Endothelial cell cultures grown on SPDS have higher angiotensin-converting enzyme than those grown on FBS. This work was supported by grants HL 22486 and HL 24660 from the National Institutes of Health, Bethesda, Maryland. Dr. Slakey is an Established Investigator of the American Heart Association. Portions of this work were presented at the 31st Annual Meeting of the Tissue Culture Association in St. Louis, Missouri.     

Influence of a lathyric agent and of hypercholesterolemic serum on cell cultures of fetal rabbit aorta]

Cell cultures of foetal rabbit aorta are cultivated with a lathyric agent (beta-amino-propio-nitrile) or with an hypercholesterolemic serum; if morphological features, in these two cases, correspond with modifications observed, in vivo, when adult rabbits are respectively submitted to the same treatment, enzymatic activities of collagen metabolism vary in opposite way. Therefore, the influence of different parameters to be studied on vascular cell functions become easier.     

An affordable method to obtain cultured endothelial cells from peripheral blood

The culture of endothelial progenitor cells (EPC) provides an excellent tool to research on EPC biology and vascular regeneration and vasculogenesis. The use of different protocols to obtain EPC cultures makes it difficult to obtain comparable results in different groups. This work offers a systematic comparison of the main variables of most commonly used protocols for EPC isolation, culture and functional evaluation. Peripheral blood samples from healthy individuals were recovered and mononuclear cells were cultured. Different recovery and culture conditions were tested: blood volume, blood anticoagulant, coating matrix and percentage of foetal bovine serum (FBS) in culture media. The success of culture procedure, first colonies of endothelial cells appearance time, correlation with number of circulating EPC (cEPC) and functional comparison with human umbilical vein endothelial cells (HUVEC) were studied. The use of heparin, a minimum blood volume of 30 ml, fibronectin as a coating matrix and endothelial growing media‐2 supplemented with 20% FBS increased the success of obtaining EPC cultures up to 80% of the processed samples while reducing EPC colony appearance mean time to a minimum of 13 days. Blood samples exhibiting higher cEPC numbers resulted in reduced EPC colony appearance mean time. Cells isolated by using this combination were endothelial cell‐like EPCs morphological and phenotypically. Functionally, cultured EPC showed decreased growing and vasculogenic capacity when compared to HUVEC. Thus, above‐mentioned conditions allow the isolation and culture of EPC with smaller blood volumes and shorter times than currently used protocols.     

Strategy to obtain axenic cultures from field-collected samples of the cyanobacterium Phormidium animalis

An efficient strategy, based on a combination of procedures, was developed to obtain axenic cultures from field-collected samples of the cyanobacterium Phormidium animalis. Samples were initially cultured in solid ASN-10 medium, and a crude separation of major contaminants from P. animalis filaments was achieved by washing in a series of centrifugations and resuspensions in liquid medium. Then, manageable filament fragments were obtained by probe sonication. Fragmentation was followed by forceful washing, using vacuum-driven filtration through an 8-microm pore size membrane and an excess of water. Washed fragments were cultured and treated with a sequential exposure to four different antibiotics. Finally, axenic cultures were obtained from serial dilutions of treated fragments. Monitoring under microscope examination and by inoculation in Luria-Bertani (LB) agar plates indicated either axenicity or the degree of contamination throughout the strategy.     

Streptomycin as a selective agent to facilitate recovery and isolation of introduced and indigenous Sphingomonas from environmental samples

Sphingomonas is an organism of major interest for the degradation of organic contaminants in soils and other environments. A medium based on the aminoglycoside antibiotic streptomycin (Sm) was developed, which, together with the yellow pigmentation of Sphingomonas, facilitated the detection, recovery and quantification of culturable Sphingomonas from soils. All 29 previously described bacterial strains belonging to 17 different Sphingomonas species were able to grow on mineral media containing 200 microg ml(-1) streptomycin, showing that the capacity to resist high concentrations of Sm is a common characteristic within Sphingomonas. Incorporation of Sm into the mineral medium led to a significant reduction in the background microbial population and a concomitant 100 times more sensitive detection of Sphingomonas inoculated in non-sterile soil matrices. The Sm-containing medium was used to examine a variety of hydrocarbon-contaminated soils for the presence and biodiversity of Sphingomonas. Incorporation of Sm in the medium led to a significant increase in the number of yellow-pigmented colonies. Comparison of contaminated and non-contaminated soils derived from the same site revealed colonization by culturable yellow-pigmented Sm-resistant bacteria of the polluted location solely. Both yellow and non-yellow-pigmented colonies were purified from plates containing glucose and Sm, and BOX-polymerase chain reaction (PCR) was used to sort out clonally related strains. Representative strains from the major BOX-PCR clusters were identified using FAME and partial 16S rRNA gene sequencing. Forty-eight of 58 Sm-resistant isolates were identified as Sphingomonas sp. Streptomycin-resistant Sphingomonas isolates generated BOX-PCR diversity patterns that were site dependent and represented different species mainly belonging to Sphingomonas subgroups containing species formerly designated as Sphingopyxis and Sphingobium. The ability to degrade phenanthrene was only found in a minority of the Sphingomonas isolates, which all originated from soils containing high phenanthrene concentrations.     

Use of paromomycin as a selective agent for oat transformation

Summary Friable, embryogenic oat (Avena sativa L.) tissue cultures were stably transformed with two different plasmids containing the E. coli tn5 neomycin phosphotransferase II gene (npt II). Selection was accomplished using the antibiotic paromomycin sulfate following microprojectile bombardment. From two independent experiments, 88 paromomycin-resistant tissue cultures were shown to be transgenic based on Southern blot analysis and detection of the neomycin phosphotransferase (NPT II) protein using ELISA. Copy numbers of the npt II gene ranged from one to eight copies per haploid oat genome integrated into high molecular weight DNA of the paromomycin-resistant cultures. Plants were regenerated from 32 of the 88 transgenic tissue cultures. Plants from 17 of the 32 regenerable cultures exhibited fertility. Stable transformation was shown by segregation patterns of the NPT II protein in R₁ seedlings produced from 16 fertile culture lines that were tested. The overall results demonstrate that the combination of the npt II gene and paromomycin provides efficient selection of transgenic oat tissue cultures. Oat plants transformed with the npt II gene present reduced ecological risk compared to the previously used herbicide-resistance selection system.Abbreviations GUS beta-glucuronidase - uid A E. coli gene coding for GUS - NPT II neomycin phosphotransferase II of Tn 5 - npt II gene for NPT II - 2,4-D 2,4-dichlorophenoxy acid - X-gluc 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid cyclohexyl-ammonium salt - NOS nopaline synthase - NAA naphthalene acetic acid - BAP benzylaminopurine - ELISA enzyme-linked immunosorbant assay     

3-hydroxy-quinazoline-2,4-dione as a useful scaffold to obtain selective Gly/NMDA and AMPA receptor antagonists

The synthesis and Gly/NMDA, AMPA and KA receptor binding activities of some 3-hydroxy-quinazoline-2,4-dione derivatives are reported. The binding data, together with functional antagonism studies, showed that the 3-hydroxy-quinazoline-2,4-dione moiety can be considered a useful scaffold to obtain selective Gly/NMDA and AMPA receptor antagonists. In fact, introduction of chlorine atom(s) on precise position(s) of the benzofused moiety yielded Gly/NMDA selective antagonists, while the presence of the 6-(1,2,4-triazol-4-yl) group shifted the affinity and selectivity towards the AMPA receptor.     

A simple method to obtain pure cultures of multiciliated ependymal cells from adult rodents

Ependymal cells form an epithelium lining the ventricular cavities of the vertebrate brain. Numerous methods to obtain primary culture ependymal cells have been developed. Most of them use foetal or neonatal rat brain and the few that utilize adult brain hardly achieve purity. Here, we describe a simple and novel method to obtain a pure non-adherent ependymal cell culture from explants of the striatal and septal walls of the lateral ventricles. The combination of a low incubation temperature followed by a gentle enzymatic digestion allows the detachment of most of the ependymal cells from the ventricular wall in a period of 6 h. Along with ependymal cells, a low percentage (less than 6 %) of non-ependymal cells also detaches. However, they do not survive under two restrictive culture conditions: (1) a simple medium (alpha-MEM with glucose) without any supplement; and (2) a low density of 1 cell/µl. This purification method strategy does not require cell labelling with antibodies and cell sorting, which makes it a simpler and cheaper procedure than other methods previously described. After a period of 48 h, only ependymal cells survive such conditions, revealing the remarkable survival capacity of ependymal cells. Ependymal cells can be maintained in culture for up to 7–10 days, with the best survival rates obtained in Neurobasal supplemented with B27 among the tested media. After 7 days in culture, ependymal cells lose most of the cilia and therefore the mobility, while acquiring radial glial cell markers (GFAP, BLBP, GLAST). This interesting fact might indicate a reprogramming of the cell identity.     

Oxolinic acid as a selective agent for the isolation of non-sporing anaerobes from clinical material

The use of oxolinic acid as a selective agent for the isolation of non-sporing anaerobes from clinical material was investigated. At a concentration of 5 mg/l it compared favourably with nalidixic acid for this purpose and had the advantage of inhibiting the growth of staphylococci.     

Rifampin as a selective agent for the enumeration and isolation of spirochetes from salt marsh habitats

Spirochetes from a variety of salt marsh sediments were enumerated and isolated directly by using a prereduced complex medium containing rifampin as a selective agent. Based on cell morphology, the spirochetal strains isolated were divided into two groups, a group consisting of tightly coiled spirochetes and a group consisting of loosely coiled spirochetes. Generally, spirochete colony-forming units ranged between 10⁴ and 10⁶ per gram wet weight of the top 1 cm of sediment. Spirochete colony-forming units were in greater abundance in the uppermost 1–2 cm of sediment than in sediment taken from depths of 3–5 cm. Apparently, the top 1 or 2 cm of salt marsh sediment can provide a favorable habitat for certain spirochetal species.     

Potential for use of nicotinic acid as a selective agent for isolation of high nicotine-producing lines of Nicotiana rustica hairy root cultures

The addition of exogenous nicotinic acid, nicotinamide or nicotine was studied with reference to their effects on growth and alkaloid production by hairy root cultures of Nicotiana rustica. Nicotinic acid and nicotinamide were toxic (50% phytostatic dose being 2.4 and 9 mM respectively) while nicotine was not toxic below 10 mM. Nicotinic acid (up to 5 mM) was found to be phytostatic rather than phytotoxic. Roots exposed to increasing nicotinic acid or nicotinamide levels had altered alkaloid accumulation patterns relative to the controls. The principal effects were to increase the intracellular and extracellular levels of anatabine and nicotine, with a markedly greater proportion of anatabine being produced. The use of nicotinic acid as a selection agent for the recovery of higher alkaloid-producing lines is identified and discussed.     

Detection of bifidobacteria by using propionic acid as a selective agent.

This study introduces the use of the filtration membrane technique and the enrichment method to detect and enumerate bifidobacteria from various animal species.     

Devices and techniques used to obtain and analyze three-dimensional cell cultures

Cell cultures are indispensable for both basic and applied research. Advancements in cell culture and analysis increase their utility for basic research and translational applications. A marked development in this direction is advent of three-dimensional (3D) cultures. The extent of advancement in 3D cell culture methods over the past decade has warranted referring to a single cell type being cultured as an aggregate or spheroid using simple scaffolds as “traditional.” In recent years, the development of “next-generation” devices has enabled cultured cells to mimic their natural environments much better than the traditional 3D culture systems. Automated platforms like chip-based devices, magnetic- and acoustics-based assembly devices, di-electrophoresis (DEP), micro pocket cultures (MPoC), and 3D bio-printing provide a dynamic environment compared to the rather static conditions of the traditional simple scaffold-based 3D cultures. Chip-based technologies, which are centered on principles of microfluidics, are revolutionizing the ways in which cell culture and analysis can be compacted into table-top instruments. A parallel evolution in analytical devices enabled efficient assessment of various complex physiological and pathological endpoints. This is augmented by concurrent development of software enabling rapid large-scale automated data acquisition and analysis like image cytometry, elastography, optical coherence tomography, surface-enhanced Raman scattering (SERS), and biosensors. The techniques and devices utilized for the purpose of 3D cell culture and subsequent analysis depend primarily on the requirement of the study. We present here an in-depth account of the devices for obtaining and analyzing 3D cell cultures.     

Novel method to obtain highly enriched cultures of adult rat Schwann cells

Schwann cells (SCs) can be used to repair both the peripheral and central nervous systems. Therefore, establishment of a procedure to obtain activated, highly proliferative SCs, in an appropriate time for clinical applications, is a prerequisite. Purification is complicated by contamination with fibroblasts which often become the predominant cell type in an in vitro SC culture. This study describes a novel and efficient method to enrich SCs by utilizing the differential detachment properties of the two cell types. In culture, cells were treated with two different media and the chelator, EGTA, which detached SCs faster than fibroblasts and allowed for easy isolation of SCs. Within seven days, high yields of SCs with a purity of greater than 99% were achieved. This was confirmed by immunostaining characterization and flow-cytometric analyses using an antibody against the p75 low affinity nerve growth factor receptor (p75LNGFR). The entire procedure was completed in approximately 21 days. This method has the advantage of being technically easier, faster, and more efficient than other previously described methods. An SC culture that was about 99% homogenous was achieved.     

An efficient method to obtain axenic cultures of Alexandrium tamarense--a PSP-producing dinoflagellate

The fact that species of harmful algae maintained in the laboratory harbor a complex bacterial flora increases the difficulties involved in the study of the relationship between bacteria and algae. An efficient method to remove bacteria from a laboratory culture of the marine dinoflagellate Alexandrium tamarense is presented in this paper. The alga was subjected to repeated washing, lysozyme/SDS and antibiotic treatment with a mixture of gentamycin, streptomycin, cephalothin and rifampicin. Axenic status was confirmed after subculturing three times in sterile f/2 medium without antibiotics. Bacteria could not be detected in various media, both solid and liquid, nor by epifluorescence microscopy and PCR amplification of 16S rDNA of both eubacteria and archaea. Bacterial presence was monitored throughout a full growth cycle and, following subculture, no bacteria were detected using the above methods. This method is more efficient and less time-consuming than other methods and the resultant axenic A. tamarense cultures would provide a simpler system for further study of bacteria-alga interactions.     

Discovery of a novel selective water-soluble SMAD3 inhibitor as an antitumor agent

Targeting the SMAD3 protein is an attractive therapeutic strategy for treating cancer, as it avoids the potential toxicities due to targeting the TGF-β signaling pathway upstream. Compound SIS3 was the first selective SMAD3 inhibitor developed that had acceptable activity, but its poor water solubility limited its development. Here, a series of SIS3 analogs was created to investigate the structure–activity relationship for inhibiting the activation of SMAD3. On the basis of this SAR, further optimization generated a water-soluble compound, 16d, which was capable of effectively blocking SMAD3 activation in vitro and had similar NK cell-mediated anticancer effects in vivo to its parent SIS3. This study not only provided a preferable lead compound, 16d, for further drug discovery or a potential tool to study SMAD3 biology, but also proved the effectiveness of our strategy for water-solubility driven optimization.