Colorectal cancer metastasis: in the surgeon's hands?

BACKGROUND: Lymphovascular ligation before tumour manipulation during colorectal cancer resection is termed the 'no-touch isolation' technique. It aims to reduce the intra-operative dissemination of colorectal cancer cells. Recently, the detection of circulating tumour cells has been enhanced by molecular biology techniques. This paper reviews the evidence for the no-touch isolation technique in light of the recent developments in circulating tumour cell detection. METHODS: Studies investigating the effect of colorectal cancer surgery on circulating tumour cells were identified by a Medline search using the subject headings colorectal neoplasms and neoplasm circulating cells together with the map term 'no-touch isolation technique'. Further references were obtained from key articles. RESULTS: Molecular biological techniques have improved the detection of circulating colorectal cancer cells. There is a trend towards reduced tumour cell dissemination with the no-touch technique compared with the conventional method. However the benefit in terms of improved patient survival remains unproven. CONCLUSION: The no-touch isolation technique reduces circulating tumour cell dissemination but further work is needed to determine the significance of this with regards to patient survival.     

In vitro isolation of Plasmodium chabaudi merozoites by continuous flow ultrasound, cell sieving, concanavalin A-affinity chromatography and poly-L-lysine coated bead support columns

Techniques of merozoite isolation based on continuous flow ultrasound, cell sieving, Concanavalin A-affinity chromatography and cationically charged bead support columns were compared using the rodent malaria parasite, Plasmodium chabaudi. While each technique proved useful in isolating merozoites in reasonable numbers, the use of Con A-Sepharose 4B columns consistently provided the greatest numbers which were free of host cell contaminating membrane material and which were invasive to cells both in vivo and in vitro. In addition, Con A-Sepharose columns could be regenerated using alpha methyl-D mannoside. These results, when considered in light of merozoite isolation procedures used for other malarial species, indicated that the host-parasite model system had a bearing on which merozoite isolation technique was most likely to be successful.     

小鼠原代呼吸道上皮细胞分离培养的新方法

现有的呼吸道上皮细胞系大多来源于肿瘤组织或成系时与肿瘤细胞融合,其生物学行为与正常呼吸道上皮细胞差异较大.为更准确反映呼吸道疾病条件下该类细胞的生物学效应,本文对小鼠呼吸道上皮细胞分离的新技术和培养方法进行了探索.利用链霉蛋白酶消化法分离获得小鼠呼吸道上皮细胞,利用特殊的完全培养基和Ⅰ型胶原包被的培养皿进行原代培养.镜...     

Atrial muscle cells from hearts of adult guinea-pigs in culture: a new preparation for cardiac cellular electrophysiology

By means of a modified Langendorff perfusion technique using collagenase and elastase cell suspensions of viable myocytes from atria of adult guinea-pigs can be obtained. If the cell isolation is performed aseptically the myocytes can be kept in long term cell culture. Under these conditions the cells attach to the bottom of the culture dish within 12 to 24 h after plating. Thereafter they round up forming spherical 'cardioballs'. These cardioballs are highly suitable for electrophysiological experiments using different configurations (cell-attached and cell-free) of the patch-clamp technique. They can be employed for these experiments for up to 8 days after isolation. Thereafter they tend to flatten resembling embryonic heart cells in tissue culture.     

Progress in human hepatocytes: isolation,culture & cryopreservation

The quality of the hepatocytes used for clinical cell transplantation is very important, and depends to a large extent on the nature of the tissue used for isolation. The collagenase perfusion technique to isolate hepatocytes from animal livers has been further developed for isolation of human hepatocytes. As the donor organ pool is a scarce resource, marginal livers unsuitable for transplantation and segments from reduced grafts remain the main source of tissue for cell isolation. Use of livers from non-heart beating donors and foetal livers may further increase the tissue pool. With the limited supply of available tissue, improvements in the cryopreservation protocols are required to maintain cell viability on thawing and establish hepatocyte banks.     

A rapid method for the isolation of human peripheral null lymphocytes.

We describe here a new technique for the isolation of human peripheral null lymphocytes: cells lacking detectable surface immunoglobulin as well as receptors for sheep erythrocytes. The double rosette technique described employs a combined negative selection for Ig⁺ cells by specific anti-Ig-coated sheep erythrocytes and E rosette selection for T cells. This singlestep procedure facilitates the isolation of null cells from large cell populations and can be completed in less than 2 hr. The isolated null subpopulation represents less than 5% of the total peripheral mononuclear cells and is at least 85–90% pure by sensitive surface marker analysis. The null subpopulation is shown to be highly enriched for effectors of both antibody-dependent cytotoxicity against autologous lymphocytes and spontaneous cytotoxicity against the K562 leukemia blast cell line. Application of this technique will allow further characterization of the effector populations for ADCC and NK as well as differentiation of T and B cell precursors.     

Isolation of bovine retinal pigment epithelial cells using adhesion to agarose: demonstration of cellular and regional heterogeneity.

The retinal pigment epithelium (RPE) shows cell heterogeneity in morphology and enzymatic activity. Routine isolation procedures for RPE cells may reduce enzymatic activity and prevent the quantification of regional enzymatic differences in vivo. We developed a new technique for the isolation of RPE cells based on adhesion of the cells to agarose. The morphology of the isolated cells resembled that of RPE cells in vivo. The cells were viable in the dye exclusion test and showed a histochemical staining pattern as RPE cells in vivo. With this technique, quantitative regional differences in the enzymatic activities were detected.     

Novel Applications of Magnetic Cell Sorting to Analyze Cell-Type Specific Gene and Protein Expression in the Central Nervous System

The isolation and study of cell-specific populations in the central nervous system (CNS) has gained significant interest in the neuroscience community. The ability to examine cell-specific gene and protein expression patterns in healthy and pathological tissue is critical for our understanding of CNS function. Several techniques currently exist to isolate cell-specific populations, each having their own inherent advantages and shortcomings. Isolation of distinct cell populations using magnetic sorting is a technique which has been available for nearly 3 decades, although rarely used in adult whole CNS tissue homogenate. In the current study we demonstrate that distinct cell populations can be isolated in rodents from early postnatal development through adulthood. We found this technique to be amendable to customization using commercially available membrane-targeted antibodies, allowing for cell-specific isolation across development and animal species. This technique yields RNA which can be utilized for downstream applications—including quantitative PCR and RNA sequencing—at relatively low cost and without the need for specialized equipment or fluorescently labeled cells. Adding to its utility, we demonstrate that cells can be isolated largely intact, retaining their processes, enabling analysis of extrasomatic proteins. We propose that magnetic cell sorting will prove to be a highly useful technique for the examination of cell specific CNS populations.     

Isolation by a replica-plating technique of chinese hamster temperature-sensitive cell cycle mutants

Isolation of a wide variety of temperature-sensitive (ts) cell cycle mutants in mammalian cells has previously proved to be a very difficult task. The various procedures used for the isolation of such mutants included a mutant enrichment step based on exposure of the cells to the restrictive temperatures in order to kill the growing wild-type cells with agents that kill DNA-synthesizing cells. Hence, these methods favored the isolation of ts mutants that do not lose viability rapidly at the restrictive temperatures, We have treated cells of the Chinese hamster established cell line E36 with the mutagen ethyl-methane-sulfonate (EMS) and used a replicaplating technique that we developed to screen the ts mutants for growth. This technique enabled us to recover all ts mutants for growth including the ts cell cycle mutants. Screening of the ts cell cycle mutants among the ts mutants for growth was performed by the flow microfluorimetry technique and the premature chromosome condensation technique. Our results show that 1.3% of the survivors of the mutagenic treatment are ts mutants for growth. Six of 84 ts mutants analyzed were found to be ts cell cycle mutants. They include ts mutants arrested in phases G1, S, and G2. Many of the ts mutants for growth including the ts cell cycle mutants arrested in S and G2 lose viability very fast when incubated at the restrictive temperature. As a consequence they could not have been isolated by any method that includes a mutant enrichment step based on the exposure of the cells to the restrictive temperature.     

Mouse blastocyst immunosurgery with commercial antiserum to mouse erythrocytes

Summary Immunosurgery is a useful technique for the isolation of inner cell masses from murine blastocysts. Conventionally, rabbit antisera made ad hoc against murine splenic or fetal cells or fibroblasts have been used as antibody sources. We investigated the feasibility of using commercially available rabbit antiserum to murine erythrocytes (anti-RBC) and compared it with rabbit antiserum generated ad hoc to murine L-cells (anti-L-cell). Our results indicate that anti-RBC is at least as effective as anti-L-cell serum for the immunosurgical isolation of inner cell masses, which became either miniblastocysts (later forming outgrowths) or embryoid bodies (undergoing ectoderm-endodermlike differentiation within 48 h). Because anti-RBC is commercially available, the technical modification described herein increases the accessibility of the immunosurgical protocol for the isolation of murine inner cell masses.     

Rapid, high-yield purification of cell surface membrane using colloidal magnetite coated with polyvinylamine: sedimentation versus magnetic isolation

A new technique for the magnetic isolation of external plasma membrane from Dictyostelium discoideum is described and compared to a previously published procedure employing sedimentation of silica-coated plasma membrane. The magnetic isolation technique involves coating intact cells with a polyvinylamine-magnetite colloid and overcoating with polyacrylate to form a dense pellicle. The magnetite pellicle totally coated the cells and was not internalized. Coated cells were lysed and membrane fragments retrieved from the cell homogenate using a diverging field electromagnet. The membrane obtained in such a manner was analyzed for marker enzyme activity and cell surface label. The plasma membrane was obtained in high yield (42%) with an average purification of 8-fold. The polyvinylamine-magnetite pellicle shielded the external plasma membrane face to proteolysis by papain and pronase. It also acted as a barrier to alpha-methylmannoside in concanavalin A-carbohydrate competition studies.     

A comparison of cell wall disruption techniques for the isolation of intracellular metabolites from Pleurotus and Lepista sp

Different techniques were compared for their effectiveness in the disruption of the rigid cell walls of Basidiomycetes. Grinding under liquid nitrogen, stirred glass bead milling and enzymatic cell lysis were applied to the mycelia of Pleurotus sapidus and Lepista irina grown submerged. Each of the disruption procedures was evaluated by testing the quantity and quality of released intracellular metabolites: DNA, RNA, enzymes, and secondary metabolites. The most suitable method for nucleic acid isolation was grinding under liquid nitrogen, while bead mill homogenization was the superior technique for isolation of active enzymes. A new effective method is proposed for isolation of secondary metabolites with the aid of bead milling of fungal mycelia.     

外泌体提取及保存技术研究进展

外泌体是多种活细胞经过"内吞-融合-外排"等一系列过程主动向胞外分泌的纳米级双层膜结构小囊泡,广泛存在于血液和尿液等生物体液中.因其携带着多种蛋白质、核酸和脂质等生物活性分子,所以外泌体不仅在细胞间物质交换和信息传递中发挥重要作用,而且对疾病诊断、预后预测和治疗管理等均具有提示意义.外泌体的高效提取、分离和完整保存是研...     

Bacterial adherence to polystyrene: a replica method of screening for bacterial hydrophobicity.

A simple replica method is described for the rapid identification of colonies of bacteria which adhere to polystyrene. A correlation was found between the adherence of bacterial strains to polystyrene and cell surface hydrophobicity, suggesting the use of this technique in screening for cell surface mutants and in the isolation of hydrophobic bacteria from nature.     

Corneal endothelium: A modified method for cultivation

Summary A modified method for establishing cultures of rabbit corneal cells is described. The new technique utilized a Lucite disc in combination with a Tygon ring for growth of pure cell cultures and was compared with an explant method for growing cells. Each method provided adequate cell cultures for biochemical or ultrastructure studies of rabbit corneal cells, but the ring and disc method described here allowed the isolation of specific cell types without the interference of stromal cell contamination.     

An improved technique for the isolation of holotrich protozoa from rumen contents by differential filtration with defined aperture textiles

An improved differential filtration method for the isolation of preparations of the holotrich protozoa Dasytricha ruminantium and mixed Isotricha prostoma and I. intestinalis from rumen contents is described. The technique utilizes combinations of defined aperture Nylon or polyester textiles, which are superior to the sintered glass filters previously used in the isolation of rumen protozoa. Data obtained in a comparative study of some aspects of the metabolic activity exhibited by cell preparations isolated by the two filtration procedures are presented.     

Corneal endothelium: a modified method for cultivation

A modified method for establishing cultures of rabbit corneal cells is described. The new technique utilized a Lucite disc in combination with a Tygon ring for growth of pure cell cultures and was compared with an explant method for growing cells. Each method provided adequate cell cultures for biochemical or ultrastructure studies of rabbit corneal cells, but the ring and disc method described here allowed the isolation of specific cell types without the interference of stromal cell contamination.     

Use of mammalian cell expression cloning systems to identify genes for cytokines, receptors, and regulatory proteins.

Mammalian cell expression cloning has become a standard technique for the isolation of mammalian genes or cDNAs. Its advantage is that the biological functions of the gene of interest are used for cloning. Therefore, the identified cDNAs or genes should be functional in vivo, and there is no need for physical or chemical information about the gene products, so that protein purification in sufficient quantity to raise antibodies or to obtain amino acid sequences is not necessary. Here, we summarize recent progress in mammalian cell cloning systems, and discuss the possible directions in which this technique will lead.     

A universally applicable method of isolating somatic cell hybrids by two-colour flow sorting

A generally applicable technique is described that permits easy identification and isolation of heterokaryons a few hours after fusion. It is based on the labelling of living cells with different fluorochromes, which, at appropriate concentrations do not affect viability or gene expression. Both fluorochromes are relatively stable and do not cross-contaminate unlabelled cells. The technique has a powerful potential in studies on gene regulation in somatic cell hybrids since heterofluorescent hybrids between any type of cells can be isolated directly from large populations of monofluorescent parental cells by using a cell sorter equipped with a single laser. Thus the technique avoids the need for genetically marked parental cells for selection.