Improving the biosafety of cell sorting by adaptation of a cell sorting system to a biosafety cabinet.

BACKGROUND: The jet-in-air cell sorters currently available are not very suitable for sorting potentially biohazardous material under optimal conditions because they do not protect operators and samples as recommended in the guidelines for safe biotechnology. To solve this problem we have adapted a cell sorting system to a special biosafety cabinet that satisfies the requirements for class II cabinets. With aid of this unit, sorting can be performed in conformance with the recommendations for biosafety level 2. METHODS: After integrating a modified fluorescence-activated cell sorter (FACS) Vantage into a special biosafety cabinet, we investigated the influence of the laminar air flow (LAF) inside the cabinet on side stream stability and the analytical precision of the cell sorter. In addition to the routine electronic counting of microparticles, we carried out tests on the containment of aerosols, using T4 bacteriophage as indicators, to demonstrate the efficiency of the biosafety cabinet for sorting experiments performed under biosafety level 2 conditions. RESULTS: The experiments showed that LAF, which is necessary to build up sterile conditions in a biosafety cabinet, does not influence the conditions for side stream stability or the analytical precision of the FACS Vantage cell sorting system. In addition, tests performed to assess aerosol containment during operation of the special biosafety cabinet demonstrated that the cabinet-integrated FACS Vantage unit (CIF) satisfies the conditions for class II cabinets. In the context of gene transfer experiments, the CIF facility was used to sort hematopoietic progenitor cells under biosafety level 2 conditions. CONCLUSIONS: The newly designed biosafety cabinet offers a practical modality for improving biosafety for operators and samples during cell sorting procedures. It can thus also be used for sorting experiments with genetically modified organisms in conformance with current biosafety regulations and guidelines.     

Sophistications of cell sorting

The self-sorting of early embryonic cells is mediated not only by pure differential adhesion but also involves other processes. Direct force measurements reveal the role of cell-cortical tension, whereas epithelial-wrapping dominates the sorting of enclosed mesenchymal cells.     

Raman activated cell sorting

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Flow cytometry and cell sorting

Flow cytometry has become an important research tool in cytology, genetics, immunology, and microbiology. The information gained from cytometric instruments is quantitative and of high statistical precision, enabling resolution of cell subpopulations. Although increasing, application to cytology is hindered by inadequate appreciation of the nature of flow cytometry and the information obtained. Many cytologic questions can be reexamined from the perspective of this technology to obtain knowledge not accessible with conventional techniques. A flow cytometer and cell sorter are described. The physical, biochemical, and functional properties measurable by these systems are discussed.     

A model of cell sorting

Voronoi polygons are introduced as a suitable representation for a two-dimensional cell sheet. These polygons are defined in terms of a finite number of points, making numerical simulations tractable and yet allowing cells to change neighbors and their shape in response to deforming forces without leaving gaps in the tissue. Using this geometry and an extension of the equilibrium theory proposed by Steinberg to drive the motion, simulations of rounding of uneven tissue and engulfment of two intact tissues are carried out.     

Applications of cell sorting in biotechnology

Due to its unique capability to analyze a large number of single cells for several parameters simultaneously, flow cytometry has changed our understanding of the behavior of cells in culture and of the population dynamics even of clonal populations. The potential of this method for biotechnological research, which is based on populations of living cells, was soon appreciated. Sorting applications, however, are still less frequent than one would expect with regard to their potential. This review highlights important contributions where flow cytometric cell sorting was used for physiological research, protein engineering, cell engineering, specifically emphasizing selection of overproducing cell lines. Finally conclusions are drawn concerning the impact of cell sorting on inverse metabolic engineering and systems biology.     

Developmental age-related cell sorting inDictyostelium discoideum

Summary The sorting behavior of mixtures ofD. discoideum cells which had been developed for different lengths of time was examined. Cells developed for 4 and 8 h were mixed together and allowed to form slugs. Within the slugs, 8 h cells sorted to the anterior prestalk region while 4 h cells sorted to the posterior prespore region. These results indicate that the more developed a cell is, the more likely it is to become part of the prestalk zone in the slug. They are also consistent with the differential adhesion and chemotaxis hypotheses as a mechanism for cell sorting since cells become more adhesive and chemotactically responsive as development proceeds.     

Cadherin-mediated cell sorting not determined by binding or adhesion specificity.

Cadherin adhesion molecules play important roles in the establishment of tissue boundaries. Cells expressing different cadherins sort out from each other in cell aggregation assays. To determine the contribution of cadherin binding and adhesion specificity to the sorting process, we examined the adhesion of cells to different purified cadherin proteins. Chinese hamster ovary cell lines expressing one of four different cadherins were allowed to bind to the purified cadherin extracellular domains of either human E-cadherin or Xenopus C-cadherin, and the specificity of adhesion was compared with cell-sorting assays. None of the different cadherin-expressing cells exhibited any adhesive specificity toward either of the two purified cadherin substrates, even though these cadherins differ considerably in their primary sequence. In addition, all cells exhibited similar strengthening of adhesion on both substrates. However, this lack of adhesive specificity did not determine whether different cadherin-expressing cells would sort from each other, and the tendency to sort was not predictable by the extent of sequence diversity in their extracellular domains. These results show that cadherins are far more promiscuous in their adhesive-binding capacity than had been expected and that the ability to sort out must be determined by mechanisms other than simple adhesive-binding specificity.     

Fluorescence-activated cell sorting for aptamer SELEX with cell mixtures

Aptamers that target a specific cell subpopulation within composite mixtures represent invaluable tools in biomedical research and in the development of cell-specific therapeutics. Here we describe a detailed protocol for a modular and generally applicable scheme to select aptamers that target the subpopulations of cells in which you are interested. A fluorescence-activated cell-sorting device is used to simultaneously differentiate and separate those subpopulations of cells having bound and unbound aptamers. There are fewer false positives when using this approach in comparison with other cell-selection approaches in which unspecific binding of nucleic acids to cells with reduced membrane integrity or their unselective uptake by dead cells occurs more often. The protocol provides a state-of-the-art approach for identifying aptamers that selectively target virtually any cell type under investigation. As an example, we provide the step-by-step protocol targeting CD19(+) Burkitt's lymphoma cells, starting from the pre-SELEX (systematic evolution of ligands by exponential amplification) measurements to establish suitable SELEX conditions and ending at completion of the SELEX procedure, which reveals the enriched single-stranded DNA library.     

Single cell Raman spectroscopy for cell sorting and imaging

Single cell Raman spectroscopy (SCRS) is a non-invasive and label-free technology, allowing in vivo and multiple parameter analysis of individual living cells. A single cell Raman spectrum usually contains more than 1000 Raman bands which provide rich and intrinsic information of the cell (e.g. nucleic acids, protein, carbohydrates and lipids), reflecting cellular genotypes, phenotypes and physiological states. A Raman spectrum serves as a molecular 'fingerprint' of a single cell, making it possible to differentiate various cells including bacterial, protistan and animal cells without prior knowledge of the cells. However, a key drawback of SCRS is the fact that spontaneous Raman signals are naturally weak; this review discusses recent research progress in significantly enhancing and improving the signal of spontaneous Raman spectroscopy, including resonance Raman spectroscopy (RRS), coherent anti-Stokes Raman spectroscopy (CARS), stimulated Raman spectroscopy (SRS) and surface enhanced Raman scattering (SERS). This review focuses on the biotechnological development and the associated applications of SCRS, including Raman activated cell sorting (RACS) and Raman imaging and mapping.     

Automatic cell identification and enrichment in lung cancer. II. Acridine orange for cell sorting of sputum.

Fluorescence spectra were obtained from cells from sputum and pleural effusions stained with different fluorescent dyes and fixed by alternate methods. The spectra were referenced to a standard allowing for fluorescence comparisons of unstained and stained cells under various conditions. The metachromasia of acridine orange-stained cells offers nuclear/cytoplasmic differentiation in a single stain; mithramycin and propidium iodide do not. Unstained cells have an appreciable amount of green (546 nm) fluorescence, as does Carbowax in Saccomanno's preservative. Cytoplasm stained with acidine orange also has appreciable green fluorescence. Consequently, cells with much cytoplasm have high total fluorescence. Cytoplasmic fluorescence is negligible with mithramycin or propidium iodide. The metachromasia of acridine orange-stained cells is altered by alcohol and Carbowax levels in fixatives, keeping other factors constant.     

Multiplexed labeling system for high-throughput cell sorting

Flow cytometry and fluorescence activated cell sorting techniques were designed to realize configurable classification and separation of target cells. A number of cell phenotypes with different functionalities have recently been revealed. Before simultaneous selective capture of cells, it is desirable to label different samples with the corresponding dyes in a multiplexing manner to allow for a single analysis. However, few methods to obtain multiple fluorescent colors for various cell types have been developed. Even when restricted laser sources are employed, a small number of color codes can be expressed simultaneously. In this study, we demonstrate the ability to manifest DNA nanostructure-based multifluorescent colors formed by a complex of dyes. Highly precise self-assembly of fluorescent dye-conjugated oligonucleotides gives anisotropic DNA nanostructures, Y- and tree-shaped DNA (Y-DNA and T-DNA, respectively), which may be used as platforms for fluorescent codes. As a proof of concept, we have demonstrated seven different fluorescent codes with only two different fluorescent dyes using T-DNA. This method provides maximum efficiency for current flow cytometry. We are confident that this system will provide highly efficient multiplexed fluorescent detection for bioanalysis compared with one-to-one fluorescent correspondence for specific marker detection.     

The cellular basis of cell sorting kinetics

Cell sorting is a dynamical cooperative phenomenon that is fundamental for tissue morphogenesis and tissue homeostasis. According to Steinberg's differential adhesion hypothesis, the structure of sorted cell aggregates is determined by physical characteristics of the respective tissues, the tissue surface tensions. Steinberg postulated that tissue surface tensions result from quantitative differences in intercellular adhesion. Several experiments in cell cultures as well as in developing organisms support this hypothesis.The question of how tissue surface tension might result from differential adhesion was addressed in some theoretical models. These models describe the cellular interdependence structure once the temporal evolution has stabilized. In general, these models are capable of reproducing sorted patterns. However, the model dynamics at the cellular scale are defined implicitly and are not well-justified. The precise mechanism describing how differential adhesion generates the observed sorting kinetics at the tissue level is still unclear.It is necessary to formulate the concepts of cell level kinetics explicitly. Only then it is possible to understand the temporal development at the cellular and tissue scales. Here we argue that individual cell mobility is reduced the more the cells stick to their neighbors. We translate this assumption into a precise mathematical model which belongs to the class of stochastic interacting particle systems. Analyzing this model, we are able to predict the emergent sorting behavior at the population level. We describe qualitatively the geometry of cell segregation depending on the intercellular adhesion parameters. Furthermore, we derive a functional relationship between intercellular adhesion and surface tension and highlight the role of cell mobility in the process of sorting. We show that the interaction between the cells and the boundary of a confining vessel has a major impact on the sorting geometry.     

Characterization of stem cells and progenitors of hemopoiesis by cell sorting.

Cell sorting has been used as a method for characterizing hemopoietic stem cells and progenitors. Fluorescent antibody-surface labels and changes in fluorescence polarization induced by in vitro stimulation with potential hemopoietic regulators were used. As detected by significant enrichment of CFU-S (pluripotent stem cells) in fluorescence-activated cell sorting, some CFU-S bear 'unique antigens' recognized by rabbit anti-human brain sera, human anti-human sperm sera, and 129 anti-F9 serum, but not A . TH anti-A . TL (Ia) ascites. Significant changes in fluorescence polarization induced by in vitro stimulation of mouse bone marrow with potential hemopoietic regulators were also observed; further, progenitors of human T-lymphocyte colonies were observed to exhibit a significantly decreased mean polarization value after short-term stimulation with PHA-LCM (phytohemagglutinin-stimulated leukocyte conditioned medium).     

Purification of cybrids by fluorescence-activated cell sorting

A general method to isolate and purify substantial numbers of viable cybrids from cultured mammalian cells immediately following cytoplast-cell fusion is described. This method uses cytoplasts whose mitochondria are selectively stained in vivo by the cationic fluorescent rhodamine dye, rhodamine 123. Large numbers of highly purified, rhodamine-stained cytoplasts are fused to appropriate recipient cell lines and then the fusion mixture is sorted based on forward angle scatter and fluorescence parameters. Plating the positively sorted population in culture for as short as 12 h eliminates contaminating cytoplasts which, lacking a nucleus, are unable to adhere or survive. The resultant population, based on an analysis of genetic markers, is 75-100% cybrids, an enrichment of 1000- to 10,000-fold over the initial fusion mixture. Cybrids purified by cell sorting may be useful for detailed molecular studies of mitochondrial DNA gene expression and in the specific induction of new mitochondrial DNA mutants.     

Flow sorting of antigen-binding B cell subsets

Antigen binding was used as a probe in the definition of functional B cell heterogeneity. Unprimed, anti-Thy 1 and complement-treated spleen cells were stained with fluorescent trinitrophenylated bovine serum albumin (FL-TNP-BSA). These cells were sorted, fluorescence negative from fluorescence positive, by using a multiparameter cell sorter and assayed for precursor frequency in antibody responses to TNP by limiting dilution analysis. The cells that bound FL-TNP-BSA were demonstrated to be enriched for antibody-forming precursors to the antigens TNP-lipopolysaccharide (LPS), TNP-sheep red blood cells (SRBC), and TNP- or DNP-Ficoll, whereas the fluorescence negative cells were depleted for these responses. B cells that bound FL-TNP-BSA were then sorted into populations that bound a moderate or high amount of FL-TNP-BSA. The B cells responsive to TNP-LPS and TNP-SRBC were present in both the moderate and high binding populations. In contrast, the B cells responsive to TNP- or DNP-Ficoll were present only in the cells that bound a moderate amount of FL-TNP-BSA. These experiments suggest that there is a population of B cells in adult mouse spleen that binds large amounts of antigen, and that can respond to antigen carried on LPS or SRBC but not carried on Ficoll.     

Fluorescence-activated cell sorting of specific affibody-displaying staphylococci

Efficient enrichment of staphylococcal cells displaying specific heterologous affinity ligands on their cell surfaces was demonstrated by using fluorescence-activated cell sorting. Using bacterial surface display of peptide or protein libraries for the purpose of combinatorial protein engineering has previously been investigated by using gram-negative bacteria. Here, the potential for using a gram-positive bacterium was evaluated by employing the well-established surface expression system for Staphylococcus carnosus. Staphylococcus aureus protein A domains with binding specificity to immunoglobulin G or engineered specificity for the G protein of human respiratory syncytial virus were expressed as surface display on S. carnosus cells. The surface accessibility and retained binding specificity of expressed proteins were demonstrated in whole-cell enzyme and flow cytometry assays. Also, affibody-expressing target cells could be sorted essentially quantitatively from a moderate excess of background cells in a single step by using a high-stringency sorting mode. Furthermore, in a simulated library selection experiment, a more-than-25,000-fold enrichment of target cells could be achieved through only two rounds of cell sorting and regrowth. The results obtained indicate that staphylococcal surface display of affibody libraries combined with fluoresence-activated cell sorting might indeed constitute an attractive alternative to existing technology platforms for affinity-based selections.     

Reversal of tissue position after cell sorting

In most tissue combinations which have been studied, cell sorting results in highly reproducible tissue patterns. In a given combination of two cell types, one cell type always occupies the perimeter of the aggregate and the other always occupies the interior. In some tissue combinations, however, position reversals have been observed to occur. The present study deals with position reversals in chick embryo heart-pigmented retinal epithelium and heart-limb bud mesenchyme tissue combinations. Evidence is presented which implies that this reversal of tissue position results from changes in cellular adhesiveness during cell culture.     

The mechanics of cell sorting and envelopment

Aggregates of embryonic cells undergo a variety of intriguing processes including sorting by histological type and envelopment of cell masses of one type by another. It has long been held that these processes were driven by differential adhesions, as embodied in the famous differential adhesion hypothesis (DAH). Here, we use analytical mechanics to investigate the forces that are generated by various sub-cellular structures including microfilaments, cell membranes and their associated proteins, and by sources of cell-cell adhesions. We consider how these forces cause the triple junctions between cells to move, and how these motions ultimately give rise to phenomena such as cell sorting and tissue envelopment. The analyses show that, contrary to the widely accepted DAH, differential adhesions alone are unable to drive sorting and envelopment. They show, instead, that these phenomena are driven by the combined effect of several force generators, as embodied in an equivalent surface or interfacial tension. These unconventional findings follow directly from the relevant surface physics and mechanics, and are consistent with well-known cell sorting and envelopment experiments, and with recent computer simulations.