Abstract: We describe a novel microfluidic cell sorter which operates in continuous flow at high sorting rates. The device is based on a surface acoustic wave cell-sorting scheme and combines many advantages of fluorescence activated cell sorting (FACS) and fluorescence activated droplet sorting (FADS) in microfluidic channels. It is fully integrated on a PDMS device, and allows fast electronic control of cell diversion. We direct cells by acoustic streaming excited by a surface acoustic wave which deflects the fluid independently of the contrast in material properties of deflected objects and the continuous phase; thus the device underlying principle works without additional enhancement of the sorting by prior labelling of the cells with responsive markers such as magnetic or polarizable beads. Single cells are sorted directly from bulk media at rates as fast as several kHz without prior encapsulation into liquid droplet compartments as in traditional FACS. We have successfully directed HaCaT cells (human keratinocytes), fibroblasts from mice and MV3 melanoma cells. The low shear forces of this sorting method ensure that cells survive after sorting.

Abstract: We propose a unique method for cell sorting, “Ephesia,” using columns of biofunctionalized superparamagnetic beads self-assembled in a microfluidic channel onto an array of magnetic traps prepared by microcontact printing. It combines the advantages of microfluidic cell sorting, notably the application of a well controlled, flow-activated interaction between cells and beads, and those of immunomagnetic sorting, notably the use of batch-prepared, well characterized antibody-bearing beads. On cell lines mixtures, we demonstrated a capture yield better than 94%, and the possibility to cultivate in situ the captured cells. A second series of experiments involved clinical samples—blood, pleural effusion, and fine needle aspirates— issued from healthy donors and patients with B-cell hematological malignant tumors (leukemia and lymphoma). The immunophenotype and morphology of B-lymphocytes were analyzed directly in the microfluidic chamber, and compared with conventional flow cytometry and visual cytology data, in a blind test. Immunophenotyping results using Ephesia were fully consistent with those obtained by flow cytometry. We obtained in situ high resolution confocal three-dimensional images of the cell nuclei, showing intranuclear details consistent with conventional cytological staining. Ephesia thus provides a powerful approach to cell capture and typing allowing fully automated high resolution and quantitative immunophenotyping and morphological analysis. It requires at least 10 times smaller sample volume and cell numbers than cytometry, potentially increasing the range of indications and the success rate of microbiopsy-based diagnosis, and reducing analysis time and cost.

Abstract: The detection, isolation and sorting of cells are important tools in both clinical diagnostics and fundamental research. Advances in microfluidic cell sorting devices have enabled scientists to attain improved separation with comparative ease and considerable time savings. Despite the great potential of Lab-on-Chip cell sorting devices for targeting cells with desired specificity and selectivity, this field of research remains unexploited. The challenge resides in the detection techniques which has to be specific, fast, cost-effective, and implementable within the fabrication limitations of microchips. Adhesion-based microfluidic devices seem to be a reliable solution compared to the sophisticated detection techniques used in other microfluidic cell sorting systems. It provides the specificity in detection, label-free separation without requirement for a preprocessing step, and the possibility of targeting rare cell types. This review elaborates on recent advances in adhesion-based microfluidic devices for sorting, detection and enrichment of different cell lines, with a particular focus on selective adhesion of desired cells on surfaces modified with ligands specific to target cells. The effect of shear stress on cell adhesion in flow conditions is also discussed. Recently published applications of specific adhesive ligands and surface functionalization methods have been presented to further elucidate the advances in cell adhesive microfluidic devices.

Abstract: Toll-like receptors (TLRs) are critical components in the regulation of pulmonary immune responses and the recognition of respiratory pathogens such as Mycobacterium Tuberculosis (M.tb). Through examination of human alveolar macrophages this study attempts to better define the expression profiles of TLR2, TLR4 and TLR9 in the human lung compartment which are as yet still poorly defined. Sixteen healthy subjects underwent venipuncture, and eleven subjects underwent additional bronchoalveolar lavage to obtain peripheral blood mononuclear and bronchoalveolar cells, respectively. Surface and intracellular expression of TLRs was assessed by fluorescence-activated cell sorting and qRT-PCR. Cells were stimulated with TLR-specific ligands and cytokine production assessed by ELISA and cytokine bead array. Surface expression of TLR2 was significantly lower on alveolar macrophages than on blood monocytes (1.2 ± 0.4% vs. 57 ± 11.1%, relative mean fluorescence intensity [rMFI]: 0.9 ± 0.1 vs. 3.2 ± 0.1, p < 0.05). The proportion of TLR4 and TLR9-expressing cells and the rMFIs of TLR4 were comparable between alveolar macrophages and monocytes. The surface expression of TLR9 however, was higher on alveolar macrophages than on monocytes (rMFI, 218.4 ± 187.3 vs. 4.4 ± 1.4, p < 0.05) while the intracellular expression of the receptor and the proportion of TLR9 positive cells were similar in both cell types. TLR2, TLR4 and TLR9 mRNA expression was lower in bronchoalveolar cells than in monocytes. Pam3Cys, LPS, and M.tb DNA upregulated TLR2, TLR4 and TLR9 mRNA in both, bronchoalveolar cells and monocytes. Corresponding with the reduced surface and mRNA expression of TLR2, Pam3Cys induced lower production of TNF-α, IL-1β and IL-6 in bronchoalveolar cells than in monocytes. Despite comparable expression of TLR4 on both cell types, LPS induced higher levels of IL-10 in monocytes than in alveolar macrophages. M.tb DNA, the ligand for TLR9, induced similar levels of cytokines in both cell types. The TLR expression profile of autologous human alveolar macrophages and monocytes is not identical, therefore perhaps contributing to compartmentalized immune responses in the lungs and systemically. These dissimilarities may have important implications for the design and efficacy evaluation of vaccines with TLR-stimulating adjuvants that target the respiratory tract.

Abstract: Background: Human postnatal stem cells have been identified in periodontal ligaments (PDLs). In this study, the in vitro biologic properties of CD105+ enriched cell subsets from PDLs harvested from deciduous (DePDL) and permanent (PePDL) teeth are comparatively assessed.Methods: PDL tissue was obtained from 12 teeth (six primary and six permanent) from which CD105+ CD34− CD45− cells were isolated by magnetic cell sorting. To identify and quantitatively compare the stem cell markers, DePDL and PePDL cells were assessed for CD166 surface antigen expression by flow cytometry, real-time polymerase chain reaction, and immunostaining for Stro-1 and Oct-4, osteogenic and adipogenic differentiation, and proliferation rate by trypan blue method.Results: Magnetic cell sorting isolated cell populations containing 23.87% (± 11.98%) and 11.68% (± 6.27%) of CD105+ expressing cells from PePDL and DePDL, respectively. Flow cytometric analysis demonstrated a higher proportion of CD105+ cells coexpressing CD166 surface ant...

Abstract: Despite many years of intensive effort, there is surprisingly little consensus on the most suitable markers with which to locate and isolate stem cells from adult tissues. By comparison, the study of cancer stem cells is still in its infancy; so, unsurprisingly, there is great uncertainty as to the identity of these cells. Stem cell markers can be broadly categorized into molecular determinants of self-renewal, clonogenicity, multipotentiality, adherence to the niche, and longevity. This review assesses the utility of recognizing cancer stem cells by virtue of high expression of aldehyde dehydrogenases (ALDHs), probably significant determinants of cell survival through their ability to detoxify many potentially cytotoxic molecules, and contributing to drug resistance. Antibodies are available against the ALDH enzyme family, but the vast majority of studies have used cell sorting techniques to enrich for cells expressing these enzymes. Live cells expressing high ALDH activity are usually identified by the ALDEFLUOR kit and sorted by fluorescence activated cell sorting (FACS). For many human tumours, but notably breast cancer, cell selection based upon ALDH activity appears to be a useful marker for enriching for cells with tumour-initiating activity (presumed cancer stem cells) in immunodeficient mice, and indeed the frequency of so-called ALDH(bri) cells in many tumours can be an independent prognostic indicator.

Abstract: To achieve an integrated understanding of the stem cell system of planarians at both the cellular and molecular levels, we developed a new method by combining "fluorescent activated cell sorting (FACS) index sorting" analysis and single-cell reverse transcription-polymerase chain reaction (RT-PCR) to detect the gene expression and cell cycle state of stem cells simultaneously. Single cells were collected using FACS, and cDNAs of each cell were used for semi-quantitative RT-PCR. The results were plotted on the FACS sorting profile using the "index sorting" function, which enabled us to analyze the gene expression in combination with cell biological data (such as cell cycle phase) for each cell. Here we investigated the adult stem cells of planarians using this method and obtained findings suggesting that the stem cells might undergo commitment during S to G2/M phase. This method could be a powerful and straightforward tool for examining the stem cell biology of not only planarians but also other organisms, including vertebrates.

Abstract: MSCs are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and can be easily separated from the hematopoietic stem cells (HSCs) due to their plastic adherence. This protocol describes how to isolate MSCs from rat femurs and tibias. The isolated cells were further enriched against two MSCs surface markers CD54 and CD90 by magnetic cell sorting. Expression of surface markers CD54 and CD90 were then confirmed by flow cytometry analysis. HSC marker CD45 was also included to check if the sorted MSCs were depleted of HSCs. MSCs are naturally quite heterogeneous. There are subpopulations of cells that have different shapes, proliferation and differentiation abilities. These subpopulations all express the known MSCs markers and no unique marker has yet been identified for the different subpopulations. Therefore, an alternative approach to separate out the different subpopulations is using cloning cylinders to separate out single-colony derived cells. The cells derived from the single-colonies can then be cultured and evaluated separately.

Abstract: Tumor stem cells or cancer initiating cells (CICs) are single tumor cells that can regenerate a tumor or a metastasis. The identification and isolation of CICs remain challenging, and a variety of putative CIC markers have been described. We hypothesized that cell lines of the NCI60 panel contain CICs and express putative CIC markers. We investigated expression of putative CIC surface markers (CD15, CD24, CD44, CD133, CD166, CD326, PgP) and the activity of aldehyde dehydrogenase in the NCI60 panel singly and in combination by six-color fluorescence-activated cell sorting analysis. All investigated markers were expressed in cell lines of the NCI60 panel. Expression levels of individual markers varied widely across the 60 cell lines, and neither single marker expression nor simple combinations nor co-expression patterns correlated with the colony-formation capacity of cell lines. Rather, marker expression patterns correlated with tumor types in multidimensional analysis. Whereas some expression patterns correlated with tumor entities such as basal breast cancer, other expression patterns occurred across different tumor types and largely related to expression of a more mesenchymal phenotype in individual breast, lung, renal, and melanoma cell lines. Our data for the first time demonstrate that tumor cell lines display CIC markers in a complex pattern that relates to the tumor type. The complexity and tumor type specificity of marker display creates challenges for the application of cell sorting and other approaches to isolation of putative tumor stem cell populations and suggests that therapeutic targeting strategies will need to take this into account.

Abstract: Background and Aim: MicroRNAs are a class of small non-coding RNAs that negatively regulate the expression of their target genes. The aim of the present study was to explore the effects of microRNA on biological behaviors of HepG2 cells and further analyze its characteristics. Methods: We detected different expression profiles of miRNAs in HepG2 and L02 cell lines by microRNA microarray. Northern blot, quantitative real-time polymerase chain reaction, methylthiazolyl tetrazolium, fluorescence-activated cell sorting, scratch wound, transwell migration and Matrigel invasion assays and western blot were carried out to determine whether or not microRNA-224 (miR-224) can influence the biological behaviors of HepG2 cells. Results: MiR-224 was significantly upregulated in HepG2 cells. Cell proliferation, migration and invasion, but not cell cycles, were altered after changing the expression of miR-224. Taking invasion and migration as a breakthrough, a close relationship between the expression of miR-224 and its proteins such as PAK4 and MMP9, which were involved in the invasion of tumor, was found. Conclusions: Overexpression of miR-224 was involved in the malignant phenotype of HepG2 cells, and it may be an important factor in regulating the migration and invasion of HepG2 cells.

Abstract: Background Skeletal muscle satellite cells are myogenic progenitors that reside on myofiber surface beneath the basal lamina. In recent years satellite cells have been identified and isolated based on their expression of CD34, a sialomucin surface receptor traditionally used as a marker of hematopoietic stem cells. Interestingly, a minority of satellite cells lacking CD34 has been described. Methodology/Principal Findings In order to elucidate the relationship between CD34+ and CD34- satellite cells we utilized fluorescence-activated cell sorting (FACS) to isolate each population for molecular analysis, culture and transplantation studies. Here we show that unless used in combination with α7 integrin, CD34 alone is inadequate for purifying satellite cells. Furthermore, the absence of CD34 marks a reversible state of activation dependent on muscle injury. Conclusions/Significance Following acute injury CD34- cells become the major myogenic population whereas the percentage of CD34+ cells remains constant. In turn activated CD34- cells can reverse their activation to maintain the pool of CD34+ reserve cells. Such activation switching and maintenance of reserve pool suggests the satellite cell compartment is tightly regulated during muscle regeneration.

Abstract: Purpose: Receptor-interacting protein of 140 kDa (RIP140) is a transcriptional cofactor for nuclear receptors involved in reproduction and energy homeostasis. Our aim was to investigate its role in the regulation of E2F1 activity and target genes both in breast cancer cell lines and in tumor biopsies. Experimental Design: Glutathione S-transferase pull-down assays, coimmunoprecipitation experiments, and chromatin immunoprecipitation analysis were used to evidence interaction between RIP140 and E2F1. The effects of RIP140 expression on E2F1 activity were determined using transient transfection and quantification of E2F target mRNAs by quantitative real-time PCR. The effect on cell cycle was assessed by fluorescence-activated cell sorting analysis on cells overexpressing green fluorescent protein–tagged RIP140. A tumor microarray data set was used to investigate the expression of RIP140 and E2F1 target genes in 170 breast cancer patients. Results: We first evidenced the complex interaction between RIP140 and E2F1 and showed that RIP140 represses E2F1 transactivation on various transiently transfected E2F target promoters and inhibits the expression of several E2F1 target genes (such as CCNE1 and CCNB2). In agreement with a role for RIP140 in the control of E2F activity, we show that increasing RIP140 levels results in a reduction in the proportion of cells in S phase in various human cell lines. Finally, analysis of human breast cancers shows that low RIP140 mRNA expression was associated with high E2F1 target gene levels and basal-like tumors. Conclusion: This study shows that RIP140 is a regulator of the E2F pathway, which discriminates luminal- and basal-like tumors, emphasizing the importance of these regulations for a clinical cancer phenotype. Clin Cancer Res; 16(11); 2959–70. ©2010 AACR.

Abstract: The epithelium of the intestinal crypt is a dynamic tissue undergoing constant regeneration through cell growth, cell division, cell differentiation and apoptosis. How the epithelial cells maintain correct positioning and how they migrate in a directed and collective fashion are still not well understood. In this paper, we developed a computational model to elucidate these processes. We show that differential adhesion between epithelial cells, caused by the differential activation of EphB receptors and ephrinB ligands along the crypt axis, is necessary to regulate cell positioning. Differential cell adhesion has been proposed previously to guide cell movement and cause cell sorting in biological tissues. The proliferative cells and the differentiated post-mitotic cells do not intermingle as long as differential adhesion is maintained. We also show that, without differential adhesion, Paneth cells are randomly distributed throughout the intestinal crypt. In addition, our model suggests that, with differential adhesion, cells migrate more rapidly as they approach the top of the intestinal crypt. Finally, by calculating the spatial correlation function of the cell velocities, we observe that differential adhesion results in the differentiated epithelial cells moving in a coordinated manner, where correlated velocities are maintained at large distances, suggesting that differential adhesion regulates coordinated migration of cells in tissues.

Abstract: Background Several laboratories have shown that cells with a memory B-cell phenotype can have the same clonotype as multiple myeloma tumor cells. Design and Methods The aim of this study was to determine whether some memory B cells have the same genetic alterations as their corresponding multiple myeloma malignant plasma cells. The methodology included sorting multiple myeloma or memory B cells into RNA stabilizing medium for generation of subset-specific polymerase chain reaction complementary DNA libraries from one or 100 cells. Results Cells with the phenotype of tumor plasma cells (CD38++CD19−CD45−/+CD56−/+/++) or memory B cells (CD38−/CD19+/CD27+) were isolated by flow activated cell sorting. In samples from all four patients with multiple myeloma and from two of the three with monoclonal gammopathy of undetermined significance, we identified memory B cells expressing multiple myeloma-specific oncogenes ( FGFR3; IGH-MMSET; CCND1 high) dysregulated by an IGH translocation in the respective tumor plasma cells. By contrast, in seven patients with multiple myeloma, each of whom had tumor plasma cells with a K- RAS61 mutation, a total of 32,400 memory B cells were analyzed using a sensitive allele-specific, competitive blocker polymerase chain reaction assay, but no K- RAS mutations were identified. Conclusions The increased expression of a specific “early” oncogene of multiple myeloma (monoclonal gammopathy of undetermined significance) in some memory B cells suggests that dysregulation of the oncogene occurs in a precursor B-cell that can generate memory B cells and transformed plasma cells. However, if memory B cells lack “late” oncogene (K- RAS ) mutations but express the “early” oncogene, they cannot be involved in maintaining the multiple myeloma tumor, but presumably represent a clonotypic remnant that is only partially transformed.

Abstract: Understanding the development of an organ requires knowledge of gene, protein, and metabolite expression in the specific cell types and tissues that comprise the organ. Fluorescence-activated cell sorting (FACS) is an efficient method to isolate specific cells of interest, and the information gained from this approach has been integral to plant developmental biology. The Benfey lab has developed this method to examine gene expression profiles of different cell types in the Arabidopsis root under both standard and stress conditions. In addition to gene expression, downstream applications of FACS include proteomic and metabolite analysis. This is a powerful method to examine biological functions of specific cell types and tissues with a systems biology approach.

Abstract: Background Isolation of spermatogonial stem cells (SSCs) could enable in vitro approaches for exploration of spermatogonial physiology and therapeutic approaches for fertility preservation. SSC isolation from adult testes is difficult due to low cell numbers and lacking cell surface markers. Glial cell-derived neurotrophic factor family receptor alpha-1 (GFRa1) plays a crucial role for the maintenance of SSCs in rodents and is expressed in monkey spermatogonia. Methods Magnetic activated cell sorting was employed for the enrichment of GFRa1+ spermatogonia from adult primate testes. Results Magnetic activated cell sorting of monkey cells enriched GFRa1+ cells threefold. 11.4% of GFRa1+ cells were recovered. 42.9% of GFRa1+ cells were recovered in sorted fractions of human testicular cells, representing a fivefold enrichment. Interestingly, a high degree of morphological heterogeneity among the GFRa1+ cells from human testes was observed. Conclusions Magnetic activated cell sorting using anti-GFRa1 antibodies provides an enrichment strategy for spermatogonia from monkey and human testes.

Abstract: This study reports an integrated microfluidic system capable of isolation, counting, and sorting of hematopoietic stem cells (HSCs) from cord blood in an automatic format by utilizing a magnetic-bead-based immunoassay. Three functional modules, including cell isolation, cell counting, and cell sorting modules are integrated on a single chip by using microfluidic technology. The cell isolation module is comprised of a four-membrane-type micromixer for binding of target stem cells and magnetic beads, two pneumatic micropumps for sample transport, and an S-shaped channel for isolation of HSCs using a permanent magnet underneath. The counting and sorting of HSCs are performed by utilizing the cell counting and sorting modules. Experimental results show that a separation efficiency as high as 88% for HSCs from cord blood is achieved within 40 min for a sample volume of 100 μl. Therefore, the development of this integrated microfluidic system may be promising for various applications such as stem cell research and cell therapy.

Abstract: Prostate-specific membrane antigen (PSMA) targeting compounds are described. Uses of the compounds for imaging, therapy, cell sorting, and tumor mapping are also described.

Abstract: Endothelial cells are remarkably heterogeneous in both morphology and function, and they play critical roles in the formation of multiple organ systems. In addition endothelial cell dysfunction can contribute to disease processes, including diabetic nephropathy, which is a leading cause of end stage renal disease. In this report we define the comprehensive gene expression programs of multiple types of kidney endothelial cells, and analyze the differences that distinguish them. Endothelial cells were purified from Tie2-GFP mice by cell dissociation and fluorescent activated cell sorting. Microarrays were then used to provide a global, quantitative and sensitive measure of gene expression levels. We examined renal endothelial cells from the embryo and from the adult glomerulus, cortex and medulla compartments, as well as the glomerular endothelial cells of the db/db mutant mouse, which represents a model for human diabetic nephropathy. The results identified the growth factors, receptors and transcription factors expressed by these multiple endothelial cell types. Biological processes and molecular pathways were characterized in exquisite detail. Cell type specific gene expression patterns were defined, finding novel molecular markers and providing a better understanding of compartmental distinctions. Further, analysis of enriched, evolutionarily conserved transcription factor binding sites in the promoters of co-activated genes begins to define the genetic regulatory network of renal endothelial cell formation. Finally, the gene expression differences associated with diabetic nephropathy were defined, providing a global view of both the pathogenic and protective pathways activated. These studies provide a rich resource to facilitate further investigations of endothelial cell functions in kidney development, adult compartments, and disease.

Abstract: Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and leukemogenesis. They have the capacity to differentiate into lymphoid and myeloid lineages. The colony forming cell (CFC) assay is used to study the proliferation and differentiation pattern of hematopoietic progenitors by their ability to form colonies in a semisolid medium. The number and the morphology of the colonies formed by a fixed number of input cells provide preliminary information about the ability of progenitors to differentiate and proliferate. Cells can be harvested from individual colonies or from the whole plate to further assess their numbers and differentiation states using flow cytometry and morphologic evaluation of Giemsa-stained slides. This assay is useful for assessing myeloid but not lymphoid differentiation. The term myeloid in this context is used in its wider sense to encompass granulocytic, monocytic, erythroid, and megakaryocytic lineages. We have used this assay to assess the effects of oncogenes on the differentiation of primary human CD34+ cells derived from peripheral blood. For this purpose cells are transduced with either control retroviral construct or a construct expressing the oncogene of interest, in this case NUP98-HOXA9. We employ a commonly used retroviral vector, MSCV-IRES-GFP, that expresses a bicistronic mRNA that produces the gene of interest and a GFP marker. Cells are pre-activated by growing in the presence of cytokines for two days prior to retroviral transduction. After another two days, GFP+ cells are isolated by fluorescence-activated cell sorting (FACS) and mixed with a methylcellulose-containing semisolid medium supplemented with cytokines and incubated till colonies appear on the surface, typically 14 days. The number and morphology of the colonies are documented. Cells are then removed from the plates, washed, counted, and subjected to flow cytometry and morphologic examination. Flow cytometry with antibodies specific to the cell surface markers expressed during hematopoiesis provides information about lineage and maturation stage. Morphological studies of individual cells under a microscope after Wright- Giemsa staining provide further information with regard to lineage and maturation. Comparison of cells transduced with control empty vector to those transduced with an oncogene reveals the effects of the oncogene on hematopoietic differentiation.

Abstract: Objectives: High-risk human papillomaviruses (HPVs) are the major causative agents of cervical cancer, and the E6 and E7 genes encode the major HPV oncoproteins. The E7 protein of high-risk HPV types disturbs cell cycle control and down-regulates components of the antigen presentation pathway, suggesting a role for E7 in tumor immune evasion. We previously reported that HPV-16 E7 expression and down-regulation of HLA class I was highly correlated in cervical lesions. This study was aimed to determine whether HPV-16 E7 oncoprotein could down-regulate surface HLA class I antigen in HPV-16 E7-transfected cells, and whether it had correlation with the expression of the transporter associated with antigen processing (TAP). Methods: The HPV-16 E7 open reading frame was transfected into HaCaT cells. After G418 selection, resistant colonies were individually picked and expanded into clonal cell lines. Using the fluoresence-activated cell sorting analysis, the levels of cell surface HLA class I antigen and intracellular TAP-1 and TAP-2 expressions were detected. Results Compared with the empty vector control, a statistical significant decrease of approximately 50% in cell surface HLA class I expression was observed in HPV-16 E7 expressing HaCaT cells (P Conclusions: Our finding demonstrates that HPV-16 E7 down-regulates surface HLA class I antigen, which in part correlates with the decrease of TAP-1.