Selection of mammalian cells based on their cell-cycle phase using dielectrophoresis

An effective, noninvasive means of selecting cells based on their phase within the cell cycle is an important capability for biological research. Current methods of producing synchronous cell populations, however, tend to disrupt the natural physiology of the cell or suffer from low synchronization yields. In this work, we report a microfluidic device that utilizes the dielectrophoresis phenomenon to synchronize cells by exploiting the relationship between the cell's volume and its phase in the cell cycle. The dielectrophoresis activated cell synchronizer (DACSync) device accepts an asynchronous mixture of cells at the inlet, fractionates the cell populations according to the cell-cycle phase (G₁/S and G₂/M), and elutes them through different outlets. The device is gentle and efficient; it utilizes electric fields that are 1–2 orders of magnitude below those used in electroporation and enriches asynchronous tumor cells in the G₁ phase to 96% in one round of sorting, in a continuous flow manner at a throughput of 2 × 10⁵ cells per hour per microchannel. This work illustrates the feasibility of using laminar flow and electrokinetic forces for the efficient, noninvasive separation of living cells.     

An efficient method of sorting liver stem cells by using immuno-magnetic microbeads

AIM: To develop a method to isolate liver stem cells fast and efficiently. METHODS: Fetal mouse liver cells were characterized by cell surface antigens (c-Kit and CD45/TER119) using flow cytometry. The candidate liver stem cells were sorted by using immuno-magnetic microbeads and identified by lone-forming culture, RT-PCR and immunofluorescence says. RESULTS: The c-Kit-(CD45/TER119)- cell population with 97.9% of purity were purified by immuno-magnetic microbeads at one time. The yield of this separation was about 6% of the total sorting cells and the cell viability was above 98%. When cultured in vitro these cells had high clone-forming and self-renewing ability and expressed markers of hepatocytes and bile duct cells. Functionally mature hepatocytes were observed after 21 d of culture. CONCLUSION: This method offers an excellent tool for the enrichment of liver stem cells with high purity and viability, which could be used for further studies. It is fast, efficient, simple and not expensive.     

Detection of disseminated tumor cells in bone marrow of gastric cancer using magnetic activated cell sorting and fluorescent activated cell sorting

Background and Aim: Magnetic activated cell sorting (MACS) and fluorescent activated cell sorting (FACS) were employed to enrich and detect the gastric cancer cells from a cell line in a model system, and to enrich and detect disseminated tumor cells (DTCs) from bone marrow (BM) of patients with gastric cancer. Methods: Fifteen patients with benign gastric lesions and 35 patients with gastric cancer who received curative operations between December 2002 and June 2003 were selected. Mononuclear cells were separated from their BM. Cells from cell line OCUM‐2M were seeded with 10‐grade ratio into mononuclear cells from patients with benign gastric lesion. After labeling by MACS minibeads conjugated with cytokeratin (CK) 7/8 antibodies, anti‐CK‐fluorescein isothiocyanate (FITC), and anti‐CD45‐perdinin chlorophyll protein (PerCP), the samples were enriched twice using an MS+/RS+ positive separation column. The FACS analysis was conducted on these samples before and after MACS enrichment. The results were analyzed using clinopathological parameters. Results: Disseminated tumor cells were detected in the BM of 25 (71.43%) patients with gastric cancer. The frequencies of DTCs were 1.38 × 10^?8–2.40 × 10^?5, 2.19 × 10^?7–3.70 × 10^?5, 4.01 × 10^?6–8.57 × 10^?5 in patients with well, moderately, and poorly differentiated carcinoma, respectively (P = 0.026). Disseminated tumor cells in BM had close correlation with tumor tumor‐node‐metastasis (TNM) stage (P = 0.034) and cancer‐free survival (P = 0.035). Conclusion: Disseminated tumor cells are very common in the BM of gastric cancer patients. Poor histological differentiation and more advanced TNM stage have more DTCs in the BM of gastric cancer patients. Patients with DTCs tend to have a poor prognosis.     

Optimization of immunomagnetic selection of myeloma cells from bone marrow using magnetic activated cell sorting

Plasma cells (PCs) enrichment from bone marrow samples of multiple myeloma (MM) patients is frequently performed by immunomagnetic separation (magnetic activated cell sorting, MACS) using anti-CD138 MicroBeads. The aim of our work was to find optimal strategy for immunomagnetic separation of PCs and determine optimal algorithm of separation techniques for samples with various percentage of neoplastic cells. From 2007 to 2008, selection of PCs using separation programs Possels and Posseld₂ was carried out on 234 bone marrow samples obtained from 208 MM patients. In 2008, an optimal algorithm for separation programs was introduced based on the analysis of the previous experiments. The Possels program is applicable for samples with >10% PCs in the mononuclear fraction, while the Posseld₂ program is used for samples with 5–10% PCs in the mononuclear fraction. Median purity of 92.6% for the positive fraction of cells (range 14.5–99.6%) and median recovery of 60.4% (range 25.7–99.5%) were obtained when the Possels program was applied (n = 45). A total of 80% (36/45) of processed samples had purity of >70%. Median purity for the positive fraction of 83.7% (range 14.3–99.7%) and median recovery of 14.3% (range 3.6–50.0%) were achieved using the Posseld₂ program (n = 99). A total of 68% (67/99) of processed samples reached >70% purity. This separation strategy enabled us to obtain sufficient amounts of highly purified PCs required for subsequent research purposes. The MACS method has been unsuccessful if the percentage of PCs in the initial sample was <5%. These samples were processed by fluorescence activated cell sorting (FACS).     

Purification of hemopoietic progenitor cells from human marrow using a fucose-binding lectin and cell sorting

Human peripheral blood granulocytes, but not lymphocytes, erythrocytes, or monocytes, bound the fucose-binding lectin from Lotus tetragonolobus (FBP), and this binding was competitively inhibited by the sugar alpha- L-fucose. The fluorescence-activated cell sorter was used to study the appearance of this receptor on human marrow cells during granulocyte differentiation and to prepare fractions enriched for granulocyte- macrophage progenitor cells (granulocyte-macrophage colony-forming cells--GM-CFC). Cell binding of fluoresceinated FBP increased for bone marrow cells in the sequence--lymphocytes, blast cells, promyelocytes and myelocytes, monocytes, and polymorphonuclear cells. Selection of cells with appropriate low-angle or high-angle light scatter characteristics achieved a 10-fold or 2-3-fold enrichment of progenitor cells, respectively. By selecting cells with intermediate fluorescence intensity, a further 2-3-fold enrichment for GM-CFC was obtained. Cell sorting using the optimal selection of these three parameters produced up to 36-fold enrichment of the progenitor cells from human bone marrow. The most enriched fraction was composed of 23% progenitor cells (colony- and cluster-forming cells) with a yield of 36%. In populations most highly enriched by GM-CFC, immature cells (blast cells, promyelocytes, and myelocytes) made up 95% of the cells present.     

Fluorescence-activated cell sorting purification of pancreatic progenitor cells

Here we review progress on isolation and characterization of progenitor cells in the pancreas. We discuss advantages and current limitations of experiments with purified pancreatic cells, and areas where future growth in our understanding is needed to advance experiments in pancreas biology based on cell purification.     

Biosynthesis and sorting of myeloperoxidase in hematopoietic cells

The neutrophil granulocytes have a critical role in innate immunity through killing of phagocytized microorganisms, in which myeloperoxidase (MPO) participates. MPO is stored in cytoplasmic azurophil lysosome-like granules together with other antibiotic proteins and digestive enzymes. During passage in the secretory pathway pro-MPO is folded, subjected to oligosaccharide modification, and retrieval from constitutive secretion to become targeted to azurophil granules for final processing and storage. Propeptide-deleted MPO precursor was found not to be processed to mature MPO and not to be targeted for storage but instead degraded or secreted. This indicated that the propeptide of the MPO precursor was a prerequisite for the final processing and granule targeting of proMPO. When the MPO propeptide was expressed as a chimera with a normally secretory protein, the ER retention of the chimera was prolonged compared with that of the native protein. Thus, the propeptide of MPO precursor may also mediate the normally long ER-residence of proMPO. Both mature MPO and secreted proMPO contained complex oligosaccharide side chains indicating that proMPO and, thus, mature MPO has passed the medial Golgi stack where complex oligosaccharides are formed, and exited at TGN like other proteins targeted for azurophil granules.     

Enrichment of rare cancer cells through depletion of normal cells using density and flow-through, immunomagnetic cell separation

OBJECTIVE: To develop a reliable technique to enrich for rare cells in blood suspensions using only negative selection steps including a flow-through immunomagnetic cell separations system and by optimizing variables normally encountered during such enrichment processes. METHODS: A human breast cancer cell line was cultivated and spiked at a ratio of 1 cancer cell to 10(5) total leukocytes in buffy coat or 1 cancer cell to 10(8) total cells in whole blood samples. The final, optimized process consisted of: a red cell lysis step, immunomagnetically staining leukocytes with an anti-CD45 PE, anti- MACS sandwich, immunomagnetic sorting using a flow-through system (QMS), and a final cell analysis step using either an automated cell counter, filtration, and visual counting or a cytospin analysis. RESULTS: The final, optimized process produced a final enrichment of the rare cancer cells of 5.17 log(10) and an average, final recovery of 46%. It should be noted that a negative depletion protocol was used (i.e., no labeling of the rare cancer cells was used). CONCLUSIONS: To the authors' knowledge, no examples in the literature exist of a 5.17 log(10) enrichment of cancer cells in human blood using a negative depletion protocol. The closest example is a 4 log(10) enrichment in which two positive magnetic cell separation steps were used (none were used in this study). Ongoing studies are investigating further modifications of the precommercial, prototype flow-through immunmagnetic separation system to increase both the enrichment and recovery rate. However, even at current performance levels, the presented process could significantly improve visual and molecular analysis of rare cells in blood.     

Isolating highly enriched populations of circulating epithelial cells and other rare cells from blood using a magnetic sweeper device

The enumeration of rare circulating epithelial cells (CEpCs) in the peripheral blood of metastatic cancer patients has shown promise for improved cancer prognosis. Moving beyond enumeration, molecular analysis of CEpCs may provide candidate surrogate endpoints to diagnose, treat, and monitor malignancy directly from the blood samples. Thorough molecular analysis of CEpCs requires the development of new sample preparation methods that yield easily accessible and purified CEpCs for downstream biochemical assays. Here, we describe a new immunomagnetic cell separator, the MagSweeper, which gently enriches target cells and eliminates cells that are not bound to magnetic particles. The isolated cells are easily accessible and can be extracted individually based on their physical characteristics to deplete any cells nonspecifically bound to beads. We have shown that our device can process 9 mL of blood per hour and captures >50% of CEpCs as measured in spiking experiments. We have shown that the separation process does not perturb the gene expression of rare cells. To determine the efficiency of our platform in isolating CEpCs from patients, we have isolated CEpCs from all 47 tubes of 9-mL blood samples collected from 17 women with metastatic breast cancer. In contrast, we could not find any circulating epithelial cells in samples from 5 healthy donors. The isolated CEpCs are all stored individually for further molecular analysis.     

Purification of adult hepatic progenitor cells using green fluorescent protein (GFP)-transgenic mice and fluorescence-activated cell sorting

BACKGROUND/AIMS: Recent advances in stem cell research have revealed that hepatic stem/progenitor cells may play an important role in liver development and regeneration. However, a lack of detectable definitive markers in viable cells has hindered their primary culture from adult livers. METHODS: Enzymatically dissociated liver cells from green fluorescent protein (GFP)-transgenic mice, which express GFP highly in liver endodermal cells, were sorted by GFP expression using a fluorescence-activated cell sorter. Sorted cells were characterized, and also low-density cultured for extended periods to determine their proliferation and clonal differentiation capacities. RESULTS: When CD45(-)TER119(-) side-scatter(low) GFP(high) cells were sorted, alpha-fetoprotein-positive immature endoderm-characterized cells, having high growth potential, were present in this population. Clonal analysis and electron microscopic evaluation revealed that each single cell of this population could differentiate not only into hepatocytes, but also into biliary epithelial cells, showing their bilineage differentiation activity. When surface markers were analyzed, they were positive for Integrin-alpha6 and -beta1, but negative for c-Kit and Thy1.1. CONCLUSIONS: Combination of GFP-transgenic mice and fluorescence-activated cell sorting enabled purification of hepatic progenitor cells from adult mouse liver. Further analysis of this population may lead to purification of their human correspondence that would be an ideal cell-source candidate for regenerative medicine.     

大肠癌干细胞分选与鉴定的研究进展

肿瘤干细胞理论认为结肠癌的发病、转移与复发与大肠癌干细胞(colorectal cancer stem cells,CCSCs)有关,因此分离纯化大肠癌干细胞对于结肠癌的预防,诊断及治疗具有重要意义.本文就大肠癌干细胞的分选与鉴定方法作一综述.     

Enrichment of murine hemopoietic clonogenic cells by multivariate analyses and sorting

Multivariate analyses, dual beam flow cytometry and sorting, and list mode data processing were used to distinguish and enrich committed and pluripotent stem cells in mouse bone marrow. These cells were discriminated on the basis of their forward angle and perpendicular light scatter characteristics and their Hoechst 33342 fluorescence intensity. Myeloid committed progenitors (CFU-GM) and spleen colony-forming units (CFU-S) (day 9 and day 13) were enriched 100-fold by sorting on the basis of high forward angle light scatter, intermediate perpendicular light scatter, and very low HO fluorescence intensity. Approximately 10% of the sorted cells formed colonies in the CFU-GM assay and 2% formed CFU-S colonies. Morphologic analysis of the sorted subpopulation revealed 92% blast immature cell types. The DNA distribution of the sorted subpopulation, assessed by propidium iodide staining, indicated that 98% of the progenitor-enriched subpopulations contained 2N DNA content. This separation procedure offers a simple method to obtain preparations highly enriched in clonogenic cells in one pass through the cell sorter.     

Platelet particle processing: an example of surface sorting on single cells

The ability of glycoprotein IIb-IIIa (GPIIb-IIIa) receptors on fully spread, surface-activated platelets to bind specific antibodies or ligands, singly or sequentially, and clear them toward cell centres has been established in several investigations. However, the basic mechanism involved in receptor-ligand translocation remains unclear. The present study has attempted to provide additional information by adding two different electron-dense probes simultaneously to surface-activated cells. Over a 5 min period of incubation small latex particles were cleared more rapidly from platelet margins to cell centres than simultaneously added particles of colloidal gold coupled to fibrinogen (Fgn/Au). Large and small latex spherules mixed together and added to spread platelets under the same conditions were moved at the same rate and concentrated together in the cell centres. Results of this investigation indicate that simple diffusion is unlikely to be the generating force for movement of receptor complexes on platelet plasma membranes.     

Microfluidic sorting and multimodal typing of cancer cells in self-assembled magnetic arrays

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.     

Endocytosis, intracellular sorting, and processing of exosomes by dendritic cells

Exosomes are nanovesicles released by leukocytes and epithelial cells. Although their function remains enigmatic, exosomes are a source of antigen and transfer functional major histocompatibility complex (MHC)-I/peptide complexes to dendritic cells (DCs) for CD8(+) T-cell activation. Here we demonstrate that exosomes also are internalized and processed by immature DCs for presentation to CD4(+) T cells. Endocytosed exosomes are sorted into the endocytic compartment of DCs for processing, followed by loading of exosome-derived peptides in MHC-II molecules for presentation to CD4(+) T cells. Targeting of exosomes to DCs is mediated via milk fat globule (MFG)-E8/lactadherin, CD11a, CD54, phosphatidylserine, and the tetraspanins CD9 and CD81 on the exosome and alpha(v)/beta(3) integrin, and CD11a and CD54 on the DCs. Circulating exosomes are internalized by DCs and specialized phagocytes of the spleen and by hepatic Kupffer cells. Internalization of blood-borne allogeneic exosomes by splenic DCs does not affect DC maturation and is followed by loading of the exosome-derived allopeptide IEalpha(52-68) in IA(b) by host CD8alpha(+) DCs for presentation to CD4(+) T cells. These data imply that exosomes present in circulation or extracellular fluids constitute an alternative source of self- or allopeptides for DCs during maintenance of peripheral tolerance or initiation of the indirect pathway of allorecognition in transplantation.     

Purification of functional lactotrophs and somatotrophs from female rats using fluorescence-activated cell sorting

As the secretory granules of anterior pituitary cells fuse with the cell surface, there would appear to be sufficient hormone present on the cell surface to be labelled by polyclonal hormone antibodies and thus analysed by flow cytometry. We have therefore applied fluorescence-activated cell sorting to these labelled pituitary cells. Percentage purity and depletion of other cell types was assessed by immunocytochemistry and the reverse haemolytic plaque assay (RHPA). Results demonstrate that fluorescence-activated cell sorting allows almost complete purification of functional lactotrophs and somatotrophs to 96.7 +/- 1.7 (S.E.M.)% and 98 +/- 1.0% respectively by immunocytochemistry, and to 95.8 +/- 1.1% and 97 +/- 0.8% respectively by RHPA. Depletion of other anterior pituitary cell types to less than 2% was demonstrated by both immunocytochemistry and RHPA. Fluorescence-activated cell sorting to this degree of purity was routinely possible with cell yields of 91 +/- 3.4%. To obtain such purity/depletion, it was necessary to use specific antisera of high titre, at concentrations which ensured maximal cell-surface labelling associated with maximal stimulation of hormonal secretion by the appropriate hypothalamic stimulatory factor. Separating cells on the basis of the intensity of prolactin cell-surface labelling demonstrated a low level of binding of the prolactin antibody to gonadotrophs (but not of sufficient fluorescence intensity to be sorted into the prolactin enriched population), raising the possibility of prolactin receptors on gonadotrophs. We were unable to demonstrate the presence of mammosomatotrophs in the normal female rat, since purified lactotrophs did not contain or secrete GH nor did purified somatotrophs contain or secrete prolactin.     

Opposite sorting of tissue factor in human umbilical vein endothelial cells and Madin-Darby canine kidney epithelial cells

Tissue factor (TF) is a 48-kD transmembrane glycoprotein that triggers the extrinsic pathway of blood coagulation by interacting with the plasma coagulation factor VII (FVII). TF is also a true receptor in that a cellular signal is generated when activated FVII (FVIIa) binds to TF. For both of these functions, the cellular surface distribution of TF is important, since FVII is primarily available on the apical side of vascular endothelial cells and on the basolateral side of epithelial cells lining the internal and external surfaces. We show that in endothelial cells, TF (both antigen and procoagulant activity) is sorted to the apical surface, whereas in wild-type and stably transfected Madin-Darby canine kidney epithelial cells (MDCK), which form tight junctions and express TF constitutively, TF antigen is on the basolateral surface. No significant clotting activity is detectable on this surface. Truncated TF (cytoplasmic tail residues 246 to 263 deleted) is sorted as wild-type in MDCK cells.     

Characterization and purification of osteogenic cells from murine bone marrow by two-color cell sorting using anti-Sca-1 monoclonal antibody and wheat germ agglutinin

Osteogenic cells were sorted from bone marrow of 5-fluorouracil (5-FU)- treated mice based on light scatter characteristics, Sca-1 expression, and their binding to wheat germ agglutinin (WGA). Four sort gates were established using forward (FSC) and perpendicular (SSC) light scatter and were denominated as FSChigh SSClow, FSClow SSChigh, FSClow SSClow, and FSChigh SSChigh cell. Cells from the FSChigh SSChigh gate, but not from the other gates, synthesized alkaline phosphatase, collagen, and osteocalcin and formed a mineralized matrix in culture. The number of osteoprogenitor cells was significantly enriched after depleting the 5- FU bone marrow from cells of the lymphoid and myeloid lineage, eg, T cells, B cells, natural killer cells, granulocytes, macrophages, and erythrocytes. Approximately 95% of the FSChigh SSChigh cell population of this "lineage-negative" (Lin-) marrow expressed the Sca-1 antigen (Sca-1+) and bound WGA. Three additional sort windows were established based on WGA binding intensity and were denominated as Sca-1+ WGAdull, Sca-1+ WGAmedium, and Sca-1+ WGAbright. Cells from the Sca-1+ WGAbright gate, but not from the other gates, synthesized bone proteins and formed a mineralized matrix. However, they lost this capacity upon subcultivation. Further immunophenotypic characterization showed that FSChigh SSChigh Lin- Sca-1+ WGAbright cells expressed stromal (KM16) and endothelial (Sab-1 and Sab-2) markers, but not hematopoietic surface markers such as c-kit and Thy1.2. Sorted FSChigh SSChigh Lin- Sca-1+ WGAbright cells form three-dimensional nodules that stain with the von Kossa technique and contain osteoblast and osteocyte-like cells.     

Fluorescence-activated sorting of totipotent embryonic stem cells expressing developmentally regulated lacZ fusion genes.

Murine embryonic stem (ES) cells were infected with a retrovirus promoter trap vector, and clones expressing lacZ fusion genes (LacZ+) were isolated by fluorescence-activated cell sorting (FACS). Of 12 fusion genes tested, 1 was repressed when ES cells were allowed to differentiate in vitro. Two of three lacZ fusion genes tested were passed into the germ line, indicating that FACS does not significantly affect stem cell totipotency. The pattern of lacZ expression observed in vivo was consistent with that seen in vitro. Both fusion genes were expressed in preimplantation blastulas. However, a fusion gene whose expression was unaffected by in vitro differentiation was ubiquitously expressed in day-10 embryos, while the other, which showed regulated expression in vitro, was restricted to cells located along the posterior neural fold, the optic chiasm, and within the fourth ventricle. These results demonstrate the utility of using promoter trap vectors in conjunction with fluorescence sorting to disrupt developmentally regulated genes in mice.