免疫磁珠纯化小鼠精原干细胞的研究

精原干细胞（spermatogonial stem cells,SSCs）富集纯化是利用SSCs进行基因修饰新方法等研究的前提基础。采用免疫磁珠分选法,使用干细胞抗体CD90.2进行小鼠SSCs的纯化富集,并采用流式细胞分析法和定量PCR验证了磁珠分选效率。流式细胞分析结果：免疫磁珠分选后SSCs纯度为50.11%。荧光定量PCR检测结果：磁珠分选后支持细胞特异表达基因 GATA4 显著下调（6倍）、SSCs表达基因 GFRα-1 上调（6.5倍）、生殖干细胞特异表达基因 OCT4 极显著上调（5.9倍）,3个基因相对表达量的变化说明,免疫磁珠分选效率为6倍。流式细胞分析法所产生的偏差可能是受到了未解离磁珠及SSCs本身转基因荧光的影响。     

Fetal nucleated erythrocyte recovery: fluorescence activated cell sorting-based positive selection using anti-gamma globin versus magnetic activated cell sorting using anti-CD45 depletion and anti-gamma globin positive selection

BACKGROUND: Fluorescence activated cell sorting (FACS)-based anti-gamma (gamma) positive selection and magnetic activated cell sorting (MACS)-based anti-CD45 depletion followed by anti-gamma positive staining have been two of the most frequently used methods to isolate fetal cells from maternal blood. To date, there has been no direct comparison of fetal cell recovery by these two methods. This study was designed to address this issue. METHODS: Fluorescence in situ hybridization (FISH) was performed on nucleated anti-gamma positive cells using X and Y probes. Twenty-four maternal blood samples were obtained immediately after elective termination of pregnancy to ensure a detectable number of fetal cells. RESULTS: The yield and purity of fetal nucleated erythrocytes (FNRBCs) was statistically higher in FACS sorted samples (P < 0.01). The specificity of staining for FNRBCs was statistically higher in MACS sorted samples (P < 0.01). CONCLUSIONS: The data from this study demonstrate that both techniques have benefits and limitations. FACS has the advantage of having higher yield, higher purity, higher FISH efficiency and ease in microscope analysis, and MACS has the advantage of having higher specificity and less cell loss during FISH.     

Screening of peptide libraries against protective antigen of Bacillus anthracis in a disposable microfluidic cartridge

Bacterial surface peptide display has gained popularity as a method of affinity reagent generation for a wide variety of applications ranging from drug discovery to pathogen detection. In order to isolate the bacterial clones that express peptides with high affinities to the target molecule, multiple rounds of manual magnetic activated cell sorting (MACS) followed by multiple rounds of fluorescence activated cell sorting (FACS) are conventionally used. Although such manual methods are effective, alternative means of library screening which improve the reproducibility, reduce the cost, reduce cross contamination, and minimize exposure to hazardous target materials are highly desired for practical application. Toward this end, we report the first semi-automated system demonstrating the potential for screening bacterially displayed peptides using disposable microfluidic cartridges. The Micro-Magnetic Separation platform (MMS) is capable of screening a bacterial library containing 3×10¹⁰ members in 15 minutes and requires minimal operator training. Using this system, we report the isolation of twenty-four distinct peptide ligands that bind to the protective antigen (PA) of Bacilus anthracis in three rounds of selection. A consensus motif WXCFTC was found using the MMS and was also found in one of the PA binders isolated by the conventional MACS/FACS approach. We compared MMS and MACS rare cell recovery over cell populations ranging from 0.1% to 0.0000001% and found that both magnetic sorting methods could recover cells down to 0.0000001% initial cell population, with the MMS having overall lower standard deviation of cell recovery. We believe the MMS system offers a compelling approach towards highly efficient, semi-automated screening of molecular libraries that is at least equal to manual magnetic sorting methods and produced, for the first time, 15-mer peptide binders to PA protein that exhibit better affinity and specificity than peptides isolated using conventional MACS/FACS.     

Pulsed Low-Frequency Magnetic Fields Induce Tumor Membrane Disruption and Altered Cell Viability

Tumor cells express a unique cell surface glycocalyx with upregulation of sulfated glycosaminoglycans and charged glycoproteins. Little is known about how electromagnetic fields interact with this layer, particularly with regard to harnessing unique properties for therapeutic benefit. We applied a pulsed 20-millitesla (mT) magnetic field with rate of rise (dB/dt) in the msec range to cultured tumor cells to assess whether this affects membrane integrity as measured using cytolytic assays. A 10-min exposure of A549 human lung cancer cells to sequential 50- and 385-Hz oscillating magnetic fields was sufficient to induce intracellular protease release, suggesting altered membrane integrity after the field exposure. Heparinase treatment, which digests anionic sulfated glycan polymers, before exposure rendered cells insensitive to this effect. We further examined a non-neoplastic human primary cell line (lung lymphatic endothelial cells) as a typical normal host cell from the lung cancer microenvironment and found no effect of field exposure on membrane integrity. The field exposure was also sufficient to alter proliferation of tumor cells in culture, but not that of normal lymphatic cells. Pulsed magnetic field exposure of human breast cancer cells that express a sialic-acid rich glycocalyx also induced protease release, and this was partially abrogated by sialidase pretreatment, which removes cell surface anionic sialic acid. Scanning electron microscopy showed that field exposure may induce unique membrane “rippling” along with nanoscale pores on A549 cells. These effects were caused by a short exposure to pulsed 20-mT magnetic fields, and future work may examine greater magnitude effects. The proof of concept herein points to a mechanistic basis for possible applications of pulsed magnetic fields in novel anticancer strategies.     

High-efficiency immunomagnetic isolation of solid tissue-originated integrin-expressing adult stem cells

Isolation of highly pure specific cell types is crucial for successful adult stem cell-based therapy. As the number of such cells in adult tissue is low, an extremely efficient method is needed for their isolation. Here, we describe cell-separation methodologies based on magnetic-affinity cell sorting (MACS) MicroBeads with monoclonal antibodies against specific membrane proteins conjugated to superparamagnetic particles. Cells labeled with MACS MicroBeads are retained in a magnetic field within a MACS column placed in a MACS separator, allowing fast and efficient separation. Both positively labeled and non-labeled fractions can be used directly for downstream applications as the separated cell fractions remain viable with no functional impairment. As immunomagnetic separation depends on the interaction between a cell's membrane and the magnetically labeled antibody, separation of specific cells originating from solid tissues is more complex and demands a cell-dissociating pretreatment. In this paper, we detail the use of immunomagnetic separation for the purpose of regenerating damaged salivary gland (SG) function in animal and human models of irradiated head and neck cancer. Each year 500,000 new cases of head and neck cancer occur worldwide. Most of these patients lose SG function following irradiation therapy. SGs contain integrin α6β1-expressing epithelial stem cells. We hypothesized that these cells can be isolated, multiplied in culture and auto-implanted into the irradiated SGs to regenerate damaged SG function.     

A heterologous 3-D coculture model of breast tumor cells and fibroblasts to study tumor-associated fibroblast differentiation

The objective of our study was to establish spheroid cocultures as a valid 3-D in vitro model mimicking tumor-fibroblast interactions in scirrhous breast tumors. The experimental setup was designed to verify if in cocultures (a) adherence and migration reflect the invasive potential of breast tumor cells, (b) breast tumor cells induce tumor-associated fibroblast differentiation, and (c) tumor-derived fibroblasts better reflect the in vivo situation than normal skin fibroblasts. Only one (SK-BR-3) out of five tumor cell types showed extensive fibroblast infiltration, MCF-7 cells frequently invaded fibroblast spheroids; BT474, T47D, and ZR-75-1 were noninvasive. While tumor cell invasion was independent of fibroblast origin, tumor-associated myofibroblast differentiation defined by alpha-SMA expression was demonstrated for tumor-derived but not normal skin fibroblasts in coculture indicating that (a) tumor cell invasion and myofibroblast differentiation are autonomous processes and (b) cocultures with tumor-derived fibroblasts resemble advanced stages of desmoplastic carcinomas while cocultures with normal skin fibroblasts rather reflect the early tumor development. The latter is also implied by fibroblast-associated alterations in tumor cell morphology and ECM distribution in the system. By using RNA arbitrarily primed PCR and cells isolated from cocultures by fluorescence-activated and magnetic cell separation, peripheral myelin protein PMP22/SR13 has been identified as a novel candidate with potential relevance in the interaction between tumor cell and normal fibroblast since PMP22 mRNA was significantly reduced in normal skin fibroblasts in coculture with BT474 cells.     

Morphological evaluation of sperm from infertile men selected by magnetic activated cell sorting (MACS)

Electron microscopy analysis performed in five infertile human subjects after sperm selection by swim-up followed by magnetic activated cell sorting (MACS) demonstrated a decrease in the number of spermatozoa with characteristics compatible with cell death. However, no significant differences were found when the swim-up/MACS semen fraction was compared with swim-up fraction alone.     

The isolation of reticulocyte-free human red blood cells

We depleted reticulocytes from erythrocytes of both sickle cell disease (SCD) subjects and healthy controls by four methods: fluorescence-activated cell sorting (FACS), Miltenyi immunomagnetic depletion (MACS), a combination of these methods (FACS + MACS) and Percoll density separation. The efficiency of these methods was assessed by new methylene blue staining and manual enumeration of the reticulocytes. FACS sorted erythrocytes from reticulocytes based on size and granularity, as well as the absence of dsDNA staining. MACS depleted reticulocytes from erythrocytes based on the immunoaffinity to CD36 and CD71. Reticulocytes from healthy controls were depleted to 相似文献   

免疫磁珠法分离鼻咽癌基质的T淋巴细胞

肿瘤内环境与肿瘤的发生密切相关.肿瘤细胞周围的组织在癌变发生时不会是一个沉默的旁观者,可能在肿瘤的发生和发展中扮演十分重要的角色.本研究分别采用不同的磁珠分选技术分离T淋巴细胞.采用CK LMP1,CD105和成纤维细胞表面蛋白,结合全血总T细胞试剂盒,间接法分离鼻咽癌基质的T淋巴细胞;采用CD3直接磁分选法分离鼻咽癌基质的T淋巴细胞,然后用免疫组化法鉴定分选的效果和细胞的质量.结果表明,免疫组化显示间接磁分选法分离出来的T淋巴细胞不能完全去除肿瘤细胞,RNA的质量不佳;而直接磁分选分离出来的T淋巴细胞为纯净的T淋巴细胞,而且RNA的质量良好.提示直接磁分选技术是分离鼻咽癌基质T淋巴细胞的首选方法.     

Efficient enrichment of undifferentiated GFR alpha 1+ spermatogonia from immature rat testis by magnetic activated cell sorting

Spermatogonial stem cells (SSCs) are a documented source for adult multipotent stem cells. Thus, the isolation of SSCs is of great interest. However, the isolation of spermatogonia from mammalian testes is difficult because of their low total numbers and the lack of well-characterized cell surface markers. Glial-cell-derived neurotrophic factor family receptor alpha-1 (GFRα1) is expressed on undifferentiated mouse spermatogonia (including SSCs) and plays a crucial role, in rodents, for the maintenance of SSCs mediated by the Sertoli cell product GDNF. The present study has aimed to optimize the sorting efficiency and total cell yield of magnetic activated cell sorting (MACS) with anti-GFRα1 antibodies. Because of the technical limitations intrinsic to the magnetic columns, various sorting setups and strategies were compared. Use of Mini-MACS (MS) columns for single cell suspensions from 7-day-old rat testes resulted in a three-fold enrichment of GFRα1-positive cells in sorted fractions versus presorted fractions. However, with this method, only 1.77% of cells loaded onto the column were recovered in the sorted fraction. A sequential two-step sorting approach did not improve this poor yield. We therefore evaluated cell separation by using larger volume Midi-MACS (LS) columns. Enrichment of GFRα1-positive cells in sorted fractions was four-fold, and 14.5% of cells loaded onto the column were directed to the sorted fraction. With this method, approximately half of all GFRα1-positive cells present in the sample were found in the sorted fraction. We conclude that GFRα1 serves as a suitable surface marker for the enrichment of rat spermatogonia, and that the large-volume Midi-MACS separation system is superior to the routinely used small-volume Mini-MACS separation system. This work was financially supported by startup funds from the University Münster, NIH grant U54 HD 008610, Center grant, project 1 (to S.S.), a doctoral scholarship from the Ernst Schering Research Foundation (to K.G.), and a Young Investigator Grant from the Lance Armstrong Foundation (to J.E.).     

Akt, MAPK (Erk1/2), and p38 act in concert to promote apoptosis in response to ErbB receptor family inhibition

The ErbB receptor family is implicated in the malignant transformation of several tumor types and is overexpressed frequently in breast, ovarian, and other tumors. The mechanism by which CI-1033 and gemcitabine, either singly or in combination, kill tumor cells was examined in two breast lines, MDA-MB-453 and BT474; both overexpress the ErbB-2 receptor. CI-1033, a potent inhibitor of the ErbB family of receptor tyrosine kinases, reduced levels of activated Akt in MDA-MB-453 cells. This effect alone, however, did not induce apoptosis in these cells. Gemcitabine treatment resulted in a moderate increase in the percentage of apoptotic cells that was accompanied by activation of p38 and MAPK (ERK1/2). CI-1033 given 24 h after gemcitabine produced a significant increase in the apoptotic fraction over treatment with either drug alone. During the combined treatment p38 remained activated, whereas Akt and activated MAPK were suppressed. Substitution of CI-1033 with the phosphatidylinositol 3-kinase inhibitor LY294002 and the MAPK/ERK kinase inhibitor PD 098059 in combination with gemcitabine produced the same results as the combination of CI-1033 and gemcitabine. p38 suppression by SB203580 prevented the enhanced cell kill by CI-1033. In contrast to MDA-MB-453, BT474 cells exhibited activated p38 under unstressed conditions as well as activated Akt and MAPK. Treatment of BT474 cells with CI-1033 inhibited both the phosphorylation of Akt and MAPK and resulted in a 47% apoptotic fraction. Gemcitabine did not cause apoptosis in the BT474 cells. These data indicate that suppression of Akt and MAPK in the presence of activated p38 results in cell death and a possible mechanism for the enhanced apoptosis produced by the combination of CI-1033 and gemcitabine in MDA-MB-453 cells. Furthermore, tumors that depend on ErbB receptor signaling for survival and exhibit activated p38 in the basal state may be susceptible to apoptosis by CI-1033 as a single agent.     

Selection of nonapoptotic spermatozoa as a new tool for enhancing assisted reproduction outcomes: an in vitro model

Magnetic cell sorting (MACS) using annexin V-conjugated microbeads eliminates apoptotic spermatozoa based on the externalization of phosphatidylserine residues. The procedure delivers two sperm fractions: annexin V-negative (nonapoptotic) and annexin V-positive (apoptotic). Our aim was to determine whether the sperm fertilizing potential can be improved by selecting a nonapoptotic fraction using MACS. Semen samples (n = 35) were subjected to separation on a density gradient followed by MACS. Extent of apoptosis was assessed by measuring levels of activated caspase 3 using fluorescein-labeled inhibitors of caspase, alterations in mitochondrial membrane potential (MMP) using a lipophilic cationic dye, and DNA fragmentation using terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling assay. The sperm fertilization potential was assessed using hamster oocyte penetration assay and hamster oocyte-intracytoplasmic sperm injection (ICSI). Annexin V-negative sperm displayed superior quality in terms of high motility, low caspase 3 activation, MMP integrity, and small extent of DNA fragmentation. Annexin V-negative sperm demonstrated higher oocyte penetration capacity but comparable sperm chromatin decondensation (SCD) following ICSI. Conversely, the annexin V-positive sperm presented with poor quality and fertilization potential. The oocyte penetration rate was negatively correlated with apoptotic marker expression, whereas SCD following ICSI was only associated with apoptosis on sperm-damaged membranes. We conclude that apoptosis appears to impact sperm-oocyte penetration rate; however, it does not seem to affect early stages of fertilization such as SCD in spermatozoa of healthy donors. The selection of nonapoptotic sperm by MACS may be used to enhance results of in vitro fertilization by increasing sperm-oocyte penetration.     

Differential impact of Cetuximab, Pertuzumab and Trastuzumab on BT474 and SK-BR-3 breast cancer cell proliferation

OBJECTIVES: The potential of epidermal growth factor receptor (EGFR)- and Her2-targeted antibodies Cetuximab, Pertuzumab and Trastuzumab, used in combination to inhibit cell proliferation of breast cancer cells in vitro, has not been extensively investigated. It is anticipated that there would be differences between specific erbB receptor co-expression profiles that would affect tumour cell growth. MATERIALS AND METHODS: We have examined the effects of Cetuximab, Pertuzumab and Trastuzumab, applied separately or in combination, on cell proliferation of BT474 and SK-BR-3 breast cancer cell lines. Cell cycle progression of BT474 and SK-BR-3 cells was statically and dynamically assessed using flow cytometry. In order to discover a potential influence of differential EGFR co-expression on sensitivity to antibody treatment, EGFR was down-regulated by siRNA in SK-BR-3. An annexinV/propidium iodide assay was used to identify potential induction of apoptosis. RESULTS: Treatment with Pertuzumab and Trastuzumab, both targeted to Her2, resulted in a reduced fraction of proliferating cells, prolongation of G(1) phase and a great increase in quiescent BT474 cells. Cetuximab had no additional contribution to the effect of either Pertuzumab or Trastuzumab when administered simultaneously. Treatment with the antibodies did not induce an appreciable amount of apoptosis in either BT474 or SK-BR-3 cells. In contrast to SK-BR-3, the BT474 cell line appears to be more sensitive to antibody treatment due to low EGFR content besides Her2 overexpression. CONCLUSION: The extent of decelerated or blocked cell proliferation after antibody treatment that is targeted to EGFR and to Her2 depends both on EGFR and Her2 co-expression and on antibody combination used in the treatment setting. Cetuximab did not enhance any inhibitory effect of Trastuzumab or Pertuzumab, most probably due to the dominant overexpression of Her2. Cell susceptibility to Trastuzumab/Pertuzumab, both targeted to Her2, was defined by the ratio of EGFR/Her2 co-expression.     

2D LC/MS analysis of membrane proteins from breast cancer cell lines MCF7 and BT474

Membrane proteins play a central role in the interaction of the cell with its environment and in the function of subcellular organelles. The current study focused on developing a better understanding of the membrane proteome of two well-characterized breast cancer cell lines. Membranes from osmotically lysed BT474 and MCF7 cells were treated with cyanogen bromide followed by a combination of trypsin and Staphylococcus V8 protease to obtain hydrophilic peptides from membrane proteins. The complex peptide mixtures obtained were separated by 2-dimensional liquid chromatography coupled online with a nano-electrospray ionization ion trap mass spectrometer (2D LC/nanoESI-MS). The strong cation exchange column used in the first dimension of the separation was eluted in an automated fashion using a series of salt steps of increasing concentration. Peptides eluted from each of the salt steps were separated using a capillary reversed-phase HPLC column, the output of which was directed through a nano-electrospray fused silica tip into the mass spectrometer. Peptides were fragmented by collision-induced dissociation (CID) and analyzed by data-dependent MS/MS followed by database searching using the Sequest algorithm. Analysis of the data revealed both similarities and expected differences between proteins identified from these cell lines. As demonstrated by others, mRNA and the HER2/neu protein tyrosine kinase-linked receptor in BT474 cells is up regulated compared to its level in MCF7, while the expression of the estrogen receptor alpha is known to be up regulated in MCF7 cells. As expected, our studies showed identification of peptides from HER2 in BT474 while estrogen receptor peptides were detected in the MCF7 line. A total of 604 proteins were identified from BT474 membranes while 313 proteins were found from MCF7. The results are discussed in terms of the known differences in both protein and mRNA expression between these two breast cancer cell lines and also in the context of other known phenotypic differences between these cells.     

Responses of Escherichia coli, Listeria monocytogenes, and Staphylococcus aureus to simulated food processing treatments, determined using fluorescence-activated cell sorting and plate counting

Three common food pathogenic microorganisms were exposed to treatments simulating those used in food processing. Treated cell suspensions were then analyzed for reduction in growth by plate counting. Flow cytometry (FCM) and fluorescence-activated cell sorting (FACS) were carried out on treated cells stained for membrane integrity (Syto 9/propidium iodide) or the presence of membrane potential [DiOC2(3)]. For each microbial species, representative cells from various subpopulations detected by FCM were sorted onto selective and nonselective agar and evaluated for growth and recovery rates. In general, treatments giving rise to the highest reductions in counts also had the greatest effects on cell membrane integrity and membrane potential. Overall, treatments that impacted cell membrane permeability did not necessarily have a comparable effect on membrane potential. In addition, some bacterial species with extensively damaged membranes, as detected by FCM, appeared to be able to replicate and grow after sorting. Growth of sorted cells from various subpopulations was not always reflected in plate counts, and in some cases the staining protocol may have rendered cells unculturable. Optimized FCM protocols generated a greater insight into the extent of the heterogeneous bacterial population responses to food control measures than did plate counts. This study underlined the requirement to use FACS to relate various cytometric profiles generated by various staining protocols with the ability of cells to grow on microbial agar plates. Such information is a prerequisite for more-widespread adoption of FCM as a routine microbiological analytical technique.     

Enrichment using antibody-coated microfluidic chambers in shear flow: model mixtures of human lymphocytes

Isolation of phenotypically-pure cell subpopulations from heterogeneous cell mixtures such as blood is a difficult yet fundamentally important task. Current techniques such as fluorescent activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) require pre-incubation with antibodies which lead to processing times of at least 15-60 min. In this study, we explored the use of antibody-coated microfluidic chambers to negative deplete undesired cell types, thus obtaining an enriched cell subpopulation at the outlet. We used human lymphocyte cell lines, MOLT-3 and Raji, as a model system to examine the dynamic cell binding behavior on antibody coated surfaces under shear flow. Shear stress ranging between 0.75 and 1.0 dyn/cm2 was found to provide most efficient separation. Cell adhesion was shown to follow pseudo-first order kinetics, and an anti-CD19 coated (Raji-depletion) device with approximately 2.6 min residence time was demonstrated to produce 100% pure MOLT-3 cells from 50-50 MOLT-3/Raji mixture. We have developed a mathematical model of the separation device based on the experimentally determined kinetic parameters that can be extended to design future separation modules for other cell mixtures. We conclude that we can design microfluidic devices that exploits the kinetics of dynamic cell adhesion to antibody coated surfaces to provide enriched cell subpopulations within minutes of total processing time.     

Epidermal growth factor receptor coexpression modulates susceptibility to Herceptin in HER2/neu overexpressing breast cancer cells via specific erbB-receptor interaction and activation

BACKGROUND: Growth factors and Herceptin specifically and differentially modulate cell proliferation of tumor cells. However, the mechanism of action on erbB-receptor level is incompletely understood. We evaluated Herceptin's capacity to modulate erbB-receptor activation and interaction on the cell surface level and thereby potentially impair cell proliferation of HER2/neu (c-erbB2) overexpressing breast cancer cells, both in the presence and absence of relevant growth factors. METHODS: BT474 and SK-BR-3 breast cancer cell lines were treated with Epidermal Growth Factor (EGF), Heregulin, and with Herceptin in different combinations. Kinetics of cell proliferation were evaluated flow cytometrically based on BrdU-labeling. Fluorescence Resonance Energy Transfer, ELISAs and phosphorylation site specific Western Blotting was performed to investigate erbB-receptor interaction and activation. RESULTS: EGF induced EGFR/EGFR and EGFR/c-erbB2 interactions correlate with stimulation of cell proliferation in BT474 cells. Both homo- and heterodimerization are considerably less pronounced in SK-BR-3 cells and heterointeraction is additionally reduced by EGF treatment, causing inhibition of cell proliferation. Heregulin stimulates cell proliferation extensively in both cell lines. Herceptin drives BT474 cells more efficiently into quiescence than it does with SK-BR-3 cells and thereby blocks cell cycle progress. In SK-BR-3 Herceptin treatment causes c-erbB2 phosphorylation of Y877 and Y1248, EGF induces Y877 and Y1112 phosphorylation. The Y1112 phosphorylation site, activated by EGF in SK-BR-3 cell, is bypassed in BT474. In addition the inhibitory capacity of Herceptin on BT474 and SK-BR-3 cell proliferation depends on the presence and absence of growth factors to a various extent. CONCLUSION: The growth inhibitory effect of Herceptin on c-erbB2 overexpressing breast cancer cells is considerably modulated by EGFR coexpression and consequently EGFR/c-erbB2 homo- and heterointeractions, as well as the presence or absence of growth factors. C-erbB2 overexpression alone is insufficient to predict the impact of growth factors and antibodies on cell proliferation. The optimization and specification of therapeutic approaches based on erbB-receptor targeting requires to account for EGFR coexpression as well as the potential presence of erbB-receptor relevant growth factors.     

Rapid purification of human ductal cells from human pancreatic fractions with surface antibody CA19-9

Generating human insulin-secreting cells for cell therapy of diabetes represents a highly competitive world challenge. Human ductal cells can give rise to islets in vivo and in vitro. The goal of this study was to devise a rapid sorting method to highly purify human ductal cells from pancreatic tissue using a pan-ductal membrane antibody carbohydrate antigen 19-9 (CA19-9). Human pancreatic sections confirmed antibody specificity. The human exocrine fraction (30% ductal cells) was sorted with magnetic bead technology or by FACS. Immunocytochemistry post-sorting determined ductal cell content. The manual magnetic bead technique resulted in 74%+/-2 (n = 4) CA19 positive cells. Whereas the automated AutoMACS technique (n = 5) yielded 92.6%+/-0.5 CA19-9 positive cells with only a minor beta cell contamination (0.2%+/-0.03); cell yield post-sorting was 12.9%+/-2.5 (1.69+/-0.41 x 10(6) cells) with 51.7%+6.5 (n = 5) viability post-sorting. The FACS (n = 6) resulted in 97.1%+/-0.82 CA19-9 positive cells, a cell yield of 25.5%+/-5.6 (5.03+/-1.0 x 10(6)), with 72.1%+/-6.1 viability post-sorting.     

Fetal erythroblast isolation up to purity from cord blood and their culture in vitro

BACKGROUND: Erythroblasts have been the most encouraging candidate cell type for noninvasive prenatal genetic investigation. We previously showed that human erythroblasts can be recovered from bone marrow and blood bank buffy coats by a physical cell separation. In the present study, we modified our previous methodology, taking into account the peculiar behavior of erythroblasts in response to modifications of pH and osmolality of the separation medium. METHODS: Twenty to forty milliters of cord blood were initially centrifuged on Ficoll/diatrizoate (1.085 g/ml). The interphase cells were further separated on a continuous density gradient (1.040-1.085 g/ml). Two different gradients were initially compared: the first was iso-osmolar and neutral, whereas the second also contained an ionic strength gradient and a pH gradient (triple gradient). A subsequent monocyte depletion was performed by using magnetic microbeads coated with anti-CD14 monoclonal antibody (mAb), and erythroblasts were purified by sedimentation velocity. Purified cells were investigated by analyses with fluorescence-activated cell sorting (FACS) and fluorescence in situ hybridization (FISH) and immunocytochemistry with mAb against fetal hemoglobin and were cultured in vitro. RESULTS: When nucleated cells were spun on an iso-osmolar and neutral continuous density gradient, two separated bands of nucleated red blood cells (NRBCs) were obtained: a light fraction banding at 1.062 g/ml and an heavy fraction banding at 1.078 g/ml. Conversely, when cells were spun in the triple gradient, NRBCs were shifted to the low-density region. Monocyte depletion by immunomagnetic microbeads and velocity sedimentation provided a pure erythroblast population. FACS and FISH analyses and immunocytochemistry substantiated the purity of the isolated cell fraction, which was successfully cultured in vitro. CONCLUSIONS: We have shown that fetal erythroblasts can be purified up to homogeneity from cord blood, but further refinements of the isolation procedure are necessary before the same results can be obtained from maternal peripheral blood.     

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations. FACS is a technique of choice to purify cell populations of known phenotype. Other bulk methods of purification include panning, complement depletion and magnetic bead separation. However, FACS has several advantages over other available methods. FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density. In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker. FACS allows the purification of individual cells based on size, granularity and fluorescence. In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1). Negative selection of unstained cells is also possible. FACS purification requires a flow cytometer with sorting capacity and the appropriate software. For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser. The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells. The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2). The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb. Sorting parameters can be adjusted depending on the requirement of purity and yield. Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.