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1.
Recent Results Cancer Res ; 215: 25-55, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31605222

RESUMO

Circulating tumor cells (CTCs) are responsible for the metastatic spread of cancer and therefore are extremely valuable not only for basic research on cancer metastasis but also as potential biomarkers in diagnosing and managing cancer in the clinic. While relatively non-invasive access to the blood tissue presents an opportunity, CTCs are mixed with approximately billion-times more-populated blood cells in circulation. Therefore, the accuracy of technologies for reliable enrichment of the rare CTC population from blood samples is critical to the success of downstream analyses. The focus of this chapter is to provide the reader an overview of significant advances made in the development of diverse CTC enrichment technologies by presenting the strengths of individual techniques in addition to specific challenges remaining to be addressed.


Assuntos
Separação Celular/métodos , Células Neoplásicas Circulantes , Humanos
2.
Recent Results Cancer Res ; 215: 77-88, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31605224

RESUMO

Circulating tumor cells (CTCs) provide valuable information about the molecular evolution of cancers, as they may initially respond and ultimately progress on therapy. As intact tumor cells isolated from the bloodstream, CTCs also enable assessment of heterogeneous subpopulations, and their analysis may include DNA, RNA, and protein biomarkers. New microfluidic cell isolation strategies greatly facilitate the challenge of enriching viable tumor cells from the billions of hematopoietic cells within a standard blood specimen. While counting and characterization of enriched CTCs have primarily relied on immunostaining for tumor cell-specific antigens, new RNA-based analytic platforms are providing new insight into the identity of CTCs and providing new tools for clinical applications. Single-cell RNA sequencing of CTCs reveals a high degree of heterogeneity among cancer cells from a single individual, while new digital RNA-based amplification platforms may now allow high-sensitivity and high-throughput quantitative scoring of CTCs for clinical applications. Here, we focus on transcriptomic analysis of CTCs and its relevance in understanding metastatic cancer progression and in developing diagnostic assays to monitor cancer.


Assuntos
Separação Celular/métodos , Neoplasias/genética , Neoplasias/patologia , Células Neoplásicas Circulantes , RNA Neoplásico/análise , Progressão da Doença , Humanos , Neoplasias/diagnóstico , Células Neoplásicas Circulantes/metabolismo , RNA Neoplásico/genética
3.
Immunology ; 159(1): 63-74, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31573680

RESUMO

Monocyte-derived macrophages (MDMs) generated from peripheral blood monocytes are widely used to model human macrophages for in vitro studies. However, the possible impact of different isolation methods on the resulting MDM phenotype is poorly described. We aimed to investigate the effects of three commonly used monocyte isolation techniques on the resulting MDM phenotype. Plastic adhesion, negative selection, and CD14pos selection were compared. Monocyte-derived macrophages were generated by 5-day culture with macrophage and granulocyte-macrophage colony-stimulating factors. We investigated monocyte and MDM yields, purity, viability, and cell phenotype. CD14pos selection resulted in highest monocyte yield (19·8 × 106 cells, equivalent to 70% of total) and purity (98·7%), compared with negative selection (17·7 × 106 cells, 61% of total, 85·0% purity), and plastic adhesion (6·1 × 106 cells, 12·9% of total, 44·2% purity). Negatively selected monocytes were highly contaminated with platelets. Expression of CD163 and CD14 were significantly lower on CD14pos selection and plastic adhesion monocytes, compared with untouched peripheral blood mononuclear cells. After maturation, CD14pos selection also resulted in the highest MDM purity (98·2%) compared with negative selection (94·5%) and plastic adhesion (66·1%). Furthermore, MDMs from plastic adhesion were M1-skewed (CD80high  HLA-DRhigh  CD163low ), whereas negative selection MDMs were M2-skewed (CD80low  HLA-DRlow  CD163high ). Choice of monocyte isolation method not only significantly affects yield and purity, but also impacts resulting phenotype of cultured MDMs. These differences may partly be explained by the presence of contaminating cells when using plastic adherence or negative selection. Careful considerations of monocyte isolation methods are important for designing in vitro assays on MDMs.


Assuntos
Diferenciação Celular , Separação Celular/métodos , Citometria de Fluxo , Receptores de Lipopolissacarídeos/metabolismo , Macrófagos/fisiologia , Monócitos/fisiologia , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Biomarcadores , Adesão Celular , Células Cultivadas , Humanos , Interleucina-6/metabolismo , Lectinas Tipo C/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Lectinas de Ligação a Manose/metabolismo , Monócitos/imunologia , Monócitos/metabolismo , Fenótipo , Receptores de Superfície Celular/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
4.
Biosci Biotechnol Biochem ; 84(1): 198-207, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31566090

RESUMO

High glycosidase-producing strains of Aspergillus luchuensis were isolated from 2-deoxyglucose (2-DG) resistant mutants. α-Amylase, exo-α-1,4-glucosidase, ß-glucosidase and ß-xylosidase activity in the mutants was ~3, ~2, ~4 and ~2.5 times higher than the parental strain RIB2604 on koji-making conditions, respectively. Citric acid production and mycelia growth of the mutants, however, approximately halved to that of the parent. Compared to the parent, the alcohol yield from rice and sweet potato shochu mash of the mutant increased ~5.7% and 3.0%, respectively. The mutant strains showed significantly low glucose assimilability despite the fructose one was almost normal, and they had a single missense or nonsense mutation in the glucokinase gene glkA. The recombinant strain that was introduced at one of the mutations, glkA Q300K, demonstrated similar but not identical phenotypes to the mutant strain. This result indicates that glkA Q300K is one of the major mutations in 2-DG resistant strains.


Assuntos
Aspergillus/genética , Aspergillus/isolamento & purificação , Separação Celular/métodos , Códon sem Sentido/genética , Genes Fúngicos/genética , alfa-Glucosidases/metabolismo , Aspergillus/classificação , Aspergillus/metabolismo , Catepsina A/metabolismo , Ácido Cítrico/metabolismo , Desoxiglucose/farmacologia , Farmacorresistência Fúngica , Etanol/metabolismo , Fermentação , Frutose/metabolismo , Glucoquinase/genética , Glucose/metabolismo , Ipomoea batatas/química , Oryza/química , Fenótipo , Saccharomyces cerevisiae/classificação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/isolamento & purificação , Saccharomyces cerevisiae/metabolismo , Xilosidases/metabolismo , alfa-Amilases/metabolismo , beta-Glucosidase/metabolismo
5.
Sheng Li Xue Bao ; 71(6): 883-893, 2019 Dec 25.
Artigo em Chinês | MEDLINE | ID: mdl-31879744

RESUMO

In this study, we improved the culture method of mouse hippocampal primary microglia to obtain hippocampal ramified microglia with high activity and purity, which were resemble to the resting status of normal microglia in healthy brain in vivo. Hippocampal tissue was excised from 2-4-week-old SPF C57BL/6J mice and cut into pieces after PBS perfusion, and then manually dissociated into the single-cell suspension by using Miltenyi Biotec's Adult Brain Dissociation Kit. The tissue fragments such as myelin in the supernatant were removed by debris removal solution in the kit. The cell suspension was incubated with CD11b immunomagnetic beads for 15 min at 4 °C. To obtain high-purity microglia, we used two consecutive cell-sorting steps by magnetic activated cell sorting (MACS). After centrifugation, the cells were resuspended and seeded in a 24-well culture plate. The primary microglia were cultured with complete medium (CM) or TIC medium (a serum-free medium with TGF-ß, IL-34 and cholesterol as the main nutritional components) for 4 days, and then were used for further experiments. The results showed that: (1) The cell viability was (56.03 ± 2.10)% by manual dissociation of hippocampus; (2) Compared with immunopanning, two-step MACS sorting allowed for efficient enrichment of microglia with higher purity of (86.20 ± 0.68)%; (3) After being incubated in TIC medium for 4 d, microglia exhibited branching, quiescent morphology; (4) The results from qRT-PCR assay showed that the levels of TNF-α, IL-1ß and CCL2 mRNA in TIC cultured-microglia were similar to freshly isolated microglia, while those were much higher in CM cultured-microglia after incubation for 4 d and 7 d (P < 0.05). Taken together, compared to the conventional approaches, this modified protocol of mouse hippocampal primary microglia culture by using MACS and TIC medium enables the increased yield and purity of microglia in the quiescent state, which is similar to normal ramified microglia in healthy brain in vivo.


Assuntos
Técnicas de Cultura de Células , Separação Celular , Hipocampo , Magnetismo , Microglia , Animais , Técnicas de Cultura de Células/métodos , Separação Celular/métodos , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BL , Microglia/citologia
6.
Cell Prolif ; 52(6): e12674, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31517418

RESUMO

OBJECTIVES: Chemotherapeutic drugs induce senescence in cancer cells but, unlike replicative senescence or oncogene-induced senescence, do so rather inefficiently and depending on DNA damage. A thorough understanding of the biology of chemotherapy-induced senescent cells requires their isolation from a mixed population of adjacent senescent and non-senescent cancer cells. MATERIALS AND METHODS: We have developed and optimized a rapid iodixanol (OptiPrep)-based gradient centrifugation system to identify, isolate and characterize doxorubicin (DXR)-induced senescent hepatocellular carcinoma (HCC) cells (HepG2 and Huh-7) in vitro. RESULTS: After cellular exposure to DXR, we used iodixanol gradient-based centrifugation to isolate and re-plate cells on collagen-coated flasks, despite their low or null proliferative capacity. The isolated cell populations were enriched for DXR-induced senescent HCC cells, as confirmed by proliferation arrest assay, and ß-galactosidase and DNA damage-dependent γH2A.X staining. CONCLUSIONS: Analysing pure cultures of chemotherapy-induced senescent versus non-responsive cancer cells will increase our knowledge on chemotherapeutic mechanisms of action, and help refine current therapeutic strategies.


Assuntos
Carcinoma Hepatocelular/patologia , Separação Celular , Neoplasias Hepáticas/patologia , Ácidos Tri-Iodobenzoicos/farmacologia , Separação Celular/métodos , Senescência Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Doxorrubicina/farmacologia , Humanos
7.
Anal Chim Acta ; 1082: 136-145, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31472702

RESUMO

Circulating tumor cells (CTCs) are expected to serve as a blood-based biomarker in the diagnosis of cancers at an early stage, providing an opportunity to increase the survival of cancer patients. Current techniques for CTC detection were designed for some particular types of cancer with confirmed primary tumor origin. In this work, a platform for the detection of two cancer types and the identification of the primary tumor origin of CTCs was established to meet the requirement of cancer diagnosis and clinical application. A combined strategy based on in vivo capture method using antibody cocktail and multicolor fluorescence imaging using aptamer was designed to achieve the high-efficiency capture of CTCs and the accurate location of the primary tumor. An antibody cocktail of epithelial cell adhesion molecule (EpCAM) and epidermal growth factor receptor (EGFR) was applied to capture breast cancer CTCs and hepatocellular CTCs in vivo. The capture efficiency of hepatocellular CTCs was significantly increased from 3.17% to 26.67% and the capture efficiency of breast cancer CTCs slightly increased from 27.00% to 29.84% compared with EpCAM-based capture of CTCs. Meanwhile, the primary tumor origins of breast cancer CTCs and hepatocellular CTCs were simultaneously distinguished by specific aptamer-based fluorescence probes without any signal crosstalk. The results of in vivo experiments using the dual tumor-bearing mouse model confirmed the feasibility of this method to capture CTCs and identify primary tumor origins. This simple and efficient approach has potential for future applications in cancer diagnosis and prognosis.


Assuntos
Aptâmeros de Nucleotídeos/química , Neoplasias da Mama/diagnóstico , Neoplasias Hepáticas/diagnóstico , Células Neoplásicas Circulantes/patologia , Adenocarcinoma/diagnóstico , Adenocarcinoma/patologia , Animais , Anticorpos Imobilizados/imunologia , Neoplasias da Mama/patologia , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Separação Celular/instrumentação , Separação Celular/métodos , Molécula de Adesão da Célula Epitelial/imunologia , Receptores ErbB/imunologia , Feminino , Corantes Fluorescentes/química , Humanos , Neoplasias Hepáticas/patologia , Camundongos Endogâmicos BALB C , Camundongos Nus , Microscopia de Fluorescência/métodos , Células Neoplásicas Circulantes/imunologia , Coelhos
8.
BMC Mol Cell Biol ; 20(1): 32, 2019 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-31409295

RESUMO

BACKGROUND: Endothelial cells (ECs) play a key role in tissue homeostasis, in several pathological conditions, and specifically in the control of vascular functions. ECs are frequently used as in vitro model systems for cardiovascular studies and vascular biology. The porcine model is commonly used in human clinical cardiovascular studies. Currently, however, there is no robust protocol for the isolation of porcine heart ECs. We have developed a fast isolation protocol, which is cost effective, takes only 1-2 h, and produces EC purity of over 97%. This protocol is optimized for porcine hearts but can be adapted for use with other large animals. METHODS: Heart is washed by flushing with PBS, whereafter endothelial cells are detached by collagenase incubation and the cells can then be collected immediately after the incubation and plated within an hour after the heart is isolated from a pig. RESULTS: The swiftness of the protocol limits changes in the phenotype and RNA expression profile of the cells. Cells were identified as ECs with CD31 (PECAM-1) antibody immunostaining. Functionality of ECs were ensured with in vitro angiogenesis assay. The purity of the ECs was verified by using fluorescence assisted cell sorting (FACS) with the CD31 antibody. CONCLUSION: We developed a new, fast, and cost-effective isolation method for pig heart ECs. Successful isolation of pure ECs is a prerequisite for several cardiovascular and vascular biology studies.


Assuntos
Doenças Cardiovasculares/patologia , Biologia Celular , Separação Celular/métodos , Células Endoteliais/citologia , Genômica , Miocárdio/citologia , Transcriptoma/genética , Animais , Células Cultivadas , Feminino , Suínos
9.
Biosens Bioelectron ; 143: 111604, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31466047

RESUMO

Despite the fact that the identification and detection of circulating tumor cells (CTCs) plays a critical role in cancer monitoring and diagnosis, it remains a major challenge to isolate and detect these cells, due to their extreme scarcity in peripheral blood. In this work, by coupling a dual recognition strategy and the commercial personal glucose meter, we established a point-of-care approach for detecting rare CTCs in whole blood with high sensitivity and selectivity. The antibody-conjugated magnetic beads lead to the capture and isolation of the CTCs while the enzyme- and second antibody-modified microspheres yield the signal for detection. Because of the dual recognition format, the developed method is highly selective, and a low detection limit of 7 cells can be realized as well, owing to the great signal amplification through the enzyme-loaded microbead labels. More importantly, the detection of CTCs in whole blood can be achieved in a point-of-care fashion with the using of the glucose meter transducer, offering our method a convenient and attractive alternative to traditional biopsy for the diagnosis of various cancers.


Assuntos
Técnicas Biossensoriais , Imunoconjugados/imunologia , Neoplasias/sangue , Células Neoplásicas Circulantes , Contagem de Células , Separação Celular/métodos , Glucose/metabolismo , Humanos , Imunoconjugados/química , Microesferas , Sistemas Automatizados de Assistência Junto ao Leito
10.
Nat Commun ; 10(1): 3417, 2019 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-31366909

RESUMO

High costs and technical limitations of cell sorting and single-cell techniques currently restrict the collection of large-scale, cell-type-specific DNA methylation data. This, in turn, impedes our ability to tackle key biological questions that pertain to variation within a population, such as identification of disease-associated genes at a cell-type-specific resolution. Here, we show mathematically and empirically that cell-type-specific methylation levels of an individual can be learned from its tissue-level bulk data, conceptually emulating the case where the individual has been profiled with a single-cell resolution and then signals were aggregated in each cell population separately. Provided with this unprecedented way to perform powerful large-scale epigenetic studies with cell-type-specific resolution, we revisit previous studies with tissue-level bulk methylation and reveal novel associations with leukocyte composition in blood and with rheumatoid arthritis. For the latter, we further show consistency with validation data collected from sorted leukocyte sub-types.


Assuntos
Separação Celular/métodos , Biologia Computacional/métodos , Metilação de DNA/genética , Epigênese Genética/genética , Análise de Célula Única/métodos , Artrite Reumatoide/sangue , Ilhas de CpG/genética , Humanos , Contagem de Leucócitos , Leucócitos/classificação , Leucócitos/citologia
11.
Int J Nanomedicine ; 14: 4187-4209, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31289440

RESUMO

Circulating tumor cells (CTCs) are disseminated cancer cells. The occurrence and circulation of CTCs seem key for metastasis, still the major cause of cancer-associated deaths. As such, CTCs are investigated as predictive biomarkers. However, due to their rarity and heterogeneous biology, CTCs' practical use has not made it into the clinical routine. Clearly, methods for the effective isolation and reliable detection of CTCs are urgently needed. With the development of nanotechnology, various nanosystems for CTC isolation and enrichment and CTC-targeted cancer therapy have been designed. Here, we summarize the relationship between CTCs and tumor metastasis, and describe CTCs' unique properties hampering their effective enrichment. We comment on nanotechnology-based systems for CTC isolation and recent achievements in microfluidics and lab-on-a-chip technologies. We discuss recent advances in CTC-targeted cancer therapy exploiting the unique properties of nanomaterials. We conclude by introducing developments in CTC-directed nanosystems and other advanced technologies currently in (pre)clinical research.


Assuntos
Biomarcadores Tumorais/análise , Separação Celular/métodos , Nanomedicina/métodos , Células Neoplásicas Circulantes/patologia , Biomarcadores Tumorais/isolamento & purificação , Materiais Biomiméticos , Grafite , Humanos , Dispositivos Lab-On-A-Chip , Microfluídica/instrumentação , Microfluídica/métodos , Nanoestruturas/química , Nanotecnologia/métodos , Nanotubos de Carbono
12.
Hematol Oncol ; 37(4): 401-408, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31291481

RESUMO

Multiple myeloma (MM) is malignant tumor with abnormal proliferation of bone marrow plasma cells. The existing clinical tools used to determine treatment response and tumor relapse are limited in sensitivity. We investigated the CD138+ microparticles (MPs) of MM patients to find out whether MPs could provide a novel means to monitor the malignant cells in MM patients. Our study showed that the levels of MPs were significantly elevated in MM patients. The MP counts in peripheral blood from new diagnosed MM patients were significantly higher than patients in CR and HD. Consist with the total MPs, the number of the PC-derived MPs (CD138+) increased in BM from MM patients compared with CR and HD. The ratio of the PC-derived MPs (CD138+) in BM increased in MM patients compared with CR and HD. The correlation test revealed that the CD138+ MPs in BM and PB were all positively correlated with the plasmacyte ratio in bone marrow (BMPC) and the ß2 -MG. New diagnosed MM patients and controls were compared, and ROC curves were used to identify cutoff points with optimal sensitivity and specificity concerning the ratios and counts of CD138+ MPs in BM and PB. The AUC of the CD138+ MP counts in BM was 0.767, and in PB was 0.680. The AUC of the CD138+ MP ratios in BM was 0.714, and in PB was 0.666. According to this, the counts of CD138+ MPs in BM showed to be a powerful marker of diagnosis. We demonstrated that CD138+ MPs from the plasma provide support for a potential monitoring biomarker of MM.


Assuntos
Células da Medula Óssea/química , Medula Óssea/patologia , Micropartículas Derivadas de Células/química , Mieloma Múltiplo/sangue , Proteínas de Neoplasias/sangue , Sindecana-1/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Área Sob a Curva , Biomarcadores Tumorais/sangue , Separação Celular/métodos , Feminino , Citometria de Fluxo/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/diagnóstico , Mieloma Múltiplo/patologia , Plasmócitos/patologia , Curva ROC , Sensibilidade e Especificidade , Sindecana-1/análise , Microglobulina beta-2/análise
13.
Hematology ; 24(1): 533-537, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31280705

RESUMO

OBJECTIVE: Buffy coat and ficoll of bone marrow (BM) are viable options for the study of minimal residual disease (MRD) in multiple myeloma (MM). As yet, there is no data about the superiority of either sample types. Herein, we aimed to address this issue. METHODS: Forty pairs of ficolled BMs and BM buffy coats of 19 MM patients were studied for MRD by allele-specific oligonucleotide real-time quantitative PCR, with patient-specific primers/probes whenever appropriate. RESULTS: There were 41 pairs of MRD data for comparison analysis due to one patient with biclonal disease. MRD levels in ficolls and buffy coats were highly concordant (rs = 0.936, P < 0.0001), with 31 (76%) and seven (17%) pairs being concomitantly MRD-positive or -negative. On the other hand, apart from the 16 pairs being both MRD-negative, or -positive but not quantifiable in ficolls and buffy coats, majority (n = 22, 88%) had higher MRD levels in ficolled BMs than BM buffy coats. Furthermore, in 17 pairs, in which MRD was quantifiable in both, MRD levels in ficolled BMs were 3.1 times those of BM buffy coats (median, 567/105 vs. 184/105, P = 0.001). CONCLUSION: Taken together, ficolled BM is more sensitive than BM buffy coat for MRD detection in MM, hence should be recommended.


Assuntos
Exame de Medula Óssea/métodos , Separação Celular/métodos , Centrifugação/métodos , Ficoll , Mieloma Múltiplo/patologia , Centrifugação com Gradiente de Concentração/métodos , Células Clonais , Primers do DNA , DNA de Neoplasias/análise , DNA de Neoplasias/isolamento & purificação , Rearranjo Gênico , Genes de Imunoglobulinas , Humanos , Leucócitos Mononucleares , Mieloma Múltiplo/genética , Neoplasia Residual , Células-Tronco Neoplásicas , Plasmócitos , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Manejo de Espécimes
14.
Talanta ; 204: 731-738, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31357358

RESUMO

Blood is a routinely tested biological fluid for diagnosis and monitoring of diseases as many diseases would trigger a change in white blood cell count. Thus, several methods have been established to isolate or enrich white blood cells from patient blood samples for such analyses. One method of preparing an enriched white blood cell sample is through the selective lysis of red blood cells by hypotonic shock and restoration of osmolarity to maintain viability of target white blood cells. An inherent problem with this approach is the loss of target cells during sample handling. We report a two-stage separation system that can perform lysis and restoration of osmolarity of blood on-chip and direct the resultant sample to the second step of the analysis. Hence, there is no loss of sample. The post-lysis makeup features a protein-rich buffer to help stabilize cells. As proof of concept, we spiked HL-60 cells into a whole blood and a pre-lysed blood sample and compared capture metrics of each method using a downstream affinity separation. The capture efficiency of the whole blood sample ranged between 40 and 80% using <7 µL of sample compared to 10-52% from 60 µL of pre-lysed blood required for similar analysis. In addition, both pre-lysed and whole blood samples showed no significant difference in purity and viability. This two-stage separation system has demonstrated the capacity to replace centrifugation and wash steps required for the preparation of lysed blood, for white blood cell analyses.


Assuntos
Separação Celular/métodos , Dispositivos Lab-On-A-Chip , Leucócitos/citologia , Separação Celular/instrumentação , Eritrócitos/metabolismo , Células HL-60 , Hemólise , Humanos , Concentração Osmolar , Estudo de Prova de Conceito
15.
Nat Commun ; 10(1): 2880, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253799

RESUMO

Cell state-specific promoters constitute essential tools for basic research and biotechnology because they activate gene expression only under certain biological conditions. Synthetic Promoters with Enhanced Cell-State Specificity (SPECS) can be superior to native ones, but the design of such promoters is challenging and frequently requires gene regulation or transcriptome knowledge that is not readily available. Here, to overcome this challenge, we use a next-generation sequencing approach combined with machine learning to screen a synthetic promoter library with 6107 designs for high-performance SPECS for potentially any cell state. We demonstrate the identification of multiple SPECS that exhibit distinct spatiotemporal activity during the programmed differentiation of induced pluripotent stem cells (iPSCs), as well as SPECS for breast cancer and glioblastoma stem-like cells. We anticipate that this approach could be used to create SPECS for gene therapies that are activated in specific cell states, as well as to study natural transcriptional regulatory networks.


Assuntos
Aprendizado de Máquina , Regiões Promotoras Genéticas , Software , Neoplasias da Mama , Linhagem Celular Tumoral , Separação Celular/métodos , Feminino , Regulação da Expressão Gênica , Biblioteca Gênica , Glioblastoma , Humanos , Células-Tronco Pluripotentes Induzidas , Lentivirus , Células-Tronco Neoplásicas , Organoides , Elementos Reguladores de Transcrição
16.
Opt Express ; 27(10): 13706-13720, 2019 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-31163830

RESUMO

An outstanding challenge for immunology is the classification of immune cells in a label-free fashion with high speed. For this purpose, optical techniques such as Raman spectroscopy or digital holographic microscopy have been used successfully to identify immune cell subsets. To achieve high accuracy, these techniques require a post-processing step using linear methods of multivariate processing, such as principal component analysis. Here we demonstrate for the first time a comparison between artificial neural networks and principal component analysis (PCA) to classify the key granulocyte cell lineages of neutrophils and eosinophils using both digital holographic microscopy and Raman spectroscopy. Artificial neural networks can offer advantages in terms of classification accuracy and speed over a PCA approach. We conclude that digital holographic microscopy with convolutional neural networks based analysis provides a route to a robust, stand-alone and high-throughput hemogram with a classification accuracy of 91.3 % at a throughput rate of greater than 100 cells per second.


Assuntos
Eosinófilos/citologia , Holografia/métodos , Neutrófilos/citologia , Análise Espectral Raman/métodos , Linhagem da Célula , Separação Celular/métodos , Citometria de Fluxo , Humanos , Análise de Componente Principal
17.
Nat Protoc ; 14(7): 1991-2014, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31160788

RESUMO

Ploidy represents the number of chromosome sets in a cell. Although gametes have a haploid genome (n), most mammalian cells have diploid genomes (2n). The diploid status of most cells correlates with the number of probable alleles for each autosomal gene and makes it difficult to target these genes via mutagenesis techniques. Here, we describe a 7-week protocol for the derivation of mouse haploid embryonic stem cells (hESCs) from female gametes that also outlines how to maintain the cells once derived. We detail additional procedures that can be used with cell lines obtained from the mouse Haplobank, a biobank of >100,000 individual mouse hESC lines with targeted mutations in 16,970 genes. hESCs can spontaneously diploidize and can be maintained in both haploid and diploid states. Mouse hESCs are genomically and karyotypically stable, are innately immortal and isogenic, and can be derived in an array of differentiated cell types; they are thus highly amenable to genetic screens and to defining molecular connectivity pathways.


Assuntos
Técnicas de Cultura de Células/métodos , Haploidia , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/fisiologia , Animais , Blastocisto/citologia , Linhagem Celular , Separação Celular/métodos , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Fluxo de Trabalho
18.
Methods Mol Biol ; 1982: 39-59, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31172465

RESUMO

Determination of the structure of human neutrophil (PMN) flavocytochrome b (Cytb) is a necessary step for the understanding of the structure-function essentials of NADPH oxidase activity. This understanding is crucial for structure-driven therapeutic approaches addressing control of inflammation and infection. Our work on purification and sample preparation of Cytb has facilitated progress toward the goal of structure determination. Here we describe exploiting immunoaffinity purification of Cytb for initial examination of its size and shape by a combination of classical and cryoelectron microscopic (EM) methods. For these evaluations, we used conventional negative-stain transmission electron microscopy (TEM) to examine both detergent-solubilized Cytb as single particles and Cytb in phosphatidylcholine reconstituted membrane vesicles as densely packed random, partially ordered, and subcrystalline arrays. In preliminary trials, we also examined single particles by cryoelectron microscopy (cryoEM) methods. We conclude that Cytb in detergent and reconstituted in membrane is a relatively compact, symmetrical protein of about 100 Å in maximum dimension. The negative stain, preliminary cryoEM, and crude molecular models suggest that the protein is probably a heterotetramer of two p22phox and gp91phox subunits in both detergent micelles and membrane vesicles. This exploratory study also suggests that high-resolution 2D electron microscopic approaches may be accessible to human material collected from single donors.


Assuntos
Separação Celular/métodos , Grupo dos Citocromos b/metabolismo , Microscopia Eletrônica , NADPH Oxidases/metabolismo , Neutrófilos/metabolismo , Neutrófilos/ultraestrutura , Anticorpos Monoclonais , Biomarcadores , Microscopia Crioeletrônica , Grupo dos Citocromos b/química , Grupo dos Citocromos b/isolamento & purificação , Estabilidade Enzimática , Humanos , Lipossomos/química , Lipossomos/metabolismo , Lipossomos/ultraestrutura , Microscopia Eletrônica/métodos , NADPH Oxidases/química , NADPH Oxidases/isolamento & purificação , Neutrófilos/imunologia
19.
Biomater Sci ; 7(8): 3359-3372, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31231724

RESUMO

Magnetic nanomaterials have drawn ample attention in the field of biotechnology due to their excellent magnetic properties and biocompatibility. These materials have been widely used for exosome isolation, DNA separation, magnetic resonance imaging, and drug delivery. However, their application in cell isolation has been limited due to the lack of efficient antibody conjugation and instability in aqueous solutions. In this study, we produced hybrid maghemite nanorod/immuno-microgels with high capturing capacity for cell isolation and enumeration. Lepidocrocite (γ-FeOOH) and maghemite (γ-Fe2O3) nanorods with controlled morphology are synthesized using hydrolysis method. The effects of the different synthesis conditions on morphology, phase composition, and magnetic properties of lepidocrocite are studied to determine the best synthesis conditions. We coat the nanorods with chitosan and attach them to the poly(N-isopropylacrylamide-co-acrylic acid) (PNIPAM-AA) microgel through chemical bonding to form a nano/hybrid microstructure. Our results suggest that the hybrid magnetic microgels have more antibody binding capacity and higher cancer cell capturing rate compared to pristine maghemite nanorods. In addition, new cell magnetometery method was applied for cancer cell quantification after capturing step in which different magnetized labelled cells were correlated to the saturation magnetization. In this method, higher concentrations of the primary cell suspension resulted in more binding of the magnetic immuno-microgels to the cells which was shown as saturation magnetization drop in the microgel-cell complex.


Assuntos
Contagem de Células/métodos , Separação Celular/métodos , Compostos Férricos/química , Nanoestruturas/química , Resinas Acrílicas/química , Géis , Nanotubos/química , Propriedades de Superfície
20.
Int J Dev Biol ; 63(6-7): 295-299, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31250913

RESUMO

We have successfully isolated cells with stem-like properties from bottlenose dolphin (Tursiops truncatus) umbilical cord. Our results show that this cetacean species has embryonic fetal and adult stem cells as do humans and other studied mammals. This accomplishment allows to eventually investigate whether dolphins, due to their unique adaptations to aquatic environments, have special stem cell lineages or distinctive mechanisms of cell programming. Further characterization of their potency to differentiate into multiple cell lineages would fulfill numerous applicative purposes. We characterized, developed and refined a new protocol for obtaining potential stem cells from umbilical cord tissues of the bottlenose dolphin. Tissue samples were taken from umbilical cords of successful deliveries immediately after placenta ejection and collection from the water. Umbilical cord samples (2-3 cm3) were excised and subjected to enzymatic digestion and mechanical dissociation. Viable cells from specimens resident in the Oceanografic Valencia were cultured and subsequently isolated and tested for pluripotent characteristics (cell morphology, phenotype and expression of surface markers). Cell viability was confirmed also after freezing/thawing. The established protocol is suitable for collection/isolation/culture of dolphin potential mesenchymal stem cells from dolphin umbilical cord, which can be deposited in cell banks for future research needs.


Assuntos
Células-Tronco Adultas/citologia , Golfinho Nariz-de-Garrafa/metabolismo , Separação Celular/métodos , Células-Tronco Embrionárias/citologia , Células-Tronco Fetais/citologia , Células-Tronco Mesenquimais/citologia , Cordão Umbilical/citologia , Células-Tronco Adultas/metabolismo , Animais , Biomarcadores/metabolismo , Diferenciação Celular , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Feminino , Células-Tronco Fetais/metabolismo , Células-Tronco Mesenquimais/metabolismo , Cordão Umbilical/metabolismo
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