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1.
Proc Natl Acad Sci U S A ; 117(29): 16839-16847, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32641515

RESUMO

Circulating tumor cell (CTC)-based liquid biopsies provide unique opportunities for cancer diagnostics, treatment selection, and response monitoring, but even with advanced microfluidic technologies for rare cell detection the very low number of CTCs in standard 10-mL peripheral blood samples limits their clinical utility. Clinical leukapheresis can concentrate mononuclear cells from almost the entire blood volume, but such large numbers and concentrations of cells are incompatible with current rare cell enrichment technologies. Here, we describe an ultrahigh-throughput microfluidic chip, LPCTC-iChip, that rapidly sorts through an entire leukapheresis product of over 6 billion nucleated cells, increasing CTC isolation capacity by two orders of magnitude (86% recovery with 105 enrichment). Using soft iron-filled channels to act as magnetic microlenses, we intensify the field gradient within sorting channels. Increasing magnetic fields applied to inertially focused streams of cells effectively deplete massive numbers of magnetically labeled leukocytes within microfluidic channels. The negative depletion of antibody-tagged leukocytes enables isolation of potentially viable CTCs without bias for expression of specific tumor epitopes, making this platform applicable to all solid tumors. Thus, the initial enrichment by routine leukapheresis of mononuclear cells from very large blood volumes, followed by rapid flow, high-gradient magnetic sorting of untagged CTCs, provides a technology for noninvasive isolation of cancer cells in sufficient numbers for multiple clinical and experimental applications.


Assuntos
Separação Celular/métodos , Ensaios de Triagem em Larga Escala/métodos , Microfluídica/métodos , Células Neoplásicas Circulantes/classificação , Linhagem Celular Tumoral , Separação Celular/instrumentação , Ensaios de Triagem em Larga Escala/instrumentação , Humanos , Leucaférese/métodos , Campos Magnéticos , Microfluídica/instrumentação
2.
Oncol Rep ; 43(6): 1975-1985, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32236590

RESUMO

Circulating tumor cells (CTCs) or CTC clusters are considered as suitable and relevant targets for liquid biopsy as they more accurately indicate cancer progression, the therapeutic effects of treatment and allows for monitoring of cancer metastasis in real­time. Among the various methods for isolating CTCs, size­based filtration is one of the most convenient methods. However, cell clogging makes the filtration process less efficient. In the present study, an electromagnetic vibration­based filtration (eVBF) device was developed that efficiently isolated rare CTCs and CTC clusters from clinical blood samples of patients with gastric cancer. Using human blood samples spiked with human gastric cancer cells, the parameters of this device such as vibrating amplitude and flow rate were optimized. Putative CTCs were detected using a conventional filtration method and the eVBF device from the peripheral blood samples of patients with gastric cancer. Continuous flow isolation of CTCs was evaluated by a simulated blood flow system. The eVBF device utilized the electromagnetic force to generate a periodic vibration that prevented the cell clogging and improved the filtering efficiency. The optimized eVBF device with the high­amplitude vibration exhibited a recovery efficiency of 80­90% from whole blood samples spiked with 100 or 1,000 gastric cancer cells per ml. Using the eVBF device, CTCs were detected in 100% of patients (10/10) with gastric cancer, and the positive detection rate of the eVBF device was 30% higher compared with the conventional filtration method. Furthermore, CTC clusters were detected in 40% (4/10) of CTC­positive patient samples, and the integrity of CTC clusters was preserved using the eVBF device. The eVBF device allowed for high­throughput (1 ml/min) and continuous flow isolation of CTCs without the addition of any antibodies, any chemical reagents or any pretreatment processes. Thus, the eVBF device provides an efficient tool for isolating rare CTCs and CTC clusters from patients with cancer, highlighting its potential for use in cancer diagnosis, treatment and cancer biology research.


Assuntos
Separação Celular/instrumentação , Filtração/instrumentação , Células Neoplásicas Circulantes/patologia , Neoplasias Gástricas/patologia , Idoso , Contagem de Células , Linhagem Celular Tumoral , Fenômenos Eletromagnéticos , Desenho de Equipamento , Feminino , Ensaios de Triagem em Larga Escala/instrumentação , Humanos , Masculino , Pessoa de Meia-Idade , Vibração
3.
Talanta ; 207: 120261, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31594567

RESUMO

Significant progress on circulating tumor cells (CTCs) has profound impact for noninvasive tumor profiling including early diagnosis, treatment monitoring, and metastasis recognition. Therefore, CTCs based liquid biopsy technology is taking a rapid growth in the field of precision oncology. The label-free approaches relied on microfluidic chip stand out from a crowd of methods that suffer from time consuming, extensive blood samples, lost target cells and labor-intensive operation. In this paper, a label-free separation microfluidic device was developed using multistage channel, which took full advantage of inertial lift force. Our strategy demonstrated CTCs were efficiently isolated from untreated human blood samples including antibody conjugation and erythrocyte lysis. This device was applied for isolating human brain malignant glioma cells that were spiked in human peripheral blood samples. The experimental condition was optimized and exhibited an average separation efficiency of ≥ 90% across cell morphological analysis, up to 84.96% purity of collected CTCs and the viability of all cells is >95%, which was better than other one-step CTCs separation methods. Furthermore, the CTCs were successfully separated from untreated clinical blood sample of cancer patient on the proposed microfluidic device. The entire experimental procedures are extremely low-cost and easy manipulation. It is believed that the proposed multistage microfluidic chip can become a promising tool for CTCs separation and early diagnosis of cancer.


Assuntos
Separação Celular/instrumentação , Hidrodinâmica , Dispositivos Lab-On-A-Chip , Células Neoplásicas Circulantes/patologia , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Desenho de Equipamento , Eritrócitos/patologia , Glioma/patologia , Humanos
4.
Ann Clin Lab Sci ; 49(6): 740-747, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31882424

RESUMO

OBJECTIVE: To explore the application of carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) and MACSiBeads™ Particles within in vitro suppression assays of regulatory T (Treg) cells. METHODS: CD4+CD25+ Treg cells and CD8+ T cells were sorted using magnetically activated cell sorting. CD8+ T cells were subjected to CFDA-SE staining to determine their optimal staining concentration. MACSi-Beads™ Particles, a component of the Treg expansion kit, were used as stimulators in suppression assays. Five experimental groups were set based on the condition of MACSiBeads™ Particles, CFDA-SE staining and cell composition of co-culture system, which were stimulated CD8+ cells without CFDA-SE staining, unstimulated CD8+ cells with CFDA-SE staining, stimulated CD8+ cells with CFDA-SE staining, co-cultured with Treg cells at a ratio of 1:0.25, 1:0.125 and 1:0, respectively. Flow cytometry was performed using the BD FACS Canto™ and flow data was analyzed using FCS Express 4 Plus software. RESULTS: CFSE fluorescence intensity correlated with cell type and culture time. The final CFDA-SE staining concentration was 0.5µM. Within in vitro suppression assays, Treg cells showed a significant inhibitory effect on the proliferation of CD8+ T cells increasing as its concentration increased. CONCLUSION: CFDA-SE combined with MACSiBeads™ Particles can be used to evaluate the in vitro inhibition effect of Treg cells.


Assuntos
Linfócitos T CD8-Positivos/citologia , Separação Celular/métodos , Fluoresceínas/farmacologia , Succinimidas/farmacologia , Linfócitos T Reguladores/efeitos dos fármacos , Proliferação de Células , Separação Celular/instrumentação , Técnicas de Cocultura , Citometria de Fluxo , Fluorescência , Corantes Fluorescentes , Humanos , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Leucócitos Mononucleares/citologia , Coloração e Rotulagem
5.
Sensors (Basel) ; 19(22)2019 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-31766178

RESUMO

There are a huge number, and abundant types, of microalgae in the ocean; and most of them have various values in many fields, such as food, medicine, energy, feed, etc. Therefore, how to identify and separation of microalgae cells quickly and effectively is a prerequisite for the microalgae research and utilization. Herein, we propose a microfluidic system that comprised microalgae cell separation, treatment and viability characterization. Specifically, the microfluidic separation function is based on the principle of deterministic lateral displacement (DLD), which can separate various microalgae species rapidly by their different sizes. Moreover, a concentration gradient generator is designed in this system to automatically produce gradient concentrations of chemical reagents to optimize the chemical treatment of samples. Finally, a single photon counter was used to evaluate the viability of treated microalgae based on laser-induced fluorescence from the intracellular chlorophyll of microalgae. To the best of our knowledge, this is the first laboratory prototype system combining DLD separation, concentration gradient generator and chlorophyll fluorescence detection technology for fast analysis and treatment of microalgae using marine samples. This study may inspire other novel applications of micro-analytical devices for utilization of microalgae resources, marine ecological environment protection and ship ballast water management.


Assuntos
Separação Celular/instrumentação , Microalgas/citologia , Microfluídica/instrumentação , Sobrevivência Celular , Fluorescência , Movimento , Reologia , Soluções
6.
Commun Biol ; 2: 393, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31701022

RESUMO

Cell detachment is essential in culturing adherent cells. Trypsinization is the most popular detachment technique, even though it reduces viability due to the damage to the membrane and extracellular matrix. Avoiding such damage would improve cell culture efficiency. Here we propose an enzyme-free cell detachment method that employs the acoustic pressure, sloshing in serum-free medium from intermittent traveling wave. This method detaches 96.2% of the cells, and increases its transfer yield to 130% of conventional methods for 48 h, compared to the number of cells detached by trypsinization. We show the elimination of trypsinization reduces cell damage, improving the survival of the detached cells. Acoustic pressure applied to the cells and media sloshing from the intermittent traveling wave were identified as the most important factors leading to cell detachment. This proposed method will improve biopharmaceutical production by expediting the amplification of tissue-cultured cells through a more efficient transfer process.


Assuntos
Separação Celular/métodos , Animais , Células CHO , Adesão Celular , Proliferação de Células , Separação Celular/instrumentação , Cricetulus , Meios de Cultura Livres de Soro , Dano ao DNA , Desenho de Equipamento , Proteínas de Membrana/metabolismo , Microscopia Eletrônica de Varredura , Tripsina , Ondas Ultrassônicas
7.
Int J Mol Sci ; 20(21)2019 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-31684107

RESUMO

Autologous therapies using adipose-derived stromal vascular fraction (AD-SVFs) and adult adipose-derived mesenchymal stem cells (AD-MSCs) warrant careful preparation of the harvested adipose tissue. Currently, no standardized technique for this preparation exists. Processing quantitative standards (PQSs) define manufacturing quantitative variables (such as time, volume, and pressure). Processing qualitative standards (PQLSs) define the quality of the materials and methods in manufacturing. The purpose of the review was to use PQSs and PQLSs to report the in vivo and in vitro results obtained by different processing kits that use different procedures (enzymatic vs. non-enzymatic) to isolate human AD-SVFs/AD-MSCs. PQSs included the volume of fat tissue harvested and reagents used, the time/gravity of centrifugation, and the time, temperature, and tilt level/speed of incubation and/or centrifugation. PQLSs included the use of a collagenase, a processing time of 30 min, kit weight, transparency of the kit components, the maintenance of a closed sterile processing environment, and the use of a small centrifuge and incubating rocker. Using a kit with the PQSs and PQLSs described in this study enables the isolation of AD-MSCs that meet the consensus quality criteria. As the discovery of new critical quality attributes (CQAs) of AD-MSCs evolve with respect to purity and potency, adjustments to these benchmark PQSs and PQLs will hopefully isolate AD-MSCs of various CQAs with greater reproducibility, quality, and safety. Confirmatory studies will no doubt need to be completed.


Assuntos
Tecido Adiposo/citologia , Vasos Sanguíneos/citologia , Separação Celular/métodos , Células-Tronco Mesenquimais/citologia , Células Estromais/citologia , Tecido Adiposo/irrigação sanguínea , Proliferação de Células , Separação Celular/instrumentação , Células Cultivadas , Centrifugação , Colagenases/metabolismo , Humanos
8.
Biosens Bioelectron ; 146: 111746, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31586761

RESUMO

Different circulating tumor cells (CTCs) in blood were separated and detected through the decoration of anti-cancer drug on the target cells, along with chemical modification of the microfluidic channel walls using a lipid attached covalently to the conducting polymer. The working principle of the electrochemical microfluidic device was evaluated with experimental parameters affecting on the separation, in terms of mass and surface charge of target species, fluid flow rate, AC amplitude, and AC frequency. The separated CTCs were selectively detected via the oxidation of daunomycin adsorbed specifically at the cells using an electrochemical sensor installed at the channel end. The fluorescence microscopic examination also confirmed the separation of CTCs in the channel. To evaluate the reliability of the method, blood samples from 37 cancer patients were tested. The device was able to separate the CTCs with 92.0 ±â€¯0.5 % efficiency and 90.9% detection rate.


Assuntos
Separação Celular/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Neoplasias/diagnóstico , Células Neoplásicas Circulantes/patologia , Técnicas Biossensoriais/instrumentação , Desenho de Equipamento , Células HEK293 , Células HeLa , Humanos , Lipídeos/química , Neoplasias/sangue , Polímeros/química
9.
Analyst ; 144(20): 5934-5946, 2019 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-31483419

RESUMO

We propose a novel microfluidic device for continuous, label-free and size-selective particle separation. The process consists of two stages: the particle separation based on the pre-focus of sheath flow and the size-selective interface between a Newtonian sample fluid and non-Newtonian poly(ethylene oxide) (PEO) solution (1st stage), and separation distance expansion due to the contraction-expansion structure (2nd stage). The force balance has been analyzed to explore the mechanism and the factors in the particle migration. In the 1st stage, the inertial lift force and the interfacial elastic lift force are a couple of counter forces which only allow the target particles to penetrate the interface. By controlling the flow rate ratio and the PEO concentration, all unwanted particles can be confined to the sample layer. In the 2nd stage, the elastic lift force is used to counteract the inertial lift force, which increases the predomination of the Dean drag force in the lateral migration of the target particles. We conclude that the separation distance is not monotonically increasing with the elastic lift force but peaks at 50-150 ppm. Thus, an optimal parameter for particle separation in our device is obtained. Compared to a similar method without the 2nd stage and the same device without the 1st stage, the distance between the target particles and the unwanted particles could increase by approximately 35.8% and 101.2%, respectively. Finally, a sensitive, time saving and no background-interfering cell smear method is approved to diagnose the malignant pleural effusion efficiently.


Assuntos
Separação Celular/instrumentação , Separação Celular/métodos , Micropartículas Derivadas de Células/patologia , Técnicas Analíticas Microfluídicas/instrumentação , Derrame Pleural Maligno/patologia , Polietilenoglicóis/química , Elasticidade , Humanos , Células Tumorais Cultivadas , Viscosidade
10.
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
11.
Electrophoresis ; 40(22): 2988-2995, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31538669

RESUMO

We present a novel technique for continuous label-free separation of particles based on their dielectrophoretic crossover frequencies. Our technique relies on our unique microfluidic geometry which performs hydrodynamic focusing, generates a stagnation flow with two outlets, and simultaneously produces an isomotive dielectrophoretic field via wall-situated electrodes. To perform particle separation, we hydrodynamically focus particles onto stagnation streamlines and use isomotive dielectrophoretic force to nudge the particles off these streamlines and direct them into appropriate outlets. Focusing particles onto stagnation streamlines obviates the need for large forces to be applied to the particles and therefore increases system throughput. The use of isomotive (spatially uniform) dielectrophoretic force increases system reliability. To guide designers, we develop and describe a simple scaling model for the particle separation dynamics of our technique. The model predicts the range of particle sizes that can be separated as well as the processing rate that can be achieved as a function of system design parameters: channel size, flow rate, and applied potential. Finally, as a proof-of-principle, we use this technique to separate polystyrene bead and cell mixtures of the same diameters as well as mixtures of both particles with varying diameters.


Assuntos
Separação Celular/instrumentação , Separação Celular/métodos , Eletroforese/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Desenho de Equipamento , Humanos , Células Jurkat , Tamanho da Partícula
12.
Andrologia ; 51(10): e13403, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31434165

RESUMO

The most recent technologies for sperm sorting involve microfluidics. However, the most important question whether their use is of any advantage in terms of laboratory and clinical IVF/ICSI outcomes still remains controversy. In this study, we aimed to investigate whether a microfluidic sperm sorting device (Fertile Plus® ) has a positive add-on effect on laboratory and clinical outcomes. Sibling oocytes of 81 patients were assigned to two sperm sorting groups including swim up and Fertile Plus® . All embryos were cultured until day 5/6. Fertilisation, embryo quality and blastocyst development were assessed as primary outcomes among 81 patients; clinical pregnancy, implantation and live birth rates were analysed as secondary outcomes as a subgroup analysis due to transfer cancellations. No statistically significant differences were found between groups in terms of all outcomes analysed in laboratory and clinical terms (p > .05 for all). The results of this study suggest that sorting spermatozoa through Fertile chip does not improve laboratory outcomes significantly and does not seem to have a positive contribution to clinical outcomes.


Assuntos
Separação Celular/instrumentação , Infertilidade Masculina/terapia , Dispositivos Lab-On-A-Chip , Injeções de Esperma Intracitoplásmicas/métodos , Espermatozoides , Adulto , Coeficiente de Natalidade , Separação Celular/métodos , Feminino , Humanos , Masculino , Oócitos , Gravidez , Taxa de Gravidez , Irmãos , Resultado do Tratamento , Adulto Jovem
13.
Talanta ; 205: 120129, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31450438

RESUMO

Self-assembly of building blocks for constructing multifunctional materials has opened prospects for sensing applications in the biomedical fields. In particular, the combination of aptamer with DNA assembly-based nanotechnology has greatly improved the performance of cancer cell detection. Nevertheless, the cancer cell detection strategies of integrating aptamer with protein are relatively sparse. So we have developed a self-assembled aptamer method to realize the efficient capture and rapid detection of cancer cells by ingeniously combining aptamer modified magnetic nanoparticles as capture nanoprobes with self-assembled aptamer/protein hybrid probes (SAPPs) as signal amplification probes. By merely mixing the component materials together simultaneously, the SAPPs, integrating aptamer for cancer cell recognition with protein for amplifying signal, were fabricated by DNA-governed one-step assembly. In addition, the SAPPs-based method exhibits efficient capture, rapid (about 45 min) and specific CCRF-CEM detection performance, with limits of detection down to 75 cells/mL in buffer and 200 cells/mL in whole blood.


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Separação Celular/métodos , Imãs/química , Células Neoplásicas Circulantes/patologia , Proteínas/metabolismo , Adsorção , Aptâmeros de Nucleotídeos/genética , Sequência de Bases , Linhagem Celular Tumoral , Separação Celular/instrumentação , Humanos , Nanopartículas/química , Fatores de Tempo
14.
Transfus Clin Biol ; 26(3): 164-170, 2019 Sep.
Artigo em Francês | MEDLINE | ID: mdl-31400933

RESUMO

BACKGROUND: The collection of granulocytes by apheresis requires volunteer donor stimulation by corticoids and the use of HES, a compound which is currently challenged by potential safety issues. Preparation of pooled granulocytes concentrates from whole blood buffy coats (PGC) represent an alternative to apheresis with a better benefit/risk for the donors. METHOD: Whole blood is collected in a bottom and top blood bag for buffy coat preparation. After centrifugation and separation, buffy coat are obtained. Twenty ABO matched buffy coats are selected for processing into one PGC. Four pools of five buffy coats were made, platelet additive solution is added to each pool, mixed gently and centrifuged. The red cell residue, supernatant and granulocyte rich layer are separated. Two granulocyte rich layers are pooled and added with 70mL of ABO matched plasma from the initial donations (=PGC10). The final PGC (=PGC20) is obtained by pooling two PGC10 into a platelet storage bag. Neutrophil content and in-vitro functionality are assessed at day of preparation (D1) and at expiry hour, 48 hours after collection (D2). RESULTS: On N=18, mean: Volume=408±4mL, 2.2*1010±0.24 neutrophils, Hematocrit=18%±3%, 4.7*1011platelets. Viability is well preserved: 95%±6% day of PGC preparation, 85%±7% after 24h of storage (D2). Functionality (ROS production measurement) is well preserved: 1.36±0.25 at D1 and 1.38±0.18 at D2. Expression and modulation of adhesion molecules after stimulation are normal at D1 and slightly decreased at D2 but still normal. CONCLUSIONS: PGC20 in vitro characteristics are in conformance with the EDQM guide (V19) and similar to apheresis for granulocytes content and hematocrit. The viability and two mean indicators which explore neutrophil function are well maintained during PGC preparation and after 24 hours of storage.


Assuntos
Buffy Coat/citologia , Separação Celular/métodos , Granulócitos , Sistema ABO de Grupos Sanguíneos/análise , Remoção de Componentes Sanguíneos , Doadores de Sangue , Plaquetas , Preservação de Sangue/instrumentação , Preservação de Sangue/métodos , Separação Celular/instrumentação , Centrifugação , Granulócitos/imunologia , Humanos , Masculino
15.
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
16.
Electrophoresis ; 40(21): 2845-2852, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31267582

RESUMO

Circulating tumor cells are specifically referred as cells that detached from the primary tumor and are present in the bloodstream. They could be isolated from blood and used as representative biomarker for predicting cancer prognoses. Here, we developed a microfluidic chip with multiple curved channels, in which DNA fragments and antibody-based enrichment are exploited to capture circulating tumor cells in blood sample. By introducing DNA fragments as long tentacles, the active antibody could be extended into the microchannel stereoscopically, which could greatly increase the chances of adhesion in a multidirectional way and improve the capture efficacy. Several pivotal factors for cell capturing were optimized to the best state. Compared to conventional chips for planar capturing, the capture efficiency of MCF-7 cells was greatly increased from 37.17 to 85.10%. For the detection of MCF-7-containing artificial blood sample detection, the capture efficiency of tumor cells was about 74.19 ± 2.13%, which was obviously better than the result of flow cytometry (29.67 ± 4.02%). Captured cells were easily released from the surface of microfluidic chip with high cell viability, which could be investigated for the molecular analysis in the field of tumor diagnosis.


Assuntos
Separação Celular , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas , Células Neoplásicas Circulantes , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Separação Celular/instrumentação , Separação Celular/métodos , Sobrevivência Celular , DNA/química , DNA/metabolismo , Desenho de Equipamento , Humanos , Células MCF-7 , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Células Neoplásicas Circulantes/química , Células Neoplásicas Circulantes/metabolismo
17.
Biomed Microdevices ; 21(3): 59, 2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-31227912

RESUMO

Particle/cell sorting has great potential in medical diagnosis and chemical analysis. Two kinds of microfluidic sorting chips (sequential sorting chip and direct sorting chip) are designed, which combine hydraulic force and acoustic radiation force to achieve continuous sorting of multiple particles. Firstly, the optimal values of the angle (α) between the interdigital transducer (IDT) and the main channel, the peak-to-peak voltage (Vpp), the main flow velocity (Vmax) and the flow ratio (A) are determined by simulation and experiments, the related optimal parameters were obtained that the α = 15°, Vpp = 25 V, Vmax = 4 mm/s, flow ratio A1 = 0.2, and A2 = 0.5, respectively. Then, the corresponding sorting experiments were carried out using two kinds of sorting chips to sort the polystyrene (PS) particles with diameters of 1 µm, 5 µm, and 10 µm, and the sorting rate and purity of particles were calculated and analyzed. Experimental results show that the two kinds of sorting chips can achieve continuous sorting of multiple particles, and the sorting effect of sequential sorting chip (control flow ratio) is better than that of direct sorting chip. In addition, the sorting chips in our research have the advantages of simple structure, high sorting efficiency, and the ability to sort multiple particles, which can be applied in medical and chemical research fields, such as cell sorting and chemical analysis.


Assuntos
Separação Celular/instrumentação , Dispositivos Lab-On-A-Chip , Som , Poliestirenos/isolamento & purificação
18.
Electrophoresis ; 40(20): 2718-2727, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31206722

RESUMO

Microelectrode arrays are used to sort single fluorescently labeled cells and particles as they flow through a microfluidic channel using dielectrophoresis. Negative dielectrophoresis is used to create a "Dielectrophoretic virtual channel" that runs along the center of the microfluidic channel. By switching the polarity of the electrodes, the virtual channel can be dynamically reconfigured to direct particles along a different path. This is demonstrated by sorting particles into two microfluidic outlets, controlled by an automated system that interprets video data from a color camera and makes complex sorting decisions based on color, intensity, size, and shape. This enables the rejection of particle aggregates and other impurities, and the system is optimized to isolate high purity populations from a heterogeneous sample. Green beads are isolated from an excess of red beads with 100% purity at a rate of up to 0.9 particles per second, in addition application to the sorting of osteosarcoma and human bone marrow cells is evidenced. The extension of Dielectrophoretic Virtual Channels to an arbitrary number of sorting outputs is examined, with design, simulation, and experimental verification of two alternate geometries presented and compared.


Assuntos
Separação Celular , Eletroforese , Processamento de Imagem Assistida por Computador/métodos , Técnicas Analíticas Microfluídicas/instrumentação , Linhagem Celular Tumoral , Separação Celular/instrumentação , Separação Celular/métodos , Eletroforese/instrumentação , Eletroforese/métodos , Desenho de Equipamento , Humanos , Tamanho da Partícula
19.
Biomed Microdevices ; 21(3): 54, 2019 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-31203429

RESUMO

Aptamers have been widely used to recognize and capture their targets in sensitive detection applications, such as in detections of circulating tumor cells. In this study, we investigate the effects of different lengths of oligo-T spacers on surface tethered sgc8 aptamers and their target capturing performances. To achieve this, sgc8 aptamers were immobilized onto microfluidic channel surfaces via oligo-T spacers of different lengths, and the target capturing performances of these immobilized aptamers were studied using CCRF-CEM cells. We demonstrate that the capturing performances of the immobilized aptamers were significantly affected by steric hindrance. Our results also show that aptamers immobilized on surfaces via spacers of ten Ts demonstrated the best cell capturing performances; aptamers with either too short or too long oligo-T spacers showed reduced cell capturing performances. Therefore it can be concluded that spacer optimizations are critically important for surface tethered aptamers that are commonly used in microfluidic devices for sensitive target sensing and detections.


Assuntos
Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Separação Celular/instrumentação , Dispositivos Lab-On-A-Chip , Aptâmeros de Nucleotídeos/genética , Sequência de Bases , Linhagem Celular Tumoral , Dimetilpolisiloxanos/química , Humanos , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patologia , Nylons/química , Propriedades de Superfície
20.
Talanta ; 200: 169-176, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31036170

RESUMO

Circulating tumor cells (CTCs) are rare cancer cells that are shed from the tumors into the peripheral blood and are instrumental in distant metastasis. Early detection of CTCs can therefore improve prognoses and help design patient-specific treatment regimen. However, the current CTC isolation techniques have poor efficacy and selectivity, owing to the rarity and heterogeneity of the CTCs. We designed a microchip for integrated single-cell isolation of CTCs - based on cell size and immuno-phenotype - and analysis. Each isolation unit consisted of a trap channel, a bypass channel, and a release channel. The larger cells were preferentially captured at the trap channels and flushed out selectively via release microvalves according to their immuno-phenotype. The average recovery rate and purity of lung cancer cells isolated from a spiked WBC population were respectively 92.5% and 94% using the microchip, which were significantly higher compared to that obtained using anti-CD45 magnetic beads. In addition, the isolated cancer cells were analyzed on chip for the surface markers of epithelial mesenchymal transition. Taken together, the integrated microchip is a promising tool for the isolation and analysis of CTCs in the clinical setting.


Assuntos
Separação Celular/instrumentação , Separação Celular/métodos , Neoplasias Pulmonares/patologia , Técnicas Analíticas Microfluídicas/instrumentação , Células Neoplásicas Circulantes/patologia , Análise de Célula Única/instrumentação , Análise de Célula Única/métodos , Linhagem Celular Tumoral , Humanos
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