RESUMO
A layer-by-layer gelatin nanocoating is presented for use as a tunable, dual response biomaterial for the capture and release of circulating tumor cells (CTCs) from cancer patient blood. The entire nanocoating can be dissolved from the surface of microfluidic devices through biologically compatible temperature shifts. Alternatively, individual CTCs can be released through locally applied mechanical stress.
Assuntos
Separação Celular/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Nanoestruturas , Células Neoplásicas Circulantes , Materiais Biocompatíveis/química , Biópsia por Agulha Fina , Neoplasias da Mama/sangue , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Separação Celular/métodos , Sobrevivência Celular , Desenho de Equipamento , Gelatina/química , Humanos , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/genética , Masculino , Teste de Materiais , Técnicas Analíticas Microfluídicas/métodos , Modelos Teóricos , Nanoestruturas/química , Células Neoplásicas Circulantes/química , Neoplasias da Próstata/sangue , Análise de Célula Única/instrumentação , Análise de Célula Única/métodos , Estresse Mecânico , TemperaturaRESUMO
Controlled manipulation of particles from very large volumes of fluid at high throughput is critical for many biomedical, environmental and industrial applications. One promising approach is to use microfluidic technologies that rely on fluid inertia or elasticity to drive lateral migration of particles to stable equilibrium positions in a microchannel. Here, we report on a hydrodynamic approach that enables deterministic focusing of beads, mammalian cells and anisotropic hydrogel particles in a microchannel at extremely high flow rates. We show that on addition of micromolar concentrations of hyaluronic acid, the resulting fluid viscoelasticity can be used to control the focal position of particles at Reynolds numbers up to Re≈10,000 with corresponding flow rates and particle velocities up to 50 ml min(-1) and 130 m s(-1). This study explores a previously unattained regime of inertio-elastic fluid flow and demonstrates bioparticle focusing at flow rates that are the highest yet achieved.
Assuntos
Ensaios de Triagem em Larga Escala/métodos , Hidrodinâmica , Microquímica/métodos , Técnicas Analíticas Microfluídicas/métodos , Células , Ácido Hialurônico , Hidrogéis , Microesferas , Substâncias ViscoelásticasRESUMO
The ability to isolate and analyze rare circulating tumor cells (CTCs) has the potential to further our understanding of cancer metastasis and enhance the care of cancer patients. In this protocol, we describe the procedure for isolating rare CTCs from blood samples by using tumor antigen-independent microfluidic CTC-iChip technology. The CTC-iChip uses deterministic lateral displacement, inertial focusing and magnetophoresis to sort up to 107 cells/s. By using two-stage magnetophoresis and depletion antibodies against leukocytes, we achieve 3.8-log depletion of white blood cells and a 97% yield of rare cells with a sample processing rate of 8 ml of whole blood/h. The CTC-iChip is compatible with standard cytopathological and RNA-based characterization methods. This protocol describes device production, assembly, blood sample preparation, system setup and the CTC isolation process. Sorting 8 ml of blood sample requires 2 h including setup time, and chip production requires 2-5 d.
Assuntos
Separação Celular/métodos , Técnicas Analíticas Microfluídicas/métodos , Células Neoplásicas Circulantes , Humanos , Proteínas de Insetos , ImãsRESUMO
Inertial microfluidics has demonstrated the potential to provide a rich range of capabilities to manipulate biological fluids and particles to address various challenges in biomedical science and clinical medicine. Various microchannel geometries have been used to study the inertial focusing behavior of particles suspended in simple buffer solutions or in highly diluted blood. One aspect of inertial focusing that has not been studied is how particles suspended in whole or minimally diluted blood respond to inertial forces in microchannels. The utility of imaging techniques (i.e., high-speed bright-field imaging and long exposure fluorescence (streak) imaging) primarily used to observe particle focusing in microchannels is limited in complex fluids such as whole blood due to interference from the large numbers of red blood cells (RBCs). In this study, we used particle trajectory analysis (PTA) to observe the inertial focusing behavior of polystyrene beads, white blood cells, and PC-3 prostate cancer cells in physiological saline and blood. Identification of in-focus (fluorescently labeled) particles was achieved at mean particle velocities of up to 1.85 m s(-1). Quantitative measurements of in-focus particles were used to construct intensity maps of particle frequency in the channel cross-section and scatter plots of particle centroid coordinates vs. particle diameter. PC-3 cells spiked into whole blood (HCT = 45%) demonstrated a novel focusing mode not observed in physiological saline or diluted blood. PTA can be used as an experimental frame of reference for understanding the physical basis of inertial lift forces in whole blood and discover inertial focusing modes that can be used to enable particle separation in whole blood.