RESUMO
The clinical potential of circulating tumor cells (CTCs) in managing cancer metastasis is significant. However, low CTC isolation purities from patient blood have hindered sensitive molecular assays of these rare cells. Described herein is the ultra-pure isolation of CTCs from patient blood samples and how this platform has enabled highly specific molecular (mRNA and miRNA) profiling of patient CTCs.
RESUMO
Improved detection and therapy of breast neoplasia might benefit from nanodevices traveling inside mammary ducts. However, the decreasing size of branched mammary ducts prevents access to remote areas of the ductal system using a pressure-driven fluid-based approach. Magnetic field guidance of superparamagnetic submicron particles (SMPs) in a stationary fluid might provide a possible alternative but it is critical to first reproduce the breast ductal system to assess the use of such devices for future therapeutic & diagnostic ("theranostic") purposes. Here we describe the engineering of a portion of a breast ductal system using polydimethylsiloxane (PDMS) microfluidic channels with a total volume of 0.09 µl. A magnet was used to move superparamagnetic/fluorescent SMPs through a static fluid inside the microchannels. Non-neoplastic mammary epithelial S1 cells developed basoapical polarity as a flat monolayer on the PDMS surface when cultured in the presence of laminin 111, and incubation with SMPs did not result in detectable toxicity. Cells could not withstand the fluid pressure if microinjected directly in completed channels. Whereas, they readily covered laminin 111-coated PDMS surfaces when cultured in U-shaped "hemichannels" before completing the channels. This breast-on-chip model represents a critical step towards the mimicry of the tree-like ductal system of the breast for further testing and targeting of SMPs.
Assuntos
Neoplasias da Mama/diagnóstico , Neoplasias da Mama/terapia , Dispositivos Lab-On-A-Chip , Glândulas Mamárias Humanas , Técnicas de Cultura de Células/métodos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Polaridade Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Colágeno/química , Colágeno/farmacologia , Colágeno Tipo IV/química , Colágeno Tipo IV/farmacologia , Dimetilpolisiloxanos/química , Combinação de Medicamentos , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/farmacologia , Feminino , Humanos , Laminina/química , Laminina/farmacologia , Magnetismo , Microscopia de Fluorescência , Microesferas , Movimento (Física) , Proteoglicanas/química , Proteoglicanas/farmacologiaRESUMO
Current methods to screen for bacterial contamination involve using costly reagents such as antibodies or PCR reagents or time-costly growth in cultures. There is need for portable, real-time, multiplex pathogen detection technology that can predict the safety of food. Surface plasmon resonance (SPR) imaging is a sensitive, label-free method that can detect the binding of an analyte to a surface by the changes in refractive index that occur upon binding. We have designed a hybrid microfluidic biochip to perform multiplexed detection of single-celled pathogens using a combination of SPR and fluorescence imaging. The device consists of an array of gold spots, each functionalized with a capture biomolecule targeting a specific pathogen. This biosensor array is enclosed by a polydimethylsiloxane microfluidic flow chamber that delivers a magnetically concentrated sample to be tested. The sample is imaged by SPR on the bottom of the biochip and epi-fluorescence on the top. The prototype instrument was successfully able to image antibody-captured E. coli O157:H7 bacteria by SPR and fluorescence imaging. The efficiency of capture of these bacteria by the magnetic particles was determined using spectrophotometric ferric oxide absorbance measurements. The binding of the E. coli to each spot was quantified by measuring the percent of the gold spot area upon which the bacteria was bound and analyzed using NIH ImageJ software. This hybrid imaging approach of pathogenic E. coli detection coupled with an estimate of relative infectivity is shown to be a working example of a testing device for potential foodborne pathogens.