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In JoVE (1)
Other Publications (9)
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Articles by Larry J. Millet in JoVE
अलग और मल्टी चैनल microfluidic उपकरणों में मोती और कक्ष dielectrophoresis और लैिमनार फ्लो का उपयोग
Larry J. Millet1,2, Kidong Park1,2, Nicholas N. Watkins1,2, K. Jimmy Hsia2,3, Rashid Bashir1,2,4
1Electrical and Computer Engineering, University of Illinois at Urbana-Champaign, 2Micro and Nanotechnology Lab, University of Illinois at Urbana-Champaign, 3Mechanical Science and Engineering, University of Illinois at Urbana-Champaign, 4Bioengineering, University of Illinois at Urbana-Champaign
Dielectrophoresis (रवानगी) कोशिकाओं में हेरफेर करने के लिए एक प्रभावी तरीका है. मुद्रित सर्किट बोर्ड (पीसीबी) संपर्क मुक्त microfluidic उपकरणों के भीतर सेल हेरफेर के लिए सस्ती, पुन: प्रयोज्य और प्रभावी इलेक्ट्रोड प्रदान कर सकते हैं. PCBs पर coverslips साथ PDMS आधारित microfluidic चैनलों के संयोजन से, हम multichannel microfluidic उपकरणों के भीतर मनका और सेल हेरफेर और जुदाई प्रदर्शित करता है.
Other articles by Larry J. Millet on PubMed
Lab on a Chip. Aug, 2007 | Pubmed ID: 17653340
Microfluidic devices have been used to study high-density cultures of many cell types. Because cell-to-cell signaling is local, however, there exists a need to develop culture systems that sustain small numbers of neurons and enable analyses of the microenvironments. Such cultures are hard to maintain in stable form, and it is difficult to prevent cell death when using primary mammalian neurons. We demonstrate that postnatal primary hippocampal neurons from rat can be cultured at low densities within nanoliter-volume microdevices fabricated using polydimethylsiloxane (PDMS). Doing so requires an additional fabrication step, serial extractions/washes of PDMS with several solvents, which removes uncrosslinked oligomers, solvent and residues of the platinum catalyst used to cure the polymer. We found this step improves the biocompatibility of the PDMS devices significantly. Whereas neurons survive for > or = 7 days in open channel microdevices, the ability to culture neurons in closed-channel devices made of untreated, native PDMS is limited to < or = 2 days. When the closed-channel PDMS devices are extracted, biocompatibility improves allowing for reliable neuron cultures at low densities for > or = 7 days. Comparisons made to autoclaved PDMS and native, untreated PDMS reveal that the solvent-treated polymer is superior in sustaining low densities of primary neurons in culture. When neuronal affinity for local substrates is observed directly, we find that axons localize to channel corners and prefer PDMS surfaces to glass in hybrid devices. When perfusing the channels with media by gravity flow, cultured hippocampal neurons survive for > or = 11 days. Extracting PDMS improves biocompatibility of microfluidic devices and thus enables the study of differentiation of identifiable neurons and the characterization of local extracellular signals.
Optics Letters. Jun, 2008 | Pubmed ID: 18516197
We developed an interferometric microscopy technique, referred to as Jones phase microscopy, capable of extracting the spatially resolved Jones polarization matrix associated with transparent and anisotropic samples. This is a generalization of quantitative phase imaging, which is recovered from one diagonal element of the measured matrix. The principle of the technique is demonstrated with measurements of a liquid crystal spatial light modulator and the potential for live cell imaging with experiments on live neurons in culture.
Journal of Proteome Research. Nov, 2008 | Pubmed ID: 18816085
The mammalian supraoptic nucleus (SON) is a neuroendocrine center in the brain regulating a variety of physiological functions. Within the SON, peptidergic magnocellular neurons that project to the neurohypophysis (posterior pituitary) are involved in controlling osmotic balance, lactation, and parturition, partly through secretion of signaling peptides such as oxytocin and vasopressin into the blood. An improved understanding of SON activity and function requires identification and characterization of the peptides used by the SON. Here, small-volume sample preparation approaches are optimized for neuropeptidomic studies of isolated SON samples ranging from entire nuclei down to single magnocellular neurons. Unlike most previous mammalian peptidome studies, tissues are not immediately heated or microwaved. SON samples are obtained from ex vivo brain slice preparations via tissue punch and the samples processed through sequential steps of peptide extraction. Analyses of the samples via liquid chromatography mass spectrometry and tandem mass spectrometry result in the identification of 85 peptides, including 20 unique peptides from known prohormones. As the sample size is further reduced, the depth of peptide coverage decreases; however, even from individually isolated magnocellular neuroendocrine cells, vasopressin and several other peptides are detected.
Guiding Neuron Development with Planar Surface Gradients of Substrate Cues Deposited Using Microfluidic Devices
Lab on a Chip. Jun, 2010 | Pubmed ID: 20390196
Wiring the nervous system relies on the interplay of intrinsic and extrinsic signaling molecules that control neurite extension, neuronal polarity, process maturation and experience-dependent refinement. Extrinsic signals establish and enrich neuron-neuron interactions during development. Understanding how such extrinsic cues direct neurons to establish neural connections in vitro will facilitate the development of organized neural networks for investigating the development and function of nervous system networks. Producing ordered networks of neurons with defined connectivity in vitro presents special technical challenges because the results must be compliant with the biological requirements of rewiring neural networks. Here we demonstrate the ability to form stable, instructive surface-bound gradients of laminin that guide postnatal hippocampal neuron development in vitro. Our work uses a three-channel, interconnected microfluidic device that permits the production of adlayers of planar substrates through the combination of laminar flow, diffusion and physisorption. Through simple flow modifications, a variety of patterns and gradients of laminin (LN) and fluorescein isothiocyanate-conjugated poly-l-lysine (FITC-PLL) were deposited to present neurons with an instructive substratum to guide neuronal development. We present three variations in substrate design that produce distinct growth regimens for postnatal neurons in dispersed cell cultures. In the first approach, diffusion-mediated gradients of LN were formed on cover slips to guide neurons toward increasing LN concentrations. In the second approach, a combined gradient of LN and FITC-PLL was produced using aspiration-driven laminar flow to restrict neuronal growth to a 15 microm wide growth zone at the center of the two superimposed gradients. The last approach demonstrates the capacity to combine binary lines of FITC-PLL in conjunction with surface gradients of LN and bovine serum albumin (BSA) to produce substrate adlayers that provide additional levels of control over growth. This work demonstrates the advantages of spatio-temporal fluid control for patterning surface-bound gradients using a simple microfluidics-based substrate deposition procedure. We anticipate that this microfluidics-based patterning approach will provide instructive patterns and surface-bound gradients to enable a new level of control in guiding neuron development and network formation.
Direct Cellular Peptidomics of Supraoptic Magnocellular and Hippocampal Neurons in Low-density Co-cultures
ACS Chemical Neuroscience. Jan, 2010 | Pubmed ID: 20401326
Genomic and proteomic studies of brain regions of specialized function provide evidence that communication among neurons is mediated by systems of diverse chemical messengers. These analyses are largely tissue- or population-based, whereas the actual communication is from cell-to-cell. To understand the complement of intercellular signals produced by individual neurons, new methods are required. We have developed a novel neuron-to-neuron, serum-free, co-culture approach that was used to determine the higher-level cellular peptidome of individual primary mammalian neurons. We isolated magnocellular neurons from the supraoptic nucleus of early postnatal rat and maintained them in serum-free low density cultures without glial support layers; under these conditions they required low-density co-cultured neurons. Co-culturing magnocellular neurons with hippocampal neurons permitted local access to individual neurons within the culture for mass spectrometry. Using direct sampling, peptide profiles were obtained for spatially distinct, identifiable neurons within the co-culture. We repeatedly detected 10 peaks that we assign to previously characterized peptides and 17 peaks that remain unassigned. Peptides from the vasopressin prohormone and secretogranin-2 are attributed to magnocellular neurons, whereas neurokinin A, peptide J, and neurokinin B are attributed to cultured hippocampal neurons. This approach enables the elucidation of cell-specific prohormone processing and the discovery of cell-cell signaling peptides.
Proceedings of the National Academy of Sciences of the United States of America. Nov, 2010 | Pubmed ID: 21068372
The characterization of physical properties of cells such as their mass and stiffness has been of great interest and can have profound implications in cell biology, tissue engineering, cancer, and disease research. For example, the direct dependence of cell growth rate on cell mass for individual adherent human cells can elucidate the mechanisms underlying cell cycle progression. Here we develop an array of micro-electro-mechanical systems (MEMS) resonant mass sensors that can be used to directly measure the biophysical properties, mass, and growth rate of single adherent cells. Unlike conventional cantilever mass sensors, our sensors retain a uniform mass sensitivity over the cell attachment surface. By measuring the frequency shift of the mass sensors with growing (soft) cells and fixed (stiff) cells, and through analytical modeling, we derive the Young's modulus of the unfixed cell and unravel the dependence of the cell mass measurement on cell stiffness. Finally, we grew individual cells on the mass sensors and measured their mass for 50+ hours. Our results demonstrate that adherent human colon epithelial cells have increased growth rates with a larger cell mass, and the average growth rate increases linearly with the cell mass, at 3.25%/hr. Our sensitive mass sensors with a position-independent mass sensitivity can be coupled with microscopy for simultaneous monitoring of cell growth and status, and provide an ideal method to study cell growth, cell cycle progression, differentiation, and apoptosis.
Biomedical Optics Express. 2010 | Pubmed ID: 21258463
We applied the newly developed Fourier transform light scattering (FTLS) to study dynamic light scattering in single live cells, at a temporal scale of seconds to hours. The nanoscale cell fluctuations were measured with and without the active actin contribution. We found experimentally that the spatio-temporal signals rendered by FTLS reveal interesting cytoskeleton dynamics in glial cells (the predominant cell type in the nervous system). The active contribution of actin cytoskeleton was obtained by modulating its dynamic properties via Cytochalasin-D, a drug that inhibits actin polymerization/depolymerization.
Optics Express. Oct, 2011 | Pubmed ID: 21996999
We present spatial light interference tomography (SLIT), a label-free method for 3D imaging of transparent structures such as live cells. SLIT uses the principle of interferometric imaging with broadband fields and combines the optical gating due to the micron-scale coherence length with that of the high numerical aperture objective lens. Measuring the phase shift map associated with the object as it is translated through focus provides full information about the 3D distribution associated with the refractive index. Using a reconstruction algorithm based on the Born approximation, we show that the sample structure may be recovered via a 3D, complex field deconvolution. We illustrate the method with reconstructed tomographic refractive index distributions of microspheres, photonic crystals, and unstained living cells.
Integrative Biology : Quantitative Biosciences from Nano to Macro. Dec, 2011 | Pubmed ID: 22057472
The nervous system is a complex, highly-ordered, integrated network of cells. Dispersed cultures of neurons enable investigations into intrinsic cellular functions without the complexities inherent in the intact nervous system. This culture process generates a homogeneously dispersed population that is assumed to be spatially random. Despite the vast number of studies utilizing dispersed neurons, few studies address the spatial distribution of large populations of neurons, in vitro. We used ink-jet printing and surface chemistry to define patterned areas of poly-lysine adhesion (∼50 μm spots) juxtaposed against a fluorinated-silane background. We quantitatively analysed populations of patterned neurons on printed protein spots, and unpatterned neurons. Using a microarray scanner, we acquired large images (72 mm × 22 mm) of patterns, and neurons with and without patterns. Fast Fourier transformation (FFT) image analysis was used to determine global alignment of neurons to patterns. Through point pattern analysis, we described the spatial organization of dispersed neurons with, or without, patterned substrates. Patterned neurons show spatial organization characteristics reminiscent of printed patterns, with spatial distributions representative of unpatterned neurons. Most notably, both patterned and unpatterned neurons show departure from null models of complete spatial randomness (CSR; a homogeneous Poisson process) at shorter distances with conformity to CSR occurring at longer distances. Cellular morphometrics show that when compared to their unpatterned counterparts, spot-patterned neurons exhibit a significant increase (p < 0.0001) in the mean dendritic circularity and an increase in the number of more circular neurons. Through neurite tracing, we show that dendritic processes are also highly confined to patterned areas, and that they are on average 58% shorter than dendrites of neurons without patterns. Our findings show that patterned areas change the spatial organization of the somata and dendrites of cultured neurons, and that traditional neuronal cultures deviate from CSR.