Biomedical subjects

Noo Li Jeon

Publications and source records attributed to Noo Li Jeon.

14 recordsLinked to original sources

PubMed2007

External force-assisted cell positioning inside microfluidic devices.

This paper describes straightforward approaches to positioning cells within microfluidic devices that can be implemented without special equipment or fabrication steps. External forces can effectively transport and position cells in preferred locations inside microfluidic channels. Except for centrifugal force-based positioning that can be used with any microfluidic channels, hydrodynamic and gravitational force-based positioning yield reproducible and biocompatible results when implemented with a microfluidic "module" that contains a barrier with embedded microgrooves. Primary rat cortical neurons, metastatic human breast cancer cells MDA-MB-231, NIH 3T3 mouse fibroblasts, and human umbilical vein endothelial cells (HUVECs) were compatible with the positioning processes. After positioning, cells attached, proliferated and migrated like control cells that were cultured on tissue culture dishes or glass coverslips. No apparent morphological differences were observed in positioned cells compared with control cells. Finally, to demonstrate a practical application of the methods, cells were placed in a single row along a wall inside a microfluidic chemotaxis chamber (MCC), and were exposed to stable concentration gradient of chemoattractant. Cell positioning allows that all cells get exposed to the same level of chemoattractant at the start of the experiment helping standardize cellular response.

Animals↗

PubMed2006

A microfluidic multi-injector for gradient generation.

This paper describes a microfluidic multi-injector (MMI) that can generate temporal and spatial concentration gradients of soluble molecules. Compared to conventional glass micropipette-based methods that generate a single gradient, the MMI exploits microfluidic integration and actuation of multiple pulsatile injectors to generate arbitrary overlapping gradients that have not previously been possible. The MMI device is fabricated in poly(dimethylsiloxane) (PDMS) using multi-layer soft lithography and consists of fluidic channels and control channels with pneumatically actuated on-chip barrier valves. Repetitive actuation of on-chip valves control pulsatile release of solution that establishes microscopic chemical gradients around the orifice. The volume of solution released per actuation cycle ranged from 30 picolitres to several hundred picolitres and increased linearly with the duration of valve opening. The shape of the measured gradient profile agreed closely with the simulated diffusion profile from a point source. Steady state gradient profiles could be attained within 10 minutes, or less with an optimized pulse sequence. Overlapping gradients from 2 injectors were generated and characterized to highlight the advantages of MMI over conventional micropipette assays. The MMI platform should be useful for a wide range of basic and applied studies on chemotaxis and axon guidance.

Dimethylpolysiloxanes↗

PubMed2006

Gene targeting of GAN in mouse causes a toxic accumulation of microtubule-associated protein 8 and impaired retrograde axonal transport.

Mutations in gigaxonin were identified in giant axonal neuropathy (GAN), an autosomal recessive disorder. To understand how disruption of gigaxonin's function leads to neurodegeneration, we ablated the gene expression in mice using traditional gene targeting approach. Progressive neurological phenotypes and pathological lesions that developed in the GAN null mice recapitulate characteristic human GAN features. The disruption of gigaxonin results in an impaired ubiquitin-proteasome system leading to a substantial accumulation of a novel microtubule-associated protein, MAP8, in the null mutants. Accumulated MAP8 alters the microtubule network, traps dynein motor protein in insoluble structures and leads to neuronal death in cultured wild-type neurons, which replicates the process occurring in GAN null mutants. Defective axonal transport is evidenced by the in vitro assays and is supported by vesicular accumulation in the GAN null neurons. We propose that the axonal transport impairment may be a deleterious consequence of accumulated, toxic MAP8 protein.

Animals↗

PubMed2006

A parallel-gradient microfluidic chamber for quantitative analysis of breast cancer cell chemotaxis.

Growth factor-induced chemotaxis of cancer cells is believed to play a critical role in metastasis, directing the spread of cancer from the primary tumor to secondary sites in the body. Understanding the mechanistic and quantitative behavior of cancer cell migration in growth factor gradients would greatly help in future treatment of metastatic cancers. Using a novel microfluidic chemotaxis chamber capable of simultaneously generating multiple growth factor gradients, we examined the migration of the human metastatic breast cancer cell line MDA-MB-231 in various conditions. First, we quantified and compared the migration in two gradients of epidermal growth factor (EGF) spanning different concentrations: 0-50 ng/ml and 0.1-6 ng/ml. Cells showed a stronger response in the 0-50 ng/ml gradient. However, the fact that even a shallow gradient of EGF can induce chemotaxis, and that EGF can direct migration over a large dynamic range of gradients, confirms the potency of EGF as a chemoattractant. Second, we investigated the effect of antibody against the EGF receptor (EGFR) on MDA-MB-231 chemotaxis. Quantitative analysis indicated that anti-EGFR antibody impaired both motility and directional orientation (CI = 0.03, speed = 0.71 microm/min), indicating that cell motility was induced by the activation of EGFR. The ability to compare, in terms of quantitative parameters, the effects of different pharmaceutical inhibitors, as well as subtle differences in experimental conditions, will aid in our understanding of mechanisms that drive metastasis. The microfluidic chamber described in this work will provide a platform for cell-based assays that can be used to compare the effectiveness of different pharmaceutical compounds targeting cell migration and metastasis.

Breast Neoplasms↗

PubMed2006

Microfluidic chambers for cell migration and neuroscience research.

This chapter describes the fabrication and use microfluidic chambers for cell migration and neuroscience research. Both microfluidic chambers are made using soft lithography and replica molding. The main advantages of using soft lithography to create microfluidic chambers are reproducibility, ease of use, and straightforward fabrication procedures. The devices can be fabricated in biology and chemistry laboratories with minimal access to clean-room facilities. First, a microfluidic chemotaxis chamber, which has been used in investigating chemotaxis of neutrophils, human breast cancer cells, and other cell types, is described. Precise and stable gradients of chemoattractants with arbitrary shapes can be generated for different applications. Second, a multicompartment culture chamber that can fluidically isolate neuronal processes from cell bodies is described. The design of this chamber is such that only neurites grow through a series of microgrooves embedded in a physical barrier. Both devices are compatible with phase, differential interference contrast, and fluorescence microscopy.

Animals↗

PubMed2005

Human neural stem cell growth and differentiation in a gradient-generating microfluidic device.

This paper describes a gradient-generating microfluidic platform for optimizing proliferation and differentiation of neural stem cells (NSCs) in culture. Microfluidic technology has great potential to improve stem cell (SC) cultures, whose promise in cell-based therapies is limited by the inability to precisely control their behavior in culture. Compared to traditional culture tools, microfluidic platforms should provide much greater control over cell microenvironment and rapid optimization of media composition using relatively small numbers of cells. Our platform exposes cells to a concentration gradient of growth factors under continuous flow, thus minimizing autocrine and paracrine signaling. Human NSCs (hNSCs) from the developing cerebral cortex were cultured for more than 1 week in the microfluidic device while constantly exposed to a continuous gradient of a growth factor (GF) mixture containing epidermal growth factor (EGF), fibroblast growth factor 2 (FGF2) and platelet-derived growth factor (PDGF). Proliferation and differentiation of NSCs into astrocytes were monitored by time-lapse microscopy and immunocytochemistry. The NSCs remained healthy throughout the entire culture period, and importantly, proliferated and differentiated in a graded and proportional fashion that varied directly with GF concentration. These concentration-dependent cellular responses were quantitatively similar to those measured in control chambers built into the device and in parallel cultures using traditional 6-well plates. This gradient-generating microfluidic platform should be useful for a wide range of basic and applied studies on cultured cells, including SCs.

Astrocytes↗

PubMed2005

Neutrophil migration in opposing chemoattractant gradients using microfluidic chemotaxis devices.

Neutrophils migrating in tissue respond to complex overlapping signals generated by a variety of chemotactic factors (CFs). Previous studies suggested a hierarchy between bacteria-derived CFs and host-derived CFs but could not differentiate neutrophil response to potentially equal host-derived CFs (IL-8 and LTB4). This paper reports neutrophil migration in conflicting gradients of IL-8 and LTB4 using a microfluidic chemotaxis device that can generate stable and well-defined gradients. We quantitatively characterized the movement of cells from time-lapse images. Neutrophils migrate more efficiently toward single IL-8 gradients than single LTB4 gradients as measured by the effective chemotactic index (ECI). In opposing gradients of IL-8 and LTB4, neutrophils show obvious chemotaxis toward a distant gradient, consistent with previous reports. When an opposing gradient of LTB4 is present, neutrophils show less effective chemotaxis toward IL-8 than when they are in a gradient of IL-8 alone. In contrast, the chemotactic response of neutrophils to LTB4 is not reduced in opposing gradients as compared to that in a single LTB4 gradient. These results indicate that the presence of one host-derived CF modifies the response of neutrophils to a second CF suggesting a subtle hierarchy between them.

Cell Culture Techniques↗

PubMed2005

A microfluidic culture platform for CNS axonal injury, regeneration and transport.

Investigation of axonal biology in the central nervous system (CNS) is hindered by a lack of an appropriate in vitro method to probe axons independently from cell bodies. Here we describe a microfluidic culture platform that polarizes the growth of CNS axons into a fluidically isolated environment without the use of targeting neurotrophins. In addition to its compatibility with live cell imaging, the platform can be used to (i) isolate CNS axons without somata or dendrites, facilitating biochemical analyses of pure axonal fractions and (ii) localize physical and chemical treatments to axons or somata. We report the first evidence that presynaptic (Syp) but not postsynaptic (Camk2a) mRNA is localized to developing rat cortical and hippocampal axons. The platform also serves as a straightforward, reproducible method to model CNS axonal injury and regeneration. The results presented here demonstrate several experimental paradigms using the microfluidic platform, which can greatly facilitate future studies in axonal biology.

Animals↗

PubMed2004

Differential effects of EGF gradient profiles on MDA-MB-231 breast cancer cell chemotaxis.

Chemotaxis, directed cell migration in a gradient of chemoattractant, is an important biological phenomenon that plays pivotal roles in cancer metastasis. Newly developed microfluidic chemotaxis chambers (MCC) were used to study chemotaxis of metastatic breast cancer cells, MDA-MB-231, in EGF gradients of well-defined profiles. Migration behaviors of MDA-MB-231 cells in uniform concentrations of EGF (0, 25, 50, and 100 ng/ml) and EGF (0-25, 0-50, and 0-100 ng/ml) with linear and nonlinear polynomial profiles were investigated. MDA-MB-231 cells exhibited increased speed and directionality upon stimulation with uniform concentrations of EGF. The cells were viable and motile for over 24 h, confirming the compatibility of MCC with cancer cells. Linear concentration gradients of different ranges were not effective in inducing chemotactic movement as compared to nonlinear gradients. MDA-MB-231 cells migrating in EGF gradient of 0-50 ng/ml nonlinear polynomial profile exhibited marked directional movement toward higher EGF concentration. This result suggests that MDA-MB-231 cancer cell chemotaxis depends on the shape of gradient profile as well as on the range of EGF concentrations.

Breast Neoplasms↗

PubMed2004

Patterned cell culture inside microfluidic devices.

This paper describes a simple plasma-based dry etching method that enables patterned cell culture inside microfluidic devices by allowing patterning, fluidic bonding and sterilization steps to be carried out in a single step. This plasma-based dry etching method was used to pattern cell-adhesive and non-adhesive areas on the glass and polystyrene substrates. The patterned substrate was used for selective attachment and growth of human umbilical vein endothelial cells, MDA-MB-231 human breast cancer cells, NIH 3T3 mouse fibroblasts, and primary rat cortical neurons. Finally, we have successfully combined the dry-patterned substrate with a microfluidic device. Patterned primary rat neurons were maintained for up to 6 days inside the microfluidic devices and the neurons' somas and processes were confined to the cell-adhesive region. The method developed in this work offers a convenient way of micropatterning biomaterials for selective attachment of cells on the substrates, and enables culturing of patterned cells inside microfluidic devices for a number of biological research applications where cells need to be exposed to well-controlled fluidic microenvironment.

Animals↗

PubMed2004

Effective neutrophil chemotaxis is strongly influenced by mean IL-8 concentration.

Neutrophils need to correctly interpret gradients of chemotactic factors (CFs) such as interleukin 8 (IL-8) to migrate to the site of infection and perform immune functions. Because diffusion-based chemotaxis assays used in previous studies suffer from temporally changing gradients, it is difficult to distinguish the influence of CF gradient steepness from mean CF concentration on chemotaxis. To better understand the roles of mean CF concentration and CF gradient steepness, we developed a microfluidic device that can maintain stable IL-8 gradients. We report that the random motility of neutrophils is a biphasic function of IL-8 concentration and its magnitude plays a decisive role in effective chemotaxis, a quantitative measure of migration. We show that the concentrations for the optimum chemotaxis in linear IL-8 gradients and for the maximum random motility in uniform IL-8 coincide. In contrast, we find that the steepness of IL-8 gradients has no significant effect on effective chemotaxis.

Chemotaxis, Leukocyte↗

PubMed2004

Generation of dynamic temporal and spatial concentration gradients using microfluidic devices.

This paper describes a microfluidic approach to generate dynamic temporal and spatial concentration gradients using a single microfluidic device. Compared to a previously described method that produced a single fixed gradient shape for each device, this approach combines a simple "mixer module" with gradient generating network to control and manipulate a number of different gradient shapes. The gradient profile is determined by the configuration of fluidic inputs as well as the design of microchannel network. By controlling the relative flow rates of the fluidic inputs using separate syringe pumps, the resulting composition of the inlets that feed the gradient generator can be dynamically controlled to generate temporal and spatial gradients. To demonstrate the concept and illustrate this approach, examples of devices that generate (1) temporal gradients of homogeneous concentrations, (2) linear gradients with dynamically controlled slope, baseline, and direction, and (3) nonlinear gradients with controlled nonlinearity are shown and their limitations are described.

Equipment Design↗

PubMed2002

Neutrophil chemotaxis in linear and complex gradients of interleukin-8 formed in a microfabricated device.

Although a wealth of knowledge about chemotaxis has accumulated in the past 40 years, these studies have been hampered by the inability of researchers to generate simple linear gradients instantaneously and to maintain them at steady state. Here we describe a device microfabricated by soft lithography and consisting of a network of microfluidic channels that can generate spatially and temporally controlled gradients of chemotactic factors. When human neutrophils are positioned within a microchannel, their migration in simple and complex interleukin-8 (IL-8) gradients can be tested. The cells exhibit strong directional migration toward increasing concentrations of IL-8 in linear gradients. Neutrophil migration halts abruptly when cells encounter a sudden drop in the chemoattractant concentration to zero ("cliff" gradient). When neutrophils are challenged with a gradual increase and decrease in chemoattractant ("hill" gradient), however, the cells traverse the crest of maximum concentration and migrate further before reversing direction. The technique described in this paper provides a robust method to investigate migratory cells under a variety of conditions not accessible to study by earlier techniques.

Biological Assay↗

PubMedDate not supplied

Diffusion limits of an in vitro thick prevascularized tissue.

Although tissue engineering promises to replace or restore lost function to nearly every tissue in the body, successful applications are currently limited to tissue less than 2 mm in thickness. in vivo capillary networks deliver oxygen and nutrients to thicker (> 2 mm) tissues, suggesting that introduction of a preformed in vitro vascular network may be a useful strategy for engineered tissues. This article describes a system for generating capillary-like networks within a thick fibrin matrix. Human umbilical vein endothelial cells, growing on the surface of microcarrier beads, were embedded in fibrin gels a known distance (Delta = 1.8-4.5 mm) from a monolayer of human dermal fibroblasts. The distance of the growth medium, which contained vascular endothelial growth factor and basic fibroblast growth factor, from the beads, C, was varied from 2.7 to 7.2 mm. Capillaries with visible lumens sprouted in 2-3 days, reaching lengths that exceeded 500 microm within 6-8 days. On day 7, capillary network formation was largely independent of C; however, a strong inverse correlation with Delta was observed, with the maximum network formation at Delta = 1.8 mm. Surprisingly, the thickness of the gel was not a limiting factor for oxygen diffusion as these tissue constructs retained a relatively high oxygen tension of > 125 mmHg. We conclude that diffusion of oxygen in vitro is not limiting, allowing the development of tissue constructs on the order of centimeters in thickness. In addition, diffusion of fibroblast-derived soluble mediators is necessary for stable capillary formation, but is significantly impeded relative to that of nutrients present in the medium.

Capillaries↗