Parallel probing of drug uptake of single cancer cells on a microfluidic device.

Drug resistance is frequently developing during treatment of cancer patients. Intracellular drug uptake is one of the important characteristics to understand mechanism of drug resistance. However, the heterogeneity of cancer cells requires the investigation of drug uptake at the single cell level. Here, we developed a microfluidic device for parallel probing of drug uptake. We combined a v-type valve and peristaltic pumping to select individual cells from a pool of prostate cancer cells (PC3) and place them successively in separate cell chambers in which they were exposed to the drug. Six different concentrations of doxorubicin, a naturally fluorescent anti-cancer drug, were created in loop-shaped reactors and exposed to the cell in closed 2 nL volume chambers. Monitoring every single cell over time in 18 parallel chambers revealed increased intracellular fluorescence intensity according to the dose of doxorubicin, as well as nuclear localization of the fluorescent drug after 2 h of incubation. The herein proposed technology demonstrated a first series of proof of concept experiments and it shows high potential to use for probing drug sensitivity of single cancer cell.

Differences in structural connectome organization across sleep quality.

Sleep quality, which measures satisfaction with overall sleep, is an important factor affecting an individual's health. Although previous neuroimaging studies based on magnetic resonance imaging (MRI) have observed altered regional morphology and functional activation related to poor sleep quality, the impact of sleep quality on whole-brain structural connectome organization is relatively under-investigated. To address this gap, we utilized dimensionality reduction techniques to estimate low-dimensional eigenvectors of structural connectivity derived from diffusion MRI tractography, and assessed their associations with measures of sleep quality. We found significant effects in the unimodal association and limbic regions. Additionally, the meta-analytic cognitive decoding analysis revealed associations with negative terms, including nociceptive and pain. Our findings suggest a link between alterations in the whole-brain structural organization and sleep quality, providing insights into the relationship between sleep quality and brain structure.

Safety and feasibility of countering neurological impairment by intravenous administration of autologous cord blood in cerebral palsy.

BACKGROUNDS: We conducted a pilot study of the infusion of intravenous autologous cord blood (CB) in children with cerebral palsy (CP) to assess the safety and feasibility of the procedure as well as its potential efficacy in countering neurological impairment. METHODS: Patients diagnosed with CP were enrolled in this study if their parents had elected to bank their CB at birth. Cryopreserved CB units were thawed and infused intravenously over 10~20 minutes. We assessed potential efficacy over 6 months by brain magnetic resonance imaging (MRI)-diffusion tensor imaging (DTI), brain perfusion single-photon emission computed tomography (SPECT), and various evaluation tools for motor and cognitive functions. RESULTS: Twenty patients received autologous CB infusion and were evaluated. The types of CP were as follows: 11 quadriplegics, 6 hemiplegics, and 3 diplegics. Infusion was generally well-tolerated, although 5 patients experienced temporary nausea, hemoglobinuria, or urticaria during intravenous infusion. Diverse neurological domains improved in 5 patients (25%) as assessed with developmental evaluation tools as well as by fractional anisotropy values in brain MRI-DTI. The neurologic improvement occurred significantly in patients with diplegia or hemiplegia rather than quadriplegia. CONCLUSIONS: Autologous CB infusion is safe and feasible, and has yielded potential benefits in children with CP.

Correlation between chemokines released from umbilical cord blood-derived mesenchymal stem cells and engraftment of hematopoietic stem cells in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice.

Umbilical cord blood (UCB)-derived mesenchymal stem cells (MSCs) enhance the engraftment of human hematopoietic stem cells (HSCs) when they are cotransplanted in animal and human studies. However, the type of MSCs that preferentially facilitate the engraftment and homing of HSCs is largely unknown. The authors categorized UCB-MSCs as the least-effective MSCs (A) or most-effective MSCs (B) at enhancing the engraftment of HSCs, and compared the gene expression profiles of various cytokines and growth factors in the UCB-MSC populations. The most-effective UCB-MSCs (B) secreted higher levels of several factors, including chemokine (C-X-C motif) ligand 12 (CXCL12), regulated upon activation, normal T cells expressed and secreted (RANTES), epithelial growth factor (EGF), and stem cell factor (SCF), which are required for the engraftment and homing of HSCs. By contrast, levels of growth-related oncogene (GRO), insulin-like growth factor-binding protein 1 (IGFBP1), and interleukin-8 (IL-8), which are associated with immune inflammation, were secreted at higher levels in UCB-MSCs (A). In addition, there were no differences between the transcripts of the 2 UCB-MSC populations after interferon-gamma (IFN-γ) stimulation, except for cyclooxygenase (COX)-1. Based on these findings, the authors propose that these chemokines may be useful for modulating these cells in a clinical setting and potentially for enhancing the effectiveness of the engraftment and homing of HSCs.

Headpost Surgery for in vivo Electrophysiological Recording in the Mouse Inferior Colliculus during Locomotion.

The inferior colliculus (IC) is a critical midbrain integration center for auditory and non-auditory information. Although much is known about the response properties of the IC neurons to auditory stimuli, how the IC neural circuits function during movement such as locomotion remains poorly understood. Mice offer a valuable model in this respect, but previous studies of the mouse IC were performed in anesthetized or restrained preparations, making it difficult to study the IC function during behavior. Here we describe a neural recording protocol for the mouse IC in which mice are head-fixed, but can run on a passive treadmill. Mice first receive a headpost surgery, and become habituated to head-fixing while being on a treadmill. Following a few days of habituation, neural recordings of the IC neuron activity are performed. The neural activity can be compared across different behavioral conditions, such as standing still versus running on a treadmill. We describe how to overcome the challenges of headpost surgery for awake IC recording, presented by the location and overlying bones. This protocol allows investigations of the IC function in behaving mice, while allowing precise stimulus control and the use of recording methods similar to those for anesthetized preparations.

Integration of locomotion and auditory signals in the mouse inferior colliculus.

The inferior colliculus (IC) is the major midbrain auditory integration center, where virtually all ascending auditory inputs converge. Although the IC has been extensively studied for sound processing, little is known about the neural activity of the IC in moving subjects, as frequently happens in natural hearing conditions. Here, by recording neural activity in walking mice, we show that the activity of IC neurons is strongly modulated by locomotion, even in the absence of sound stimuli. Similar modulation was also found in hearing-impaired mice, demonstrating that IC neurons receive non-auditory, locomotion-related neural signals. Sound-evoked activity was attenuated during locomotion, and this attenuation increased frequency selectivity across the neuronal population, while maintaining preferred frequencies. Our results suggest that during behavior, integrating movement-related and auditory information is an essential aspect of sound processing in the IC.

Superporous agarose beads as a solid support for microfluidic immunoassay.

We demonstrate here with the feasibility of superporous agarose (SA) beads as a solid support in microfluidic immunoassay by detecting goat IgG. In our procedure, SA beads containing superpores were covalently conjugated to protein A. The conjugated beads were introduced into a polydimethyl siloxane microfluidic device. The sandwich immunoassay was performed in the microfluidic device by subsequently introducing anti-goat IgG as the primary antibodies, goat IgG as analytes, alkaline phosphatase-conjugated F(ab')2 anti-goat IgG as detection antibodies, and 5-bromo-4-chloro-3-indolylphosphate/nitroblue tetrazolium as substrate in a flow. Depending on the goat IgG concentration, dark and pinky precipitates appeared inside the microchannel immediately after the introduction of all the reagents. The minimum detection limit, 100 pg goat IgG/mL in PBS, was achieved with the naked eye. This enhanced sensitivity is mainly because analytical reagents were allowed to access the outer surface as well as the inner matrices of the beads. This is supported by the facts that the binding of fluorescein isothiocyanate IgG happened throughout the inside matrices of protein A-conjugated SA beads but was limited to the outer surface of protein A-conjugated homogeneous agarose beads. These results suggest that SA beads are highly suitable as a solid support for microfluidic immunoassays.

Autologous bone marrow cell transplantation combined with off-pump coronary artery bypass grafting in patients with ischemic cardiomyopathy.

BACKGROUND: Clinical studies have demonstrated that intracoronary or intramyocardial transplantation of bone marrow mononuclear cells (BMMNCs) into ischemic myocardium improves cardiac function. The objective of the present study was to evaluate the safety and feasibility of intramyocardial BMMNC transplantation into nongraftable areas in combination with off-pump coronary artery bypass grafting in patients with ischemic cardiomyopathy. METHODS: Five male patients with myocardial infarction lasting for more than 1 month and with nongraftable myocardium received autologous mononuclear cell transplantation during off-pump coronary artery bypass grafting. Autologous bone marrow was aspirated from the iliac crest. BMMNCs (mean 1.6, standard error [SE] 0.3 x 10(9)) including CD34-positive cells (mean 6.8, SE 1.1 x 10(6)) and AC133-positive cells (mean 3.1, SE 1.7 x 10(6)) were injected into the nongraftable ischemic myocardium. Heart function was evaluated with the use of echocardiography, and myocardial perfusion was examined with single photon emission computed tomography technetium-99mTc sestamibi scans. RESULTS: Two months after cell transplantation, the mean ejection fraction had increased by 7.4%, SE 1.9% (p = 0.016) compared with that before cell transplantation and off-pump coronary artery bypass grafting. The increase in ejection fraction was not correlated with the number of transplanted total mononuclear cells, CD34-positive cells and AC133-positive cells. Myocardial perfusion at the cell-transplanted area increased after cell transplantation and off-pump coronary artery bypass grafting. No arrhythmia was observed. CONCLUSION: The present clinical study suggests that intramyocardial transplantation of autologous BMMNCs into the ischemic area during off-pump coronary artery bypass grafting is both feasible and safe and has beneficial effects on cardiac function.

A biological sensor platform using a pneumatic-valve controlled microfluidic device containing Tetrahymena pyriformis.

In this study, we introduce a microfluidic device equipped with pneumatically actuated valves, generating a linear gradient of chemoeffectors to quantify the chemotactic response of Tetrahymena pyriformis, a freshwater ciliate. The microfluidic device was fabricated from an elastomer, poly(dimethylsiloxane) (PDMS), using multi-layer soft lithography. The components of the device include electronically controlled pneumatic microvalves, microchannels and microchambers. The linear gradient of the chemoeffectors was established by releasing a chemical from a ciliate-free microchamber into a microchamber containing the ciliate. The ciliate showed chemotactic behaviours by either swimming toward or avoiding the gradient. By counting the number of ciliates residing in each microchamber, we obtained a precise time-response curve. The ciliates in the microfluidic device were sensitive enough to be attracted to 10 pmol glycine-proline, which indicates a 10(5) increase in the ciliate's known sensitivity. With the use of blockers, such as DL-2-amino-5-phosphonopentanoic acid (APPA) or lanthanum chloride (LaCl3), we have demonstrated that the NMDA (N-methyl-d-aspartate) receptor plays a critical role in the perception of chemoeffectors, whereas the Ca2+ channel is related to the motility of the ciliate. These results demonstrate that our microfluidic chemotaxis assay system is useful not only for the study of ciliate chemotaxis but also for a better understanding of the signal transduction mechanism on their receptors.

Combined therapy with human cord blood cell transplantation and basic fibroblast growth factor delivery for treatment of myocardial infarction.

BACKGROUND: Transplanting cord blood-derived cells has been shown to augment neovascularization in ischaemic tissue. AIM: To test whether sustained delivery of basic fibroblast growth factor (bFGF) enhances the efficacy of angiogenic cord blood mononuclear cell (CBMNC) transplantation therapy in treating myocardial infarction. METHODS: Three weeks after myocardial infarction, Sprague-Dawley rats were randomised to either injection of medium only (control), CBMNC transplantation, sustained bFGF delivery, or combined CBMNC transplantation and sustained bFGF delivery. Six weeks after treatment, tissue formation, neovascularization, and apoptotic activity in the infarct regions were evaluated by histology and immunohistochemistry. Left ventricular (LV) dimensions and function were evaluated by magnetic resonance imaging. RESULTS: Combined bFGF delivery and CBMNC transplantation significantly enhanced neovascularization in the ischaemic myocardium, as compared with either therapy alone. The enhanced neovascularization was likely due to increased VEGF and bFGF expression. The combined therapy also exhibited a reduced infarct area and apoptosis in the ischaemic myocardium, as compared with either individual therapy. The combined therapy did not attenuate LV dilation or increase ejection fraction significantly over either individual therapy. CONCLUSION: This study demonstrates that sustained bFGF delivery enhances the angiogenic efficacy of CBMNC transplantation in rat myocardial infarction models.

Enhancement of angiogenic efficacy of human cord blood cell transplantation.

We tested the hypotheses that angiogenic efficacy of cord blood mononuclear cell (CBMNC) transplantation would be enhanced by using matrix and that combined therapy of CBMNC transplantation using matrix and sustained delivery of basic fibroblast growth factor (bFGF) would be synergistic in angiogenesis induction in ischemic limbs. One day after surgical induction of hindlimb ischemia, C57BL/6J mice were randomized to receive either medium injection, CBMNC transplantation using medium, CBMNC transplantation using fibrin matrix, sustained delivery of bFGF, or a combination of sustained delivery of bFGF and CBMNC transplantation using fibrin matrix. Four weeks after treatment, the angiogenic efficacy of the treatments was evaluated by immunohistochemical examinations and microvessel density determination in the ischemic sites. Transplanted CBMNCs survived, proliferated, and participated in capillary formation in ischemic limbs. CBMNC transplantation using fibrin matrix significantly increased the densities of capillaries and arterioles compared with CBMNC transplantation using medium. Importantly, combined therapy of sustained delivery of bFGF and CBMNC transplantation using fibrin matrix further increased the densities of capillaries and arterioles compared with either therapy alone. The angiogenic efficacy of angiogenic cell transplantation is enhanced by cell transplantation using matrix. Combined therapy of sustained release of angiogenic protein and angiogenic cell transplantation synergistically enhances angiogenesis in ischemic limbs compared to each therapy separately.

Programmable v-type valve for cell and particle manipulation in microfluidic devices.

A new microfluidic valve or a "v-type valve" which can be flexibly actuated to focus a fluid flow and block a specific area of a microchannel is demonstrated. Valves with different design parameters were fabricated by multilayer soft lithography and characterized at various operating pressures. To evaluate the functionality of the valve, single microparticles (∅ 7 µm and ∅ 15 µm) and single cells were trapped from flowing suspensions. Continuous processes of particle capture and release were achieved by controlling the actuation and deactuation of the valve. Integration of the v-type valve with poly(dimethyl siloxane) (PDMS) monolithic valves in microfluidic devices was demonstrated to illustrate the potential of the system in various applications such as the creation of a solid phase column, the isolation of a specific number of particles in reactors, and the capture and release of particles or cells in the flow of two immiscible liquids. We believe that this new valve system will be suitable for manipulating particles and cells in a broad range of applications.

Microfluidic device for DNA amplification of single cancer cells isolated from whole blood by self-seeding microwells.

Self-seeding microwell chips can sort single cells into 6400 wells based on cell size and their identity verified by immunofluorescence staining. Here, we developed a microfluidic device in which these single cells can be placed, lysed and their DNA amplified for further interrogation. Whole blood spiked with MCF7 tumor cells was passed through the microwell chips after leukocyte depletion and 37% of the MCF7 cells were identified by epithelial cell adhesion molecule (EpCAM) staining in the microwells. Identified single cells were punched into the reaction chamber of the microfluidic device and reagents for cell lysis and DNA amplification introduced sequentially by peristaltic pumping of micro-valves. On-chip lysis and amplification was performed in 8 parallel chambers yielding a 10,000 fold amplification of DNA. Accessibility of the sample through the reaction chamber allowed for easy retrieval and interrogation of target-specific genes to characterize the tumor cells.

Parallel single cancer cell whole genome amplification using button-valve assisted mixing in nanoliter chambers.

The heterogeneity of tumor cells and their alteration during the course of the disease urges the need for real time characterization of individual tumor cells to improve the assessment of treatment options. New generations of therapies are frequently associated with specific genetic alterations driving the need to determine the genetic makeup of tumor cells. Here, we present a microfluidic device for parallel single cell whole genome amplification (pscWGA) to obtain enough copies of a single cell genome to probe for the presence of treatment targets and the frequency of its occurrence among the tumor cells. Individual cells were first captured and loaded into eight parallel amplification units. Next, cells were lysed on a chip and their DNA amplified through successive introduction of dedicated reagents while mixing actively with the help of integrated button-valves. The reaction chamber volume for scWGA 23.85 nl, and starting from 6-7 pg DNA contained in a single cell, around 8 ng of DNA was obtained after WGA, representing over 1000-fold amplification. The amplified products from individual breast cancer cells were collected from the device to either directly investigate the amplification of specific genes by qPCR or for re-amplification of the DNA to obtain sufficient material for whole genome sequencing. Our pscWGA device provides sufficient DNA from individual cells for their genetic characterization, and will undoubtedly allow for automated sample preparation for single cancer cell genomic characterization.

Synergistic administration of photothermal therapy and chemotherapy to cancer cells using polypeptide-based degradable plasmonic matrices.

AIM: Resistance of cancer cells to hyperthermic temperatures and spatial limitations of nanoparticle-induced hyperthermia necessitates the identification of effective combination treatments that can enhance the efficacy of this treatment. Here we show that novel polypeptide-based degradable plasmonic matrices can be employed for simultaneous administration of hyperthermia and chemotherapeutic drugs as an effective combination treatment that can overcome cancer cell resistance to hyperthermia. METHOD: Novel gold nanorod elastin-like polypeptide matrices were generated and characterized. The matrices were also loaded with the heat-shock protein (HSP)90 inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17-AAG), currently in clinical trials for different malignancies, in order to deliver a combination of hyperthermia and chemotherapy. RESULTS: Laser irradiation of cells cultured over the plasmonic matrices (without 17-AAG) resulted in the death of cells directly in the path of the laser, while cells outside the laser path did not show any loss of viability. Such spatial limitations, in concert with expression of prosurvival HSPs, reduce the efficacy of hyperthermia treatment. 17-AAG-gold nanorod-polypeptide matrices demonstrated minimal leaching of the drug to surrounding media. The combination of hyperthermic temperatures and the release of 17-AAG from the matrix, both induced by laser irradiation, resulted in significant (>90%) death of cancer cells, while 'single treatments' (i.e., hyperthermia alone and 17-AAG alone) demonstrated minimal loss of cancer cell viability (<10%). CONCLUSION: Simultaneous administration of hyperthermia and HSP inhibitor release from plasmonic matrices is a powerful approach for the ablation of malignant cells and can be extended to different combinations of nanoparticles and chemotherapeutic drugs for a variety of malignancies.

Enhancement of in vivo bone regeneration efficacy of osteogenically undifferentiated human cord blood mesenchymal stem cells.

We hypothesized that bone morphogenetic protein-2 (BMP-2) would significantly enhance in vivo bone formation efficacy of osteogenically undifferentiated human cord blood mesenchymal stem cells (hCBMSCs). To test this hypothesis, poly(lactic-co-glycolic acid)/hydroxyapatite (PLGA/HA) scaffolds (group 1), BMP-2-loaded PLGA/HA scaffolds (group 2), undifferentiated hCBMSCs seeded on PLGA/HA scaffolds (group 3), undifferentiated hCBMSCs seeded on BMP-2-loaded PLGA/HA scaffolds (group 4), and osteogenically differentiated hCBMSCs seeded on PLGA/HA scaffolds (group 5) were implanted into dorsal, subcutaneous spaces of athymic mice for 8 weeks. Histological analysis showed that group 4 exhibited the largest bone formation area. RT-PCR analysis showed that human mRNA expression of osteoblastic markers such as ALP and osteocalcin in group 4 was higher than that of the other groups. Mouse osteoblastic markers of the host cells in the implants were also expressed more in group 4 than in the other groups. This study demonstrated that hCBMSCs that were not differentiated osteogenically in vitro prior to transplantation regenerate bone negligibly in vivo and that transplantation of osteogenically undifferentiated hCBMSCs with BMP-2 delivery results in much more extensive bone formation in vivo than that of undifferentiated or osteogenically differentiated hCBMSCs.

Cotransplanted bone marrow derived mesenchymal stem cells (MSC) enhanced engraftment of hematopoietic stem cells in a MSC-dose dependent manner in NOD/SCID mice.

Transplantation of marrow-derived mesenchymal stem cells (MSCs), expanded by culture in addition to whole bone marrow, has been shown to enhance engraftment of human hematopoietic stem cells (HSCs). Our hypothesis was that there might be an optimum ratio range that could enhance engraftment. We examined the percent donor chimerism according to the ratio of HSCs to MSCs in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. We tested a series of ratios of co-transplanted CD34(+) -selected bone marrow cells, and marrow-derived MSCs into sublethally irradiated NOD/SCID mice. In all experiments, 1x10(5) bone marrow derived human CD34(+) cells were administered to each mouse and human MSCs from different donors were infused concomitantly. We repeated the procedure three times and evaluated engraftment with flow cytometry four weeks after each transplantation. Serial ratios of HSCs to MSCs were 1:0, 1:1, 1:2 and 1:4, in the first experiment, 1:0, 1:1, 1:2, 1:4 and 1:8 in the second and 1:0, 1:1, 1:4, 1:8 and 1:16 in the third. Cotransplantation of HSCs and MSCs enhanced engraftment as the dose of MSCs increased. Our results suggest that the optimal ratio of HSCs and MSCs for cotransplantation might be in the range of 1:8-1:16; whereas, an excessive dose of MSCs might decrease engraftment efficiency.

Gene expression profile of cytokine and growth factor during differentiation of bone marrow-derived mesenchymal stem cell.

Mesenchymal stem cells (MSCs), which are adherent stromal cells of a nonhematopoietic origin, have the ability to give rise to various differentiated cell types. MSCs regulate localization, self-renewal and differentiation of hematopoietic stem cells (HSCs) due to MSCs' secretion of cytokines and growth factors, the cell-to-cell interactions and the influence of the extracellular matrix proteins. Using RT-PCR analysis, we examined the expression levels of cytokines and growth factors from MSCs and their differentiated cell types, including osteoblasts, adipocytes and endothelial cells. Cytokine and growth factor genes, including IL-6, IL-8, IL-11, IL-12, IL-14, IL-15, LIF, G-CSF, GM-CSF, M-SCF, FL and SCF, were found to be expressed in the MSCs. In contrast, there was no IL-1alpha, IL-1beta, or IL-7 expression observed. The IL-12, IL-14, G-CSF, and GM-CSF mRNA expression levels either disappeared or decreased after the MSCs differentiated into osteoblasts, adipocytes, and endothelial cells. Among the differentiated cells derived from MSCs, osteoblasts, adipocytes, and endothelial cells expressed the osteopontin, aP2, and the VEGFR-2 gene, respectively. These profiles could help determine future clinical applications of MSCs and their derivatives for cell therapy.