Sealed Microwell Research Articles

Abstract 644: Angiogenic Blood Vessels Reconfigure/ Attenuate Hypoxic Tumor Microenvironments In A Quantitative In Vitro Model

Introduction: Angiogenesis is crucial in the spread of cancer cells, and hypoxic tumor microenvironment (TME) plays a vital role in its initiation. Hypoxia stimulates the release of pro-angiogenic factors, promoting the growth of new blood vessels towards the tumor, which further alters the TME. The variation of in vivo hypoxic TME have been studied recently. However, due to technical limitations such as highly invasive procedure, high cost, tedious process, and poor signal-to-noise ratio, it is challenging to perform quantitative oxygen sensing. Here we develop an advanced in vitro model directly investigating the interaction between a solid tumor and blood vessels for an extended period. Hypothesis: The angiogenic blood vessels can reconfigure and attenuate the hypoxic TME, which can be directly measured Methods: The model is built using a microfluidic device with a sealed microwell for 3D tumor spheroid culture (human colon cancer cell line, HCT116), and a microfluidic channel beneath the microwell for endothelium formation (human umbilical vein endothelial cell, HUVEC), connected by a small hole for interaction between the cell types. Oxygen sensitive fluorescence particles are distributed in the hydrogel matrix, and the widefield frequency domain fluorescence lifetime imaging microscopy (FD-FLIM) is exploited to reliably characterize the oxygen tension around the tumor spheroid with minimal sensitivity to ambient optical noise. Results: The oxygen tension of TME around the tumor spheroid within the distance of 0 to 0.7 mm rapidly decreases during tumor growth, from 15% at day 1 to less than 6% at day 14. The presence of endothelial cells slows down this decreasing rate and maintains it in a stable range, from 16% to 14%. The average oxygen tension difference between the two experiments, with and without co-culture of endothelial cells, is statistically significant after 5 days of observation (p<0.01). Conclusions: We develop a quantitative in vitro method to study the hypoxic TME for its dynamic pattern and as a target for cancer treatment. By observing the effect of angiogenesis on it, this method can aid in exploring optimal cancer management strategies, such as chemotherapy, immunotherapy, tailored anti-angiogenic agents and other targeted therapies.

Simultaneous monitoring of oxygen consumption and acidification rates of a single zebrafish embryo during embryonic development within a microfluidic device

A microfluidic device with a light modulation system was developed to simultaneously measure the oxygen consumption rate (OCR) and acid extrusion rate (AER) of a single zebrafish embryo during embryonic development. The device combines two components: an array of acrylic microwells containing two sensing layers as the dual luminescent sensor for oxygen (O2) and acid (pH) detection, and a microfluidic module with pneumatically actuated glass lids to controllably seal the microwells. The continuous blue LED and modulated UV LED lights were simultaneously used to excite the dual luminescent sensor, with the emission detected by a single photodetector. The detection signals were then split into DC and AC components to measure the time variations in fluorescence intensity and phosphorescence lifetime for pH and O2 detection, respectively. We have successfully measured the OCR and AER of a single developing zebrafish embryo inside a sealed microwell from the blastula stage (3 h post-fertilization, 3 hpf) through the hatching stage of 48 hpf. We also demonstrated the measurement of the OCR and AER of a single 48 hpf zebrafish that experienced acute hypoxia by using our device to monitor the transition between aerobic and anaerobic metabolism. We observed that the AER began to significantly increase, while the OCR rapidly decreased after 20 min of hypoxia, indicating the time point of transition where the non-mitochondrial metabolism subsequently dominated the energy production. Our proposed methodology provides the potential for studying the bioenergetic metabolism in a developing organism that relates mitochondrial physiology and disease.

Red Blood Cell Deformability, Age, Ethnicity and Susceptibility to Malaria in Africa

Introduction: malaria is one of the most frequent hematological diseases worldwide. Because the malaria parasite Plasmodium falciparum develops mainly in red blood cells (RBC), splenic retention of infected and uninfected RBC is likely a key player in the variable susceptibility of humans to malaria. Age and ethnicity are important determinants of the manifestations of malaria in Africa (Reyburn JAMA 2005, Dolo Am J Trop Med Hyg 2005; Greenwood Ann Trop Med Parasitol 1987; Torcia PNAS 2008), Asia (Price Am J Trop Med Hyg 2001), and in travelers (Seringe Emerg Infect Dis 2011). We had speculated that variations in the splenic sensing of RBC contribute to the innate protection/susceptibility of infants against distinct forms of severe malaria and to the pathogenesis of chronic malaria (Buffet Curr Opin Hematol 2009; Buffet Blood 2011). Here, we explore the deformability and morphology of circulating RBC in populations living in a malaria-endemic area.Materials and methods: experiments were embedded in an integrated study driven by Institut de Recherche pour le Développement, which aims at the identification of genetic, epidemiologic and anthropologic determinants of susceptibility to malaria. IRB approval was obtained from Institut des Sciences Biomédicales Appliquées, Benin. Clinical and biological data were collected at the beginning of the rainy season from 627 individuals, belonging to 4 different ethnic groups living in sympatry in Atakora, North Benin and included age, gender, ethnicity, body temperature, presence and grade of splenomegaly, rapid diagnostic test for malaria (RDT), thick film and rapid hemoglobin determination with HemoCue©. Venous blood was collected for determination of RBC morphology and deformability. Using microsphiltration, a RBC filtering method that uses microsphere layers to mimic the mechanical retention of RBC in the splenic red pulp (Deplaine Blood 2011) we quantified the ability of a mix of labeled and non-labeled RBCs to squeeze between calibrated slits, results being expressed as retention or enrichment rates (RER) of subject’s RBC compared to normal RBC (from a single French O-positive donor) stored in blood bank conditions. Microsphiltration has been adapted to high-throughput experimentation using microplates (Duez AAC 2015). Experiments were performed in a field laboratory established on site; microplates were prepared in Paris and brought to North Benin in luggage with constant care to avoid shocks during transportation. All RBC samples were filtered in triplicate less than 8 hours after blood collection. Up- and downstream samples were brought back to France at 4°C in sealed micro-well plates and analyzed for individual RER calculation in the next 2 weeks by flow cytometry.Results: over 10 days, 262 adults and 249 children were included, 31% Bariba, 17% Gando (genetically related to Bariba), 24% Otamari, 27% Peulhs. Prevalences of splenomegaly, positive RDT, and fever were 13%, 27%, and 2%, respectively. Of 629 blood samples collected, 511 could be analyzed. RER of controls remained stable with time and across 17 microfiltering plates, with a median (IQR) retention rate of 12% (5% - 21%). Ethnicity and age were the only two factors associated with statistically significant differences in RER (figures 1 and 2). Infants (less than 2 year-old) had a more important enrichment than older children and adults (median in 2 years old or less 287%; 3 to 5 years 103%; 6 to 10 years: 64%; more than ten years: 91%; p=0.0161). Peulhs and Otamari also had higher median enrichment rates than Bariba and Gando (RER: 122% and 118%, 75%, 64%, respectively; p=0.0246). Conversely, splenomegaly, gender, positivity of RDT or anemia at the time of sampling were not associated with RER.Discussion: higher averaged enrichment rates in specific ethnic subgroups, namely Peulhs and Otamari, likely result from a more stringent splenic retention, leaving more deformable RBC in circulation. An innate spleen-RBC interaction process was also observed in infants, which is consistent with the higher incidence of severe malarial anemia and splenomegaly observed in this population (Reyburn JAMA 2005; Price Am J Trop Med Hyg 2001). Our results show that innate factors (e.g. ethnicity and age) tend to influence the deformability of RBC, and therefore the phenotypic expression of malaria in Africa. Ongoing experiments aim at deciphering the mechanisms responsible for these differences. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

An Automated Microwell Platform for Large-Scale Single Cell RNA-Seq.

Recent developments have enabled rapid, inexpensive RNA sequencing of thousands of individual cells from a single specimen, raising the possibility of unbiased and comprehensive expression profiling from complex tissues. Microwell arrays are a particularly attractive microfluidic platform for single cell analysis due to their scalability, cell capture efficiency, and compatibility with imaging. We report an automated microwell array platform for single cell RNA-Seq with significantly improved performance over previous implementations. We demonstrate cell capture efficiencies of >50%, compatibility with commercially available barcoded mRNA capture beads, and parallel expression profiling from thousands of individual cells. We evaluate the level of cross-contamination in our platform by both tracking fluorescent cell lysate in sealed microwells and with a human-mouse mixed species RNA-Seq experiment. Finally, we apply our system to comprehensively assess heterogeneity in gene expression of patient-derived glioma neurospheres and uncover subpopulations similar to those observed in human glioma tissue.

Selective infiltration and storage of picoliter volumes of liquids into sealed SU-8 microwells

AbstractThis paper describes the selective infiltration and storage of picoliter volumes of water and IPA in arrays of sealed SU-8 microwells. Microwells, with a volume of approximately 300 picoliters, are fabricated employing photolithography and a polymer onto polymer lamination method to seal the structures with a thin cover of SU-8 and PDMS in order to suppress the evaporation of the infiltrated liquids. A glass capillary is used to punch through the SU-8/PDMS cover and to infiltrate the liquid of interest into the microwells. The influence of the mixing ratio of the PDMS and its curing agent is studied and the results show that a lower ratio of 2:1 suppresses the evaporation more when compared to the standard mixing ratio of 10:1. In regards to water and IPA, the dwell time in the reservoirs was increased by approximately 50 % and 450 % respectively. Depending on the physical properties of the microwells and the liquids, the SU-8/PDMS cover suppresses the evaporation up to 32 mins for water and 463 mins for IPA, respectively, until the microwell is completely empty again. Additionally, multiple infiltrations of the same microwell are demonstrated using two immiscible liquids IPA and paraffin oil. Based on the popular polymers SU-8 and PDMS, the sealed microwell structures are scalable and combinable with different glass capillaries according to the needs of future analytical research and medical diagnostics.

Hermetically sealed microwell with a lipid bilayer created using a self-assembled monolayer

To provide a platform for biodevices designed to characterize membrane proteins, we fabricated a new type of microwell sealed with a lipid membrane on a SiO2 substrate. The microwell is surrounded by a Au ring with a self-assembled monolayer, on which a lipid membrane is formed to create a seal. Fluorescence and electrophysiological studies reveal that the structure prevents unfavorable ion leakage from/into microwells. By separating the microwells and outer regions, ion diffusion through the water layer between the membrane and the substrate is reduced. This study offers a promising approach for the functional analysis of membrane proteins.

Respiration Rate Measurements of Single Bacterial Cells

One of the limitations of a population-based analysis method is that it averages over a large number of cells. While this helps average out fluctuations in the measured signal, it potentially can cover up subpopulations that could be functionally important. Using a single-cell approach, one can individually measure cells and look at the variation within the isogenic cell population.Respiration can be an indicator of physiological state and therefore is an excellent target for analysis of heterogeneity within the sample. Single-cells are isolated in microwells containing Pt-porphyrin embedded microspheres on a glass chip. These microwells are diffusionally sealed with a lid actuator which can be raised at the end of the measurement to reoxygenate the sample. Since the phosphorescence lifetime of the Pt-porphyrin depends on the oxygen concentration, the Pt-porphyrin embedded microspheres can be used as an oxygen sensor. Monitoring the consumption of oxygen in the sealed microwells over time allows a respiration rate to be calculated for the individual cells. This method is compatible with other optical imaging techniques.

Biological system development for GraviSat: A new platform for studying photosynthesis and microalgae in space

Microalgae have great potential to be used as part of a regenerative life support system and to facilitate in-situ resource utilization (ISRU) on long-duration human space missions. Little is currently known, however, about microalgal responses to the space environment over long (months) or even short (hours to days) time scales. We describe here the development of biological support subsystems for a prototype “3U” (i.e., three conjoined 10-cm cubes) nanosatellite, called GraviSat, designed to experimentally elucidate the effects of space microgravity and the radiation environment on microalgae and other microorganisms. The GraviSat project comprises the co-development of biological handling-and-support technologies with implementation of integrated measurement hardware for photosynthetic efficiency and physiological activity in support of long-duration (3–12 months) space missions. It supports sample replication in a fully autonomous system that will grow and analyze microalgal cultures in 120 μL wells around the circumference of a microfluidic polymer disc; the cultures will be launched while in stasis, then grown in orbit. The disc spins at different rotational velocities to generate a range of artificial gravity levels in space, from microgravity to multiples of Earth gravity. Development of the biological support technologies for GraviSat comprised the screening of more than twenty microalgal strains for various physical, metabolic and biochemical attributes that support prolonged growth in a microfluidic disc, as well as the capacity for reversible metabolic stasis. Hardware development included that necessary to facilitate accurate and precise measurements of physical parameters by optical methods (pulse amplitude modulated fluorometry) and electrochemical sensors (ion-sensitive microelectrodes). Nearly all microalgal strains were biocompatible with nanosatellite materials; however, microalgal growth was rapidly inhibited (∼1 week) within sealed microwells that did not include dissolved bicarbonate due to CO2 starvation. Additionally, oxygen production by some microalgae resulted in bubble formation within the wells, which interfered with sensor measurements. Our research achieved prolonged growth periods (>10 months) without excess oxygen production using two microalgal strains, Chlorella vulgaris UTEX 29 and Dunaliella bardawil 30 861, by lowering light intensities (2–10 μmol photons m−2 s−1) and temperature (4–12 °C). Although the experiments described here were performed to develop the GraviSat platform, the results of this study should be useful for the incorporation of microalgae in other satellite payloads with low-volume microfluidic systems.

Metabolic profile analysis of a single developing zebrafish embryo via monitoring of oxygen consumption rates within a microfluidic device

A combination of a microfluidic device with a light modulation system was developed to detect the oxygen consumption rate (OCR) of a single developing zebrafish embryo via phase-based phosphorescence lifetime detection. The microfluidic device combines two components: an array of glass microwells containing Pt(II) octaethylporphyrin as an oxygen-sensitive luminescent layer and a microfluidic module with pneumatically actuated glass lids above the microwells to controllably seal the microwells of interest. The total basal respiration (OCR, in pmol O2/min/embryo) of a single developing zebrafish embryo inside a sealed microwell has been successfully measured from the blastula stage (3 h post-fertilization, 3 hpf) through the hatching stage (48 hpf). The total basal respiration increased in a linear and reproducible fashion with embryonic age. Sequentially adding pharmacological inhibitors of bioenergetic pathways allows us to perform respiratory measurements of a single zebrafish embryo at key developmental stages and thus monitor changes in mitochondrial function in vivo that are coordinated with embryonic development. We have successfully measured the metabolic profiles of a single developing zebrafish embryo from 3 hpf to 48 hpf inside a microfluidic device. The total basal respiration is partitioned into the non-mitochondrial respiration, mitochondrial respiration, respiration due to adenosine triphosphate (ATP) turnover, and respiration due to proton leak. The changes in these respirations are correlated with zebrafish embryonic development stages. Our proposed platform provides the potential for studying bioenergetic metabolism in a developing organism and for a wide range of biomedical applications that relate mitochondrial physiology and disease.

Light-addressable measurements of cellular oxygen consumption rates in microwell arrays based on phase-based phosphorescence lifetime detection

A digital light modulation system that utilizes a modified commercial digital micromirror device (DMD) projector, which is equipped with a UV light-emitting diode as a light modulation source, has been developed to spatially direct excited light toward a microwell array device to detect the oxygen consumption rate (OCR) of single cells via phase-based phosphorescence lifetime detection. The microwell array device is composed of a combination of two components: an array of glass microwells containing Pt(II) octaethylporphine (PtOEP) as the oxygen-sensitive luminescent layer and a microfluidic module with pneumatically actuated glass lids set above the microwells to controllably seal the microwells of interest. By controlling the illumination pattern on the DMD, the modulated excitation light can be spatially projected to only excite the sealed microwell for cellular OCR measurements. The OCR of baby hamster kidney-21 fibroblast cells cultivated on the PtOEP layer within a sealed microwell has been successfully measured at 104 ± 2.96 amol s(-1) cell(-1). Repeatable and consistent measurements indicate that the oxygen measurements did not adversely affect the physiological state of the measured cells. The OCR of the cells exhibited a good linear relationship with the diameter of the microwells, ranging from 400 to 1000 μm and containing approximately 480 to 1200 cells within a microwell. In addition, the OCR variation of single cells in situ infected by Dengue virus with a different multiplicity of infection was also successfully measured in real-time. This proposed platform provides the potential for a wide range of biological applications in cell-based biosensing, toxicology, and drug discovery.

Strand Displacement Amplification and Homogeneous Real-Time Detection Incorporated in a Second-Generation DNA Probe System, BDProbeTecET

Amplified DNA probes provide powerful tools for the detection of infectious diseases, cancer, and genetic diseases. Commercially available amplification systems suffer from low throughput and require decontamination schemes, significant hands-on time, and specially trained laboratory staff. Our objective was to develop a DNA probe system to overcome these limitations. We developed a DNA probe system, the BDProbeTecTMET, based on simultaneous strand displacement amplification and real-time fluorescence detection. The system uses sealed microwells to minimize the release of amplicons to the environment. To avoid the need for specially trained labor, the system uses a simple workflow with predispensed reagent devices; a programmable, expandable-spacing pipettor; and the 96-microwell format. Amplification and detection time was 1 h, with potential throughput up to 564 patient results per shift. We tested 122 total patient specimens obtained from a family practice clinic with the BD ProbeTecET and the Abbott LCx(R) amplified system for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae. Based on reportable results, the BDProbeTecET results for both organisms were 100% sensitive and 100% specific relative to the LCx. The BDProbeTecET is an easy-to-use, high-throughput, closed amplification system for the detection of nucleic acid from C. trachomatis and N. gonorrhoeae and other organisms.