Perfusion System for Modification of Luminal Contents of Human Intestinal Organoids and Realtime Imaging Analysis of Microbial Populations

Intestinal organoids are 3D cell structures that replicate some aspects of organ function and are organized with a polarized epithelium facing a central lumen. To enable more applications, new technologies are needed to access the luminal cavity and apical cell surface of organoids. We developed a perfusion system utilizing a double-barrel glass capillary with a pressure-based pump to access and modify the luminal contents of a human intestinal organoid for extended periods of time while applying cyclic cellular strain. Cyclic injection and withdrawal of fluorescent FITC-Dextran coupled with real-time measurement of fluorescence intensity showed discrete changes of intensity correlating with perfusion cycles. The perfusion system was also used to modify the lumen of organoids injected with GFP-expressing E. coli. Due to the low concentration and fluorescence of the E. coli, a novel imaging analysis method utilizing bacteria enumeration and image flattening was developed to monitor E. coli within the organoid. Collectively, this work shows that a double-barrel perfusion system provides constant luminal access and allows regulation of luminal contents and luminal mixing.

Improvement of Nanostructured Polythiophene Film Uniformity Using a Cruciform Electrode and Substrate Rotation in Atmospheric Pressure Plasma Polymerization

In atmospheric pressure (AP) plasma polymerization, increasing the effective volume of the plasma medium by expanding the plasma-generating region within the plasma reactor is considered a simple method to create regular and uniform polymer films. Here, we propose a newly designed AP plasma reactor with a cruciform wire electrode that can expand the discharge volume. Based on the plasma vessel configuration, which consists of a wide tube and a substrate stand, two tungsten wires crossed at 90 degrees are used as a common powered electrode in consideration of two-dimensional spatial expansion. In the wire electrode, which is partially covered by a glass capillary, discharge occurs at the boundary where the capillary terminates, so that the discharge region is divided into fourths along the cruciform electrode and the discharge volume can successfully expand. It is confirmed that although a discharge imbalance in the four regions of the AP plasma reactor can adversely affect the uniformity of the polymerized, nanostructured polymer film, rotating the substrate using a turntable can significantly improve the film uniformity. With this AP plasma reactor, nanostructured polythiophene (PTh) films are synthesized and the morphology and chemical properties of the PTh nanostructure, as well as the PTh-film uniformity and electrical properties, are investigated in detail.

A simple surface modification to generate atomically-flat and hydrophobic substrates for gliding assays with protein motors

In vitro gliding assay is a well-established assay for determining the activity of protein motors, such as actin-associated myosins and microtubule-associated kinesins and dyneins. In one of the conventional methods, protein motors are immobilized onto a nitrocellulose-coated coverslip and it propels actin filaments in the presence of ATP. Gliding assays also serve as the foundation for protein-motor-based nanotechnological devices such as biosensing and sorting. However, the preparation of nitrocellulose-coated coverslips is time-consuming and produces rough surfaces. Furthermore, the nitrocellulose film exhibits high background autofluorescence, which can be a problem in single-molecule measurements. Here, we investigated the use of hexamethyldisilazane (HMDS) to study actomyosin function and characterized its physical properties on glass coverslips and glass capillary tubes. We showed that the total preparation time to coat a coverslip with HMDS is <30 minutes, which is 1 order of magnitude faster than the >12-hour protocol for coating glass surfaces with nitrocellulose. In contrast to nitrocellulose film, HMDS vapor deposition is effortless and provides an atomically flat surface with low autofluorescence. In addition, HMDS does not interfere with myosin function, which is indicated by the similar actin gliding speed when compared with nitrocellulose. Our results show that HMDS vapor deposition is a more favorable surface treatment to nitrocellulose for in vitro gliding assay.

Characteristics of megahertz resonant platform for evaluating sensitivity of photoacoustic contrast agent

A platform designed exclusively for evaluating photoacoustic contrast agents is required to prevent deviations in the results caused by differences in the architecture of photoacoustic imaging systems. In this paper, we managed to develop an evaluation system using a cost-efficient laser diode running in megahertz frequency band. To increase the output signal, an acoustic resonance structure is introduced where the sample liquid is filled in a glass capillary embedded in a soft phantom. The optimal width and interval of the excitation pulse were investigated and found to be 230 ns and 4.92 μs, respectively. Experimental results on inks and Indocyanine green verified the feasibility and effectiveness of the platform. Next, as a non-resonant platform, the glass capillary was replaced with a soft tube to examine the signal enhancement effect of the resonance. The signal-to-noise ratio was on average improved 2.5-fold by the resonance occurring in the glass capillary. The success in improving the signal-to-noise ratio may ensure the lower requirement on the laser power, which could consequently suppress the cost of the evaluation platform.

Emergence of uniform linearly-arranged micro-droplets entrapping DNA and living cells through water/water phase-separation

Living cells maintain their lives through self-organization in an environment crowded with a rich variety of biological species. Recently, it was found that micro-droplets containing biomacromolecules, which vary widely in size, are generated accompanied by water/water phase-separation by simple mechanical mixing of an aqueous solution with binary polymers. Here, we report that cell-sized droplets of nearly the same size are generated as a linear array within a glass capillary upon the introduction of a binary polymer solution of polyethylene glycol (PEG) and dextran (DEX). Interestingly, when DNA molecules are added to the polymer solution, stable droplets entrapping DNA molecules are obtained. Similarly, living cells are entrapped spontaneously for the linearly-arranged cell-sized droplets. This simple method for generating micro-droplets entrapping DNA and also living cells is expected to stimulate further study on the self-construction of protocells and micro organoids.

Perturbations of the Depth of Liquid Penetration Into the Capillary During Bubble Departures

In the present paper, the influence of bubble size on liquid penetration into the capillary was experimentally and numerically studied. In the experiment, bubbles were generated from a glass capillary (with an inner diameter equal to 1 mm) in a glass tank containing distilled water, tap water or an aqueous solution of calcium carbonate. These liquids differ in the value of their surface tension, which influences the bubble size. During experimental investigations, air pressure fluctuations in the gas supply system were measured. Simultaneously, the videos showing the liquids’ penetration into the capillary were recorded. Based on the videos, the time series of liquid movements inside the capillary were recovered. The numerical models were used to study the influence of bubble size on the velocity of liquid flow above the capillary and the depth of liquid penetration into the capillary. It was shown that the air volume flow rate and the surface tension have the greatest impact on the changes of pressure during a single cycle of bubble departure (Δp). The changes in pressure during a single cycle of bubble departure determine the depth of liquid penetration into the capillary. Moreover, the values of Δp and, consequently, the depth of liquid penetration can be modified by perturbations in the liquid velocity above the capillary outlet.

The choice of materials for the production of new reference materials of liquid viscosity certified in the interval of allowed temperature values from -40 °C to -5 °C

The article presents the results of research aimed at the selection of materials for the production of new reference materials intendedfor verification, calibration, and testing for type approval of means of measuring the viscosity of liquids, as well as certification of methods for measuring the viscosity of liquid media at temperatures below 0 °C. The developed reference materials are certified in the interval of allowed temperature values from -40 °C to -5 °C. This allows providing a wide range of modern methods for measuring the viscosity of liquids such as glass capillary viscometers, rotational viscometers, rheometers, etc.

Sub-Minute Analysis of Lactate from a Single Blood Drop Using Capillary Electrophoresis with Contactless Conductivity Detection in Monitoring of Athlete Performance

A simple and fast method for the analysis of lactate from a single drop of blood was developed. The finger-prick whole blood sample (10 µL) was diluted (1:20) with a 7% (w/v) solution of [tris(hydroxymethyl)methylamino] propanesulfonic acid and applied to a blood plasma separation device. The device accommodates a membrane sandwich composed of an asymmetric polysulfone membrane and a supporting textile membrane that allows the collection of blood plasma into a narrow glass capillary in less than 20 s. Separated and simultaneously diluted blood plasma was directly injected into a capillary electrophoresis instrument with a contactless conductivity detector (CE-C4D) and analyzed in less than one minute. A separation electrolyte consisted of 10 mmol/L l-histidine, 15 mmol/L dl-glutamic acid, and 30 µmol/L cetyltrimethylammonium bromide. The whole procedure starting from the finger-prick sampling until the CE-C4D analysis was finished, took less than 5 min and was suitable for monitoring lactate increase in blood plasma during incremental cycling exercise. The observed lactate increase during the experiments measured by the developed CE-C4D method correlated well with the results from a hand-held lactate analyzer (R = 0.9882). The advantage of the developed CE method is the speed, significant savings per analysis, and the possibility to analyze other compounds from blood plasma.