Continuous Medium Flow Research Articles

Fast formation and maturation enhancement of human liver organoids using a liver-organoid-on-a-chip.

Background: Spatial and functional hepatic zonation, established by the heterogeneous tissue along the portal-central axis of the liver, is important for ensuring optimal liver function. Researchers have attempted to develop reliable hepatic models to mimic the liver microenvironment and analyze liver function using hepatocytes cultured in the developed systems. However, mimicking the liver microenvironment in vitro remains a great challenge owing to the lack of perfusable vascular networks in the model systems and the limitation in maintaining hepatocyte function over time. Methods: In this study, we established a microphysiological system that operated under continuous flush medium flow, thereby allowing the supply of nutrients and oxygen to liver organoids and the removal of waste and release of cytokines therefrom, similar to the function of blood vessels. Results: The application of microphysiological system to organoid culture was advantageous for reducing the differentiation time and enhancing the functional maturity of human liver organoid. Conclusion: Hence, our microphysiological culture system might open the possibility of the miniaturized liver model system into a single device to enable more rational in vitro assays of liver response.

A closed 3D printed microfluidic device for automated growth and differentiation of cerebral organoids from single-cell suspension.

The development of 3D organoids has provided a valuable tool for studying human tissue and organ development in vitro. Cerebral organoids, in particular, offer a unique platform for investigating neural diseases. However, current methods for generating cerebral organoids suffer from limitations such as labor-intensive protocols and high heterogeneity among organoids. To address these challenges, we present a microfluidic device designed to automate and streamline the formation and differentiation of cerebral organoids. The device utilizes microwells with two different shapes to promote the formation of a single aggregate per well and incorporates continuous medium flow for optimal nutrient exchange. In silico simulations supported the effectiveness of the microfluidic chip in replicating cellular microenvironments. Our results demonstrate that the microfluidic chip enables uniform growth of cerebral organoids, significantly reducing the hands-on time required for maintenance. Importantly, the performance of the microfluidic system is comparable to the standard 96-well plate format even when using half the amount of culture medium, and the resulting organoids exhibit substantially developed neuroepithelial buds and cortical structures. This study highlights the potential of custom-designed microfluidic technology in improving the efficiency of cerebral organoid culture.

Droplet Microfluidic Method for Estimating the Dynamic Interfacial Tension of Ion-Crosslinked Sodium Alginate Microspheres.

The research focuses on optimizing the production of hydrogel microspheres using droplet microfluidics for pharmaceutical and bioengineering applications. A semiempirical method has been developed to predict the dynamic interfacial tension at the interface of ion-cross-linked sodium alginate microsphere-sunflower oil modified with glacial acetic acid and Tween 80 surfactant. These microspheres are produced in a small-scale coaxial device that is manufactured using affordable DLP/LCD 3D printing technology with a transparent photopolymer. The method was tested to design the minireactor in the device, which allows for the production of fully cross-linked microspheres that are ready for use at the output of the reactor without additional cross-linking steps in the microsphere collector. The mathematical expression for estimating the interfacial tension at the moment of formation of a hydrogel microsphere includes the Reynolds number for a two-phase liquid, the Ohnesorge number, and the surface tension at the liquid-air interface for continuous medium flow (modified oil). The reliability of the prediction is confirmed for continuous medium and dispersed phase flow rates of 0.8-3.2 and 0.01-0.08 mL/min, respectively. The evolution of the interfacial tension from the moment the microspheres formed and the estimated ultimate interfacial tension in a cross-linked hydrogel-modified oil system contributed to the reliable determination of the linear size of a minireactor. The ultimate interfacial tension of 76.5 ± 0.3 mN/m was determined using the Young-Laplace equation, which is based on measuring the surface free energy of the hydrogel as soft matter using the Owens-Wendt method. Additionally, the equilibrium static contact angle of the fully cross-linked hydrogel surface wetted with oil is measured using the sessile drop method. From a practical perspective, a method for optimizing and streamlining the high-tech manufacturing of cross-linked polymer microspheres and mini- and microchannel devices for use in bioengineering and pharmaceutical applications is suggested.

Dynamic flow for efficient partial decellularization of tracheal grafts: A preliminary rabbit study.

Bioengineered tracheal grafts are a potential solution for the repair of long-segment tracheal defects. A recent advancement is partially decellularized tracheal grafts (PDTGs) which enable regeneration of host epithelium and retain viable donor chondrocytes for hypothesized benefits to mechanical properties. We propose a novel and tunable 3D-printed bioreactor for creating large animal PDTG that brings this technology closer to the bedside. Conventional agitated immersion with surfactant and enzymatic activity was used to partially decellularize New Zealand white rabbit (Oryctolagus cuniculus) tracheal segments (n = 3). In parallel, tracheal segments (n = 3) were decellularized in the bioreactor with continuous extraluminal flow of medium and alternating intraluminal flow of surfactant and medium. Unprocessed tracheal segments (n = 3) were also collected as a control. The grafts were assessed using the H&E stain, tissue DNA content, live/dead assay, Masson's trichrome stain, and mechanical testing. Conventional processing required 10 h to achieve decellularization of the epithelium and submucosa with poor chondrocyte viability and mechanical strength. Using the bioreactor reduced processing time by 6 h and resulted in chondrocyte viability and mechanical strength similar to that of native trachea. Large animal PDTG created using our novel 3D printed bioreactor is a promising approach to efficiently produce tracheal grafts. The bioreactor offers flexibility and adjustability favorable to creating PDTG for clinical research and use. Future research includes optimizing flow conditions and transplantation to assess post-implant regeneration and mechanical properties. NA.

Ultrasound directed self-assembly of filler in continuous flow of a viscous medium through an extruder nozzle for additive manufacturing

Ultrasound directed self-assembly of filler in continuous flow of a viscous medium through an extruder nozzle for additive manufacturing

Metabolites from scutellarin alleviating deferoxamine-induced hypoxia injury in BV2 cells cultured on microfluidic chip combined with a mass spectrometer

The changes of metabolites of tricarboxylic acid (TCA) cycle in cells under hypoxia play a key role in drug screening. In order to dynamically monitor the drug metabolism changes of Scutellarin in the hypoxia environment induced by deferoxamine (DFO), a microfluidic-chip mass spectrometry method was used to study the real-time monitoring of drug metabolism changes under hypoxia conditions. This system has six drug-loading units, cell culture chamber, metabolite collection, filtration, HPLC separation and mass spectrometer. The cells in each microchannel were incubated with continuous flow of culture medium, metabolites will be collected by the fixed card slot, automatic sampling needle will be precise positioned and sampled. Through this new system combined with molecular biological methods, the changes of metabolites in TCA cycle of BV2 cells and drug metabolism of Scutellarin can be determined in real-time. In general, we illustrated a new mechanism of Scutellarin for reducing BV2 cell hypoxia injury and presented a novel analysis strategy that opened a way for real-time online monitoring of the energy metabolic mechanism of the effect of drugs on cells and further provided a superior strategy to screen natural drug candidates for hypoxia-related brain disease treatment.

On Stationary Flow of Dense Plasma under Localized Energy Deposition

The paper presents a one-dimensional gas-dynamic model that gives an opportunity to establish the necessary conditions for the emergence and determine characteristics of a stationary flow of a compressible continuous medium with nonlinear thermal conductivity, an example of which is a fully or partially ionized plasma, in the presence of a localized heat source with a given power.

Reynolds Analogy Coefficient in the Longitudinal Cylindrical Couette Problem: from the Continuous Medium to Free Molecular Flow

The gas Couette flow for a cylindrical geometry of bounding surfaces that move in the longitudinal direction relative to their symmetry axis is considered. For a monoatomic gas, the relation between the shear stress and the energy flux transferred to the longitudinal-flow surface (Reynolds analogy) is studied. In the case of continuous medium and free molecular flow regimes, simple explicit analytical expressions for the Reynolds analogy coefficient are obtained, which depend only on the Eckert number and are independent of the ratio of the cylinder radii. The transitional regime for various values of the Knudsen number is studied using the direct simulation Monte Carlo (DSMC) method. It is shown that, in this case, the Reynolds analogy coefficient at a fixed ratio of the radii and the Knudsen number depends on the relative velocity and temperatures of the surfaces mainly through the Eckert number. A relationship between the linear energy fluxes transferred to the cylindrical surfaces is found.

Experimental study and numerical analysis of vacuum thermal characteristics of brush DC motor

Brush direct current (DC) motors have several qualities that make them very attractive for space flight applications. Considering the high reliability requirements of aerospace missions, the thermal characteristics and thermal failure of the brush DC motor in the space environment were studied. Using a motor thermal resistance network model, a special thermal test method was determined and combined with a thermal conductivity analysis model, the thermal parameters were obtained via item-by-item stripping, and the motor temperature field was constructed. By introducing the arc discharge factor to evaluate the electric-corrosion heat consumption, the numerical analysis results were in good agreement with the test results under the conditions of stalled rotor, normal rotation, single brush, and multiple brushes. The analysis and test results show that continuous operation for 110 s will lead to melting of the brush solder joints, and electrical corrosion heat consumption is one of the main factors that cannot be ignored. The reliability model of vacuum applications should be established in the normal working mode of at least two brushes in both the positive and negative electrodes. To improve the reliability, a sealed air-filled structure of the motor was proposed, a heat-flow co-simulation model of a continuous medium flow with a large curvature and constant without a gravity field was established, and the temperature and velocity fields under different sealed pressures were obtained. The results show that the temperature of the single brush reduced to below 140 °C from 204.5 °C in vacuum, which can meet the long-term continuous working requirement of high reliability of brush motors in space missions. In addition, it was found that with the decrease in pressure, the effect of convective heat transfer gradually weakens, the temperature gradually increases and converges to the unique heat conduction process of the gas, while the effect of convection is negligible. As the pressure continues to decrease, the sealed gas evolves from continuous medium flow to transitional and free molecular flow, and the heat conduction effect of the gas weakens again until it approaches the singleness solid conduction process.

Impact of separately located obstacles on air flow movement calculation

In hydrodynamic calculations, the confederation of the right set of coordinates of a continuous medium (gas, liquid) becomes complicated because it makes the calculation mechanismspecific and tied only to a specific object. The paper is devoted to an improved method to calculate the small-scale circulation of the surface layer of the atmosphere in urban areas with many isolated buildings from each other. The proposed method will work for explicit and implicit schemes for calculating the transport equations and conservation of momentum of motion. It is designed to calculate the small-scale circulation of the surface layer of the atmosphere in urban areas with many isolated buildings from each other. The method allows for consideration of the interaction of a continuous medium flow with impenetrable obstacles discretely and randomly located inside the calculation area. This method will be helpful when calculating microcirculation in urban development.

ДОСЛІДЖЕННЯ НЕРЕЗОНАНСНИХ КОЛИВАНЬ СИСТЕМИ «ТЕЛЕСКОПІЧНИЙ ГВИНТ – СИПКЕ СЕРЕДОВИЩЕ»

In the article it is substantiated the value of the angular speeds of rotation of the auger screw, which leads to the breakdown of its lateral vibrations. The dependences describing the law of change of amplitude or natural frequency at slowly variable length of the telescopic screw are deduced. Based on the Van der Paul’s method, in the developed system differential equations are obtained that determine the laws of change of amplitude and frequency of the wave process in the system of a telescopic propeller. It is established that for nonresonant oscillations for this system the main parameters of bending oscillations are a continuous flow of bulk medium - the screw does not depend on its small torsional oscillations and external periodic perturbation. The analysis of the given regression equations shows that to reduce the torque of the auger it is necessary to reduce the frequency of its rotation and the angle of the conveyor. The constructive diagram and the results of theoretical calculations for assessing the influence of constructive-kinematic parameters on the torque indicators of the telescopic screw conveyor are presented.

Adhesion of Yeast and Bacteria to Oral Surfaces.

Colonization of surfaces in the human body by microorganisms is an early, essential, step in the initiation of infectious disease. We have developed in vitro assays to investigate interactions between yeast or bacterial cells and human tissues, fluids, or prostheses. Such assays can be used to identify the adhesins, ligands, and receptors involved in these interactions, for example, by determining which components of the microbe or human tissue/fluid interfere with adherence in the assay. The assays can also be applied to find ways of preventing adhesion, and subsequent disease, by investigating the effects of different conditions and added compounds on adherence in the in vitro assays. Here we describe assays for measuring adhesion of the oral yeast Candida albicans, a common commensal and opportunistic pathogen, or the bacterium Staphylococcus epidermidis, which is not normally pathogenic but is known to form biofilms on medical prostheses. The assays described belong to two approaches to investigating adhesion and biofilm formation: (i) retention at a fixed time point following liquid washes, and (ii) retention against a continuous flow of medium.

Generation of 3D Spheroids Using a Thiol-Acrylate Hydrogel Scaffold to Study Endocrine Response in ER+ Breast Cancer.

Culturing cancer cells in a three-dimensional (3D) environment better recapitulates in vivo conditions by mimicking cell-to-cell interactions and mass transfer limitations of metabolites, oxygen, and drugs. Recent drug studies have suggested that a high rate of preclinical and clinical failures results from mass transfer limitations associated with drug entry into solid tumors that 2D model systems cannot predict. Droplet microfluidic devices offer a promising alternative to grow 3D spheroids from a small number of cells to reduce intratumor heterogeneity, which is lacking in other approaches. Spheroids were generated by encapsulating cells in novel thiol–acrylate (TA) hydrogel scaffold droplets followed by on-chip isolation of single droplets in a 990- or 450-member trapping array. The TA hydrogel rapidly (∼35 min) polymerized on-chip to provide an initial scaffold to support spheroid development followed by a time-dependent degradation. Two trapping arrays were fabricated with 150 or 300 μm diameter traps to investigate the effect of droplet size and cell seeding density on spheroid formation and growth. Both trapping arrays were capable of ∼99% droplet trapping efficiency with ∼90% and 55% cellular encapsulation in trapping arrays containing 300 and 150 μm traps, respectively. The oil phase was replaced with media ∼1 h after droplet trapping to initiate long-term spheroid culturing. The growth and viability of MCF-7 3D spheroids were confirmed for 7 days under continuous media flow using a customized gravity-driven system to eliminate the need for syringe pumps. It was found that a minimum of 10 or more encapsulated cells are needed to generate a growing spheroid while fewer than 10 parent cells produced stagnant 3D spheroids. As a proof of concept, a drug susceptibility study was performed treating the spheroids with fulvestrant followed by interrogating the spheroids for proliferation in the presence of estrogen. Following fulvestrant exposure, the spheroids showed significantly less proliferation in the presence of estrogen, confirming drug efficacy.

A microfluidic-based approach to investigate the inflammatory response of macrophages to pristine and drug-loaded nanostructured hydroxyapatite.

The in vitro biological characterization of biomaterials is largely based on static cell cultures. However, for highly reactive biomaterials such as calcium-deficient hydroxyapatite (CDHA), this static environment has limitations. Drastic alterations in the ionic composition of the cell culture medium can negatively affect cell behavior, which can lead to misleading results or data that is difficult to interpret. This challenge could be addressed by a microfluidics-based approach (i.e. on-chip), which offers the opportunity to provide a continuous flow of cell culture medium and a potentially more physiologically relevant microenvironment. The aim of this work was to explore microfluidic technology for its potential to characterize CDHA, particularly in the context of inflammation. Two different CDHA substrates (chemically identical, but varying in microstructure) were integrated on-chip and subsequently evaluated. We demonstrated that the on-chip environment can avoid drastic ionic alterations and increase protein sorption, which was reflected in cell studies with RAW 264.7 macrophages. The cells grown on-chip showed a high cell viability and enhanced proliferation compared to cells maintained under static conditions. Whereas no clear differences in the secretion of tumor necrosis factor alpha (TNF-α) were found, variations in cell morphology suggested a more anti-inflammatory environment on-chip. In the second part of this study, the CDHA substrates were loaded with the drug Trolox. We showed that it is possible to characterize drug release on-chip and moreover demonstrated that Trolox affects the TNF-α secretion and morphology of RAW 264.7 ​cells. Overall, these results highlight the potential of microfluidics to evaluate (bioactive) biomaterials, both in pristine form and when drug-loaded. This is of particular interest for the latter case, as it allows the biological characterization and assessment of drug release to take place under the same dynamic in vitro environment.

Evaluation of the Feasibility of Endothelial Colony-Forming Cells to Develop Tissue-Engineered Vascular Grafts Based on the Gene Expression Profile Analysis.

In the experiment, we used the endothelial colony-forming cells (ECFC) obtained from the peripheral blood of patients who underwent percutaneous coronary intervention. The cells were isolated on a Histopaque 1077 density gradient (Sigma-Aldrich, USA), and then cultured in EGM-2MV culture medium (Lonza, Switzerland). A commercial culture of primary human coronary artery endothelial cells (HCAEC) was used as a control. The cells were unfrozen and cultured according to the manufacturer's recommendations in MesoEndo Cell Growth Medium (Cell Applications, USA).The experiment was carried out in specialized μ-Luer plates in the perfusion system (IBIDI, Germany), which provided a continuous unidirectional flow of the culture medium with a shear stress of 5 dyn/cm2. Control plates were cultured under standard conditions for a similar period of time. Total RNA was isolated from cell samples. The expression of the genes NOTCH4, NRP2, PLAT, PLAU, NOTCH1, FLT1, COL4A2, CD34, SERPINE1, HEY2, MKI67, KLF4, LYVE1, FLT4 was assessed using a quantitative real-time polymerase chain reaction. The expression of the genes was calculated by the ΔCt method and expressed on a logarithmic (log10) scale as a fold change relating to the control samples. In mature endothelial cells HCAEC when exposed to a laminar flow, only the transcription factor KLF4 and venous differentiation NRP2 marker values increased significantly. ECFC showed statistically significant growth in KLF4, NRP2, CD34, and LYVE1, as well as PLAU expression decrease. In addition, we observed the overexpression of FLT4, LYVE1, NOTCH4, and NRP2 in ECFC in relation to HCAEC and HEY2 hypoexpression. CD34 overexpression characteristic of progenitor cells was also found. An increase in COL4A2 expression associated with type IV collagen synthesis was a characteristic feature of ECFC. The gene expression profile of endothelial colony-forming cells is quite close to that of primary endothelial cells of the human coronary artery, and thus, the cells obtained from patients' peripheral blood can be used to develop personalized tissue-engineered constructs.

Calculated estimation of influence of convection rate of molten working and heating medium during hardening of restored parts

The paper presents a computational and mathematical assessment of the convection rate of the molten working and heating medium during the strengthening of the restored parts. The calculated estimate is based on the theory of multidimensional heat and mass transfer processes occurring in the electrode furnace during the strengthening of the restored parts. Computational modeling is carried out based on the choice of initial data and characteristics. The calculation results made it possible to establish the effect of the convection rate of the molten working and heating medium on the hardening of the restored parts. The theoretical description of the conditions for the onset of convective motion of a molten continuous medium is based on the Rayleigh–Bénard, Navier–Stokes, and Lorenz equations for the flow of a viscous continuous medium using the Oberbeck-Boussinesq approximation. The description of the internal space of the tank furnace is carried out by a system of differential equations of the balances of elementary volumes into which it is divided. Transformations into a system of differential equations are carried out on the basis of the Galerkin method. The use of this transition method leads to the fact that the sought functions are represented in a variable-separated form that exactly satisfies the boundary conditions. The introduction of assumptions and boundary conditions made it possible to obtain the equations of motion of mass forces and a viscous medium in a polar coordinate system. A formula describing the configuration of the temperature field is obtained.

Real-time monitoring the efficacy of 7-hydroxycoumarin to cells cultured on microfluidics in different extracellular pH environments by chip-mass spectrometry.

The extracellular microenvironments play a key role in tumor metabolism. To online dynamic monitoring the efficacy of 7-hydroxycoumarin (7-OHC) to cells cultured on microfluidics in acidic microenvironment, we developed an integrated multi-channel chip-mass spectrometry system. This system has six drug-loading units, cell culture chamber, metabolite collection, filtration, HPLC separation and MS detection. The cells in each microchannel will be incubated with continuous flow of culture medium, metabolites will be collected by the fixed card slot, automatic sampling needle will be precise positioned and sampled. Through this new system, the 7-hydroxycoumarin-sulfonate (7-OHC-sulfonate) and 7-hydroxycoumarin-glucuronide (7-OHC-glucuronide) can be determined in real-time. The results revealed that the addition of lactic acid promoted the formation of inactive 7-OHC-sulfonate and 7-OHC-glucuronide metabolites. Besides, acidic extracellular environment amplified cancer cell proliferation, indicating the anticancer effect of 7-OHC was weakened by low extracellular pH.

Enrichment of cancer stem-like cells by controlling oxygen, glucose and fluid shear stress in a microfluidic spheroid culture device

Current chemotherapies can often kill fast-growing cancer cells but are ineffective in destroying cancer stem cells (CSCs). This study aimed to investigate the effect of in vitro cell culture conditions on the features of lung adenocarcinoma cells (represented by A549, non-small cell type (NSCLC), cell lines) and the induction of cancer stem-like cells. For this purpose, a microfluidic system containing U-shaped arrays was designed and numerically optimized. This system was used for the three-dimensional culture of cells under a continuous laminar flow of culture medium. Numerical simulations were also performed to estimate the quiescent and necrotic zones inside the spheroids and the fluid shear stress on the cultured spheroids. Moreover, the effects of culture medium flow rate, oxygen and glucose concentrations and anoikis phenomenon on the number of CD90+ cells and expression of stemness, epithelial-mesenchymal transition (EMT) and ATP-binding cassette (ABC)-transporters genes were investigated. The results showed that all these parameters substantially affected the enrichment of A549 cancer stem-like cells. We also investigated the effect of the CSC enrichment method, i.e., shear stress and oxygen and glucose concentrations, on resistance to cisplatin treatment. Increasing shear stress and glucose concentration and decreasing oxygen concentration led to a sharp increase in chemoresistance of cells. Interestingly, changing oxygen concentration was more significant than changing the other factors. The results of this paper can be helpful for the effective enrichment of CSCs by adjusting in vitro culture conditions.

NMR Analysis Method of Gas Flow Pattern in the Process of Shale Gas Depletion Development

Shale gas reservoirs have pores of various sizes, in which gas flows in different patterns. The coexistence of multiple gas flow patterns is common. In order to quantitatively characterize the flow pattern in the process of shale gas depletion development, a physical simulation experiment of shale gas depletion development was designed, and a high-pressure on-line NMR analysis method of gas flow pattern in this process was proposed. The signal amplitudes of methane in pores of various sizes at different pressure levels were calculated according to the conversion relationship between the NMR T 2 relaxation time and pore radius, and then, the flow patterns of methane in pores of various sizes under different pore pressure conditions were analyzed as per the flow pattern determination criteria. It is found that there are three flow patterns in the process of shale gas depletion development, i.e., continuous medium flow, slip flow, and transitional flow, which account for 73.5%, 25.8%, and 0.7% of total gas flow, respectively. When the pore pressure is high, the continuous medium flow is dominant. With the gas production in shale reservoir, the pore pressure decreases, the Knudsen number increases, and the pore size range of slip flow zone and transitional flow zone expands. When the reservoir pressure is higher than the critical desorption pressure, the adsorbed gas is not desorbed intensively, and the produced gas is mainly free gas. When the reservoir pressure is lower than the critical desorption pressure, the adsorbed gas is gradually desorbed, and the proportion of desorbed gas in the produced gas gradually increases.

A Simple Method for Growth of Candida albicans Biofilms Under Continuous Media Flow and for Recovery of Biofilm Dispersed Cells.

Majority of nosocomial infections are associated with biofilms growing on indwelling medical devices. These biofilms are nourished by a continuous flow of body fluids and subjected to shear stress forces. Cells dispersed from C. albicans biofilms are highly virulent and developmentally distinct from their parent biofilms. To study biofilm dispersed cells, it becomes imperative to isolate newly dispersed cells using a flow biofilm model. In this chapter, we detail the methods underlying assembly and workings of a simple flow biofilm model using materials commonly available in most microbiological laboratories. Biofilms developed using this system are robust and particularly suitable for studies requiring large amounts of biofilm (dispersed) cells for downstream analyses. Importantly, this apparatus mimics in vivo flow conditions, thereby making it a physiologically relevant model.