Medium Exchange Research Articles

Optimization of VSV-ΔG-spike production process with the Ambr15 system for a SARS-COV-2 vaccine.

To face the coronavirus disease 2019 pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) virus, our institute has developed the rVSV‐ΔG‐spike vaccine, in which the glycoprotein of vesicular stomatitis virus (VSV) was replaced by the spike protein of SARS‐CoV‐2. Many process parameters can influence production yield. To maximize virus vaccine yield, each parameter should be tested independently and in combination with others. Here, we report the optimization of the production of the VSV‐ΔG‐spike vaccine in Vero cells using the Ambr15 system. This system facilitates high‐throughput screening of process parameters, as it contains 24 individually controlled, single‐use stirred‐tank minireactors. During optimization, critical parameters were tested. Those parameters included: cell densities; the multiplicity of infection; virus production temperature; medium addition and medium exchange; and supplementation of glucose in the virus production step. Virus production temperature, medium addition, and medium exchange were all found to significantly influence the yield. The optimized parameters were tested in the BioBLU 5p bioreactors production process and those that were found to contribute to the vaccine yield were integrated into the final process. The findings of this study demonstrate that an Ambr15 system is an effective tool for bioprocess optimization of vaccine production using macrocarriers and that the combination of production temperature, rate of medium addition, and medium exchange significantly improved virus yield.

Numerical and experimental simulation of a dynamic-rotational 3D cell culture for stratified living tissue models

Human tissues and organs are inherently heterogeneous, and their functionality is determined by the interplay between different cell types, their secondary architecture, and gradients of signalling molecules and metabolites. To mimic the dynamics of native tissues, perfusion bioreactors and microfluidic devices are widely used in tissue engineering (TE) applications for enhancing cell culture viability in the core of 3D constructs. Still, most in vitro screening methods for compound efficacy and toxicity assessment include cell or tissue exposure to constant and homogeneous compound concentrations over a defined testing period. Moreover, a prevalent issue inhibiting the large-scale adoption of microfluidics and bioreactor is the tubing dependence to induce a perfusion regime. Here, we propose a compartmentalized rotational (CR) 3D cell culture platform for a stable control over gradient tissue culture conditions. Using the CR bioreactor, adjacent lanes of constructs are patterned by controlled flow dynamics to enable tissue stratification. Numerical and experimental simulations demonstrate cell seeding dynamics, as well as culture media rotational perfusion and gradient formations. Additionally, the developed system induces vertical and horizontal rotations, which increase medium exchange and homogeneous construct maturation, allowing both perfused tubing-based and tubing-free approaches. As a proof-of-concept, experiments and accompanying simulation of cellular inoculation and growth in 3D scaffold and hydrogel were performed, before the examination of a blood-brain-barrier model, demonstrating the impact of a heterotypic culture on molecular permeability under mimetic dynamic conditions. Briefly, the present work discloses the simulation of 3D dynamic cultures, and a semi-automated platform for heterotypic tissues in vitro modelling, for broad TE and drug discovery/screening applications.

A cloud-based buyer-seller watermarking protocol (CB-BSWP) using semi-trusted third party for copy deterrence and privacy preserving.

Nowadays, cloud computing provides a platform infrastructure for the secure dealing of digital data, but privacy and copy control are the two important issues in it over a network. Cloud data is available to the end user and requires enormous security and privacy techniques to protect the data. Moreover, the access control mechanism with encryption-based technique protects the digital rights for participants in a transaction, but they do not protect the media from being illegally redistributed and do not restrict an authorized user to reveal their secret information this is referred to as you can access but you cannot leak. This brought out a need for controlling copy deterrence and preserving the privacy of digital media over the internet. To overlook this, we proposed a cloud-based buyer-seller watermarking protocol (CB-BSWP) with the use of a semi-trusted third party for copy deterrence and privacy-preserving in the cloud environment. The suggested scheme uses 1) a privacy homomorphism cryptosystem with Diffie-Hellman key exchange algorithm to provide an encrypted domain for the secure exchange of digital media 2) adopt robust and fair watermarking techniques to ensure high imperceptibility and robustness for the watermarked images against attacks 3) two services of cloud Infrastructure as a service (IaaS) to support virtualized computing infrastructure and Watermarking as a service (WaaS) to execute the speedy process of watermarking, this process is supported by watermarking generation and signing phase (WGSP) and watermark extraction and verifying phase reported in 4th section. 4) cloud service provider (CSP) considered as a “semi-trusted” third party to reduce the burden from the trusted third party (TTP) server and provide storage for the encrypted digital media on cloud databases, this frees content owner from not having a separate storage infrastructure. The proposed scheme encrypts the digital content by using SHA-512 algorithm with key size 512-bits to ensure that it doesn’t affect computational time during the process of encryption. The suggested scheme addresses the problems of piracy tracing, anonymity, tamper resistance, non-framing, customer rights problem. The role of cloud is crucial because it reduces communication overhead, provides unlimited storage, supports the watermarking process and offers a solution for the secure distribution of end-to-end security of digital content over cloud. To check the performance of the suggested CB-BSWP protocol against common image processing attacks, we have conducted experiments in which the perceptual quality of watermarked digital media was found enhanced, resulting in a robust watermark.

Mapping Bacterial Biofilm on Features of Orthopedic Implants In Vitro

Implant-associated infection is a major complication of orthopedic surgery. One of the most common organisms identified in periprosthetic joint infections is Staphylococcus aureus, a biofilm-forming pathogen. Orthopedic implants are composed of a variety of materials, such as titanium, polyethylene and stainless steel, which are at risk for colonization by bacterial biofilms. Little is known about how larger surface features of orthopedic hardware (such as ridges, holes, edges, etc.) influence biofilm formation and attachment. To study how biofilms might form on actual components, we submerged multiple orthopedic implants of various shapes, sizes, roughness and material type in brain heart infusion broth inoculated with Staphylococcus aureus SAP231, a bioluminescent USA300 strain. Implants were incubated for 72 h with daily media exchanges. After incubation, implants were imaged using an in vitro imaging system (IVIS) and the metabolic signal produced by biofilms was quantified by image analysis. Scanning electron microscopy was then used to image different areas of the implants to complement the IVIS imaging. Rough surfaces had the greatest luminescence compared to edges or smooth surfaces on a single implant and across all implants when the images were merged. The luminescence of edges was also significantly greater than smooth surfaces. These data suggest implant roughness, as well as large-scale surface features, may be at greater risk of biofilm colonization.

High-Titer Hepatitis C Virus Production in a Scalable Single-Use High Cell Density Bioreactor.

Hepatitis C virus (HCV) infections pose a major public health burden due to high chronicity rates and associated morbidity and mortality. A vaccine protecting against chronic infection is not available but would be important for global control of HCV infections. In this study, cell culture-based HCV production was established in a packed-bed bioreactor (CelCradle™) aiming to further the development of an inactivated whole virus vaccine and to facilitate virological and immunological studies requiring large quantities of virus particles. HCV was produced in human hepatoma-derived Huh7.5 cells maintained in serum-free medium on days of virus harvesting. Highest virus yields were obtained when the culture was maintained with two medium exchanges per day. However, increasing the total number of cells in the culture vessel negatively impacted infectivity titers. Peak infectivity titers of up to 7.2 log10 focus forming units (FFU)/mL, accumulated virus yields of up to 5.9 × 1010 FFU, and a cell specific virus yield of up to 41 FFU/cell were obtained from one CelCradle™. CelCradle™-derived and T flask-derived virus had similar characteristics regarding neutralization sensitivity and buoyant density. This packed-bed tide-motion system is available with larger vessels and may thus be a promising platform for large-scale HCV production.

Medium and long-term electricity market trading strategy considering renewable portfolio standard in the transitional period of electricity market reform in Jiangsu, China

The Energy Consumption Guarantee Mechanism (ECGM) of renewable energy requires the prior consumption ratio of renewable energy, and regulates the proportion of renewable energy power in each province. The implementation of Renewable Portfolio Standard (RPS) will have a great impact on the energy portfolio management and trading strategy of electricity in the medium and long-term electricity market. Grid companies (or power exchange center), as the organizers of the medium and long-term market and the primary entities responsible for renewable energy consumption, need a set of scientific trading strategies for the overall scheduling of inter- and intra-provincial power exchange arrangements. Firstly, based on the medium and long-term power market trading rules of Jiangsu, China, this paper analyzes the risk of uncertainty in the power output of renewable energy, and introduces the optimization model into the cost of risk to schedule the consumption ratio of renewable energy in the province, namely arranging the amount of renewable and non-renewable energy electricity as well as that of electricity outside the province. Secondly, the risk levels and their response are determined, and the electricity arrangements inside and outside the province in the medium and long-term market are updated based on a time window rolling method and the proposed mechanism. Finally, illustrative cases are performed to verify the effectiveness of the model and to provide a reference for the organizers' decision-making of medium and long-term power exchange.

Technology Impact in Modern Political Communications: Psychological Grounds

The author writes about the manipulative technologies used in political communications and an attempt to interpret through the prism of psychological and anthropological concepts (cognitive psychology, evolutionary psychology). The article discusses such political technologies as “Wait Zone”, “Victimisational Snow”, “semantic differentiation”, “Fear trade”, “false theatricalisation”, “red herring”, and “Creating a victim”. Political technologists analyse them quite superficially, not trying to understand the deep psychological foundations of their effectiveness, so an attempt is made to understand this issue with support for social, cognitive, and evolutionary psychology. The author does not claim that his explanations will be exhaustive, but it suggests that they can shed light on the particular effectiveness of media exchange techniques. This issue is particularly relevant in connection with the thesis of many specialists that humanity has entered a new media manipulation era — The post-truth era. It seems that this determines the special relevance of this article.

Feed Digestion, Growth and Disease Prevalence in Nile Tilapia (Oreochromis niloticus) Cultured at Different Water Exchange Rates in a Recirculating Aquaculture System

The quality of culture water and the rate of water flow within a culture system can affect fish growth and health. In this study, the effects of different water flow rates on digestion efficiency, growth and welfare of juvenile Nile tilapia (Oreochromis niloticus) (Initial weight: 25.75±0.47 g) were investigated. Fish (15 per tank) were randomly stocked into twelve 150 L thermoplastic tanks of a recirculating aquaculture system and subjected in quadruplicate groups to water flow rates of 50 Lh-1 (Low exchange; LE), 100 Lh-1 (Medium exchange; ME) and 150 Lh-1 (High exchange; HE) for 72 days. Water quality parameters including total ammonia nitrogen, nitrate and alkalinity, dry matter, protein and lipid digestibilities, somatic growth and some welfare indicators including haematology and disease prevelance were monitored. There were no significant differences in growth among the treatments. Higher prevalence of cataract, fin erosion, oral and dermal lesions were observed in the LE and ME treatments indicating compromised welfare. There were no statistical differences among the treatments in terms of nutrient digestibility. This study has shown that culturing Nile tilapia under moderately high water flow rates of at least 100% tank water replacement per hour in RAS ensures good growth and feed utilization.

A reconfigurable microfluidic building block platform for high-throughput nonhormonal contraceptive screening.

Identifying nonhormonal contraceptives will have profound impacts on avoiding side effects of hormonal birth control methods, minimizing pregnancy complications and infant mortality rates, and promoting family planning. However, phenotypic screening of contraceptives is challenging due to the diverse procedures associated with oocyte culture, biochemical assays, and molecular imaging. This study reports a multifunctional microfluidic platform comprising reconfigurable building blocks and interfaces to implement various cell-based drug screening protocols. This versatile platform has three major layers. The top layer consists of interchangeable 3D microfluidic building blocks (e.g., branching microchannels, chemical gradient generators, pumpless flow controllers, and emulsion generators) or an open interface. The middle layer incorporates a multiwell array with embedded membrane filters for live cell culture, medium exchange, enzymatic cumulus cell removal, washing, and fluorescence staining. The bottom layer is also reconfigurable for waste collection, oocyte culture, plate reader measurement, and high-resolution microscopy. We demonstrate an 8 by 16 (128 wells) system for performing the cumulus-oocyte complex (COC) expansion and oocyte maturation assays for screening nonhormonal contraceptives. The microfluidic building block platform is scalable and can be reconfigured for a variety of drug screening applications in the future.

Induction of Human Natural Killer Cells Under Defined Conditions by Seamless Transition from Maintenance Culture of Pluripotent Stem Cells.

The use of pluripotent stem cells (PSCs) as a source of natural killer cells (NK cells) can improve reproducibility in the analysis of the pathogenesis of NK cell-associated diseases and in the production of off-the-shelf cellular medicines. We have developed a method for the differentiation of NK cells from human PSCs under serum-free and two-dimensional condition. Our method enables the seamless transition from maintenance of PSCs to differentiation of NK cells, without the use of any techniques other than medium exchange and whole culture passage.

Analysis of influence factors of casing heat transfer in middle-deep geothermal wells in severe cold region

The medium and deep coaxial casing heat exchange technology uses the fluid circulation inside the casing to improve the deep geothermal energy. Due to the high outlet temperature at the source side， it is a good choice for the heat source of buildings in severe cold areas in winter. We establish a two-dimensional unsteady numerical heat transfer model of fluid-thermal-structure coupling based on standard <inline-formula><mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M1"><mml:mi>κ</mml:mi></mml:math></inline-formula>-<inline-formula><mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M2"><mml:mi>ε</mml:mi></mml:math></inline-formula> model and Simple pressure velocity solution method by analyzing the heat transfer process between coaxial casing and rock mass at source side. According to the climate and stratum characteristics of Changchun City， we analyze the heat transfer performance of heat exchanger and rock and soil mass in full heating cycle by simulation. The results show that due to the cold winter in Changchun City， during the operation of the heat pump， the shallow formation temperature is lower than the fluid temperature at the inlet of the heat exchanger， and the rock and soil within 3.9 m below the ground is a heat source funnel， which gradually changes into a heat sink funnel with the increase of formation depth. In the first week of operation of the heat pump， the outlet temperature and heat exchange of the heat exchanger decrease rapidly and tend to be stable after one month of operation. In the whole operation cycle， the unit time decline of the outlet temperature of the buried pipe becomes smaller and smaller， and the relationship between the change of the outlet temperature and time decreases in the form of a power function with a slow speed. With the increase of thermal conductivity of cementing cement， the heat exchange first increases， and tends to be stable when the thermal conductivity of cementing cement is greater than that of rock and soil. The thermal conductivity of the inner tube increases， the thermal short circuit is serious， and the outlet water temperature and heat exchange of the heat exchanger decrease. When the heat pump unit 24 hours on and off time （start-stop ratio） is 16 h∶8 h， 12 h∶12 h， 8 h∶16 h， respectively， the heat exchanger outlet temperature and heat transfer are irregular， but the overall trend is gradually reduced. The longer the heat pump stops running， the better the temperature recovery of rock and soil body on the source side， and the greater the water outlet temperature and heat transfer when it is turned on again. Through the numerical model， the variation law of influence factors of casing heat exchanger can be obtained. <fig fig-type="abstract-image" id="F1" orientation="portrait" position="float"><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="SP.J.1249-2022-39-1-42/D20AE2D9-DE77-4b51-BEC5-8F4EB83A9A4A-F001.jpg"/></fig>

Effects of light wavelength exposure during in vitro blastocyst production on preimplantation development of mouse embryos.

Despite the absence of light within the body, the application of microscopy during stages of in vitro embryo production has led to the discovery of light irradiation effects on embryo preimplantation development. To determine the optimal light irradiation wavelengths at various embryo stages for improving the preimplantation development of mouse embryos and the quality (total cell number) of blastocysts. All in vitro procedures of zygote or 2-cell embryo manipulation, embryo monitoring, and culture medium exchange were conducted under visible (390-750nm), blue (445-500nm), green (500-575nm), yellow (575-585nm), or red (620-750nm) light irradiation wavelength. We found that blue, green, and yellow light irradiation during in vitro blastocyst production from zygotes significantly improved blastocyst production and quality, compared to visible and red light irradiation. However, 2-cell embryos exposed to yellow light during in vitro blastocyst production produced significantly more high-quality blastocysts than did 2-cell embryos exposed to visible, blue, green, or red light. After exposure to blue and green - but not yellow - light during in vitro zygote manipulation, yellow light irradiation during embryo monitoring and culture medium exchange triggered significant retardation of preimplantation development. These results demonstrate that yellow light irradiation during in vitro blastocyst production, regardless of embryo stage, improves preimplantation development of mouse embryos. The present study will contribute to produce greater high-quality blastocysts and reduce experimental errors generated by light exposure during mouse embryo-related studies.

A Cerebral Organoid Connectivity Apparatus to Model Neuronal Tract Circuitry.

Mental disorders have high prevalence, but the efficacy of existing therapeutics is limited, in part, because the pathogenic mechanisms remain enigmatic. Current models of neural circuitry include animal models and post-mortem brain tissue, which have allowed enormous progress in understanding the pathophysiology of mental disorders. However, these models limit the ability to assess the functional alterations in short-range and long-range network connectivity between brain regions that are implicated in many mental disorders, e.g., schizophrenia and autism spectrum disorders. This work addresses these limitations by developing an in vitro model of the human brain that models the in vivo cerebral tract environment. In this study, microfabrication and stem cell differentiation techniques were combined to develop an in vitro cerebral tract model that anchors human induced pluripotent stem cell-derived cerebral organoids (COs) and provides a scaffold to promote the formation of a functional connecting neuronal tract. Two designs of a Cerebral Organoid Connectivity Apparatus (COCA) were fabricated using SU-8 photoresist. The first design contains a series of spikes which anchor the CO to the COCA (spiked design), whereas the second design contains flat supporting structures with open holes in a grid pattern to anchor the organoids (grid design); both designs allow effective media exchange. Morphological and functional analyses reveal the expression of key neuronal markers as well as functional activity and signal propagation along cerebral tracts connecting CO pairs. The reported in vitro models enable the investigation of critical neural circuitry involved in neurodevelopmental processes and has the potential to help devise personalized and targeted therapeutic strategies.

Detection of Galactose and Glycerophospholipids by Galactose Biosensors and Advancement Their Proficiency Using Berkelium Colloidal Nanoparticles and Poly(3,4-Ethylenedioxythiophene)-Poly(Styrenesulfonate)-Based Biosensors

In the current paper, galactose-oxidase enzyme is used as stabilization medium due to its more proficiency, ability for more accurate controlling the enzyme reaction, protecting against wasting of enzyme as well as simple and easy use and exchange of enzyme medium after performing some levels of surface modification and developing multi-walled carbon nanotubes (MWCNTs) on Berkelium plate. For better connecting and stabilizing the enzyme on the medium, the prepared medium is washed by high concentration sulfuric acid and nitric acid and a large volume of deionized water and for protecting enzyme from devastating effect of Berkelium and prohibiting them to become inactive, surface is covered with cystamine before stabilization. Regarding the large size of galactose-oxidase enzyme compared to surface of medium, a connective material with amid at one end and pyrine at the other end is used as transfer agent and for stabilizing this connection, the prepared medium is placed into dimethylformamide (DMF) solution for a couple of hours. Activity of stabilized enzyme at 460 (nm) wavelength recorded by spectroscope was depicted against time to evaluate its stability in various times. The prepared medium, which have a large amount of galactose-oxidase enzyme, can be used as electrode in sensors. Furthermore, galactose-oxidase electrochemical sensor is one of the best methods for detecting low amount of galactose and applying Berkelium colloidal nanoparticles as a supplementary material in the structure of biosensor can be effective for advancement its proficiency and optimum proficiency. On the other hand, in the current study, electrode biosensor entitled as modified carbon paste electrode with Berkelium colloidal nanoparticles (Bknano/CPE) is produced by carbon graphite powder, paraffin oil and Berkelium colloidal nanoparticles (24 nm) and it is compared with carbon paste electrode (CP). In semi-permeable membranes, a combination of 1 (ml) of 0.1 (M) phosphate buffer with specified pH and 10 (mg) of galactose-oxidase enzyme is placed around each electrode. In the same potential of 0.7 (V), biosensors are tested with galactose in concentration range of (0-1) (mM) and various amounts of pH (4,6,8) which lead to producing the maximum current and tracing galactose in pH=6 and concentration of 1 (M) as the optimum condition. Currentmetry induced from both biosensors are compared and it is confirmed that using Berkelium colloidal nanoparticles in the structure of (Bknano/CPE) electrode leads to increasing the conductivity and currentmerty of biosensor. In addition, qualitative and quantitative measurement of food components is of great importance due to high cost of traditional methods, in addition to tendency for more accurate and sensitive detecting of these components. galactose and glycerophospholipids are such compounds that they frequently measure. Various methods are used to detect these food elements. However, the necessity for accurate measurement of these two compounds with high sensitivity, especially for food health issue, leads to developing biological methods, especially biosensors. Among them, biosensors based on conductive polymer nanostructures, especially Poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate), have been recently interested due to their unique characteristics. The current paper aims to introduce and investigate the previously performed studies about Poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate)-based biosensors for detecting galactose and glycerophospholipids.

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Abstract 13067: Recapitulating Long QT Syndrome Phenotype in Induced Pluripotent Stem Cells-Derived Cardiomyocytes Using CRISPR/Cas9 Technology

Introduction: Long QT syndrome (LQTS) is an inherited arrhythmia syndrome with a hallmark of QT prolongation secondary to delayed repolarization of cardiomyocytes (CM) on 12-lead ECG. Hypothesis: Studying LQTS in an induced pluripotent stem cell (iPSC)-derived CM model allows for patient-specific phenotype characterization in vitro . We report such a strategy (fig. A) in a patient with LQT type 3 (ECG QTc 503 msec) who harbored a heterozygous pathogenic variant in SCN5A (c.1231G&gt;A), and in a healthy control for comparison (ECG QTc 388 msec). Methods: IPSCs were generated from patient and control dermal fibroblasts using a non-integrational Sendai reprogramming method. The patient’s SCN5A (c.1231G&gt;A) variant was edited into the healthy control using CRISPR/Cas9 technology. CM were subsequently differentiated and beating CM clusters were seeded onto Maestro CytoView multi-electrode array plates. Serial corrected field potential duration (FPD) measurements (n=12), equivalent to QTc values on ECG, were recorded on days 24 to 39 of CM differentiation per sample. CM were exposed to varying concentrations of Nifedipine and Sotalol through cell culture media exchange. Paired t-tests (non-parametric) were utilized for p&lt;0.05. Results: Reprogrammed iPSCs displayed pluripotency characteristics (fig.B). IPSC-derived CMs had confirmed cardiac lineage (fig. C). Prolonged FPD values, corrected for beating rate, were observed in both the LQT3 patient-derived iPSC-CMs (p&lt;0.0001) and the genetically induced LQT3 iPSC-CMs (p&lt;0.0001) compared to control (fig. D &amp; E). Nifedipine and sotalol altered FPD in a dose-dependent response (fig. F &amp; G). Conclusions: An iPSC-derived CM model reliably recapitulates the phenotype of LQTS. Induced LQT3 iPSC-CMs genetically edited from a healthy control accurately recapitulate the LQT3 disease phenotype. Drug effects on CM repolarization were demonstrable in the iPSC-CM model.

Clearance of oxytocin and its potentially enzyme resistant analogues in the OXT-receptor compartment of the potassium depolarized rat myometrium.

The time–response behaviour of a group of oxytocin analogues structurally modified on potential sites of oxytocin splitting by tissue inactivation enzymes (“enzyme probes”) was investigated ex vivo on the potassium depolarized rat myometrium (at 30°C) and compared with the data obtained in the in vivo experiments. The modified oil‐immersion method by Kalsner and Nickerson was used to record time profiles after cessation of a steady state myometrium contraction triggered by analogues in a high potassium tissue medium. An exchange of the aqueous medium for mineral oil enables to suppress return diffusion of the peptide and to record its irreversible clearance near the corresponding receptor compartment. Response records were analysed by a nonlinear numeric procedure based on combination of steady state and kinetic terms that allows concomitant estimations of affinities from time–response measurements, in the given case for analogues on depolarized myometrium. Potential inactivation‐sensitive sites in the oxytocin chain are the Ν‐terminal peptide bond Cys1‐Tyr2 (aminopeptidase splitting), the intramolecular disulphide bridge (reduction and formation of the practically inactive linear peptide) and the C‐terminal Leu8‐GlyNH2 9 or the Pro7‐Leu8 (postprolin cleaving enzyme) bond, respectively. Clearance rate constants of single peptides in the OXT‐receptor compartment were in an interval of 0.025 to 0.28 min−1. The fragment contribution analysis reveals a significant linear additivity of individual structural changes and thus a predictivity of irreversible inactivation rate in the receptor compartment. The most potent inactivation of oxytocin is associated with aminopeptidase splitting; other enzymes may play some though nondecisive role. Less significant differences within the peptide group were found for rate constants for peptide transport between receptor compartment and its external aqueous medium. Besides rate constants, the evaluation of time–response data yields affinity values of the tested peptides and indicates a 25‐times desensitation of depolarized compared with a native state.

A microfluidic chip carrier including temperature control and perfusion system for long-term cell imaging

Microfluidic devices are widely used for biomedical applications but there is still a lack of affordable, reliable and user-friendly systems for transferring microfluidic chips from an incubator to a microscope while maintaining physiological conditions when performing microscopy. The presented carrier represents a cost-effective option for sustaining environmental conditions of microfluidic chips in combination with minimizing the device manipulation required for reagent injection, media exchange or sample collection. The carrier, which has the outer dimension of a standard well plate size, contains an integrated perfusion system that can recirculate the media using piezo pumps, operated in either continuous or intermittent modes (50–1000 µl/min). Furthermore, a film resistive heater made from 37 µm-thick copper wires, including temperature feedback control, was used to maintain the microfluidic chip temperature at 37 °C when outside the incubator. The heater characterisation showed a uniform temperature distribution along the chip channel for perfusion flow rates up to 10 µl/min. To demonstrate the feasibility of our platform for long term cell culture monitoring, mouse brain endothelial cells (bEnd.3) were repeatedly monitored for a period of 10 days, demonstrating a system with both the versatility and the potential for long imaging in microphysiological system cell cultures.

P-076: Evaluating the changes treatment elicit on the immune repertoire of the myeloma niche using patient-derived multiple myeloma organoid models.

<h3>Background</h3> Multiple myeloma (MM) cells have an intricate interaction with immune components of the bone marrow (BM) niche that are key for survival and drug resistance. Evaluating the immune system in ex-vivo models has been a challenge that is being partially overcome by xenografts or 3D cultures that replicate the tumor niche. The purpose of this study is to use an established 3D myeloma organoid platform to evaluate changes in cellular immunity within a tumor niche caused by different therapeutic agents using patient-derived BM aspirate samples. <h3>Methods</h3> MM patients scheduled for BM biopsy were consented and 3-5 ml of aspirate was collected. Mononuclear cells from BM aspirates were co-cultured and suspended in Matrigel. Individual 40-50μm 3D organoids were placed in each well of a 48-well plate. Partial exchange of media containing RPMI, GM-CSF, IL6, IL7, and EGF (cocktail #3) was performed every 2-3 days. Organoids from the same patient were exposed to different drugs on day 5 for 48 hours and the following tests performed at different time points: viability with ATP, immunohistochemistry, flow-cytometry, chemosensitivity assays, AlamarBlue, RayBio, and cell sequencing (Proteona). The primary objective was to maintain viability of the 3D organoid components long enough to study the immune components in the MM tumor niche over an extended period of time. Secondary objective was to evaluate the impact therapies may have on the immune cell function within the tumor niche to identify targetable mechanisms of resistance/sensitization. <h3>Results</h3> Patient-derived BM aspirate samples were collected and used for this study. Number of organoids made and conditions tested varied based on sample quality and quantity. Organoid lifespan using Matrigel and partial exchange of cocktail #3 allowed for heterogeneous and adequate viability for up to 21 days as seen by flow and histology. Expansion of immune cells was seen during the course of the experiments with variability based on the conditions exposed to (see graph attached). When analyzing for chemosensitivity, there was not a significant difference seen between some agents likely due to the low drug concentrations used. Despite of that, cytokines on supernatant and gene expression profiling at different time points showed distinct changes in immune cell activity from the same patient when exposed to different conditions (see image). These findings suggest off-tumor targets drugs may have on the tumor niche. <h3>Conclusion</h3> This platform maintained viability of MM cells and its stroma for up to 21 days with good representation of its immune compartment. Immune cells such as dendritic cells, NK cells, and T-cell showed changes in activity and gene expression after exposure to different therapeutic agents using this organoid model. Further studies to evaluate how to optimize the activity of the immune cells within the MM tumor niche and better understand the impact current therapies have are needed.

A Sessile Drop Method for Facile and Robust Spheroid Cultures (Adv. Mater. Interfaces 20/2021)

A Sessile Drop Method for 3D Cell Spheroid Culture In article number 2100972, Bangyong Sun, Gang Li, and co-workers demonstrate a sessile drop platform for long-term spheroid culture. Benefitting from its open, upward, and non-wettable features, the sessile drop platform provides not only high mechanical stability and easiness of medium exchange but also excellent antifouling ability and high oxygen availability, thus enabling a robust long-term spheroid culture in a high-throughput manner.