Medium Exchange Research Articles

Development of an end-to-end platform for the controlled, closed, scalable and cost-effective manufacture of allogeneic therapies derived from pluripotent stem cells in aggregate-based cultures

Background & Aim Pluripotent stem cell (PSC)-derived therapies have the potential to serve as “off-the-shelf” allogenic treatments for a wide range of chronic indications including cardiovascular and neurodegenerative diseases. These therapies target large patient populations and require high doses of ≥109 cells making them unsuited to traditional 2D culture-ware that are manual, open and poorly scalable technologies with limited in-process monitoring and increased risk and cost of batch failure. The commercial success of allogeneic PSC-derived therapies relies on the design and development of controlled, robust and cost-efficient processing platforms for large-scale manufacture. Methods, Results & Conclusion Here we present the development of a closed, scalable, automated and affordable upstream process for the expansion of PSCs in high-density, aggregate based culture in stirred tank reactors (STR). Production of PSCs at scale requires a seed train to generate the numbers needed to seed a bioreactor. This step is typically open, manual and labour intensive and at high risk for contamination and concomitant batch failure. We show this process step can be closed and automated effectively using a hollow fiber bioreactor system, able to produce up to 2 × 109 cells. Resulting cells were single seeded onto the STR and expanded using periodic settling to allow for automated medium exchange. An effective 3D-aggregate expansion was achieved with a 12-fold cell expansion over six days, with retention of pluripotency markers and differentiation potential. However, we found periodic settling reduces the ability to control aggregate size- a critical parameter that impacts expansion and differentiation. To overcome this, we investigated an acoustic perfusion system to enable STR process intensification with controlled and automated media exchange. We show cell retention ability (91.5±2.5%), increased control on aggregate size and maintenance of pluripotency markers. We also show the ability of the resulting aggregates to differentiate into the three germ layers. In parallel, different impeller speeds were screened at a 1500-mL scale to identify a suitable scaling factor between stirred tank reactors for the baseline process developed at 130-mL. Finally, integrated technologies were used for process concentration and wash. We, at Cell and Gene Therapy Catapult, have developed a closed, scalable and automated process for the expansion of PSCs up to 1500-mL scale.

Coating strategies for expansion of mesenchymal stromal cells in a hollow-fibre bioreactor

Background & Aim Background The increasing request for culture expanded mesenchymal stromal cells (MSC) in the last years discloses the need to reduce costs and man power in the manufacturing process. This can be achieved by the use of closed, (semi-)automated bioreactor systems. We have decided to test a functionally closed hollow-fibre bioreactor system (Quantum®). Surveillance of metabolic parameters and adaptation of feeding in dependence of these is possible. However, the nature of the hollow-fibre surface does not support attachment of MSC without coating. Several biological substances for coating are available, either as isolated proteins (e.g. fibronectin, vitronectin), or as complex protein mixtures derived from human blood (e.g. cryoprecipitate, platelet lysate). The use of such closed systems additionally reduces the risk of contaminations. Methods, Results & Conclusion Methods MSC were cultured as conventional cell culture or in a platelet lysate (PL), cryoprecipitate (CP), vitronectin (VN), or fibronectin (FN) coated Quantum® bioreactor (TerumoBCT) for 5 - 15 days in alphaMEM (Lonza), supplemented with 5% - 10% platelet lysate (PL from IKT Ulm) and 1 i.u. heparin (B.Braun). The surface of the Quantum® bioreactor is 21,000 cm2. For Quantum®, the feed rate was adjusted based on daily measured lactate or glucose levels. For conventional cell culture, mainly 2 chamber CellSTACK® (Corning) with a surface of 1272 cm2 were used and complete medium exchange was performed twice a week. TrypZEAN® (Lonza) was used for detachment of cells. Results For results of the cell expansion experiments (N ≥ 3) using the different coating reagents refer to Table 1. Conclusion We showed that the Quantum® bioreactor is suitable for expansion of MSC using PL, CP, FN or VN as coating reagent. Despite higher doubling times for cells grown in the Quantum®, this system may help to save costs since less man power is needed to manage the system and several steps do not necessarily have to be performed in a class A cabinet. In addition, clinically relevant cell numbers could be achieved by the use of a single bioreactor (data not shown). Perspective Protocols for cell expansion in the Quantum® still may have to be optimized in order to increase its potential in saving man power and consumables.

Optimizing a micro-fluidic slide for in vivo imaging of larval Zebrafish.

Larval zebrafish (Danio rerio) are optically transparent at six- and seven-days post fertilization (dpf) which makes them useful research models for the study of metabolic and physiological processes. Previous methods to image zebrafish were limited to embryogenesis and early larval stages. Our goal was to design a novel micro-fluidic slide for mounting zebrafish that greatly extends the duration of time for in vivo imaging of zebrafish larvae that are six to seven days post fertilization (dpf). The ideal mounting slide will have channels to provide media exchange, nourishment, and/or drug treatments and channels to remove waste and flush the system of previous test conditions. We had a microfluidic slide manufactured to our specifications. Initial testing revealed that the larval bed was too large. To optimize our previous prototype, we used the Zeiss Discovery V8 microscope to recalculate our measurements for the larvae bed. We have now re-imaged five each of six dpf larvae and five each of seven dpf larvae in order to adjust the measurements to the new average larval size. This work was supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence and NIH Grant P20GM103434 awarded to Bluefield State College

The study of selective primary culture and determination of a breast cancer cell line in vitro*

Abstract Objective The successful establishment of a tumor cell bank is based on the premise that the target cells can be cultured by a legitimate approach. In this experiment, we used primary culture to select and detect breast cancer cells in vitro, which can provide experimental ideas and methods for the establishment of a living tumor tissue cell bank. Methods Fifty-two specimens were collected over a two-year period from people with breast cancer who needed surgical treatment in our hospital. Cells were isolated and used to establish successful cell culture. Cell activity and cell purity were measured before liquid nitrogen cryopreservation. Results (1) At the initial culture stage, cells grew with adherence. Cell multiplication could be seen after the cell medium was exchanged three times. Cell viability was above 86%, while the viability of the target cells was above 75%, as detected by hematoxylin and eosin (HE) staining. (2) The number of breast cancer cells decreased, while the number of fibroblasts increased after five rounds of passage. (3) The success rate was 73.08%, which did not include polluted cells and those that were not successfully cryopreserved. Conclusion (1) breast cancer cells could be selected from primary culture in vitro through an appropriate method. (2) Exchange of the cell medium and further cell passage improved cell multiplication. (3) The experimental results could be monitored using trypan blue and HE staining. (4) The success of breast cancer cell culture in vitro could be used as a reference for other cell culture, so as to establish a tumor tissue cell bank.

Effects of Mesenchymal Stem Cell Coculture on Human Lung Small Airway Epithelial Cells.

Mesenchymal stem cells (MSCs) and their secreted extracellular vesicles have been used effectively in different lung disease animal models and clinical trials. Their specific beneficial effects, the potential differences between MSCs derived from different organs, and interactions between MSC products and target cells still need to be studied further. Therefore, we investigated the effects of secreted products of human MSCs derived from the bone marrow and adipose tissue on human lung small airway epithelial (AE) cells in vitro. AE cells were cocultured with MSCs in inserts that allowed the free exchange of medium but did not allow direct cell-to-cell contact. We examined the effects on AE cell viability, proliferation, cell numbers, expression of AE cell-specific genes, and CD54 (intercellular adhesion molecule 1 (ICAM1)) surface positivity, as well as the secretion/uptake of growth factors relevant for AE cell. We found that coculture increased the viability of AE cells. The majority of AE cells expressed CD54 on their surface, but the percentage of cells being positive for CD54 did not increase in coculture. However, ICAM1 gene expression was increased in coculture. Also, we observed increased gene expression of mucin (MUC1), a lung-enriched cell surface glycoprotein. These observed effects were the same between bone marrow and adipose tissue MSCs. However, MSCs derived from adipose tissue reduced angiopoietin concentrations in coculture, whereas those from the bone marrow did not. Conclusively, MSCs influenced AE cells positively by increasing their viability and affecting gene expression, with some effects being specific for the tissue origin of MSCs.

Effect of culture condition on cell viability and gel contraction in a skin chip

The importance of physiologically realistic in vitro skin models in development of cosmetics and drugs for skin disorders have been well recognized, but current in vitro skin models carry several limitations in recapitulating the physiology of the human skin. Recently, in vitro skin models in a microfluidic platform have been proposed with improved physiological relevance. Survival and differentiation of skin tissue construct requires accurate refinement of cell culture environment, which can be different for static and dynamic culture conditions. In this study, we studied the effect of different culture conditions on the skin construct in a microfluidic skin chip. Our observation suggests that different seeding cell concentrations, thickness of the collagen matrix, volume of cell culture media and the frequency of media exchange affects the cell viability, extent of collagen gel contraction, and adherence of keratinocytes to the gel matrix. Our study provides valuable information about optimization of cell culture conditions in a microfluidic skin chip.

BCL‑3 promotes cyclooxygenase‑2/prostaglandinE2 signalling in colorectal cancer.

First discovered as an oncogene in leukaemia, recent reports highlight an emerging role for the proto‑oncogene BCL‑3 in solid tumours. Importantly, BCL‑3 expression is upregulated in >30% of colorectal cancer cases and is reported to be associated with a poor prognosis. However, the mechanism by which BCL‑3 regulates tumorigenesis in the large intestine is yet to be fully elucidated. In the present study, it was shown for the first time that knocking down BCL‑3 expression suppressed cyclooxygenase‑2 (COX‑2)/prostaglandinE2 (PGE2) signalling in colorectal cancer cells, a pathway known to drive several of the hallmarks of cancer. RNAi‑mediated suppression of BCL‑3 expression decreased COX‑2 expression in colorectal cancer cells both at the mRNA and protein level. This reduction in COX‑2 expression resulted in a significant and functional reduction (30‑50%) in the quantity of pro‑tumorigenic PGE2 produced by the cancer cells, as shown by enzyme linked immunoassays and medium exchange experiments. In addition, inhibition of BCL‑3 expression also significantly suppressed cytokine‑induced (TNF‑α or IL‑1β) COX‑2 expression. Taken together, the results of the present study identified a novel role for BCL‑3 in colorectal cancer and suggested that expression of BCL‑3 may be a key determinant in the COX‑2‑meditated response to inflammatory cytokines in colorectal tumour cells. These results suggest that targeting BCL‑3 to suppress PGE2 synthesis may represent an alternative or complementary approach to using non‑steroidal anti‑inflammatory drugs [(NSAIDs), which inhibit cyclooxygenase activity and suppress the conversion of arachidonic acid to prostaglandin], for prevention and/or recurrence in PGE2‑driven tumorigenesis.

Selection of human induced pluripotent stem cells lines optimization of cardiomyocytes differentiation in an integrated suspension microcarrier bioreactor

BackgroundThe production of large quantities of cardiomyocyte is essential for the needs of cellular therapies. This study describes the selection of a human-induced pluripotent cell (hiPSC) line suitable for production of cardiomyocytes in a fully integrated bioprocess of stem cell expansion and differentiation in microcarrier stirred tank reactor.MethodsFive hiPSC lines were evaluated first for their cardiac differentiation efficiency in monolayer cultures followed by their expansion and differentiation compatibility in microcarrier (MC) cultures under continuous stirring conditions.ResultsThree cell lines were highly cardiogenic but only one (FR202) of them was successfully expanded on continuous stirring MC cultures. FR202 was thus selected for cardiac differentiation in a 22-day integrated bioprocess under continuous stirring in a stirred tank bioreactor. In summary, we integrated a MC-based hiPSC expansion (phase 1), CHIR99021-induced cardiomyocyte differentiation step (phase 2), purification using the lactate-based treatment (phase 3) and cell recovery step (phase 4) into one process in one bioreactor, under restricted oxygen control (< 30% DO) and continuous stirring with periodic batch-type media exchanges. High density of undifferentiated hiPSC (2 ± 0.4 × 106 cells/mL) was achieved in the expansion phase. By controlling the stirring speed and DO levels in the bioreactor cultures, 7.36 ± 1.2 × 106 cells/mL cardiomyocytes with > 80% Troponin T were generated in the CHIR99021-induced differentiation phase. By adding lactate in glucose-free purification media, the purity of cardiomyocytes was enhanced (> 90% Troponin T), with minor cell loss as indicated by the increase in sub-G1 phase and the decrease of aggregate sizes. Lastly, we found that the recovery period is important for generating purer and functional cardiomyocytes (> 96% Troponin T). Three independent runs in a 300-ml working volume confirmed the robustness of this process.ConclusionA streamlined and controllable platform for large quantity manufacturing of pure functional atrial, ventricular and nodal cardiomyocytes on MCs in conventional-type stirred tank bioreactors was established, which can be further scaled up and translated to a good manufacturing practice-compliant production process, to fulfill the quantity requirements of the cellular therapeutic industry.

The pivotal role of micro-environmental cells in a human blood\u2013brain barrier in vitro model of cerebral ischemia: functional and transcriptomic analysis

BackgroundThe blood–brain barrier (BBB) is altered in several diseases of the central nervous system. For example, the breakdown of the BBB during cerebral ischemia in stroke or traumatic brain injury is a hallmark of the diseases’ progression. This functional damage is one key event which is attempted to be mimicked in in vitro models. Recent studies showed the pivotal role of micro-environmental cells such as astrocytes for this barrier damage in mouse stroke in vitro models. The aim of this study was to evaluate the role of micro-environmental cells for the functional, paracellular breakdown in a human BBB cerebral ischemia in vitro model accompanied by a transcriptional analysis.MethodsTranswell models with human brain endothelial cell line hCMEC/D3 in mono-culture or co-culture with human primary astrocytes and pericytes or rat glioma cell line C6 were subjected to oxygen/glucose deprivation (OGD). Changes of transendothelial electrical resistance (TEER) and FITC-dextran 4000 permeability were recorded as measures for paracellular tightness. In addition, qPCR and high-throughput qPCR Barrier chips were applied to investigate the changes of the mRNA expression of 38 relevant, expressed barrier targets (tight junctions, ABC-transporters) by different treatments.ResultsIn contrast to the mono-culture, the co-cultivation with human primary astrocytes/pericytes or glioma C6 cells resulted in a significantly increased paracellular permeability after 5 h OGD. This indicated the pivotal role of micro-environmental cells for BBB breakdown in the human model. Hierarchical cluster analysis of qPCR data revealed differently, but also commonly regulated clustered targets dependent on medium exchange, serum reduction, hydrocortisone addition and co-cultivations.ConclusionsThe co-cultivation with micro-environmental cells is necessary to achieve a functional breakdown of the BBB in the cerebral ischemia model within an in vivo relevant time window. Comprehensive studies by qPCR revealed that distinct expression clusters of barrier markers exist and that these are regulated by different treatments (even by growth medium change) indicating that controls for single cell culture manipulation steps are crucial to understand the observed effects properly.

Effect of CO2 injection on interfacial tension of oil-formation water system under high temperature and pressure

The interfacial interaction between CO2 and oil and water is one of the important factors affecting the effect of CO2 enhanced oil recovery. Taking dead oil in Zhoucheng Oilfield as an example, the variation of interfacial tension between crude oil-carbonated water two-phase system and oil-formation water two-phase system was studied by pendant drop method. The influence of different CO2 saturation on the equilibrium interfacial tension of oil-formation water system and changes of equilibrium interfacial tension between crude oil-carbonated water system and crude oil-formation water system were analyzed. Moreover, previous studies did not reflect the experimental phenomena of extracting light hydrocarbons from oil droplets by CO2 through gas-water interface. This paper provides experimental research and analysis for this experimental phenomenon. The results show that the equilibrium interfacial tension of crude oil-carbonated water system decreases with the increase of pressure, and the dissolution and diffusion of the system becomes more and more obvious with the increase of pressure. The medium exchange occurs between oil and carbonated water, and the phenomenon of partial miscibility occurs. There is a threshold pressure value between crude oil and formation water with different CO2 saturation. Each group of samples has its threshold pressure and optimum CO2 injection rate. It’s not that the more CO2 injection, the better oil displacement effect. This research provides a reference for us to save the cost of CO2 flooding.

A novel quantum steganography-Steganalysis system for audio signals

As a substitute for classical solutions, quantum information hiding techniques have become an essential issue in the field of quantum communications by utilizing the inherent features of quantum mechanics and creating more secured communications for the more reliable exchange of digital media within the context of quantum communications networks. Quantum steganography has been considered as one of these approaches in recent years, but the importance of investigating and discovering these hidden communications within the context of quantum communication networks that require the use of quantum steganalysis methods has not been addressed so far. Therefore, in this paper, a novel quantum steganography-steganalysis system for digital audio signals is proposed, which can accurately detect audio steganography methods in the context of quantum communication networks. The proposed model consists of two separate sections: steganography and steganalysis; in the steganography part, to minimize the impacts of the embedding process and increasing the Signal to Noise Ratio (SNR), the embedding operation is carried out within the Least Significant Fractional Qubit (LSFQ) of the amplitude information of the audio signal samples. Then, a universal steganalyzer in the steganalysis part distinguishes the stego audio signals using the extracted statistical features from the audio signals. The universal steganalyzer consists of a mean feature extraction module to extract features from the audio signal frames and the quantum circuits for implementing the K-Nearest Neighbor (KNN) algorithm and the Hamming distance criterion. The simulation-based quantum circuits of the proposed system tested and evaluated using different audio files. Over 80% accuracy in detecting stego audio signals indicates high accuracy and efficiency of the proposed scheme and its applicability in quantum communication networks. Along with the higher efficiency and security of quantum steganography methods when compared with the classical one, the results show that the proposed quantum steganography-steganalysis scheme is also capable of competing with classical methods in terms of accurately detecting steganography methods.

Sulfide-binding to Cu(II) in wine: Impact on oxygen consumption rates

This study investigates the relationship between the two main forms of Cu in wine and their impact on the rate of oxygen consumption. The Cu forms were differentiated by medium exchange constant current stripping potentiometry, which classified the Cu as either bound to sulfide or not. Oxygen consumption rates were determined in red, white and model wines after saturation with oxygen. The results for white wines showed that the oxygen consumption was sensitive to the non-sulfide-bound Cu concentration when ascorbic acid was present, and the first order rates ranged from 0.02 to 0.11 h−1. However, the same was not true for wines without added ascorbic acid that showed little influence of Cu form on oxygen decay rates. Cu forms were also found to significantly change in some wines during the oxygen decay experiment. Ascorbic acid is critical in enabling the form of Cu to significantly influence the oxygen reaction rate in wine.

Lipid Nanotubes and Double-Membrane Sheets Induced by Osmotic Deflation of Giant Unilamellar Vesicles Encapsulating Aqueous Two-Phase Solutions

Membraneless organelles and liquid-liquid condensates in cells are gaining increasing interest in the last years. Here, we mimic them using giant vesicles encapsulating aqueous two-phase polymer solutions. The vesicles undergo morphological transformations upon osmotic deflation, accompanied by phase separation of the encapsulated solution (Adv. Mater. Interfaces 4:1600451, 2017). The vesicles exhibit budding, inward nanotube and double-membrane sheet formation. The structures are confined to the liquid-liquid interface and have a typical thickness below conventional optical resolution (ACS Nano 10:463, 2016). We use super-resolution microscopy (2D and 3D STED) combined with a microfluidic technique (Lab Chip 19:626, 2019) to resolve their shape and dimension. The nanotubes can transform into double-membrane sheets reminiscent of endoplasmic reticulum cisternae. They are connected to the mother vesicle surface via a small membrane neck which allows medium exchange with the external environment. The tube-to-sheet transformation can either start from the highly curved end of a nanotube, or proceed via coalescence of the orifices of multiple nanotubes on the mother vesicle surface. The transformation process is prohibited when tubes intertwine into knots. The double-membrane sheets provide a more efficient way of storing excess membrane area compared to nanotubes. Understanding the coexistence of nanotubes and double-membrane sheets as in the endoplasmic reticulum and the tube-to-sheet transformation may help to shed light on the origin and evolution of similar structures in the cell. This work is part of the MaxSynBio consortium, jointly funded by the Federal Ministry of Education and Research of Germany and the Max Planck Society.

Micropatterned Adhesion Sites for Spheroid Cultivation Under Flow

The focus of cell based assays in recent years switched from 2D to 3D applications using spheroids and organoid cell assemblies due to the higher validity of the experimental outcome concerning in vivo relevance. However, spheroid generation, continues media exchange for good nutrients supply and high quality imaging over a long period of time is often very laborious. We use a perfusable channel system with micropatterned adhesion ligands on a highly passivated coverslip, which enables high resolution fluorescence microscopy, to generate numerous homogenously distributed spheroids in parallel within one channel. In this system spheroids can either be directly generated by simple injection of a large number of cells and a self-sorting process or by addition of preformed spheroids, which will bind to the patterned adhesion sites. Due to the immobilization of spheroids to the adhesion spots, long-term observation of the same spheroids can be performed with many spheroids in parallel. Perfusion of the spheroid chip at low flow rates not only enables the homogenous supply of all spheroids with nutrients, but also the application of a defined shear stress, metabolite analysis together with toxicological screenings or even co-culture of multiple spheroid types. With our micropatterned channel system we were able to show that by applying a flow the overall spheroid growth increases over time and spheroid size is very homogenous. By applying very high flow rates spheroids can be harvested from the surfaces, collected and further analyzed. The flow also influences the drug response of spheroids towards a more in vivo like reaction, showing that 3D spheroid cultivation under flow is the method of choice for generating relevant experimental results.

Technical aspects of quality assurance for intravitreal injections (IVI)

Successful quality assurance in intravitreal injection (IVI) of medications requires acomplex information technology infrastructure. The main challenges are data availability independent of location, standardization of clinical data, integration of extensive and currently non-standardized image documentation from coherence tomography and compliance with data protection regulations. In this article the technical implementation and data protection principles are reviewed. Essential aspects in the implementation of quality assurance in the field of IVI are discussed in asystematic approach. In the field of network architectures web-based applications supplemented by local virtual private networks (VPN) and/or other software instances have recently replaced the previously commonly used physical data medium exchange. The standardization of the data, e.g. by converting the visual acuity into logMAR, plays an important role in the collection of treatment data. Multiple non-standardized data formats in optical coherence tomography complicate the general quality assurance structure and comparability of data. International standards will probably facilitate this in the near future. Until then individual solutions have to be found on site.

Microfluidic perfusion modulates growth and motor neuron differentiation of stem cell aggregates.

Microfluidic technologies provide many advantages for studying differentiation of three-dimensional (3D) stem cell aggregates, including the ability to control the culture microenvironment, isolate individual aggregates for longitudinal tracking, and perform imaging-based assays. However, applying microfluidics to studying mechanisms of stem cell differentiation requires an understanding of how microfluidic culture conditions impact cell phenotypes. Conventional cell culture techniques cannot directly be applied to the microscale, as microscale culture varies from macroscale culture in multiple aspects. Therefore, the objective of this work was to explore key parameters in microfluidic culture of 3D stem cell aggregates and to understand how these parameters influence stem cell behavior and differentiation. These studies were done in the context of differentiation of embryonic stem cells (ESCs) to motor neurons (MNs). We assessed how media exchange frequency modulates the biochemical microenvironment, including availability of exogenous factors (e.g. nutrients, small molecule additives) and cell-secreted molecules, and thereby impacts differentiation. The results of these studies provide guidance on how key characteristics of 3D cell cultures can be considered when designing microfluidic culture parameters. We demonstrate that discontinuous perfusion is effective at supporting stem cell aggregate growth. We find that there is a balance between the frequency of media exchange, which is needed to ensure that cells are not nutrient-limited, and the need to allow accumulation of cell-secreted factors to promote differentiation. Finally, we show how microfluidic device geometries can influence transport of biomolecules and potentially promote asymmetric spatial differentiation. These findings are instructive for future work in designing devices and experiments for culture of cell aggregates.

A method to simplify bioreactor processing for recombinant protein production in rice cell suspension cultures

Transgenic plant cell suspension culture is a promising platform for recombinant protein production. Rice cell suspension culture is one of the systems that has been developed due to its unique metabolically-regulated promoter, rice alpha-amylase 3D (RAmy3D), that is up-regulated in sugar-deprived medium. Using the RAmy3D promoter system in transgenic rice cell suspensions results in two phases of the culture, the growth phase and the induction phase. Conventionally, medium exchange is performed to remove residual sugar and induce recombinant protein. In this work, a simplified production process is demonstrated in a 5-L bioreactor, including reduction of sugar concentration in the initial culture medium, elimination of the media exchange operation, and uncontrolled dissolved oxygen (DO) with constant aeration. The simplified method significantly improves the accumulation level of a recombinant protein, protein purity, and productivity compared to the conventional method. This method also reduces costs associated with material and labor.• The method of simplified bioreactor processing includes single-stage culture, uncontrolled dissolved oxygen (DO) but controlled inlet air flowrate, and lower (50% reduction) initial sucrose concentration in the culture medium.• This method improves recombinant protein production level and productivity compared to the conventional method.• This method reduces material and labor costs.

Gas circulation rate and medium exchange ratio as influential factors affecting ethanol production in carbon monoxide fermentation using a packed-bed reactor

A packed-bed reactor (PBR) which has recyclable internal gas and medium exchange functions for carbon monoxide (CO) fermentation was operated using an ethanol producing acetogen, Clostridium autoethanogenum DSM 10061.

Silicone Chambers for Pollen Tube Imaging in Microstructured In Vitro Environments.

Live cell imaging at high resolution of pollen tubes growing in vitro requires an experimental setup that maintains the elongated cells in a single optical plane and allows for controlled exchange of growth medium. As a low-cost alternative to lithography-based microfluidics, we developed a silicone-based spacer system that allows introducing spatial features and flexible design. These growth chambers can be cleaned and reused repeatedly.

CXCL-13 Regulates Resistance to 5-Fluorouracil in Colorectal Cancer.

Purpose5-Fluorouracil (5-Fu) is used as a conventional chemotherapy drug in chemotherapy for patients with advanced colorectal cancer, but many patients still suffer from treatment failure due to 5-Fu resistance. Emerging observations revealed the important role of chemokine (C-X-C motif) ligand 13 (CXCL-13) in tumor microenvironment and its relationship with prognosis in patients with colorectal cancer. This study is designed to reveal the important role of CXCL-13 in causing colorectal cancer resistance to 5-Fu.Materials and MethodsCXCL-13 levels of patient's serum or cell culture supernatants were measured separately by enzyme-linked immunosorbent assay. In cell assays, cell viability is detected by Cell Counting Kit-8. Therefore, the recombinant human CXCL-13 was used to simulate its high expression in cells while its antibody and siRNA were used to reduce CXCL-13 expression in cells.ResultsIn this study, we demonstrated that CXCL-13 is associated with 5-Fu resistance by culture medium exchange experiments and cytokine arrays of colorectal cancer resistant and non-resistant cells. Clinical studies showed that CXCL-13 is highly expressed in the serum of 5-Fu–resistant patients. High levels of serum CXCL-13 also predict a worse clinical outcome. The addition of recombinant CXCL-13 cytokine resulted in 5-Fu resistance, while its antibody overcame 5-Fu resistance, and knockdown of CXCL-13 expression by siRNA also reduced 5-Fu resistance, which can be saved by added recombination CXCL-13.ConclusionThese results not only identify a CXCL-13 mediated 5-Fu resistance mechanism but also provide a novel target for 5-Fu–resistant colorectal cancer in prevention and treatment strategies.