Membrane Extraction Research Articles

Fuel-cell parameter estimation based on improved gorilla troops technique

The parameter extraction of the proton exchange membrane fuel cells (PEMFCs) is an active study area over the past few years to achieve accurate current/voltage (I/V) curves. This work proposes an advanced version of an improved gorilla troops technique (IGTT) to precisely estimate the PEMFC’s model parameters. The GTT's dual implementation of the migration approach enables boosting the exploitation phase and preventing becoming trapped in the local minima. Besides, a Tangent Flight Strategy (TFS) is incorporated with the exploitation stage for efficiently searching the search space. Using two common PEMFCs stacks of BCS 500W, and Modular SR-12, the developed IGTT is effectively applied. Furthermore, the two models are evaluated under varied partial temperature and pressure. In addition to this, different new recently inspired optimizers are employed for comparative validations namely supply demand optimization (SDO), flying foxes optimizer (FFO) and red fox optimizer (RFO). Also, a comparative assessment of the developed IGTT and the original GTT are tested to ten unconstrained benchmark functions following to the Congress on Evolutionary Computation (CEC) 2017. The proposed IGTT outperforms the standard GTT, grey wolf algorithm (GWA) and Particle swarm optimizer (PSO) in 92.5%, 87.5% and 92.5% of the statistical indices. Moreover, the viability of the IGTT is proved in comparison to various previously published frameworks-based parameter's identification of PEMFCs stacks. The obtained sum of squared errors (SSE) and the standard deviations (STD) are among the difficult approaches in this context and are quite competitive. For the PEMFCs stacks being studied, the developed IGTT achieves exceedingly small SSE values of 0.0117 and 0.000142 for BCS 500 and SR-12, respectively. Added to that, the IGTT gives superior performance compared to GTT, SDO, FFO and RFO obtaining the smallest SSE objective with the least STD ever.

Synthesis of trioctyl polyvinyl chloride ammonium, membrane extraction properties, and electrodriven mass transfer behavior of Chromium (VI)

Trioctyl polyvinyl chloride ammonium (PVCTQAS) with quaternary ammonium salt content (QAS) of 25 % was synthesized by a nucleophilic substitution reaction of between polyvinyl chloride (PVC) and trioctylamine (TOA) for the first time. The mass transfer behavior of HCrO4−, CdCl3−, CoCl42−, FeCl4−, and CuCl42− metal complex anions within the plasticized PVCTQAS membrane followed a first-order kinetic rate equation. Taking the Cr(Ⅵ)-PVCTQAS system as a representative, the influences of plasticizer content and membrane thickness on the mass transfer performance were investigated, and the electrodriven mass transfer behavior of Cr(VI) was studied. The results showed that the mass transfer of HCrO4− in PVCTQASD followed a jumping mechanism, with a QAS content threshold of 22 %. In the Cr(VI)-PVCTQAS electrodriven mass transfer system, the PVCTQASD exhibited uniform electrochemical properties, and the membrane thickness had little influence on the mass transfer rate. The response of HCrO4− mass transfer rate to voltage depended on the distribution density of QAS in the membrane. The mass transfer performance of PVCTQASD with QAS between 15 % and 20 % had a high dependence on the voltage, while the permeability coefficient of Cr(VI)-PVCTQAS system became stable when the voltage exceeded 10 V after surpassing the QAS content threshold. 50 μm QAS 25 % Cr(VI)-PVCTQAS system had a treatment efficiency of 710 mg·m−2·h−1 for HCrO4− at 10 V, while maintaining exceptional selectivity and stability.

Thyroid hormone membrane receptor binding and transcriptional regulation in the sea urchin Strongylocentrotus purpuratus.

Thyroid hormones (THs) are small amino acid derived signaling molecules with broad physiological and developmental functions in animals. Specifically, their function in metamorphic development, ion regulation, angiogenesis and many others have been studied in detail in mammals and some other vertebrates. Despite extensive reports showing pharmacological responses of invertebrate species to THs, little is known about TH signaling mechanisms outside of vertebrates. Previous work in sea urchins suggests that non-genomic mechanisms are activated by TH ligands. Here we show that several THs bind to sea urchin (Strongylocentrotus purpuratus) cell membrane extracts and are displaced by ligands of RGD-binding integrins. A transcriptional analysis across sea urchin developmental stages shows activation of genomic and non-genomic pathways in response to TH exposure, suggesting that both pathways are activated by THs in sea urchin embryos and larvae. We also provide evidence associating TH regulation of gene expression with TH response elements in the genome. In ontogeny, we found more differentially expressed genes in older larvae compared to gastrula stages. In contrast to gastrula stages, the acceleration of skeletogenesis by thyroxine in older larvae is not fully inhibited by competitive ligands or inhibitors of the integrin membrane receptor pathway, suggesting that THs likely activate multiple pathways. Our data confirms a signaling function of THs in sea urchin development and suggests that both genomic and non-genomic mechanisms play a role, with genomic signaling being more prominent during later stages of larval development.

Efficient and scalable generation of primordial germ cells in 2D culture using basement membrane extract overlay

Current methods to generate human primordial germ cell-like cells (hPGCLCs) from human pluripotent stem cells (hPSCs) can be inefficient, and it is challenging to generate sufficient hPGCLCs to optimize invitro gametogenesis. We present a differentiation method that uses diluted basement membrane extract (BMEx) and low BMP4 concentration to efficiently induce hPGCLC differentiation in scalable 2D cell culture. We show that BMEx overlay potentiated BMP/SMAD signaling, induced lumenogenesis, and increased expression of key hPGCLC-progenitor markers such as TFAP2A and EOMES. hPGCLCs that were generated using the BMEx overlay method were able to upregulate more mature germ cell markers, such as DAZL and DDX4, in human fetal ovary reconstitution culture. These findings highlight the importance of BMEx during hPGCLC differentiation and demonstrate the potential of the BMEx overlay method to interrogate the formation of PGCs and amnion in humans, as well as to investigate the next steps to achieve invitro gametogenesis.

Development of Matrix-Embedded Bovine Tracheal Organoids to Study the Innate Immune Response against Bovine Respiratory Disease

Bovine respiratory disease (BRD) is the leading cause of morbidity and mortality in feedlot cattle. Bovine herpesvirus-1 (BHV-1) is one of the main culprits of BRD; however, research on BHV-1 is hampered by the lack of suitable models for infection and drug testing. In this study, we established a novel bovine tracheal organoid culture grown in a basement membrane extract type 2 (BME2) matrix and compared it with the air–liquid interface (ALI) culture system. After differentiation, the matrix-embedded organoids developed beating cilia and demonstrated a transcriptomic profile similar to the ALI culture system. The matrix-embedded organoids were also highly susceptible to BHV-1 infection and immune stimulation by Pam2Cys, an immunomodulator, which resulted in robust cytokine production and tracheal antimicrobial peptide mRNA upregulation. However, treatment of bovine tracheal organoid cultures with Pam2Cys was not sufficient to inhibit viral infection or replication, suggesting a role of the non-epithelial cellular microenvironment in vivo.

Divalent nanobodies to platelet CLEC-2 can serve as agonists or antagonists

CLEC-2 is a target for a new class of antiplatelet agent. Clustering of CLEC-2 leads to phosphorylation of a cytosolic YxxL and binding of the tandem SH2 domains in Syk, crosslinking two receptors. We have raised 48 nanobodies to CLEC-2 and crosslinked the most potent of these to generate divalent and tetravalent nanobody ligands. Fluorescence correlation spectroscopy (FCS) was used to show that the multivalent nanobodies cluster CLEC-2 in the membrane and that clustering is reduced by inhibition of Syk. Strikingly, the tetravalent nanobody stimulated aggregation of human platelets, whereas the divalent nanobody was an antagonist. In contrast, in human CLEC-2 knock-in mouse platelets, the divalent nanobody stimulated aggregation. Mouse platelets express a higher level of CLEC-2 than human platelets. In line with this, the divalent nanobody was an agonist in high-expressing transfected DT40 cells and an antagonist in low-expressing cells. FCS, stepwise photobleaching and non-detergent membrane extraction show that CLEC-2 is a mixture of monomers and dimers, with the degree of dimerisation increasing with expression thereby favouring crosslinking of CLEC-2 dimers. These results identify ligand valency, receptor expression/dimerisation and Syk as variables that govern activation of CLEC-2 and suggest that divalent ligands should be considered as partial agonists.

Extraction of Copper from Sulfuric Acid Solutions Based on Pseudo-Liquid Membrane Technology

Pseudo-liquid membranes are extraction devices in which a liquid membrane phase is retained in an apparatus consisting of two interconnected chambers while feed and stripping phases pass through the stationary liquid membrane phase as mobile phases. The organic phase of the liquid membrane sequentially contacts the aqueous phases of the feed and stripping solutions in the extraction and stripping chambers, recirculating between them. This extraction separation method, called multiphase pseudo-liquid membrane extraction, can be implemented using traditional extraction equipment: extraction columns and mixer-settlers. In the first case, the three-phase extraction apparatus consists of two extraction columns connected at the top and bottom by recirculation tubes. In the second case, the three-phase apparatus consists of a recycling close-loop, which includes two mixer-settler extractors. In this study, the extraction of copper from sulfuric acid solutions in two-column three-phase extractors was experimentally studied. A 20% solution of LIX-84 in dodecane was used as the membrane phase in the experiments. It was shown that the extraction of copper from sulfuric acid solutions in the apparatuses studied was controlled by the interfacial area in the extraction chamber. The possibility of the purification of sulfuric acid wastewaters from copper using three-phase extractors is shown. To increase the degree of extraction of metal ions, it is proposed to equip two-column three-phase extractors with perforated vibrating discs. To further increase the efficiency of extraction using the pseudo-liquid membrane method, it is proposed to use multistage processes. The mathematical description of multistage three-phase pseudo-liquid membrane extraction is discussed.

Abstract 829: Evaluation of SK-N-DZ CLENs as a potential drug delivery system for selective targeting of neuroblastoma cells

Abstract Background: Neuroblastoma (NB) is a malignancy of the neuroendocrine system affecting infants. NB is highly resistant to drug therapy, which leads to ineffective treatments and increased refractory. Current treatments have low specificity to targeted NB cells resulting in unwanted side effects. A drug delivery system that can overcome these problems is urgently needed. Incorporating drugs in optimized lipid nanoparticles such as liposomes have proven to be an effective method for drug targeting. To achieve better cellular selectivity, lipid membrane extract (LE) is derived from NB cells using a modified Dyer and Blight lipid extraction procedure. The objective of the study was to determine an optimized cell membrane lipid-extracted nanoliposomes (CLENs) for the NB cell model while considering physicochemical properties (i.e. size and molecular charge) and composition of the preparation. Methods: The neuroblastoma cell line, SK-N-DZ, was used for drug selectivity study. Expanded culture of SK-N-DZ was employed for cellular extraction purposes. The method for lipid membrane extraction was previously reported by Alharbi and Campbell, (AAPS Open 4,5(2018)). SK-N-DZ lipid extract was the main ingredient to prepare SK-N-DZ CLENs. Phase I of the study is to prepare CLENs with various compositions of DOPC, cholesterol, DPPE-PEG5000 and SK-N-DZ lipid extract. DPPE-Rhodamine was used for fluorescence and cellular uptake studies. Phase II of the study evaluated the effect of SK-N-DZ lipid extraction inclusion on drug incorporation, cellular uptake and selectivity, and cytotoxicity. Invitrogen alamarBlue HS in vitro assay was used to determine cell viability for cytotoxicity studies. SK-N-SH (brain tissue), BT-20 (breast tissue) and G401 (kidney tissue) cell lines served as negative controls. Results: The results from the cellular uptake optimization studies revealed that the optimized composition and ratio for SK-N-DZ CLENs was DOPC/Cholesterol/LE/PEG (65/10/20/5). The average particle size and value for zeta potential was 164 nm +/- 1.2 and -7.92 +/- 1.55 mV, respectively. The inclusion of SK-N-DZ lipid membrane extract (2, 10, 20 and 40 mol%) in preparation of CLENs increased cellular uptake by SK-N-DZ cells compared to corresponding control (no LE included). No difference observed in cellular uptake of preparations containing 20 and 40 mol% of LE. The additional inclusion of cholesterol (10, 20, 40 mol%) increased overall cellular uptake of preparations compared to CLENs without the additional cholesterol content. Overall, the inclusion of PEG (5, 10, and 20 mol%) in SK-N-DZ CLENs increased their uptake by the target cell population. Conclusion: In conclusion, the inclusion of SK-N-DZ lipid membrane extract in the nanoliposome preparations increased their overall cellular uptake by SK-N-DZ target cells. Additional cellular selectively and cytotoxicity studies are currently underway. Citation Format: Kevin Bumanglag, Thi Tran, Robert Campbell, Yishan Li. Evaluation of SK-N-DZ CLENs as a potential drug delivery system for selective targeting of neuroblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 829.

Abstract 5720: Combination therapies in matched 3D in vitro and in vivo preclinical models of rare and recalcitrant cancers from the National Cancer Institute’s Patient-Derived Models Repository

Abstract There is a major need in oncology drug development to establish predictive preclinical assays with high translational relevance to patient responses. The National Cancer Institute’s Patient-Derived Models Repository (https://pdmr.cancer.gov) offers a collection of highly characterized models from a variety of cancer types including rare and recalcitrant malignancies and tumors from patients of diverse ancestry. This collection includes matched sets of patient-derived cell lines, organoids, and xenografts (PDXs), which allows comparisons of drug responses from in vitro and in vivo assays performed with the same patient-derived tumor model. A high-throughput screen was conducted using matched sets of patient-derived organoids and cell lines. Patient-derived cell lines were grown as 3D multicellular spheroids mixed with endothelial cells and mesenchymal stem cells. The patient-derived organoids were 100% tumor cells and were plated in 5% basement membrane extract supplemented with growth factors and cytokines. All drugs were tested at concentrations up to their reported clinical Cmax values and cell viability for individual drug treatments and drug combinations were assayed using CellTiter-Glo 3D after seven days drug exposure. Prior to the endpoint viability measurements, growth curves for spheroid median volume and organoid median surface area were calculated from a series of non-invasive brightfield images collected every 12 hours. For some drug combinations, differential responses were observed between the matched organoids and multicellular spheroids, potentially reflecting the contribution of the stromal component in the spheroids. Overall, the drug-dependent growth responses observed from the two 3D in vitro models (i.e., multicellular spheroids and organoids) were frequently comparable to those observed in vivo from PDXs. For example, the in vitro activities of several drug combinations including: BAY1895344 + temozolomide, erlotinib + cediranib, entinostat + talazoparib, and selumetinib + abemaciclib, demonstrated good agreement with the responses observed in vivo. However, among the drug combinations tested ixazomib + panobinostat showed the greatest cytotoxicity in vitro but had no activity in the matched PDX models. The availability of matched patent-derived cell lines, organoids and PDXs provides an opportunity to learn about the features of assay methodologies and data analyses that influence the successful translation of preclinical results between in vitro and in vivo systems. The results of this study are encouraging, but also highlight discrepancies that will be important to investigate, understand and address in order to improve translational capacity of future assays. This project was funded in part with federal funds from the NCI, NIH, under contract no. HHSN261201500003I. Citation Format: Thomas S. Dexheimer, Thomas Silvers, Rene Delosh, Russell Reinhart, Chad Ogle, Zahra Davoudi, Eric Jones, Debbie Trail, John Carter, Justine Mills, Kyle Georgius, Howard Stotler, Michelle Norris, Shannon Uzelac, Suzanne Borgel, Tiffanie Minor, Luke Stockwin, Michael Mullendore, Kevin Plater, Keegan Kalmbach, Jessica Steed, Matthew Murphy, Gareth Bliss, Carrie Bonomi, Kelly Dougherty, Marion Gibson, Kevin Cooper, Dianne Newton, Cindy R. Timme, Yvonne A. Evrard, Melinda G. Hollingshead, Nathan P. Coussens, Ralph E. Parchment, James H. Doroshow, Beverly A. Teicher. Combination therapies in matched 3D in vitro and in vivo preclinical models of rare and recalcitrant cancers from the National Cancer Institute’s Patient-Derived Models Repository. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5720.

Abstract 194: Ovarian cancer patient-derived organoids and tumor xenografts as preclinical models for precision medicine

Abstract Background: Patient-derived organoids (PDO) and patient-derived xenografts (PDX) has been investigated as preclinical models for drug development and precision medicine. In this study, we established ovarian cancer PDOs and PDX models from Aug 2020 to Nov 2022, and developed protocols. Methods: We obtained tumor tissue from several sites (ovary, omentum, diaphragm, and peritoneum) or ascites from surgery of ovarian cancer patients. The tissue samples were dissociated for single cells and ascitic samples were centrifuged and washed to concentrate the tumor cells. Then, it is embedded in matrigel or basement membrane extract (BME) and seeded on a 24-well plate supplemented with culture medium containing modified growth factors. The association with clinical characteristics with success of model establishment were evaluated. Drug screening for organoids were designed on the 384-well plate using Celltiter-Glo 3D cell viability assay (Promega). The list of drugs consists of alkylating agent, monoclonal antibody, taxane, tyrosine kinase inhibitor (TKI), and poly(ADP-ribose) polymerase inhibitor (PARPi). For increase of success rate for xenograft, we compared intrabursa and subcutaneous injection xenograft models using ES-2 cell line then patient tumors in NOG mouse. Results: A total of 397 organoids passed first passage, however 32 (8%) organoids were continuously cultured over 5 passages. The associated clinical factors for establishment of organoids were stage (p=0.020, low stage 20% of success vs. high stage 2.4% of success) and pathologic types (p=0.035, low grade serous cancer (LGSC) 30% and clear cell 16.7% of success vs. high grade serous cancer (HGSC) 5.6% of success). Thirteen drugs were evaluated and AUC represented various range according to patient from 0.0004 to 1.3876. Three sites (ovary, omentum and ascites) established organoids from one patient represented difference in drug sensitivity which suggest role of cancer microenvironment. The growth upto 100mm3 of intrabursa injection (12.33 ± 0.6667) was faster than that of subcutaneous injection model (15.00 ± 1.000, p-value = 0.09) using ES-2 cell. Then comparison of patients tumor (N=10) into intrabursa vs. subcutaneous are undergoing with one completed case showing fast intrabursa growing. Conclusions: During relative short period we developed many PDOs and PDXs for future study including drug development. Moreover, we focused to develop better model for PDXs and it will give a new approach for successful model establishment. (This work was supported by National Research Foundation of Korea grant, founded by the Korea government (MSIT) [No.2020R1A2C2010566]) Citation Format: Yebeen Yu, Hye Ju Park, Cha Yeon Kang, Myong Cheol Lim, Sang Yoon Park, Yo Han Woo, Yun-Hee Kim, Sun-Young Kong. Ovarian cancer patient-derived organoids and tumor xenografts as preclinical models for precision medicine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 194.

Abstract 172: The impact of extracellular matrix modulation and cancer-associated fibroblast presence on patient-derived tumor organoid growth rate, morphology, and chemoresistance

Abstract The tumor microenvironment (TME) of colorectal cancer (CRC) plays an important role in tumor progression and chemoresistance. Therefore, it is important to incorporate elements of the TME, such as extracellular matrix (ECM) composition and stromal cells, into preclinical models to better recapitulate tumor dynamics in vivo. Particularly, cancer-associated fibroblasts (CAFs) are a dominant stromal cell type within the TME that secrete and remodel the ECM and whose abundance can correlate with poor overall survival. Patient-derived tumor organoids (PDTOs) are an innovative preclinical model that has recently emerged as a biomarker for predicting patient-specific drug responses in CRC. However, traditional PDTO cultures fail to account for the aforementioned elements of the TME. Here, we present a basement membrane extract (BME) and collagen type I-based co-culture system utilizing patient-matched CRC PDTOs and CAFs to investigate the effects of the TME on PDTO growth rate, morphology, and drug response.First, we compared the growth rate, morphology, and drug response of PDTO-only cultures in 2 ECMs: 100% BME and 90% BME + 10% collagen type I. PDTOs were seeded 4 days prior to treatment with clinically relevant therapeutics 5-Fluorouracil (5-FU) and SN-38 (active metabolite of irinotecan). Cell viability was assessed 5 days after treatment using the CellTiter-Glo 3D assay. In addition, brightfield and fluorescent images of the PDTOs were taken on days -1, 0, 3, and 5 of the drug treatment. GFP-labeled PDTOs were utilized for the image-based analysis to improve segmentation and quantification of PDTO growth rate and morphology as well as to help distinguish between PDTOs and CAFs in the co-culture experiments. The co-culture experiments of PDTOs and CAFs in 90% BME + 10% collagen type I were performed and analyzed using the same assays. To enhance tumor-stromal interactions, the PDTOs and CAFs were suspended together in the ECM. By culturing CRC PDTOs in the BME + collagen I matrix, as well as co-culturing with patient-matched CAFs, we observed differential responses in PDTO growth rate, morphology, and drug response compared to the 100% BME, PDTO-only condition. In conclusion, our initial findings warrant further investigation into the importance of incorporating elements of the TME into PDTO models for drug discovery applications to more closely mimic in vivo tumor conditions. Citation Format: Scott Valena, Pratiksha Kshetri, Yuyuan Zhao, Elizabeth Elton, Shohei Imamura, Nolan Ung, Seungil Kim, Michael E. Doche, Shannon M. Mumenthaler. The impact of extracellular matrix modulation and cancer-associated fibroblast presence on patient-derived tumor organoid growth rate, morphology, and chemoresistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 172.

GRK6 palmitoylation increasing its membrance translocation promotes LPS-induced inflammation by PI3K/ AKT pathway in kuppfer cells.

G protein-coupled receptor kinases 6 (GRK6) is one kinase of GPCRs, previous studies have shown that GRK6 is involved in the regulation of inflammatory processes. However, the role of GRK6 in inflammation is not well understood and what is the effect of its palmitoylation modification on inflammatory response in macrophage are still largely unknown. LPS stimulated Kupffer cells to simulate inflammatory injury model. SiGRK6 and GRK6 lentiviral plasmids were used to alter cellular GRK6 levels. Subcellular localization of GRK6 was detected using Membrane and Cytoplasmic Protein Extraction Kit and immunofluorescence. Palmitoylated Protein Assay Kit (Red) and modified Acyl-RAC method were used to detect palmitoylation levels. GRK6 mRNA and protein expression decreased in LPS-induced inflammatory response in Kupffer cells (P<0.05). Overexpression of GRK6 promoted inflammatory response, while silencing GRK6 reduced inflammatory response (P<0.05). In terms of molecular mechanisms, LPS induced increased palmitoylation of GRK6 and promoted the translocation of GRK6 to cell membranes (P<0.05). Subsequently, GRK6 functioned through the PI3K/ AKT signaling pathway (P<0.05). Inhibition of palmitoylation level of GRK6 can inhibit its membrane translocation and reduce inflammatory response (P<0.05). Inhibition of palmitoylation level of GRK6 might relieve LPS-induced inflammation in Kupffer cells by blocking GRK6 membrane translocation and subsequent inflammatory signaling pathway, providing a theoretical basis for targeting GRK6 to regulate inflammation.

Cell-matrix interactions control biliary organoid polarity, architecture, and differentiation.

Cholangiopathies are an important cause of morbidity and mortality. Their pathogenesis and treatment remain unclear in part because of the lack of disease models relevant to humans. Three-dimensional biliary organoids hold great promise; however, the inaccessibility of their apical pole and the presence of extracellular matrix (ECM) limits their application. We hypothesized that signals coming from the extracellular matrix regulate organoids' 3-dimensional architecture and could be manipulated to generate novel organotypic culture systems. Biliary organoids were generated from human livers and grown embedded into Culturex Basement Membrane Extract as spheroids around an internal lumen (EMB). When removed from the EMC, biliary organoids revert their polarity and expose the apical membrane on the outside (AOOs). Functional, immunohistochemical, and transmission electron microscope studies, along with bulk and single-cell transcriptomic, demonstrate that AOOs are less heterogeneous and show increased biliary differentiation and decreased expression of stem cell features. AOOs transport bile acids and have competent tight junctions. When cocultured with liver pathogenic bacteria (Enterococcus spp.), AOOs secrete a range of proinflammatory chemokines (ie, MCP1, IL8, CCL20, and IP-10). Transcriptomic analysis and treatment with a beta-1-integrin blocking antibody identified beta-1-integrin signaling as a sensor of the cell-extracellular matrix interaction and a determinant of organoid polarity. This novel organoid model can be used to study bile transport, interactions with pathobionts, epithelial permeability, cross talk with other liver and immune cell types, and the effect of matrix changes on the biliary epithelium and obtain key insights into the pathobiology of cholangiopathies.

Polymer film-based microwell array platform for long-term culture and research of human bronchial organoids

The culture of lung organoids relies on drops of basement membrane matrices. This comes with limitations, for example, concerning the microscopic monitoring and imaging of the organoids in the drops. Also, the culture technique is not easily compatible with micromanipulations of the organoids. In this study, we investigated the feasibility of the culture of human bronchial organoids in defined x-, y- and z-positions in a polymer film-based microwell array platform. The circular microwells have thin round/U-bottoms. For this, single cells are first precultured in drops of basement membrane extract (BME). After they form cell clusters or premature organoids, the preformed structures are then transferred into the microwells in a solution of 50% BME in medium. There, the structures can be cultured toward differentiated and mature organoids for several weeks. The organoids were characterized by bright-field microscopy for size growth and luminal fusion over time, by scanning electron microscopy for overall morphology, by transmission electron microscopy for the existence of microvilli and cilia, by video microscopy for beating cilia and swirling fluid, by live-cell imaging, by fluorescence microscopy for the expression of cell-specific markers and for proliferating and apoptotic cells, and by ATP measurement for extended cell viability. Finally, we demonstrated the eased micromanipulation of the organoids in the microwells by the example of their microinjection.

Fabrication of Cell-Laden AME-Loaded Collagen-Based Hydrogel Promotes Fibroblast Proliferation and Wound Healing In Vitro.

The biological factors secreted from cells and cell-based products stimulate growth, proliferation, and migration of the cells in their microenvironment, and play vital roles in promoting wound healing. The amniotic membrane extract (AME), which is rich in growth factors (GFs), can be loaded into a cell-laden hydrogel and released to a wound site to promote the healing of the wound. The present study was conducted to optimize the concentration of the loaded AME that induces secretion of GFs and structural collagen protein from cell-laden AME-loaded collagen-based hydrogels, to promote wound healing in vitro. In this experimental study, fibroblast-laden collagen-based hydrogel loaded with different concentrations of AME (0.1, 0.5, 1, and 1.5 mg/mL, as test groups) and without AME (as control group), were incubated for 7 days. The total proteins secreted by the cells from the cell-laden hydrogel loaded with different concentrations of AME were collected and the levels of GFs and type I collagen were assessed using ELISA method. Cell proliferation and scratch assay were done to evaluate the function of the construct. The results of ELISA showed that the concentrations of GFs in the conditioned medium (CM) secreted from the cell-laden AME-loaded hydrogel were significantly higher than those secreted by only the fibroblast group. Interestingly, the metabolic activity of fibroblasts and the ability of the cells to migrate in scratch assay significantly increased in the CM3-treated fibroblast culture compared to other groups. The concentrations of the cells and the AME for preparation of CM3 group were 106 cell/mL and 1 mg/mL, respectively. We showed that 1 mg/ml of AME loaded in fibroblast-laden collagen hydrogel significantly enhanced the secretion of EGF, KGF, VEGF, HGF, and type I collagen. The CM3 secreted from the cell-laden AME-loaded hydrogel promoted proliferation and scratch area reduction in vitro.

Msp1-mediated proofreading mechanism for localization of tail-anchored membrane proteins.

Protein targeting to organelles has been thought to be a very precise process, and proteins that fail to localize correctly are rapidly degraded. Tail-anchored proteins are posttranslationally targeted to the endoplasmic reticulum membrane via guided entry of tail-anchored (TA) proteins pathway. However, these proteins can be mislocalized to the mitochondrial outer membrane. We found that the AAA-ATPase Msp1 on the mitochondrial outer membrane extracts mislocalized TA proteins to the cytosol, passing them to the guided entry of the TA proteins pathway to facilitate their transfer to the endoplasmic reticulum membrane. After the transfer to the endoplasmic reticulum, such TA proteins are directed to degradation if they are recognized by the quality control system on the endoplasmic reticulum. If not recognized, they are retargeted to their original destination along the secretory pathway. Thus, we have identified an intracellular proofreading system that corrects the localization of TA proteins.

Nitrogen-doped metal-organic framework derived porous carbon/polymer membrane for the simultaneous extraction of four benzotriazole ultraviolet stabilizers in environmental water

Benzotriazole ultraviolet stabilizers (BUVSs) that are added to pharmaceutical and personal care products (PPCPs) have raised global concerns because of their high toxicity. An efficient method to monitor its pollution level is urgently imperative. Here, a nitrogen-doped metal-organic framework (MOF) derived porous carbon (UiO-66-NH2/DC) was prepared and integrated into polyvinylidene fluoride mixed matrix membrane (PVDF MMM) as an adsorbent for the first time. The hydrophobic UiO-66-NH2/DC with a pore size of 162 Å exhibited outstanding extraction performance for BUVSs, which solves the problem of difficult enrichment of large-size and hydrophobic targets. Notably, the density functional theory simulation was employed to reveal the structure of the derived carbon material and explored the recognition and enrichment mechanism (synergy of π-π conjugation, hydrogen bond, coordination, hydrophobic interaction and mesoporous channel) of BUVSs by UiO-66-NH2/DC-PVDF MMM. And then, an influential method based on dispersive membrane extraction (DME) coupled with ultrahigh-performance liquid chromatography−tandem mass spectrometry (UHPLC−MS/MS) was developed for the simultaneous analysis of four BUVSs in environmental water samples. The validated method benefited from the high sensitivity (the limits of detection within 0.25−1.40 ng/L), accuracy (recoveries of 71.9−102.8% for wastewater) and rapidity (50 min to enrich 9 samples). This study expands the application prospects of porous carbon derived from MOF for sample pretreatment of pollutants in water.

The covalent organic framework based nylon membrane extraction coupled with UHPLC-MS/MS for highly efficiency determination of hexabromocyclododecanes in environmental water

Hexabromocyclododecanes (HBCDs) have given their adverse effects on environment and human health, and highly sensitive analysis of HBCDs in water is urgent. In this study, a new method for the determination of trace HBCDs in water was established by covalent organic framework (COF) based nylon membrane extraction (ME) coupled with ultrahigh-performance liquid chromatography−tandem mass spectrometry (UHPLC−MS/MS). The COF had been self-assembled onto the nylon membrane in a gentle strategy to fabricate COF nylon membrane. Several important ME parameters including the dosage of COF, pH, eluent condition and salinity were systematically investigated. The limits of detection and quantification were 0.011–0.014 and 0.038–0.047 ng/L for three HBCDs, respectively. The linear ranges were from 0.04 to 20 ng/L, and the relative standard deviations were 5.7–17.8 % (intra-day) and 5.2–14.1 % (inter-day). In addition, density functional theory (DFT) calculations on adsorption energy proved that the introduction of halogen bond (XB) made a key contribution to high extraction efficiency and excellent selectivity of COF nylon membrane for HBCDs. The 500 mL of samples, including tap water and reservoir water, could be extracted only in 23 min. The established method presented highly sensitive for ultra-trace analysis of HBCDs in environmental water.

Extracellular matrix drives tumor organoids toward desmoplastic matrix deposition and mesenchymal transition.

Patient-derived tumor organoids have been established as promising tools for in vitro modelling of multiple tumors, including cholangiocarcinoma (CCA). However, organoids are commonly cultured in basement membrane extract (BME) which does not recapitulate the intricacies of the extracellular matrix (ECM). We combined CCA organoids (CCAOs) with native tumor and liver scaffolds, obtained by decellularization, to effectuate a model to study the interaction between epithelial tumor cells and their surrounding ECM. Decellularization resulted in removal of cells while preserving ECM structure and retaining important characteristics of the tissue origin, including stiffness and presence of desmoplasia. The transcriptome of CCAOs in a tumor scaffold much more resembled that of patient-paired CCA tissue in vivo compared to CCAOs cultured in BME or liver scaffolds. This was accompanied by an increase in chemoresistance to clinically-relevant chemotherapeutics. CCAOs in decellularized scaffolds revealed environment-dependent proliferation dynamics, driven by the occurrence of epithelial-mesenchymal transition. Furthermore, CCAOs initiated an environment-specific desmoplastic reaction by increasing production of multiple collagen types. In conclusion, convergence of organoid-based models with native ECM scaffolds will lead to better understanding of the in vivo tumor environment. STATEMENT OF SIGNIFICANCE: The extracellular matrix (ECM) influences various facets of tumor behavior. Understanding the exact role of the ECM in controlling tumor cell fate is pertinent to understand tumor progression and develop novel therapeutics. This is particularly the case for cholangiocarcinoma (CCA), whereby the ECM displays a distinct tumor environment, characterized by desmoplasia. However, current models to study the interaction between epithelial tumor cells and the environment are lacking. We have developed a fully patient-derived model encompassing CCA organoids (CCAOs) and human decellularized tumor and tumor-free liver ECM. The tumor ECM induced recapitulation of various aspects of CCA, including migration dynamics, transcriptome and proteome profiles, and chemoresistance. Lastly, we uncover that epithelial tumor cells contribute to matrix deposition, and that this phenomenon is dependent on the level of desmoplasia already present.

A Fibrous and Porous Nature of Hydrogels Promotes Directed Migration of Schwann Cells

Introduction: Hydrogel fillings have been successful in enhancing artificial conduits for nerve injuries, but the ideal hydrogel is yet to be found. Its discovery is impeded by an overflow of available products as well as a lack of in vitro experiments that fully analyze the effects of these hydrogels on cells crucial for nerve regeneration. Materials and Methods: This study investigated various hydrogels advertised for peripheral nerve regeneration. Schwann cells were seeded on the hydrogels and their morphology, viability, proliferation and migration were examined in vitro. Results: Our results demonstrated that the basement membrane extract hydrogel Cultrex® 3D Culture Matrix® (Trevigen) promoted elongated morphology and directed migration in SCs. To elucidate the possible reasons behind this, detailed material characterizations of the hydrogels were conducted and identified highly bundled networks of fibres in Cultrex® 3D Culture Matrix® as well as strain-stiffening. In a next step, we further analyzed two more hydrogels consisting of solely one component of Cultrex® 3D Culture Matrix® respectively namely laminin and collagen and found that the SCs also migrated more directed in the laminin hydrogel but not in the collagen hydrogel. Interestingly, out of the two hydrogels only the laminin hydrogel showed a similar fibrous surface roughness to Cultrex® 3D Culture Matrix® and also exhibited strain-stiffening. This indicates a correlation between a fibrous nature of hydrogels, strain-stiffening and directed migration in Schwann cells. Conclusion: These experiments provide a unique systematic assessment of various hydrogels in respect to their material properties and their effect on cells thereby paving the path for the future manufacturing of tailored biomaterials.