bims-orenst 2022-08-21 papers

Issue of 2022–08–21
five papers selected by

iScience. 2022 Aug 19. 25(8): 104813

A microengineered Brain-Chip to model neuroinflammation in humans.

Iosif Pediaditakis, Konstantia R Kodella, Dimitris V Manatakis, Christopher Y Le, Sonalee Barthakur, Alexander Sorets, Achille Gravanis, Lorna Ewart, Lee L Rubin, Elias S Manolakos, Christopher D Hinojosa, Katia Karalis.

Species differences in brain and blood-brain barrier (BBB) biology hamper the translation of findings from animal models to humans, impeding the development of therapeutics for brain diseases. Here, we present a human organotypic microphysiological system (MPS) that includes endothelial-like cells, pericytes, glia, and cortical neurons and maintains BBB permeability at in vivo relevant levels. This human Brain-Chip engineered to recapitulate critical aspects of the complex interactions that mediate neuroinflammation and demonstrates significant improvements in clinical mimicry compared to previously reported similar MPS. In comparison to Transwell culture, the transcriptomic profiling of the Brain-Chip displayed significantly advanced similarity to the human adult cortex and enrichment in key neurobiological pathways. Exposure to TNF-α recreated the anticipated inflammatory environment shown by glia activation, increased release of proinflammatory cytokines, and compromised barrier permeability. We report the development of a robust brain MPS for mechanistic understanding of cell-cell interactions and BBB function during neuroinflammation.

Keywords: Biomedical engineering; Cellular neuroscience; Molecular neuroscience

DOI: https://doi.org/10.1016/j.isci.2022.104813

J Biosci Bioeng. 2022 Aug 11. pii: S1389-1723(22)00189-X. [Epub ahead of print]

Perfusion culture of multi-layered HepG2 hepatocellular carcinoma cells in a pressure-driven microphysiological system.

Shinji Sugiura, Taku Satoh, Kazumi Shin, Reiko Onuki-Nagasaki, Toshiyuki Kanamori.

Here we report the perfusion culture of a multi-layered tissue composed of HepG2 cells (a human hepatoma line) in a pressure-driven microphysiological system (PD-MPS), which we developed previously as a multi-throughput perfusion culture platform. The perfusion culture of multi-layered tissue model was constructed by inserting a modified commercially available permeable membrane insert into the PD-MPS. HepG2 cells were layered on the membrane, and culture medium was perfused both through and below the membrane. The seeded density (number of cells/cm2) of the culture model is 70 times that of static culture in a conventional 35-mm culture dish. Pressure-driven circulation of the medium in our compact device (8.6 × 7.0 × 4.5 cm3), which comprised two perfusion-culture modules and a pneumatic connection port, enabled perfusion culture of two multi-layered tissues (initially 1 × 105 cells). To obtain insight into the basic functionality of the multi-layered tissues as hepatocytes, we compared albumin production and urea synthesis between perfusion cultures and static cultures. The HepG2 cells grew and secreted increasing amounts of albumin throughout 20 days of perfusion culture, whereas albumin secretion did not increase under static culture conditions. In addition, on day 20, the amount of albumin secreted by the HepG2 cells in the microfluidic device was 68% of that in the conventional culture dish, which was seeded with the same number of cells but had a 70 times larger culture area. These features of high-density culture of functioning cells in a compact device support the application of PD-MPS in single- and multi-organ MPS.

Keywords: Hepatocyte; Liver-on-a-chip; Microfluidic device; Microphysiological systems; Multi-layered culture; Organ-on-a-chip; Perfusion culture

DOI: https://doi.org/10.1016/j.jbiosc.2022.07.001

ACS Appl Mater Interfaces. 2022 Aug 19.

PDMS-PEG Block Copolymer and Pretreatment for Arresting Drug Absorption in Microphysiological Devices.

Devin B Mair, Marcus Alonso Cee Williams, Jeffrey Fanzhi Chen, Alex Goldstein, Alex Wu, Peter H U Lee, Nathan J Sniadecki, Deok-Ho Kim.

Poly(dimethylsiloxane) (PDMS) is a commonly used polymer in organ-on-a-chip devices and microphysiological systems. However, due to its hydrophobicity and permeability, it absorbs drug compounds, preventing accurate drug screening applications. Here, we developed an effective and facile method to prevent the absorption of drugs by utilizing a PDMS-PEG block copolymer additive and drug pretreatment. First, we incorporated a PDMS-PEG block copolymer into PDMS to address its inherent hydrophobicity. Next, we addressed the permeability of PDMS by eliminating the concentration gradient via pretreatment of the PDMS with the drug prior to experimentally testing drug absorption. The combined use of a PDMS-PEG block copolymer with drug pretreatment resulted in a mean reduction of drug absorption by 91.6% in the optimal condition. Finally, we demonstrated that the proposed method can be applied to prevent drug absorption in a PDMS-based cardiac microphysiological system, enabling more accurate drug studies.

Keywords: PDMS; PDMS−PEG; drug absorption; microphysiological systems; organ-on-a-chip

DOI: https://doi.org/10.1021/acsami.2c10669

Gastroenterology. 2022 Aug 10. pii: S0016-5085(22)00912-X. [Epub ahead of print]

Identification of Quiescent LGR5+ Stem Cells in the Human Colon.

BACKGROUND & AIMS: In the mouse intestinal epithelium, Lgr5+ stem cells are vulnerable to injury owing to their predominantly cycling nature, and their progenies de-differentiate to replenish the stem cell pool. However, how human colonic stem cells behave in homeostasis and during regeneration remains unknown.
METHODS: Transcriptional heterogeneity among colonic epithelial cells was analyzed by scRNA-seq analysis of human and mouse colonic epithelial cells. To trace the fate of human colonic stem or differentiated cells, we generated LGR5-tdTomato, LGR5-iCT, LGR5-split-Cre, and KRT20-ERCreER knock-in human colon organoids via genome engineering. p27+ dormant cells were further visualized with the p27-mVenus reporter. To analyze the dynamics of human colonic stem cells in vivo, we orthotopically xenotransplanted fluorescence-labeled human colon organoids into immune-deficient mice. The cell cycle dynamics in xenograft cells was evaluated using EdU pulse-chase analysis. The clonogenic capacity of slow-cycling human stem cells or differentiated cells was analyzed in the context of homeostasis, LGR5 ablation and 5-FU-induced mucosal injury.
RESULTS: ScRNA-seq analysis illuminated the presence of non-dividing LGR5+ stem cells in the human colon. Visualization and lineage tracing of slow-cycling LGR5+p27+ cells and orthotopic xenotransplantation validated their homeostatic lineage-forming capability in vivo, which was augmented by 5-FU-induced mucosal damage. TGF-β signaling regulated the quiescent state of LGR5+ cells. Despite the plasticity of differentiated KRT20+ cells, they did not display clonal growth following 5-FU-induced injury, suggesting that occupation of the niche environment by LGR5+p27+ cells prevented neighboring differentiated cells from de-differentiating.
CONCLUSIONS: Our results highlighted the quiescent nature of human LGR5+ colonic stem cells and their contribution to post-injury regeneration.

Keywords: Intestinal stem cells; Organoids; Slow-cycling stem cell; TGF-β signaling

DOI: https://doi.org/10.1053/j.gastro.2022.07.081

Sci Rep. 2022 Aug 17. 12(1): 13988

Differential gene expression in iPSC-derived human intestinal epithelial cell layers following exposure to two concentrations of butyrate, propionate and acetate.

Menno Grouls, Aafke W F Janssen, Loes P M Duivenvoorde, Guido J E J Hooiveld, Hans Bouwmeester, Meike van der Zande.

Intestinal epithelial cells and the intestinal microbiota are in a mutualistic relationship that is dependent on communication. This communication is multifaceted, but one aspect is communication through compounds produced by the microbiota such as the short-chain fatty acids (SCFAs) butyrate, propionate and acetate. Studying the effects of SCFAs and especially butyrate in intestinal epithelial cell lines like Caco-2 cells has been proven problematic. In contrast to the in vivo intestinal epithelium, Caco-2 cells do not use butyrate as an energy source, leading to a build-up of butyrate. Therefore, we used human induced pluripotent stem cell derived intestinal epithelial cells, grown as a cell layer, to study the effects of butyrate, propionate and acetate on whole genome gene expression in the cells. For this, cells were exposed to concentrations of 1 and 10 mM of the individual short-chain fatty acids for 24 h. Unique gene expression profiles were observed for each of the SCFAs in a concentration-dependent manner. Evaluation on both an individual gene level and pathway level showed that butyrate induced the biggest effects followed by propionate and then acetate. Several known effects of SCFAs on intestinal cells were confirmed, such as effects on metabolism and immune responses. The changes in metabolic pathways in the intestinal epithelial cell layers in this study demonstrate that there is a switch in energy homeostasis, this is likely associated with the use of SCFAs as an energy source by the induced pluripotent stem cell derived intestinal epithelial cells similar to in vivo intestinal tissues where butyrate is an important energy source.

DOI: https://doi.org/10.1038/s41598-022-17296-8