bims-orenst 2022-04-17 papers

bims-orenst

Biomed News

on Organs-on-chips and engineered stem cell models

Issue of 2022–04–17
seven papers selected by

Contractility analysis of human engineered 3D heart tissues by an automatic tracking technique using a standalone application.
A Microfluidic Platform to Monitor Real-Time Effects of Extracellular Vesicle Exchange between Co-Cultured Cells across Selectively Permeable Barriers.
Construction and Functional Evaluation of a Three-Dimensional Blood-Brain Barrier Model Equipped With Human Induced Pluripotent Stem Cell-Derived Brain Microvascular Endothelial Cells.
Sprayable nanomicelle hydrogels and inflammatory bowel disease patient cell chips for development of intestinal lesion-specific therapy.
Extending the viability of human precision-cut intestinal slice model for drug metabolism studies.
Streptococcus pneumoniae Affects Endothelial Cell Migration in Microfluidic Circulation.
Multi-omics analysis based on 3D-bioprinted models innovates therapeutic target discovery of osteosarcoma.

PLoS One. 2022 ;17(4): e0266834

Contractility analysis of human engineered 3D heart tissues by an automatic tracking technique using a standalone application.

José M Rivera-Arbeláez, Carla Cofiño-Fabres, Verena Schwach, Tom Boonen, Simone A Ten Den, Kim Vermeul, Albert van den Berg, Loes I Segerink, Marcelo C Ribeiro, Robert Passier.

The use of Engineered Heart Tissues (EHT) as in vitro model for disease modeling and drug screening has increased, as they provide important insight into the genetic mechanisms, cardiac toxicity or drug responses. Consequently, this has highlighted the need for a standardized, unbiased, robust and automatic way to analyze hallmark physiological features of EHTs. In this study we described and validated a standalone application to analyze physiological features of EHTs in an automatic, robust, and unbiased way, using low computational time. The standalone application "EHT Analysis" contains two analysis modes (automatic and manual) to analyzes the contractile properties and the contraction kinetics of EHTs from high speed bright field videos. As output data, the graphs of displacement, contraction force and contraction kinetics per file will be generated together with the raw data. Additionally, it also generates a summary file containing all the data from the analyzed files, which facilitates and speeds up the post analysis. From our study we highlight the importance of analyzing the axial stress which is the force per surface area (μN/mm2). This allows to have a readout overtime of tissue compaction, axial stress and leave the option to calculate at the end point of an experiment the physiological cross-section area (PSCA). We demonstrated the utility of this tool by analyzing contractile properties and compaction over time of EHTs made out of a double reporter human pluripotent stem cell (hPSC) line (NKX2.5EGFP/+-COUP-TFIImCherry/+) and different ratios of human adult cardiac fibroblasts (HCF). Our standalone application "EHT Analysis" can be applied for different studies where the physiological features of EHTs needs to be analyzed under the effect of a drug compound or in a disease model.

DOI: https://doi.org/10.1371/journal.pone.0266834
Int J Mol Sci. 2022 Mar 24. pii: 3534. [Epub ahead of print]23(7):

A Microfluidic Platform to Monitor Real-Time Effects of Extracellular Vesicle Exchange between Co-Cultured Cells across Selectively Permeable Barriers.

Hunter G Mason, Joshua Bush, Nitin Agrawal, Ramin M Hakami, Remi Veneziano.

  Exosomes and other extracellular vesicles (EVs) play a significant yet poorly understood role in cell-cell communication during homeostasis and various pathological conditions. Conventional in vitro and in vivo approaches for studying exosome/EV function depend on time-consuming and expensive vesicle purification methods to obtain sufficient vesicle populations. Moreover, the existence of various EV subtypes with distinct functional characteristics and submicron size makes their analysis challenging. To help address these challenges, we present here a unique chip-based approach for real-time monitoring of cellular EV exchange between physically separated cell populations. The extracellular matrix (ECM)-mimicking Matrigel is used to physically separate cell populations confined within microchannels, and mimics tissue environments to enable direct study of exosome/EV function. The submicron effective pore size of the Matrigel allows for the selective diffusion of only exosomes and other smaller EVs, in addition to soluble factors, between co-cultured cell populations. Furthermore, the use of PEGDA hydrogel with a very small pore size of 1.2 nm in lieu of Matrigel allows us to block EV migration and, therefore, differentiate EV effects from effects that may be mediated by soluble factors. This versatile platform bridges purely in vitro and in vivo assays by enabling studies of EV-mediated cellular crosstalk under physiologically relevant conditions, enabling future exosome/EV investigations across multiple disciplines through real-time monitoring of vesicle exchange.

Keywords:  Matrigel; PEGDA; exosomes; extracellular matrix; extracellular vesicles; functional EV assays; intercellular communication; lab-on-a-chip; microfluidic device

DOI:  https://doi.org/10.3390/ijms23073534
Pharm Res. 2022 Apr 11.

Construction and Functional Evaluation of a Three-Dimensional Blood-Brain Barrier Model Equipped With Human Induced Pluripotent Stem Cell-Derived Brain Microvascular Endothelial Cells.

Toshiki Kurosawa, Daiki Sako, Yuma Tega, Yasuyuki Debori, Yumi Tomihara, Kazunobu Aoyama, Yoshiyuki Kubo, Nobuyuki Amano, Yoshiharu Deguchi.

   PURPOSE: The purpose of this study was to construct and validate an in vivo three-dimensional blood-brain barrier (3D-BBB) model system equipped with brain microvascular endothelial cells derived from human induced pluripotent stem cells (hiPS-BMECs).
METHODS: The 3D-BBB system was constructed by seeding hiPS-BMECs onto the capillary lane of a MIMETAS OrganoPlate® 3-lane coated with fibronectin/collagen IV. hiPS-BMECs were incubated under continuous switchback flow with an OrganoFlow® for 2 days. The 3D capillary structure and expression of tight-junction proteins and transporters were confirmed by immunocytochemistry. The mRNA expression of transporters in the 3D environment was determined using qRT-PCR, and the permeability of endogenous substances and drugs was evaluated under various conditions.
RESULTS AND DISCUSSION: The expression of tight-junction proteins, including claudin-5 and ZO-1, was confirmed by immunohistochemistry. The permeability rate constant of lucifer yellow through hiPS-BMECs was undetectably low, indicating that paracellular transport is highly restricted by tight junctions in the 3D-BBB system. The mRNA expression levels of transporters and receptors in the 3D-BBB system differed from those in the 2D-culture system by 0.2- to 5.8-fold. The 3D-cultured hiPS-BMECs showed asymmetric transport of substrates of BCRP, CAT1 and LAT1 between the luminal (blood) and abluminal (brain) sides. Proton-coupled symport function of MCT1 was also confirmed.
CONCLUSION: The 3D-BBB system constructed in this study mimics several important characteristics of the human BBB, and is expected to be a useful high-throughput evaluation tool in the development of CNS drugs.

Keywords:  3D culture; blood–brain barrier; human iPS cells; organ-on-a-chip; transporter

DOI:  https://doi.org/10.1007/s11095-022-03249-3
Bioact Mater. 2022 Dec;18 433-445

Sprayable nanomicelle hydrogels and inflammatory bowel disease patient cell chips for development of intestinal lesion-specific therapy.

Hyo-Jin Yoon, Songhyun Lee, Tae Young Kim, Seung Eun Yu, Hye-Seon Kim, Young Shin Chung, Seyong Chung, Suji Park, Yong Cheol Shin, Eun Kyung Wang, Jihye Noh, Hyun Jung Kim, Cheol Ryong Ku, Hong Koh, Chang-Soo Kim, Joon-Sang Park, Young Min Shin, Hak-Joon Sung.

  All-in-one treatments represent a paradigm shift in future medicine. For example, inflammatory bowel disease (IBD) is mainly diagnosed by endoscopy, which could be applied for not only on-site monitoring but also the intestinal lesion-targeted spray of injectable hydrogels. Furthermore, molecular conjugation to the hydrogels would program both lesion-specific adhesion and drug-free therapy. This study validated this concept of all-in-one treatment by first utilizing a well-known injectable hydrogel that underwent efficient solution-to-gel transition and nanomicelle formation as a translatable component. These properties enabled spraying of the hydrogel onto the intestinal walls during endoscopy. Next, peptide conjugation to the hydrogel guided endoscopic monitoring of IBD progress upon adhesive gelation with subsequent moisturization of inflammatory lesions, specifically by nanomicelles. The peptide was designed to mimic the major component that mediates intestinal interaction with Bacillus subtilis flagellin during IBD initiation. Hence, the peptide-guided efficient adhesion of the hydrogel nanomicelles onto Toll-like receptor 5 (TLR5) as the main target of flagellin binding and Notch-1. The peptide binding potently suppressed inflammatory signaling without drug loading, where TLR5 and Notch-1 operated collaboratively through downstream actions of tumor necrosis factor-alpha. The results were produced using a human colorectal cell line, clinical IBD patient cells, gut-on-a-chip, a mouse IBD model, and pig experiments to validate the translational utility.

Keywords:  All-in-one treatment; DLS, dynamic light scattering; DSS, dextran sodium sulfate; HPLC, high-performance liquid chromatography; IBD, inflammatory bowel disease; Inflammatory bowel disease; Injectable hydrogel; Nanomicelle; P(CL), poly(caprolactone); Pe, peptide; Peptide display; R&D, research and development; SEM, scanning electron microscopy; TEER, transepithelial electrical resistance; TEM, transmission electron microscopy; TLR5, Toll-like receptor 5; TNF-α, tumor necrosis factor-alpha; Theranostic; mPEG, methoxy poly (ethylene glycol)

DOI:  https://doi.org/10.1016/j.bioactmat.2022.03.031
Arch Toxicol. 2022 Apr 15.

Extending the viability of human precision-cut intestinal slice model for drug metabolism studies.

C Biel, O Martinec, B Sibering, K van Summeren, A M A Wessels, D J Touw, K P de Jong, V E de Meijer, K N Faber, J P Ten Klooster, I A M de Graaf, P Olinga.

  Human Precision-cut intestinal slices (hPCIS) are used to study intestinal physiology, pathophysiology, drug efficacy, toxicology, kinetics, and metabolism. However, the use of this ex vivo model is restricted to approximately a 24 h timeframe because of declining viability of the hPCIS during traditional culture. We hypothesized that we could extend the hPCIS viability by using organoid medium. Therefore, we cultured hPCIS for up to 72 h in organoid media [expansion medium (Emed) and differentiation medium (Dmed)]. After incubation, we assessed culture-induced changes on viability markers, specific cell type markers and we assessed the metabolic activity of enterocytes by measuring midazolam metabolite formation. We show that the adenosine triphosphate (ATP)/protein ratio of Emed-cultured hPCIS and morphology of both Emed- and Dmed-cultured hPCIS was improved compared to WME-cultured hPCIS. Emed-cultured hPCIS showed an increased expression of proliferation and stem cell markers, whereas Dmed-cultured hPCIS showed an increased expression of proliferation and enterocyte markers, along with increased midazolam metabolism. Using the Emed, the viability of hPCIS could be extended for up to 72 h, and proliferating stem cells remained preserved. Using Dmed, hPCS also remained viable for up to 72 h, and specifically rescued the metabolizing enterocytes during culture. In conclusion, by using two different organoid culture media, we could extend the hPCIS viability for up to 72 h of incubation and specifically steer stem cells or enterocytes towards their original function, metabolism, and proliferation, potentially allowing pharmacokinetic and toxicology studies beyond the 24 h timeframe.

Keywords:  Drug metabolism; Ex vivo model; Human precision-cut intestinal slices; Organoid medium; Viability

DOI:  https://doi.org/10.1007/s00204-022-03295-1
Front Microbiol. 2022 ;13 852036

Streptococcus pneumoniae Affects Endothelial Cell Migration in Microfluidic Circulation.

Anna Kopenhagen, Isabell Ramming, Belinda Camp, Sven Hammerschmidt, Marcus Fulde, Mathias Müsken, Michael Steinert, Simone Bergmann.

  Bloodstream infections caused by Streptococcus pneumoniae induce strong inflammatory and procoagulant cellular responses and affect the endothelial barrier of the vascular system. Bacterial virulence determinants, such as the cytotoxic pore-forming pneumolysin, increase the endothelial barrier permeability by inducing cell apoptosis and cell damage. As life-threatening consequences, disseminated intravascular coagulation followed by consumption coagulopathy and low blood pressure is described. With the aim to decipher the role of pneumolysin in endothelial damage and leakage of the vascular barrier in more detail, we established a chamber-separation cell migration assay (CSMA) used to illustrate endothelial wound healing upon bacterial infections. We used chambered inlets for cell cultivation, which, after removal, provide a cell-free area of 500 μm in diameter as a defined gap in primary endothelial cell layers. During the process of wound healing, the size of the cell-free area is decreasing due to cell migration and proliferation, which we quantitatively determined by microscopic live cell monitoring. In addition, differential immunofluorescence staining combined with confocal microscopy was used to morphologically characterize the effect of bacterial attachment on cell migration and the velocity of gap closure. In all assays, the presence of wild-type pneumococci significantly inhibited endothelial gap closure. Remarkably, even in the presence of pneumolysin-deficient pneumococci, cell migration was significantly retarded. Moreover, the inhibitory effect of pneumococci on the proportion of cell proliferation versus cell migration within the process of endothelial gap closure was assessed by implementation of a fluorescence-conjugated nucleoside analogon. We further combined the endothelial CSMA with a microfluidic pump system, which for the first time enabled the microscopic visualization and monitoring of endothelial gap closure in the presence of circulating bacteria at defined vascular shear stress values for up to 48 h. In accordance with our CSMA results under static conditions, the gap remained cell free in the presence of circulating pneumococci in flow. Hence, our combined endothelial cultivation technique represents a complex in vitro system, which mimics the vascular physiology as close as possible by providing essential parameters of the blood flow to gain new insights into the effect of pneumococcal infection on endothelial barrier integrity in flow.

Keywords:  Streptococcus pneumoniae; cell migration; endothelium; microfluidic; pneumolysin; wound healing

DOI:  https://doi.org/10.3389/fmicb.2022.852036
Bioact Mater. 2022 Dec;18 459-470

Multi-omics analysis based on 3D-bioprinted models innovates therapeutic target discovery of osteosarcoma.

Yixuan Lin, Yiqi Yang, Kai Yuan, Shengbing Yang, Shuhong Zhang, Hanjun Li, Tingting Tang.

  Current in vitro models for osteosarcoma investigation and drug screening, including two-dimensional (2D) cell culture and tumour spheroids (i.e. cancer stem-like cells), lack extracellular matrix (ECM). Therefore, results from traditional models may not reflect real pathological processes in genuine osteosarcoma histological structures. Here, we report a three-dimensional (3D) bioprinted osteosarcoma model (3DBPO) that contains osteosarcoma cells and shrouding ECM analogue in a 3D frame. Photo-crosslinkable bioinks composed of gelatine methacrylamide and hyaluronic acid methacrylate mimicked tumour ECM. We performed multi-omics analysis, including transcriptomics and DNA methylomics, to determine differences between the 3DBPO model and traditional models. Compared with 2D models and tumour spheroids, our 3DBPO model showed significant changes in cell cycle, metabolism, adherens junctions, and other pathways associated with epigenetic regulation. The 3DBPO model was more sensitive to therapies targeted to the autophagy pathway. We showed that simulating ECM yielded different osteosarcoma cell metabolic characteristics and drug sensitivity in the 3DBPO model compared with classical models. We suggest 3D printed osteosarcoma models can be used in osteosarcoma fundamental and translational research, which may contribute to novel therapeutic strategy discovery.

Keywords:  3D culture; Bioprinting; Drug screening; In vitro model; Multi-omics; Osteosarcoma

DOI:  https://doi.org/10.1016/j.bioactmat.2022.03.029