bims-orenst 2021-07-11 papers

bims-orenst

Biomed News

on Organs-on-chips and engineered stem cell models

Issue of 2021–07–11
nine papers selected by
Joram Mooiweer, University of Groningen

Multicellular modeling of ciliopathy by combining iPS cells and microfluidic airway-on-a-chip technology.
Three dimensional modeling of biologically relevant fluid shear stress in human renal tubule cells mimics in vivo transcriptional profiles.
An organ-on-a-chip model for pre-clinical drug evaluation in progressive non-genetic cardiomyopathy.
A versatile microfluidic tool for the 3D culture of HepaRG cells seeded at various stages of differentiation.
Parallel and large-scale antitumor investigation using stable chemical gradient and heterotypic three-dimensional tumor coculture in a multi-layered microfluidic device.
On-Chip Construction of Liver Lobules with Self-Assembled Perfusable Hepatic Sinusoid Networks.
Functional assistance for stress distribution in cell culture membrane under periodically stretching.
Retinal organoids on-a-chip: a micro-millifluidic bioreactor for long-term organoid maintenance.
Coordination of endothelial cell positioning and fate specification by the epicardium.

Sci Transl Med. 2021 Jul 07. pii: eabb1298. [Epub ahead of print]13(601):

Multicellular modeling of ciliopathy by combining iPS cells and microfluidic airway-on-a-chip technology.

Naoyuki Sone, Satoshi Konishi, Koichi Igura, Koji Tamai, Satoshi Ikeo, Yohei Korogi, Shuhei Kanagaki, Toshinori Namba, Yuki Yamamoto, Yifei Xu, Kazuhiko Takeuchi, Yuichi Adachi, Toyofumi F Chen-Yoshikawa, Hiroshi Date, Masatoshi Hagiwara, Sachiko Tsukita, Toyohiro Hirai, Yu-Suke Torisawa, Shimpei Gotoh.

Mucociliary clearance is an essential lung function that facilitates the removal of inhaled pathogens and foreign matter unidirectionally from the airway tract and is innately achieved by coordinated ciliary beating of multiciliated cells. Should ciliary function become disturbed, mucus can accumulate in the airway causing subsequent obstruction and potentially recurrent pneumonia. However, it has been difficult to recapitulate unidirectional mucociliary flow using human-derived induced pluripotent stem cells (iPSCs) in vitro and the mechanism governing the flow has not yet been elucidated, hampering the proper humanized airway disease modeling. Here, we combine human iPSCs and airway-on-a-chip technology, to demonstrate the effectiveness of fluid shear stress (FSS) for regulating the global axis of multicellular planar cell polarity (PCP), as well as inducing ciliogenesis, thereby contributing to quantifiable unidirectional mucociliary flow. Furthermore, we applied the findings to disease modeling of primary ciliary dyskinesia (PCD), a genetic disease characterized by impaired mucociliary clearance. The application of an airway cell sheet derived from patient-derived iPSCs and their gene-edited counterparts, as well as genetic knockout iPSCs of PCD causative genes, made it possible to recapitulate the abnormal ciliary functions in organized PCP using the airway-on-a-chip. These findings suggest that the disease model of PCD developed here is a potential platform for making diagnoses and identifying therapeutic targets and that airway reconstruction therapy using mechanical stress to regulate PCP might have therapeutic value.

DOI: https://doi.org/10.1126/scitranslmed.abb1298
Sci Rep. 2021 Jul 07. 11(1): 14053

Three dimensional modeling of biologically relevant fluid shear stress in human renal tubule cells mimics in vivo transcriptional profiles.

Emily J Ross, Emily R Gordon, Hanna Sothers, Roshan Darji, Oakley Baron, Dustin Haithcock, Balabhaskar Prabhakarpandian, Kapil Pant, Richard M Myers, Sara J Cooper, Nancy J Cox.

The kidney proximal tubule is the primary site for solute reabsorption, secretion and where kidney diseases can originate, including drug-induced toxicity. Two-dimensional cell culture systems of the human proximal tubule cells (hPTCs) are often used to study these processes. However, these systems fail to model the interplay between filtrate flow, fluid shear stress (FSS), and functionality essential for understanding renal diseases and drug toxicity. The impact of FSS exposure on gene expression and effects of FSS at differing rates on gene expression in hPTCs has not been thoroughly investigated. Here, we performed RNA-sequencing of human RPTEC/TERT1 cells in a microfluidic chip-based 3D model to determine transcriptomic changes. We measured transcriptional changes following treatment of cells in this device at three different fluidic shear stress. We observed that FSS changes the expression of PTC-specific genes and impacted genes previously associated with renal diseases in genome-wide association studies (GWAS). At a physiological FSS level, we observed cell morphology, enhanced polarization, presence of cilia, and transport functions using albumin reabsorption via endocytosis and efflux transport. Here, we present a dynamic view of hPTCs response to FSS with increasing fluidic shear stress conditions and provide insight into hPTCs cellular function under biologically relevant conditions.

DOI: https://doi.org/10.1038/s41598-021-93570-5
J Mol Cell Cardiol. 2021 Jun 30. pii: S0022-2828(21)00134-6. [Epub ahead of print]

An organ-on-a-chip model for pre-clinical drug evaluation in progressive non-genetic cardiomyopathy.

Erika Yan Wang, Uros Kuzmanov, Jacob B Smith, Wenkun Dou, Naimeh Rafatian, Benjamin Fook Lun Lai, Rick Xing Ze Lu, Qinghua Wu, Joshua Yazbeck, Xiao-Ou Zhang, Yu Sun, Anthony Gramolini, Milica Radisic.

  Angiotensin II (Ang II) presents a critical mediator in various pathological conditions such as non-genetic cardiomyopathy. Osmotic pump infusion in rodents is a commonly used approach to model cardiomyopathy associated with Ang II. However, profound differences in electrophysiology and pharmacokinetics between rodent and human cardiomyocytes may limit predictability of animal-based experiments. This study investigates the application of an Organ-on-a-chip (OOC) system in modeling Ang II-induced progressive cardiomyopathy. The disease model is constructed to recapitulate myocardial response to Ang II in a temporal manner. The long-term tissue cultivation and non-invasive functional readouts enable monitoring of both acute and chronic cardiac responses to Ang II stimulation. Along with mapping of cytokine secretion and proteomic profiles, this model presents an opportunity to quantitatively measure the dynamic pathological changes that could not be otherwise identified in animals. Further, we present this model as a testbed to evaluate compounds that target Ang II-induced cardiac remodeling. Through assessing the effects of losartan, relaxin, and saracatinib, the drug screening data implicated multifaceted cardioprotective effects of relaxin in restoring contractile function and reducing fibrotic remodeling. Overall, this study provides a controllable platform where cardiac activities can be explicitly observed and tested over the pathological process. The facile and high-content screening can facilitate the evaluation of potential drug candidates in the pre-clinical stage.

Keywords:  Angiotensin II; Cardiomyopathy; Compound evaluation; Organ-on-a-chip; Progressive disease modeling; Relaxin

DOI:  https://doi.org/10.1016/j.yjmcc.2021.06.012
Sci Rep. 2021 Jul 07. 11(1): 14075

A versatile microfluidic tool for the 3D culture of HepaRG cells seeded at various stages of differentiation.

Manon Boul, Nassima Benzoubir, Antonietta Messina, Rasta Ghasemi, Ismail Ben Mosbah, Jean-Charles Duclos-Vallée, Anne Dubart-Kupperschmitt, Bruno Le Pioufle.

The development of livers-on-a-chip aims to provide pharmaceutical companies with reliable systems to perform drug screening and toxicological studies. To that end, microfluidic systems are engineered to mimic the functions and architecture of this organ. In this context we have designed a device that reproduces series of liver microarchitectures, each permitting the 3D culture of hepatocytes by confining them to a chamber that is separated from the medium conveying channel by very thin slits. We modified the structure to ensure its compatibility with the culture of hepatocytes from different sources. Our device was adapted to the migratory and adhesion properties of the human HepaRG cell line at various stages of differentiation. Using this device, it was possible to keep the cells alive for more than 14 days, during which they achieved a 3D organisation and acquired or maintained their differentiation into hepatocytes. Albumin secretion as well as functional bile canaliculi were confirmed on the liver-on-a-chip. Finally, an acetaminophen toxicological assay was performed. With its multiple micro-chambers for hepatocyte culture, this microfluidic device architecture offers a promising opportunity to provide new tools for drug screening applications.

DOI: https://doi.org/10.1038/s41598-021-92011-7
Biotechnol J. 2021 Jul 04. e2000655

Parallel and large-scale antitumor investigation using stable chemical gradient and heterotypic three-dimensional tumor coculture in a multi-layered microfluidic device.

Wenming Liu, Rui Hu, Kai Han, Meilin Sun, Dan Liu, Jinwei Zhang, Jinyi Wang.

   BACKGROUND: Cancer has been responsible for a large number of human deaths in the 21st century. Establishing a controllable, biomimetic, and large-scale analytical platform to investigate the tumor-associated pathophysiological and preclinical events, such as oncogenesis and chemotherapy, is necessary.
METHODS AND RESULTS: This study presents antitumor investigation in a parallel, large-scale, and tissue-mimicking manner based on well-constructed chemical gradients and heterotypic three-dimensional (3D) tumor cocultures using a multifunction-integrated device. The integrated microfluidic device was engineered to produce a controllable and steady chemical gradient by manipulative optimization. Array-like and size-homogeneous production of heterotypic 3D tumor cocultures with in vivo-like features, including similar tumor-stromal composition and functional phenotypic gradients of metabolic activity and viability, was successfully established. Furthermore, temporal, parallel, and high-throughput analyses of tumor behaviors in different antitumor stimulations were performed in a device based on the integrated operations involving gradient generation and coculture.
CONCLUSION: This achievement holds great potential for applications in the establishment of multifunctional tumor platforms to perform tissue-biomimetic neoplastic research and therapy assessment in the fields of oncology, bioengineering, and drug discovery. This article is protected by copyright. All rights reserved.

Keywords:  biomimetic microplatform; heterotypic coculture; integrated microfluidics; three-dimensional tumor; throughput analysis

DOI:  https://doi.org/10.1002/biot.202000655
ACS Appl Mater Interfaces. 2021 Jul 06.

On-Chip Construction of Liver Lobules with Self-Assembled Perfusable Hepatic Sinusoid Networks.

Shengnan Ya, Weiping Ding, Shibo Li, Kun Du, Yuanyuan Zhang, Chengpan Li, Jing Liu, Fenfen Li, Ping Li, Tianzhi Luo, Liqun He, Ao Xu, Dayong Gao, Bensheng Qiu.

  Although various liver chips have been developed using emerging organ-on-a-chip techniques, it remains an enormous challenge to replicate the liver lobules with self-assembled perfusable hepatic sinusoid networks. Herein we develop a lifelike bionic liver lobule chip (LLC), on which the perfusable hepatic sinusoid networks are achieved using a microflow-guided angiogenesis methodology; additionally, during and after self-assembly, oxygen concentration is regulated to mimic physiologically dissolved levels supplied by actual hepatic arterioles and venules. This liver lobule design thereby produces more bionic liver microstructures, higher metabolic abilities, and longer lasting hepatocyte function than other liver-on-a-chip techniques that are able to deliver. We found that the flow through the unique micropillar design in the cell coculture zone guides the radiating assembly of the hepatic sinusoid, the oxygen concentration affects the morphology of the sinusoid by proliferation, and the oxygen gradient plays a key role in prolonging hepatocyte function. The expected breadth of applications our LLC is suited to is demonstrated by means of preliminarily testing chronic and acute hepatotoxicity of drugs and replicating growth of tumors in situ. This work provides new insights into designing more extensive bionic vascularized liver chips, while achieving longer lasting ex-vivo hepatocyte function.

Keywords:  hepatic sinusoid; hepatocyte function; liver chip; microfluidics; oxygen concentration

DOI:  https://doi.org/10.1021/acsami.1c00794
J Biomech. 2021 Jun 30. pii: S0021-9290(21)00344-4. [Epub ahead of print]125 110564

Functional assistance for stress distribution in cell culture membrane under periodically stretching.

Zhi-Xuan Dai, Po-Jen Shih, Jia-Yush Yen, I-Jong Wang.

  Dynamic cell cultures simulate the in vivo cell environment for a regular loading system with curtain strains. However, it is difficult to obtain strains that are suitable for cells without conducting multiple trials. This study develops a device that increases the strain gradient by changing the tensile section, in order to determine the effect of various cyclic strains on cultured human keratinocytes (HK) cells. This device is used to determine the effect of 3% and 5% cyclic strain and shear strain on cell proliferation and arrangement at 1 Hz. The results show that compared with static and 3% strain, a 5% cyclic strain better inhibits the proliferation of HK cells. Compared to the initial cell attachment when there is no specific directionality, the cells are aligned in the vertical stretching direction after cyclic stretching. This equipment increases the efficiency of the experiment and more intuitively maps the cell behavior and shape to the strain field and the response to the shear strain.

Keywords:  Cyclic stretching; Finite element analysis (FEA); Human keratinocytes; Stress distribution

DOI:  https://doi.org/10.1016/j.jbiomech.2021.110564
Lab Chip. 2021 Jul 08.

Retinal organoids on-a-chip: a micro-millifluidic bioreactor for long-term organoid maintenance.

Yuntian Xue, Magdalene J Seiler, William C Tang, Jasmine Y Wang, Jeffrey Delgado, Bryce T McLelland, Gabriel Nistor, Hans S Keirstead, Andrew W Browne.

Retinal degeneration is a leading cause of vision impairment and blindness worldwide and medical care for advanced disease does not exist. Stem cell-derived retinal organoids (RtOgs) became an emerging tool for tissue replacement therapy. However, existing RtOg production methods are highly heterogeneous. Controlled and predictable methodology and tools are needed to standardize RtOg production and maintenance. In this study, we designed a shear stress-free micro-millifluidic bioreactor for nearly labor-free retinal organoid maintenance. We used a stereolithography (SLA) 3D printer to fabricate a mold from which Polydimethylsiloxane (PDMS) was cast. We optimized the chip design using in silico simulations and in vitro evaluation to optimize mass transfer efficiency and concentration uniformity in each culture chamber. We successfully cultured RtOgs at three different differentiation stages (day 41, 88, and 128) on an optimized bioreactor chip for more than 1 month. We used different quantitative and qualitative techniques to fully characterize the RtOgs produced by static dish culture and bioreactor culture methods. By analyzing the results from phase contrast microscopy, single-cell RNA sequencing (scRNA seq), quantitative polymerase chain reaction (qPCR), immunohistology, and electron microscopy, we found that bioreactor-cultured RtOgs developed cell types and morphology comparable to static cultured ones and exhibited similar retinal genes expression levels. We also evaluated the metabolic activity of RtOgs in both groups using fluorescence lifetime imaging (FLIM), and found that the outer surface region of bioreactor cultured RtOgs had a comparable free/bound NADH ratio and overall lower long lifetime species (LLS) ratio than static cultured RtOgs during imaging. To summarize, we validated an automated micro-millifluidic device with significantly reduced shear stress to produce RtOgs of comparable quality to those maintained in conventional static culture.

DOI: https://doi.org/10.1039/d1lc00011j
Nat Commun. 2021 07 06. 12(1): 4155

Coordination of endothelial cell positioning and fate specification by the epicardium.

Pearl Quijada, Michael A Trembley, Adwiteeya Misra, Jacquelyn A Myers, Cameron D Baker, Marta Pérez-Hernández, Jason R Myers, Ronald A Dirkx, Ethan David Cohen, Mario Delmar, John M Ashton, Eric M Small.

The organization of an integrated coronary vasculature requires the specification of immature endothelial cells (ECs) into arterial and venous fates based on their localization within the heart. It remains unclear how spatial information controls EC identity and behavior. Here we use single-cell RNA sequencing at key developmental timepoints to interrogate cellular contributions to coronary vessel patterning and maturation. We perform transcriptional profiling to define a heterogenous population of epicardium-derived cells (EPDCs) that express unique chemokine signatures. We identify a population of Slit2+ EPDCs that emerge following epithelial-to-mesenchymal transition (EMT), which we term vascular guidepost cells. We show that the expression of guidepost-derived chemokines such as Slit2 are induced in epicardial cells undergoing EMT, while mesothelium-derived chemokines are silenced. We demonstrate that epicardium-specific deletion of myocardin-related transcription factors in mouse embryos disrupts the expression of key guidance cues and alters EPDC-EC signaling, leading to the persistence of an immature angiogenic EC identity and inappropriate accumulation of ECs on the epicardial surface. Our study suggests that EC pathfinding and fate specification is controlled by a common mechanism and guided by paracrine signaling from EPDCs linking epicardial EMT to EC localization and fate specification in the developing heart.

DOI: https://doi.org/10.1038/s41467-021-24414-z