Effect of biochemical and biomechanical factors on vascularization of kidney organoid-on-a-chip

AbstractKidney organoids derived from the human pluripotent stem cells (hPSCs) recapitulating human kidney are the attractive tool for kidney regeneration, disease modeling, and drug screening. However, the kidney organoids cultured by static conditions have the limited vascular networks and immature nephron-like structures unlike human kidney. Here, we developed a kidney organoid-on-a-chip system providing fluidic flow mimicking shear stress with optimized extracellular matrix (ECM) conditions. We demonstrated that the kidney organoids cultured in our microfluidic system showed more matured podocytes and vascular structures as compared to the static culture condition. Additionally, the kidney organoids cultured in microfluidic systems showed higher sensitivity to nephrotoxic drugs as compared with those cultured in static conditions. We also demonstrated that the physiological flow played an important role in maintaining a number of physiological functions of kidney organoids. Therefore, our kidney organoid-on-a-chip system could provide an organoid culture platform for in vitro vascularization in formation of functional three-dimensional (3D) tissues.

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Maturation of Nephrons by Implanting hPSC-Derived Kidney Progenitors Under Kidney Capsules of Unilaterally Nephrectomized Mice

10.21203/rs.3.rs-847264/v1 ◽

2021 ◽

Author(s):

Xin Yu ◽

Shan Jiang ◽

Kailin Li ◽

Xianzhen Yang ◽

Zhihe Xu ◽

...

Keyword(s):

Disease Modeling ◽

Vascular System ◽

Human Kidney ◽

Target Cells ◽

Mice Model ◽

Immunodeficient Mice ◽

Filtration Barrier ◽

Kidney Organoids

Abstract Background Human pluripotent stem cell (hPSCs)-derived kidney organoids may contribute to disease modeling and generation of kidney replacement tissues. However, realization of such applications requires the induction of hPSCs into functional mature organoids. One of the key questions for this process is whether a specific vascular system exists for nephrogenesis. Our previous study showed that implantation of hPSC-derived organoids below the kidney capsules of unilaterally nephrectomized immunodeficient mice for a short-term (2 weeks) resulted in the enlargement of organoids and production of vascular cells, although signs of maturation were lacking. Methods In this study, organoids are induced in vitro during 15 days and then sub-capsularly grafted into kidneys, we used the same unilaterally nephrectomized immunodeficient mice model to examine whether a medium -term (4 weeks) implantation could improve organoid maturation and vascularization, as evaluated by immunofluorescence and transmission electron microscopy(TEM). Results We demonstrate that after 2–4 weeks implantation, implanted renal organoids can form host-derived vascularization and mature in the absence of any exogenous vascular endothelial growth factor. Glomerular filtration barrier maturation was evidenced by glomerular basement membrane deposition, perforated glomerular endothelial cell development, as well as apical to basal podocyte polarization. A polarized monolayer epithelium and extensive brush border were also observed for tubular epithelial cells. Conclusions Our results indicate that the in vivo microenvironment is important for the maturation of human kidney organoids. Stromal expansion and a reduction of nephron structures were observed following longer-term (12 weeks) implantation,suggesting effects on off-target cells during the induction process. Accordingly, induction efficiency and transplantation models should be improved in the future.

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Rapid target validation in a Cas9-inducible hiPSC derived kidney model

Scientific Reports ◽

10.1038/s41598-021-95986-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Yasaman Shamshirgaran ◽

Anna Jonebring ◽

Anna Svensson ◽

Isabelle Leefa ◽

Mohammad Bohlooly-Y ◽

...

Keyword(s):

High Efficiency ◽

Disease Modeling ◽

Genetic Manipulation ◽

Disease Model ◽

Genetically Engineered ◽

Model Systems ◽

Target Validation ◽

Direct Differentiation ◽

Kidney Organoids

AbstractRecent advances in induced pluripotent stem cells (iPSCs), genome editing technologies and 3D organoid model systems highlight opportunities to develop new in vitro human disease models to serve drug discovery programs. An ideal disease model would accurately recapitulate the relevant disease phenotype and provide a scalable platform for drug and genetic screening studies. Kidney organoids offer a high cellular complexity that may provide greater insights than conventional single-cell type cell culture models. However, genetic manipulation of the kidney organoids requires prior generation of genetically modified clonal lines, which is a time and labor consuming procedure. Here, we present a methodology for direct differentiation of the CRISPR-targeted cell pools, using a doxycycline-inducible Cas9 expressing hiPSC line for high efficiency editing to eliminate the laborious clonal line generation steps. We demonstrate the versatile use of genetically engineered kidney organoids by targeting the autosomal dominant polycystic kidney disease (ADPKD) genes: PKD1 and PKD2. Direct differentiation of the respective knockout pool populations into kidney organoids resulted in the formation of cyst-like structures in the tubular compartment. Our findings demonstrated that we can achieve > 80% editing efficiency in the iPSC pool population which resulted in a reliable 3D organoid model of ADPKD. The described methodology may provide a platform for rapid target validation in the context of disease modeling.

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Human iPSC-Based Modeling of Central Nerve System Disorders for Drug Discovery

International Journal of Molecular Sciences ◽

10.3390/ijms22031203 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1203

Author(s):

Lu Qian ◽

Julia TCW

Keyword(s):

Drug Discovery ◽

Disease Modeling ◽

Three Dimensional ◽

Structural Complexity ◽

Induced Pluripotent Stem Cell ◽

Cell Types ◽

Central Nerve System ◽

Human Ipscs ◽

Nerve System

A high-throughput drug screen identifies potentially promising therapeutics for clinical trials. However, limitations that persist in current disease modeling with limited physiological relevancy of human patients skew drug responses, hamper translation of clinical efficacy, and contribute to high clinical attritions. The emergence of induced pluripotent stem cell (iPSC) technology revolutionizes the paradigm of drug discovery. In particular, iPSC-based three-dimensional (3D) tissue engineering that appears as a promising vehicle of in vitro disease modeling provides more sophisticated tissue architectures and micro-environmental cues than a traditional two-dimensional (2D) culture. Here we discuss 3D based organoids/spheroids that construct the advanced modeling with evolved structural complexity, which propels drug discovery by exhibiting more human specific and diverse pathologies that are not perceived in 2D or animal models. We will then focus on various central nerve system (CNS) disease modeling using human iPSCs, leading to uncovering disease pathogenesis that guides the development of therapeutic strategies. Finally, we will address new opportunities of iPSC-assisted drug discovery with multi-disciplinary approaches from bioengineering to Omics technology. Despite technological challenges, iPSC-derived cytoarchitectures through interactions of diverse cell types mimic patients’ CNS and serve as a platform for therapeutic development and personalized precision medicine.

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Three-dimensional bone formation including vascular networks derived from dental pulp stem cells in vitro

Human Cell ◽

10.1007/s13577-018-00228-y ◽

2018 ◽

Vol 32 (2) ◽

pp. 114-124

Author(s):

Miho Watanabe ◽

Akihiro Ohyama ◽

Hiroshi Ishikawa ◽

Akira Tanaka

Keyword(s):

Stem Cells ◽

Bone Formation ◽

Dental Pulp ◽

Three Dimensional ◽

Dental Pulp Stem Cells ◽

Vascular Networks

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Gravity-Based Flow Efficient Perfusion Culture System for Spheroids Mimicking Liver Inflammation

Biomedicines ◽

10.3390/biomedicines9101369 ◽

2021 ◽

Vol 9 (10) ◽

pp. 1369

Author(s):

Young-Su Kim ◽

Arun Asif ◽

Abdul Rahim Chethikkattuveli Salih ◽

Jae-Wook Lee ◽

Ki-Nam Hyun ◽

...

Keyword(s):

Culture System ◽

Disease Modeling ◽

Perfusion Culture ◽

Drug Analysis ◽

Culture Conditions ◽

Current Approach ◽

Spheroid Culture ◽

Organ Specific ◽

Static Conditions

The spheroid culture system provides an efficient method to emulate organ-specific pathophysiology, overcoming the traditional two-dimensional (2D) cell culture limitations. The intervention of microfluidics in the spheroid culture platform has the potential to enhance the capacity of in vitro microphysiological tissues for disease modeling. Conventionally, spheroid culture is carried out in static conditions, making the media nutrient-deficient around the spheroid periphery. The current approach tries to enhance the capacity of the spheroid culture platform by integrating the perfusion channel for dynamic culture conditions. A pro-inflammatory hepatic model was emulated using a coculture of HepG2 cell line, fibroblasts, and endothelial cells for validating the spheroid culture plate with a perfusable channel across the spheroid well. Enhanced proliferation and metabolic capacity of the microphysiological model were observed and further validated by metabolic assays. A comparative analysis of static and dynamic conditions validated the advantage of spheroid culture with dynamic media flow. Hepatic spheroids were found to have improved proliferation in dynamic flow conditions as compared to the static culture platform. The perfusable culture system for spheroids is more physiologically relevant as compared to the static spheroid culture system for disease and drug analysis.

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3D kidney organoids for bench-to-bedside translation

Journal of Molecular Medicine ◽

10.1007/s00109-020-01983-y ◽

2020 ◽

Author(s):

Navin Gupta✉ ◽

Emre Dilmen ◽

Ryuji Morizane

Keyword(s):

Collecting Duct ◽

Developmental Toxicity ◽

Human Kidney ◽

Great Promise ◽

Duct Systems ◽

Recent Developments ◽

Induced Pluripotent ◽

Kidney Organoids

Abstract The kidneys are essential organs that filter the blood, removing urinary waste while maintaining fluid and electrolyte homeostasis. Current conventional research models such as static cell cultures and animal models are insufficient to grasp the complex human in vivo situation or lack translational value. To accelerate kidney research, novel research tools are required. Recent developments have allowed the directed differentiation of induced pluripotent stem cells to generate kidney organoids. Kidney organoids resemble the human kidney in vitro and can be applied in regenerative medicine and as developmental, toxicity, and disease models. Although current studies have shown great promise, challenges remain including the immaturity, limited reproducibility, and lack of perfusable vascular and collecting duct systems. This review gives an overview of our current understanding of nephrogenesis that enabled the generation of kidney organoids. Next, the potential applications of kidney organoids are discussed followed by future perspectives. This review proposes that advancement in kidney organoid research will be facilitated through our increasing knowledge on nephrogenesis and combining promising techniques such as organ-on-a-chip models.

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Soft robotic constrictor for in vitro modeling of dynamic tissue compression

Scientific Reports ◽

10.1038/s41598-021-94769-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Jungwook Paek ◽

Joseph W. Song ◽

Ehsan Ban ◽

Yuma Morimitsu ◽

Chinedum O. Osuji ◽

...

Keyword(s):

Three Dimensional ◽

Physiological Changes ◽

Vascular Networks ◽

Human Endothelial Cells ◽

In Vitro Modeling ◽

Tissue Microenvironment ◽

Cyclic Compression ◽

Applied Forces ◽

New Research

AbstractHere we present a microengineered soft-robotic in vitro platform developed by integrating a pneumatically regulated novel elastomeric actuator with primary culture of human cells. This system is capable of generating dynamic bending motion akin to the constriction of tubular organs that can exert controlled compressive forces on cultured living cells. Using this platform, we demonstrate cyclic compression of primary human endothelial cells, fibroblasts, and smooth muscle cells to show physiological changes in their morphology due to applied forces. Moreover, we present mechanically actuatable organotypic models to examine the effects of compressive forces on three-dimensional multicellular constructs designed to emulate complex tissues such as solid tumors and vascular networks. Our work provides a preliminary demonstration of how soft-robotics technology can be leveraged for in vitro modeling of complex physiological tissue microenvironment, and may enable the development of new research tools for mechanobiology and related areas.

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Renal reabsorption in 3D vascularized proximal tubule models

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1815208116 ◽

2019 ◽

Vol 116 (12) ◽

pp. 5399-5404 ◽

Cited By ~ 68

Author(s):

Neil Y. C. Lin ◽

Kimberly A. Homan ◽

Sanlin S. Robinson ◽

David B. Kolesky ◽

Nathan Duarte ◽

...

Keyword(s):

Proximal Tubule ◽

Disease Modeling ◽

Three Dimensional ◽

Kidney Tissue ◽

Renal Reabsorption ◽

Native Kidney ◽

Glucose Reabsorption ◽

Diseased State ◽

And Function

Three-dimensional renal tissues that emulate the cellular composition, geometry, and function of native kidney tissue would enable fundamental studies of filtration and reabsorption. Here, we have created 3D vascularized proximal tubule models composed of adjacent conduits that are lined with confluent epithelium and endothelium, embedded in a permeable ECM, and independently addressed using a closed-loop perfusion system to investigate renal reabsorption. Our 3D kidney tissue allows for coculture of proximal tubule epithelium and vascular endothelium that exhibits active reabsorption via tubular–vascular exchange of solutes akin to native kidney tissue. Using this model, both albumin uptake and glucose reabsorption are quantified as a function of time. Epithelium–endothelium cross-talk is further studied by exposing proximal tubule cells to hyperglycemic conditions and monitoring endothelial cell dysfunction. This diseased state can be rescued by administering a glucose transport inhibitor. Our 3D kidney tissue provides a platform for in vitro studies of kidney function, disease modeling, and pharmacology.

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Spheroids as a Type of Three-Dimensional Cell Cultures—Examples of Methods of Preparation and the Most Important Application

International Journal of Molecular Sciences ◽

10.3390/ijms21176225 ◽

2020 ◽

Vol 21 (17) ◽

pp. 6225 ◽

Cited By ~ 1

Author(s):

Kamila Białkowska ◽

Piotr Komorowski ◽

Maria Bryszewska ◽

Katarzyna Miłowska

Keyword(s):

Cell Cultures ◽

Cell Biology ◽

Disease Modeling ◽

Three Dimensional ◽

3D Culture ◽

Matrix Interactions ◽

Vitro Model ◽

Extracellular Matrix Interactions ◽

Natural Cell

Cell cultures are very important for testing materials and drugs, and in the examination of cell biology and special cell mechanisms. The most popular models of cell culture are two-dimensional (2D) as monolayers, but this does not mimic the natural cell environment. Cells are mostly deprived of cell–cell and cell–extracellular matrix interactions. A much better in vitro model is three-dimensional (3D) culture. Because many cell lines have the ability to self-assemble, one 3D culturing method is to produce spheroids. There are several systems for culturing cells in spheroids, e.g., hanging drop, scaffolds and hydrogels, and these cultures have their applications in drug and nanoparticles testing, and disease modeling. In this paper we would like to present methods of preparation of spheroids in general and emphasize the most important applications.

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