Bioengineered Cystinotic Kidney Tubules Recapitulate a Nephropathic Phenotype

Nephropathic cystinosis is a rare and severe disease caused by disruptions in the CTNS gene. Cystinosis is characterized by lysosomal cystine accumulation, vesicle trafficking impairment, oxidative stress, and apoptosis. Additionally, cystinotic patients exhibit weakening and leakage of the proximal tubular segment of the nephrons, leading to renal Fanconi syndrome and kidney failure early in life. Current in vitro cystinotic models cannot recapitulate all clinical features of the disease which limits their translational value. Therefore, the development of novel, complex in vitro models that better mimic the disease and exhibit characteristics not compatible with 2-dimensional cell culture is of crucial importance for novel therapies development. In this study, we developed a 3-dimensional bioengineered model of nephropathic cystinosis by culturing conditionally immortalized proximal tubule epithelial cells (ciPTECs) on hollow fiber membranes (HFM). Cystinotic kidney tubules showed lysosomal cystine accumulation, increased autophagy and vesicle trafficking deterioration, the impairment of several metabolic pathways, and the disruption of the epithelial monolayer tightness as compared to control kidney tubules. In particular, the loss of monolayer organization and leakage could be mimicked with the use of the cystinotic kidney tubules, which has not been possible before, using the standard 2-dimensional cell culture. Overall, bioengineered cystinotic kidney tubules recapitulate better the nephropathic phenotype at a molecular, structural, and functional proximal tubule level compared to 2-dimensional cell cultures.

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Drug toxicity in the proximal tubule: new models, methods and mechanisms

Pediatric Nephrology ◽

10.1007/s00467-021-05121-9 ◽

2021 ◽

Author(s):

Andrew M. Hall ◽

Francesco Trepiccione ◽

Robert J. Unwin

Keyword(s):

Proximal Tubule ◽

Drug Toxicity ◽

Clinical Manifestations ◽

Kidney Injury ◽

In Vitro Models ◽

Cellular Mechanisms ◽

Renal Fanconi Syndrome ◽

Systemic Complications ◽

New Biomarkers

AbstractThe proximal tubule (PT) reabsorbs most of the glomerular filtrate and plays an important role in the uptake, metabolism and excretion of xenobiotics. Some therapeutic drugs are harmful to the PT, and resulting nephrotoxicity is thought to be responsible for approximately 1 in 6 of cases of children hospitalized with acute kidney injury (AKI). Clinically, PT dysfunction leads to urinary wasting of important solutes normally reabsorbed by this nephron segment, leading to systemic complications such as bone demineralization and a clinical scenario known as the renal Fanconi syndrome (RFS). While PT defects can be diagnosed using a combination of blood and urine markers, including urinary excretion of low molecular weight proteins (LMWP), standardized definitions of what constitutes clinically significant toxicity are lacking, and identifying which patients will go on to develop progressive loss of kidney function remains a major challenge. In addition, much of our understanding of cellular mechanisms of drug toxicity is still limited, partly due to the constraints of available cell and animal models. However, advances in new and more sophisticated in vitro models of the PT, along with the application of high-content analytical methods that can provide readouts more relevant to the clinical manifestations of nephrotoxicity, are beginning to extend our knowledge. Such technical progress should help in discovering new biomarkers that can better detect nephrotoxicity earlier and predict its long-term consequences, and herald a new era of more personalized medicine.

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Interstitial Flow Recapitulates Gemcitabine Chemoresistance in A 3D Microfluidic Pancreatic Ductal Adenocarcinoma Model by Induction of Multidrug Resistance Proteins

International Journal of Molecular Sciences ◽

10.3390/ijms20184647 ◽

2019 ◽

Vol 20 (18) ◽

pp. 4647 ◽

Cited By ~ 6

Author(s):

Bart Kramer ◽

Luuk de Haan ◽

Marjolein Vermeer ◽

Thomas Olivier ◽

Thomas Hankemeier ◽

...

Keyword(s):

Cell Culture ◽

Multidrug Resistance ◽

Pancreatic Ductal Adenocarcinoma ◽

3D Cell Culture ◽

In Vitro Models ◽

Ductal Adenocarcinoma ◽

Interstitial Flow ◽

Multidrug Resistance Proteins ◽

And Function

Pancreatic Ductal Adenocarcinoma (PDAC) is one of the most lethal cancers due to a high chemoresistance and poor vascularization, which results in an ineffective systemic therapy. PDAC is characterized by a high intratumoral pressure, which is not captured by current 2D and 3D in vitro models. Here, we demonstrated a 3D microfluidic interstitial flow model to mimic the intratumoral pressure in PDAC. We found that subjecting the S2-028 PDAC cell line to interstitial flow inhibits the proliferation, while maintaining a high viability. We observed increased gemcitabine chemoresistance, with an almost nine-fold higher EC50 as compared to a monolayer culture (31 nM versus 277 nM), and an alleviated expression and function of the multidrug resistance protein (MRP) family. In conclusion, we developed a 3D cell culture modality for studying intratissue pressure and flow that exhibits more predictive capabilities than conventional 2D cell culture and is less time-consuming, and more scalable and accessible than animal models. This increase in microphysiological relevance might support improved efficiency in the drug development pipeline.

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In vitro Hair Follicle Regeneration by Three-Dimensional Cell Culture Method Simulating in vivo cellular organization

The Proceedings of the Bioengineering Conference Annual Meeting of BED/JSME ◽

10.1299/jsmebio.2017.29.2d35 ◽

2017 ◽

Vol 2017.29 (0) ◽

pp. 2D35

Author(s):

Takahiro SUMI ◽

Aki SUGENO ◽

Shogo MIYATA ◽

Hanako YAZAWA ◽

Hajime INOUE

Keyword(s):

Cell Culture ◽

Hair Follicle ◽

Three Dimensional ◽

Culture Method ◽

Cellular Organization ◽

Dimensional Cell

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Tailoring 3D cell culture templates with common hydrogels

10.1101/370882 ◽

2018 ◽

Cited By ~ 1

Author(s):

Aurélien Pasturel ◽

Pierre-Olivier Strale ◽

Vincent Studer

Keyword(s):

Cell Culture ◽

3D Cell Culture ◽

In Vitro Models ◽

Manufacturing Method ◽

Biologically Relevant ◽

Physiological Conditions ◽

Subtractive Manufacturing ◽

User Friendly

3D cell culture aims at reconciliating the simplicity of in vitro models with the human like properties encountered in vivo. Soft permeable hydrogels have emerged as user-friendly materials to grow cells in more physiological conditions. With the intent on turning these homogeneous substrates into biomimetic templates, we introduce a generic solution compatible with the most biologically relevant and often frail materials. Here we take control of the chemical environment driving generic radical reactions to craft common gels with patterned light. In a simple microreactor, we harness the well-known inhibition of radicals by oxygen to enable topographical photopolymerization. Strikingly, by sustaining an oxygen rich environment, we can also induce hydrogel photo-scission which turns out to be a powerful and generic subtractive manufacturing method. We finally introduce a flexible patterned functionalization protocol based on available photo-linkers. Using these common tools on the most popular hydrogels, we tailored soft templates where cells grow or self-organize into standardized structures. The platform we describe has the potential to set a standard in future 3D cell culture experiments.

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Biomaterials for Three-Dimensional Cell Culture: From Applications in Oncology to Nanotechnology

Nanomaterials ◽

10.3390/nano11020481 ◽

2021 ◽

Vol 11 (2) ◽

pp. 481

Author(s):

Tarek Saydé ◽

Omar El Hamoui ◽

Bruno Alies ◽

Karen Gaudin ◽

Gaëtane Lespes ◽

...

Keyword(s):

Cell Culture ◽

Complex Structure ◽

Tumor Development ◽

Three Dimensional ◽

3D Culture ◽

Supramolecular Assembly ◽

Hydrogel Scaffold ◽

Scale Characterization ◽

Dimensional Cell

Three-dimensional cell culture has revolutionized cellular biology research and opened the door to novel discoveries in terms of cellular behavior and response to microenvironment stimuli. Different types of 3D culture exist today, including hydrogel scaffold-based models, which possess a complex structure mimicking the extracellular matrix. These hydrogels can be made of polymers (natural or synthetic) or low-molecular weight gelators that, via the supramolecular assembly of molecules, allow the production of a reproducible hydrogel with tunable mechanical properties. When cancer cells are grown in this type of hydrogel, they develop into multicellular tumor spheroids (MCTS). Three-dimensional (3D) cancer culture combined with a complex microenvironment that consists of a platform to study tumor development and also to assess the toxicity of physico-chemical entities such as ions, molecules or particles. With the emergence of nanoparticles of different origins and natures, implementing a reproducible in vitro model that consists of a bio-indicator for nano-toxicity assays is inevitable. However, the maneuver process of such a bio-indicator requires the implementation of a repeatable system that undergoes an exhaustive follow-up. Hence, the biggest challenge in this matter is the reproducibility of the MCTS and the associated full-scale characterization of this system’s components.

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Alginate-Based Three-Dimensional In Vitro Tumor Models: A Better Alternative to Current Two-Dimensional Cell Culture Models

Springer Series in Biomaterials Science and Engineering - Alginates and Their Biomedical Applications ◽

10.1007/978-981-10-6910-9_6 ◽

2017 ◽

pp. 157-183 ◽

Cited By ~ 3

Author(s):

Amit Khurana ◽

Chandraiah Godugu

Keyword(s):

Cell Culture ◽

Three Dimensional ◽

Two Dimensional ◽

Tumor Models ◽

Culture Models ◽

Cell Culture Models ◽

Dimensional Cell

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A novel in vitro permeability assay using three-dimensional cell culture system

Journal of Biotechnology ◽

10.1016/j.jbiotec.2014.12.019 ◽

2015 ◽

Vol 205 ◽

pp. 93-100 ◽

Cited By ~ 10

Author(s):

Jong Bong Lee ◽

Sung Hwa Son ◽

Min Chul Park ◽

Tae Hwan Kim ◽

Min Gi Kim ◽

...

Keyword(s):

Cell Culture ◽

Culture System ◽

Three Dimensional ◽

Cell Culture System ◽

Permeability Assay ◽

Dimensional Cell

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A Simple Aspect Ratio Dependent Method of Patterning Microwells for Selective Cell Attachment

2018 Design of Medical Devices Conference ◽

10.1115/dmd2018-6811 ◽

2018 ◽

Author(s):

Erik A. Zavrel ◽

Michael L. Shuler ◽

Xiling Shen

Keyword(s):

Cell Culture ◽

Cell Attachment ◽

In Vitro Models ◽

Contact Printing ◽

Deep Well ◽

Biomimetic Approach ◽

Authentic Environment ◽

And Control

3-D culture has been shown to provide cells with a more physiologically authentic environment than traditional 2-D (planar) culture [1, 2]. 3-D cues allow cells to exhibit more realistic functions and behaviors, e.g., adhesion, spreading, migration, metabolic activity, and differentiation. Knowledge of changes in cell morphology, mechanics, and mobility in response to geometrical cues and topological stimuli is important for understanding normal and pathological cell development [3]. Microfabrication provides unique in vitro approaches to recapitulating in vivo conditions due to the ability to precisely control the cellular microenvironment [4, 5]. Microwell arrays have emerged as robust alternatives to traditional 2D cell culture substrates as they are relatively simple and compatible with existing laboratory techniques and instrumentation [6, 7]. In particular, microwells have been adopted as a biomimetic approach to modeling the unique micro-architecture of the epithelial lining of the gastrointestinal (GI) tract [8–10]. The inner (lumen-facing) surface of the intestine has a convoluted topography consisting of finger-like projections (villi) with deep well-like invaginations (crypts) between them. The dimensions of villi and crypts are on the order of hundreds of microns (100–700 μm in height and 50–250 μm in diameter) [11]. While microwells have proven important in the development of physiologically realistic in vitro models of human intestine, existing methods of ensuring their surface is suitable for cell culture are lacking. Sometimes it is desirable to selectively seed cells within microwells and confine or restrict them to the microwells in which they are seeded. Existing methods of patterning microwells for cell attachment either lack selectivity, meaning cells can adhere and migrate anywhere on the microwell array, i.e., inside microwells or outside of them, or necessitate sophisticated techniques such as micro-contact printing, which requires precise alignment and control to selectively pattern the bottoms of microwells for cell attachment [12, 13].

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