Three-dimensional bioprinted hepatorganoids prolong survival of mice with liver failure

ObjectiveShortage of organ donors, a critical challenge for treatment of end-stage organ failure, has motivated the development of alternative strategies to generate organs in vitro. Here, we aim to describe the hepatorganoids, which is a liver tissue model generated by three-dimensional (3D) bioprinting of HepaRG cells and investigate its liver functions in vitro and in vivo.Design3D bioprinted hepatorganoids (3DP-HOs) were constructed using HepaRG cells and bioink, according to specific 3D printing procedures. Liver functions of 3DP-HOs were detected after 7 days of differentiation in vitro, which were later transplanted into Fah-deficient mice. The in vivo liver functions of 3DP-HOs were evaluated by survival time and liver damage of mice, human liver function markers and human-specific debrisoquine metabolite production.Results3DP-HOs broadly acquired liver functions, such as ALBUMIN secretion, drug metabolism and glycogen storage after 7 days of differentiation. After transplantation into abdominal cavity of Fah-/-Rag2-/- mouse model of liver injury, 3DP-HOs further matured and displayed increased synthesis of liver-specific proteins. Particularly, the mice acquired human-specific drug metabolism activities. Functional vascular systems were also formed in transplanted 3DP-HOs, further enhancing the material transport and liver functions of 3DP-HOs. Most importantly, transplantation of 3DP-HOs significantly improved the survival of mice.ConclusionsOur results demonstrated a comprehensive proof of principle, which indicated that 3DP-HO model of liver tissues possessed in vivo hepatic functions and alleviated liver failure after transplantation, suggesting that 3D bioprinting could be used to generate human liver tissues as the alternative transplantation donors for treatment of liver diseases.

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Molecular Networking for Drug Toxicities Studies: The Case of Hydroxychloroquine in COVID-19 Patients

International Journal of Molecular Sciences ◽

10.3390/ijms23010082 ◽

2021 ◽

Vol 23 (1) ◽

pp. 82

Author(s):

Pierre-Jean Ferron ◽

Brendan Le Daré ◽

Julie Bronsard ◽

Clara Steichen ◽

Elodie Babina ◽

...

Keyword(s):

Drug Metabolism ◽

High Resolution Mass Spectrometry ◽

Study Drug ◽

University Hospital ◽

Obese Patients ◽

Molecular Networking ◽

Heparg Cells ◽

In Vitro Data

Using drugs to treat COVID-19 symptoms may induce adverse effects and modify patient outcomes. These adverse events may be further aggravated in obese patients, who often present different illnesses such as metabolic-associated fatty liver disease. In Rennes University Hospital, several drug such as hydroxychloroquine (HCQ) have been used in the clinical trial HARMONICOV to treat COVID-19 patients, including obese patients. The aim of this study is to determine whether HCQ metabolism and hepatotoxicity are worsened in obese patients using an in vivo/in vitro approach. Liquid chromatography high resolution mass spectrometry in combination with untargeted screening and molecular networking were employed to study drug metabolism in vivo (patient’s plasma) and in vitro (HepaRG cells and RPTEC cells). In addition, HepaRG cells model were used to reproduce pathophysiological features of obese patient metabolism, i.e., in the condition of hepatic steatosis. The metabolic signature of HCQ was modified in HepaRG cells cultured under a steatosis condition and a new metabolite was detected (carboxychloroquine). The RPTEC model was found to produce only one metabolite. A higher cytotoxicity of HCQ was observed in HepaRG cells exposed to exogenous fatty acids, while neutral lipid accumulation (steatosis) was further enhanced in these cells. These in vitro data were compared with the biological parameters of 17 COVID-19 patients treated with HCQ included in the HARMONICOV cohort. Overall, our data suggest that steatosis may be a risk factor for altered drug metabolism and possibly toxicity of HCQ.

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Preparation of Three-dimensional (3-D) Human Liver (HepaRG) Cultures for Histochemical and Immunohistochemical Staining and Light Microscopic Evaluation

Toxicologic Pathology ◽

10.1177/0192623318789069 ◽

2018 ◽

Vol 46 (6) ◽

pp. 653-659 ◽

Cited By ~ 3

Author(s):

Natasha P. Clayton ◽

Alanna Burwell ◽

Heather Jensen ◽

Barbara F. Williams ◽

Quashana D. Brown ◽

...

Keyword(s):

Immunohistochemical Staining ◽

Human Liver ◽

Three Dimensional ◽

Culture Systems ◽

Discovery Research ◽

Glass Slides ◽

Drug Discovery Research ◽

Microscopic Evaluation

The use of three-dimensional (3-D) in vitro culture systems (spheroids, organoids) in biomolecular and drug discovery research has become increasingly popular. The popularity is due, in part, to a diminished reliance on animal bioassays and a desire to develop physiologically relevant cell culture systems that simulate the in vivo tissue microenvironment. Most evaluations of 3-D cultures are by confocal microscopy and high-content imaging; however, these technologies do not allow for detailed cellular morphologic assessments or permit basic hematoxylin and eosin histologic evaluations. There are few studies that have reported detailed processes for preparing 3-D cultures for paraffin embedding and subsequent use for histochemical or immunohistochemical staining. In an attempt to do so, we have developed a protocol to paraffin-embed human liver spheroids that can be sectioned with a microtome and mounted onto glass slides for routine histochemical and immunohistochemical staining and light microscopic evaluations.

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3D Bioprinting for Vascularized Tissue-Engineered Bone Fabrication

Materials ◽

10.3390/ma13102278 ◽

2020 ◽

Vol 13 (10) ◽

pp. 2278 ◽

Cited By ~ 1

Author(s):

Fei Xing ◽

Zhou Xiang ◽

Pol Maria Rommens ◽

Ulrike Ritz

Keyword(s):

Three Dimensional ◽

Current Status ◽

Layer By Layer ◽

3D Bioprinting ◽

Vascular Networks ◽

Waste Products ◽

Tissue Microenvironment ◽

Spatial Architecture

Vascularization in bone tissues is essential for the distribution of nutrients and oxygen, as well as the removal of waste products. Fabrication of tissue-engineered bone constructs with functional vascular networks has great potential for biomimicking nature bone tissue in vitro and enhancing bone regeneration in vivo. Over the past decades, many approaches have been applied to fabricate biomimetic vascularized tissue-engineered bone constructs. However, traditional tissue-engineered methods based on seeding cells into scaffolds are unable to control the spatial architecture and the encapsulated cell distribution precisely, which posed a significant challenge in constructing complex vascularized bone tissues with precise biomimetic properties. In recent years, as a pioneering technology, three-dimensional (3D) bioprinting technology has been applied to fabricate multiscale, biomimetic, multi-cellular tissues with a highly complex tissue microenvironment through layer-by-layer printing. This review discussed the application of 3D bioprinting technology in the vascularized tissue-engineered bone fabrication, where the current status and unique challenges were critically reviewed. Furthermore, the mechanisms of vascular formation, the process of 3D bioprinting, and the current development of bioink properties were also discussed.

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3D Bioprinting of Vascularized Tissues for in vitro and in vivo Applications

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2021.664188 ◽

2021 ◽

Vol 9 ◽

Author(s):

Earnest P. Chen ◽

Zeren Toksoy ◽

Bruce A. Davis ◽

John P. Geibel

Keyword(s):

Tissue Engineering ◽

Drug Testing ◽

Rapid Development ◽

Three Dimensional ◽

3D Bioprinting ◽

Vascular Networks ◽

Main Challenge ◽

3D Printed

With a limited supply of organ donors and available organs for transplantation, the aim of tissue engineering with three-dimensional (3D) bioprinting technology is to construct fully functional and viable tissue and organ replacements for various clinical applications. 3D bioprinting allows for the customization of complex tissue architecture with numerous combinations of materials and printing methods to build different tissue types, and eventually fully functional replacement organs. The main challenge of maintaining 3D printed tissue viability is the inclusion of complex vascular networks for nutrient transport and waste disposal. Rapid development and discoveries in recent years have taken huge strides toward perfecting the incorporation of vascular networks in 3D printed tissue and organs. In this review, we will discuss the latest advancements in fabricating vascularized tissue and organs including novel strategies and materials, and their applications. Our discussion will begin with the exploration of printing vasculature, progress through the current statuses of bioprinting tissue/organoids from bone to muscles to organs, and conclude with relevant applications for in vitro models and drug testing. We will also explore and discuss the current limitations of vascularized tissue engineering and some of the promising future directions this technology may bring.

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Brain Organoids—A Bottom-Up Approach for Studying Human Neurodevelopment

Bioengineering ◽

10.3390/bioengineering6010009 ◽

2019 ◽

Vol 6 (1) ◽

pp. 9 ◽

Cited By ~ 10

Author(s):

Eyal Karzbrun ◽

Orly Reiner

Keyword(s):

Human Brain ◽

Three Dimensional ◽

Human Stem Cells ◽

In Vitro Systems ◽

Brain Organoid ◽

Specific Disorders ◽

Bioengineering Techniques ◽

Human Specific

Brain organoids have recently emerged as a three-dimensional tissue culture platform to study the principles of neurodevelopment and morphogenesis. Importantly, brain organoids can be derived from human stem cells, and thus offer a model system for early human brain development and human specific disorders. However, there are still major differences between the in vitro systems and in vivo development. This is in part due to the challenge of engineering a suitable culture platform that will support proper development. In this review, we discuss the similarities and differences of human brain organoid systems in comparison to embryonic development. We then describe how organoids are used to model neurodevelopmental diseases. Finally, we describe challenges in organoid systems and how to approach these challenges using complementary bioengineering techniques.

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Development and Application of an Additively Manufactured Calcium Chloride Nebulizer for Alginate 3D-Bioprinting Purposes

Journal of Functional Biomaterials ◽

10.3390/jfb9040063 ◽

2018 ◽

Vol 9 (4) ◽

pp. 63 ◽

Cited By ~ 9

Author(s):

Lukas Raddatz ◽

Antonina Lavrentieva ◽

Iliyana Pepelanova ◽

Janina Bahnemann ◽

Dominik Geier ◽

...

Keyword(s):

Cell Culture ◽

Culture Media ◽

Single Cells ◽

Matrix Material ◽

Three Dimensional ◽

Culture Cell ◽

3D Bioprinting ◽

Cacl2 Solution

Three-dimensional (3D)-bioprinting enables scientists to mimic in vivo micro-environments and to perform in vitro cell experiments under more physiological conditions than is possible with conventional two-dimensional (2D) cell culture. Cell-laden biomaterials (bioinks) are precisely processed to bioengineer tissue three-dimensionally. One primarily used matrix material is sodium alginate. This natural biopolymer provides both fine mechanical properties when gelated and high biocompatibility. Commonly, alginate is 3D bioprinted using extrusion based devices. The gelation reaction is hereby induced by a CaCl2 solution in the building chamber after material extrusion. This established technique has two main disadvantages: (1) CaCl2 can have toxic effects on the cell-laden hydrogels by oxygen diffusion limitation and (2) good printing resolution in the CaCl2 solution is hard to achieve, since the solution needs to be removed afterwards and substituted by cell culture media. Here, we show an innovative approach of alginate bioprinting based on a CaCl2 nebulizer. The device provides CaCl2 mist to the building platform inducing the gelation. The necessary amount of CaCl2 could be decreased as compared to previous gelation strategies and limitation of oxygen transfer during bioprinting can be reduced. The device was manufactured using the MJP-3D printing technique. Subsequently, its digital blueprint (CAD file) can be modified and additive manufactured easily and mounted in various extrusion bioprinters. With our approach, a concept for a more gentle 3D Bioprinting method could be shown. We demonstrated that the concept of an ultrasound-based nebulizer for CaCl2 mist generation can be used for 3D bioprinting and that the mist-induced polymerization of alginate hydrogels of different concentrations is feasible. Furthermore, different cell-laden alginate concentrations could be used: Cell spheroids (mesenchymal stem cells) and single cells (mouse fibroblasts) were successfully 3D printed yielding viable cells and stable hydrogels after 24 h cultivation. We suggest our work to show a different and novel approach on alginate bioprinting, which could be useful in generating cell-laden hydrogel constructs for e.g., drug screening or (soft) tissue engineering applications.

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Prediction of in vivo drug metabolism in the human liver from in vitro metabolism data

Pharmacology & Therapeutics ◽

10.1016/s0163-7258(96)00184-2 ◽

1997 ◽

Vol 73 (2) ◽

pp. 147-171 ◽

Cited By ~ 315

Author(s):

Takafumi Iwatsubo ◽

Noriko Hirota ◽

Tsuyoshi Ooie ◽

Hiroshi Suzuki ◽

Noriaki Shimada ◽

...

Keyword(s):

Drug Metabolism ◽

Human Liver ◽

In Vitro Metabolism ◽

In Vivo Drug Metabolism

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3D bioprinting dual-factor releasing and gradient-structured constructs ready to implant for anisotropic cartilage regeneration

Science Advances ◽

10.1126/sciadv.aay1422 ◽

2020 ◽

Vol 6 (37) ◽

pp. eaay1422 ◽

Cited By ~ 2

Author(s):

Ye Sun ◽

Yongqing You ◽

Wenbo Jiang ◽

Bo Wang ◽

Qiang Wu ◽

...

Keyword(s):

Large Scale ◽

Structural Integrity ◽

Three Dimensional ◽

Cartilage Regeneration ◽

Cartilage Tissue ◽

3D Bioprinting ◽

Deep Layers ◽

Joint Prostheses

Cartilage injury is extremely common and leads to joint dysfunction. Existing joint prostheses do not remodel with host joint tissue. However, developing large-scale biomimetic anisotropic constructs mimicking native cartilage with structural integrity is challenging. In the present study, we describe anisotropic cartilage regeneration by three-dimensional (3D) bioprinting dual-factor releasing and gradient-structured constructs. Dual-factor releasing mesenchymal stem cell (MSC)–laden hydrogels were used for anisotropic chondrogenic differentiation. Together with physically gradient synthetic biodegradable polymers that impart mechanical strength, the 3D bioprinted anisotropic cartilage constructs demonstrated whole-layer integrity, lubrication of superficial layers, and nutrient supply in deep layers. Evaluation of the cartilage tissue in vitro and in vivo showed tissue maturation and organization that may be sufficient for translation to patients. In conclusion, one-step 3D bioprinted dual-factor releasing and gradient-structured constructs were generated for anisotropic cartilage regeneration, integrating the feasibility of MSC- and 3D bioprinting–based therapy for injured or degenerative joints.

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A Novel Microplate 3D Bioprinting Platform for the Engineering of Muscle and Tendon Tissues

SLAS TECHNOLOGY Translating Life Sciences Innovation ◽

10.1177/2472630318776594 ◽

2018 ◽

Vol 23 (6) ◽

pp. 599-613 ◽

Cited By ~ 12

Author(s):

Sandra Laternser ◽

Hansjoerg Keller ◽

Olivier Leupin ◽

Martin Rausch ◽

Ursula Graf-Hausner ◽

...

Keyword(s):

Drug Screening ◽

Musculoskeletal Diseases ◽

Marker Gene ◽

Three Dimensional ◽

In Vitro Models ◽

3D Bioprinting ◽

Promising Tool ◽

Screening Platform

Two-dimensional (2D) cell cultures do not reflect the in vivo situation, and thus it is important to develop predictive three-dimensional (3D) in vitro models with enhanced reliability and robustness for drug screening applications. Treatments against muscle-related diseases are becoming more prominent due to the growth of the aging population worldwide. In this study, we describe a novel drug screening platform with automated production of 3D musculoskeletal-tendon-like tissues. With 3D bioprinting, alternating layers of photo-polymerized gelatin-methacryloyl-based bioink and cell suspension tissue models were produced in a dumbbell shape onto novel postholder cell culture inserts in 24-well plates. Monocultures of human primary skeletal muscle cells and rat tenocytes were printed around and between the posts. The cells showed high viability in culture and good tissue differentiation, based on marker gene and protein expressions. Different printing patterns of bioink and cells were explored and calcium signaling with Fluo4-loaded cells while electrically stimulated was shown. Finally, controlled co-printing of tenocytes and myoblasts around and between the posts, respectively, was demonstrated followed by co-culture and co-differentiation. This screening platform combining 3D bioprinting with a novel microplate represents a promising tool to address musculoskeletal diseases.

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