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You are researching: In Vitro Models
Cell Type
Tissue and Organ Biofabrication
Skin Tissue Engineering
Drug Delivery
Biological Molecules
Solid Dosage Drugs
Stem Cells
Personalised Pharmaceuticals
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Drug Discovery
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All Groups
- Review Paper
- Printing Technology
- Biomaterial
- Non-cellularized gels/pastes
- Carbopol
- Epoxy
- poly (ethylene-co -vinyl acetate) (PEVA)
- Poly(itaconate-co-citrate-cooctanediol) (PICO)
- Poly(N-isopropylacrylamide) (PNIPAAm)
- Mineral Oil
- poly(octanediol-co-maleic anhydride-co-citrate) (POMaC)
- Poly(Oxazoline)
- Poly(trimethylene carbonate)
- 2-hydroxyethyl) methacrylate (HEMA)
- Zein
- Acrylamide
- Pluronic – Poloxamer
- Polyisobutylene
- Paraffin
- Silicone
- Konjac Gum
- Polyphenylene Oxide
- Ionic Liquids
- Polyvinylpyrrolidone (PVP)
- Gelatin-Sucrose Matrix
- Salt-based
- Chlorella Microalgae
- Acrylates
- Poly(Vinyl Formal)
- 2-hydroxyethyl-methacrylate (HEMA)
- Phenylacetylene
- Magnetorheological fluid (MR fluid – MRF)
- Salecan
- Poly(vinyl alcohol) (PVA)
- PEDOT
- Jeffamine
- Polyethylene
- SEBS
- Micro/nano-particles
- Biological Molecules
- Bioinks
- Methacrylated hyaluronic acid (HAMA)
- Pectin
- Silk Fibroin
- Pyrogallol
- Xanthan Gum
- Fibrinogen
- Fibrin
- Paeoniflorin
- Fibronectin
- (2-Hydroxypropyl)methacrylamide (HPMA)
- Methacrylated Collagen (CollMA)
- Carrageenan
- Glucosamine
- Chitosan
- Glycerol
- Poly(glycidol)
- Alginate
- Agarose
- Gelatin-Methacryloyl (GelMA)
- methacrylated chondroitin sulfate (CSMA)
- Cellulose
- Novogel
- Hyaluronic Acid
- Peptide gel
- Methacrylated Silk Fibroin
- Polyethylene glycol (PEG) based
- α-Bioink
- Collagen
- Elastin
- Heparin
- Gelatin
- Matrigel
- Gellan Gum
- Methacrylated Chitosan
- Ceramics
- Decellularized Extracellular Matrix (dECM)
- Metals
- Solid Dosage Drugs
- Thermoplastics
- Non-cellularized gels/pastes
- Bioprinting Technologies
- Bioprinting Applications
- Cell Type
- Endothelial
- CardioMyocites
- Melanocytes
- Retinal
- Chondrocytes
- Embrionic Kidney (HEK)
- Corneal Stromal Cells
- Fibroblasts
- β cells
- Myoblasts
- Pericytes
- Hepatocytes
- Cancer Cell Lines
- Bacteria
- Articular cartilage progenitor cells (ACPCs)
- Tenocytes
- Osteoblasts
- Monocytes
- Mesothelial cells
- Epithelial
- Neutrophils
- Adipocytes
- Human Umbilical Vein Endothelial Cells (HUVECs)
- Organoids
- Stem Cells
- Spheroids
- Meniscus Cells
- Synoviocytes
- Keratinocytes
- Skeletal Muscle-Derived Cells (SkMDCs)
- Neurons
- Macrophages
- Human Trabecular Meshwork Cells
- Institution
- University of Manchester
- University of Bucharest
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- Hong Kong University
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- University of Nottingham
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- Rice University
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- University of Central Florida
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- Chalmers University of Technology
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- University of Freiburg
- Helmholtz Institute for Pharmaceutical Research Saarland
- AO Research Institute (ARI)
- Shanghai University
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- University of Toronto
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- University of Nantes
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- University of Michigan – School of Dentistry
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- Harbin Institute of Technology
- University of Amsterdam
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- National Institutes of Health (NIH)
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- Rizzoli Orthopaedic Institute
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- Biomaterials & Bioinks
- Application
- Bioelectronics
- Biomaterial Processing
- Tissue Models – Drug Discovery
- Industrial
- Drug Discovery
- In Vitro Models
- Robotics
- Electronics – Robotics – Industrial
- Medical Devices
- Tissue and Organ Biofabrication
- Nerve – Neural Tissue Engineering
- Meniscus Tissue Engineering
- Heart – Cardiac Patches Tissue Engineering
- Adipose Tissue Engineering
- Trachea Tissue Engineering
- Ocular Tissue Engineering
- Intervertebral Disc (IVD) Tissue Engineering
- Muscle Tissue Engineering
- Liver tissue Engineering
- Cartilage Tissue Engineering
- Bone Tissue Engineering
- Drug Delivery
- Skin Tissue Engineering
- Vascularization
- BioSensors
- Personalised Pharmaceuticals
AUTHOR
Title
Shaping Synthetic Multicellular and Complex Multimaterial Tissues via Embedded Extrusion-Volumetric Printing of Microgels
[Abstract]
Year
2023
Journal/Proceedings
Advanced Materials
Reftype
Groups
AbstractAbstract In living tissues, cells express their functions following complex signals from their surrounding microenvironment. Capturing both hierarchical architectures at the micro- and macroscale, and anisotropic cell patterning remains a major challenge in bioprinting, and a bottleneck towards creating physiologically-relevant models. Addressing this limitation, we introduced a novel technique, termed Embedded Extrusion-Volumetric Printing (EmVP), converging extrusion-bioprinting and layer-less, ultra-fast volumetric bioprinting, allowing to spatially pattern multiple inks/cell types. Light-responsive microgels were developed for the first time as bioresins (μResins) for light-based volumetric bioprinting, providing a microporous environment permissive for cell homing and self-organization. Tuning the mechanical and optical properties of gelatin-based microparticles enables their use as support bath for suspended extrusion printing, in which features containing high cell densities can be easily introduced. μResins can be sculpted within seconds with tomographic light projections into centimetre-scale, granular hydrogel-based, convoluted constructs. Interstitial microvoids enhanced differentiation of multiple stem/progenitor cells (vascular, mesenchymal, neural), otherwise not possible with conventional bulk hydrogels. As proof-of-concept, EmVP was applied to create complex synthetic biology-inspired intercellular communication models, where adipocyte differentiation is regulated by optogenetic-engineered pancreatic cells. Overall, EmVP offers new avenues for producing regenerative grafts with biological functionality, and for developing engineered living systems and (metabolic) disease models. This article is protected by copyright. All rights reserved
AUTHOR
Title
3D bioprinted, vascularized neuroblastoma tumor environment in fluidic chip devices for precision medicine drug testing
[Abstract]
Year
2022
Journal/Proceedings
Biofabrication
Reftype
DOI/URL
DOI
Groups
AbstractNeuroblastoma is an extracranial solid tumor which develops in early childhood and still has a poor prognosis. One strategy to increase cure rates is the identification of patient-specific drug responses in tissue models that mimic the interaction between patient cancer cells and tumor environment. We therefore developed a perfused and micro-vascularized tumor-environment model that is directly bioprinted into custom-manufactured fluidic chips. A gelatin-methacrylate/fibrin-based matrix containing multiple cell types mimics the tumor-microenvironment that promotes spontaneous micro-vessel formation by embedded endothelial cells. We demonstrate that both, adipocyte- and iPSC-derived mesenchymal stem cells can guide this process. Bioprinted channels are coated with endothelial cells post printing to form a dense vessel - tissue barrier. The tissue model thereby mimics structure and function of human soft tissue with endothelial cell-coated larger vessels for perfusion and micro-vessel networks within the hydrogel-matrix. Patient-derived neuroblastoma spheroids are added to the matrix during the printing process and grown for more than two weeks. We demonstrate that micro-vessels are attracted by and grow into tumor spheroids and that neuroblastoma cells invade the tumor-environment as soon as the spheroids disrupt. In summary, we describe the first bioprinted, micro-vascularized neuroblastoma – tumor-environment model directly printed into fluidic chips and a novel medium-throughput biofabrication platform suitable for studying tumor angiogenesis and metastasis in precision medicine approaches in future.
AUTHOR
Title
Macromolecular crowding tuned extracellular matrix deposition in a bioprinted human rhabdomyosarcoma model
[Abstract]
Year
2022
Journal/Proceedings
Bioprinting
Reftype
Groups
AbstractThe role of the extracellular matrix (ECM) in tumor recurrence and metastasis has been gaining attention. Indeed, not only cellular, but also structural proteins influence migratory and invasive capacity of tumor cells, including growth and resistance to drugs. Therefore, new in vitro tumor models that entail improved ECM formation and deposition are needed. Here, we are developed three-dimensional (3D) models of pediatric soft tissue sarcoma (Rhabdomyosarcoma [RMS]) with the two major subgroups, the embryonal (ERMS) and the alveolar (ARMS) form. We applied macromolecular crowding (MMC) technology to monolayer cultures, spheroids, and 3D bioprinted constructs. In all culture models, exposure to MMC significantly increased ECM deposition. Interestingly, bioprinted constructs showed a collagen and fibronectin matrix architecture that was comparable to that of tumor xenografts. Furthermore, the bioprinted model not only showed tumor cell growth inside the structure but also displayed cell clusters leaving the edges of the bioprinted construct, probably emulating a metastatic mechanism. ARMS and ERMS cells reacted differently in the bioprinted structure. Indeed, the characteristic metastatic behavior was much more pronounced in the more aggressive ARMS subtype. This promising approach opens new avenues for studying RMS microenvironment and creating a platform for cancer drug testing including the native tumor ECM.
AUTHOR
Year
2021
Journal/Proceedings
Macromolecular Bioscience
Reftype
DOI/URL
DOI
Groups
AbstractAbstract There is a need for long-lived hepatic in vitro models to better predict drug induced liver injury (DILI). Human liver-derived epithelial organoids are a promising cell source for advanced in vitro models. Here, organoid technology is combined with biofabrication techniques, which holds great potential for the design of in vitro models with complex and customizable architectures. Here, porous constructs with human hepatocyte-like cells derived from organoids are generated using extrusion-based printing technology. Cell viability of bioprinted organoids remains stable for up to ten days (88–107% cell viability compared to the day of printing). The expression of hepatic markers, transporters, and phase I enzymes increased compared to undifferentiated controls, and is comparable to non-printed controls. Exposure to acetaminophen, a well-known hepatotoxic compound, decreases cell viability of bioprinted liver organoids to 21–51% (p < 0.05) compared to the start of exposure, and elevated levels of damage marker miR-122 are observed in the culture medium, indicating the potential use of the bioprinted constructs for toxicity testing. In conclusion, human liver-derived epithelial organoids can be combined with a biofabrication approach, thereby paving the way to create perfusable, complex constructs which can be used as toxicology- and disease-models.
AUTHOR
Title
Fabrication and Characterization of 3D Bioprinted Triple-layered Human Alveolar Lung Models
[Abstract]
Year
2021
Journal/Proceedings
International journal of bioprinting
Reftype
DOI/URL
URL
Groups
AbstractThe global prevalence of respiratory diseases caused by infectious pathogens has resulted in an increased demand for realistic in-vitro alveolar lung models to serve as suitable disease models. This demand has resulted in the fabrication of numerous two-dimensional (2D) and three-dimensional (3D) in-vitro alveolar lung models. The ability to fabricate these 3D in-vitro alveolar lung models in an automated manner with high repeatability and reliability is important for potential scalable production. In this study, we reported the fabrication of human triple-layered alveolar lung models comprising of human lung epithelial cells, human endothelial cells, and human lung fibroblasts using the drop-on-demand (DOD) 3D bioprinting technique. The polyvinylpyrrolidone-based bio-inks and the use of a 300 mm nozzle diameter improved the repeatability of the bioprinting process by achieving consistent cell output over time using different human alveolar lung cells. The 3D bioprinted human triple-layered alveolar lung models were able to maintain cell viability with relative similar proliferation profile over time as compared to non-printed cells. This DOD 3D bioprinting platform offers an attractive tool for highly repeatable and scalable fabrication of 3D in-vitro human alveolar lung models.
AUTHOR
Title
Matrigel 3D bioprinting of contractile human skeletal muscle models recapitulating exercise and pharmacological responses
[Abstract]
Year
2021
Journal/Proceedings
Communications Biology
Reftype
Alave Reyes-Furrer2021
DOI/URL
DOI
Groups
AbstractA key to enhance the low translatability of preclinical drug discovery are in vitro human three-dimensional (3D) microphysiological systems (MPS). Here, we show a new method for automated engineering of 3D human skeletal muscle models in microplates and functional compound screening to address the lack of muscle wasting disease medication. To this end, we adapted our recently described 24-well plate 3D bioprinting platform with a printhead cooling system to allow microvalve-based drop-on-demand printing of cell-laden Matrigel containing primary human muscle precursor cells. Mini skeletal muscle models develop within a week exhibiting contractile, striated myofibers aligned between two attachment posts. As an in vitro exercise model, repeated high impact stimulation of contractions for 3 h by a custom-made electrical pulse stimulation (EPS) system for 24-well plates induced interleukin-6 myokine expression and Akt hypertrophy pathway activation. Furthermore, the known muscle stimulators caffeine and Tirasemtiv acutely increase EPS-induced contractile force of the models. This validated new human muscle MPS will benefit development of drugs against muscle wasting diseases. Moreover, our Matrigel 3D bioprinting platform will allow engineering of non-self-organizing complex human 3D MPS.
AUTHOR
Title
Utilization of patterned bioprinting for heterogeneous and physiologically representative reconstructed epidermal skin models
[Abstract]
Year
2021
Journal/Proceedings
Scientific Reports
Reftype
Madiedo-Podvrsan2021
DOI/URL
DOI
Groups
AbstractOrganotypic skin tissue models have decades of use for basic research applications, the treatment of burns, and for efficacy/safety evaluation studies. The complex and heterogeneous nature of native human skin however creates difficulties for the construction of physiologically comparable organotypic models. Within the present study, we utilized bioprinting technology for the controlled deposition of separate keratinocyte subpopulations to create a reconstructed epidermis with two distinct halves in a single insert, each comprised of a different keratinocyte sub-population, in order to better model heterogonous skin and reduce inter-sample variability. As an initial proof-of-concept, we created a patterned epidermal skin model using GPF positive and negative keratinocyte subpopulations, both printed into 2 halves of a reconstructed skin insert, demonstrating the feasibility of this approach. We then demonstrated the physiological relevance of this bioprinting technique by generating a heterogeneous model comprised of dual keratinocyte population with either normal or low filaggrin expression. The resultant model exhibited a well-organized epidermal structure with each half possessing the phenotypic characteristics of its constituent cells, indicative of a successful and stable tissue reconstruction. This patterned skin model aims to mimic the edge of lesions as seen in atopic dermatitis or ichthyosis vulgaris, while the use of two populations within a single insert allows for paired statistics in evaluation studies, likely increasing study statistical power and reducing the number of models required per study. This is the first report of human patterned epidermal model using a predefined bioprinted designs, and demonstrates the relevance of bioprinting to faithfully reproduce human skin microanatomy.
AUTHOR
Title
3D-Printed Soft Lithography for Complex Compartmentalized Microfluidic Neural Devices
[Abstract]
Year
2020
Journal/Proceedings
Advanced Science
Reftype
DOI/URL
DOI
Groups
AbstractAbstract Compartmentalized microfluidic platforms are an invaluable tool in neuroscience research. However, harnessing the full potential of this technology remains hindered by the lack of a simple fabrication approach for the creation of intricate device architectures with high-aspect ratio features. Here, a hybrid additive manufacturing approach is presented for the fabrication of open-well compartmentalized neural devices that provides larger freedom of device design, removes the need for manual postprocessing, and allows an increase in the biocompatibility of the system. Suitability of the method for multimaterial integration allows to tailor the device architecture for the long-term maintenance of healthy human stem-cell derived neurons and astrocytes, spanning at least 40 days. Leveraging fast-prototyping capabilities at both micro and macroscale, a proof-of-principle human in vitro model of the nigrostriatal pathway is created. By presenting a route for novel materials and unique architectures in microfluidic systems, the method provides new possibilities in biological research beyond neuroscience applications.
AUTHOR
Title
A 3D biofabricated cutaneous squamous cell carcinoma tissue model with multi-channel confocal microscopy imaging biomarkers to quantify antitumor effects of chemotherapeutics in tissue
[Abstract]
Year
2020
Journal/Proceedings
Oncotarget; Vol 11, No 27
Reftype
DOI/URL
URL
Groups
Abstract// James R. Browning 1 , Paige Derr 2 , Kristy Derr 2 , Nicole Doudican 3 , Sam Michael 2 , Samantha R. Lish 1 , Nicholas A. Taylor 3 , James G. Krueger 1 , Marc Ferrer 2 , John A. Carucci 3 and Daniel S. Gareau 1 1 Laboratory for Investigative Dermatology, The Rockefeller University, New York, New York, USA 2 National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland, USA 3 The Ronald O. Perelman Department of Dermatology, New York University School of Medicine, New York, New York, USA Correspondence to: Daniel S. Gareau, email: dgareau@rockefeller.edu Keywords: squamous cell carcinoma; screening; 3D printing; in vitro model; confocal microscopy Received: January 05, 2020 Accepted: April 03, 2020 Published: July 07, 2020 ABSTRACT Cutaneous squamous cell carcinoma (cSCC) causes approximately 10,000 deaths annually in the U. S. Current therapies are largely ineffective against metastatic and locally advanced cSCC. There is a need to identify novel, effective, and less toxic small molecule cSCC therapeutics. We developed a 3-dimensional bioprinted skin (3DBPS) model of cSCC tumors together with a microscopy assay to test chemotherapeutic effects in tissue. The full thickness SCC tissue model was validated using hematoxylin and eosin (H&E) and immunohistochemical histological staining, confocal microscopy, and cDNA microarray analysis. A nondestructive, 3D fluorescence confocal imaging assay with tdTomato-labeled A431 SCC and ZsGreen-labeled keratinocytes was developed to test efficacy and general toxicity of chemotherapeutics. Fluorescence-derived imaging biomarkers indicated that 50% of cancer cells were killed in the tissue after 1?M 5-Fluorouracil 48-hour treatment, compared to a baseline of 12% for untreated controls. The imaging biomarkers also showed that normal keratinocytes were less affected by treatment (11% killed) than the untreated tissue, which had no significant killing effect. Data showed that 5-Fluorouracil selectively killed cSCC cells more than keratinocytes. Our 3DBPS assay platform provides cellular-level measurement of cell viability and can be adapted to achieve nondestructive high-throughput screening (HTS) in bio-fabricated tissues.
AUTHOR
Title
A three-dimensional bioprinted model to evaluate the effect of stiffness on neuroblastoma cell cluster dynamics and behavior
[Abstract]
Year
2020
Journal/Proceedings
Scientific Reports
Reftype
Monferrer2020
DOI/URL
DOI
Groups
AbstractThree-dimensional (3D) bioprinted culture systems allow to accurately control microenvironment components and analyze their effects at cellular and tissue levels. The main objective of this study was to identify, quantify and localize the effects of physical-chemical communication signals between tumor cells and the surrounding biomaterial stiffness over time, defining how aggressiveness increases in SK-N-BE(2) neuroblastoma (NB) cell line. Biomimetic hydrogels with SK-N-BE(2) cells, methacrylated gelatin and increasing concentrations of methacrylated alginate (AlgMA 0%, 1% and 2%) were used. Young’s modulus was used to define the stiffness of bioprinted hydrogels and NB tumors. Stained sections of paraffin-embedded hydrogels were digitally quantified. Human NB and 1% AlgMA hydrogels presented similar Young´s modulus mean, and orthotopic NB mice tumors were equally similar to 0% and 1% AlgMA hydrogels. Porosity increased over time; cell cluster density decreased over time and with stiffness, and cell cluster occupancy generally increased with time and decreased with stiffness. In addition, cell proliferation, mRNA metabolism and antiapoptotic activity advanced over time and with stiffness. Together, this rheological, optical and digital data show the potential of the 3D in vitro cell model described herein to infer how intercellular space stiffness patterns drive the clinical behavior associated with NB patients.
AUTHOR
Title
Two-Dimensional Cellular and Three-Dimensional Bio-Printed Skin Models to Screen Topical-Use Compounds for Irritation Potential
[Abstract]
Year
2020
Journal/Proceedings
Frontiers in Bioengineering and Biotechnology
Reftype
DOI/URL
DOI
Groups
AbstractAssessing skin irritation potential is critical for the safety evaluation of topical drugs and other consumer products such as cosmetics. The use of advanced cellular models, as an alternative to replace animal testing in the safety evaluation for both consumer products and ingredients, is already mandated by law in the European Union (EU) and other countries. However, there has not yet been a large-scale comparison of the effects of topical-use compounds in different cellular skin models. This study assesses the irritation potential of topical-use compounds in different cellular models of the skin that are compatible with high throughput screening (HTS) platforms. A set of 451 topical-use compounds were first tested for cytotoxic effects using two-dimensional (2D) monolayer models of primary neonatal keratinocytes and immortalized human keratinocytes. Forty-six toxic compounds identified from the initial screen with the monolayer culture systems were further tested for skin irritation potential on reconstructed human epidermis (RhE) and full thickness skin (FTS) three-dimensional (3D) tissue model constructs. Skin irritation potential of the compounds was assessed by measuring tissue viability, trans-epithelial electrical resistance (TEER), and secretion of cytokines interleukin 1 alpha (IL-1α) and interleukin 18 (IL-18). Among known irritants, high concentrations of methyl violet and methylrosaniline decreased viability, lowered TEER, and increased IL-1α secretion in both RhE and FTS models, consistent with irritant properties. However, at low concentrations, these two compounds increased IL-18 secretion without affecting levels of secreted IL-1α, and did not reduce tissue viability and TEER, in either RhE or FTS models. This result suggests that at low concentrations, methyl violet and methylrosaniline have an allergic potential without causing irritation. Using both HTS-compatible 2D cellular and 3D tissue skin models, together with irritation relevant activity endpoints, we obtained data to help assess the irritation effects of topical-use compounds and identify potential dermal hazards.
AUTHOR
Title
Fully 3D Bioprinted Skin Equivalent Constructs with Validated Morphology and Barrier Function
[Abstract]
Year
2019
Journal/Proceedings
Tissue Engineering Part C: Methods
Reftype
DOI/URL
DOI
Groups
AbstractDevelopment of high throughput, reproducible, three-dimensional bioprinted skin equivalents that are morphologically and functionally comparable to native skin tissue is advancing research in skin diseases, and providing a physiologically relevant platform for the development of therapeutics, transplants for regenerative medicine, and testing of skin products like cosmetics. Current protocols for the production of engineered skin rafts are limited in their ability to control three dimensional geometry of the structure and contraction leading to variability of skin function between constructs. Here we describe a method for the biofabrication of skin equivalents that are fully bioprinted using an open market bioprinter, made with commercially available primary cells and natural hydrogels. The unique hydrogel formulation allows for the production of a human-like skin equivalent with minimal lateral tissue contraction in a multiwell plate format, thus making them suitable for high throughput bioprinting in a single print with fast print and relatively short incubation times. The morphology and barrier function of the fully three-dimensional bioprinted skin equivalents are validated by immunohistochemistry staining, optical coherence tomography, and permeation assays.
AUTHOR
Title
A Novel Microplate 3D Bioprinting Platform for the Engineering of Muscle and Tendon Tissues
[Abstract]
Year
2018
Journal/Proceedings
SLAS TECHNOLOGY: Translating Life Sciences Innovation
Reftype
DOI/URL
DOI
Groups
AbstractTwo-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.
AUTHOR
Title
High-throughput production of liver parenchymal microtissues and enrichment of organ-specific functions in gelatin methacrylamide microenvironment
[Abstract]
Year
2022
Journal/Proceedings
Biotechnology and Bioengineering
Reftype
DOI/URL
DOI
Groups
AbstractAbstract Liver parenchymal microtissues (LPMTs) are three-dimensional (3D) aggregates of hepatocytes that recapitulate in vivo-like cellular assembly. They are considered as a valuable model to study drug metabolism, disease biology, and serve as ideal building blocks for liver tissue engineering. However, their integration into the mainstream drug screening process has been hindered due to the lack of simple, rapid techniques to produce a large number of uniform microtissues and preserve their structural–functional integrity over the long term. Here, we present a high-throughput methodology to produce LPMTs in a novel, economic, and reusable Hanging-drop Culture Chamber (HdCC). A drop-on-demand bioprinting approach was optimized to generate droplets of HepG2 cell suspension on a polyethylene terephthalate substrate. The substrates carrying droplets were placed inside a novel HdCC and incubated to obtain 1600 LPMTs having a size of 200–300 μm. Tissue size, cell viability, cellular arrangement and polarity, and insulin-mediated glucose uptake by LPMTs were analyzed. The microtissues were viable and exhibited an active response to insulin stimulation. Cells within the microtissue reorganized to form hepatic plate-like structures and expressed apical (Multidrug Resistance Protein 2 [MRP2]) and epithelial (Zonula Occludens 1 [ZO1]) markers. Further to maintain the structural integrity and enhance the functional capabilities, LPMTs were sandwiched within gelatin methacrylamide (GelMA) hydrogel and the liver-specific functions were monitored for 2 weeks. The results showed that the 3D structure of LPMTs in GelMA sandwich was maintained while the albumin secretion, urea synthesis, and cytochrome P450 activity were enhanced compared with LPMTs in suspension. In conclusion, this study presents a novel culture chamber for mass production of microtissues and a method for enhancing organ-specific functions of LPMTs in vitro.
AUTHOR
Title
Microvalve bioprinting as a biofabrication tool to decipher tumor and endothelial cell crosstalk: Application to a simplified glioblastoma model
[Abstract]
Year
2021
Journal/Proceedings
Bioprinting
Reftype
Groups
AbstractBioprinting technologies are powerful new bioengineering tools that can spatially reproduce multiple microenvironmental cues in a highly controlled, tunable, and precise manner. In this study, microvalve bioprinting technology was successfully used to print in close proximity endothelial and tumor cells at higher concentrations than previously thought possible, while preserving their viability. We propose that the resulting multicellular models, bioprinted in a controlled extracellular matrix microenvironment, are well-suited to study endothelial and cancer cell crosstalk within a cancer niche. As proof of concept, microvalve bioprinting was applied to the bioengineering of a simplified glioblastoma model in which biological processes involved in tumor expansion, such as tumor cell invasion patterns, cell proliferation, and senescence could be easily visualized and quantified. In this model, U251 glioblastoma cells and primary human umbilical vein endothelial cells (HUVECs) exhibited good printability and high viability after printing. U251 cells formed physiologically relevant clusters and invasion margins, while HUVECs generated vascular-like networks when primary fibroblasts were added to the model. An oxidative stress mimicking the one encountered within a tumor microenvironment during radiotherapy or genotoxic chemotherapy was shown to both diminish endothelial cells proliferation and to increase their senescence. Results also suggested that stressed glioblastoma cells may alter normal endothelial cell proliferation but not impact their senescence. This data demonstrates the potential of microvalve bioprinting to fabricate in vitro models that can help decipher endothelial and tumor cell crosstalk, within controlled and modulable microenvironments, and can then be used to address critical questions in the context of cancer recurrence.
AUTHOR
Title
A biofabricated vascularized skin model of atopic dermatitis for preclinical studies
[Abstract]
Year
2020
Journal/Proceedings
Biofabrication
Reftype
DOI/URL
DOI
Groups
AbstractThree-dimensional (3D) biofabrication techniques enable the production of multicellular tissue models as assay platforms for drug screening. The increased cellular and physiological complexity in these 3D tissue models should recapitulate the relevant biological environment found in the body. Here we describe the use of 3D bioprinting techniques to fabricate skin equivalent tissues of varying physiological complexity, including human epidermis, non-vascularized and vascularized full-thickness skin tissue equivalents, in a multi-well platform to enable drug screening. Human keratinocytes, fibroblasts, and pericytes, and induced pluripotent stem cell (iPSC)-derived endothelial cells were used in the biofabrication process to produce the varying complexity. The skin equivalents exhibit the correct structural markers of dermis and epidermis stratification, with physiological functions of the skin barrier. The robustness, versatility and reproducibility of the biofabrication techniques are further highlighted by the generation of atopic dermatitis (AD)-disease like tissues. These AD models demonstrate several clinical hallmarks of the disease, including: (i) spongiosis and hyperplasia; (ii) early and terminal expression of differentiation proteins; and (iii) increases in levels of pro-inflammatory cytokines. We show the pre-clinical relevance of the biofabricated AD tissue models to correct disease phenotype by testing the effects of dexamethasone, an anti-inflammatory corticosteroid, and three Janus Kinase inhibitors from clinical trials for AD. This study demonstrates the development of a versatile and reproducible bioprinting approach to create human skin equivalents with a range of cellular complexity for disease modelling. In addition, we establish several assay readouts that are quantifiable, robust, AD relevant, and can be scaled up for compound screening. The results show that the cellular complexity of the tissues develops a more physiologically relevant AD disease model. Thus, the skin models in this study offer an in vitro approach for the rapid understanding of pathological mechanisms, and testing for efficacy of action and toxic effects of drugs.
AUTHOR
Year
2018
Journal/Proceedings
Biofabrication
Reftype
DOI/URL
DOI
Groups
AbstractThree-dimensional (3D) pigmented human skin constructs have been fabricated using a 3D bioprinting approach. The 3D pigmented human skin constructs are obtained from using three different types of skin cells (keratinocytes, melanocytes and fibroblasts from three different skin donors) and they exhibit similar constitutive pigmentation (pale pigmentation) as the skin donors. A two-step drop-on-demand bioprinting strategy facilitates the deposition of cell droplets to emulate the epidermal melanin units (pre-defined patterning of keratinocytes and melanocytes at the desired positions) and manipulation of the microenvironment to fabricate 3D biomimetic hierarchical porous structures found in native skin tissue. The 3D bioprinted pigmented skin constructs are compared to the pigmented skin constructs fabricated by conventional a manual-casting approach; in-depth characterization of both the 3D pigmented skin constructs has indicated that the 3D bioprinted skin constructs have a higher degree of resemblance to native skin tissue in term of the presence of well-developed stratified epidermal layers and the presence of a continuous layer of basement membrane proteins as compared to the manually-cast samples. The 3D bioprinting approach facilitates the development of 3D in vitro pigmented human skin constructs for potential toxicology testing and fundamental cell biology research.
AUTHOR
Title
3D bioprinting of E. coli MG1655 biofilms on human lung epithelial cells for building complex in vitro infection models
[Abstract]
Year
2023
Journal/Proceedings
Biofabrication
Reftype
DOI/URL
DOI
Groups
AbstractBiofilm-associated infections are causing over half a million deaths each year, raising the requirement for innovative therapeutic approaches. For developing novel therapeutics against bacterial biofilm infections, complex in vitro models that allow to study drug effects on both pathogens and host cells as well as their interaction under controlled, physiologically relevant conditions appear as highly desirable. Nonetheless, building such models is quite challenging because (1) rapid bacterial growth and release of virulence factors may lead to premature host cell death and (2) maintaining the biofilm status under suitable co-culture requires a highly controlled environment. To approach that problem, we chose 3D bioprinting. However, printing living bacterial biofilms in defined shapes on human cell models, requires bioinks with very specific properties. Hence, this work aims to develop a 3D bioprinting biofilm method to build robust in vitro infection models. Based on rheology, printability and bacterial growth, a bioink containing 3% gelatin and 1% alginate in Luria-Bertani-medium was found optimal for Escherichia coli MG1655 biofilms. Biofilm properties were maintained after printing, as shown visually via microscopy techniques as well as in antibiotic susceptibility assays. Metabolic profile analysis of bioprinted biofilms showed high similarity to native biofilms. After printing on human bronchial epithelial cells (Calu-3), the shape of printed biofilms was maintained even after dissolution of non-crosslinked bioink, while no cytotoxicity was observed over 24 h. Therefore, the approach presented here may provide a platform for building complex in vitro infection models comprising bacterial biofilms and human host cells.
AUTHOR
Title
Embedded 3D Printing in Self-Healing Annealable Composites for Precise Patterning of Functionally Mature Human Neural Constructs
[Abstract]
Year
2022
Journal/Proceedings
Advanced Science
Reftype
DOI/URL
DOI
Groups
AbstractAbstract Human in vitro models of neural tissue with tunable microenvironment and defined spatial arrangement are needed to facilitate studies of brain development and disease. Towards this end, embedded printing inside granular gels holds great promise as it allows precise patterning of extremely soft tissue constructs. However, granular printing support formulations are restricted to only a handful of materials. Therefore, there has been a need for novel materials that take advantage of versatile biomimicry of bulk hydrogels while providing high-fidelity support for embedded printing akin to granular gels. To address this need, Authors present a modular platform for bioengineering of neuronal networks via direct embedded 3D printing of human stem cells inside Self-Healing Annealable Particle-Extracellular matrix (SHAPE) composites. SHAPE composites consist of soft microgels immersed in viscous extracellular-matrix solution to enable precise and programmable patterning of human stem cells and consequent generation mature subtype-specific neurons that extend projections into the volume of the annealed support. The developed approach further allows multi-ink deposition, live spatial and temporal monitoring of oxygen levels, as well as creation of vascular-like channels. Due to its modularity and versatility, SHAPE biomanufacturing toolbox has potential to be used in applications beyond functional modeling of mechanically sensitive neural constructs.
AUTHOR
Title
Precision Plating of Human Electrogenic Cells on Microelectrodes Enhanced With Precision Electrodeposited Nano-Porous Platinum for Cell-Based Biosensing Applications
[Abstract]
Year
2019
Journal/Proceedings
Journal of Microelectromechanical Systems
Reftype
Groups
AbstractMicroelectrode Arrays are established platforms for biosensing applications; however, limitations in electrode impedance and cell-electrode coupling still exist. In this paper, the SNR of 25 μm diameter gold (Au) microelectrodes was improved by decreasing the impedance with precision electrodeposition. SEM determined that N-P Pt. microelectrodes had nanoporous structures that filled the insulation cylinders. EIS, CV, and RMS noise measurements concluded that the optimized electrodeposition of N-P Pt. led to a lowered impedance of 18.36 kΩ ± 2.6 kΩ at 1 kHz, a larger double layer capacitance of 73 nF, and lowered RMS noise of 2.08±0.16 μV as compared to the values for Au of 159 kΩ ± 28 kΩ at 1 kHz, 17nF, and 3.14 ± 0.42 μV, respectively. Human motoneurons and human cardiomyocytes were cultured on N-P Pt. devices to assess their biocompatibility and signal quality. In order to improve the cell-electrode coupling, a precision plating technique was used. Both cell types were electrically active on devices for up to 10 weeks, demonstrated improved SNR, and expected responses to precision chemical and electrical stimulation. The modification of Au microelectrodes with nanomaterials in combination with precision culturing of human cell types provides cost effective, highly sensitive, well coupled and relevant biosensing platforms for medical and pharmaceutical research.
AUTHOR
Title
3D bioprinted functional skeletal muscle models have potential applications for studies of muscle wasting in cancer cachexia
[Abstract]
Year
2023
Journal/Proceedings
Biomaterials Advances
Reftype
Groups
AbstractAcquired muscle diseases such as cancer cachexia are responsible for the poor prognosis of many patients suffering from cancer. In vitro models are needed to study the underlying mechanisms of those pathologies. Extrusion bioprinting is an emerging tool to emulate the aligned architecture of fibers while implementing additive manufacturing techniques in tissue engineering. However, designing bioinks that reconcile the rheological needs of bioprinting and the biological requirements of muscle tissue is a challenging matter. Here we formulate a biomaterial with dual crosslinking to modulate the physical properties of bioprinted models. We design 3D bioprinted muscle models that resemble the mechanical properties of native tissue and show improved proliferation and high maturation of differentiated myotubes suggesting that the GelMA-AlgMA-Fibrin biomaterial possesses myogenic properties. The electrical stimulation of the 3D model confirmed the contractile capability of the tissue and enhanced the formation of sarcomeres. Regarding the functionality of the models, they served as platforms to recapitulate skeletal muscle diseases such as muscle wasting produced by cancer cachexia. The genetic expression of 3D models demonstrated a better resemblance to the muscular biopsies of cachectic mouse models. Altogether, this biomaterial is aimed to fabricate manipulable skeletal muscle in vitro models in a non-costly, fast and feasible manner.
AUTHOR
Title
A 3D multi-cellular tissue model of the human omentum to study the formation of ovarian cancer metastasis
[Abstract]
Year
2023
Journal/Proceedings
Biomaterials
Reftype
Groups
AbstractReliable and predictive experimental models are urgently needed to study metastatic mechanisms of ovarian cancer cells in the omentum. Although models for ovarian cancer cell adhesion and invasion were previously investigated, the lack of certain omental cell types, which influence the metastatic behavior of cancer cells, limits the application of these tissue models. Here, we describe a 3D multi-cellular human omentum tissue model, which considers the spatial arrangement of five omental cell types. Reproducible tissue models were fabricated combining permeable cell culture inserts and bioprinting technology to mimic metastatic processes of immortalized and patient-derived ovarian cancer cells. The implementation of an endothelial barrier further allowed studying the interaction between cancer and endothelial cells during hematogenous dissemination and the impact of chemotherapeutic drugs. This proof-of-concept study may serve as a platform for patient-specific investigations in personalized oncology in the future.
AUTHOR
Title
A culture model to analyze the acute biomaterial-dependent reaction of human primary neutrophils in vitro
[Abstract]
Year
2023
Journal/Proceedings
Bioactive Materials
Reftype
Groups
AbstractNeutrophils play a pivotal role in orchestrating the immune system response to biomaterials, the onset and resolution of chronic inflammation, and macrophage polarization. However, the neutrophil response to biomaterials and the consequent impact on tissue engineering approaches is still scarcely understood. Here, we report an in vitro culture model that comprehensively describes the most important neutrophil functions in the light of tissue repair. We isolated human primary neutrophils from peripheral blood and exposed them to a panel of hard, soft, naturally- and synthetically-derived materials. The overall trend showed increased neutrophil survival on naturally derived constructs, together with higher oxidative burst, decreased myeloperoxidase and neutrophil elastase and decreased cytokine secretion compared to neutrophils on synthetic materials. The culture model is a step to better understand the immune modulation elicited by biomaterials. Further studies are needed to correlate the neutrophil response to tissue healing and to elucidate the mechanism triggering the cell response and their consequences in determining inflammation onset and resolution.
AUTHOR
Year
2023
Journal/Proceedings
Advanced NanoBiomed Research
Reftype
DOI/URL
DOI
Groups
AbstractThe demand for high-throughput and scalable cell expansion platforms that can accommodate diverse cell types remains a critical requirement across various biomedical fields. Fibronectin (Fn), an essential component of the extracellular matrix (ECM), has been used as a conformal surface coating for two-dimensional (2D) cell culture systems. However, the soluble, globular Fn used for 2D coatings differs structurally from the native Fn, which possesses a three-dimensional (3D) fibrillar structure. Herein, a large-scale engineered ECM (EECM) cell expansion platform based on a 3D fibrillar Fn network spanning over centimeters is presented. Extended fibrillar networks are formed by shearing dilute Fn solutions over tessellated polymeric scaffolds, which are conveniently prepared by 3D printing. The structure and size of the Fn-based 3D EECM scaffold are optimized by evaluating the proliferation of a colorectal tumor cell line, CT26, commonly used in the in vivo tumor immunotherapy models. The 3D EECM scaffolds support a fourfold more efficient tumor cell expansion than a conventional 2D culture system, demonstrating the potential efficacy in supporting the robust expansion of cancer cells ex vivo with an eye on cancer immunotherapy.
AUTHOR
Title
Flexible 3D printed microwires and 3D microelectrodes for heart-on-a-chip engineering
[Abstract]
Year
2023
Journal/Proceedings
Biofabrication
Reftype
DOI/URL
DOI
Groups
AbstractWe developed a heart-on-a-chip platform that integrates highly flexible, vertical, 3D micropillar electrodes for electrophysiological recording and elastic microwires for the tissue’s contractile force assessment. The high aspect ratio microelectrodes were 3D-printed into the device using a conductive polymer, poly(3,4-ethylenedioxythiophene): poly(styrene sulfonate) (PEDOT:PSS). A pair of flexible, quantum dots/thermoplastic elastomer nanocomposite microwires were 3D printed to anchor the tissue and enable continuous contractile force assessment. The 3D microelectrodes and flexible microwires enabled unobstructed human iPSC-based cardiac tissue formation and contraction, suspended above the device surface, under both spontaneous beating and upon pacing with a separate set of integrated carbon electrodes. Recording of extracellular field potentials using the PEDOT:PSS micropillars was demonstrated with and without epinephrine as a model drug, non-invasively, along with in situ monitoring of tissue contractile properties and calcium transients. Uniquely, the platform provides integrated profiling of electrical and contractile tissue properties, which is critical for proper evaluation of complex, mechanically and electrically active tissues, such as the heart muscle under both physiological and pathological conditions.
AUTHOR
Year
2023
Journal/Proceedings
Applied Sciences
Reftype
Groups
Abstract(1) Background: Synovial tissue plays a fundamental role in inflammatory processes. Therefore, understanding the mechanisms regulating healthy and diseased synovium functions, as in rheumatic diseases, is crucial to discovering more effective therapies to minimize or prevent pathological progress. The present study aimed at developing a bioartificial synovial tissue as an in vitro model for drug screening or personalized medicine applications using 3D bioprinting technology. (2) Methods: The volumetric extrusion technique has been used to fabricate cell-laden scaffolds. Gelatin Methacryloyl (GelMA), widely applied in regenerative medicine and tissue engineering, was selected as a bioink and combined with an immortalized cell line of fibroblast-like synoviocytes (K4IM). (3) Results: Three different GelMA formulations, 7.5–10–12.5% w/v, were tested for the fabrication of the scaffold with the desired morphology and internal architecture. GelMA 10% w/v was chosen and combined with K4IM cells to fabricate scaffolds that showed high cell viability and negligible cytotoxicity for up to 14 days tested by Live & Dead and lactate dehydrogenase assays. (4) Conclusions: We successfully 3D bioprinted synoviocytes-laden scaffolds as a proof-of-concept (PoC) towards the fabrication of a 3D synovial membrane model suitable for in vitro studies. However, further research is needed to reproduce the complexity of the synovial microenvironment to better mimic the physiological condition.
AUTHOR
Title
Revolutionizing drug development: harnessing the potential of organ-on-chip technology for disease modeling and drug discovery
[Abstract]
Year
2023
Journal/Proceedings
Frontiers in Pharmacology
Reftype
DOI/URL
DOI
AbstractThe inefficiency of existing animal models to precisely predict human pharmacological effects is the root reason for drug development failure. Microphysiological system/organ-on-a-chip technology (organ-on-a-chip platform) is a microfluidic device cultured with human living cells under specific organ shear stress which can faithfully replicate human organ-body level pathophysiology. This emerging organ-on-chip platform can be a remarkable alternative for animal models with a broad range of purposes in drug testing and precision medicine. Here, we review the parameters employed in using organ on chip platform as a plot mimic diseases, genetic disorders, drug toxicity effects in different organs, biomarker identification, and drug discoveries. Additionally, we address the current challenges of the organ-on-chip platform that should be overcome to be accepted by drug regulatory agencies and pharmaceutical industries. Moreover, we highlight the future direction of the organ-on-chip platform parameters for enhancing and accelerating drug discoveries and personalized medicine.
AUTHOR
Title
A 3D Collagen-Based Bioprinted Model to Study Osteosarcoma Invasiveness and Drug Response
[Abstract]
Year
2022
Journal/Proceedings
Polymers
Reftype
Groups
AbstractThe biological and therapeutic limits of traditional 2D culture models, which only partially mimic the complexity of cancer, have recently emerged. In this study, we used a 3D bioprinting platform to process a collagen-based hydrogel with embedded osteosarcoma (OS) cells. The human OS U-2 OS cell line and its resistant variant (U-2OS/CDDP 1 μg) were considered. The fabrication parameters were optimized to obtain 3D printed constructs with overall morphology and internal microarchitecture that accurately match the theoretical design, in a reproducible and stable process. The biocompatibility of the 3D bioprinting process and the chosen collagen bioink in supporting OS cell viability and metabolism was confirmed through multiple assays at short- (day 3) and long- (day 10) term follow-ups. In addition, we tested how the 3D collagen-based bioink affects the tumor cell invasive capabilities and chemosensitivity to cisplatin (CDDP). Overall, we developed a new 3D culture model of OS cells that is easy to set up, allows reproducible results, and better mirrors malignant features of OS than flat conditions, thus representing a promising tool for drug screening and OS cell biology research.
AUTHOR
Title
A platform of assays for the discovery of anti-Zika small-molecules with activity in a 3D-bioprinted outer-blood-retina model
[Abstract]
Year
2022
Journal/Proceedings
PLOS ONE
Reftype
DOI/URL
DOI
Groups
AbstractThe global health emergency posed by the outbreak of Zika virus (ZIKV), an arthropod-borne flavivirus causing severe neonatal neurological conditions, has subsided, but there continues to be transmission of ZIKV in endemic regions. As such, there is still a medical need for discovering and developing therapeutical interventions against ZIKV. To identify small-molecule compounds that inhibit ZIKV disease and transmission, we screened multiple small-molecule collections, mostly derived from natural products, for their ability to inhibit wild-type ZIKV. As a primary high-throughput screen, we used a viral cytopathic effect (CPE) inhibition assay conducted in Vero cells that was optimized and miniaturized to a 1536-well format. Suitably active compounds identified from the primary screen were tested in a panel of orthogonal assays using recombinant Zika viruses, including a ZIKV Renilla luciferase reporter assay and a ZIKV mCherry reporter system. Compounds that were active in the wild-type ZIKV inhibition and ZIKV reporter assays were further evaluated for their inhibitory effects against other flaviviruses. Lastly, we demonstrated that wild-type ZIKV is able to infect a 3D-bioprinted outer-blood-retina barrier tissue model and disrupt its barrier function, as measured by electrical resistance. One of the identified compounds (3-Acetyl-13-deoxyphomenone, NCGC00380955) was able to prevent the pathological effects of the viral infection on this clinically relevant ZIKV infection model.
AUTHOR
Title
A simple and scalable 3D printing methodology for generating aligned and extended human and murine skeletal muscle tissues
[Abstract]
Year
2022
Journal/Proceedings
Biomedical Materials
Reftype
DOI/URL
DOI
Groups
AbstractPreclinical biomedical and pharmaceutical research on disease causes, drug targets, and side effects increasingly relies on in vitro models of human tissue. 3D printing offers unique opportunities for generating models of superior physiological accuracy, as well as for automating their fabrication. Towards these goals, we here describe a simple and scalable methodology for generating physiologically relevant models of skeletal muscle. Our approach relies on dual-material micro-extrusion of two types of gelatin hydrogel into patterned soft substrates with locally alternating stiffness. We identify minimally complex patterns capable of guiding the large-scale self-assembly of aligned, extended, and contractile human and murine skeletal myotubes. Interestingly, we find high-resolution patterning is not required, as even patterns with feature sizes of several hundred micrometers is sufficient. Consequently, the procedure is rapid and compatible with any low-cost extrusion-based 3D printer. The generated myotubes easily span several millimeters, and various myotube patterns can be generated in a predictable and reproducible manner. The compliant nature and adjustable thickness of the hydrogel substrates, serves to enable extended culture of contractile myotubes. The method is further readily compatible with standard cell-culturing platforms as well as commercially available electrodes for electrically induced exercise and monitoring of the myotubes.
AUTHOR
Title
Bioprinted 3D outer retina barrier uncovers RPE-dependent choroidal phenotype in advanced macular degeneration
[Abstract]
Year
2022
Journal/Proceedings
Nature Methods
Reftype
Song2022
DOI/URL
DOI
Groups
AbstractAge-related macular degeneration (AMD), a leading cause of blindness, initiates in the outer-blood-retina-barrier (oBRB) formed by the retinal pigment epithelium (RPE), Bruch’s membrane, and choriocapillaris. The mechanisms of AMD initiation and progression remain poorly understood owing to the lack of physiologically relevant human oBRB models. To this end, we engineered a native-like three-dimensional (3D) oBRB tissue (3D-oBRB) by bioprinting endothelial cells, pericytes, and fibroblasts on the basal side of a biodegradable scaffold and establishing an RPE monolayer on top. In this 3D-oBRB model, a fully-polarized RPE monolayer provides barrier resistance, induces choriocapillaris fenestration, and supports the formation of Bruch’s-membrane-like structure by inducing changes in gene expression in cells of the choroid. Complement activation in the 3D-oBRB triggers dry AMD phenotypes (including subRPE lipid-rich deposits called drusen and choriocapillaris degeneration), and HIF-α stabilization or STAT3 overactivation induce choriocapillaris neovascularization and type-I wet AMD phenotype. The 3D-oBRB provides a physiologically relevant model to studying RPE-choriocapillaris interactions under healthy and diseased conditions.
AUTHOR
Title
Bioprinting Decellularized Breast Tissue for the Development of Three-Dimensional Breast Cancer Models
[Abstract]
Year
2022
Journal/Proceedings
ACS Appl. Mater. Interfaces
Reftype
DOI/URL
DOI
Groups
AbstractThe tumor extracellular matrix (ECM) plays a vital role in tumor progression and drug resistance. Previous studies have shown that breast tissue-derived matrices could be an important biomaterial to recreate the complexity of the tumor ECM. We have developed a method for decellularizing and delipidating a porcine breast tissue (TDM) compatible with hydrogel formation. The addition of gelatin methacrylamide and alginate allows this TDM to be bioprinted by itself with good printability, shape fidelity, and cytocompatibility. Furthermore, this bioink has been tuned to more closely recreate the breast tumor by incorporating collagen type I (Col1). Breast cancer cells (BCCs) proliferate in both TDM bioinks forming cell clusters and spheroids. The addition of Col1 improves the printability of the bioink as well as increases BCC proliferation and reduces doxorubicin sensitivity due to a downregulation of HSP90. TDM bioinks also allow a precise three-dimensional printing of scaffolds containing BCCs and stromal cells and could be used to fabricate artificial tumors. Taken together, we have proven that these novel bioinks are good candidates for biofabricating breast cancer models.
AUTHOR
Title
Deep Learning for Automated Analysis of Cellular and Extracellular Components of the Foreign Body Response in Multiphoton Microscopy Images
[Abstract]
Year
2022
Journal/Proceedings
Frontiers in Bioengineering and Biotechnology
Reftype
DOI/URL
DOI
Groups
AbstractThe Foreign body response (FBR) is a major unresolved challenge that compromises medical implant integration and function by inflammation and fibrotic encapsulation. Mice implanted with polymeric scaffolds coupled to intravital non-linear multiphoton microscopy acquisition enable multiparametric, longitudinal investigation of the FBR evolution and interference strategies. However, follow-up analyses based on visual localization and manual segmentation are extremely time-consuming, subject to human error, and do not allow for automated parameter extraction. We developed an integrated computational pipeline based on an innovative and versatile variant of the U-Net neural network to segment and quantify cellular and extracellular structures of interest, which is maintained across different objectives without impairing accuracy. This software for automatically detecting the elements of the FBR shows promise to unravel the complexity of this pathophysiological process.
AUTHOR
Title
Development and evaluation of a multicomponent bioink consisting of alginate, gelatin, diethylaminoethyl cellulose and collagen peptide for 3D bioprinting of tissue construct for drug screening application
[Abstract]
Year
2022
Journal/Proceedings
International Journal of Biological Macromolecules
Reftype
Groups
AbstractThree dimensional (3D) bioprinting technology has been making a progressive advancement in the field of tissue engineering to produce tissue constructs that mimic the shape, framework, and microenvironment of an organ. The technology has not only paved the way to organ development but has been widely studied for its application in drug and cosmetic testing using 3D bioprinted constructs. However, not much has been explored on the utilization of bioprinting technology for the development of tumor models to test anti-cancer drug efficacy. The conventional methodology involves a two dimensional (2D) monolayer model to test cellular drug response which has multiple limitations owing to its inability to mimic the natural tissue environment. The choice of bioink for 3D bioprinting is critical as cell morphology and proliferation depend greatly on the property of bioink. In this study, we developed a multicomponent bioink composed of alginate, diethylaminoethyl cellulose, gelatin, and collagen peptide to generate a 3D bioprinted construct. The bioink has been characterised and validated for its printability, shape fidelity and biocompatibility to be used for generating tumor models. Further, a bioprinted tumor model was developed using lung cancer cell line and the efficacy of 3D printed construct for drug screening application was established.
AUTHOR
Title
Dissecting the recruitment and self-organization of αSMA-positive fibroblasts in the foreign body response
[Abstract]
Year
2022
Journal/Proceedings
Science Advances
Reftype
DOI/URL
DOI
Groups
AbstractThe foreign body response (FBR) is a clinically relevant issue that can cause malfunction of implanted medical devices by fibrotic encapsulation. Whereas inflammatory aspects of the FBR have been established, underlying fibroblast-dependent mechanisms remain unclear. We here combine multiphoton microscopy with ad hoc reporter mice expressing α–smooth muscle actin (αSMA) protein to determine the locoregional fibroblast dynamics, activation, and fibrotic encapsulation of polymeric materials. Fibroblasts invaded as individual cells and established a multicellular network, which transited to a two-compartment fibrotic response displaying an αSMA cold external capsule and a long-lasting, inner αSMA hot environment. The recruitment of fibroblasts and extent of fibrosis were only incompletely inhibited after depletion of macrophages, implicating coexistence of macrophage-dependent and macrophage-independent mediators. Furthermore, neither altering material type or porosity modulated αSMA+ cell recruitment and distribution. This identifies fibroblast activation and network formation toward a two-compartment FBR as a conserved, self-organizing process partially independent of macrophages. Fibroblast recruitment in the foreign body response is a conserved, self-organizing process partially independent of macrophages.
AUTHOR
Title
Exploring the Potential of Alginate-Gelatin-Diethylaminoethyl Cellulose-Fibrinogen based Bioink for 3D Bioprinting of Skin Tissue Constructs
[Abstract]
Year
2022
Journal/Proceedings
Carbohydrate Polymer Technologies and Applications
Reftype
Groups
AbstractDesigning printable bioinks for 3D bioprinting capable of supporting cellular viability with post-printing functionality remains challenging. Native ECM offers several physical, chemical, and biological cues that are difficult to restore using only a single component. Herein, we have optimized a multicomponent-based bioink formulation comprising alginate (ALG), gelatin (GEL), diethylaminoethyl cellulose (DCEL) and fibrinogen (FIB), termed as ALG-GEL-DCEL-FIB bioink for potential application in bioprinting and biofabrication of skin tissue equivalents. The designed formulation was extensively studied for its printability, physico-chemical, rheological, and biocompatibility properties. Excellent printability, shape fidelity and cell-laden tissue equivalent printing were established using the RegenHu 3D Discovery Bioprinter. The human primary fibroblast and keratinocyte-laden bioprinted constructs exhibited good cell viability. Long term culture of 4 weeks comprising 5 days of air-liquid-interphase followed by 21 days of submerged culture produced biomimetic tissue histology in the ALG-GEL-DCEL-FIB bioink printed constructs. Specific epidermal-dermal marker expressions proving functionality were evident in immunohistochemical, biochemical and gene expression analysis. The ALG-GEL-DCEL-FIB bioink may be explored further for potential biofabrication and therapeutic applications.
AUTHOR
Title
Impact of microstructure on cell behavior and tissue mechanics in collagen and dermal decellularized extra-cellular matrices
[Abstract]
Year
2022
Journal/Proceedings
Acta Biomaterialia
Reftype
Groups
AbstractSkin models are used for many applications such as research and development or grafting. Unfortunately, most lack a proper microenvironment producing poor mechanical properties and inaccurate extra-cellular matrix composition and organization. In this report we focused on mechanical properties, extra-cellular matrix organization and cell interactions in human skin samples reconstructed with pure collagen or dermal decellularized extra-cellular matrices (S-dECM) and compared them to native human skin. We found that Full-thickness S-dECM samples presented stiffness two times higher than collagen gel and similar to ex vivo human skin, and proved for the first time that keratinocytes also impact dermal mechanical properties. This was correlated with larger fibers in S-dECM matrices compared to collagen samples and with a differential expression of F-actin, vinculin and tenascin C between S-dECM and collagen samples. This is clear proof of the microenvironment's impact on cell behaviors and mechanical properties. Statement of significance In vitro skin models have been used for a long time for clinical applications or in vitro knowledge and evaluation studies. However, most lack a proper microenvironment producing a poor combination of mechanical properties and appropriate biological outcomes, partly due to inaccurate extra-cellular matrix (ECM) composition and organization. This can lead to limited predictivity and weakness of skin substitutes after grafting. This study shows, for the first time, the importance of a complex and rich microenvironment on cell behaviors, matrix macro- and micro-organization and mechanical properties. The increased composition and organization complexity of dermal skin decellularized extra-cellular matrix populated with differentiated cells produces in vitro skin models closer to native human skin physiology.
AUTHOR
Title
Melt Electrowriting of Graded Porous Scaffolds to Mimic the Matrix Structure of the Human Trabecular Meshwork
[Abstract]
Year
2022
Journal/Proceedings
ACS Biomaterials Science & Engineering
Reftype
DOI/URL
DOI
Groups
AbstractThe permeability of the human trabecular meshwork (HTM) regulates eye pressure via a porosity gradient across its thickness modulated by stacked layers of matrix fibrils and cells. Changes in HTM porosity are associated with increases in intraocular pressure and the progress of diseases such as glaucoma. Engineered HTMs could help to understand the structure–function relation in natural tissues and lead to new regenerative solutions. Here, melt electrowriting (MEW) is explored as a biofabrication technique to produce fibrillar, porous scaffolds that mimic the multilayer, gradient structure of native HTM. Poly(caprolactone) constructs with a height of 125–500 μm and fiber diameters of 10–12 μm are printed. Scaffolds with a tensile modulus between 5.6 and 13 MPa and a static compression modulus in the range of 6–360 kPa are obtained by varying the scaffold design, that is, the density and orientation of the fibers and number of stacked layers. Primary HTM cells attach to the scaffolds, proliferate, and form a confluent layer within 8–14 days, depending on the scaffold design. High cell viability and cell morphology close to that in the native tissue are observed. The present work demonstrates the utility of MEW for reconstructing complex morphological features of natural tissues.
AUTHOR
Year
2022
Journal/Proceedings
ACS Appl. Bio Mater.
Reftype
DOI/URL
DOI
Groups
AbstractHuman mesenchymal stem cells (HMSCs) are important for cell-based therapies. However, the success of HMSC therapy requires large-scale in vitro expansion of these multipotent cells. The traditional expansion of HMSCs on tissue-culture-treated stiff polystyrene induces significant changes in their shape, multipotency, and secretome, leading to early senescence and subdued paracrine activity. To enhance their therapeutic potential, here, we have developed two-dimensional soft hydrogels with imprinted microscale aligned grooves for use as HMSC culture substrates. We showed that, depending on the dimensions of the topographical features, these substrates led to lower cellular spreading and cytoskeletal tension, maintaining multipotency and osteogenic and adipogenic differentiate potential, while lowering cellular senescence. We also observed a greater capacity of HMSCs to produce anti-inflammatory cytokines after short-term priming on these hydrogel substrates. Overall, these soft hydrogels with unique surface topography have shown great promise as in vitro culture substrates to maximize the therapeutic potential of HMSCs.
AUTHOR
Title
Organ-on-a-chip microengineering for bio-mimicking disease models and revolutionizing drug discovery
[Abstract]
Year
2022
Journal/Proceedings
Biosensors and Bioelectronics: X
Reftype
AbstractThe core of the drug research and screening processes is predicting the effect of drugs prior to human clinical trials. Due to the 2D cell culture and animal models' poor predictability, the cost of drug discovery is continuously rising. The development of organ-on-a-chip technology, an alternative to traditional preclinical drug testing models, resulted from the intersection of microfabrication & tissue engineering. Preclinical safety and effectiveness testing is improved by the ability of organ-on-a-chip technologies to mimic important human physiological functions necessary for understanding drug effects. Organ-on-a-chip could drastically improve the success rate of the preclinical testing thereby better predicting how the drug will act on the clinical trials. Organ-on-a-chip is a term used to describe a microengineered biomimetic device that mimics the structure and functionality of human tissue. It integrates engineering, cell biology, & biomaterial technologies on a miniature platform. To reflect human physiology in vitro and bridge the gap between in vivo and in vitro data, simplification shouldn't compromise physiological relevance. At this level of organ-on-a-chip technological development, biomedical engineers specializing in device engineering are more important than ever to expedite the transfer of technology from the academic lab bench to specialized product development institutions and an ever-growing market. This review focuses on the recent advancements in the organ-on-a-chip technology and discusses the potential of this technology based on the current available literature.
AUTHOR
Title
Radical scavenging gelatin methacrylamide based bioink formulation for three dimensional bioprinting of parenchymal liver construct
[Abstract]
Year
2022
Journal/Proceedings
Bioprinting
Reftype
Groups
AbstractMethacrylated gelatin (GelMA) in the form of methacryloyl, methacrylate, and methacrylamide is an established and widely accepted photocrosslinkable bioink, for three dimensional bioprinting of various tissues. One of the limitations of photocrosslinkable bioinks is the inability to control the free radicals generated by photoinitiators and ultraviolet (UV) rays. The presence of excess free radicals compromises the viability and functionality of cells during crosslinking. In this study, ascorbic acid, a known free radical scavenger (FRS) molecule, was introduced into the GelMA bioink formulation to protect the cell viability, proliferation, and tissue functions of 3D bioprinted parenchymal liver constructs. The concentration of FRS in the bioink was optimized and used for 3D bioprinting of HepG2 cells. The results confirmed that the inclusion of 3.4 mM FRS in the GelMA bioink formulation nullified the excess ROS formed inside the cells. Furthermore, the optimized GelMA formulation containing FRS preserved and improved the cell activity, albumin, and urea synthesis in the 3D construct over 7 days in culture. In the future, this concept could be implemented in the biofabrication of large liver constructs that require multiple or longer durations of UV irradiation.
AUTHOR
Year
2022
Journal/Proceedings
Bioengineered
Reftype
DOI/URL
DOI
Groups
AbstractABSTRACTArtificial skins have been used as skin substitutes for wound healing in the clinic, and as in vitro models for safety assessment in cosmetic and pharmaceutical industries. The three-dimensional (3D) bioprinting technique provides a promising strategy in the fabrication of artificial skins. Despite the technological advances, many challenges remain to be conquered, such as the complicated preparation conditions for bio-printed skin and the unavailability of stability and robustness of skin bioprinting. Here, we formulated a novel bio-ink composed of gelatin, sodium alginate and fibrinogen. By optimizing the ratio of components in the bio-ink, the design of the 3D model and the printing conditions, a fibroblasts-containing dermal layer construct was firstly fabricated, on the top of which laminin and keratinocytes were sequentially placed. Through air-liquid interface (ALI) culture by virtue of sterile wire mesh, a full-thickness skin tissue was thus prepared. HE and immunofluorescence staining showed that the bio-printed skin was not only morphologically representative of the human skin, but also expressed the specific markers related to epidermal differentiation and stratum corneum formation. The presented easy and robust preparation of full-thickness skin constructs provides a powerful tool for the establishment of artificial skins, holding critical academic significance and application value.
AUTHOR
Title
Three-dimensional (3D) liver cell models - a tool for bridging the gap between animal studies and clinical trials when screening liver accumulation and toxicity of nanobiomaterials
[Abstract]
Year
2022
Journal/Proceedings
Drug Delivery and Translational Research
Reftype
Tutty2022
DOI/URL
DOI
AbstractDespite the exciting properties and wide-reaching applications of nanobiomaterials (NBMs) in human health and medicine, their translation from bench to bedside is slow, with a predominant issue being liver accumulation and toxicity following systemic administration. In vitro 2D cell-based assays and in vivo testing are the most popular and widely used methods for assessing liver toxicity at pre-clinical stages; however, these fall short in predicting toxicity for NBMs. Focusing on in vitro and in vivo assessment, the accurate prediction of human-specific hepatotoxicity is still a significant challenge to researchers. This review describes the relationship between NBMs and the liver, and the methods for assessing toxicity, focusing on the limitations they bring in the assessment of NBM hepatotoxicity as one of the reasons defining the poor translation for NBMs. We will then present some of the most recent advances towards the development of more biologically relevant in vitro liver methods based on tissue-mimetic 3D cell models and how these could facilitate the translation of NBMs going forward. Finally, we also discuss the low public acceptance and limited uptake of tissue-mimetic 3D models in pre-clinical assessment, despite the demonstrated technical and ethical advantages associated with them.
AUTHOR
Title
Computational modeling identifies multitargeted kinase inhibitors as effective therapies for metastatic, castration-resistant prostate cancer
[Abstract]
Year
2021
Journal/Proceedings
Proceedings of the National Academy of Sciences
Reftype
Groups
AbstractMetastatic, castration-resistant prostate cancer (mCRPC) is an advanced prostate cancer with limited therapeutic options and poor patient outcomes. To investigate whether multitargeted kinase inhibitors (KIs) represent an opportunity for mCRPC drug development, we applied machine learning{textendash}based functional screening and identified two KIs, PP121 and SC-1, which demonstrated strong suppression of CRPC growth in vitro and in vivo. Furthermore, we show the marked ability of these KIs to improve on standard-of-care chemotherapy in both tumor response and survival, suggesting that combining multitargeted KIs with chemotherapy represents a promising avenue for mCRPC treatment. Overall, our findings demonstrate the application of a multidisciplinary strategy that blends bench science with machine-learning approaches for rapidly identifying KIs that result in desired phenotypic effects.Castration-resistant prostate cancer (CRPC) is an advanced subtype of prostate cancer with limited therapeutic options. Here, we applied a systems-based modeling approach called kinome regularization (KiR) to identify multitargeted kinase inhibitors (KIs) that abrogate CRPC growth. Two predicted KIs, PP121 and SC-1, suppressed CRPC growth in two-dimensional in vitro experiments and in vivo subcutaneous xenografts. An ex vivo bone mimetic environment and in vivo tibia xenografts revealed resistance to these KIs in bone. Combining PP121 or SC-1 with docetaxel, standard-of-care chemotherapy for late-stage CRPC, significantly reduced tibia tumor growth in vivo, decreased growth factor signaling, and vastly extended overall survival, compared to either docetaxel monotherapy. These results highlight the utility of computational modeling in forming physiologically relevant predictions and provide evidence for the role of multitargeted KIs as chemosensitizers for late-stage, metastatic CRPC.All study data are included in the article and/or supporting information.
AUTHOR
Year
2021
Journal/Proceedings
Journal of Nuclear Medicine
Reftype
Groups
AbstractRadium 223 (223Ra) is an α-emitter approved for the treatment of bone metastatic prostate cancer (PCa), which exerts direct cytotoxicity towards PCa cells near the bone interface, whereas cells positioned in the core respond poorly, due to short α-particle penetrance. β1 integrin (β1I) interference has been shown to increase radiosensitivity and significantly enhance external beam radiation efficiency. We hypothesized that targeting β1I would improve 223Ra outcome. We tested the effect of combining 223Ra and anti-β1I antibody treatment in PC3 and C4-2B PCa cell models expressing high and low β1I levels, respectively. In vivo tumor growth was evaluated through bioluminescence. Cellular and molecular determinants of response were analyzed by ex vivo three-dimensional imaging of bone lesions, proteomic analysis and further confirmed by computational modeling and in vitro functional analysis in tissue-engineered bone mimetic systems. Interference with β1I combined with 223Ra reduced PC3 cell growth in bone and significantly improved overall mouse survival, while no change was achieved in C4-2B tumors. Anti-β1I treatment decreased PC3 tumor cell mitosis index and spatially expanded 223Ra lethal effects two-fold, in vivo and in silico. Regression was paralleled by decreased expression of radio-resistance mediators. Targeting β1I significantly improves 223Ra outcome and points towards combinatorial application in PCa tumors with high β1I expression.
AUTHOR
Year
2020
Journal/Proceedings
Reftype
DOI/URL
DOI
Groups
AbstractIncreasing ethical and biological concerns require a paradigm shift toward animal-free testing strategies for drug testing and hazard assessments. To this end, the application of bioprinting technology in the field of biomedicine is driving a rapid progress in tissue engineering. In particular, standardized and reproducible in vitro models produced by three-dimensional (3D) bioprinting technique represent a possible alternative to animal models, enabling in vitro studies relevant to in vivo conditions. The innovative approach of 3D bioprinting allows a spatially controlled deposition of cells and biomaterial in a layer-by-layer fashion providing a platform for engineering reproducible models. However, despite the promising and revolutionizing character of 3D bioprinting technology, standardized protocols providing detailed instructions are lacking. Here, we provide a protocol for the automatized printing of simple alveolar, bronchial, and intestine epithelial cell layers as the basis for more complex respiratory and gastrointestinal tissue models. Such systems will be useful for high-throughput toxicity screening and drug efficacy evaluation.
AUTHOR
Title
Collagen-rich omentum is a premetastatic niche for integrin α2-mediated peritoneal metastasis
[Abstract]
Year
2020
Journal/Proceedings
eLife
Reftype
DOI/URL
DOI
Groups
AbstractThe extracellular matrix (ECM) plays critical roles in tumor progression and metastasis. However, the contribution of ECM proteins to early metastatic onset in the peritoneal cavity remains unexplored. Here, we suggest a new route of metastasis through the interaction of integrin alpha 2 (ITGA2) with collagens enriched in the tumor coinciding with poor outcome in patients with ovarian cancer. Using multiple gene-edited cell lines and patient-derived samples, we demonstrate that ITGA2 triggers cancer cell adhesion to collagen, promotes cell migration, anoikis resistance, mesothelial clearance, and peritoneal metastasis in vitro and in vivo. Mechanistically, phosphoproteomics identify an ITGA2-dependent phosphorylation of focal adhesion kinase and mitogen-activated protein kinase pathway leading to enhanced oncogenic properties. Consequently, specific inhibition of ITGA2-mediated cancer cell-collagen interaction or targeting focal adhesion signaling may present an opportunity for therapeutic intervention of metastatic spread in ovarian cancer.
AUTHOR
Title
Impact of extracellular matrix stiffness on genomic heterogeneity in MYCN-amplified neuroblastoma cell line
[Abstract]
Year
2020
Journal/Proceedings
Journal of Experimental & Clinical Cancer Research
Reftype
López-Carrasco2020
DOI/URL
DOI
Groups
AbstractIncreased tissue stiffness is a common feature of malignant solid tumors, often associated with metastasis and poor patient outcomes. Vitronectin, as an extracellular matrix anchorage glycoprotein related to a stiff matrix, is present in a particularly increased quantity and specific distribution in high-risk neuroblastoma. Furthermore, as cells can sense and transform the proprieties of the extracellular matrix into chemical signals through mechanotransduction, genotypic changes related to stiffness are possible.
AUTHOR
Title
Investigation of drug dissolution and uptake from low-density DPI formulations in an impactor–integrated cell culture model
[Abstract]
Year
2020
Journal/Proceedings
European Journal of Pharmaceutics and Biopharmaceutics
Reftype
Groups
AbstractBesides deposition, pulmonary bioavailability is determined by dissolution of particles in the scarce epithelial fluid and by cellular API uptake. In the present work, we have developed an experimental in vitro model, which is combining the state-of-the-art next generation impactor (NGI), used for aerodynamic performance assessment of inhalation products, with a culture of human alveolar A549 epithelial cells to study the fate of inhaled drugs following lung deposition. The goal was to investigate five previously developed nano-milled and spray-dried budesonide formulations and to examine the suitability of the in vitro test model. The NGI dissolution cups of stages 3, 4, and 5 were transformed to accommodate cell culture inserts while assuring minimal interference with the air flow. A549 cells were cultivated at the air–liquid interface on Corning® Matrigel® -coated inserts. After deposition of aerodynamically classified powders on the cell cultures, budesonide amount was determined on the cell surface, in the interior of the cell monolayer, and in the basal solution for four to eight hours. Significant differences in the total deposited drug amount and the amount remaining on the cell surface at the end of the experiment were found between different formulations and NGI stages. Roughly 50% of budesonide was taken up by the cells and converted to a large extent to its metabolic conjugate with oleic acid for all formulations and stages. Prolonged time required for complete drug dissolution and cell uptake in case of large deposited powder amounts suggested initial drug saturation of the surfactant layer of the cell surface. Discrimination between formulations with respect to time scale of dissolution and cell uptake was possible with the present test model providing useful insights into the biopharmaceutical performance of developed formulations that may be relevant for predicting local bioavailability. The absolute quantitative result of cell uptake and permeation into the systemic compartment is unreliable, though, because of partly compromised cell membrane integrity due to particle impaction and professed leakiness of A549 monolayer tight junctions, respectively.
AUTHOR
Year
2020
Journal/Proceedings
Advanced Functional Materials
Reftype
DOI/URL
DOI
Groups
AbstractAbstract The composition and intercellular interactions of tumor cells in the tissues dictate the biochemical and metabolic properties of the tumor microenvironment. The metabolic rewiring has a profound impact on the properties of the microenvironment, to an extent that monitoring such perturbations could harbor diagnostic and therapeutic relevance. A growing interest in these phenomena has inspired the development of novel technologies with sufficient sensitivity and resolution to monitor metabolic alterations in the tumor microenvironment. In this context, surface-enhanced Raman scattering (SERS) can be used for the label-free detection and imaging of diverse molecules of interest among extracellular components. Herein, the application of nanostructured plasmonic substrates comprising Au nanoparticles, self-assembled as ordered superlattices, to the precise SERS detection of selected tumor metabolites, is presented. The potential of this technology is first demonstrated through the analysis of kynurenine, a secreted immunomodulatory derivative of the tumor metabolism and the related molecules tryptophan and purine derivatives. SERS facilitates the unambiguous identification of trace metabolites and allows the multiplex detection of their characteristic fingerprints under different conditions. Finally, the effective plasmonic SERS substrate is combined with a hydrogel-based three-dimensional cancer model, which recreates the tumor microenvironment, for the real-time imaging of metabolite alterations and cytotoxic effects on tumor cells.
AUTHOR
Title
Printed elastic membranes for multimodal pacing and recording of human stem-cell-derived cardiomyocytes
[Abstract]
Year
2020
Journal/Proceedings
npj Flexible Electronics
Reftype
Athanasiadis2020
DOI/URL
DOI
Groups
AbstractBioelectronic interfaces employing arrays of sensors and bioactuators are promising tools for the study, repair and engineering of cardiac tissues. They are typically constructed from rigid and brittle materials processed in a cleanroom environment. An outstanding technological challenge is the integration of soft materials enabling a closer match to the mechanical properties of biological cells and tissues. Here we present an algorithm for direct writing of elastic membranes with embedded electrodes, optical waveguides and microfluidics using a commercial 3D printing system and a palette of silicone elastomers. As proof of principle, we demonstrate interfacing of cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs), which are engineered to express Channelrhodopsin-2. We demonstrate electrical recording of cardiomyocyte field potentials and their concomitant modulation by optical and pharmacological stimulation delivered via the membrane. Our work contributes a simple prototyping strategy with potential applications in organ-on-chip or implantable systems that are multi-modal and mechanically soft.
AUTHOR
Title
Three-dimensional liver models: state of the art and their application for hepatotoxicity evaluation
[Abstract]
Year
2020
Journal/Proceedings
Critical Reviews in Toxicology
Reftype
DOI/URL
DOI
AbstractAbstractWhile alternative methods for toxicity testing using re-constructed human skin and cornea have been written into guidelines and adopted by regulatory authorities, three-dimensional (3D) liver models are currently applied in the industrial settings for hepatotoxicity screening and prediction. These 3D liver models can recapitulate the architecture, functionality and toxicity response of the native liver, demonstrated by a set of related hallmarks. In this comprehensive review, non-scaffold and scaffold-based methods available for 3D liver model formation are introduced, with an emphasis on their advantages and drawbacks. We then focus on the characteristics of primary human hepatocytes, stem cell derived hepatocyte like cells, and immortalized hepatic cell lines as cell resources for model reconstruction. Primary hepatocytes are generally regarded to be superior to other cell types due to their comparable metabolic profiles to the native liver. Additionally, the application of 3D liver models (mostly liver spheroids) on the evaluation of drug induced liver injury and chronic liver diseases (steatosis, cirrhosis, cholestasis), as well as the potential of nanomaterials to introduce hepatotoxicity are summarized. Finally, the global 3D cell market from 3D liver model manufacturing to the contract service of in vitro hepatotoxicity testing using the models is extensively explored. However, 3D liver models face cultural and regulatory barriers in different countries, and therefore the business development of 3D liver models is not easy. Toxicologists, material scientists, engineers should work together to develop, validate and apply 3D liver models for hepatotoxicity testing under the support from industrial organizations and governmental agencies.
AUTHOR
Title
A bilayer photoreceptor‐retinal tissue model with gradient cell density design: A study of microvalve‐based bioprinting
[Abstract]
Year
2018
Journal/Proceedings
Journal of Tissue Engineering and Regenerative Medicine
Reftype
DOI/URL
DOI
Groups
AbstractAbstract ARPE‐19 and Y79 cells were precisely and effectively delivered to form an in vitro retinal tissue model via 3D cell bioprinting technology. The samples were characterized by cell viability assay, haematoxylin and eosin and immunofluorescent staining, scanning electrical microscopy and confocal microscopy, and so forth. The bioprinted ARPE‐19 cells formed a high‐quality cell monolayer in 14 days. Manually seeded ARPE‐19 cells were poorly controlled during and after cell seeding, and they aggregated to form uneven cell layer. The Y79 cells were subsequently bioprinted on the ARPE‐19 cell monolayer to form 2 distinctive patterns. The microvalve‐based bioprinting is efficient and accurate to build the in vitro tissue models with the potential to provide similar pathological responses and mechanism to human diseases, to mimic the phenotypic endpoints that are comparable with clinical studies, and to provide a realistic prediction of clinical efficacy.
AUTHOR
Title
Composite Biomaterials as Long-Lasting Scaffolds for 3D Bioprinting of Highly Aligned Muscle Tissue
Year
2018
Journal/Proceedings
Macromolecular Bioscience
Reftype
DOI/URL
DOI
Groups
AUTHOR
Year
2018
Journal/Proceedings
Nanomaterials
Reftype
Groups
AbstractPolydopamine can form biocompatible particles that convert light into heat. Recently, a protocol has been optimized to synthesize polydopamine/protein hybrid nanoparticles that retain the biological function of proteins, and combine it with the stimuli-induced heat generation of polydopamine. We have utilized this novel system to form polydopamine particles, containing transferrin (PDA/Tf). Mouse melanoma cells, which strongly express the transferrin receptor, were exposed to PDA/Tf nanoparticles (NPs) and, subsequently, were irradiated with a UV laser. The cell death rate was monitored in real-time. When irradiated, the melanoma cells exposed to PDA/Tf NPs underwent apoptosis, faster than the control cells, pointing towards the ability of PDA/Tf to mediate UV-light-induced cell death. The system was also validated in an organotypic, 3D-printed tumor spheroid model, comprising mouse melanoma cells, and the exposure and subsequent irradiation with UV-light, yielded similar results to the 2D cell culture. The process of apoptosis was found to be targeted and mediated by the lysosomal membrane permeabilization. Therefore, the herein presented polydopamine/protein NPs constitute a versatile and stable system for cancer cell-targeting and photothermal apoptosis induction.
AUTHOR
Year
2017
Journal/Proceedings
Circulation Research
Reftype
AbstractCurrent strategies for engineering cardiovascular cells and tissues have yielded a variety of sophisticated tools for studying disease mechanisms, for development of drug therapies, and for fabrication of tissue equivalents that may have application in future clinical use. These efforts are motivated by the need to extend traditional 2-dimensional (2D) cell culture systems into 3D to more accurately replicate in vivo cell and tissue function of cardiovascular structures. Developments in microscale devices and bioprinted 3D tissues are beginning to supplant traditional 2D cell cultures and preclinical animal studies that have historically been the standard for drug and tissue development. These new approaches lend themselves to patient-specific diagnostics, therapeutics, and tissue regeneration. The emergence of these technologies also carries technical challenges to be met before traditional cell culture and animal testing become obsolete. Successful development and validation of 3D human tissue constructs will provide powerful new paradigms for more cost effective and timely translation of cardiovascular tissue equivalents.
AUTHOR
Title
Increased lipid accumulation and adipogenic gene expression of adipocytes in 3D bioprinted nanocellulose scaffolds
[Abstract]
Year
2017
Journal/Proceedings
Biofabrication
Reftype
DOI/URL
URL
Groups
AbstractCompared to standard 2D culture systems, new methods for 3D cell culture of adipocytes could provide more physiologically accurate data and a deeper understanding of metabolic diseases such as diabetes. By resuspending living cells in a bioink of nanocellulose and hyaluronic acid, we were able to print 3D scaffolds with uniform cell distribution. After one week in culture, cell viability was 95%, and after two weeks the cells displayed a more mature phenotype with larger lipid droplets than standard 2D cultured cells. Unlike cells in 2D culture, the 3D bioprinted cells did not detach upon lipid accumulation. After two weeks, the gene expression of the adipogenic marker genes PPAR γ and FABP4 was increased 2.0- and 2.2-fold, respectively, for cells in 3D bioprinted constructs compared with 2D cultured cells. Our 3D bioprinted culture system produces better adipogenic differentiation of mesenchymal stem cells and a more mature cell phenotype than conventional 2D culture systems.
AUTHOR
Title
Layer-by-Layer 3D Constructs of Fibroblasts in Hydrogel for Examining Transdermal Penetration Capability of Nanoparticles
[Abstract]
Year
2016
Journal/Proceedings
Journal of Laboratory Automation
Reftype
Groups
AbstractNanoparticles are emerging transdermal delivery systems. Their size and surface properties determine their efficacy and efficiency to penetrate through the skin layers. This work utilizes three-dimensional (3D) bioprinting technology to generate a simplified artificial skin model to rapidly screen nanoparticles for their transdermal penetration ability. Specifically, this model is built through layer-by-layer alternate printing of blank collagen hydrogel and fibroblasts. Through controlling valve on-time, the spacing between printing lines could be accurately tuned, which could enable modulation of cell infiltration in the future. To confirm the effectiveness of this platform, a 3D construct with one layer of fibroblasts sandwiched between two layers of collagen hydrogel is used to screen silica nanoparticles with different surface charges for their penetration ability, with positively charged nanoparticles demonstrating deeper penetration, consistent with the observation from an existing study involving living skin tissue.
AUTHOR
Title
3D Bioprinted Muscle and Tendon Tissues for Drug Development
Year
2015
Journal/Proceedings
{CHIMIA} International Journal for Chemistry
Reftype
DOI/URL
DOI
AUTHOR
Year
2015
Journal/Proceedings
Scientific reports
Reftype
DOI/URL
URL
Groups
AbstractIntensive efforts in recent years to develop and commercialize in vitro alternatives in the field of risk assessment have yielded new promising two- and three dimensional (3D) cell culture models. Nevertheless, a realistic 3D in vitro alveolar model is not available yet. Here we report on the biofabrication of the human air-blood tissue barrier analogue composed of an endothelial cell, basement membrane and epithelial cell layer by using a bioprinting technology. In contrary to the manual method, we demonstrate that this technique enables automatized and reproducible creation of thinner and more homogeneous cell layers, which is required for an optimal air-blood tissue barrier. This bioprinting platform will offer an excellent tool to engineer an advanced 3D lung model for high-throughput screening for safety assessment and drug efficacy testing.
AUTHOR
Year
2015
Journal/Proceedings
Journal of laboratory automation
Reftype
DOI/URL
DOI
Groups
AbstractCells grown in 3D are more physiologically relevant than cells cultured in 2D. To use 3D models in substance testing and regenerative medicine, reproducibility and standardization are important. Bioprinting offers not only automated standardizable processes but also the production of complex tissue-like structures in an additive manner. We developed an all-in-one bioprinting solution to produce soft tissue models. The holistic approach included (1) a bioprinter in a sterile environment, (2) a light-induced bioink polymerization unit, (3) a user-friendly software, (4) the capability to print in standard labware for high-throughput screening, (5) cell-compatible inkjet-based printheads, (6) a cell-compatible ready-to-use BioInk, and (7) standard operating procedures. In a proof-of-concept study, skin as a reference soft tissue model was printed. To produce dermal equivalents, primary human dermal fibroblasts were printed in alternating layers with BioInk and cultured for up to 7 weeks. During long-term cultures, the models were remodeled and fully populated with viable and spreaded fibroblasts. Primary human dermal keratinocytes were seeded on top of dermal equivalents, and epidermis-like structures were formed as verified with hematoxylin and eosin staining and immunostaining. However, a fully stratified epidermis was not achieved. Nevertheless, this is one of the first reports of an integrative bioprinting strategy for industrial routine application.