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AUTHOR Nothdurfter, Daniel and Ploner, Christian and Coraça-Huber, Débora C. and Wilflingseder, Doris and Müller, Thomas and Hermann, Martin and Hagenbuchner, Judith and Ausserlechner, Michael J.
Title 3D bioprinted, vascularized neuroblastoma tumor environment in fluidic chip devices for precision medicine drug testing [Abstract]
Year 2022
Journal/Proceedings Biofabrication
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Neuroblastoma 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 D'Agostino, Stefania and Rimann, Markus and Gamba, Piergiorgio and Perilongo, Giorgio and Pozzobon, Michela and Raghunath, Michael
Title Macromolecular crowding tuned extracellular matrix deposition in a bioprinted human rhabdomyosarcoma model [Abstract]
Year 2022
Journal/Proceedings Bioprinting
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The 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 Bouwmeester, Manon C. and Bernal, Paulina N. and Oosterhoff, Loes A. and van Wolferen, Monique E. and Lehmann, Vivian and Vermaas, Monique and Buchholz, Maj-Britt and Peiffer, Quentin C. and Malda, Jos and van der Laan, Luc J. W. and Kramer, Nynke I. and Schneeberger, Kerstin and Levato, Riccardo and Spee, Bart
Title Bioprinting of Human Liver-Derived Epithelial Organoids for Toxicity Studies [Abstract]
Year 2021
Journal/Proceedings Macromolecular Bioscience
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Abstract 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 Ng, Wei Long and Ayi, Teck Choon and Liu, Yi-Chun and Sing, Swee Leong and Yeong, Wai Yee and Tan, Boon-Huan
Title Fabrication and Characterization of 3D Bioprinted Triple-layered Human Alveolar Lung Models [Abstract]
Year 2021
Journal/Proceedings International journal of bioprinting
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The 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 Alave Reyes-Furrer, Angela and De Andrade, Sonia and Bachmann, Dominic and Jeker, Heidi and Steinmann, Martin and Accart, Nathalie and Dunbar, Andrew and Rausch, Martin and Bono, Epifania and Rimann, Markus and Keller, Hansjoerg
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
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A 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 Madiedo-Podvrsan, Sabrina and Belaïdi, Jean-Philippe and Desbouis, Stephanie and Simonetti, Lucie and Ben-Khalifa, Youcef and Soeur, Jérémie and Rielland, Maïté
Title Utilization of patterned bioprinting for heterogeneous and physiologically representative reconstructed epidermal skin models [Abstract]
Year 2021
Journal/Proceedings Scientific Reports
Reftype Madiedo-Podvrsan2021
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Organotypic 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 Kajtez, Janko and Buchmann, Sebastian and Vasudevan, Shashank and Birtele, Marcella and Rocchetti, Stefano and Pless, Christian Jonathan and Heiskanen, Arto and Barker, Roger A. and Martínez-Serrano, Alberto and Parmar, Malin and Lind, Johan Ulrik and Emnéus, Jenny
Title 3D-Printed Soft Lithography for Complex Compartmentalized Microfluidic Neural Devices [Abstract]
Year 2020
Journal/Proceedings Advanced Science
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Abstract 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 Browning, James R. and Derr, Paige and Derr, Kristy and Doudican, Nicole and Michael, Sam and Lish, Samantha R. and Taylor, Nicholas A. and Krueger, James G. and Ferrer, Marc and Carucci, John A. and Gareau, Daniel S.
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
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// 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 Monferrer, Ezequiel and Martín-Vañó, Susana and Carretero, Aitor and García-Lizarribar, Andrea and Burgos-Panadero, Rebeca and Navarro, Samuel and Samitier, Josep and Noguera, Rosa
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
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Three-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 Wei, Zhengxi and Liu, Xue and Ooka, Masato and Zhang, Li and Song, Min Jae and Huang, Ruili and Kleinstreuer, Nicole C. and Simeonov, Anton and Xia, Menghang and Ferrer, Marc
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
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Assessing 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 Derr, Kristy and Zou, Jinyun and Luo, Keren and Song, Min Jae and Sittampalam, G. Sitta and Zhou, Chao and Michael, Samuel and Ferrer, Marc and Derr, Paige
Title Fully 3D Bioprinted Skin Equivalent Constructs with Validated Morphology and Barrier Function [Abstract]
Year 2019
Journal/Proceedings Tissue Engineering Part C: Methods
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Development 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 Laternser, Sandra and Keller, Hansjoerg and Leupin, Olivier and Rausch, Martin and Graf-Hausner, Ursula and Rimann, Markus
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
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Two-dimensional (2D) cell cultures do not reflect the in vivo situation, and thus it is important to develop predictive three-dimensional (3D) in vitro models with enhanced reliability and robustness for drug screening applications. Treatments against muscle-related diseases are becoming more prominent due to the growth of the aging population worldwide. In this study, we describe a novel drug screening platform with automated production of 3D musculoskeletal-tendon-like tissues. With 3D bioprinting, alternating layers of photo-polymerized gelatin-methacryloyl-based bioink and cell suspension tissue models were produced in a dumbbell shape onto novel postholder cell culture inserts in 24-well plates. Monocultures of human primary skeletal muscle cells and rat tenocytes were printed around and between the posts. The cells showed high viability in culture and good tissue differentiation, based on marker gene and protein expressions. Different printing patterns of bioink and cells were explored and calcium signaling with Fluo4-loaded cells while electrically stimulated was shown. Finally, controlled co-printing of tenocytes and myoblasts around and between the posts, respectively, was demonstrated followed by co-culture and co-differentiation. This screening platform combining 3D bioprinting with a novel microplate represents a promising tool to address musculoskeletal diseases.
AUTHOR Roopesh, Ramesh Pai and Muthusamy, Senthilkumar and Velayudhan, Shiny and Sabareeswaran, Arumugham and Anil Kumar, Pallickaveedu RajanAsari
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
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Abstract 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 Dusserre, Nathalie and Stachowicz, Marie-Laure and Medina, Chantal and Henri, Baptiste and Fricain, Jean-Christophe and Paris, François and Oliveira, Hugo
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
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Bioprinting 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 Liu, Xue and Michael, Samuel and Bharti, Kapil and Ferrer, Marc and Song, Min Jae
Title A biofabricated vascularized skin model of atopic dermatitis for preclinical studies [Abstract]
Year 2020
Journal/Proceedings Biofabrication
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Three-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 Ng, Wei Long and Qi, Jovina Tan Zhi and Yeong, Wai Yee and Naing, May Win
Title Proof-of-concept: 3D bioprinting of pigmented human skin constructs [Abstract]
Year 2018
Journal/Proceedings Biofabrication
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Three-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 Azim, N. and Hart, C. and Sommerhage, F. and Aubin, M. and Hickman, J. J. and Rajaraman, S.
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
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Microelectrode 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 Dorjsuren, Dorjbal and Eastman, Richard T. and Song, Min Jae and Yasgar, Adam and Chen, Yuchi and Bharti, Kapil and Zakharov, Alexey V. and Jadhav, Ajit and Ferrer, Marc and Shi, Pei-Yong and Simeonov, Anton
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
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The 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 Cakal, Selgin D. and Radeke, Carmen and Alcala, Juan F. and Ellman, Ditte G. and Butdayev, Sarkhan and Andersen, Ditte C. and Calloe, Kirstine and Lind, Johan U.
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
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Abstract
Preclinical 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 Sarti, Mattia and Parlani, Maria and Diaz-Gomez, Luis and Mikos, Antonios G. and Cerveri, Pietro and Casarin, Stefano and Dondossola, Eleonora
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
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DOI/URL DOI
Abstract
The 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 Geevarghese, Rency and Somasekharan, Lakshmi T. and Bhatt, Anugya and Kasoju, Naresh and Nair, Renjith P.
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
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DOI/URL URL DOI
Abstract
Three 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 Ramakrishnan, Rashmi and Kasoju, Naresh and Raju, Riya and Geevarghese, Rency and Gauthaman, Ashna and Bhatt, Anugya
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
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DOI/URL URL DOI
Abstract
Designing 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 Girardeau-Hubert, Sarah and Lynch, Barbara and Zuttion, Francesca and Label, Rabab and Rayee, Chrystelle and Brizion, Sébastien and Ricois, Sylvie and Martinez, Anthony and Park, Eunhye and Kim, Changhwan and Marinho, Paulo André and Shim, Jin-Hyung and Jin, Songwan and Rielland, Maïté and Soeur, Jérémie
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
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DOI/URL URL DOI
Abstract
Skin 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 Liu, Jing and Zhou, Zhengtong and Zhang, Min and Song, Feng and Feng, Chong and Liu, Haochen
Title Simple and robust 3D bioprinting of full-thickness human skin tissue [Abstract]
Year 2022
Journal/Proceedings Bioengineered
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Abstract
ABSTRACTArtificial 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 Tutty, Melissa Anne and Movia, Dania and Prina-Mello, Adriele
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
Abstract
Despite 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 Seydel, Caroline
Title 3D-Bioprinted Cell Therapy and Disease Modeling Applications
Year 2021
Journal/Proceedings Genetic Engineering & Biotechnology News
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DOI/URL DOI
AUTHOR Bello, Thomas and Paindelli, Claudia and Diaz-Gomez, Luis A. and Melchiorri, Anthony and Mikos, Antonios G. and Nelson, Peter S. and Dondossola, Eleonora and Gujral, Taranjit S.
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
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DOI/URL URL DOI
Abstract
Metastatic, 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 Paindelli, Claudia and Casarin, Stefano and Wang, Feng and Diaz-Gomez, Luis and Zhang, Jianhua and Mikos, Antonios G. and Logothetis, Christopher J. and Friedl, Peter and Dondossola, Eleonora
Title Enhancing Radium 223 treatment efficacy by anti-beta 1 integrin targeting [Abstract]
Year 2021
Journal/Proceedings Journal of Nuclear Medicine
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Abstract
Radium 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 Estermann, Manuela and Bisig, Christoph and Septiadi, Dedy and Petri-Fink, Alke and Rothen-Rutishauser, Barbara
Title Bioprinting for Human Respiratory and Gastrointestinal In Vitro Models [Abstract]
Year 2020
Journal/Proceedings
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DOI/URL DOI
Abstract
Increasing 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 Huang, Yen-Lin and Liang, Ching-Yeu and Ritz, Danilo and Coelho, Ricardo and Septiadi, Dedy and Estermann, Manuela and Cumin, Cécile and Rimmer, Natalie and Schötzau, Andreas and Núñez López, Mónica and Fedier, André and Konantz, Martina and Vlajnic, Tatjana and Calabrese, Diego and Lengerke, Claudia and David, Leonor and Rothen-Rutishauser, Barbara and Jacob, Francis and Heinzelmann-Schwarz, Viola
Title Collagen-rich omentum is a premetastatic niche for integrin α2-mediated peritoneal metastasis [Abstract]
Year 2020
Journal/Proceedings eLife
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DOI/URL DOI
Abstract
The 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 Steier, Anke and Schmieg, Barbara and Irtel von Brenndorff, Yannic and Meier, Manuel and Nirschl, Hermann and Franzreb, Matthias and Lahann, Joerg
Title Enzyme Scaffolds with Hierarchically Defined Properties via 3D Jet Writing [Abstract]
Year 2020
Journal/Proceedings Macromolecular Bioscience
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DOI/URL DOI
Abstract
Abstract The immobilization of enzymes into polymer hydrogels is a versatile approach to improve their stability and utility in biotechnological and biomedical applications. However, these systems typically show limited enzyme activity, due to unfavorable pore dimensions and low enzyme accessibility. Here, 3D jet writing of water-based bioinks, which contain preloaded enzymes, is used to prepare hydrogel scaffolds with well-defined, tessellated micropores. After 3D jet writing, the scaffolds are chemically modified via photopolymerization to ensure mechanical stability. Enzyme loading and activity in the hydrogel scaffolds is fully retained over 3 d. Important structural parameters of the scaffolds such as pore size, pore geometry, and wall diameter are controlled with micrometer resolution to avoid mass-transport limitations. It is demonstrated that scaffold pore sizes between 120 µm and 1 mm can be created by 3D jet writing approaching the length scales of free diffusion in the hydrogels substrates and resulting in high levels of enzyme activity (21.2% activity relative to free enzyme). With further work, a broad range of applications for enzyme-laden hydrogel scaffolds including diagnostics and enzymatic cascade reactions is anticipated.
AUTHOR López-Carrasco, Amparo and Martín-Vañó, Susana and Burgos-Panadero, Rebeca and Monferrer, Ezequiel and Berbegall, Ana P. and Fernández-Blanco, Beatriz and Navarro, Samuel and Noguera, Rosa
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
Abstract
Increased 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 Šimková, Kateřina and Thormann, Ursula and Imanidis, Georgios
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
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DOI/URL URL DOI
Abstract
Besides 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 Plou, Javier and García, Isabel and Charconnet, Mathias and Astobiza, Ianire and García-Astrain, Clara and Matricardi, Cristiano and Mihi, Agustín and Carracedo, Arkaitz and Liz-Marzán, Luis M.
Title Multiplex SERS Detection of Metabolic Alterations in Tumor Extracellular Media [Abstract]
Year 2020
Journal/Proceedings Advanced Functional Materials
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Abstract
Abstract 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 Chen, Grona and Xu, Yihua and Chi Lip Kwok, Philip and Kang, Lifeng
Title Pharmaceutical Applications of 3D Printing [Abstract]
Year 2020
Journal/Proceedings Additive Manufacturing
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Abstract
Although 3D printing (3DP) has long been an integral part of industries such as aviation and automotive, its use in healthcare, especially the pharmaceutical industry, is relatively new and currently receiving close attention. At the beginning of 2018, we reviewed the applications of 3DP for drug delivery and drug testing [1]. Due to the rapid development of this field, it is necessary to summarize the latest development in this field after 2 years. In this article, we reviewed the three major areas in pharmaceutical applications. First, drug delivery system is the most studied subject, including controlled release, polypills, gastrofloating, orodispersibles and microneedles. Second, 3DP also helped the development of pharmaceutical devices, including pharmacy dispensing aids and drug eluting devices. Lastly, we reviewed the pharmaceutical models for drug testing, covering acellular and cellular models. We also summarized the materials used in the mentioned articles and their regulatory status for pharmaceutical applications to provide references for future research.
AUTHOR Athanasiadis, Markos and Afanasenkau, Dzmitry and Derks, Wouter and Tondera, Christoph and Murganti, Francesca and Busskamp, Volker and Bergmann, Olaf and Minev, Ivan R.
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
Abstract
Bioelectronic 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 Zhang, Xihui and Jiang, Tianyan and Chen, Dandan and Wang, Qi and Zhang, Leshuai W.
Title Three-dimensional liver models: state of the art and their application for hepatotoxicity evaluation [Abstract]
Year 2020
Journal/Proceedings Critical Reviews in Toxicology
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Abstract
AbstractWhile 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 Jiang, Tao and Munguía López, Jose and Flores-Torres, Salvador and Kort-Mascort, Jacqueline and Kinsella, Joseph
Title Extrusion bioprinting of soft materials: An emerging technique for biological model fabrication
Year 2019
Journal/Proceedings Applied Physics Reviews
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AUTHOR Shi, Pujiang and Tan, Yong Sheng Edgar and Yeong, Wai Yee and Li, Hoi Yeung and Laude, Augustinus
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
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Abstract
Abstract 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 García-Lizarribar, Andrea and Fernández-Garibay, Xiomara and Velasco-Mallorquí, Ferran and G. Castaño, Albert and Samitier, Josep and Ramón-Azcón, Javier
Title Composite Biomaterials as Long-Lasting Scaffolds for 3D Bioprinting of Highly Aligned Muscle Tissue
Year 2018
Journal/Proceedings Macromolecular Bioscience
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AUTHOR Gill, Elisabeth L. and Li, Xia and Birch, Mark A. and Huang, Yan Yan Shery
Title Multi-length scale bioprinting towards simulating microenvironmental cues [Abstract]
Year 2018
Journal/Proceedings Bio-Design and Manufacturing
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DOI/URL DOI
Abstract
It is envisaged that the creation of cellular environments at multiple length scales, that recapitulate in vivo bioactive and structural roles, may hold the key to creating functional, complex tissues in the laboratory. This review considers recent advances in biofabrication and bioprinting techniques across different length scales. Particular focus is placed on 3D printing of hydrogels and fabrication of biomaterial fibres that could extend the feature resolution and material functionality of soft tissue constructs. The outlook from this review discusses how one might create and simulate microenvironmental cues in vitro. A fabrication platform that integrates the competencies of different biofabrication technologies is proposed. Such a multi-process, multiscale fabrication strategy may ultimately translate engineering capability into an accessible life sciences toolkit, fulfilling its potential to deliver in vitro disease models and engineered tissue implants.
AUTHOR Hauser, Daniel and Estermann, Manuela and Milosevic, Ana and Steinmetz, Lukas and Vanhecke, Dimitri and Septiadi, Dedy and Drasler, Barbara and Petri-Fink, Alke and Ball, Vincent and Rothen-Rutishauser, Barbara
Title Polydopamine/Transferrin Hybrid Nanoparticles for Targeted Cell-Killing [Abstract]
Year 2018
Journal/Proceedings Nanomaterials
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DOI/URL URL DOI
Abstract
Polydopamine 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 Kumari, Sushma and Bargel, Hendrik and Anby, Mette U. and Lafargue, David and Scheibel, Thomas
Title Recombinant Spider Silk Hydrogels for Sustained Release of Biologicals [Abstract]
Year 2018
Journal/Proceedings ACS Biomaterials Science and Engineering
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DOI/URL DOI
Abstract
Therapeutic biologics (i.e., proteins) have been widely recognized for the treatment, prevention, and cure of a variety of human diseases and syndromes. However, design of novel protein-delivery systems to achieve a nontoxic, constant, and efficient delivery with minimal doses of therapeutic biologics is still challenging. Here, recombinant spider silk-based materials are employed as a delivery system for the administration of therapeutic biologicals. Hydrogels made of the recombinant spider silk protein eADF4(C16) were used to encapsulate the model biologicals BSA, HRP, and LYS by direct loading or through diffusion, and their release was studied. Release of model biologicals from eADF4(C16) hydrogels is in part dependent on the electrostatic interaction between the biological and the recombinant spider silk protein variant used. In addition, tailoring the pore sizes of eADF4(C16) hydrogels strongly influenced the release kinetics. In a second approach, a particles-in-hydrogel system was used, showing a prolonged release in comparison with that of plain hydrogels (from days to week). The particle-enforced spider silk hydrogels are injectable and can be 3D printed. These initial studies indicate the potential of recombinant spider silk proteins to design novel injectable hydrogels that are suitable for delivering therapeutic biologics.
AUTHOR Peng, Weijie and Datta, Pallab and Ayan, Bugra and Ozbolat, Veli and Sosnoski, Donna and Ozbolat, Ibrahim T.
Title 3D bioprinting for drug discovery and development in pharmaceutics [Abstract]
Year 2017
Journal/Proceedings Acta Biomaterialia
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Abstract
Successful launch of a commercial drug requires significant investment of time and financial resources wherein late-stage failures become a reason for catastrophic failures in drug discovery. This calls for infusing constant innovations in technologies, which can give reliable prediction of efficacy, and more importantly, toxicology of the compound early in the drug discovery process before clinical trials. Though computational advances have resulted in more rationale in silico designing, in vitro experimental studies still require gaining industry confidence and improving in vitro-in vivo correlations. In this quest, due to their ability to mimic the spatial and chemical attributes of native tissues, three-dimensional (3D) tissue models have now proven to provide better results for drug screening compared to traditional two-dimensional (2D) models. However, in vitro fabrication of living tissues has remained a bottleneck in realizing the full potential of 3D models. Recent advances in bioprinting provide a valuable tool to fabricate biomimetic constructs, which can be applied in different stages of drug discovery research. This paper presents the first comprehensive review of bioprinting techniques applied for fabrication of 3D tissue models for pharmaceutical studies. A comparative evaluation of different bioprinting modalities is performed to assess the performance and ability of fabricating 3D tissue models for pharmaceutical use as the critical selection of bioprinting modalities indeed plays a crucial role in efficacy and toxicology testing of drugs and accelerates the drug development cycle. In addition, limitations with current tissue models are discussed thoroughly and future prospects of the role of bioprinting in pharmaceutics are provided to the reader. Present advances in tissue biofabrication have crucial role to play in aiding the pharmaceutical development process achieve its objectives. Advent of three-dimensional (3D) models, in particular, is viewed with immense interest by the community due to their ability to mimic in vivo hierarchical tissue architecture and heterogeneous composition. Successful realization of 3D models will not only provide greater in vitro-in vivo correlation compared to the two-dimensional (2D) models, but also eventually replace pre-clinical animal testing, which has their own shortcomings. Amongst all fabrication techniques, bioprinting- comprising all the different modalities (extrusion-, droplet- and laser-based bioprinting), is emerging as the most viable fabrication technique to create the biomimetic tissue constructs. Notwithstanding the interest in bioprinting by the pharmaceutical development researchers, it can be seen that there is a limited availability of comparative literature which can guide the proper selection of bioprinting processes and associated considerations, such as the bioink selection for a particular pharmaceutical study. Thus, this work emphasizes these aspects of bioprinting and presents them in perspective of differential requirements of different pharmaceutical studies like in vitro predictive toxicology, high-throughput screening, drug delivery and tissue-specific efficacies. Moreover, since bioprinting techniques are mostly applied in regenerative medicine and tissue engineering, a comparative analysis of similarities and differences are also expounded to help researchers make informed decisions based on contemporary literature.
AUTHOR Borovjagin, Anton V. and Ogle, Brenda M. and Berry, Joel L. and Zhang, Jianyi
Title From Microscale Devices to 3D Printing [Abstract]
Year 2017
Journal/Proceedings Circulation Research
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Abstract
Current 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 Henriksson, I. and Gatenholm, P. and Hägg, D. A.
Title Increased lipid accumulation and adipogenic gene expression of adipocytes in 3D bioprinted nanocellulose scaffolds [Abstract]
Year 2017
Journal/Proceedings Biofabrication
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Abstract
Compared 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 Hou, Xiaochun and Liu, Shiying and Wang, Min and Wiraja, Christian and Huang, Wei and Chan, Peggy and Tan, Timothy and Xu, Chenjie
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
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Abstract
Nanoparticles 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 Arslan-Yildiz, Ahu and Assal, Rami El and Chen, Pu and Guven, Sinan and Inci, Fatih and Demirci, Utkan
Title Towards artificial tissue models: past, present, and future of 3D bioprinting [Abstract]
Year 2016
Journal/Proceedings Biofabrication
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Abstract
Regenerative medicine and tissue engineering have seen unprecedented growth in the past decade, driving the field of artificial tissue models towards a revolution in future medicine. Major progress has been achieved through the development of innovative biomanufacturing strategies to pattern and assemble cells and extracellular matrix (ECM) in three-dimensions (3D) to create functional tissue constructs. Bioprinting has emerged as a promising 3D biomanufacturing technology, enabling precise control over spatial and temporal distribution of cells and ECM. Bioprinting technology can be used to engineer artificial tissues and organs by producing scaffolds with controlled spatial heterogeneity of physical properties, cellular composition, and ECM organization. This innovative approach is increasingly utilized in biomedicine, and has potential to create artificial functional constructs for drug screening and toxicology research, as well as tissue and organ transplantation. Herein, we review the recent advances in bioprinting technologies and discuss current markets, approaches, and biomedical applications. We also present current challenges and provide future directions for bioprinting research.
AUTHOR Rimann, Markus and Laternser, Sandra and Keller, Hansj{"{o}}rg and Leupin, Olivier and Graf-Hausner, Ursula
Title 3D Bioprinted Muscle and Tendon Tissues for Drug Development
Year 2015
Journal/Proceedings {CHIMIA} International Journal for Chemistry
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AUTHOR Horvath, Lenke and Umehara, Yuki and Jud, Corinne and Blank, Fabian and Petri-Fink, Alke and Rothen-Rutishauser, Barbara
Title Engineering an in vitro air-blood barrier by 3D bioprinting. [Abstract]
Year 2015
Journal/Proceedings Scientific reports
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Abstract
Intensive 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 Rimann, Markus and Bono, Epifania and Annaheim, Helene and Bleisch, Matthias and Graf-Hausner, Ursula
Title Standardized 3D Bioprinting of Soft Tissue Models with Human Primary Cells. [Abstract]
Year 2015
Journal/Proceedings Journal of laboratory automation
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Abstract
Cells 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.