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1.
Tissue Eng Part A ; 25(13-14): 1023-1036, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30412045

RESUMO

IMPACT STATEMENT: The ability to freeze, revive, and prolong the lifetime of tissue-engineered skeletal muscle without incurring any loss of function represents a significant advancement in the field of tissue engineering. Cryopreservation enables the efficient fabrication, storage, and shipment of these tissues. This in turn facilitates multidisciplinary collaboration between research groups, enabling advances in skeletal muscle regenerative medicine, organ-on-a-chip models of disease, drug testing, and soft robotics. Furthermore, the observation that freezing undifferentiated skeletal muscle enhances functional performance may motivate future studies developing stronger and more clinically relevant engineered muscle.

2.
Dev Dyn ; 248(1): 21-33, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30016584

RESUMO

Astrocytes exhibit dynamic and complex reactions to various insults. Recently, investigations into the transitions that occur during cellular specification, differentiation, maturation, and disease responses have provided insights into understanding the mechanisms that underlie these altered states of reactivity and function. Here we summarize current concepts in how astrocyte state transitions, termed astroplasticity, are regulated, as well as how this affects neural circuit function through extracellular signaling. We postulate that a promising future approach toward enhancing functional repair after injury and disease would be to steer astrocytes away from an inhibitory response and toward one that is beneficial to neuroplasticity and neuroregeneration. Toward this goal, we discuss emerging biotechnological advancements, with a focus on human pluripotent stem cell bioengineering, which has high potential for effective manipulation and control of astroplasticity. Highlights include innovations in cellular transdifferentiation techniques, nanomedicine, organoid and three-dimensional (3D) spheroid microcircuit development, and the use of biomaterials to influence the extracellular environment. Current barriers and future applications are also summarized in order to augment the design of future preclinical trials aimed toward astrocyte-targeted neuroregeneration with a concept termed astrocellular therapeutics. Developmental Dynamics 248:21-33, 2019. © 2018 Wiley Periodicals, Inc.


Assuntos
Astrócitos/citologia , Bioengenharia/tendências , Plasticidade Celular , Regeneração Nervosa , Animais , Bioengenharia/métodos , Transdiferenciação Celular , Humanos , Células-Tronco Pluripotentes , Terapêutica/métodos , Terapêutica/tendências
3.
J Vis Exp ; (138)2018 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-30176009

RESUMO

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying the human brain. One emerging technology to address this issue is the use of three dimensional (3D) neural cell cultures, termed 'organoids' or 'spheroids', for long term preservation of intercellular interactions including extracellular adhesion molecules. However, these culture systems are time consuming and not systematically generated. Here, we detail a method to rapidly and consistently produce 3D cocultures of neurons and astrocytes from human pluripotent stem cells. First, pre-differentiated astrocytes and neuronal progenitors are dissociated and counted. Next, cells are combined in sphere-forming dishes with a Rho-Kinase inhibitor and at specific ratios to produce spheres of reproducible size. After several weeks of culture as floating spheres, cocultures ('asteroids') are finally sectioned for immunostaining or plated upon multielectrode arrays to measure synaptic density and strength. In general, it is expected that this protocol will yield 3D neural spheres that display mature cell-type restricted markers, form functional synapses, and exhibit spontaneous synaptic network burst activity. Together, this system permits drug screening and investigations into mechanisms of disease in a more suitable model compared to monolayer cultures.


Assuntos
Astrócitos/citologia , Pareamento Cromossômico/fisiologia , Técnicas de Cocultura/métodos , Neurônios/citologia , Células-Tronco Pluripotentes/citologia , Astrócitos/metabolismo , Diferenciação Celular/fisiologia , Humanos , Neurônios/metabolismo , Células-Tronco Pluripotentes/metabolismo
4.
Biomed Microdevices ; 20(3): 65, 2018 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-30078059

RESUMO

Surgeons typically rely on their past training and experiences as well as visual aids from medical imaging techniques such as magnetic resonance imaging (MRI) or computed tomography (CT) for the planning of surgical processes. Often, due to the anatomical complexity of the surgery site, two dimensional or virtual images are not sufficient to successfully convey the structural details. For such scenarios, a 3D printed model of the patient's anatomy enables personalized preoperative planning. This paper reviews critical aspects of 3D printing for preoperative planning and surgical training, starting with an overview of the process-flow and 3D printing techniques, followed by their applications spanning across multiple organ systems in the human body. State of the art in these technologies are described along with a discussion of current limitations and future opportunities.


Assuntos
Simulação por Computador , Neurocirurgia/educação , Cuidados Pré-Operatórios/educação , Impressão Tridimensional , Osso e Ossos/anatomia & histologia , Osso e Ossos/cirurgia , Encéfalo/anatomia & histologia , Encéfalo/cirurgia , Procedimentos Cirúrgicos Cardiovasculares/educação , Sistema Cardiovascular/anatomia & histologia , Ponte de Artéria Coronária/educação , Ponte de Artéria Coronária/métodos , Humanos , Imagem Tridimensional , Imagem por Ressonância Magnética , Modelos Anatômicos , Neurocirurgia/métodos , Tomografia Computadorizada por Raios X
5.
Stem Cell Reports ; 9(6): 1745-1753, 2017 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-29198827

RESUMO

Human astrocytes network with neurons in dynamic ways that are still poorly defined. Our ability to model this relationship is hampered by the lack of relevant and convenient tools to recapitulate this complex interaction. To address this barrier, we have devised efficient coculture systems utilizing 3D organoid-like spheres, termed asteroids, containing pre-differentiated human pluripotent stem cell (hPSC)-derived astrocytes (hAstros) combined with neurons generated from hPSC-derived neural stem cells (hNeurons) or directly induced via Neurogenin 2 overexpression (iNeurons). Our systematic methods rapidly produce structurally complex hAstros and synapses in high-density coculture with iNeurons in precise numbers, allowing for improved studies of neural circuit function, disease modeling, and drug screening. We conclude that these bioengineered neural circuit model systems are reliable and scalable tools to accurately study aspects of human astrocyte-neuron functional properties while being easily accessible for cell-type-specific manipulations and observations.


Assuntos
Astrócitos/citologia , Diferenciação Celular/genética , Técnicas de Cocultura , Neurônios/citologia , Astrócitos/metabolismo , Linhagem da Célula/genética , Linhagem da Célula/fisiologia , Células Cultivadas , Humanos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Sinapses/metabolismo , Sinapses/fisiologia
6.
Sci Rep ; 7(1): 3775, 2017 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-28630410

RESUMO

A combination of techniques from 3D printing, tissue engineering and biomaterials has yielded a new class of engineered biological robots that could be reliably controlled via applied signals. These machines are powered by a muscle strip composed of differentiated skeletal myofibers in a matrix of natural proteins, including fibrin, that provide physical support and cues to the cells as an engineered basement membrane. However, maintaining consistent results becomes challenging when sustaining a living system in vitro. Skeletal muscle must be preserved in a differentiated state and the system is subject to degradation by proteolytic enzymes that can break down its mechanical integrity. Here we examine the life expectancy, breakdown, and device failure of engineered skeletal muscle bio-bots as a result of degradation by three classes of proteases: plasmin, cathepsin L, and matrix metalloproteinases (MMP-2 and MMP-9). We also demonstrate the use of gelatin zymography to determine the effects of differentiation and inhibitor concentration on protease expression. With this knowledge, we are poised to design the next generation of complex biological machines with controllable function, specific life expectancy and greater consistency. These results could also prove useful for the study of disease-specific models, treatments of myopathies, and other tissue engineering applications.


Assuntos
Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Impressão Tridimensional , Proteólise , Engenharia Tecidual , Animais , Membrana Basal , Linhagem Celular , Camundongos
7.
Adv Healthc Mater ; 6(12)2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28489332

RESUMO

A deeper understanding of biological materials and the design principles that govern them, combined with the enabling technology of 3D printing, has given rise to the idea of "building with biology." Using these materials and tools, bio-hybrid robots or bio-bots, which adaptively sense and respond to their environment, can be manufactured. Skeletal muscle bioactuators are developed to power these bio-bots, and an approach is presented to make them dynamically responsive to changing environmental loads and robustly resilient to induced damage. Specifically, since the predominant cause of skeletal muscle loss of function is mechanical damage, the underlying mechanisms of damage are investigated in vitro, and an in vivo inspired healing strategy is developed to counteract this damage. The protocol that is developed yields complete recovery of healthy tissue functionality within two days of damage, setting the stage for a more robust, resilient, and adaptive bioactuator technology than previously demonstrated. Understanding and exploiting the adaptive response behaviors inherent within biological systems in this manner is a crucial step forward in designing bio-hybrid machines that are broadly applicable to grand engineering challenges.


Assuntos
Músculo Esquelético/fisiologia , Optogenética/métodos , Engenharia Tecidual/métodos , Cicatrização , Animais , Linhagem Celular , Camundongos , Estresse Mecânico
8.
Nat Protoc ; 12(3): 519-533, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28182019

RESUMO

Biological machines consisting of cells and biomaterials have the potential to dynamically sense, process, respond, and adapt to environmental signals in real time. As a first step toward the realization of such machines, which will require biological actuators that can generate force and perform mechanical work, we have developed a method of manufacturing modular skeletal muscle actuators that can generate up to 1.7 mN (3.2 kPa) of passive tension force and 300 µN (0.56 kPa) of active tension force in response to external stimulation. Such millimeter-scale biological actuators can be coupled to a wide variety of 3D-printed skeletons to power complex output behaviors such as controllable locomotion. This article provides a comprehensive protocol for forward engineering of biological actuators and 3D-printed skeletons for any design application. 3D printing of the injection molds and skeletons requires 3 h, seeding the muscle actuators takes 2 h, and differentiating the muscle takes 7 d.


Assuntos
Biomimética/instrumentação , Músculo Esquelético/fisiologia , Animais , Fenômenos Biomecânicos , Linhagem Celular , Estimulação Elétrica , Desenho de Equipamento , Humanos , Camundongos , Impressão Tridimensional , Engenharia Tecidual
9.
Microsyst Nanoeng ; 3: 17015, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-31057862

RESUMO

A complex and functional living cellular system requires the interaction of one or more cell types to perform specific tasks, such as sensing, processing, or force production. Modular and flexible platforms for fabrication of such multi-cellular modules and their characterization have been lacking. Here, we present a modular cellular system, made up of multi-layered tissue rings containing integrated skeletal muscle and motor neurons (MNs) embedded in an extracellular matrix. The MNs were differentiated from mouse embryonic stem cells through the formation of embryoid bodies (EBs), which are spherical aggregations of cells grown in a suspension culture. The EBs were integrated into a tissue ring with skeletal muscle, which was differentiated in parallel, to create a co-culture amenable to both cell types. The multi-layered rings were then sequentially placed on a stationary three-dimensional-printed hydrogel structure resembling an anatomical muscle-tendon-bone organization. We demonstrate that the site-specific innervation of a group of muscle fibers in the multi-layered tissue rings allows for muscle contraction via chemical stimulation of MNs with glutamate, a major excitatory neurotransmitter in the mammalian nervous system, with the frequency of contraction increasing with glutamate concentration. The addition of tubocurarine chloride (a nicotinic receptor antagonist) halted the contractions, indicating that muscle contraction was MN induced. With a bio-fabricated system permitting controllable mechanical and geometric attributes in a range of length scales, our novel engineered cellular system can be utilized for easier integration of other modular "building blocks" in living cellular and biological machines.

10.
Proc Natl Acad Sci U S A ; 113(13): 3497-502, 2016 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-26976577

RESUMO

Complex biological systems sense, process, and respond to their surroundings in real time. The ability of such systems to adapt their behavioral response to suit a range of dynamic environmental signals motivates the use of biological materials for other engineering applications. As a step toward forward engineering biological machines (bio-bots) capable of nonnatural functional behaviors, we created a modular light-controlled skeletal muscle-powered bioactuator that can generate up to 300 µN (0.56 kPa) of active tension force in response to a noninvasive optical stimulus. When coupled to a 3D printed flexible bio-bot skeleton, these actuators drive directional locomotion (310 µm/s or 1.3 body lengths/min) and 2D rotational steering (2°/s) in a precisely targeted and controllable manner. The muscle actuators dynamically adapt to their surroundings by adjusting performance in response to "exercise" training stimuli. This demonstration sets the stage for developing multicellular bio-integrated machines and systems for a range of applications.


Assuntos
Músculo Esquelético/fisiologia , Optogenética/métodos , Animais , Linhagem Celular , Desenho de Equipamento , Análise de Elementos Finitos , Locomoção , Camundongos , Contração Muscular/fisiologia , Optogenética/instrumentação , Impressão Tridimensional , Robótica/instrumentação , Robótica/métodos , Imagem com Lapso de Tempo , Engenharia Tecidual/instrumentação , Engenharia Tecidual/métodos
11.
Proc Natl Acad Sci U S A ; 111(28): 10125-30, 2014 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-24982152

RESUMO

Combining biological components, such as cells and tissues, with soft robotics can enable the fabrication of biological machines with the ability to sense, process signals, and produce force. An intuitive demonstration of a biological machine is one that can produce motion in response to controllable external signaling. Whereas cardiac cell-driven biological actuators have been demonstrated, the requirements of these machines to respond to stimuli and exhibit controlled movement merit the use of skeletal muscle, the primary generator of actuation in animals, as a contractile power source. Here, we report the development of 3D printed hydrogel "bio-bots" with an asymmetric physical design and powered by the actuation of an engineered mammalian skeletal muscle strip to result in net locomotion of the bio-bot. Geometric design and material properties of the hydrogel bio-bots were optimized using stereolithographic 3D printing, and the effect of collagen I and fibrin extracellular matrix proteins and insulin-like growth factor 1 on the force production of engineered skeletal muscle was characterized. Electrical stimulation triggered contraction of cells in the muscle strip and net locomotion of the bio-bot with a maximum velocity of ∼ 156 µm s(-1), which is over 1.5 body lengths per min. Modeling and simulation were used to understand both the effect of different design parameters on the bio-bot and the mechanism of motion. This demonstration advances the goal of realizing forward-engineered integrated cellular machines and systems, which can have a myriad array of applications in drug screening, programmable tissue engineering, drug delivery, and biomimetic machine design.


Assuntos
Biomimética , Bioimpressão , Locomoção , Músculo Esquelético , Animais , Linhagem Celular , Colágeno Tipo I/química , Fator de Crescimento Insulin-Like I/química , Camundongos
12.
ACS Nano ; 7(3): 1830-7, 2013 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-23527748

RESUMO

In this issue of ACS Nano, Shin et al. present their finding that the addition of carbon nanotubes (CNT) in gelatin methacrylate (GelMA) results in improved functionality of bioengineered cardiac tissue. These CNT-GelMA hybrid materials demonstrate cardiac tissue with enhanced electrophysiological performance; improved mechanical integrity; better cell adhesion, viability, uniformity, and organization; increased beating rate and lowered excitation threshold; and protective effects against cardio-inhibitory and cardio-toxic drugs. In this Perspective, we outline recent progress in cardiac tissue engineering and prospects for future development. Bioengineered cardiac tissues can be used to build "heart-on-a-chip" devices for drug safety and efficacy testing, fabricate bioactuators for biointegrated robotics and reverse-engineered life forms, treat abnormal cardiac rhythms, and perhaps one day cure heart disease with tissue and organ transplants.


Assuntos
Miocárdio/citologia , Nanotubos de Carbono , Engenharia Tecidual , Tecidos Suporte , Animais
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