Scalable 3D Printed Molds for Human Tissue Engineered Skeletal Muscle.
Ontology highlight
ABSTRACT:
SUBMITTER: Capel AJ
PROVIDER: S-EPMC6383409 | biostudies-literature | 2019
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:The size of in vitro engineered skeletal muscle tissue is limited due to the lack of a vascular network in vitro. In this article, we report tissue-engineered skeletal muscle consisting of human aligned myofibers with interspersed endothelial networks. We extend our bioartificial muscle (BAM) model by coculturing human muscle progenitor cells with human umbilical vein endothelial cells (HUVECs) in a fibrin extracellular matrix (ECM). First, the optimal medium conditions for coculturing myoblasts with HUVECs were determined in a fusion assay. Endothelial growth medium proved to be the best compromise for the coculture, without affecting the myoblast fusion index. Second, both cell types were cocultured in a BAM maintained under tension to stimulate myofiber alignment. We then tested different total cell numbers containing 50% HUVECs and found that BAMs with a total cell number of 2 × 10(6) resulted in well-aligned and densely packed myofibers while allowing for improved interspersed endothelial network formation. Third, we compared different myoblast-HUVEC ratios. Including higher numbers of myoblasts improved endothelial network formation at lower total cell density; however, improvement of network characteristics reached a plateau when 1 × 10(6) or more myoblasts were present. Finally, addition of Matrigel to the fibrin ECM did not enhance overall myofiber and endothelial network formation. Therefore, in our BAM model, we suggest the use of a fibrin extracellular matrix containing 2 × 10(6) cells of which 50-70% are muscle cells. Optimizing these coculture conditions allows for a physiologically more relevant muscle model and paves the way toward engineering of larger in vitro muscle constructs.
| S-EPMC4605445 | biostudies-literature
Project description:Cementitious structures exhibit high compression strength but suffer from inherent brittleness. Conversely, nature creates structures using mostly brittle phases that overcome the strength-toughness trade-off, mainly through internalized packaging of brittle phases with soft organic binders. Here, we develop complex architectures of cementitious materials using an inverse replica approach where a soft polymer phase emerges as an external conformal coating. Architected polymer templates are printed, cement pastes are molded into these templates, and cementitious structures with thin polymer surface coating are achieved after the solubilization of sacrificial templates. These polymer-coated architected cementitious structures display unusual mechanical behavior with considerably higher toughness compared to conventional non-porous structures. They resist catastrophic failure through delayed damage propagation. Most interestingly, the architected structures show significant deformation recovery after releasing quasi-static loading, atypical in conventional cementitious structures. This approach allows a simple strategy to build more deformation resilient cementitious structures than their traditional counterparts.
| S-EPMC7921815 | biostudies-literature
Project description:In Pompe disease, the deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA) causes skeletal and cardiac muscle weakness, respiratory failure, and premature death. While enzyme replacement therapy using recombinant human GAA (rhGAA) can significantly improve patient outcomes, detailed disease mechanisms and incomplete therapeutic effects require further studies. Here we report a three-dimensional primary human skeletal muscle ("myobundle") model of infantile-onset Pompe disease (IOPD) that recapitulates hallmark pathological features including reduced GAA enzyme activity, elevated glycogen content and lysosome abundance, and increased sensitivity of muscle contractile function to metabolic stress. In vitro treatment of IOPD myobundles with rhGAA or adeno-associated virus (AAV)-mediated hGAA expression yields increased GAA activity and robust glycogen clearance, but no improvements in stress-induced functional deficits. We also apply RNA sequencing analysis to the quadriceps of untreated and AAV-treated GAA-/- mice and wild-type controls to establish a Pompe disease-specific transcriptional signature and reveal novel disease pathways. The mouse-derived signature is enriched in the transcriptomic profile of IOPD vs. healthy myobundles and partially reversed by in vitro rhGAA treatment, further confirming the utility of the human myobundle model for studies of Pompe disease and therapy.
| S-EPMC8100136 | biostudies-literature
Project description:BackgroundTissue-engineered muscles ("myobundles") offer a promising platform for developing a human in vitro model of healthy and diseased muscle for drug development and testing. Compared to traditional monolayer cultures, myobundles better model the three-dimensional structure of native skeletal muscle and are amenable to diverse functional measures to monitor the muscle health and drug response. Characterizing the metabolic function of human myobundles is of particular interest to enable their utilization in mechanistic studies of human metabolic diseases, identification of related drug targets, and systematic studies of drug safety and efficacy.MethodsTo this end, we studied glucose uptake and insulin responsiveness in human tissue-engineered skeletal muscle myobundles in the basal state and in response to drug treatments.ResultsIn the human skeletal muscle myobundle system, insulin stimulates a 50% increase in 2-deoxyglucose (2-DG) uptake with a compiled EC50 of 0.27 ± 0.03 nM. Treatment of myobundles with 400 µM metformin increased basal 2-DG uptake 1.7-fold and caused a significant drop in twitch and tetanus contractile force along with decreased fatigue resistance. Treatment with the histone deacetylase inhibitor 4-phenylbutyrate (4-PBA) increased the magnitude of insulin response from a 1.2-fold increase in glucose uptake in the untreated state to a 1.4-fold increase after 4-PBA treatment. 4-PBA treated myobundles also exhibited increased fatigue resistance and increased twitch half-relaxation time.ConclusionAlthough tissue-engineered human myobundles exhibit a modest increase in glucose uptake in response to insulin, they recapitulate key features of in vivo insulin sensitivity and exhibit relevant drug-mediated perturbations in contractile function and glucose metabolism.
| S-EPMC7710786 | biostudies-literature
Project description:The development of laboratory-grown tissues, referred to as organoids, bio-artificial tissue or tissue-engineered constructs, is clearly expanding. We describe for the first time how engineered human muscles can be applied as a pre- or non-clinical model for intramuscular drug injection to further decrease and complement the use of in vivo animal studies. The human bio-artificial muscle (BAM) is formed in a seven day tissue engineering procedure during which human myoblasts fuse and differentiate to aligned myofibers in an extracellular matrix. The dimensions of the BAM constructs allow for injection and follow-up during several days after injection. A stereotactic setup allows controllable injection at multiple sites in the BAM. We injected several compounds; a dye, a hydrolysable compound, a reducible substrate and a wasp venom toxin. Afterwards, direct reflux, release and metabolism were assessed in the BAM constructs in comparison to 2D cell culture and isolated human muscle strips. Spectrophotometry and luminescence allowed to measure the release of the injected compounds and their metabolites over time. A release profile over 40 hours was observed in the BAM model in contrast to 2D cell culture, showing the capacity of the BAM model to function as a drug depot. We also determined compound toxicity on the BAMs by measuring creatine kinase release in the medium, which increased with increasing toxic insult. Taken together, we show that the BAM is an injectable human 3D cell culture model that can be used to measure release and metabolism of injected compounds in vitro.
| S-EPMC6093918 | biostudies-literature
Project description:We recently developed capillaric circuits (CCs) - advanced capillary microfluidic devices assembled from capillary fluidic elements in a modular manner similar to the design of electric circuits (Safavieh & Juncker, Lab Chip, 2013, 13, 4180-4189). CCs choreograph liquid delivery operations according to pre-programmed capillary pressure differences with minimal user intervention. CCs were thought to require high-precision micron-scale features manufactured by conventional photolithography, which is slow and expensive. Here we present CCs manufactured rapidly and inexpensively using 3D-printed molds. Molds for CCs were fabricated with a benchtop 3D-printer, poly(dimethylsiloxane) replicas were made, and fluidic functionality was verified with aqueous solutions. We established design rules for CCs by a combination of modelling and experimentation. The functionality and reliability of trigger valves - an essential fluidic element that stops one liquid until flow is triggered by a second liquid - was tested for different geometries and different solutions. Trigger valves with geometries up to 80-fold larger than cleanroom-fabricated ones were found to function reliably. We designed retention burst valves that encode sequential liquid delivery using capillary pressure differences encoded by systematically varied heights and widths. Using an electrical circuit analogue of the CC, we established design rules to ensure strictly sequential liquid delivery. CCs autonomously delivered eight liquids in a pre-determined sequence in <7 min. Taken together, our results demonstrate that 3D-printing lowers the bar for other researchers to access capillary microfluidic valves and CCs for autonomous liquid delivery with applications in diagnostics, research and education.
| S-EPMC5314688 | biostudies-literature
Project description:The engineering of functional skeletal muscle tissue substitutes holds promise for the treatment of various muscular diseases and injuries. However, no tissue fabrication technology currently exists for the generation of a relatively large and thick bioartificial muscle made of densely packed, uniformly aligned, and differentiated myofibers. In this study, we describe a versatile cell/hydrogel micromolding approach where polydimethylsiloxane (PDMS) molds containing an array of elongated posts were used to fabricate relatively large neonatal rat skeletal muscle tissue networks with reproducible and controllable architecture. By combining cell-mediated fibrin gel compaction and precise microfabrication of mold dimensions including the length and height of the PDMS posts, we were able to simultaneously support high cell viability, guide cell alignment along the microfabricated tissue pores, and reproducibly control the overall tissue porosity, size, and thickness. The interconnected muscle bundles within the porous tissue networks were composed of densely packed, aligned, and highly differentiated myofibers. The formed myofibers expressed myogenin, developed abundant cross-striations, and generated spontaneous tissue contractions at the macroscopic spatial scale. The proliferation of non-muscle cells was significantly reduced compared to monolayer cultures. The more complex muscle tissue architectures were fabricated by controlling the spatial distribution and direction of the PDMS posts.
| S-EPMC2726993 | biostudies-literature
Project description:Microfabricated elastomeric scaffolds with 3D structural patterns are created by semiautomated layer-by-layer assembly of planar polymer sheets with through-pores. The mesoscale interconnected pore architectures governed by the relative alignment of layers are shown to direct cell and muscle-like fiber orientation in both skeletal and cardiac muscle, enabling scale up of tissue constructs towards clinically relevant dimensions.
| S-EPMC3954574 | biostudies-literature
Project description:In vitro models of contractile human skeletal muscle hold promise for use in disease modeling and drug development, but exhibit immature properties compared to native adult muscle. To address this limitation, 3D tissue-engineered human muscles (myobundles) were electrically stimulated using intermittent stimulation regimes at 1?Hz and 10?Hz. Dystrophin in myotubes exhibited mature membrane localization suggesting a relatively advanced starting developmental maturation. One-week stimulation significantly increased myobundle size, sarcomeric protein abundance, calcium transient amplitude (?2-fold), and tetanic force (?3-fold) resulting in the highest specific force generation (19.3mN/mm2) reported for engineered human muscles to date. Compared to 1?Hz electrical stimulation, the 10?Hz stimulation protocol resulted in greater myotube hypertrophy and upregulated mTORC1 and ERK1/2 activity. Electrically stimulated myobundles also showed a decrease in fatigue resistance compared to control myobundles without changes in glycolytic or mitochondrial protein levels. Greater glucose consumption and decreased abundance of acetylcarnitine in stimulated myobundles indicated increased glycolytic and fatty acid metabolic flux. Moreover, electrical stimulation of myobundles resulted in a metabolic shift towards longer-chain fatty acid oxidation as evident from increased abundances of medium- and long-chain acylcarnitines. Taken together, our study provides an advanced in vitro model of human skeletal muscle with improved structure, function, maturation, and metabolic flux.
| S-EPMC6395553 | biostudies-literature
Project description:Plastics are one of the most commonly used materials for fabricating microfluidic devices. While various methods exist for fabricating plastic microdevices, hot embossing offers several unique advantages including high throughput, excellent compatibility with most thermoplastics and low start-up costs. However, hot embossing requires metal or silicon molds that are fabricated using CNC milling or microfabrication techniques which are time consuming, expensive and required skilled technicians. Here, we demonstrate for the first time the fabrication of plastic microchannels using 3D printed metal molds. Through optimization of the powder composition and processing parameters, we were able to generate stainless steel molds with superior material properties (density and surface finish) than previously reported 3D printed metal parts. Molds were used to fabricate poly(methyl methacrylate) (PMMA) replicas which exhibited good feature integrity and replication quality. Microchannels fabricated using these replicas exhibited leak-free operation and comparable flow performance as those fabricated from CNC milled molds. The speed and simplicity of this approach can greatly facilitate the development (i.e. prototyping) and manufacture of plastic microfluidic devices for research and commercial applications.
| S-EPMC5706547 | biostudies-literature