Long-Term Cryopreservation and Revival of Tissue-Engineered Skeletal Muscle.
Ontology highlight
ABSTRACT:
SUBMITTER: Grant L
PROVIDER: S-EPMC6916121 | biostudies-literature | 2019 Jul
REPOSITORIES: biostudies-literature
Publications
Long-Term Cryopreservation and Revival of Tissue-Engineered Skeletal Muscle.
Grant Lauren L Raman Ritu R Cvetkovic Caroline C Ferrall-Fairbanks Meghan C MC Pagan-Diaz Gelson J GJ Hadley Pierce P Ko Eunkyung E Platt Manu O MO Bashir Rashid R
Tissue engineering. Part A 20190109 13-14
<h4>Impact statement</h4>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, d ...[more]
Similar Datasets
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: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:Tissue engineered skeletal muscle allows investigation of the cellular and molecular mechanisms that regulate skeletal muscle pathology. The fabricated model must resemble characteristics of in vivo tissue and incorporate cost-effective and high content primary human tissue. Current models are limited by low throughput due to the complexities associated with recruiting tissue donors, donor specific variations, as well as cellular senescence associated with passaging. This research presents a method using fused deposition modeling (FDM) and laser sintering (LS) 3D printing to generate reproducible and scalable tissue engineered primary human muscle, possessing aligned mature myotubes reminiscent of in vivo tissue. Many existing models are bespoke causing variability when translated between laboratories. To this end, a scalable model has been developed (25-500 μL construct volumes) allowing fabrication of mature primary human skeletal muscle. This research provides a strategy to overcome limited biopsy cell numbers, enabling high throughput screening of functional human tissue.
| S-EPMC6383409 | biostudies-literature
Project description:Oesophageal tissue engineering is a therapeutic alternative when oesophageal replacement is required. Decellularised scaffolds are ideal as they are derived from tissue-specific extracellular matrix and are non-immunogenic. However, appropriate preservation may significantly affect scaffold behaviour. Here we aim to prove that an effective method for short- and long-term preservation can be applied to tissue engineered products allowing their translation to clinical application. Rabbit oesophagi were decellularised using the detergent-enzymatic treatment (DET), a combination of deionised water, sodium deoxycholate and DNase-I. Samples were stored in phosphate-buffered saline solution at 4°C (4°C) or slow cooled in medium with 10% Me2SO at -1°C/min followed by storage in liquid nitrogen (SCM). Structural and functional analyses were performed prior to and after 2 and 4 weeks and 3 and 6 months of storage under each condition. Efficient decellularisation was achieved after 2 cycles of DET as determined with histology and DNA quantification, with preservation of the ECM. Only the SCM method, commonly used for cell storage, maintained the architecture and biomechanical properties of the scaffold up to 6 months. On the contrary, 4°C method was effective for short-term storage but led to a progressive distortion and degradation of the tissue architecture at the following time points. Efficient storage allows a timely use of decellularised oesophagi, essential for clinical translation. Here we describe that slow cooling with cryoprotectant solution in liquid nitrogen vapour leads to reliable long-term storage of decellularised oesophageal scaffolds for tissue engineering purposes.
| S-EPMC5466304 | biostudies-literature
Project description:Contemporary tissue engineered skeletal muscle models display a high degree of physiological accuracy compared with native tissue, and therefore may be excellent platforms to understand how various pathologies affect skeletal muscle. Chronic obstructive pulmonary disease (COPD) is a lung disease which causes tissue hypoxia and is characterized by muscle fiber atrophy and impaired muscle function. In the present study we exposed engineered skeletal muscle to varying levels of oxygen (O2 ; 21-1%) for 24 h in order to see if a COPD like muscle phenotype could be recreated in vitro, and if so, at what degree of hypoxia this occurred. Maximal contractile force was attenuated in hypoxia compared to 21% O2 ; with culture at 5% and 1% O2 causing the most pronounced effects with 62% and 56% decrements in force, respectively. Furthermore at these levels of O2 , myotubes within the engineered muscles displayed significant atrophy which was not seen at higher O2 levels. At the molecular level we observed increases in mRNA expression of MuRF-1 only at 1% O2 whereas MAFbx expression was elevated at 10%, 5%, and 1% O2 . In addition, p70S6 kinase phosphorylation (a downstream effector of mTORC1) was reduced when engineered muscle was cultured at 1% O2 , with no significant changes seen above this O2 level. Overall, these data suggest that engineered muscle exposed to O2 levels of ≤5% adapts in a manner similar to that seen in COPD patients, and thus may provide a novel model for further understanding muscle wasting associated with tissue hypoxia. J. Cell. Biochem. 118: 2599-2605, 2017. © 2017 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.
| S-EPMC5518201 | biostudies-literature
Project description:To engineer tissue-like structures, cells must organize themselves into three-dimensional (3D) networks that mimic the native tissue microarchitecture. Microfabricated hydrogel substrates provide a potentially useful platform for directing cells into biomimetic tissue architecture in vitro. Here, we present microgrooved methacrylated gelatin hydrogels as a suitable platform to build muscle-like fibrous structures in a facile and highly reproducible fashion. Microgrooved hydrogel substrates with two different ridge sizes (50 and 100??m) were fabricated to assess the effect of the distance between engineered myofibers on the orientation of the bridging C2C12 myoblasts and the formation of the resulting multinucleated myotubes. It was shown that although the ridge size did not significantly affect the C2C12 myoblast alignment, the wider-ridged micropatterned hydrogels generated more myotubes that were not aligned to the groove direction as compared to those on the smaller-ridge micropatterns. We also demonstrated that electrical stimulation improved the myoblast alignment and increased the diameter of the resulting myotubes. By using the microstructured methacrylated gelatin substrates, we built free-standing 3D muscle sheets, which contracted when electrically stimulated. Given their robust contractility and biomimetic microarchitecture, engineered tissues may find use in tissue engineering, biological studies, high-throughput drug screening, and biorobotics.
| S-EPMC3501120 | 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:Effective models of mammalian tissues must allow and encourage physiologically (mimetic) correct interactions between co-cultured cell types in order to produce culture microenvironments as similar as possible to those that would normally occur in vivo. In the case of skeletal muscle, the development of such a culture model, integrating multiple relevant cell types within a biomimetic scaffold, would be of significant benefit for investigations into the development, functional performance, and pathophysiology of skeletal muscle tissue. Although some work has been published regarding the behaviour of in vitro muscle models co-cultured with organotypic slices of CNS tissue or with stem cell-derived neurospheres, little investigation has so far been made regarding the potential to maintain isolated motor neurons within a 3D biomimetic skeletal muscle culture platform. Here, we review the current state of the art for engineering neuromuscular contacts in vitro and provide original data detailing the development of a 3D collagen-based model for the co-culture of primary muscle cells and motor neurons. The devised culture system promotes increased myoblast differentiation, forming arrays of parallel, aligned myotubes on which areas of nerve-muscle contact can be detected by immunostaining for pre- and post-synaptic proteins. Quantitative RT-PCR results indicate that motor neuron presence has a positive effect on myotube maturation, suggesting neural incorporation influences muscle development and maturation in vitro. The importance of this work is discussed in relation to other published neuromuscular co-culture platforms along with possible future directions for the field.
| S-EPMC5175300 | biostudies-literature
Project description:Electrical impulses are necessary for proper in vivo skeletal muscle development. To fabricate functional skeletal muscle tissues in vitro, recapitulation of the in vivo niche, including physical stimuli, is crucial. Here, we report a technique to engineer skeletal muscle tissues in vitro by electrical pulse stimulation (EPS). Electrically excitable tissue-engineered skeletal muscle constructs were stimulated with continuous electrical pulses of 0.3 V/mm amplitude, 4 ms width, and 1 Hz frequency, resulting in a 4.5-fold increase in force at day 14. In myogenic differentiation culture, the percentage of peak twitch force (%Pt) was determined as the load on the tissue constructs during the artificial exercise induced by continuous EPS. We optimized the stimulation protocol, wherein the tissues were first subjected to 24.5%Pt, which was increased to 50-60%Pt as the tissues developed. This technique may be a useful approach to fabricate tissue-engineered functional skeletal muscle constructs.
| S-EPMC3998029 | biostudies-literature