Pub Date : 2024-09-10DOI: 10.1101/2024.09.06.611618
Rita Matta, Davide Reato, Alberto Lombardini, David Moreau, Rodney P. O'Connor
Electrical stimulation is a powerful tool for investigating and modulating brain activity, as well as for treating neurological disorders. However, understanding the precise effects of electrical stimulation on neural activity has been hindered by limitations in recording neuronal responses near the stimulating electrode, such as stimulation artifacts in electrophysiology or obstruction of the field of view in imaging. In this study, we introduce a novel stimulation device fabricated from conductive polymers that is transparent and therefore compatible with optical imaging techniques. The device is manufactured using a combination of microfabrication and inkjet printing techniques and is flexible, allowing better adherence to the brain's natural curvature. We characterized the electrical and optical properties of the electrode and evaluated its performance in the brain of an anesthetized mouse. Furthermore, we combined experimental data with a finite-element model of the in-vivo experimental setup to estimate the maximum electric field that the highly transparent device can generate in the mouse brain. Our findings indicate that the device can generate an electric field as high as 300 V/m, demonstrating its potential for studying and manipulating neural activity using a range of electrical stimulation techniques relevant to human applications. Overall, this work presents a promising approach for developing versatile new tools to apply and study electrical brain stimulation.
{"title":"Inkjet-printed transparent electrodes for electrical brain stimulation","authors":"Rita Matta, Davide Reato, Alberto Lombardini, David Moreau, Rodney P. O'Connor","doi":"10.1101/2024.09.06.611618","DOIUrl":"https://doi.org/10.1101/2024.09.06.611618","url":null,"abstract":"Electrical stimulation is a powerful tool for investigating and modulating brain activity, as well as for treating neurological disorders. However, understanding the precise effects of electrical stimulation on neural activity has been hindered by limitations in recording neuronal responses near the stimulating electrode, such as stimulation artifacts in electrophysiology or obstruction of the field of view in imaging. In this study, we introduce a novel stimulation device fabricated from conductive polymers that is transparent and therefore compatible with optical imaging techniques. The device is manufactured using a combination of microfabrication and inkjet printing techniques and is flexible, allowing better adherence to the brain's natural curvature. We characterized the electrical and optical properties of the electrode and evaluated its performance in the brain of an anesthetized mouse. Furthermore, we combined experimental data with a finite-element model of the in-vivo experimental setup to estimate the maximum electric field that the highly transparent device can generate in the mouse brain. Our findings indicate that the device can generate an electric field as high as 300 V/m, demonstrating its potential for studying and manipulating neural activity using a range of electrical stimulation techniques relevant to human applications. Overall, this work presents a promising approach for developing versatile new tools to apply and study electrical brain stimulation.","PeriodicalId":501308,"journal":{"name":"bioRxiv - Bioengineering","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142216588","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-10DOI: 10.1101/2024.09.05.609208
Yuankai Lu, Yi Hua, Bingrui Wang, Fugiang Zhong, Andrew Theophanous, Shaharoz Tahir, Po-Yi Lee, Ian A Sigal
Purpose: Our goal is to evaluate how lamina cribrosa (LC) oxygenation is affected by the tissue distortions resulting from elevated IOP. Design: Experimental study on monkeys Subjects: Four healthy monkey eyes with OCT scans with IOP of 10 to 50 mmHg, and then with histological sections of LC. Methods: Since in-vivo LC oxygenation measurement is not yet possible, we used 3D eye-specific numerical models of the LC vasculature which we subjected to experimentally-derived tissue deformations. We reconstructed 3D models of the LC vessel networks of 4 healthy monkey eyes from histological sections. We also obtained in-vivo IOP-induced tissue deformations from a healthy monkey using OCT images and digital volume correlation analysis techniques. The extent that LC vessels distort under a given OCT-derived tissue strain remains unknown. We therefore evaluated two biomechanics-based mapping techniques: cross-sectional and isotropic. The hemodynamics and oxygenations of the four vessel networks were simulated for deformations at several IOPs up to 60mmHg. The results were used to determine the effects of IOP on LC oxygen supply, assorting the extent of tissue mild and severe hypoxia. Main Outcome Measures: IOP-induced deformation, vasculature structure, blood supply, and oxygen supply for LC region Result: IOP-induced deformations reduced LC oxygenation significantly. More than 20% of LC tissue suffered from mild hypoxia when IOP reached 30 mmHg. Extreme IOP(>50mmHg) led to large severe hypoxia regions (>30%) in the isotropic mapping cases. Conclusion: Our models predicted that moderately elevated IOP can lead to mild hypoxia in a substantial part of the LC, which, if sustained chronically, may contribute to neural tissue damage. For extreme IOP elevations, severe hypoxia was predicted, which would potentially cause more immediate damage. Our findings suggest that despite the remarkable LC vascular robustness, IOP-induced distortions can potentially contribute to glaucomatous neuropathy.
{"title":"Impact of elevated IOP on lamina cribrosa oxygenation; A combined experimental-computational study on monkeys","authors":"Yuankai Lu, Yi Hua, Bingrui Wang, Fugiang Zhong, Andrew Theophanous, Shaharoz Tahir, Po-Yi Lee, Ian A Sigal","doi":"10.1101/2024.09.05.609208","DOIUrl":"https://doi.org/10.1101/2024.09.05.609208","url":null,"abstract":"Purpose: Our goal is to evaluate how lamina cribrosa (LC) oxygenation is affected by the tissue distortions resulting from elevated IOP. Design: Experimental study on monkeys\u0000Subjects: Four healthy monkey eyes with OCT scans with IOP of 10 to 50 mmHg, and then with histological sections of LC.\u0000Methods: Since in-vivo LC oxygenation measurement is not yet possible, we used 3D eye-specific numerical models of the LC vasculature which we subjected to experimentally-derived tissue deformations. We reconstructed 3D models of the LC vessel networks of 4 healthy monkey eyes from histological sections. We also obtained in-vivo IOP-induced tissue deformations from a healthy monkey using OCT images and digital volume correlation analysis techniques. The extent that LC vessels distort under a given OCT-derived tissue strain remains unknown. We therefore evaluated two biomechanics-based mapping techniques: cross-sectional and isotropic. The hemodynamics and oxygenations of the four vessel networks were simulated for deformations at several IOPs up to 60mmHg. The results were used to determine the effects of IOP on LC oxygen supply, assorting the extent of tissue mild and severe hypoxia. Main Outcome Measures: IOP-induced deformation, vasculature structure, blood supply, and oxygen supply for LC region\u0000Result: IOP-induced deformations reduced LC oxygenation significantly. More than 20% of LC tissue suffered from mild hypoxia when IOP reached 30 mmHg. Extreme IOP(>50mmHg) led to large severe hypoxia regions (>30%) in the isotropic mapping cases.\u0000Conclusion: Our models predicted that moderately elevated IOP can lead to mild hypoxia in a substantial part of the LC, which, if sustained chronically, may contribute to neural tissue damage. For extreme IOP elevations, severe hypoxia was predicted, which would potentially cause more immediate damage. Our findings suggest that despite the remarkable LC vascular robustness, IOP-induced distortions can potentially contribute to glaucomatous neuropathy.","PeriodicalId":501308,"journal":{"name":"bioRxiv - Bioengineering","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142216594","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-10DOI: 10.1101/2024.09.05.611289
Jesse I Gilmer, Susan K Coltman, Geraldine C Velasco, John Hutchinson, Daniel Huber, Abigail L Person, Mazen Al Borno
Mice are key model organisms in genetics, neuroscience and motor systems physiology. Fine motor control tasks performed by mice have become widely used in assaying neural and biophysical motor system mechanisms, including lever manipulation, joystick manipulation, and reach-to-grasp tasks (Becker et al., 2019; Bollu et al., 2019; Conner at al., 2021). Although fine motor tasks provide useful insights into behaviors which require complex multi-joint motor control, there is no previously developed physiological biomechanical model of the adult mouse forelimb available for estimating kinematics (including joint angles, joint velocities, fiber lengths and fiber velocities) nor muscle activity or kinetics (including forces and moments) during these behaviors. Here we have developed a musculoskeletal model based on high-resolution imaging and reconstruction of the mouse forelimb that includes muscles spanning the neck, trunk, shoulder, and limbs using anatomical data from two mice. Physics-based optimal control simulations of the forelimb model were used to estimate in vivo muscle activity present when constrained to the tracked kinematics during mouse reaching movements. The activity of a subset of muscles was recorded via electromyography and used as the ground truth to assess the accuracy of the muscle patterning in simulation. We found that the synthesized muscle patterning in the forelimb model had a strong resemblance to empirical muscle patterning, suggesting that our model has utility in providing a realistic set of estimated muscle excitations over time when provided with a kinematic template. The strength of the resemblance between empirical muscle activity and optimal control predictions increases as mice performance improves throughout learning. Our computational tools are available as open-source in the OpenSim physics and modeling platform (Seth et al., 2018). Our model can enhance research into limb control across broad research topics and can inform analyses of motor learning, muscle synergies, neural patterning, and behavioral research that would otherwise be inaccessible.
{"title":"A novel biomechanical model of the mouse forelimb predicts muscle activity in optimal control simulations of reaching movements","authors":"Jesse I Gilmer, Susan K Coltman, Geraldine C Velasco, John Hutchinson, Daniel Huber, Abigail L Person, Mazen Al Borno","doi":"10.1101/2024.09.05.611289","DOIUrl":"https://doi.org/10.1101/2024.09.05.611289","url":null,"abstract":"Mice are key model organisms in genetics, neuroscience and motor systems physiology. Fine motor control tasks performed by mice have become widely used in assaying neural and biophysical motor system mechanisms, including lever manipulation, joystick manipulation, and reach-to-grasp tasks (Becker et al., 2019; Bollu et al., 2019; Conner at al., 2021). Although fine motor tasks provide useful insights into behaviors which require complex multi-joint motor control, there is no previously developed physiological biomechanical model of the adult mouse forelimb available for estimating kinematics (including joint angles, joint velocities, fiber lengths and fiber velocities) nor muscle activity or kinetics (including forces and moments) during these behaviors. Here we have developed a musculoskeletal model based on high-resolution imaging and reconstruction of the mouse forelimb that includes muscles spanning the neck, trunk, shoulder, and limbs using anatomical data from two mice. Physics-based optimal control simulations of the forelimb model were used to estimate in vivo muscle activity present when constrained to the tracked kinematics during mouse reaching movements. The activity of a subset of muscles was recorded via electromyography and used as the ground truth to assess the accuracy of the muscle patterning in simulation. We found that the synthesized muscle patterning in the forelimb model had a strong resemblance to empirical muscle patterning, suggesting that our model has utility in providing a realistic set of estimated muscle excitations over time when provided with a kinematic template. The strength of the resemblance between empirical muscle activity and optimal control predictions increases as mice performance improves throughout learning. Our computational tools are available as open-source in the OpenSim physics and modeling platform (Seth et al., 2018). Our model can enhance research into limb control across broad research topics and can inform analyses of motor learning, muscle synergies, neural patterning, and behavioral research that would otherwise be inaccessible.","PeriodicalId":501308,"journal":{"name":"bioRxiv - Bioengineering","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142216597","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-10DOI: 10.1101/2024.09.05.611552
Michelle Feigis, Radhakrishnan Mahadevan
Biomanufacturing can play a pivotal role in the transition away from fossil fuel dependence for the production of chemicals and fuels. There is growing interest in alternative bioproduction feedstocks to conventional sugars that do not compete for land use with food production. Ethylene glycol, a C2 compound that can be recovered from plastic waste or derived from carbon dioxide with increasing efficiency, is gaining attention as a carbon source for microbial processes. Here we review the natural and synthetic metabolic pathways currently available for ethylene glycol assimilation. The pathways are compared in terms of their maximum theoretical yields for biomass and value-added products, thermodynamic favourability, minimum enzyme costs, and orthogonality to central carbon metabolism. We find that synthetic pathways outperform their natural counterparts in terms of higher thermodynamic driving forces, reduced enzyme costs, and higher theoretical yields for the majority of bioproducts analyzed as well as for biomass. However, natural assimilation pathways are equally or even more orthogonal to growth-associated reactions than synthetic pathways. Given these tradeoffs, the optimal EG assimilation pathway may depend on product and process choice.
{"title":"Synthetic metabolic pathways for ethylene glycol assimilation outperform natural counterparts","authors":"Michelle Feigis, Radhakrishnan Mahadevan","doi":"10.1101/2024.09.05.611552","DOIUrl":"https://doi.org/10.1101/2024.09.05.611552","url":null,"abstract":"Biomanufacturing can play a pivotal role in the transition away from fossil fuel dependence for the production of chemicals and fuels. There is growing interest in alternative bioproduction feedstocks to conventional sugars that do not compete for land use with food production. Ethylene glycol, a C2 compound that can be recovered from plastic waste or derived from carbon dioxide with increasing efficiency, is gaining attention as a carbon source for microbial processes. Here we review the natural and synthetic metabolic pathways currently available for ethylene glycol assimilation. The pathways are compared in terms of their maximum theoretical yields for biomass and value-added products, thermodynamic favourability, minimum enzyme costs, and orthogonality to central carbon metabolism. We find that synthetic pathways outperform their natural counterparts in terms of higher thermodynamic driving forces, reduced enzyme costs, and higher theoretical yields for the majority of bioproducts analyzed as well as for biomass. However, natural assimilation pathways are equally or even more orthogonal to growth-associated reactions than synthetic pathways. Given these tradeoffs, the optimal EG assimilation pathway may depend on product and process choice.","PeriodicalId":501308,"journal":{"name":"bioRxiv - Bioengineering","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142216591","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-10DOI: 10.1101/2024.09.05.611457
Ana P Spencer, Miguel Xavier, Sofia C Guimaraes, Adriana Vilaca, Ariel Ionescu, Rafael Santos, Maria Lazaro, Eran Perlson, Victoria Leiro, Ben M Maoz, Ana P Pego
Neurological disorders, a leading global cause of death, encompass conditions affecting the peripheral and central nervous systems (PNS and CNS, respectively). Limited axon regeneration is a significant challenge in these disorders, and it is linked to proteins like PTEN. RNA-based therapeutics, particularly siRNAs, hold potential for silencing these inhibitory pathways, but their clinical application is hindered by poor stability and cellular uptake. Our study addressed this challenge with the development of novel, fully biodegradable dendritic nanoparticles designed specifically for neuron targeting. These nanoparticles were functionalized with the neurotropic binding domain of tetanus toxin, enhancing selective neuronal targeting and cellular internalization. We demonstrated that these dendriplexes not only maintain biocompatibility and efficient siRNA delivery in neuronal cultures but also significantly enhance axonal growth, as shown in microfluidic models. In a groundbreaking PNS-CNS-on-Chip, dendriplexes exhibited effective migration from PNS to CNS neurons, highlighting their potential for targeted therapeutic delivery. This study pioneers the application of microfluidics to demonstrate the CNS targeting of dendriplexes, paving the way for innovative treatments in the field of nanomedicine.
{"title":"Unveiling the potential of neuron-targeted dendriplexes for siRNA delivery using a PNS-CNS-on-Chip","authors":"Ana P Spencer, Miguel Xavier, Sofia C Guimaraes, Adriana Vilaca, Ariel Ionescu, Rafael Santos, Maria Lazaro, Eran Perlson, Victoria Leiro, Ben M Maoz, Ana P Pego","doi":"10.1101/2024.09.05.611457","DOIUrl":"https://doi.org/10.1101/2024.09.05.611457","url":null,"abstract":"Neurological disorders, a leading global cause of death, encompass conditions affecting the peripheral and central nervous systems (PNS and CNS, respectively). Limited axon regeneration is a significant challenge in these disorders, and it is linked to proteins like PTEN. RNA-based therapeutics, particularly siRNAs, hold potential for silencing these inhibitory pathways, but their clinical application is hindered by poor stability and cellular uptake. Our study addressed this challenge with the development of novel, fully biodegradable dendritic nanoparticles designed specifically for neuron targeting. These nanoparticles were functionalized with the neurotropic binding domain of tetanus toxin, enhancing selective neuronal targeting and cellular internalization. We demonstrated that these dendriplexes not only maintain biocompatibility and efficient siRNA delivery in neuronal cultures but also significantly enhance axonal growth, as shown in microfluidic models. In a groundbreaking PNS-CNS-on-Chip, dendriplexes exhibited effective migration from PNS to CNS neurons, highlighting their potential for targeted therapeutic delivery. This study pioneers the application of microfluidics to demonstrate the CNS targeting of dendriplexes, paving the way for innovative treatments in the field of nanomedicine.","PeriodicalId":501308,"journal":{"name":"bioRxiv - Bioengineering","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142216608","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-09DOI: 10.1101/2024.09.05.611247
Laurence De Beaurepaire, Thibaud Dauphin, Quentin Pruvost, Apolline Salama, Aurelien Dupont, Laurence Dubreil, Dominique Jegou, Gregoire Mignot, Benjamin Mahieu, Julie Herve, Blandine Lieubeau, Jean-Marie Bach, Steffi Bosch, Mathilde Mosser
Small extracellular vesicles (sEV) released by healthy beta cells are promising candidates for diabetes therapy thanks to their aptitude to modulate inflammation, to induce or maintain pancreatic function and to prevent pathogenic mechanisms. To advance the clinical development of therapeutics, there is a crucial need for scalable production methods. Stirred tank bioreactors (STR) are widely used in the industry due to their ability to provide homogeneous gas and nutrient supply, online monitoring, and efficient scale up. Anchorage-dependent cells can be cultured in STR on microcarriers or as spheroids, but may experience shear stress, which can affect sEV phenotype and function. Using pancreatic beta cells, this study identifies critical cell culturing parameters, including culture mode (monolayer vs. spheroids), medium formulation (with or without serum, glucose control), and process parameters (stirring, duration, cell density). The findings show that small spheroid culture promotes beta cell maturation without decreasing the yield of sEV per cell, despite a reduced cell surface exchange area. However, stirring increased expression of cellular stress markers and decreased cell viability. Set up of a three-step bioprocess allowed to maximize cell viability and sEV yields at high cell density over short production duration. sEV produced under these conditions maintained high purity, membrane integrity, and the aptitude to reduce T-lymphocyte proliferation and IFN-γ cytokine secretion in a mixed lymphocyte reaction. Flow cytometry analysis revealed lower CD63/CD81 ratios in STR, indicating enhanced ectosome production. Switch from high glucose expansion to low glucose production medium further allowed to direct sorting of the antigen insulin into beta-sEV. This study demonstrates the feasibility of producing functional sEV from mature beta cells cultured as small spheroids, suitable for upscale. Production of sEV in STR may be particularly beneficial for ectosome-enriched compound loading for therapeutic applications.
健康β细胞释放的小细胞外囊泡（sEV）具有调节炎症、诱导或维持胰腺功能以及预防致病机制的作用，因此有望成为糖尿病治疗的候选药物。为推动治疗药物的临床开发，迫切需要可扩展的生产方法。搅拌罐生物反应器（STR）因其能够提供均匀的气体和营养供应、在线监测和高效的规模化生产而在业界得到广泛应用。依赖锚定的细胞可在 STR 中的微载体上或作为球体进行培养，但可能会受到剪切应力的影响，从而影响 sEV 的表型和功能。本研究利用胰岛β细胞确定了关键的细胞培养参数，包括培养模式（单层与球形）、培养基配方（有无血清、葡萄糖控制）和工艺参数（搅拌、持续时间、细胞密度）。研究结果表明，尽管细胞表面交换面积减少，但小球形培养可促进β细胞成熟，而不会降低每个细胞的sEV产量。然而，搅拌会增加细胞应激标记物的表达，降低细胞活力。在这些条件下生产的 sEV 保持了高纯度、膜完整性以及在混合淋巴细胞反应中减少 T 淋巴细胞增殖和 IFN-γ 细胞因子分泌的能力。流式细胞术分析表明，STR 中的 CD63/CD81 比率较低，表明外泌体生成增强。从高糖扩增培养基转换到低糖生产培养基，还能将抗原胰岛素直接分选到β-SEV中。这项研究证明了从小球体培养的成熟β细胞中生产功能性 sEV 的可行性，适合大规模生产。在 STR 中生产 sEV 可能特别有利于外显子富集化合物的治疗应用。
{"title":"Three-step scalable production of extracellular vesicles from pancreatic beta cells in stirred tank bioreactors promotes cell maturation and release of ectosomes with preserved immunomodulatory properties","authors":"Laurence De Beaurepaire, Thibaud Dauphin, Quentin Pruvost, Apolline Salama, Aurelien Dupont, Laurence Dubreil, Dominique Jegou, Gregoire Mignot, Benjamin Mahieu, Julie Herve, Blandine Lieubeau, Jean-Marie Bach, Steffi Bosch, Mathilde Mosser","doi":"10.1101/2024.09.05.611247","DOIUrl":"https://doi.org/10.1101/2024.09.05.611247","url":null,"abstract":"Small extracellular vesicles (sEV) released by healthy beta cells are promising candidates for diabetes therapy thanks to their aptitude to modulate inflammation, to induce or maintain pancreatic function and to prevent pathogenic mechanisms. To advance the clinical development of therapeutics, there is a crucial need for scalable production methods. Stirred tank bioreactors (STR) are widely used in the industry due to their ability to provide homogeneous gas and nutrient supply, online monitoring, and efficient scale up. Anchorage-dependent cells can be cultured in STR on microcarriers or as spheroids, but may experience shear stress, which can affect sEV phenotype and function. Using pancreatic beta cells, this study identifies critical cell culturing parameters, including culture mode (monolayer vs. spheroids), medium formulation (with or without serum, glucose control), and process parameters (stirring, duration, cell density). The findings show that small spheroid culture promotes beta cell maturation without decreasing the yield of sEV per cell, despite a reduced cell surface exchange area. However, stirring increased expression of cellular stress markers and decreased cell viability. Set up of a three-step bioprocess allowed to maximize cell viability and sEV yields at high cell density over short production duration. sEV produced under these conditions maintained high purity, membrane integrity, and the aptitude to reduce T-lymphocyte proliferation and IFN-γ cytokine secretion in a mixed lymphocyte reaction. Flow cytometry analysis revealed lower CD63/CD81 ratios in STR, indicating enhanced ectosome production. Switch from high glucose expansion to low glucose production medium further allowed to direct sorting of the antigen insulin into beta-sEV. This study demonstrates the feasibility of producing functional sEV from mature beta cells cultured as small spheroids, suitable for upscale. Production of sEV in STR may be particularly beneficial for ectosome-enriched compound loading for therapeutic applications.","PeriodicalId":501308,"journal":{"name":"bioRxiv - Bioengineering","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142216614","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-09DOI: 10.1101/2024.09.08.611881
Fandi Shi, william Zev Rymer, Jongsang Son
During voluntary isometric contractions of upper extremity muscles in individuals with chronic stroke, the magnitude of force variability appears to increase consistently as force increases. However, research on how such force variability changes with increasing motor impairment remains limited. This study aims to determine whether force variability is increased on the paretic side during either index finger abduction or elbow flexion in the same group of stroke survivors, and whether these changes are consistent across different muscles. Force variability was assessed using the standard deviation of force during sustained isometric contractions. Linear mixed-effects models were implemented to test whether force variability is changed on the paretic side post stroke, and whether such alterations show dependence on force level and on the degree of impairment. The results demonstrated a significant increase in force variability on the paretic side across force levels during finger abduction, while force variability for elbow flexion was increased only at high force levels. In addition, the force variability appears to increase as isometric elbow flexion force increases, whereas no clear trend was found during index finger abduction. The increase in force variability demonstrated moderate-strong dependence on the reduction in maximum muscle strength on the paretic side during elbow flexion, suggesting that monitoring force variability could potentially serve as a quantitative diagnostic tool for assessing severity of impairment in motor control and for raising potential mechanisms at the motor unit level.
{"title":"Force variability is a potential biomarker of motor impairment in hemispheric stroke survivors","authors":"Fandi Shi, william Zev Rymer, Jongsang Son","doi":"10.1101/2024.09.08.611881","DOIUrl":"https://doi.org/10.1101/2024.09.08.611881","url":null,"abstract":"During voluntary isometric contractions of upper extremity muscles in individuals with chronic stroke, the magnitude of force variability appears to increase consistently as force increases. However, research on how such force variability changes with increasing motor impairment remains limited. This study aims to determine whether force variability is increased on the paretic side during either index finger abduction or elbow flexion in the same group of stroke survivors, and whether these changes are consistent across different muscles. Force variability was assessed using the standard deviation of force during sustained isometric contractions. Linear mixed-effects models were implemented to test whether force variability is changed on the paretic side post stroke, and whether such alterations show dependence on force level and on the degree of impairment. The results demonstrated a significant increase in force variability on the paretic side across force levels during finger abduction, while force variability for elbow flexion was increased only at high force levels. In addition, the force variability appears to increase as isometric elbow flexion force increases, whereas no clear trend was found during index finger abduction. The increase in force variability demonstrated moderate-strong dependence on the reduction in maximum muscle strength on the paretic side during elbow flexion, suggesting that monitoring force variability could potentially serve as a quantitative diagnostic tool for assessing severity of impairment in motor control and for raising potential mechanisms at the motor unit level.","PeriodicalId":501308,"journal":{"name":"bioRxiv - Bioengineering","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142216613","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-09DOI: 10.1101/2024.09.04.611328
Cole D Smith, Joseph D Towles
PURPOSE. Tendon transfer surgeries that engage the flexor pollicis longus muscle (FPL) are commonly performed to enable lateral pinch grasp in persons with tetraplegia. Functional outcomes, however, have been mixed. This might be the case, in part, because FPL produces hyperflexion at the interphalangeal (IP) joint and radial deviation at the carpometacarpal (CMC) joint. Therefore, the goal of this simulation study was to investigate whether small groups of muscles could produce movement with less IP joint hyperflexion and CMC ab/adduction than FPL produces during lateral pinch movement. METHODS. We adapted a published, open-source computational musculoskeletal model of the hand to implement lateral pinch grasp. A forward-dynamics simulation approach was used to drive the thumb for 27 muscle groups being considered from an extended posture to a flexed posture to make contact with the side of the index finger. We calculated CMC ab/adduction deviation from the flexion-extension plane and IP joint flexion in the plane that all muscle groups produced, and compared those joint angle movements to those of FPL when it alone drove the thumb. RESULTS. Of the 27 simulations, three muscle groups, each consisting of three or four muscles, generated lower IP joint flexion and CMC ab/adduction than those of FPL. CONCLUSIONS. This simulation work points to the potential of novel, multi-insertion site tendon transfer surgeries to out-perform the current standard of care to restore lateral pinch grasp following tetraplegia.
目的。为使四肢瘫痪患者能够进行侧向夹抓，通常会进行肌腱转移手术，使屈肌（FPL）参与其中。然而，其功能效果却参差不齐。出现这种情况的部分原因可能是，FPL 会导致指间关节（IP）过度屈曲和腕掌关节（CMC）桡侧偏离。因此，本模拟研究的目的是探究在侧向夹持运动中，小肌肉群是否能产生比 FPL 更少的 IP 关节过度屈曲和 CMC 内收/外展运动。方法：我们对已发布的开源手部肌肉骨骼计算模型进行了改编，以实现侧向捏握。我们采用了前向动力学模拟方法来驱动 27 个肌肉群的拇指从伸展姿势变为屈曲姿势，以便与食指侧面接触。我们计算了所有肌群在屈伸平面上的 CMC 外展/内收偏差和 IP 关节在该平面上的屈曲，并将这些关节角度运动与 FPL 单独驱动拇指时的关节角度运动进行了比较。结果在 27 个模拟结果中，由三块或四块肌肉组成的三个肌群产生的 IP 关节屈曲和 CMC 外展/内收的角度低于 FPL。结论。这项模拟研究表明，新型的多插入部位肌腱转移手术在恢复四肢瘫痪后的外侧抓握能力方面有可能优于目前的标准疗法。
{"title":"Toward an understanding of the small groups of thumb muscles that produce lateral pinch movement: application to surgical restoration of grasp following neurologic impairment","authors":"Cole D Smith, Joseph D Towles","doi":"10.1101/2024.09.04.611328","DOIUrl":"https://doi.org/10.1101/2024.09.04.611328","url":null,"abstract":"PURPOSE. Tendon transfer surgeries that engage the flexor pollicis longus muscle (FPL) are commonly performed to enable lateral pinch grasp in persons with tetraplegia. Functional outcomes, however, have been mixed. This might be the case, in part, because FPL produces hyperflexion at the interphalangeal (IP) joint and radial deviation at the carpometacarpal (CMC) joint. Therefore, the goal of this simulation study was to investigate whether small groups of muscles could produce movement with less IP joint hyperflexion and CMC ab/adduction than FPL produces during lateral pinch movement. METHODS. We adapted a published, open-source computational musculoskeletal model of the hand to implement lateral pinch grasp. A forward-dynamics simulation approach was used to drive the thumb for 27 muscle groups being considered from an extended posture to a flexed posture to make contact with the side of the index finger. We calculated CMC ab/adduction deviation from the flexion-extension plane and IP joint flexion in the plane that all muscle groups produced, and compared those joint angle movements to those of FPL when it alone drove the thumb. RESULTS. Of the 27 simulations, three muscle groups, each consisting of three or four muscles, generated lower IP joint flexion and CMC ab/adduction than those of FPL. CONCLUSIONS. This simulation work points to the potential of novel, multi-insertion site tendon transfer surgeries to out-perform the current standard of care to restore lateral pinch grasp following tetraplegia.","PeriodicalId":501308,"journal":{"name":"bioRxiv - Bioengineering","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142216616","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-09DOI: 10.1101/2024.08.16.608324
Noah Eckman, Abigail K. Grosskopf, Grace Jiang, Krutarth Kamani, Michelle Huang, Brigitte Schmittlein, Sarah C Heilshorn, Simon Rogers, Eric Appel
Yielding of dynamically crosslinked hydrogels, or transition between a solid-like and liquid-like state, allows facile injection and utility in translational biomedical applications including delivery of therapeutic cells. Unfortunately, characterizing the time-varying nature of the transition has not been attempted, nor are there design rules for understanding the effects of yielding on encapsulated cells. Here, we unveil underlying molecular mechanisms governing the yielding transition of dynamically crosslinked gels currently being researched for use in cell therapy. We demonstrate through nonlinear rheological characterization that the network dynamics of the dynamic hydrogels dictate the speed and character of their yielding transition. Rheological testing of these materials reveals unexpected elastic strain stiffening during yielding, as well as characterizing the rapidity of the yielding transition. A slower yielding speed explains enhanced protection of directly injected cells from shear forces, highlighting the importance of mechanical characterization of all phases of yield-stress biomaterials.
{"title":"Dynamic Hydrogel Crosslink Strength Controls Yielding Characteristics and Cell Viability During Injection","authors":"Noah Eckman, Abigail K. Grosskopf, Grace Jiang, Krutarth Kamani, Michelle Huang, Brigitte Schmittlein, Sarah C Heilshorn, Simon Rogers, Eric Appel","doi":"10.1101/2024.08.16.608324","DOIUrl":"https://doi.org/10.1101/2024.08.16.608324","url":null,"abstract":"Yielding of dynamically crosslinked hydrogels, or transition between a solid-like and liquid-like state, allows facile injection and utility in translational biomedical applications including delivery of therapeutic cells. Unfortunately, characterizing the time-varying nature of the transition has not been attempted, nor are there design rules for understanding the effects of yielding on encapsulated cells. Here, we unveil underlying molecular mechanisms governing the yielding transition of dynamically crosslinked gels currently being researched for use in cell therapy. We demonstrate through nonlinear rheological characterization that the network dynamics of the dynamic hydrogels dictate the speed and character of their yielding transition. Rheological testing of these materials reveals unexpected elastic strain stiffening during yielding, as well as characterizing the rapidity of the yielding transition. A slower yielding speed explains enhanced protection of directly injected cells from shear forces, highlighting the importance of mechanical characterization of all phases of yield-stress biomaterials.","PeriodicalId":501308,"journal":{"name":"bioRxiv - Bioengineering","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142216615","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-09DOI: 10.1101/2024.09.05.611336
Mark Pocock, Janice Reid, Harley Robinson, Natalie Charitakis, James Krycer, Simon Foster, Natasha Tuano, Rebecca Fitzsimmons, Mary Lor, Sara Howden, Katerina Vlahos, Kevin Watt, Adam Piers, Holly Voges, Patrick Fortuna, James Rae, Robert Parton, Robert Weintraub, Igor Konstantinov, David Elliott, Mirana Ramialison, Enzo Porrello, Richard Mills, James Hudson
Cardiac maturation is an important developmental phase culminating in profound biological and functional changes to adapt to the high demand environment after birth. Maturation of human pluripotent stem cell-derived human cardiac organoids (hCO) to more closely resemble human heart tissue is critical for understanding disease pathology. Herein, we profile human heart maturation in vivo to identify key signalling pathways that drive maturation in hCOs. Transient activation of both the 5 AMP-activated kinase (AMPK) and estrogen-related receptor (ERR) promoted hCO maturation by mimicking the increased functional demands of post-natal development. hCOs cultured under these directed maturation (DM) conditions (DM-hCOs) display robust transcriptional maturation including increased expression of mature sarcomeric and oxidative phosphorylation genes resulting in enhanced metabolic capacity. DM-hCOs have functionally mature properties such as sarcoplasmic reticulum-dependent calcium handling, accurate responses to drug treatments perturbing the excitation-coupling process and ability to detect ectopy CASQ2 and RYR2 mutants. Importantly, DM-hCOs permit modelling of complex human disease processes such as desmoplakin (DSP) cardiomyopathy, which is driven by multiple cell types. Subsequently, we deploy DM-hCOs to demonstrate that bromodomain extra-terminal inhibitor INCB054329 rescues the DSP phenotype. Together, this study demonstrates that recapitulating in vivo development promotes advanced maturation enabling disease modelling and the identification of a therapeutic strategy for DSP-cardiomyopathy.
{"title":"Maturation of human cardiac organoids are required for disease modelling and drug discovery","authors":"Mark Pocock, Janice Reid, Harley Robinson, Natalie Charitakis, James Krycer, Simon Foster, Natasha Tuano, Rebecca Fitzsimmons, Mary Lor, Sara Howden, Katerina Vlahos, Kevin Watt, Adam Piers, Holly Voges, Patrick Fortuna, James Rae, Robert Parton, Robert Weintraub, Igor Konstantinov, David Elliott, Mirana Ramialison, Enzo Porrello, Richard Mills, James Hudson","doi":"10.1101/2024.09.05.611336","DOIUrl":"https://doi.org/10.1101/2024.09.05.611336","url":null,"abstract":"Cardiac maturation is an important developmental phase culminating in profound biological and functional changes to adapt to the high demand environment after birth. Maturation of human pluripotent stem cell-derived human cardiac organoids (hCO) to more closely resemble human heart tissue is critical for understanding disease pathology. Herein, we profile human heart maturation in vivo to identify key signalling pathways that drive maturation in hCOs. Transient activation of both the 5 AMP-activated kinase (AMPK) and estrogen-related receptor (ERR) promoted hCO maturation by mimicking the increased functional demands of post-natal development. hCOs cultured under these directed maturation (DM) conditions (DM-hCOs) display robust transcriptional maturation including increased expression of mature sarcomeric and oxidative phosphorylation genes resulting in enhanced metabolic capacity. DM-hCOs have functionally mature properties such as sarcoplasmic reticulum-dependent calcium handling, accurate responses to drug treatments perturbing the excitation-coupling process and ability to detect ectopy CASQ2 and RYR2 mutants. Importantly, DM-hCOs permit modelling of complex human disease processes such as desmoplakin (DSP) cardiomyopathy, which is driven by multiple cell types. Subsequently, we deploy DM-hCOs to demonstrate that bromodomain extra-terminal inhibitor INCB054329 rescues the DSP phenotype. Together, this study demonstrates that recapitulating in vivo development promotes advanced maturation enabling disease modelling and the identification of a therapeutic strategy for DSP-cardiomyopathy.","PeriodicalId":501308,"journal":{"name":"bioRxiv - Bioengineering","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142216620","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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