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1.
Bioact Mater ; 9: 358-372, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-34820576

RESUMO

To reflect human development, it is critical to create a substrate that can support long-term cell survival, differentiation, and maturation. Hydrogels are promising materials for 3D cultures. However, a bulk structure consisting of dense polymer networks often leads to suboptimal microenvironments that impedes nutrient exchange and cell-to-cell interaction. Herein, granular hydrogel-based scaffolds were used to support 3D human induced pluripotent stem cell (hiPSC)-derived neural networks. A custom designed 3D printed toolset was developed to extrude hyaluronic acid hydrogel through a porous nylon fabric to generate hydrogel granules. Cells and hydrogel granules were combined using a weaker secondary gelation step, forming self-supporting cell laden scaffolds. At three and seven days, granular scaffolds supported higher cell viability compared to bulk hydrogels, whereas granular scaffolds supported more neurite bearing cells and longer neurite extensions (65.52 ± 11.59 µm) after seven days compared to bulk hydrogels (22.90 ± 4.70 µm). Long-term (three-month) cultures of clinically relevant hiPSC-derived neural cells in granular hydrogels supported well established neuronal and astrocytic colonies and a high level of neurite extension both inside and beyond the scaffold. This approach is significant as it provides a simple, rapid and efficient way to achieve a tissue-relevant granular structure within hydrogel cultures.

2.
J Neurosci Res ; 97(7): 744-751, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30927386

RESUMO

Peripheral nerves, subject to continuous elongation and compression during everyday movement, contain neuron fibers vital for movement and sensation. At supraphysiological strains resulting from trauma, chronic conditions, aberrant limb positioning, or surgery, conduction blocks occur which may result in chronic or temporary loss of function. Previous in vitro stretch models, mainly focused on traumatic brain injury modelling, have demonstrated altered electrophysiological behavior during localized deformation applied by pipette suction. Our aim was to evaluate the changes in voltage-activated ion channel function during uniaxial straining of neurons applied by whole-cell deformation, more physiologically relevant model of peripheral nerve trauma. Here, we quantified experimentally the changes in inwards and outwards ion currents and action potential (AP) firing in dorsal root ganglion-derived neurons subject to uniaxial strains, using a custom-built device allowing simultaneous cell deformation and patch clamp recording. Peak inwards sodium currents and rectifying potassium current magnitudes were found to decrease in cells under stretch, channel reversal potentials were found to be left-shifted, and half-maximum activation potentials right-shifted. The threshold for AP firing was increased in stretched cells, although neurons retained the ability to fire induced APs. Overall, these results point to ion channels being damaged directly and immediately by uniaxial strain, affecting cell electrophysiological activity, and can help develop prevention and treatment strategies for peripheral neuropathies caused by mechanical trauma.


Assuntos
Potenciais de Ação/fisiologia , Ativação do Canal Iônico/fisiologia , Neurônios/fisiologia , Traumatismos dos Nervos Periféricos/fisiopatologia , Animais , Linhagem Celular Tumoral , Gânglios Espinais , Potenciais da Membrana/fisiologia , Neuroblastoma , Técnicas de Patch-Clamp , Ratos , Sódio
3.
Med Eng Phys ; 67: 1-10, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30878301

RESUMO

Peripheral nerves are continuously subjected to mechanical strain during everyday movements, but excessive stretch can lead to damage and neuronal cell functionality can also be impaired. To better understand cellular processes triggered by stretch, it is necessary to develop in vitro experimental methods that allow multiple concurrent measurements and replicate in vivo mechanical conditions. Current commercially available cell stretching devices do not allow flexible experimental design, restricting the range of possible multi-physics measurements. Here, we describe and characterise a custom-built uniaxial substrate-straining device, with which neurons cultured on aligned patterned surfaces (50 µm wide grooves) can be strained up to 70% and simultaneously imaged with widefield and confocal imaging (up to 100x magnification). Furthermore, direct and indirect electrophysiological measurements by patch clamping and calcium imaging can be made during strain application. We characterise the strain applied to cells cultured in deformable wells by using finite element method simulations and experimental data, showing local surface strains of up to 60% with applied strains of up to 25%. We also show how patterned substrates do not alter the mechanical properties of the system compared to unpatterned surfaces whilst still inducing a homogeneous cell response to strain. The characterisation of this device will be useful for research into investigating the effect of whole-cell mechanical stretch on neurons at both single cell and network scales, with applications found in peripheral neuropathy modelling and in platforms for preventive and regenerative studies.


Assuntos
Eletrofisiologia/instrumentação , Engenharia , Imagem Molecular/instrumentação , Neurônios/citologia , Nervos Periféricos/citologia , Estresse Mecânico , Fenômenos Biomecânicos , Cálcio/metabolismo , Humanos , Neurônios/metabolismo , Análise de Célula Única , Fatores de Tempo
4.
Neuroscience ; 404: 165-174, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30817953

RESUMO

Peripheral nerves contain neuron fibers vital for movement and sensation and are subject to continuous elongation and compression during everyday movement. At supraphysiological strains conduction blocks occur, resulting in permanent or temporary loss of function. The mechanisms underpinning these alterations in electrophysiological activity remain unclear; however, there is evidence that both ion channels and network synapses may be affected through cell membrane transmitted strain. The aim of this work was to quantify the changes in spontaneous activity resulting from application of uniaxial strain in a human iPS-derived motor neuron culture model, and to investigate the role of cell membrane mechanical properties during cell straining. Increasing strain in a custom-built cell-stretching device caused a linear decrease in spontaneous activity, and no immediate recovery of activity was observed after strain release. Imaging neuronal membranes with c-Laurdan showed changes to the lipid order in neural membranes during deformation with a decrease in lipid packing. Neural cell membrane stiffness can be modulated by increasing cholesterol content, resulting in reduced stretch-induced decrease of membrane lipid packing and in a reduced decrease in spontaneous activity caused by mechanical strain. Together these results indicate that the mechanism whereby cell injury causes impaired transmission of neural impulses may be governed by the mechanical state of the cell membrane, and contribute to establishing a direct relationship between neural uniaxial straining and loss of spontaneous neural activity.


Assuntos
Potenciais de Ação/fisiologia , Membrana Celular/fisiologia , Fenômenos Eletrofisiológicos/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Neurônios Motores/fisiologia , Estresse Mecânico , Células Cultivadas , Humanos
5.
J Tissue Eng Regen Med ; 13(3): 369-384, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30550638

RESUMO

Development of an optogenetically controllable human neural network model in three-dimensional (3D) cultures can provide an investigative system that is more physiologically relevant and better able to mimic aspects of human brain function. Light-sensitive neurons were generated by transducing channelrhodopsin-2 (ChR2) into human induced pluripotent stem cell (hiPSC) derived neural progenitor cells (Axol) using lentiviruses and cell-type specific promoters. A mixed population of human iPSC-derived cortical neurons, astrocytes and progenitor cells were obtained (Axol-ChR2) upon neural differentiation. Pan-neuronal promoter synapsin-1 (SYN1) and excitatory neuron-specific promoter calcium-calmodulin kinase II (CaMKII) were used to drive reporter gene expression in order to assess the differentiation status of the targeted cells. Expression of ChR2 and characterisation of subpopulations in differentiated Axol-ChR2 cells were evaluated using flow cytometry and immunofluorescent staining. These cells were transferred from 2D culture to 3D alginate hydrogel functionalised with arginine-glycine-aspartate (RGD) and small molecules (Y-27632). Improved RGD-alginate hydrogel was physically characterised and assessed for cell viability to serve as a generic 3D culture system for human pluripotent stem cells (hPSCs) and neuronal cells. Prior to cell encapsulation, neural network activities of Axol-ChR2 cells and primary neurons were investigated using calcium imaging. Results demonstrate that functional activities were successfully achieved through expression of ChR2- by both the CaMKII and SYN1 promoters. The RGD-alginate hydrogel system supports the growth of differentiated Axol-ChR2 cells whilst allowing detection of ChR2 expression upon light stimulation. This allows precise and non-invasive control of human neural networks in 3D.


Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Técnicas de Cultura de Células/métodos , Channelrhodopsins/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Neurônios/citologia , Optogenética , Regiões Promotoras Genéticas/genética , Sinapsinas/genética , Alginatos/farmacologia , Animais , Cálcio/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Lentivirus/metabolismo , Camundongos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Reologia , Sinapsinas/metabolismo
6.
Sci Rep ; 8(1): 15552, 2018 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-30341335

RESUMO

Microporous membranes support the growth of neurites into and through micro-channels, providing a different type of neural growth platform to conventional dish cultures. Microporous membranes are used to support various types of culture, however, the role of pore diameter in relation to neurite growth through the membrane has not been well characterised. In this study, the human cell line (SH-SY5Y) was differentiated into neuron-like cells and cultured on track-etched microporous membranes with pore and channel diameters selected to accommodate neurite width (0.8 µm to 5 µm). Whilst neurites extended through all pore diameters, the extent of neurite coverage on the non-seeded side of the membranes after 5 days in culture was found to be directly proportional to channel diameter. Neurite growth through membrane pores reduced significantly when neural cultures were non-confluent. Scanning electron microscopy revealed that neurites bridged pores and circumnavigated pore edges - such that the overall likelihood of a neurite entering a pore channel was decreased. These findings highlight the role of pore diameter, cell sheet confluence and contact guidance in directing neurite growth through pores and may be useful in applications that seek to use physical substrates to maintain separate neural populations whilst permitting neurite contact between cultures.


Assuntos
Técnicas de Cultura de Células/métodos , Proliferação de Células , Membranas , Neuritos/fisiologia , Linhagem Celular , Humanos , Microscopia Eletrônica de Varredura , Neuritos/ultraestrutura
7.
Stem Cell Res ; 32: 126-134, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30278374

RESUMO

Primary rodent neurons and immortalised cell lines have overwhelmingly been used for in vitro studies of traumatic injury to peripheral and central neurons, but have some limitations of physiological accuracy. Motor neurons (MN) derived from human induced pluripotent stem cells (iPSCs) enable the generation of cell models with features relevant to human physiology. To facilitate this, it is desirable that MN protocols both rapidly and efficiently differentiate human iPSCs into electrophysiologically active MNs. In this study, we present a simple, rapid protocol for differentiation of human iPSCs into functional spinal (lower) MNs, involving only adherent culture and use of small molecules for directed differentiation, with the ultimate aim of rapid production of electrophysiologically functional cells for short-term neural injury experiments. We show successful differentiation in two unrelated iPSC lines, by quantifying neural-specific marker expression, and by evaluating cell functionality at different maturation stages by calcium imaging and patch clamping. Differentiated neurons were shown to be electrophysiologically altered by uniaxial mechanical deformation. Spontaneous network activity decreased with applied stretch, indicating aberrant network connectivity. These results demonstrate the feasibility of this rapid, simple protocol for differentiating iPSC-derived MNs, suitable for in vitro neural injury studies focussing on electrophysiological alterations caused by mechanical deformation or trauma.


Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Neurônios Motores/citologia , Diferenciação Celular/fisiologia , Células Cultivadas , Eletrofisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Neurônios Motores/metabolismo
8.
Biotechnol Lett ; 40(3): 601-607, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29313254

RESUMO

OBJECTIVES: To test a 3D approach for neural network formation, alignment, and patterning that is reproducible and sufficiently stable to allow for easy manipulation. RESULTS: A novel cell culture system was designed by engineering a method for the directional growth of neurons. This uses NG108-15 neuroblastoma x glioma hybrid cells cultured on suspended and aligned electrospun fibers. These fiber networks improved cellular directionality, with alignment angle standard deviations significantly lower on fibers than on regular culture surfaces. Morphological studies found nuclear aspect ratios and cell projection lengths to be unchanged, indicating that cells maintained neural morphology while growing on fibers and forming a 3D network. Furthermore, fibronectin-coated fibers enhanced neurite extensions for all investigated time points. Differentiated neurons exhibited significant increases in average neurite lengths 96 h post plating, and formed neurite extensions parallel to suspended fibers, as visualized through scanning electron microscopy. CONCLUSIONS: The developed model has the potential to serve as the basis for advanced 3D studies, providing an original approach to neural network patterning and setting the groundwork for further investigations into functionality.


Assuntos
Técnicas de Cultura de Células/métodos , Técnicas Eletroquímicas/métodos , Modelos Neurológicos , Fibras Nervosas/fisiologia , Rede Nervosa/citologia , Engenharia Tecidual/métodos , Animais , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Desenho de Equipamento , Fibronectinas/farmacologia , Glioma , Neuritos/fisiologia , Neuroblastoma , Poliésteres , Ratos
9.
J Biomed Mater Res A ; 106(5): 1165-1176, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29266766

RESUMO

Research on the neuromuscular junction (NMJ) and its function and development spans over a century. However, researchers are limited in their ability to conduct experimentation on this highly specialized synapse between motor neurons and muscle fibers, as NMJs are not easily accessible outside the body. The aim of this work is to provide a reliable and reproducible muscle sheet model for in vitro NMJ study. A novel culture system was designed by engineering a method for the directional growth of myofiber sheets, using muscle progenitor cells cultured on electrospun fiber networks. Myoblastic C2C12 cells cultured on suspended aligned fibers were found to maintain directionality, with alignment angle standard deviations approximately two-thirds lower on fibers than on regular culture surfaces. Morphological studies found nuclei and cytoskeleton aspect ratios to be elongated by 20 and 150%, respectively. Furthermore, neurons were shown to form innervation patterns parallel to suspended fibers when co-cultured on developed muscle sheets, with alignment angle standard deviations three times lower compared with those on typical surfaces. The effect of agrin on samples was quantified through the slow release of agrin medium, encapsulated in alginate pellets and imbedded within culture chambers. Samples exposed to agrin showed significantly increased percentage of AChR-covered area. The developed model has potential to serve as the basis for synaptogenesis and NMJ studies, providing a novel approach to bio-artificial muscle alignment and setting the groundwork for further investigations in innervation. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1165-1176, 2018.


Assuntos
Músculos/fisiologia , Poliésteres/farmacologia , Engenharia Tecidual/métodos , Animais , Diferenciação Celular , Linhagem Celular , Forma Celular , Dextranos/química , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/química , Humanos , Camundongos , Fibras Musculares Esqueléticas/metabolismo , Receptores Colinérgicos/metabolismo , Suínos
10.
Chem Soc Rev ; 38(4): 1139-51, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19421585

RESUMO

The field of tissue engineering places complex demands on the materials it uses. The materials chosen to support the intricate processes of tissue development and maintenance need to have properties which serve both the bulk mechanical and structural requirements of the target tissue, as well as enabling interactions with cells at the molecular scale. In this critical review we explore how synthetic polymers can be utilised to meet the needs of tissue engineering applications, and how biomimetic principles can be applied to polymeric materials in order to enhance the biological response to scaffolding materials (105 references).


Assuntos
Polímeros/síntese química , Polímeros/metabolismo , Engenharia Tecidual/métodos , Sequência de Aminoácidos , Fenômenos Biomecânicos , Células/citologia , Células/metabolismo , Humanos , Nanotecnologia , Peptídeos/química , Peptídeos/metabolismo
11.
Curr Opin Chem Biol ; 10(6): 568-75, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17011226

RESUMO

Materials that enhance bone and cartilage regeneration promise to be valuable in both research and clinical applications. Both natural and synthetic polymers can be used to create scaffolds that support cells and incorporate cues which guide tissue repair. Recently, electrospinning, peptide self-assembly and biomineralisation have been employed to fabricate nanostructured scaffolds that better mimic the complex extracellular environment found within tissues, in vivo. The incorporation of peptide motifs recognised by cell receptors and the use of recombinant DNA technology have enabled the creation of scaffolds with new levels of biofunctionality. Advances in materials design will enhance our ability to create highly tailored cellular environments for bone and cartilage regeneration.


Assuntos
Materiais Biocompatíveis , Osso e Ossos/fisiologia , Cartilagem/fisiologia , Regeneração , Engenharia Tecidual
12.
Science ; 310(5751): 1135-8, 2005 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-16293749

RESUMO

Cells are inherently sensitive to local mesoscale, microscale, and nanoscale patterns of chemistry and topography. We review current approaches to control cell behavior through the nanoscale engineering of materials surfaces. Far-reaching implications are emerging for applications including medical implants, cell supports, and materials that can be used as instructive three-dimensional environments for tissue regeneration.


Assuntos
Materiais Biocompatíveis , Membrana Celular/fisiologia , Fenômenos Fisiológicos Celulares , Matriz Extracelular/fisiologia , Nanotecnologia/tendências , Animais , Previsões , Humanos , Propriedades de Superfície , Engenharia Tecidual/tendências
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