12 research outputs found

    Mapping the Complex Morphology of Cell Interactions with Nanowire Substrates Using FIB-SEM

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    Using high resolution focused ion beam scanning electron microscopy (FIB-SEM) we study the details of cell-nanostructure interactions using serial block face imaging. 3T3 Fibroblast cellular monolayers are cultured on flat glass as a control surface and on two types of nanostructured scaffold substrates made from silicon black (Nanograss) with low- and high nanowire density. After culturing for 72 hours the cells were fixed, heavy metal stained, embedded in resin, and processed with FIB-SEM block face imaging without removing the substrate. The sample preparation procedure, image acquisition and image post-processing were specifically optimised for cellular monolayers cultured on nanostructured substrates. Cells display a wide range of interactions with the nanostructures depending on the surface morphology, but also greatly varying from one cell to another on the same substrate, illustrating a wide phenotypic variability. Depending on the substrate and cell, we observe that cells could for instance: break the nanowires and engulf them, flatten the nanowires or simply reside on top of them. Given the complexity of interactions, we have categorised our observations and created an overview map. The results demonstrate that detailed nanoscale resolution images are required to begin understanding the wide variety of individual cells' interactions with a structured substrate. The map will provide a framework for light microscopy studies of such interactions indicating what modes of interactions must be considered

    Automatisation et intégration d'un réacteur de culture cellulaire pour un fonctionnement en continu

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    Over the past six decades, cell culture has become a common practice. It is a major tool in biological research for the understanding of life science, such as the study of disease and the discovery of new drugs. It plays an important role in many industries since it is involved in the production of many food, cosmetic, and pharmaceutical products.However, Research and the industry are now facing some limits and are expressing needs to be addressed. They are both associated with high costs due to a large consumption of resources (cells, reagents, qualified operators). More specifically, cell culture in research is characterized by low throughput of experiments, important variability and risk of contamination due to the recurrent manual operations performed by operators. Additionally, experiments are performed in static conditions and on models (2D cultures, animals
) which poorly resemble the human physiology. Industrial cell culture needs miniaturized systems that mimic the large scale bioreactors and offer higher screening possibilities.Microfluidic cell culture systems represent a promising tool to address the aforementioned issues and needs. The change of physical behaviors at the small-scale in microfluidic devices allow controlling temporally and spatially the cell microenvironment, unattainable with conventional cell culture methods. The level of automation and integration allows the substantial increase of the number of experience per system and considerable reduction of resource consumption. Thus, many small cellular 3D architectures grown under dynamic conditions and in high-throughput have been performed and have demonstrated their ability to quickly re-create more physiological environments. Regarding the industrial culture, miniaturized cultures have already shown their ability to reproduce the characteristics of the culture observed in macrobioreactors with higher screening capabilities.In this framework, a benchtop microfluidic bioreactor, complying with the standard microfluidic platform and format used in the host laboratory, has been successfully fabricated to perform continuous cell cultures. Integrated solutions were developed to provide continuously the adequate conditions for cell proliferation (perfusion, thermal regulation
). Integrated cell harvest was also performed with the final goal to achieve long-term cell culture in the bioreactor.The fabricated system proved to guarantee sterile conditions for cell cultures on a regular lab bench. Moreover, these cultures were achieved autonomously without requiring a cumbersome incubator. In these conditions, the bioreactor demonstrated the possibility to perform continuous cell cultures of various cell types during several days: insects cells were cultured during 5 days and mammalian cells during 3 days. Regarding the mammalian cell cultures performed, a breakthrough has been achieved compared to the cultures performed in microfluidic systems since microcarriers (diam.:175 ”m) were used as growth support.Although microcarrier cell culture is routinely performed in the industry, no autonomous microfluidic culture system has addressed this type of culture yet. Such a miniaturization is a major step forward for bioprocess applications where the need to develop scale-down bioreactors that mimic large scale operation has been clearly identified to shorten and reduce the costs associated to bioproduct development.Au cours des six derniĂšres dĂ©cennies, la culture cellulaire est devenue une pratique courante. Elle est un outil majeur de la recherche biologique pour la comprĂ©hension du vivant, l'Ă©tude de maladies et la dĂ©couverte de nouveaux mĂ©dicaments. Elle reprĂ©sente un outil trĂšs rĂ©pandu dans de nombreuses industries Ă©tant impliquĂ©es dans la production de produits alimentaires, cosmĂ©tiques et pharmaceutiques.Cependant, les cultures cellulaires en recherche et en industrie sont aujourd'hui confrontĂ©es Ă  des limites et soulĂšvent des besoins Ă  satisfaire. Elles sont toutes deux associĂ©es Ă  des coĂ»ts Ă©levĂ©s du fait des ressources nĂ©cessaires (cellules, rĂ©actifs, opĂ©rateurs qualifiĂ©s). Plus prĂ©cisĂ©ment, la culture en recherche est caractĂ©risĂ©e par le faible dĂ©bit des expĂ©riences, une variabilitĂ© importante et un risque de contamination due Ă  la rĂ©pĂ©tition d'opĂ©rations manuelles. De plus, les expĂ©riences de culture sont effectuĂ©es dans des conditions statiques et sur des modĂšles (cultures 2D, animaux...) relativement Ă©loignĂ©s de la physiologie humaine. La culture cellulaire industrielle, quant Ă  elle, a besoin de systĂšmes miniaturisĂ©s qui miment les procĂ©dĂ©s des biorĂ©acteurs Ă  grande Ă©chelle et qui offrent des possibilitĂ©s de criblage plus Ă©levĂ©s.Les systĂšmes de culture microfluidique reprĂ©sentent un outil prometteur pour rĂ©soudre ces problĂšmes et ces besoins. Le changement de comportement de la physique Ă  petite Ă©chelle dans ces dispositifs permet de contrĂŽler temporellement et spatialement le microenvironnement des cellules. Ce qui n'est pas possible avec des mĂ©thodes de culture classiques. Le degrĂ© d'automatisation et d'intĂ©gration permet une nette augmentation du nombre d'expĂ©riences par systĂšme et la rĂ©duction consĂ©quente de la consommation de ressources. Ainsi, de nombreuses petites architectures 3D cellulaires cultivĂ©es dans des conditions dynamiques et Ă  haut dĂ©bit ont Ă©tĂ© rĂ©alisĂ©es et ont dĂ©montrĂ© leur capacitĂ© Ă  recrĂ©er rapidement des environnements plus physiologiques. En ce qui concerne la culture industrielle, des cultures miniaturisĂ©es ont dĂ©jĂ  montrĂ© leur capacitĂ© Ă  reproduire les caractĂ©ristiques observĂ©es dans les macrobioreactors avec des possibilitĂ©s de criblages Ă©levĂ©es.Dans ce contexte, un biorĂ©acteur microfluidique de paillasse, se conformant aux formats standards utilisĂ©s dans le laboratoire d'accueil, a Ă©tĂ© fabriquĂ© avec succĂšs au cours de cette thĂšse pour effectuer des cultures cellulaires en continu. Des solutions intĂ©grĂ©es ont Ă©tĂ© mises au point pour fournir de façon continue les conditions adĂ©quates pour la prolifĂ©ration cellulaire (perfusion, rĂ©gulation de tempĂ©rature
). Des Ă©tudes ont Ă©galement Ă©tĂ© menĂ©es afin d'automatiser la rĂ©colte des cellules avec pour but final de cultiver ces cellules sur du long terme dans le biorĂ©acteur.Le systĂšme fabriquĂ© garantit ainsi des conditions stĂ©riles pour les cultures sur un simple banc de laboratoire. En outre, ces cultures ont Ă©tĂ© rĂ©alisĂ©es de façon autonome sans utiliser un incubateur encombrant. Dans ces conditions, le biorĂ©acteur permet de rĂ©aliser des cultures en continu de divers types cellulaires sur plusieurs jours: des cellules d'insectes ont Ă©tĂ© cultivĂ©es pendant 5 jours et des cellules de mammifĂšre pendant 3 jours. En ce qui concerne les cultures de cellules de mammifĂšre, une avancĂ©e majeure a Ă©tĂ© effectuĂ©e par rapport aux cultures rĂ©alisĂ©es dans les systĂšmes microfluidiques en utilisant comme support de culture des microporteurs (diam. : 175 ”m).Bien que la culture de cellules sur microporteurs soit rĂ©alisĂ©e en routine dans l'industrie, aucun systĂšme de culture microfluidique autonome n'a encore intĂ©grĂ© ce type de culture. Ce genre de miniaturisation est une avancĂ©e majeure pour des applications en bioprocĂ©dĂ©s oĂč il devrait permettre de raccourcir et rĂ©duire les coĂ»ts associĂ©s au dĂ©veloppement de bioproduits

    Automatisation et intégration d'un réacteur de culture cellulaire pour un fonctionnement en continu

    Get PDF
    Over the past six decades, cell culture has become a common practice. It is a major tool in biological research for the understanding of life science, such as the study of disease and the discovery of new drugs. It plays an important role in many industries since it is involved in the production of many food, cosmetic, and pharmaceutical products.However, Research and the industry are now facing some limits and are expressing needs to be addressed. They are both associated with high costs due to a large consumption of resources (cells, reagents, qualified operators). More specifically, cell culture in research is characterized by low throughput of experiments, important variability and risk of contamination due to the recurrent manual operations performed by operators. Additionally, experiments are performed in static conditions and on models (2D cultures, animals
) which poorly resemble the human physiology. Industrial cell culture needs miniaturized systems that mimic the large scale bioreactors and offer higher screening possibilities.Microfluidic cell culture systems represent a promising tool to address the aforementioned issues and needs. The change of physical behaviors at the small-scale in microfluidic devices allow controlling temporally and spatially the cell microenvironment, unattainable with conventional cell culture methods. The level of automation and integration allows the substantial increase of the number of experience per system and considerable reduction of resource consumption. Thus, many small cellular 3D architectures grown under dynamic conditions and in high-throughput have been performed and have demonstrated their ability to quickly re-create more physiological environments. Regarding the industrial culture, miniaturized cultures have already shown their ability to reproduce the characteristics of the culture observed in macrobioreactors with higher screening capabilities.In this framework, a benchtop microfluidic bioreactor, complying with the standard microfluidic platform and format used in the host laboratory, has been successfully fabricated to perform continuous cell cultures. Integrated solutions were developed to provide continuously the adequate conditions for cell proliferation (perfusion, thermal regulation
). Integrated cell harvest was also performed with the final goal to achieve long-term cell culture in the bioreactor.The fabricated system proved to guarantee sterile conditions for cell cultures on a regular lab bench. Moreover, these cultures were achieved autonomously without requiring a cumbersome incubator. In these conditions, the bioreactor demonstrated the possibility to perform continuous cell cultures of various cell types during several days: insects cells were cultured during 5 days and mammalian cells during 3 days. Regarding the mammalian cell cultures performed, a breakthrough has been achieved compared to the cultures performed in microfluidic systems since microcarriers (diam.:175 ”m) were used as growth support.Although microcarrier cell culture is routinely performed in the industry, no autonomous microfluidic culture system has addressed this type of culture yet. Such a miniaturization is a major step forward for bioprocess applications where the need to develop scale-down bioreactors that mimic large scale operation has been clearly identified to shorten and reduce the costs associated to bioproduct development.Au cours des six derniĂšres dĂ©cennies, la culture cellulaire est devenue une pratique courante. Elle est un outil majeur de la recherche biologique pour la comprĂ©hension du vivant, l'Ă©tude de maladies et la dĂ©couverte de nouveaux mĂ©dicaments. Elle reprĂ©sente un outil trĂšs rĂ©pandu dans de nombreuses industries Ă©tant impliquĂ©es dans la production de produits alimentaires, cosmĂ©tiques et pharmaceutiques.Cependant, les cultures cellulaires en recherche et en industrie sont aujourd'hui confrontĂ©es Ă  des limites et soulĂšvent des besoins Ă  satisfaire. Elles sont toutes deux associĂ©es Ă  des coĂ»ts Ă©levĂ©s du fait des ressources nĂ©cessaires (cellules, rĂ©actifs, opĂ©rateurs qualifiĂ©s). Plus prĂ©cisĂ©ment, la culture en recherche est caractĂ©risĂ©e par le faible dĂ©bit des expĂ©riences, une variabilitĂ© importante et un risque de contamination due Ă  la rĂ©pĂ©tition d'opĂ©rations manuelles. De plus, les expĂ©riences de culture sont effectuĂ©es dans des conditions statiques et sur des modĂšles (cultures 2D, animaux...) relativement Ă©loignĂ©s de la physiologie humaine. La culture cellulaire industrielle, quant Ă  elle, a besoin de systĂšmes miniaturisĂ©s qui miment les procĂ©dĂ©s des biorĂ©acteurs Ă  grande Ă©chelle et qui offrent des possibilitĂ©s de criblage plus Ă©levĂ©s.Les systĂšmes de culture microfluidique reprĂ©sentent un outil prometteur pour rĂ©soudre ces problĂšmes et ces besoins. Le changement de comportement de la physique Ă  petite Ă©chelle dans ces dispositifs permet de contrĂŽler temporellement et spatialement le microenvironnement des cellules. Ce qui n'est pas possible avec des mĂ©thodes de culture classiques. Le degrĂ© d'automatisation et d'intĂ©gration permet une nette augmentation du nombre d'expĂ©riences par systĂšme et la rĂ©duction consĂ©quente de la consommation de ressources. Ainsi, de nombreuses petites architectures 3D cellulaires cultivĂ©es dans des conditions dynamiques et Ă  haut dĂ©bit ont Ă©tĂ© rĂ©alisĂ©es et ont dĂ©montrĂ© leur capacitĂ© Ă  recrĂ©er rapidement des environnements plus physiologiques. En ce qui concerne la culture industrielle, des cultures miniaturisĂ©es ont dĂ©jĂ  montrĂ© leur capacitĂ© Ă  reproduire les caractĂ©ristiques observĂ©es dans les macrobioreactors avec des possibilitĂ©s de criblages Ă©levĂ©es.Dans ce contexte, un biorĂ©acteur microfluidique de paillasse, se conformant aux formats standards utilisĂ©s dans le laboratoire d'accueil, a Ă©tĂ© fabriquĂ© avec succĂšs au cours de cette thĂšse pour effectuer des cultures cellulaires en continu. Des solutions intĂ©grĂ©es ont Ă©tĂ© mises au point pour fournir de façon continue les conditions adĂ©quates pour la prolifĂ©ration cellulaire (perfusion, rĂ©gulation de tempĂ©rature
). Des Ă©tudes ont Ă©galement Ă©tĂ© menĂ©es afin d'automatiser la rĂ©colte des cellules avec pour but final de cultiver ces cellules sur du long terme dans le biorĂ©acteur.Le systĂšme fabriquĂ© garantit ainsi des conditions stĂ©riles pour les cultures sur un simple banc de laboratoire. En outre, ces cultures ont Ă©tĂ© rĂ©alisĂ©es de façon autonome sans utiliser un incubateur encombrant. Dans ces conditions, le biorĂ©acteur permet de rĂ©aliser des cultures en continu de divers types cellulaires sur plusieurs jours: des cellules d'insectes ont Ă©tĂ© cultivĂ©es pendant 5 jours et des cellules de mammifĂšre pendant 3 jours. En ce qui concerne les cultures de cellules de mammifĂšre, une avancĂ©e majeure a Ă©tĂ© effectuĂ©e par rapport aux cultures rĂ©alisĂ©es dans les systĂšmes microfluidiques en utilisant comme support de culture des microporteurs (diam. : 175 ”m).Bien que la culture de cellules sur microporteurs soit rĂ©alisĂ©e en routine dans l'industrie, aucun systĂšme de culture microfluidique autonome n'a encore intĂ©grĂ© ce type de culture. Ce genre de miniaturisation est une avancĂ©e majeure pour des applications en bioprocĂ©dĂ©s oĂč il devrait permettre de raccourcir et rĂ©duire les coĂ»ts associĂ©s au dĂ©veloppement de bioproduits

    Automation and integration of a bioreactor for continuous cell culture

    No full text
    Au cours des six derniĂšres dĂ©cennies, la culture cellulaire est devenue une pratique courante. Elle est un outil majeur de la recherche biologique pour la comprĂ©hension du vivant, l'Ă©tude de maladies et la dĂ©couverte de nouveaux mĂ©dicaments. Elle reprĂ©sente un outil trĂšs rĂ©pandu dans de nombreuses industries Ă©tant impliquĂ©es dans la production de produits alimentaires, cosmĂ©tiques et pharmaceutiques.Cependant, les cultures cellulaires en recherche et en industrie sont aujourd'hui confrontĂ©es Ă  des limites et soulĂšvent des besoins Ă  satisfaire. Elles sont toutes deux associĂ©es Ă  des coĂ»ts Ă©levĂ©s du fait des ressources nĂ©cessaires (cellules, rĂ©actifs, opĂ©rateurs qualifiĂ©s). Plus prĂ©cisĂ©ment, la culture en recherche est caractĂ©risĂ©e par le faible dĂ©bit des expĂ©riences, une variabilitĂ© importante et un risque de contamination due Ă  la rĂ©pĂ©tition d'opĂ©rations manuelles. De plus, les expĂ©riences de culture sont effectuĂ©es dans des conditions statiques et sur des modĂšles (cultures 2D, animaux...) relativement Ă©loignĂ©s de la physiologie humaine. La culture cellulaire industrielle, quant Ă  elle, a besoin de systĂšmes miniaturisĂ©s qui miment les procĂ©dĂ©s des biorĂ©acteurs Ă  grande Ă©chelle et qui offrent des possibilitĂ©s de criblage plus Ă©levĂ©s.Les systĂšmes de culture microfluidique reprĂ©sentent un outil prometteur pour rĂ©soudre ces problĂšmes et ces besoins. Le changement de comportement de la physique Ă  petite Ă©chelle dans ces dispositifs permet de contrĂŽler temporellement et spatialement le microenvironnement des cellules. Ce qui n'est pas possible avec des mĂ©thodes de culture classiques. Le degrĂ© d'automatisation et d'intĂ©gration permet une nette augmentation du nombre d'expĂ©riences par systĂšme et la rĂ©duction consĂ©quente de la consommation de ressources. Ainsi, de nombreuses petites architectures 3D cellulaires cultivĂ©es dans des conditions dynamiques et Ă  haut dĂ©bit ont Ă©tĂ© rĂ©alisĂ©es et ont dĂ©montrĂ© leur capacitĂ© Ă  recrĂ©er rapidement des environnements plus physiologiques. En ce qui concerne la culture industrielle, des cultures miniaturisĂ©es ont dĂ©jĂ  montrĂ© leur capacitĂ© Ă  reproduire les caractĂ©ristiques observĂ©es dans les macrobioreactors avec des possibilitĂ©s de criblages Ă©levĂ©es.Dans ce contexte, un biorĂ©acteur microfluidique de paillasse, se conformant aux formats standards utilisĂ©s dans le laboratoire d'accueil, a Ă©tĂ© fabriquĂ© avec succĂšs au cours de cette thĂšse pour effectuer des cultures cellulaires en continu. Des solutions intĂ©grĂ©es ont Ă©tĂ© mises au point pour fournir de façon continue les conditions adĂ©quates pour la prolifĂ©ration cellulaire (perfusion, rĂ©gulation de tempĂ©rature
). Des Ă©tudes ont Ă©galement Ă©tĂ© menĂ©es afin d'automatiser la rĂ©colte des cellules avec pour but final de cultiver ces cellules sur du long terme dans le biorĂ©acteur.Le systĂšme fabriquĂ© garantit ainsi des conditions stĂ©riles pour les cultures sur un simple banc de laboratoire. En outre, ces cultures ont Ă©tĂ© rĂ©alisĂ©es de façon autonome sans utiliser un incubateur encombrant. Dans ces conditions, le biorĂ©acteur permet de rĂ©aliser des cultures en continu de divers types cellulaires sur plusieurs jours: des cellules d'insectes ont Ă©tĂ© cultivĂ©es pendant 5 jours et des cellules de mammifĂšre pendant 3 jours. En ce qui concerne les cultures de cellules de mammifĂšre, une avancĂ©e majeure a Ă©tĂ© effectuĂ©e par rapport aux cultures rĂ©alisĂ©es dans les systĂšmes microfluidiques en utilisant comme support de culture des microporteurs (diam. : 175 ”m).Bien que la culture de cellules sur microporteurs soit rĂ©alisĂ©e en routine dans l'industrie, aucun systĂšme de culture microfluidique autonome n'a encore intĂ©grĂ© ce type de culture. Ce genre de miniaturisation est une avancĂ©e majeure pour des applications en bioprocĂ©dĂ©s oĂč il devrait permettre de raccourcir et rĂ©duire les coĂ»ts associĂ©s au dĂ©veloppement de bioproduits.Over the past six decades, cell culture has become a common practice. It is a major tool in biological research for the understanding of life science, such as the study of disease and the discovery of new drugs. It plays an important role in many industries since it is involved in the production of many food, cosmetic, and pharmaceutical products.However, Research and the industry are now facing some limits and are expressing needs to be addressed. They are both associated with high costs due to a large consumption of resources (cells, reagents, qualified operators). More specifically, cell culture in research is characterized by low throughput of experiments, important variability and risk of contamination due to the recurrent manual operations performed by operators. Additionally, experiments are performed in static conditions and on models (2D cultures, animals
) which poorly resemble the human physiology. Industrial cell culture needs miniaturized systems that mimic the large scale bioreactors and offer higher screening possibilities.Microfluidic cell culture systems represent a promising tool to address the aforementioned issues and needs. The change of physical behaviors at the small-scale in microfluidic devices allow controlling temporally and spatially the cell microenvironment, unattainable with conventional cell culture methods. The level of automation and integration allows the substantial increase of the number of experience per system and considerable reduction of resource consumption. Thus, many small cellular 3D architectures grown under dynamic conditions and in high-throughput have been performed and have demonstrated their ability to quickly re-create more physiological environments. Regarding the industrial culture, miniaturized cultures have already shown their ability to reproduce the characteristics of the culture observed in macrobioreactors with higher screening capabilities.In this framework, a benchtop microfluidic bioreactor, complying with the standard microfluidic platform and format used in the host laboratory, has been successfully fabricated to perform continuous cell cultures. Integrated solutions were developed to provide continuously the adequate conditions for cell proliferation (perfusion, thermal regulation
). Integrated cell harvest was also performed with the final goal to achieve long-term cell culture in the bioreactor.The fabricated system proved to guarantee sterile conditions for cell cultures on a regular lab bench. Moreover, these cultures were achieved autonomously without requiring a cumbersome incubator. In these conditions, the bioreactor demonstrated the possibility to perform continuous cell cultures of various cell types during several days: insects cells were cultured during 5 days and mammalian cells during 3 days. Regarding the mammalian cell cultures performed, a breakthrough has been achieved compared to the cultures performed in microfluidic systems since microcarriers (diam.:175 ”m) were used as growth support.Although microcarrier cell culture is routinely performed in the industry, no autonomous microfluidic culture system has addressed this type of culture yet. Such a miniaturization is a major step forward for bioprocess applications where the need to develop scale-down bioreactors that mimic large scale operation has been clearly identified to shorten and reduce the costs associated to bioproduct development

    Map of the various cell-nanowire interactions observed.

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    <p>6 cases are outlined with a schematic view and two supporting FIB-SEM images illustrating the case. Case VII, vacuolisation is to a large degree observed in images displaying Case III and Case VI.Inverted view can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053307#pone.0053307.s003" target="_blank">Figure S3</a>. The close-up images are either regions from the lower magnification image or higher resolution images from a different image.</p

    Side views of the non-tilted milling obtained image stack of a cell on glass showing the sequential processing operations’ effects.

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    <p>A) The individual slices have been aligned forming a fairly smooth image using stack-reg algorithm. B) Then the substrate is corrected such as to annul the effects of automatic E-beam shifts in the Slice and View program, resulting in a 52 degree substrate. C) Finally the image stack is rotated 52 degrees to represent the sample on the flat substrate having been cut at an angle.</p
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