WO2010149809A1 - Device and method for the three-dimensional culture of adherent cells - Google Patents

Device and method for the three-dimensional culture of adherent cells Download PDF

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Publication number
WO2010149809A1
WO2010149809A1 PCT/ES2010/000285 ES2010000285W WO2010149809A1 WO 2010149809 A1 WO2010149809 A1 WO 2010149809A1 ES 2010000285 W ES2010000285 W ES 2010000285W WO 2010149809 A1 WO2010149809 A1 WO 2010149809A1
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cells
culture
supports
dimensional
adherent cells
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PCT/ES2010/000285
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Spanish (es)
French (fr)
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Simon Marcos
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Dro Biosystems, S.L.
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Publication of WO2010149809A1 publication Critical patent/WO2010149809A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/14—Scaffolds; Matrices
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/02—Membranes; Filters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/08—Chemical, biochemical or biological means, e.g. plasma jet, co-culture
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor

Definitions

  • the present invention relates to a device and method for three-dimensional culture of adherent cells based on the use of filamentous supports immersed in a culture medium.
  • Cell culture is widely used in the biotechnology industry as a method to obtain different biological products produced by cells and also as a method to produce cells for different applications, such as cell therapy. Cell culture is also used in research as a very convenient method to study cell interactions or the effect of different substances on cell metabolism.
  • cells can be cultured in suspension or adhered to a surface, as is the case with anchor-dependent cells.
  • Anchor-dependent cells are traditionally grown as a single layer of cells on the inner surface of containers with different shapes. These surfaces are immersed in an appropriate culture medium, such as the case of rolling bottles or "T" type culture jars, where the culture medium is introduced inside the container, so that it bathes the walls where cell culture is performed. These types of cultures are used, among other things, to carry out efficacy studies of various substances on the development of certain cellular phenomena such as angiogenesis or metastasis. However, the fact that growth in monolayer does not reproduce the conditions in which cells are found in nature limits the application and interpretation of the results obtained in said studies. Other alternatives employ particulate supports with macroporous structure for cell culture. The cells grow on the outer surface of the particles and inside the internal cavities, or pores, of the support particles, which are kept in suspension by different agitation modes.
  • Spheroids prepared in an artisanal way and in which many cells grow together in suspension, adhered to each other, up to the limit imposed by the gravitational force acting on the growing spheroid that remains in suspension .
  • Spheroids are of particular interest in the study of three-dimensional cell cultures, since spheroids simulate with greater precision than surface cultures the natural conditions in which cells are found in living organisms, where rather than being structured in monolayers, The cells form three-dimensional structures and tissues.
  • the present invention describes a device and a method for three-dimensional culture of adherent cells by means of the application of a culture medium to the device, previously inoculated the culture medium with adherent cells.
  • the device comprises one or more static filamentous supports arranged so that the development of three-dimensional cultures is favored.
  • the filamentous supports are arranged parallel to the interior of the device for three-dimensional cell culture and fixed to it by its ends.
  • the filamentous supports can also be arranged in support structures that can be stackable and that are fixed to the three-dimensional cell culture device forming one or more layers of filamentous supports.
  • the filamentous supports may have undergone a surface modification treatment that affects the way in which the cells adhere to them or the way in which the cells behave once adhered.
  • the device for three-dimensional cell culture can adopt different geometries, being able to be cylindrical or prism-shaped among others.
  • the method for three-dimensional cell culture of the present invention is applicable both to the production of cells or derivatives of these cells, and to the generation of three-dimensional cultures for the study of different structural, metabolic or interaction aspects between different cell types.
  • the method begins with the filling of the device of the present invention with a suspension of adherent cells in culture medium. After D
  • the culture medium is removed and replaced by fresh medium.
  • the replacement of the culture medium can be carried out continuously by means of a filtering system.
  • the observation of the evolution of the crop can be carried out visually when the device has transparent walls or by means of the extraction of the filamentous supports when its arrangement thus allows it.
  • the cells can be recovered for other purposes by chemical treatment, by applying enzymes or other chemical agents, or by physical treatments, which alter the properties of the adhered cells and detach them from the filamentous supports. The cells are then recovered as a cell suspension through the collection port.
  • Spheroid aggregate of cells that form a homogeneous culture with spheroidal shape and diameter less than 500 micrometers. Typically a spheroid can contain a maximum of 25,000 cells.
  • Three-dimensional culture aggregate of cells that multiply in several dimensions forming layers of cells. Unlike the two-dimensional structure of monolayer cell cultures, in which the cells only interact with other cells through their side walls, three-dimensional cultures form structures in which most of the cells are in contact with other cells throughout their surface.
  • Adherent cell cell whose growth occurs adhered to a substrate or support.
  • Micro thread filament-shaped element whose cross section has a maximum distance between any two points contained in said section of less than 1000 micrometers.
  • Support surface to which the adherent cells adhere.
  • Support structure frame to which the ends of the filamentous supports are fixed.
  • the device for three-dimensional cell culture of the present invention consists of one or more static filamentous supports, preferably micro-wires, which are subject to the device that contains them.
  • the microwires are immobilized at both ends to the walls of the device, the device containing the culture medium that feeds the cells during their growth.
  • the arrangement of the micro wires inside the device for three-dimensional culture is such that contiguous micro wires are arranged in parallel, allowing the development of several layers of cells growing on each other without touching the cells that grow on adjacent micro wires.
  • the distance between adjacent microwires is equal to or greater than 100 micrometers, or even greater than 300 micrometers.
  • the ends of one or more micro-threads are fixed to a preferably stackable support structure that is introduced into the three-dimensional culture device and which is detachably fixed to the three-dimensional culture device forming at least one layer of micro threads.
  • the support structure It is a stackable frame.
  • the device may contain one or more stacked support structures, with one or more micro wires attached to each support structure forming one or more layers of micro wires.
  • the micro wires contained in the support structures may or may not have cells on their surface.
  • the support structures to which one or more micro-wires are fixed, are separated from each other by one or more spacer elements that maintain the desired distance between the successive support structures with one or more micro-wires.
  • the adjacent support structures are arranged in parallel.
  • the same three-dimensional cell culture device may have several support structures with one or more micro-wires to which cells adhere, the cells of one supporting structure being of a different type than those of another supporting structure.
  • the micro wires contained in the cell culture device may have undergone a surface modification treatment that affects the way in which the cells adhere to the micro wires or the way the cells behave once adhered.
  • the device can adopt different geometries, a cylindrical device arranged with the circular bottom parallel to the ground being preferable.
  • the microwires are arranged joined at their ends to the walls of the container in which the culture is carried out.
  • each support structure is arranged perpendicular to the axis of the hollow cylinder containing the culture medium.
  • the device for three-dimensional cell culture has a rectangular prism shape in which one or more micro wires are fixed at their ends to the walls of the container in which the culture is carried out.
  • each support structure is arranged horizontally and parallel to the ground.
  • the preferred size of the devices for cell culture in any of its geometries is 100 cm in its largest dimension, although preferably the maximum size is 50 cm or even better, 2 or even 1 cm in its largest dimension.
  • the device for three-dimensional cell culture has lids both in the lower part and in the upper part so that, together with the walls, it forms a closed container, impermeable to both liquids and gases.
  • the device for three-dimensional cell culture can have membranes of a filter that communicates the inside and outside of the container to allow the gas exchange in a sterile manner. Additionally, the device may contain filters for the input and output of culture medium and an inoculation port in the upper part and a collection port in the lower part.
  • the method for three-dimensional cell culture of the present invention begins after removing the lid of the device, with filling with a suspension of adherent cells in culture medium and restitution of the lid to close the device.
  • the cells can be introduced into the device for three-dimensional cell culture through an inoculation port disposed in the upper part thereof.
  • the device for three-dimensional cell culture remains immobile to ensure that the cells settle on the surface of the filamentous supports and adhere starting to multiply.
  • the used culture medium is removed and replaced by fresh medium.
  • the culture medium can be continuously replaced at the desired rate through a filtering system that allows the introduction of culture medium into the system while the used culture medium is removed continuously.
  • the introduction of the culture medium through the filtration system allows to reduce the turbulence generated by the replacement of the medium.
  • the evolution of the three-dimensional culture can be visually analyzed by direct observation of the growing cells on the filament supports in the case where the device for three-dimensional culture has transparent walls. In case the filamentous supports are arranged in support structures, they can be extracted to proceed to analyze the three-dimensional culture in another environment.
  • behaviors in the cells in culture are induced by the addition of chemical agents.
  • chemical agents can be introduced through the inoculation port or through the upper part of the device after removing the lid.
  • These behaviors can be, in an indicative but non-limiting manner, the induction of cell differentiation to form tissues or organs from the cells in culture, the induction or inhibition of tumors, the induction or inhibition of angiogenesis.
  • the cells can be recovered for other purposes by chemical treatment, by applying enzymes or other chemical agents, or by physical treatments, which alter the properties of the adhered cells and detach them from the filamentous supports. Then, the cells are recovered as a cell suspension through the collection port or by means of suction, of the cell suspension through the upper part of the device once the lid is removed.
  • a 30 micrometer diameter thread formed by a metal core surrounded by a vitreous roof was wrapped around a square stainless steel frame 60 mm side and 2 mm thick, leaving a space of 600 micrometers between each of the 100 turns of micro thread.
  • the free ends of the micro thread were attached to the metal frame using a cyanoacrylate glue.
  • Each frame was placed on four 2 mm high spacers inside a plastic container, with a lid, circular, 8.5 cm in diameter with a lid.
  • another frame of the same characteristics was placed separated from the previous one by four spacers placed in the corners.
  • this second frame another frame was placed separate from the previous one in the same way.
  • the set of the three frames stacked inside the container is an example of a device for the realization of three-dimensional cell cultures. - Development of a three-dimensional cell culture
  • a suspension of CHO-K1 cells at a concentration of 66,000 cells per milliliter was poured into a device for carrying out three-dimensional cell cultures that had previously been sterilized by ethanol immersion. Then the device is introduced inside an incubator at 37 0 C, 5% carbon dioxide and 95% humidity. Every 48 hours, the culture medium was replaced with fresh medium. After one week, the cultures were placed under a microscope and it was observed that the cells had grown in successive layers around the microwires to generate three-dimensional cultures of cylindrical shape and an approximate diameter of 500 micrometers. - Combination of different cell types.
  • A- Transfection of a three-dimensional culture A solution of plasmid pCMV / EGFP, which expresses the green fluorescent protein, mixed with lipofectamine, was introduced into a device for the realization of three-dimensional cell cultures containing a three-dimensional culture of CHO-K1 cells. After 24 hours, the fluorescent signal emitted by the green fluorescent protein expressed inside the cells that had been transfected was observed.
  • micrometer diameter micro wire formed by a metal core surrounded by a vitreous roof was wrapped around 35 mm diameter and 1 mm thick plastic cylindrical frames, leaving a space of 600 micrometers between each of the 100 turns of micro thread .
  • the free ends of the micro thread were glued to the respective circular frames using a cyanoacrylate-based glue.
  • Five frames so prepared were placed one above the other separated by a washer 35 mm diameter and 0.1 mm thick plastic inside a well of a multiwell plate that had 6 wells.

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Abstract

The invention relates to a device and method for the three-dimensional culture of adherent cells on filamentary substrates submerged in a culture medium.

Description

DISPOSITIVO Y MÉTODO PARA CULTIVO TRIDIMENSIONAL DE CÉLULAS DEVICE AND METHOD FOR THREE-DIMENSIONAL CELL CULTURE
ADHERENTESADHERENTS
CAMPO TÉCNICO DE LA INVENCIÓNTECHNICAL FIELD OF THE INVENTION
La presente invención se refiere a un dispositivo y método para cultivo tridimensional de células adherentes basado en el uso de soportes filamentosos inmersos en un medio de cultivo.The present invention relates to a device and method for three-dimensional culture of adherent cells based on the use of filamentous supports immersed in a culture medium.
ESTADO DE LA TÉCNICASTATE OF THE TECHNIQUE
El cultivo celular es ampliamente empleado en Ia industria biotecnológica como un método para obtener diferentes productos biológicos producidos por las células y también como un método para producir células para diferentes aplicaciones, como puede ser Ia terapia celular. El cultivo celular también es empleado en investigación como un método muy conveniente para estudiar interacciones celulares o el efecto de diferentes sustancias en el metabolismo celular.Cell culture is widely used in the biotechnology industry as a method to obtain different biological products produced by cells and also as a method to produce cells for different applications, such as cell therapy. Cell culture is also used in research as a very convenient method to study cell interactions or the effect of different substances on cell metabolism.
En los métodos de cultivo celular, las células pueden ser cultivadas en suspensión o adheridas a una superficie, como es el caso de las células dependientes de anclaje.In cell culture methods, cells can be cultured in suspension or adhered to a surface, as is the case with anchor-dependent cells.
Las células dependientes de anclaje se cultivan tradicionalmente como una única capa de células en Ia superficie interna de recipientes con diferentes formas. Estas superficies se sumergen en un medio de cultivo apropiado, como puede ser el caso de las botellas rodantes o los frascos de cultivo tipo "T", donde el medio de cultivo se introduce en el interior del recipiente, de manera que baña las paredes donde se realiza el cultivo celular. Este tipo de cultivos son empleados, entre otras cosas, para realizar estudios de eficacia de diversas sustancias sobre el desarrollo de determinados fenómenos celulares como pueden ser Ia angiogénesis o Ia metástasis. Sin embargo, el hecho de que el crecimiento en monocapa no reproduzca las condiciones en las que las células se encuentran en Ia naturaleza, limita Ia aplicación e interpretación de los resultados obtenidos en dichos estudios. Otras alternativas emplean soportes particulados con estructura macroporosa para el cultivo celular. Las células crecen en Ia superficie externa de las partículas y dentro de las cavidades internas, o poros, de las partículas del soporte, las cuales se mantienen en suspensión mediante diferentes modos de agitación.Anchor-dependent cells are traditionally grown as a single layer of cells on the inner surface of containers with different shapes. These surfaces are immersed in an appropriate culture medium, such as the case of rolling bottles or "T" type culture jars, where the culture medium is introduced inside the container, so that it bathes the walls where cell culture is performed. These types of cultures are used, among other things, to carry out efficacy studies of various substances on the development of certain cellular phenomena such as angiogenesis or metastasis. However, the fact that growth in monolayer does not reproduce the conditions in which cells are found in nature limits the application and interpretation of the results obtained in said studies. Other alternatives employ particulate supports with macroporous structure for cell culture. The cells grow on the outer surface of the particles and inside the internal cavities, or pores, of the support particles, which are kept in suspension by different agitation modes.
También es conocido Ia preparación de los llamados esferoides, preparados de manera artesanal y en los que muchas células crecen juntas en suspensión, adheridas unas a otras, hasta el límite impuesto por Ia fuerza gravitacional que actúa sobre el esferoide en crecimiento que se mantiene en suspensión. Los esferoides son de particular interés en el estudio de cultivos celulares tridimensionales, ya que los esferoides simulan con mayor precisión que los cultivos superficiales las condiciones naturales en las que las células se encuentran en los organismos vivos, donde más que estructurarse en forma de monocapas, las células forman estructuras tridimensionales y tejidos.It is also known the preparation of the so-called spheroids, prepared in an artisanal way and in which many cells grow together in suspension, adhered to each other, up to the limit imposed by the gravitational force acting on the growing spheroid that remains in suspension . Spheroids are of particular interest in the study of three-dimensional cell cultures, since spheroids simulate with greater precision than surface cultures the natural conditions in which cells are found in living organisms, where rather than being structured in monolayers, The cells form three-dimensional structures and tissues.
En Ia solicitud de patente PCT/EP2006/064483, se describe el cultivo de células en Ia superficie de microhilos en forma de monocapas. En Ia descripción de Ia invención, se describen distintas distribuciones de microhilos adecuadas para llevar a cabo el objetivo de cultivar las células en monocapas en Ia superficie de los hilos.In the patent application PCT / EP2006 / 064483, the culture of cells on the surface of micro-threads in the form of monolayers is described. In the description of the invention, different distributions of microwires suitable for carrying out the objective of culturing the cells in monolayers on the surface of the threads are described.
De especial interés es el desarrollo de cultivos tridimensionales de células adherentes en el caso de Ia producción de células terapéuticas, tanto somáticas como troncales, ya que un factor clave para el éxito de las terapias desarrolladas a partir de Ia implantación de células, es disponer de métodos de producción con suficiente productividad y que no afecten al grado de diferenciación de las células cultivadas. En estas aplicaciones, es necesario disponer de métodos de producción de células donde se consiga una elevada productividad, superior a cientos de miles de células por lote, y con elevada homogeneidad.Of special interest is the development of three-dimensional cultures of adherent cells in the case of the production of therapeutic cells, both somatic and trunk, since a key factor for the success of the therapies developed from the implantation of cells, is to have production methods with sufficient productivity and that do not affect the degree of differentiation of cultured cells. In these applications, it is necessary to have cell production methods where high productivity is achieved, greater than hundreds of thousands of cells per batch, and with high homogeneity.
Los métodos o dispositivos existentes aplicados al cultivo tridimensional de células adherentes adolecen de los siguientes problemas:The existing methods or devices applied to the three-dimensional culture of adherent cells suffer from the following problems:
- En el caso de los recipientes sobre cuya superficie interior crecen las células, estas se ven forzadas a crecer en dos dimensiones hasta que toda Ia superficie disponible para cultivo ha sido saturada por células, momento en el cual podrían iniciar el crecimiento tridimensional. Sin embargo, las células que están en contacto con Ia superficie, una vez que son cubiertas por sucesivas capas celulares, ven limitado su acceso a los nutrientes provistos por el medio de cultivo, por Io que crecen en un entorno drásticamente diferente al de las células de las capas superiores del cultivo.- In the case of containers on whose inner surface the cells, these are forced to grow in two dimensions until all the surface available for culture has been saturated by cells, at which time they could start the three-dimensional growth. However, the cells that are in contact with the surface, once they are covered by successive cell layers, are limited access to the nutrients provided by the culture medium, so they grow in a drastically different environment than the cells of the upper layers of the crop.
- En el caso de los soportes particulados macroporosos que se mantienen en suspensión mediante agitación, las células que crecen en Ia superficie de las partículas están sometidas a un choque continuo contra el resto de partículas en suspensión, por Io que son dañadas por las fuerzas de fricción. Mientras que las células que crecen en el interior de los poros, se encuentran sometidas a diferentes micro-ambientes creados en el interior de dichos poros, Io cual implica unas condiciones heterogéneas de cultivo. Además, una vez concluido el cultivo, Ia recuperación de las células está dificultada por el impedimento estérico, ya que incluso si las células son despegadas de Ia superficie interna de los poros, Ia mayoría de ellas queda atrapada en el interior de las partículas y no puede ser recuperada.- In the case of macroporous particulate supports that are kept in suspension by agitation, the cells that grow on the surface of the particles are subjected to a continuous shock against the rest of the suspended particles, due to which they are damaged by the forces of friction. While the cells that grow inside the pores, are subject to different micro-environments created inside these pores, which implies heterogeneous culture conditions. In addition, once the culture is finished, the recovery of the cells is hindered by steric hindrance, since even if the cells are detached from the internal surface of the pores, most of them are trapped inside the particles and not It can be recovered.
- En el caso de los esferoides, aunque tanto el entorno de cultivo como su similitud con un cultivo tridimensional perfecto son adecuadas, el método de preparación es extremadamente improductivo, y los tamaños de los cultivos obtenidos están limitados a pocos cientos o miles de células. Este tamaño máximo tan reducido de los cultivos tridimensionales que se pueden alcanzar con los esferoides es debido a los límites impuestos por Ia fuerza gravitacional que hace caer a los esferoides que crecen en suspensión- In the case of spheroids, although both the culture environment and its similarity with a perfect three-dimensional culture are adequate, the method of preparation is extremely unproductive, and the sizes of the cultures obtained are limited to a few hundred or thousands of cells. This very small maximum size of the three-dimensional cultures that can be reached with the spheroids is due to the limits imposed by the gravitational force that causes the spheroids that grow in suspension to fall
Para solucionar estos problemas, es necesario encontrar el modo de mantener indefinidamente en suspensión y en un ambiente homogéneo, un cultivo tridimensional, sin someterlo a fuerzas de fricción provocadas por choques entre las partículas o turbulencias provocadas por Ia agitación del medio de cultivo.In order to solve these problems, it is necessary to find a way to keep a suspension in a homogeneous environment indefinitely, three-dimensional culture, without subjecting it to friction forces caused by collisions between the particles or turbulence caused by the agitation of the culture medium.
DESCRIPCIÓN DE LA INVENCIÓNDESCRIPTION OF THE INVENTION
La presente invención describe un dispositivo y un método para cultivo tridimensional de células adherentes mediante Ia aplicación de un medio de cultivo al dispositivo, previamente inoculado el medio de cultivo con células adherentes.The present invention describes a device and a method for three-dimensional culture of adherent cells by means of the application of a culture medium to the device, previously inoculated the culture medium with adherent cells.
El dispositivo comprende uno o más soportes filamentosos estáticos dispuestos de modo que se favorece el desarrollo de cultivos tridimensionales.The device comprises one or more static filamentous supports arranged so that the development of three-dimensional cultures is favored.
Los soportes filamentosos se encuentran dispuestos de forma paralela en el interior del dispositivo para cultivo celular tridimensional y fijados a él por sus extremos. Los soportes filamentosos también pueden estar dispuestos en estructuras de soporte que pueden ser apilables y que se fijan al dispositivo de cultivo celular tridimensional formando una o más capas de soportes filamentososThe filamentous supports are arranged parallel to the interior of the device for three-dimensional cell culture and fixed to it by its ends. The filamentous supports can also be arranged in support structures that can be stackable and that are fixed to the three-dimensional cell culture device forming one or more layers of filamentous supports.
Los soportes filamentosos pueden haber sido sometidos a un tratamiento de modificación superficial que afecte al modo en que las células se adhieren a los mismos o al modo en que las células se comportan una vez adheridas.The filamentous supports may have undergone a surface modification treatment that affects the way in which the cells adhere to them or the way in which the cells behave once adhered.
El dispositivo para cultivo celular tridimensional puede adoptar diferentes geometrías, pudiendo ser de forma cilindrica o en forma de prisma entre otras.The device for three-dimensional cell culture can adopt different geometries, being able to be cylindrical or prism-shaped among others.
El método para cultivo celular tridimesional de Ia presente invención es de aplicación tanto a Ia producción de células o derivados de estas células, como a Ia generación de cultivos tridimensionales para el estudio de diferentes aspectos estructurales, metabólicos o de interacción entre diferentes tipos celulares.The method for three-dimensional cell culture of the present invention is applicable both to the production of cells or derivatives of these cells, and to the generation of three-dimensional cultures for the study of different structural, metabolic or interaction aspects between different cell types.
El método se inicia con el llenado del dispositivo de Ia presente invención con una suspensión de células adherentes en medio de cultivo. Tras DThe method begins with the filling of the device of the present invention with a suspension of adherent cells in culture medium. After D
mantener inmóvil el dispositivo y pasado el tiempo en el que las células se adhieren a Ia superficie de los soportes filamentosos, se retira el medio de cultivo y es reemplazado por medio fresco. El reemplazo del medio de cultivo puede ser realizado de forma continua mediante un sistema de filtrado.keep the device immobile and after the time in which the cells adhere to the surface of the filamentous supports, the culture medium is removed and replaced by fresh medium. The replacement of the culture medium can be carried out continuously by means of a filtering system.
La observación de Ia evolución del cultivo puede ser realizada de forma visual cuando el dispositivo dispone de paredes transparentes o mediante Ia extracción de los soportes filamentosos cuando su disposición así Io permita.The observation of the evolution of the crop can be carried out visually when the device has transparent walls or by means of the extraction of the filamentous supports when its arrangement thus allows it.
Por último y una vez que el cultivo tridimensional ha alcanzado el tamaño deseado, se inducen comportamientos en las células mediante Ia adición de agentes químicos. En caso necesario, las células pueden recuperarse para otros fines mediante tratamiento químico, aplicando enzimas u otros agentes químicos, o mediante tratamientos físicos, que alteren las propiedades de las células adheridas y las desprendan de los soportes filamentosos. A continuación, las células se recuperan como una suspensión celular a través del puerto de recolección.Finally and once the three-dimensional culture has reached the desired size, behaviors in the cells are induced by the addition of chemical agents. If necessary, the cells can be recovered for other purposes by chemical treatment, by applying enzymes or other chemical agents, or by physical treatments, which alter the properties of the adhered cells and detach them from the filamentous supports. The cells are then recovered as a cell suspension through the collection port.
El desarrollo de cultivos tridimensionales de células adherentes sobre soportes filamentosos estáticos permite imitar con más precisión que con otros sistemas existentes el entorno de crecimiento de las células y los tejidos en Ia naturaleza, por Io que es una herramienta más útil que los cultivos bidimensionales para i) analizar Ia interacción entre células del mismo o diferente tipo; ii) estudiar Ia evolución de enfermedades; iii) producir células o; iv) producir modelos experimentales de órganos y organismos.The development of three-dimensional cultures of adherent cells on static filamentous supports makes it possible to imitate more precisely than with other existing systems the environment of growth of cells and tissues in nature, which is why it is a more useful tool than two-dimensional cultures for ) analyze the interaction between cells of the same or different type; ii) study the evolution of diseases; iii) produce cells or; iv) produce experimental models of organs and organisms.
En el contexto de esta memoria serán de aplicación las siguientes definiciones:In the context of this report the following definitions will apply:
Esferoide: agregado de células que forman un cultivo homogéneo con forma esferoidal y diámetro inferior a 500 micrómetros. Típicamente un esferoide puede contener un máximo de 25.000 células.Spheroid: aggregate of cells that form a homogeneous culture with spheroidal shape and diameter less than 500 micrometers. Typically a spheroid can contain a maximum of 25,000 cells.
Cultivo tridimensional: agregado de células que se multiplican en varias dimensiones formando capas de células. A diferencia de Ia estructura bidimensional de los cultivos de células en monocapa, en los que las células únicamente interaccionan con otras células a través de sus paredes laterales, los cultivos tridimensionales forman estructuras en las que Ia mayor parte de las células está en contacto con otras células en toda su superficie.Three-dimensional culture: aggregate of cells that multiply in several dimensions forming layers of cells. Unlike the two-dimensional structure of monolayer cell cultures, in which the cells only interact with other cells through their side walls, three-dimensional cultures form structures in which most of the cells are in contact with other cells throughout their surface.
Célula adherente: célula cuyo crecimiento se produce adherida a un sustrato o soporte.Adherent cell: cell whose growth occurs adhered to a substrate or support.
Microhilo: elemento con forma filamentosa y cuya sección transversal presenta una distancia máxima entre dos puntos cualesquiera contenidos en dicha sección inferior a 1000 micrómetros.Micro thread: filament-shaped element whose cross section has a maximum distance between any two points contained in said section of less than 1000 micrometers.
Soporte: superficie a Ia que se adhieren las células adherentes.Support: surface to which the adherent cells adhere.
Estructura de soporte: armazón al que se fijan los extremos de los soportes filamentosos.Support structure: frame to which the ends of the filamentous supports are fixed.
Descripción detallada de Ia invenciónDetailed description of the invention
El dispositivo para cultivo celular tridimensional de Ia presente invención consiste en uno o más soportes filamentosos estáticos, preferiblemente microhilos, que se encuentran sujetos al dispositivo que los contiene. Preferiblemente los microhilos se encuentran inmovilizados por ambos extremos a las paredes del dispositivo, conteniendo el dispositivo el medio de cultivo que alimenta a las células durante su crecimiento.The device for three-dimensional cell culture of the present invention consists of one or more static filamentous supports, preferably micro-wires, which are subject to the device that contains them. Preferably the microwires are immobilized at both ends to the walls of the device, the device containing the culture medium that feeds the cells during their growth.
La disposición de los microhilos en el interior del dispositivo para cultivo tridimensional, es tal que microhilos contiguos se encuentran dispuestos de forma paralela, permitiendo el desarrollo de varias capas de células creciendo unas sobre otras sin tocar las células que crecen sobre microhilos contiguos. De forma preferible Ia distancia entre microhilos contiguos es igual o mayor a 100 micrómetros, o incluso mayor a 300 micrómetros.The arrangement of the micro wires inside the device for three-dimensional culture is such that contiguous micro wires are arranged in parallel, allowing the development of several layers of cells growing on each other without touching the cells that grow on adjacent micro wires. Preferably, the distance between adjacent microwires is equal to or greater than 100 micrometers, or even greater than 300 micrometers.
En un modo de realización preferente, los extremos de uno o más microhilos se encuentran fijados a una estructura de soporte preferiblemente apilable que se introduce en el dispositivo de cultivo tridimensional y que se fija de forma desmontable al dispositivo de cultivo tridimensional formando al menos una capa de microhilos. De forma preferente, Ia estructura de soporte es un marco apilable.In a preferred embodiment, the ends of one or more micro-threads are fixed to a preferably stackable support structure that is introduced into the three-dimensional culture device and which is detachably fixed to the three-dimensional culture device forming at least one layer of micro threads. Preferably, the support structure It is a stackable frame.
En otro modo de realización preferente, el dispositivo puede contener una o más estructuras de soporte apiladas, con uno o más microhilos fijados a cada estructura de soporte formando una o más capas de microhilos. Los microhilos contenidos en las estructuras de soporte pueden disponer o no de células en su superficie. Las estructuras de soporte a las que se fijan uno o más microhilos, se separan entre sí mediante uno o más elementos espaciadores que mantienen Ia distancia deseada entre las sucesivas estructuras de soporte con uno o más microhilos. De forma preferente las estructuras de soporte contiguas se disponen de forma paralela. De forma opcional, un mismo dispositivo de cultivo celular tridimensional puede disponer de varias estructuras de soporte con uno o más microhilos a los que se adhieren células siendo las células de una estrucutura de soporte de un tipo distinto a las de otra estrucutura de soporte.In another preferred embodiment, the device may contain one or more stacked support structures, with one or more micro wires attached to each support structure forming one or more layers of micro wires. The micro wires contained in the support structures may or may not have cells on their surface. The support structures to which one or more micro-wires are fixed, are separated from each other by one or more spacer elements that maintain the desired distance between the successive support structures with one or more micro-wires. Preferably, the adjacent support structures are arranged in parallel. Optionally, the same three-dimensional cell culture device may have several support structures with one or more micro-wires to which cells adhere, the cells of one supporting structure being of a different type than those of another supporting structure.
Los microhilos contenidos en el dispositivo para el cultivo celular pueden haber sido sometidos a un tratamiento de modificación superficial que afecte al modo en que las células se adhieren a los microhilos o al modo en que las células se comportan una vez adheridas.The micro wires contained in the cell culture device may have undergone a surface modification treatment that affects the way in which the cells adhere to the micro wires or the way the cells behave once adhered.
El dispositivo puede adoptar diferentes geometrías, siendo preferible un dispositivo de forma cilindrica dispuesto con el fondo circular paralelo al suelo.The device can adopt different geometries, a cylindrical device arranged with the circular bottom parallel to the ground being preferable.
En un dispositivo para cultivo celular tridimensional con geometría circular, los microhilos se disponen unidos por sus extremos a las paredes del recipiente en el que se realiza el cultivo. En el caso de que uno o más microhilos se dispongan en una o más capas mediante el apilamiento de estructuras de soporte, se dispone cada estructura de soporte de manera perpendicular al eje del cilindro hueco que contiene al medio de cultivo. En otro modo de realización, el dispositivo para cultivo celular tridimensional posee forma de prisma rectangular en el que uno o más microhilos se fijan por sus extremos a las paredes del recipiente en el que se realiza el cultivo. En el caso de que uno o más microhilos se dispongan en una o más capas mediante el apilamiento de estructuras de soporte, se dispone cada estructura de soporte de forma horizontal y paralela al suelo. OIn a device for three-dimensional cell culture with circular geometry, the microwires are arranged joined at their ends to the walls of the container in which the culture is carried out. In the event that one or more micro-threads are arranged in one or more layers by stacking support structures, each support structure is arranged perpendicular to the axis of the hollow cylinder containing the culture medium. In another embodiment, the device for three-dimensional cell culture has a rectangular prism shape in which one or more micro wires are fixed at their ends to the walls of the container in which the culture is carried out. In the event that one or more microwires are arranged in one or more layers by stacking support structures, each support structure is arranged horizontally and parallel to the ground. OR
El tamaño preferido de los dispositivos para cultivo celular en cualquiera de sus geometrías es de 100 cm en su mayor dimensión, aunque preferiblemente el tamaño máximo es de 50 cm o incluso mejor, de 2 o incluso 1 cm en su mayor dimensión.The preferred size of the devices for cell culture in any of its geometries is 100 cm in its largest dimension, although preferably the maximum size is 50 cm or even better, 2 or even 1 cm in its largest dimension.
El dispositivo para cultivo celular tridimensional dispone de tapas tanto en Ia parte inferior como en Ia superior de manera que, junto con las paredes, forma un recipiente cerrado, impermeable tanto a líquidos como a gases. El dispositivo para cultivo celular tridimensional puede disponer de membranas de un filtro que comunica el interior y el exterior del recipiente para permitir el intercambio gaseoso de manera estéril. Adicionalmente, el dispositivo puede contener filtros para Ia entrada y salida de medio de cultivo y un puerto de inoculación en Ia parte superior y otro de recolección en Ia parte inferior.The device for three-dimensional cell culture has lids both in the lower part and in the upper part so that, together with the walls, it forms a closed container, impermeable to both liquids and gases. The device for three-dimensional cell culture can have membranes of a filter that communicates the inside and outside of the container to allow the gas exchange in a sterile manner. Additionally, the device may contain filters for the input and output of culture medium and an inoculation port in the upper part and a collection port in the lower part.
El método para cultivo celular tridimensional de Ia presente invención se inicia tras retirar Ia tapa del dispositivo, con el llenado con una suspensión de células adherentes en medio de cultivo y Ia restitución de Ia tapa para cerrar el dispositivo.The method for three-dimensional cell culture of the present invention begins after removing the lid of the device, with filling with a suspension of adherent cells in culture medium and restitution of the lid to close the device.
Alternativamente, las células pueden ser introducidas en el dispositivo para cultivo celular tridimensional a través de un puerto de inoculación dispuesto en Ia parte superior del mismo.Alternatively, the cells can be introduced into the device for three-dimensional cell culture through an inoculation port disposed in the upper part thereof.
Posteriormente el dispositivo para cultivo celular tridimensional se mantiene inmóvil para garantizar que las células sedimenten sobre Ia superficie de los soportes filamentosos y se adhieran comenzando a multiplicarse.Subsequently, the device for three-dimensional cell culture remains immobile to ensure that the cells settle on the surface of the filamentous supports and adhere starting to multiply.
Transcurrido el tiempo necesario para que las células ubicadas sobre Ia superficie de los microhilos se adhieran a estos, se retira el medio de cultivo usado y es reemplazado por medio fresco. De forma opcional el medio de cultivo puede ser reemplazado continuamente al ritmo deseado a través de un sistema de filtrado que permite introducir medio de cultivo al interior del sistema mientras se retira el medio de cultivo usado de manera continua. La introducción del medio de cultivo a través del sistema de filtración permite reducir las turbulencias generadas por el recambio del medio. La evolución del cultivo tridimensional puede ser analizada visualmente mediante observación directa de las células en crecimiento sobre los soportes filamentos en el caso de que el dispositivo para cultivo tridimensional disponga de paredes transparentes. En caso de que los soportes filamentosos estén dispuestos en estructuras de soporte, éstos pueden ser extraídos para proceder a analizar el cultivo tridimensional en otro entorno.After the time necessary for the cells located on the surface of the microwires to adhere to them, the used culture medium is removed and replaced by fresh medium. Optionally, the culture medium can be continuously replaced at the desired rate through a filtering system that allows the introduction of culture medium into the system while the used culture medium is removed continuously. The introduction of the culture medium through the filtration system allows to reduce the turbulence generated by the replacement of the medium. The evolution of the three-dimensional culture can be visually analyzed by direct observation of the growing cells on the filament supports in the case where the device for three-dimensional culture has transparent walls. In case the filamentous supports are arranged in support structures, they can be extracted to proceed to analyze the three-dimensional culture in another environment.
Cuando el cultivo tridimensional ha alcanzado el tamaño deseado, se inducen comportamientos en las células en cultivo mediante Ia adición de agentes químicos. Estos agentes químicos pueden ser introducidos a través del puerto de inoculación o bien por Ia parte superior del dipositivo tras retirar Ia tapa. Estos comportamientos pueden ser, de manera orientativa aunque no limitativa, Ia inducción de Ia diferenciación celular para formar tejidos u órganos a partir de las células en cultivo, Ia inducción o inhibición de tumores, Ia inducción o inhibición de angiogénesis.When the three-dimensional culture has reached the desired size, behaviors in the cells in culture are induced by the addition of chemical agents. These chemical agents can be introduced through the inoculation port or through the upper part of the device after removing the lid. These behaviors can be, in an indicative but non-limiting manner, the induction of cell differentiation to form tissues or organs from the cells in culture, the induction or inhibition of tumors, the induction or inhibition of angiogenesis.
En caso necesario, las células pueden recuperarse para otros fines mediante tratamiento químico, aplicando enzimas u otros agentes químicos, o mediante tratamientos físicos, que alteren las propiedades de las células adheridas y las desprendan de los soportes filamentosos. A continuación, las células se recuperan como una suspensión celular a través del puerto de recolección o mediante Ia succión, de Ia suspensión celular por Ia parte superior del dispositivo una vez retirada Ia tapa.If necessary, the cells can be recovered for other purposes by chemical treatment, by applying enzymes or other chemical agents, or by physical treatments, which alter the properties of the adhered cells and detach them from the filamentous supports. Then, the cells are recovered as a cell suspension through the collection port or by means of suction, of the cell suspension through the upper part of the device once the lid is removed.
De forma explicativa y no limitativa de describen los siguientes ejemplos:Explanatory and non-limiting, describe the following examples:
EjemplosExamples
1- Preparación de un dispositivo para el desarrollo de cultivos celulares tridimensionales1- Preparation of a device for the development of three-dimensional cell cultures
Microhilo de 30 micrómetros de diámetro formado por un alma metálica rodeada de una cubierta vitrea fue enrollado alrededor de un marco cuadrado de acero inoxidable de 60 mm de lado y 2 mm de espesor, dejando un espacio de 600 micrómetros entre cada dos de las 100 vueltas de microhilo. A continuación, los extremos libres del microhilo se pegaron al marco metálico empleando un pegamento a base de cianocrilato. Cada marco se colocó sobre cuatro espaciadores de 2 mm de altura en el interior de un recipiente de plástico, con tapa, circular, de 8,5 cm de diámetro con tapa. Sobre el marco así dispuesto se colocó otro marco de iguales características separado del anterior por cuatro espaciadores colocados en las esquinas. Sobre este segundo marco se colocó otro marco separado del anterior del mismo modo. El conjunto de los tres marcos apilados en el interior del recipiente constituye un ejemplo de un dispositivo para Ia realización de cultivos celulares tridimensionales. - Desarrollo de un cultivo celular tridimensionalA 30 micrometer diameter thread formed by a metal core surrounded by a vitreous roof was wrapped around a square stainless steel frame 60 mm side and 2 mm thick, leaving a space of 600 micrometers between each of the 100 turns of micro thread. Next, the free ends of the micro thread were attached to the metal frame using a cyanoacrylate glue. Each frame was placed on four 2 mm high spacers inside a plastic container, with a lid, circular, 8.5 cm in diameter with a lid. On the frame thus arranged another frame of the same characteristics was placed separated from the previous one by four spacers placed in the corners. On this second frame another frame was placed separate from the previous one in the same way. The set of the three frames stacked inside the container is an example of a device for the realization of three-dimensional cell cultures. - Development of a three-dimensional cell culture
Una suspensión de células CHO-K1 a una concentración de 66.000 células por mililitro se vertió en el interior de un dispositivo para Ia realización de cultivos celulares tridimensionales que previamente había sido esterilizado mediante inmersión en etanol. A continuación, el dispositivo se introdujo en el interior de un incubador a 37 0C, 5 % de dióxido de carbono y 95 % de humedad. Cada 48 horas, el medio de cultivo se reemplazó con medio fresco. Transcurrida una semana, los cultivos se colocaron en un microscopio y se observó que las células habían crecido en capas sucesivas alrededor de los microhilos hasta generar cultivos tridimensionales de forma cilindrica y un diámetro aproximado de 500 micrómetros. - Combinación de distintos tipos celulares.A suspension of CHO-K1 cells at a concentration of 66,000 cells per milliliter was poured into a device for carrying out three-dimensional cell cultures that had previously been sterilized by ethanol immersion. Then the device is introduced inside an incubator at 37 0 C, 5% carbon dioxide and 95% humidity. Every 48 hours, the culture medium was replaced with fresh medium. After one week, the cultures were placed under a microscope and it was observed that the cells had grown in successive layers around the microwires to generate three-dimensional cultures of cylindrical shape and an approximate diameter of 500 micrometers. - Combination of different cell types.
Una suspensión de células CHO-K1 a una concentración deA suspension of CHO-K1 cells at a concentration of
66.000 células por mililitro se vertió en el interior de un dispositivo para Ia realización de cultivos celulares tridimensionales que previamente había sido esterilizado mediante inmersión en etanol. En otro dispositivo de las mismas características se vertió del mismo modo una suspensión celular de células HEK293 fluorescentes a una concentración de de 60.000 células por mililitro. A continuación, ambos dispositivos se introdujeron en el interior de un incubador a 37 0C, 5 % de dióxido de carbono y 95 % de humedad. Cada 48 horas, el medio de cultivo se reemplazó con medio fresco. Al cabo de dos días y empleando los marcos contenidos en los dispositivos en cultivo, se produjo un nuevo dispositivo alternando capas de marcos con células HEK293 fluorescentes y células CHO-K1 y se continuó el cultivo del mismo modo. Al cabo de una semana de cultivo se observó Ia interacción ocurrida entre las células CHO-K1 y las células HEK293 fluorescentes que interactuaban en el cultivo tridimensional.66,000 cells per milliliter was poured into a device for the realization of three-dimensional cell cultures that had previously been sterilized by immersion in ethanol. In another device of the same characteristics, a Cell suspension of fluorescent HEK293 cells at a concentration of 60,000 cells per milliliter. Then, both devices were introduced inside an incubator at 37 0 C, 5% carbon dioxide and 95% humidity. Every 48 hours, the culture medium was replaced with fresh medium. After two days and using the frames contained in the devices in culture, a new device was produced alternating layers of frames with fluorescent HEK293 cells and CHO-K1 cells and the culture was continued in the same way. After one week of culture, the interaction occurred between the CHO-K1 cells and the fluorescent HEK293 cells that interacted in the three-dimensional culture was observed.
A- Transfección de un cultivo tridimensional. Una solución del plásmido pCMV/EGFP, que expresa Ia proteína verde fluorescente, mezclada con lipofectamina, fue introducida en el interior de un dipositivo para Ia realización de cultivos celulares tridimensionales que contenía un cultivo tridimensional de células CHO-K1. Al cabo de 24 horas se observo Ia señal fluorescente emitida por Ia proteína verde fluorescente expresada en el interior de las células que habían sido transfectadas.A- Transfection of a three-dimensional culture. A solution of plasmid pCMV / EGFP, which expresses the green fluorescent protein, mixed with lipofectamine, was introduced into a device for the realization of three-dimensional cell cultures containing a three-dimensional culture of CHO-K1 cells. After 24 hours, the fluorescent signal emitted by the green fluorescent protein expressed inside the cells that had been transfected was observed.
5- Estructura de soporte circular en pocilios de placa multipocillos5- Circular support structure in multiwell plate wells
Microhilo de 30 micrómetros de diámetro formado por un alma metálica rodeada de una cubierta vitrea fue enrollado alrededor de marcos cilindricos de plástico de 35 mm de diámetro y 1 mm de espesor, dejando un espacio de 600 micrómetros entre cada dos de las 100 vueltas de microhilo. A continuación, los extremos libres del microhilo se pegaron a los respectivos marcos circulares empleando un pegamento a base de cianocrilato. Cinco marcos así preparados se colocaron uno sobre otro separados por una arandela de plástico de 35 mm de diámetro y 0,1 mm de espesor en el interior de un pocilio de una placa multipocillo que disponía de 6 pocilios. 30 micrometer diameter micro wire formed by a metal core surrounded by a vitreous roof was wrapped around 35 mm diameter and 1 mm thick plastic cylindrical frames, leaving a space of 600 micrometers between each of the 100 turns of micro thread . Next, the free ends of the micro thread were glued to the respective circular frames using a cyanoacrylate-based glue. Five frames so prepared were placed one above the other separated by a washer 35 mm diameter and 0.1 mm thick plastic inside a well of a multiwell plate that had 6 wells.

Claims

REIVINDICACIONES
1- Dispositivo para cultivo tridimensional de células adherentes que comprende uno o más soportes filamentosos, caracterizado porque Ia disposición de dichos soportes filamentosos contiguos es de forma paralela.1- Device for three-dimensional culture of adherent cells comprising one or more filamentous supports, characterized in that the arrangement of said adjacent filamentous supports is parallel.
2- Dispositivo según Ia reivindicación 1 caracterizado porque Ia distancia entre dichos soportes filamentosos contiguos es de al menos 50 micrómetros.2- Device according to claim 1 characterized in that the distance between said adjacent filamentary supports is at least 50 micrometers.
3- Dispositivo según Ia reivindicación 1 caracterizado porque dichos soportes filamentosos tienen un diámetro máximo de 1000 micrómetros.3- Device according to claim 1 characterized in that said filamentary supports have a maximum diameter of 1000 micrometers.
4- Dispositivo según cualquiera de las reivindicaciones anteriores caracterizado porque los extremos de dichos soportes filamentosos están inmovilizados por sus extremos a las paredes de dicho dispositivo.4- Device according to any of the preceding claims characterized in that the ends of said filamentary supports are immobilized at their ends to the walls of said device.
5- Dispositivo según cualquiera de las reivindicaciones anteriores caracterizado porque dichos soportes filamentos se disponen en al menos una estructura de soporte que se fija a dicho dispositivo de cultivo celular.5- Device according to any of the preceding claims characterized in that said filament supports are arranged in at least one support structure that is fixed to said cell culture device.
6- Dispositivo según reivindicación 5 caracterizado porque dichas estructuras de soporte son apilables.6- Device according to claim 5 characterized in that said support structures are stackable.
7- Dispositivo según reivindicación 6 caracterizado porque dichas estructuras de soporte se separan por al menos un elemento separador.7- Device according to claim 6 characterized in that said support structures are separated by at least one separator element.
8- Dispositivo según cualquiera de las reivindicaciones anteriores caracterizado porque comprende membranas filtrantes que permiten Ia entrada y salida de medio de cultivo estéril de manera continua.8- Device according to any of the preceding claims characterized in that it comprises filtering membranes that allow the input and output of sterile culture medium continuously.
9- Método para cultivo tridimensional de células adherentes caracterizado porque: a) se llena un dispositivo de cultivo celular según cualquiera de las reivindicaciones anteriores con una suspensión de células adherentes en medio de cultivo.9- Method for three-dimensional culture of adherent cells characterized in that: a) a cell culture device according to any of the preceding claims is filled with a suspension of adherent cells in culture medium.
c) se reemplaza el medio de cultivo agotado por las células por medio fresco.c) the depleted culture medium is replaced by the cells with fresh medium.
d) se analizan las células en crecimiento sobre los soportes filamentosos.d) the growing cells on the filamentous supports are analyzed.
e) se inducen comportamientos en las células en cultivo.e) behaviors are induced in cultured cells.
f) se recuperan las células del cultivo tridimensional.f) the cells of the three-dimensional culture are recovered.
g) se extrae Ia suspensión celular para su posterior uso en otras aplicaciones.g) the cell suspension is extracted for later use in other applications.
10- Método para cultivo tridimensional de células adherentes según reivindicación 9 caracterizado porque dicho análisis de las células en crecimiento sobre dichos soportes filamentosos se realiza de forma visual.10. Method for three-dimensional culture of adherent cells according to claim 9, characterized in that said analysis of the growing cells on said filamentous supports is performed visually.
11- Método para cultivo tridimensional de células adherentes según reivindicación 9 caracterizado porque dicho análisis se realiza mediante Ia extracción de dichas estructuras de soporte que contienen dichos soportes filamentosos.11. Method for three-dimensional culture of adherent cells according to claim 9, characterized in that said analysis is performed by means of the extraction of said support structures containing said filamentous supports.
12- Método para cultivo tridimensional de células adherentes según reivindicación 9 caracterizado porque dicha inducción de comportamientos se realiza mediante Ia adicción de agentes químicos.12- Method for three-dimensional culture of adherent cells according to claim 9, characterized in that said induction of behaviors is carried out by the addition of chemical agents.
13- Método para cultivo tridimensional de células adherentes según reivindicación 9 caracterizado porque dicha inducción de comportamientos se realiza mediante Ia combinación de estructuras de soporte con células de distinto tipo en un mismo dispositivo.13. Method for three-dimensional culture of adherent cells according to claim 9 characterized in that said induction of behaviors is performed by combining support structures with cells of different types in the same device.
14- Método para cultivo tridimensional de células adherentes según reivindicación 9 caracterizado porque dicha recuperación se realiza mediante tratamiento químico, enzimático o físico. 14. Method for three-dimensional culture of adherent cells according to claim 9 characterized in that said recovery is carried out by chemical, enzymatic or physical treatment.
PCT/ES2010/000285 2009-06-26 2010-06-24 Device and method for the three-dimensional culture of adherent cells WO2010149809A1 (en)

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