WO2014053678A1 - Device and method for encapsulating microfluidic systems - Google Patents

Device and method for encapsulating microfluidic systems Download PDF

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Publication number
WO2014053678A1
WO2014053678A1 PCT/ES2013/000219 ES2013000219W WO2014053678A1 WO 2014053678 A1 WO2014053678 A1 WO 2014053678A1 ES 2013000219 W ES2013000219 W ES 2013000219W WO 2014053678 A1 WO2014053678 A1 WO 2014053678A1
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WO
WIPO (PCT)
Prior art keywords
piece
chip
filling
external
fluidic
Prior art date
Application number
PCT/ES2013/000219
Other languages
Spanish (es)
French (fr)
Inventor
Luis José FERNÁNDEZ LEDESMA
Rosa María MONGE PRIETO
Guillermo Alejandro LLAMAZARES PRIETO
Jorge Santolaria Mazo
Ignacio OCHO GARRIDO
Aitor AGUILAR ALEJANDRE
Rebeca GUERRRERO GIMÉNEZ
Original Assignee
Universidad De Zaragoza
Centro De Investigación Biomédica En Red En Bioingeniería, Biomateriales Y Nanomedicina (Ciber-Bbn)
Moldes Y Plásticos Aragón, S.L.
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Application filed by Universidad De Zaragoza, Centro De Investigación Biomédica En Red En Bioingeniería, Biomateriales Y Nanomedicina (Ciber-Bbn), Moldes Y Plásticos Aragón, S.L. filed Critical Universidad De Zaragoza
Publication of WO2014053678A1 publication Critical patent/WO2014053678A1/en

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L9/00—Supporting devices; Holding devices
    • B01L9/52—Supports specially adapted for flat sample carriers, e.g. for plates, slides, chips
    • B01L9/527—Supports specially adapted for flat sample carriers, e.g. for plates, slides, chips for microfluidic devices, e.g. used for lab-on-a-chip
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502715—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
    • 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
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/16—Microfluidic devices; Capillary tubes
    • 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
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/48—Holding appliances; Racks; Supports
    • 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
    • C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/02—Adapting objects or devices to another
    • B01L2200/026—Fluid interfacing between devices or objects, e.g. connectors, inlet details
    • B01L2200/027—Fluid interfacing between devices or objects, e.g. connectors, inlet details for microfluidic devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0605—Metering of fluids
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0684—Venting, avoiding backpressure, avoid gas bubbles
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0689—Sealing
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0478—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure pistons

Definitions

  • the present invention refers to a device and a method for encapsulating microfluidic systems, preferably used in "laboratory chip” systems (also designated by its English term, "lab-on-chip”). More specifically, the invention proposed by the applicant relates to a device and method designed, preferably, for cell culture and / or for the circulation of fluids through cell cultures.
  • bubble traps are not useful in applications where it allows the flow once the microfluidic chips are mounted (as is the case in cell culture applications in microfluidic chips), since when the cells are inserted into the chip, the fluid inlets and outlets are open so, again, it is inevitable that new small bubbles form.
  • air bubbles cannot be removed by purging, since the cells need time to be adhered to the culture substrate before applying any liquid flow. Even waiting long enough for e! settlement of the cells, the bubbles will grow continuously if e! Chip is not integrated in a closed fluid circuit. Again, if e! chip is integrated into e! circuit, the small bubbles created during cell insertion will stop growing, but will remain at the entrances of that circuit. Finally, even if e!
  • the present invention is intended to meet said needs.
  • an object of the present invention relates to an encapsulation system of a microfiuidic system that is capable of providing a simple insertion of the particles (preferably, culture cells or nanoparticles functioning, for example gold nanoparticles) or any type of liquid that is dispensed directly into the chip, and which, in addition, completely prevents the appearance of air bubbles in said chip.
  • the term "microfiuidic” is interpreted as relative to fluids located in volumes of the order of microliter ( ⁇ ) or less, while the general term “fluidic” is interpreted as relative to fluids located in channels, chambers or chips of any volume.
  • first support piece configured as the base of the device, comprising a housing and support means for the cell culture chip, said chip being provided with, a! less, a cane! input and at least one liquid outlet channel;
  • a second filling piece comprising fluid connections to the channels of! cell culture chip; internal means of tight connection between said chip channels and fluidic connections; and two filling wells communicated upstream with said fluid connections;
  • a third encapsulation piece comprising microfluidic connectors to a fluidic or microfluidic circuit, and a purge chamber connectable to the filling wells of the second part, said chamber being also connected to a gas permeable filter, but impermeable to liquids, communicated abroad;
  • the device also includes:
  • - first position it comprises an external coupling and connection between the third encapsulation piece and the second filling part, by means of its external means of joining or coupling and its external means of sealing, but without there being an internal hermetic connection between both parts a through the internal means of hermetic connection between the microfluidic connectors of the third part and the fluidic connections of the second part;
  • - second position it comprises, in addition to the coupling and external connection between the third encapsulation piece and the second filling part of the first position, also its internal hermetic connection between the microfluidic connectors of the third piece and the fluidic connections of the second piece, through the internal means of hermetic connection.
  • the third encapsulation piece is connected to a fluidic or microfluidic circuit. In this way it is possible to obtain complete fluidic systems free of air bubbles in their channels and / or microchannels.
  • the external means of sealing between the second piece and the third part comprise one or more ring seals. In this way an effective means of connecting the parts without loss and / or air / liquid inlet in the system is obtained, by means of a single closing element.
  • the external attachment or coupling means of the first support piece, the second filling part and / or the third encapsulation part comprise clip / hole type closure systems.
  • the internal means of hermetic connection of the second filling piece or the third encapsulating part comprise o-rings. This achieves the isolation of the fluidic system at the junctions of its components, thus preventing the entry / exit of air and / or liquid.
  • the first support piece comprises a window configured for monitoring the cell culture chip by microscopy.
  • Another object of the present invention is a method of encapsulating a cell culture comprising the use of a device according to any of the preceding claims.
  • said method comprises the following steps:
  • a) a chip equipped with a plurality of inputs / outputs is housed on the first support piece of the device; b) the second filling piece is coupled to the first support piece, connecting said pieces through external means of joining or coupling said pieces and providing a tight connection between the input / output channels of the chip and the fluidic channels of the second piece, through internal means of hermetic connection arranged in said second piece;
  • a liquid filling means is used for filling the chip and its input / output channels, through the fluid connections of the second piece and both the chip and the input / output channels are purged of air, up to partially or completely saturate the filling wells of the second piece;
  • the third encapsulation piece is connected to a fluidic circuit in which the device is integrated, through the microfluidic connectors of the third piece.
  • the third encapsulation piece is connected to the second filling piece, through the application of the external sealing means and external coupling or joining means between the second piece and the third piece, and configuring said external means of hermetic closure and external coupling means in the first configuration position of the device, but without applying the internal means of hermetic connection between the second and third parts.
  • the internal hermetic connection between the third piece and the second piece is made, through the internal means of hermetic connection, in the second configuration position of the device , closing the complete fluidic circuit of said device and transferring the liquid that saturates the filling wells and, optionally, residual air in the circuit, to the purge chamber.
  • FIGURES Figure 1 shows an external perspective view of the encapsulation device of the invention, with its three parts coupled.
  • Figure 2 shows a perspective view of the first support piece of the device of the invention.
  • Figure 3 shows a perspective view of the second liquid filling part of the device of the invention.
  • Figure 4 shows a perspective view of the third encapsulation piece of the device of the invention.
  • Figure 5 shows a perspective view of the housing of a cell culture chip in the first support piece of the device.
  • Figures 6a-6b show, respectively, a side perspective view and a bottom perspective view of the external and internal connection of the second filling piece with the first support piece.
  • Figure 7 shows a top perspective view of the filling of the fluidic connections and the wells of the second piece, by means of a pipette.
  • Figure 8 shows a top perspective view of the saturation of the wells of the second piece, by filling with the pipette shown in Figure 7.
  • Figure 9 shows a perspective view of the connection of the third encapsulation piece to a fluidic or microfluidic circuit.
  • Figures 10a and 10b show, respectively, an external and internal representation (in lateral section) of the first configuration position of the device in its external coupling between the second and third pieces.
  • Figure 11 shows a side section of the device during air purging of the device, performed in its first position of external coupling configuration between the second and third pieces.
  • Figures 12a and 12b respectively show an external and internal representation of the second configuration position of the device in its external and internal coupling between the second and third parts, after purging air and encapsulating the device.
  • the device of the invention preferably comprises three attachable parts (Figure 1) that give rise to the encapsulation of the cell culture chip . These pieces comprise a first piece
  • the first piece (1) acts as the base of the device and comprises a means for receiving and supporting the chip (4), preferably in a configuration suitable for observing said chip by means of equipment of microscopy. More preferably, the first part (1) includes an open window (5), which allows the objective of the microscopy equipment used to be placed in close contact with the chip. Additionally, the first part (1) comprises external means for joining or coupling (6, 6 ') to the second part (2) of the device (in Figure 1, four joints by means of clip-like pressure).
  • the second filling part (2) ( Figure 3) is an intermediate piece intended for coupling between the first (1) and the third part (3). Said second piece (2) provides a means of isolation and filling of the chip, by means of fluidic connections (7, 7 ') with its input and output channels.
  • the second part comprises external joining or coupling means (8, 8 ') with the first part (1) of support (in the example of Figure 3, the coupling means (8, 8 ') are holes attachable to the clips of the first support piece (1)), as well as internal means of hermetic connection (9, 9' ) (not shown in Figure 3, for being in the inner region of the piece, see for example in Figure 6b), for example through O-rings, with the input and output channels of the chip (for the context of the In the present invention, the term “external” applies to those elements related to the structural components of the device not related to the fluid circuit, and the term “internal” refers to those structural elements of the device related to the fluid circuit).
  • the second piece (2) comprises filling wells (10, 10 ') communicated with the fluidic connections (7, 7 ') of the second piece (2), and located at a level upstream of said connections (7, 7').
  • Said filling wells (10, 10 ') allow the introduction of liquid into the chip, for example by means of a pipette, until both said chip and its inlet and outlet channels are filled, with saturation of the wells (10, 10 ') as a capacity limit of the fluid circuit between the first (1) and the second part (2).
  • the existence of the filling wells (10, 10 ') is of great importance, as will be seen later, to avoid any formation of bubbles during the use of the device.
  • the second part (2) comprises:
  • the coupling means (1 1, 1 1' ) are holes attachable to clips (elements (6, 6 ') in Figure 2) of the third piece (3)).
  • the third piece (3) ( Figure 4 of this document) constitutes the upper part of the device, which allows the hermetic encapsulation of the union of first (1) and second (2) pieces once e! filling of the chip, its input / output channels, fluidic connections (7, 7 ') and filling wells (10, 10').
  • said third part (3) also comprises external means of joining or coupling (12) with the second part (2) (preferably, by means of a system of clips / holes as in the joints of the first parts (1) and second (2)), and internal means of hermetic connection (13) (not shown in Figure 4 for being in the inner region of the piece, see for example in Figure 10b), for example by o-rings, to the fluidic connections (7, 7 ') of the second piece
  • the third encapsulation part (3) comprises standard microfluidic connectors (14) configured to allow a direct connection to the fluidic circuit in which the chip is integrated together with the first (1) and second (2) parts.
  • the third piece (3) also comprises a purging chamber (15) (not shown in Figure 4 as it is in the inner region of the piece, see for example in Figure 10b) connectable to the wells (10, 10 ' ) of the second filling part (2), and said chamber (15) equipped with a filter (16) to allow é! air exchange between the wells (10, 10 ') and the outside, which allows to avoid, as will be seen below, the contamination of the complete circuit.
  • the external means of connection or coupling (1 1, 11 ', 12) and the external means of sealing (11 ") of the second (2) and third pieces (3) comprise at least two configuration positions of said coupling or connection in the device:
  • the third part (3) is coupled to the second part (2) by means of the application of the external sealing means (11, 1 1 ') of the second part (2) and of the external means of coupling or joining (12) of the third piece (3), but without applying the internal means of hermetic connection (13) between both (preferably, the o-rings (13) of the third piece (3) are located at a distance between 1 and 3 mm of the fluidic connections (7, 7 ') connected to the wells (10, 10') of the second piece (2)).
  • the third part (3) is coupled to the second part (2) (preferably, the internal hermetic connection means (13) of the third part
  • the method of encapsulating microfluidic systems proposed in the present application is based on the use of a device according to the previously described embodiments, and preferably comprises one or more of the following steps: a) As shown in Figure 5, houses a chip (17) equipped with a plurality of inputs / outputs (18, 18 ') on the first support piece (1).
  • the second filling piece (2) is coupled to the first support piece (1), connecting the joining or coupling means (6, 6 ', 8, 8') of said parts ( Figure 6a) and providing an airtight connection between the input / output channels (18, 18 ') of the chip (17) (shown said channels and the chip in Figure 5) and the internal means of hermetic connection (9, 9 ') (for example, through O-rings) arranged at the bottom of the second piece (2) ( Figure 6b).
  • a liquid filling means (19) preferably a micropipette, is used for filling the chip (element (17) in Figure 5) and its input / output channels ( elements (18, 18 ') in Figure 5), through the fluidic connections (7, 7') of the second piece (2) ( Figure 7).
  • a liquid filling means (19) preferably a micropipette
  • both the chip (17) and the inlet / outlet channels (18, 18 ') are purged of air, until the fluidic circuit is completely filled with liquid, partially or totally saturating the wells of filling (10, 10 '). In this way, it is prevented that the air can access the chip (17) once it is filled with liquid.
  • any particle (for example, cells) or liquid can be inserted into the chip (17), for its location in any specific chamber thereof, by using a pipette (for example , the chip (17) could be fed by cells by inserting it through a dedicated input in it).
  • the third part (3) (encapsulation piece) is connected to the fluidic circuit (20) in which the device will be integrated.
  • the third encapsulation part (3) is connected to the second filling part (2), through the application of the external sealing means (1 1 ") of the second part (2) (preferably, a hermetic ring) and of the external means of coupling or joining (11, 1 1 ', 12) of the second piece (2) and of the third piece (3) (preferably by means of a plurality of clip / hole systems), and configuring said external sealing means (11 ") and external coupling means (1, 1 ', 12) in the" first position "of! device described above, but without applying the internal seal (13) between the second (2) and third (3) parts (see Figures 10a and 10b, where, respectively, in external and internal views, the "first position" is illustrated of referred configuration).
  • the filling wells (10, 10 ') defined in the second part (2) are only connected to the outside of the device through the filter (16) located at the top of the third part (3), thus avoiding any possible contamination of! chip (17) and filling wells (10, 10 ').
  • the fluidic connectors (14) of the third piece (3) are located at a certain distance (preferably, between 1 and 3 mm) from the fiuidic connections (7, 7 ') of the second piece (2), being furthermore preferably covered by the liquid from the filling wells (10, 10') of said second piece
  • the third piece (3) is not internally connected to the chip circuit (17) (it is only connected externally to the second piece (2), all bubbles are confined in the internal space defined in the purge chamber (15) between the second (2) and third (3) pieces, thus purging the entire device circuit
  • step (g) the chip (17) is connected to the rest of the circuit, avoiding bubbles and without the need to introduce, subsequently, flow added! of liquid through! chip (17).
  • step (g) Once closed e! Complete fluidic circuit, static or dynamic cell culture can be carried out without bubbles inside! device.

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Abstract

The invention relates to a device and a method for the encapsulation of microfluidic systems, preferably "laboratory-on-a-chip" systems. More concretely, the invention refers to a device and a method associated therewith, where the device comprises a first support part designed as the base of the device and used to house a cell culture chip, a second part for filling with liquid, and a third encapsulation part, said three parts being configurable into at least two coupling positions that allow, by means of the application thereof, the removal of air bubbles from the microfluidic system.

Description

DISPOSITIVO Y MÉTODO DE ENCAPSULADO DE SISTEMAS MICROFLUÍDICOS DEVICE AND METHOD OF ENCAPSULATED MICROFLUIDIC SYSTEMS
CAMPO DE LA INVENCIÓN La presente invención hace referencia a un dispositivo y a un método destinados al encapsulado de sistemas microfluídicos, preferentemente utilizados en sistemas "laboratorio en chip" (también designados por su término en inglés, "lab-on-chip"). Más concretamente, la invención propuesta por el solicitante se refiere a un dispositivo y a un método diseñados, preferentemente, para el cultivo de células y/o para la circulación de fluidos a través de cultivos celulares. FIELD OF THE INVENTION The present invention refers to a device and a method for encapsulating microfluidic systems, preferably used in "laboratory chip" systems (also designated by its English term, "lab-on-chip"). More specifically, the invention proposed by the applicant relates to a device and method designed, preferably, for cell culture and / or for the circulation of fluids through cell cultures.
ANTECEDENTES DE LA INVENCIÓN BACKGROUND OF THE INVENTION
Es conocido en el estado de la técnica correspondiente a los sistemas "lab-on-chip" que uno de los principales problemas, relacionados con el uso de dispositivos de microfluídica, es la aparición de burbujas de aire no deseadas en los circuitos de fluido. Típicamente, los chips, tubos, elementos de control de fluidos, depósitos, etc. de dichos sistemas necesitan ser conectados entre sí para crear un sistema fluídico y, tal y como muestra la experiencia en este campo técnico, es imposible evitar la aparición de burbujas en determinados regiones de los dispositivos, tales como los elementos conectores o las entradas y salidas de los chips. En algunas aplicaciones, sí es posible evitar la presencia de burbujas, mediante la alimentación/purgado de todo e! sistema una vez montado, con un volumen tota! de a! menos dos o tres veces el volumen muerto del circuito. Sin embargo, esta solución se puede aplicar sólo si e! sistema es operado en un circuito abierto, ya que para un sistema cerrado, al conectar los extremos de! circuito se producen nuevas burbujas de aire. It is known in the state of the art corresponding to "lab-on-chip" systems that one of the main problems, related to the use of microfluidic devices, is the appearance of unwanted air bubbles in the fluid circuits. Typically, chips, tubes, fluid control elements, deposits, etc. of these systems need to be connected to each other to create a fluidic system and, as experience in this technical field shows, it is impossible to avoid the appearance of bubbles in certain regions of the devices, such as the connecting elements or the inputs and outputs of the chips In some applications, it is possible to avoid the presence of bubbles, by feeding / purging all e! system once assembled, with a total volume! from a! minus two or three times the dead volume of the circuit. However, this solution can be applied only if e! system is operated in an open circuit, since for a closed system, when connecting the ends of! New air bubbles are produced.
El problema anterior implica ¡imitaciones graves en el campo de la microfluídica, y para resolverlo se han desarrollado, en los últimos años, dispositivos capaces de impedir el paso de las burbujas a lo largo del circuito microfluídico, confinándolas en un volumen localizado del dispositivo. Si bien estos dispositivos (conocidos con el nombre de trampas de burbujas) son capaces de ¡imitar el impacto del aire en los cultivos microfluídicos, aumentan sustancialmente la complejidad del sistema fina!, introduciendo componentes adicionales (lo que aumenta los tiempos de montaje y mantenimiento de los dispositivos) y aumentando e! volumen muerto de! sistema. Además, las trampas de burbujas no resultan de utilidad en aplicaciones donde no se permite el flujo una vez que los chips microfluídicos están montados (como es el caso en aplicaciones de cultivo celular en chips microfluídicos), ya que cuando las células son introducidas en el chip, las entradas y las salidas de fluido están abiertas por lo que, de nuevo, es inevitable que se formen nuevas burbujas de pequeño tamaño. En estos sistemas, las burbujas de aire no pueden eliminarse mediante purgado, ya que las células necesitan tiempo para ser adheridas al sustrato de cultivo antes de aplicar cualquier flujo de líquido. Incluso esperando un tiempo suficiente para e! asentamiento de las células, las burbujas crecerán continuamente si e! chip no está integrado en un circuito de fluido cerrado. De nuevo, si e! chip es integrado en e! circuito, las pequeñas burbujas creadas durante la inserción de células dejarán de crecer, pero permanecerán en las entradas de dicho circuito. Finalmente, incluso si e! flujo de purgado se aplica una vez que las células están totalmente adheridas a! sustrato, es inevitable que algunas de las burbujas migren a la cámara de cultivo celular. Es por ello que se plantea, en e! estado de la técnica, la necesidad de obtener circuitos microfluídicos capaces de evitar la presencia de burbujas, tanto en la microcámara o chip destinado a! cultivo celular sometido a estudio, como en el circuito microfiuídico destinado a la circulación de fluidos (por ejemplo, para la alimentación de las células de cultivo). Asimismo, otra necesidad derivada de las limitaciones técnicas de ios dispositivos existentes en el estado de la técnica es la de permitir una inserción cómoda y sencilla de cualquier partícula (preferentemente, de células) o de líquidos directamente en el chip, mediante el uso de, por ejemplo, una micropipeta. The previous problem involves serious imitations in the field of microfluidics, and in order to solve it, devices capable of preventing the passage of bubbles along the microfluidic circuit have been developed in recent years, confining them in a localized volume of the device. While these devices (known as bubble traps) are capable of mimicking the impact of air on microfluidic cultures, they substantially increase the complexity of the fine system !, by introducing additional components (which increases assembly and maintenance times of the devices) and increasing e! dead volume of! system. In addition, bubble traps are not useful in applications where it allows the flow once the microfluidic chips are mounted (as is the case in cell culture applications in microfluidic chips), since when the cells are inserted into the chip, the fluid inlets and outlets are open so, again, it is inevitable that new small bubbles form. In these systems, air bubbles cannot be removed by purging, since the cells need time to be adhered to the culture substrate before applying any liquid flow. Even waiting long enough for e! settlement of the cells, the bubbles will grow continuously if e! Chip is not integrated in a closed fluid circuit. Again, if e! chip is integrated into e! circuit, the small bubbles created during cell insertion will stop growing, but will remain at the entrances of that circuit. Finally, even if e! Purge flow is applied once the cells are fully adhered to! substrate, it is inevitable that some of the bubbles migrate to the cell culture chamber. That is why it arises, in e! state of the art, the need to obtain microfluidic circuits capable of preventing the presence of bubbles, both in the microcamera or chip destined to! Cell culture under study, as in the microfiuidic circuit intended for the circulation of fluids (for example, for the feeding of the culture cells). Also, another need derived from the technical limitations of the devices existing in the state of the art is that of allowing a comfortable and simple insertion of any particle (preferably, of cells) or of liquids directly into the chip, through the use of, for example, a micropipette.
La presente invención está destinada a satisfacer dichas necesidades. The present invention is intended to meet said needs.
DESCRIPCIÓN BREVE DE LA INVENCIÓN BRIEF DESCRIPTION OF THE INVENTION
Tal y como se ha descrito en párrafos precedentes, un objeto de la presente invención se refiere a un sistema de encapsulado de un sistema microfiuídico que sea capaz de proporcionar una inserción sencilla de las partículas (preferentemente, células de cultivo o nanopartículas funcionaüzadas, por ejemplo nanopartícuias de oro) o cualquier tipo de líquido que se dispense directamente en el chip, y que consiga, además, prevenir completamente la aparición de burbujas de aire en dicho chip. Para el contexto de la presente invención, el término "microfiuídico" se interpreta como relativo a fluidos localizados en volúmenes del orden del microlitro (μΙ) o inferiores, mientras que el término general "fluídico" se interpreta como relativo a fluidos localizados en canales, cámaras o chips de cualquier volumen. As described in the preceding paragraphs, an object of the present invention relates to an encapsulation system of a microfiuidic system that is capable of providing a simple insertion of the particles (preferably, culture cells or nanoparticles functioning, for example gold nanoparticles) or any type of liquid that is dispensed directly into the chip, and which, in addition, completely prevents the appearance of air bubbles in said chip. For the context of the present invention, the term "microfiuidic" is interpreted as relative to fluids located in volumes of the order of microliter (μΙ) or less, while the general term "fluidic" is interpreted as relative to fluids located in channels, chambers or chips of any volume.
El objeto de la invención anteriormente descrito se consigue mediante un dispositivo que comprende: The object of the invention described above is achieved by a device comprising:
- una primera pieza de soporte configurada como base del dispositivo, que comprende un medio de alojamiento y soporte del chip de cultivo celular, estando dicho chip provisto de, a! menos, un cana! de entrada y ai menos un canal de salida de líquido; - a first support piece configured as the base of the device, comprising a housing and support means for the cell culture chip, said chip being provided with, a! less, a cane! input and at least one liquid outlet channel;
- una segunda pieza de llenado que comprende conexiones fluídicas a los canales de! chip de cultivo celular; medios internos de conexión hermética entre dichos canales del chip y las conexiones fluídicas; y sendos pozos de llenado comunicados aguas arriba con dichas conexiones fluídicas; - a second filling piece comprising fluid connections to the channels of! cell culture chip; internal means of tight connection between said chip channels and fluidic connections; and two filling wells communicated upstream with said fluid connections;
- una tercera pieza de encapsulado, que comprende conectores microfluídicos a un circuito fluídico o microfluídico, y una cámara de purgado conectable a los pozos de llenado de la segunda pieza, estando dicha cámara, además, conectada a un filtro permeable a gases, pero impermeable a líquidos, comunicado con el exterior;  - a third encapsulation piece, comprising microfluidic connectors to a fluidic or microfluidic circuit, and a purge chamber connectable to the filling wells of the second part, said chamber being also connected to a gas permeable filter, but impermeable to liquids, communicated abroad;
donde el dispositivo comprende, asimismo: where the device also includes:
- medios externos de unión o acoplamiento entre la primera pieza y la segunda pieza; - external means of connection or coupling between the first piece and the second piece;
- medios externos de unión o acoplamiento entre la segunda pieza y la tercera pieza; - medios externos de cierre hermético entre la segunda pieza y la tercera pieza,- external means of connection or coupling between the second piece and the third piece; - external sealing means between the second piece and the third piece,
- medios internos de conexión hermética entre los canales del chip y las conexiones fluídicas de la segunda pieza; - internal means of tight connection between the channels of the chip and the fluid connections of the second part;
- medios internos de conexión hermética entre los conectores microfluídicos de la tercera pieza y las conexiones fluídicas de la segunda pieza;  - internal means of hermetic connection between the microfluidic connectors of the third part and the fluidic connections of the second part;
y donde el dispositivo, a través de los medios externos e internos de las piezas segunda y tercera, comprende, al menos, dos posiciones de configuración: and where the device, through the external and internal means of the second and third pieces, comprises at least two configuration positions:
- primera posición: comprende un acoplamiento y conexión externa entre la tercera pieza de encapsulado y la segunda pieza de llenado, mediante sus medios externos de unión o acoplamiento y sus medios externos de cierre hermético, pero sin existir una conexión hermética interna entre ambas piezas a través de los medios internos de conexión hermética entre los conectores microfluídicos de la tercera pieza y las conexiones fluídicas de la segunda pieza;  - first position: it comprises an external coupling and connection between the third encapsulation piece and the second filling part, by means of its external means of joining or coupling and its external means of sealing, but without there being an internal hermetic connection between both parts a through the internal means of hermetic connection between the microfluidic connectors of the third part and the fluidic connections of the second part;
- segunda posición: comprende, además del acoplamiento y conexión externa entre la tercera pieza de encapsulado y la segunda pieza de llenado de la primera posición, también su conexión hermética interna entre los conectores microfluídicos de la tercera pieza y las conexiones fluídicas de la segunda pieza, a través de los medios internos de conexión hermética. - second position: it comprises, in addition to the coupling and external connection between the third encapsulation piece and the second filling part of the first position, also its internal hermetic connection between the microfluidic connectors of the third piece and the fluidic connections of the second piece, through the internal means of hermetic connection.
En una realización preferente de la invención, la tercera pieza de encapsuiado se encuentra conectada a un circuito fluídico o microfluídico. De este modo se consigue obtener sistemas fluídicos completos libres de burbujas de aire en sus canales y/o microcanales. In a preferred embodiment of the invention, the third encapsulation piece is connected to a fluidic or microfluidic circuit. In this way it is possible to obtain complete fluidic systems free of air bubbles in their channels and / or microchannels.
En otra realización preferente de la invención, los medios externos de cierre hermético entre la segunda pieza y la tercera pieza comprenden una o más juntas herméticas de anillo. De este modo se obtiene un medio efectivo de conectar las piezas sin pérdida y/o entrada de aire/líquido en el sistema, mediante un único elemento de cierre. In another preferred embodiment of the invention, the external means of sealing between the second piece and the third part comprise one or more ring seals. In this way an effective means of connecting the parts without loss and / or air / liquid inlet in the system is obtained, by means of a single closing element.
En una realización preferente adicional de la invención, los medios externos de unión o acoplamiento de la primera pieza de soporte, de la segunda pieza de llenado y/o de la tercera pieza de encapsuiado comprenden sistemas de cierre de tipo clip/agujero. Se consiguen, con ello, medios eficaces de acoplamiento externo que resultan, al mismo tiempo, fáciles de utilizar y aplicar por el usuario. En otra realización de la invención los medios internos de conexión hermética de la segunda pieza de llenado o de la tercera pieza de encapsuiado comprenden juntas tóricas. Se consigue con ello el aislamiento del sistema fluídico en las uniones de sus componentes, evitando así la entrada/salida de aire y/o líquido. En una realización preferente de la invención, la primera pieza de soporte comprende una ventana configurada para la monitorización del chip de cultivo celular mediante microscopía. Se consigue con ello un medio de monitorización del chip fácilmente integrable en el sistema microfluídico del dispositivo. Otro objeto de la presente invención es un método de encapsuiado de un cultivo celular que comprende el uso de un dispositivo según cualquiera de las reivindicaciones anteriores. Preferentemente, dicho método comprende los siguientes pasos: In a further preferred embodiment of the invention, the external attachment or coupling means of the first support piece, the second filling part and / or the third encapsulation part comprise clip / hole type closure systems. In this way, effective external coupling means are achieved which are, at the same time, easy to use and apply by the user. In another embodiment of the invention the internal means of hermetic connection of the second filling piece or the third encapsulating part comprise o-rings. This achieves the isolation of the fluidic system at the junctions of its components, thus preventing the entry / exit of air and / or liquid. In a preferred embodiment of the invention, the first support piece comprises a window configured for monitoring the cell culture chip by microscopy. This achieves a means of monitoring the chip easily integrable in the microfluidic system of the device. Another object of the present invention is a method of encapsulating a cell culture comprising the use of a device according to any of the preceding claims. Preferably, said method comprises the following steps:
a) se aloja un chip equipado con una pluralidad de entradas/salidas sobre la primera pieza de soporte del dispositivo; b) se acopla la segunda pieza de llenado a la primera pieza de soporte, conectando dichas piezas a través de medios externos de unión o acoplamiento de dichas piezas y proporcionando una conexión hermética entre los canales de entrada/salida del chip y los canales fluídicos de la segunda pieza, a través de medios internos de conexión hermética dispuestos en dicha segunda pieza; a) a chip equipped with a plurality of inputs / outputs is housed on the first support piece of the device; b) the second filling piece is coupled to the first support piece, connecting said pieces through external means of joining or coupling said pieces and providing a tight connection between the input / output channels of the chip and the fluidic channels of the second piece, through internal means of hermetic connection arranged in said second piece;
c) se utiliza un medio de llenado de líquido para el llenado del chip y sus canales de entrada/salida, a través de las conexiones fluídicas de la segunda pieza y se purga de aire tanto el chip como los canales de entrada/salida, hasta saturar de líquido parcial o totalmente ios pozos de llenado de la segunda pieza;  c) a liquid filling means is used for filling the chip and its input / output channels, through the fluid connections of the second piece and both the chip and the input / output channels are purged of air, up to partially or completely saturate the filling wells of the second piece;
d) opcionalmente, una vez llenado de líquido tanto el chip como los canales de entrada/salida y los pozos de llenado con el medio de llenado, se insertan partículas (preferentemente, células de cultivo o nanopartículas funcionalizadas, por ejemplo nanopartículas de oro) y/o líquido en dicho chip; e) se conecta la tercera pieza de encapsulado a un circuito fluídico en el que se integra el dispositivo, a través de los conectares microfluídicos de la tercera pieza.  d) optionally, once both the chip and the input / output channels and the filling wells with the filling medium have been filled with liquid, particles (preferably, culture cells or functionalized nanoparticles, for example gold nanoparticles) are inserted and / or liquid in said chip; e) the third encapsulation piece is connected to a fluidic circuit in which the device is integrated, through the microfluidic connectors of the third piece.
f) se conecta la tercera pieza de encapsulado a la segunda pieza de llenado, a través de la aplicación de los medios de cierre hermético externo y de medios externos de acoplamiento o unión entre la segunda pieza y la tercera pieza, y configurando dichos medios externos de cierre hermético y medios externos de acoplamiento en la primera posición de configuración del dispositivo, pero sin aplicar los medios internos de conexión hermética entre las piezas segunda y tercera.  f) the third encapsulation piece is connected to the second filling piece, through the application of the external sealing means and external coupling or joining means between the second piece and the third piece, and configuring said external means of hermetic closure and external coupling means in the first configuration position of the device, but without applying the internal means of hermetic connection between the second and third parts.
g) manteniendo el dispositivo en la primera posición de configuración, se bombea líquido a través del circuito fluídico, para purgarlo de aire,  g) keeping the device in the first configuration position, liquid is pumped through the fluidic circuit, to purge it of air,
h) una vez que todo el aire se ha retirado del circuito fluídico o microfluídico, se realiza la conexión hermética interna entre la tercera pieza y la segunda pieza, a través de los medios internos de conexión hermética, en la segunda posición de configuración del dispositivo, cerrando el circuito fluídico completo de dicho dispositivo y trasladando el líquido que satura los pozos de llenado y, opcionalmente, aire residual en el circuito, a la cámara de purgado.  h) once all the air has been removed from the fluidic or microfluidic circuit, the internal hermetic connection between the third piece and the second piece is made, through the internal means of hermetic connection, in the second configuration position of the device , closing the complete fluidic circuit of said device and transferring the liquid that saturates the filling wells and, optionally, residual air in the circuit, to the purge chamber.
Adicionalmente a las ya planteadas, otras características y ventajas de la invención se desprenderán de la descripción que sigue, así como de las figuras que acompañan al presente documento. In addition to those already raised, other features and advantages of the invention will emerge from the description that follows, as well as from the figures accompanying the present document
DESCRIPCIÓN DE LAS FIGURAS La Figura 1 muestra una vista externa en perspectiva del dispositivo de encapsulado de la invención, con sus tres piezas acopladas. DESCRIPTION OF THE FIGURES Figure 1 shows an external perspective view of the encapsulation device of the invention, with its three parts coupled.
La Figura 2 muestra una vista en perspectiva de la primera pieza de soporte del dispositivo de !a invención. Figure 2 shows a perspective view of the first support piece of the device of the invention.
La Figura 3 muestra una vista en perspectiva de la segunda pieza de llenado de líquido del dispositivo de la invención. Figure 3 shows a perspective view of the second liquid filling part of the device of the invention.
La Figura 4 muestra una vista en perspectiva de la tercera pieza de encapsulado del dispositivo de la invención. Figure 4 shows a perspective view of the third encapsulation piece of the device of the invention.
La Figura 5 muestra una vista en perspectiva del alojamiento de un chip de cultivo celular en la primera pieza de soporte del dispositivo. Las Figuras 6a-6b muestran, respectivamente, una vista en perspectiva lateral y una vista en perspectiva inferior de la conexión externa e interna de la segunda pieza de llenado con la primera pieza de soporte. Figure 5 shows a perspective view of the housing of a cell culture chip in the first support piece of the device. Figures 6a-6b show, respectively, a side perspective view and a bottom perspective view of the external and internal connection of the second filling piece with the first support piece.
La Figura 7 muestra una vista en perspectiva superior del llenado de las conexiones fluídicas y de los pozos de la segunda pieza, mediante una pipeta. Figure 7 shows a top perspective view of the filling of the fluidic connections and the wells of the second piece, by means of a pipette.
La Figura 8 muestra una vista en perspectiva superior de la saturación de los pozos de la segunda pieza, mediante el llenado realizado con la pipeta mostrado en la Figura 7. Figure 8 shows a top perspective view of the saturation of the wells of the second piece, by filling with the pipette shown in Figure 7.
La Figura 9 muestra una vista en perspectiva de la conexión de la tercera pieza de encapsulado a un circuito fluídico o microfluídico. Figure 9 shows a perspective view of the connection of the third encapsulation piece to a fluidic or microfluidic circuit.
Las Figuras 10a y 10b muestran, respectivamente, una representación extema e interna (en sección lateral) de la primera posición de configuración del dispositivo en su acoplamiento externo entre las piezas segunda y tercera. La Figura 11 muestra una sección lateral del dispositivo durante el purgado de aire del dispositivo, realizado en su primera posición de configuración de acoplamiento externo entre las piezas segunda y tercera. Las Figuras 12a y 12b muestran respectivamente, una representación externa e interna de la segunda posición de configuración del dispositivo en su acoplamiento externo e interno entre las piezas segunda y tercera, tras el purgado de aire y el encapsulado del dispositivo. DESCRIPCIÓN DETALLADA DE LA INVENCIÓN Figures 10a and 10b show, respectively, an external and internal representation (in lateral section) of the first configuration position of the device in its external coupling between the second and third pieces. Figure 11 shows a side section of the device during air purging of the device, performed in its first position of external coupling configuration between the second and third pieces. Figures 12a and 12b respectively show an external and internal representation of the second configuration position of the device in its external and internal coupling between the second and third parts, after purging air and encapsulating the device. DETAILED DESCRIPTION OF THE INVENTION
Según se muestra en las Figuras 1-12 del presente documento y, de acuerdo a lo previamente descrito en apartados anteriores, el dispositivo de la invención comprende, preferentemente, tres piezas acoplables (Figura 1 ) que dan lugar al encapsulado del chip de cultivo celular. Dichas piezas comprenden una primera piezaAs shown in Figures 1-12 of this document and, as previously described in previous sections, the device of the invention preferably comprises three attachable parts (Figure 1) that give rise to the encapsulation of the cell culture chip . These pieces comprise a first piece
(1 ) destinada al alojamiento y fijación de un chip en el dispositivo, una segunda pieza(1) intended for housing and fixing a chip in the device, a second piece
(2) intermedia fijable a la primera pieza (1 ) y que proporciona un medio de inserción de fluido en el chip, y una tercera pieza (3) de encapsulado, acoplable a la segunda pieza (2), que proporciona un cierre hermético del chip, así como la conexión con los canales microfluídicos del circuito completo. (2) intermediate attachable to the first piece (1) and that provides a means of inserting fluid into the chip, and a third piece (3) of encapsulation, attachable to the second piece (2), which provides a tight seal of the chip, as well as the connection with the microfluidic channels of the complete circuit.
Más específicamente (según se muestra en la Figura 2), la primera pieza (1 ) hace de base del dispositivo y comprende un medio de alojamiento y soporte (4) del chip, preferentemente en una configuración apta para la observación de dicho chip mediante equipos de microscopía. Más preferentemente, la primera pieza (1 ) incluye una ventana (5) abierta, que permite que el objetivo del equipo de microscopía empleado pueda ser colocado en estrecho contacto con el chip. Adicionalmente, la primera pieza (1 ) comprende medios externos de unión o acoplamiento (6, 6') a la segunda pieza (2) del dispositivo (en la Figura 1 , cuatro uniones mediante presión de tipo clip). More specifically (as shown in Figure 2), the first piece (1) acts as the base of the device and comprises a means for receiving and supporting the chip (4), preferably in a configuration suitable for observing said chip by means of equipment of microscopy. More preferably, the first part (1) includes an open window (5), which allows the objective of the microscopy equipment used to be placed in close contact with the chip. Additionally, the first part (1) comprises external means for joining or coupling (6, 6 ') to the second part (2) of the device (in Figure 1, four joints by means of clip-like pressure).
La segunda pieza (2) de llenado (Figura 3) es una pieza intermedia destinada a su acoplamiento entre la primera (1 ) y ¡a tercera pieza (3). Dicha segunda pieza (2) proporciona un medio de aislamiento y llenado del chip, mediante conexiones fluídicas (7, 7') con sus canales de entrada y salida. Preferentemente, la segunda pieza comprende medios externos de unión o acoplamiento (8, 8') con la primera pieza (1 ) de soporte (en el ejemplo de la Figura 3, los medios de acoplamiento (8, 8') son agujeros acoplables a los clips de la primera pieza (1) de soporte), así como medios internos de conexión hermética (9, 9') (no representados en la Figura 3, por encontrarse en la región interior de la pieza, ver por ejemplo en Figura 6b), por ejemplo a través de juntas tóricas, con los canales de entrada y salida del chip (para el contexto de la presente invención, el término "externo" se aplica a aquellos elementos relativos a los componentes estructurales del dispositivo no relacionados con el circuito fluidico, y el término "interno" se refiere a aquellos elementos estructurales del dispositivo relacionados con el circuito fluidico). La ubicación de las entradas y salidas y el tamaño externo del dispositivo son las únicas restricciones impuestas al chip para que sea compatible con el sistema de encapsulado propuesto. Aparte de esta consideración, cualquier diseño de microcanales, cámara de cultivo o cualesquiera otros elementos pueden ser diseñados dentro del chip microfluídico. Adicionalmente, con el objetivo de proporcionar un medio de aislamiento y llenado del chip alojado entre las piezas primera (1 ) y segunda (2), la segunda pieza (2) comprende pozos de llenado (10, 10') comunicados con las conexiones fluídicas (7, 7') de la segunda pieza (2), y situados a un nivel aguas arriba de dichas conexiones (7, 7'). Dichos pozos de llenado (10, 10') permiten la introducción de líquido en el chip, por ejemplo mediante una pipeta, hasta que tanto dicho chip como sus canales de entrada y salida queden llenados, con la saturación de los pozos (10, 10') como límite de capacidad del circuito fluidico comprendido entre la primera (1 ) y la segunda pieza (2). La existencia de los pozos de llenado (10, 10') resulta de gran importancia, como se verá más adelante, para evitar cualquier formación de burbujas durante el uso del dispositivo. The second filling part (2) (Figure 3) is an intermediate piece intended for coupling between the first (1) and the third part (3). Said second piece (2) provides a means of isolation and filling of the chip, by means of fluidic connections (7, 7 ') with its input and output channels. Preferably, the second part comprises external joining or coupling means (8, 8 ') with the first part (1) of support (in the example of Figure 3, the coupling means (8, 8 ') are holes attachable to the clips of the first support piece (1)), as well as internal means of hermetic connection (9, 9' ) (not shown in Figure 3, for being in the inner region of the piece, see for example in Figure 6b), for example through O-rings, with the input and output channels of the chip (for the context of the In the present invention, the term "external" applies to those elements related to the structural components of the device not related to the fluid circuit, and the term "internal" refers to those structural elements of the device related to the fluid circuit). The location of the inputs and outputs and the external size of the device are the only restrictions imposed on the chip to be compatible with the proposed encapsulation system. Apart from this consideration, any design of microchannels, culture chamber or any other elements can be designed within the microfluidic chip. Additionally, in order to provide a means of isolation and filling of the chip housed between the first (1) and second (2) pieces, the second piece (2) comprises filling wells (10, 10 ') communicated with the fluidic connections (7, 7 ') of the second piece (2), and located at a level upstream of said connections (7, 7'). Said filling wells (10, 10 ') allow the introduction of liquid into the chip, for example by means of a pipette, until both said chip and its inlet and outlet channels are filled, with saturation of the wells (10, 10 ') as a capacity limit of the fluid circuit between the first (1) and the second part (2). The existence of the filling wells (10, 10 ') is of great importance, as will be seen later, to avoid any formation of bubbles during the use of the device.
Asimismo, para su conexión con la tercera pieza (3), la segunda pieza (2) comprende:Also, for connection with the third part (3), the second part (2) comprises:
- medios externos de unión o acoplamiento (11 , 11 '), configurados para acoplar la segunda pieza (2) a la tercera pieza (3) (en el ejemplo de la Figura 3, los medios de acoplamiento (1 1, 1 1') son agujeros acoplables a clips (elementos (6, 6') en la Figura 2) de la tercera pieza (3)). - external joining or coupling means (11, 11 '), configured to couple the second part (2) to the third part (3) (in the example of Figure 3, the coupling means (1 1, 1 1' ) are holes attachable to clips (elements (6, 6 ') in Figure 2) of the third piece (3)).
- medios externos de conexión hermética (11 "), configurados para proporcionar una conexión hermética entre las piezas segunda (2) y tercera (3) (en el ejemplo de la Figura 3, los medios externos de conexión hermética (1 1") comprenden un cierre hermético de anillo). Por su parte, la tercera pieza (3) (Figura 4 del presente documento) constituye la pieza superior del dispositivo, que permite el encapsulado hermético de la unión de piezas primera (1) y segunda (2) una vez se ha realizado e! llenado del chip, de sus canales de entrada/salida, de las conexiones fluídicas (7, 7') y de los pozos de llenado (10, 10'). Por otra parte, dicha tercera pieza (3) comprende también medios externos de unión o acoplamiento (12) con la segunda pieza (2) (preferentemente, mediante un sistema de clips/agujeros como en las uniones de las piezas primera (1) y segunda (2)), y medios internos de conexión hermética (13) (no representados en la Figura 4 por encontrarse en la región interior de la pieza, ver por ejemplo en Figura 10b), por ejemplo mediante juntas tóricas, a las conexiones fluídicas (7, 7') de la segunda pieza- external hermetic connection means (11 "), configured to provide a hermetic connection between the second (2) and third (3) parts (in the example of Figure 3, the external hermetic connection means (1 1") comprise a hermetic ring closure). For its part, the third piece (3) (Figure 4 of this document) constitutes the upper part of the device, which allows the hermetic encapsulation of the union of first (1) and second (2) pieces once e! filling of the chip, its input / output channels, fluidic connections (7, 7 ') and filling wells (10, 10'). On the other hand, said third part (3) also comprises external means of joining or coupling (12) with the second part (2) (preferably, by means of a system of clips / holes as in the joints of the first parts (1) and second (2)), and internal means of hermetic connection (13) (not shown in Figure 4 for being in the inner region of the piece, see for example in Figure 10b), for example by o-rings, to the fluidic connections (7, 7 ') of the second piece
(2) . (2) .
Adicionalmente, la tercera pieza (3) de encapsulado comprende conectores estándar microfluídicos (14) configurados para permitir una conexión directa al circuito fluídico en el que se integra el chip junto con las piezas primera (1) y segunda (2). Finalmente, la tercera pieza (3) comprende también una cámara de purgado (15) (no representada en la Figura 4 por encontrarse en la región interior de la pieza, ver por ejemplo en Figura 10b) conectable a los pozos (10, 10') de la segunda pieza (2) de llenado, y equipada dicha cámara (15) con un filtro (16) para permitir é! intercambio de aire entre los pozos (10, 10') y el exterior, lo que permite evitar, como se verá a continuación, la contaminación del circuito completo. Additionally, the third encapsulation part (3) comprises standard microfluidic connectors (14) configured to allow a direct connection to the fluidic circuit in which the chip is integrated together with the first (1) and second (2) parts. Finally, the third piece (3) also comprises a purging chamber (15) (not shown in Figure 4 as it is in the inner region of the piece, see for example in Figure 10b) connectable to the wells (10, 10 ' ) of the second filling part (2), and said chamber (15) equipped with a filter (16) to allow é! air exchange between the wells (10, 10 ') and the outside, which allows to avoid, as will be seen below, the contamination of the complete circuit.
Para evitar la presencia de burbujas en el interior del circuito microfiuídico, ¡os medios externos de unión o acoplamiento (1 1, 11 ', 12) y los medios externos de cierre hermético (11 ") de las piezas segunda (2) y tercera (3) comprenden, al menos, dos posiciones de configuración de dicho acoplamiento o unión en el dispositivo: To avoid the presence of bubbles inside the microfiuidic circuit, the external means of connection or coupling (1 1, 11 ', 12) and the external means of sealing (11 ") of the second (2) and third pieces (3) comprise at least two configuration positions of said coupling or connection in the device:
- Primera posición: La tercera pieza (3) se encuentra acoplada a la segunda pieza (2) mediante la aplicación de los medios de cierre hermético externo (11 , 1 1 ') de la segunda pieza (2) y de los medios externos de acoplamiento o unión (12) de la tercera pieza (3), pero sin aplicarse ¡os medios internos de conexión hermética (13) entre ambas (preferentemente, las juntas tóricas (13) de la tercera pieza (3) se sitúan a una distancia comprendida entre 1 y 3 mm de las conexiones fluídicas (7, 7') conectadas con los pozos (10, 10') de la segunda pieza (2)).  - First position: The third part (3) is coupled to the second part (2) by means of the application of the external sealing means (11, 1 1 ') of the second part (2) and of the external means of coupling or joining (12) of the third piece (3), but without applying the internal means of hermetic connection (13) between both (preferably, the o-rings (13) of the third piece (3) are located at a distance between 1 and 3 mm of the fluidic connections (7, 7 ') connected to the wells (10, 10') of the second piece (2)).
- Segunda posición: La tercera pieza (3) se encuentra acoplada a la segunda pieza (2) (preferentemente, los medios internos de conexión hermética (13) de la tercera pieza - Second position: The third part (3) is coupled to the second part (2) (preferably, the internal hermetic connection means (13) of the third part
(3) se acoplan a las conexiones fluídicas (7, 7') de la segunda pieza (2)), realizándose, además del cierre externo de la primera posición, un cierre interno entre ambas piezas (2, 3) con una conexión hermética con el chip de cultivo celular. (3) are coupled to the fluidic connections (7, 7 ') of the second piece (2)), being carried out, In addition to the external closure of the first position, an internal closure between both pieces (2, 3) with an airtight connection with the cell culture chip.
Por su parte, el método de encapsulado de sistemas microfluídicos propuesto en la presente solicitud se basa en el uso de un dispositivo según las realizaciones anteriormente descritas, y comprende preferentemente, uno o más de los siguientes pasos: a) Tal y como se muestra en la Figura 5, se aloja un chip (17) equipado con una pluralidad de entradas/salidas (18, 18') sobre la primera pieza (1) de soporte. b) (Figuras 6a-6b del documento) Se acopla la segunda pieza (2) de llenado a la primera pieza (1 ) de soporte, conectando los medios de unión o acoplamiento (6, 6', 8, 8') de dichas piezas (Figura 6a) y proporcionando una conexión hermética entre los canales de entrada/salida (18, 18') del chip (17) (mostrados dichos canales y el chip en la Figura 5) y los medios internos de conexión hermética (9, 9') (por ejemplo, a través de juntas tóricas) dispuestos en el fondo de la segunda pieza (2) (Figura 6b). c) Tal y como se muestra en la Figura 7, se utiliza un medio de llenado de líquido (19), preferentemente una micropipeta, para el llenado del chip (elemento (17) en Figura 5) y sus canales de entrada/salida (elementos (18, 18') en Figura 5), a través de las conexiones fluídicas (7, 7') de la segunda pieza (2) (Figura 7). Mediante llenados sucesivos con la micropipeta (19), se purga de aire tanto el chip (17) como los canales de entrada/salida (18, 18'), hasta llenar completamente de líquido el circuito fluídico, saturando parcial o totalmente los pozos de llenado (10, 10'). De este modo, se impide que el aire pueda acceder al chip (17) una vez llenado de líquido. d) (Figura 8) Una vez llenado de líquido tanto el chip (elemento (17) en Figura 5) como los canales de entrada/salida (elementos (18, 18') en Figura 5), y los pozos de llenado (10, 10') con la pipeta (19), cualquier partícula (por ejemplo, células) o líquido se puede insertar en el chip (17), para su ubicación en cualquier cámara específica del mismo, mediante el uso de una pipeta (por ejemplo, el chip (17) podría ser alimentado por células mediante su inserción a través de una entrada dedicada en el mismo). Tal y como muestra en la Figura 8, la presencia de líquido en los pozos de llenado (10, 10'), que se encuentran aguas arriba de las conexiones fluídicas (7, 7') de la segunda pieza (2) (Figura 7), de los canales de entrada/salida (elementos (18, 18') en Figura 5) y del chip (elemento (17) en Figura 5), evita ia inserción de burbujas durante la alimentación de dicho chip (17). e) Tal y como se muestra en la Figura 9, se conecta la tercera pieza (3) (pieza de encapsulado) al circuito fluídico (20) en el que se integrará el dispositivo. f) Se conecta la tercera pieza (3) de encapsulado a ia segunda pieza (2) de llenado, a través de !a aplicación de los medios de cierre hermético externo (1 1") de la segunda pieza (2) (preferentemente, un anillo hermético) y de los medios externos de acopiamiento o unión (11 , 1 1', 12) de la segunda pieza (2) y de ia tercera pieza (3) (preferentemente mediante una pluralidad de sistemas de clip/agujero), y configurando dichos medios externos de cierre hermético (11 ") y medios externos de acoplamiento (1 , 1 ', 12) en la "primera posición" de! dispositivo anteriormente descrita, pero sin aplicar el cierre hermético interno (13) entre las piezas segunda (2) y tercera (3) (ver Figuras 10a y 10b, donde se ilustra, respectivamente, en vistas externa e interna, la "primera posición" de configuración referida). Gracias a la aplicación de los medios de cierre hermético externo (11") (preferentemente, mediante un anillo hermético (11") en la segunda pieza(2)), los pozos de llenado (10, 10') definidos en la segunda pieza (2) sólo están conectados con ei exterior del dispositivo a través del filtro (16) ubicado en la parte superior de la tercera pieza (3), evitando así cualquier posibie contaminación de! chip (17) y de los pozos de llenado (10, 10'). En dicha primera posición, los conectares fluídicOs (14) de la tercera pieza (3) (concretamente, sus medios internos de conexión hermética (13)) se encuentran situados a cierta distancia (preferentemente, entre 1 y 3 mm) de las conexiones fiuídicas (7, 7') de la segunda pieza (2), quedando, además, preferentemente cubiertos por el líquido de los pozos de llenado (10, 10') de dicha segunda piezaFor its part, the method of encapsulating microfluidic systems proposed in the present application is based on the use of a device according to the previously described embodiments, and preferably comprises one or more of the following steps: a) As shown in Figure 5, houses a chip (17) equipped with a plurality of inputs / outputs (18, 18 ') on the first support piece (1). b) (Figures 6a-6b of the document) The second filling piece (2) is coupled to the first support piece (1), connecting the joining or coupling means (6, 6 ', 8, 8') of said parts (Figure 6a) and providing an airtight connection between the input / output channels (18, 18 ') of the chip (17) (shown said channels and the chip in Figure 5) and the internal means of hermetic connection (9, 9 ') (for example, through O-rings) arranged at the bottom of the second piece (2) (Figure 6b). c) As shown in Figure 7, a liquid filling means (19), preferably a micropipette, is used for filling the chip (element (17) in Figure 5) and its input / output channels ( elements (18, 18 ') in Figure 5), through the fluidic connections (7, 7') of the second piece (2) (Figure 7). By successive filling with the micropipette (19), both the chip (17) and the inlet / outlet channels (18, 18 ') are purged of air, until the fluidic circuit is completely filled with liquid, partially or totally saturating the wells of filling (10, 10 '). In this way, it is prevented that the air can access the chip (17) once it is filled with liquid. d) (Figure 8) Once the chip (element (17) in Figure 5) and the input / output channels (elements (18, 18 ') in Figure 5) have been filled with liquid, and the filling wells (10 , 10 ') with the pipette (19), any particle (for example, cells) or liquid can be inserted into the chip (17), for its location in any specific chamber thereof, by using a pipette (for example , the chip (17) could be fed by cells by inserting it through a dedicated input in it). As shown in Figure 8, the presence of liquid in the filling wells (10, 10 '), which are located upstream of the fluid connections (7, 7') of the second part (2) (Figure 7), of the inlet / outlet channels (elements (18, 18 ') in Figure 5) and of the chip (element (17) in Figure 5), prevents the insertion of bubbles during the feeding of said chip (17). e) As shown in Figure 9, the third part (3) (encapsulation piece) is connected to the fluidic circuit (20) in which the device will be integrated. f) The third encapsulation part (3) is connected to the second filling part (2), through the application of the external sealing means (1 1 ") of the second part (2) (preferably, a hermetic ring) and of the external means of coupling or joining (11, 1 1 ', 12) of the second piece (2) and of the third piece (3) (preferably by means of a plurality of clip / hole systems), and configuring said external sealing means (11 ") and external coupling means (1, 1 ', 12) in the" first position "of! device described above, but without applying the internal seal (13) between the second (2) and third (3) parts (see Figures 10a and 10b, where, respectively, in external and internal views, the "first position" is illustrated of referred configuration). Thanks to the application of the external sealing means (11 ") (preferably, by means of an airtight ring (11") in the second part (2)), the filling wells (10, 10 ') defined in the second part (2) they are only connected to the outside of the device through the filter (16) located at the top of the third part (3), thus avoiding any possible contamination of! chip (17) and filling wells (10, 10 '). In said first position, the fluidic connectors (14) of the third piece (3) (specifically, its internal means of hermetic connection (13)) are located at a certain distance (preferably, between 1 and 3 mm) from the fiuidic connections (7, 7 ') of the second piece (2), being furthermore preferably covered by the liquid from the filling wells (10, 10') of said second piece
(2). g) Manteniendo el dispositivo en la primera posición de acoplamiento, el líquido es bombeado a través del circuito fluídico (elemento (20), Figura 9), para asegurar que todas las burbujas puedan realizar, al menos, un ciclo completo(2). g) Keeping the device in the first coupling position, the liquid is pumped through the fluidic circuit (element (20), Figure 9), to ensure that all bubbles can carry out at least one complete cycle
(preferentemente, entre 2 y 3 veces el volumen muerto caicuiado en el circuito (20)). Como en dicha primera posición de configuración la tercera pieza (3) no se encuentra internamente conectada al circuito del chip (17) (sólo se encuentra conectada externamente a la segunda pieza (2), todas las burbujas quedan confinadas en el espacio interno definido en la cámara de purgado (15) entre ¡as piezas segunda (2) y tercera (3), purgándose así todo el circuito del dispositivo(preferably, 2 to 3 times the dead volume caicuiado in the circuit (twenty)). As in said first configuration position the third piece (3) is not internally connected to the chip circuit (17) (it is only connected externally to the second piece (2), all bubbles are confined in the internal space defined in the purge chamber (15) between the second (2) and third (3) pieces, thus purging the entire device circuit
(Figura 11 ). Además, dado que dicho espacio interno (15) está conectado con el exterior a través del filtro (16) situado en la tercera pieza (3), ia presión dentro del dispositivo no aumenta durante el proceso de encapsulado. h) Una vez que todas las burbujas se han retirado de! circuito, se realiza el acoplamiento completo de la tercera pieza (3) y de la segunda pieza (2), a través de sus medios internos de conexión hermética (13), en la "segunda posición" de configuración del dispositivo antes descrita (ver Figuras 12a y 12b, donde se ilustra, respectivamente en vistas externa e interna, la segunda posición referida), con lo que se cierra herméticamente el circuito fluídico completo del dispositivo. Como consecuencia de la eliminación del aire en dicho circuito realizada en el paso (g), el chip (17) queda conectado al resto del circuito, evitando las burbujas y sin la necesidad de introducir, posteriormente, flujo adiciona! de líquido a través de! chip (17). i) Una vez cerrado e! circuito fluídico completo, el cultivo de células estático o dinámico puede llevarse a cabo sin burbujas en el interior de! dispositivo. (Figure 11). Furthermore, since said internal space (15) is connected to the outside through the filter (16) located in the third part (3), the pressure inside the device does not increase during the encapsulation process. h) Once all the bubbles have been removed from! circuit, the complete coupling of the third part (3) and the second part (2) is carried out, through its internal means of hermetic connection (13), in the "second position" of configuration of the device described above (see Figures 12a and 12b, which illustrates, respectively in external and internal views, the second position referred to), thereby closing the complete fluidic circuit of the device. As a consequence of the elimination of the air in said circuit carried out in step (g), the chip (17) is connected to the rest of the circuit, avoiding bubbles and without the need to introduce, subsequently, flow added! of liquid through! chip (17). i) Once closed e! Complete fluidic circuit, static or dynamic cell culture can be carried out without bubbles inside! device.
Finalmente, una vez descrita la presente invención y algunas de sus realizaciones preferentes, junto con sus principales ventajas sobre e! estado de la técnica, cabe resaltar, de nuevo, que su aplicación no ha de ser entendida como limitada necesariamente a una configuración determinada de los componentes descritos, ni a las realizaciones referidas en los ejemplos de ¡a invención, sino que resulta aplicable también a otro tipo de configuraciones y procedimientos, mediante las adecuadas variaciones en sus elementos, siempre que dichas variaciones no alteren la esencia de la invención, así como el objeto de la misma. Finally, once the present invention and some of its preferred embodiments have been described, together with its main advantages over e! prior art, it should be noted again that its application should not be understood as necessarily limited to a particular configuration of the described components, nor to the embodiments referred to in the examples of the invention, but that it is also applicable to other types of configurations and procedures, by means of the appropriate variations in its elements, provided that said variations do not alter the essence of the invention, as well as the object thereof.

Claims

REIVINDICACIONES
1 .- Dispositivo de encapsulado de un sistema microfluídico, caracterizado porque comprende: 1 .- Encapsulation device of a microfluidic system, characterized in that it comprises:
- una primera pieza (1 ) de soporte configurada como base del dispositivo, que comprende un medio de alojamiento y soporte (4) de un chip (17) de cultivo celular, estando dicho chip (17) provisto de, a! menos, un canal de entrada (18) y a! menos un canal de salida (1 8') de liquido; - a first support piece (1) configured as the base of the device, comprising a housing and support means (4) of a cell culture chip (17), said chip (17) being provided with, a! less, an input channel (18) and a! minus one outlet channel (1 8 ') of liquid;
- una segunda pieza (2) de llenado que comprende conexiones fluídicas (7, T) a los canales (18, 1 8') del chip (17) de cultivo celular; medios internos de conexión hermética (9, 9') entre dichos canales (1 8, 18') del chip (17) y las conexiones fluídicas (7, 7'); y sendos pozos de llenado (10, 10') comunicados aguas arriba con dichas conexiones fluídicas (7, 7');  - a second filling piece (2) comprising fluidic connections (7, T) to the channels (18, 1 8 ') of the cell culture chip (17); internal means of hermetic connection (9, 9 ') between said channels (1, 8, 18') of the chip (17) and the fluidic connections (7, 7 '); and two filling wells (10, 10 ') communicated upstream with said fluidic connections (7, 7');
- una tercera pieza (3) de encapsulado, que comprende conectores microfluídicos (14) a un circuito (20) fluídico o microfluídico, y una cámara de purgado (15) conectabie a los pozos de llenado (10, 10') de la segunda pieza (2), estando dicha cámara (15), además, conectada a un filtro (16) permeable a gases, pero impermeable a líquidos, comunicado con el exterior;  - a third piece (3) of encapsulation, comprising microfluidic connectors (14) to a fluidic or microfluidic circuit (20), and a purge chamber (15) connected to the filling wells (10, 10 ') of the second piece (2), said chamber (15) being also connected to a filter (16) permeable to gases, but impervious to liquids, connected to the outside;
donde el dispositivo comprende, asimismo: where the device also includes:
- medios externos de unión o acoplamiento (6, 6', 8, 8') entre la primera pieza (1 ) y la segunda pieza (2); - external means of connection or coupling (6, 6 ', 8, 8') between the first part (1) and the second part (2);
- medios externos de unión o acoplamiento (1 1 , 1 1 ', 12) entre la segunda pieza (2) y la tercera pieza (3);  - external means of connection or coupling (1 1, 1 1 ', 12) between the second part (2) and the third part (3);
- medios externos de cierre hermético (1 1 ") entre la segunda pieza (2) y la tercera pieza (3),  - external sealing means (1 1 ") between the second part (2) and the third part (3),
- medios internos de conexión hermética (9, 9') entre los canales (18, 1 8') del chip (17) y las conexiones fluídicas (7, 7') de la segunda pieza (2);  - internal means of hermetic connection (9, 9 ') between the channels (18, 1 8') of the chip (17) and the fluidic connections (7, 7 ') of the second piece (2);
- medios internos de conexión hermética (13) entre los conectores microfluídicos (14) de la tercera pieza (3) y las conexiones fluídicas (7, T) de la segunda pieza (2);  - internal means of hermetic connection (13) between the microfluidic connectors (14) of the third part (3) and the fluidic connections (7, T) of the second part (2);
y donde el dispositivo, a través de los medios externos (6, 6', 8, 8', 1 1 , 1 1 ', 1 1 ", 12) e internos (1 3) de las piezas segunda (2) y tercera (3), comprende, al menos, dos posiciones de configuración: and where the device, through the external means (6, 6 ', 8, 8', 1 1, 1 1 ', 1 1 ", 12) and internal (1 3) of the second (2) and third pieces (3), comprises at least two configuration positions:
- primera posición: comprende un acoplamiento y conexión externa entre la tercera pieza (3) de encapsulado y la segunda pieza (2) de llenado, mediante sus medios externos de unión o acoplamiento (1 1 , 1 1 ', 12) y sus medios externos de cierre hermético (1 1 "), pero sin existir una conexión hermética interna entre ambas piezas (2, 3) a través de los medios internos de conexión hermética (13) entre los conectores microfluídicos (14) de la tercera pieza (3) y las conexiones fluídicas (7, 7') de la segunda pieza (2); - first position: it comprises an external coupling and connection between the third encapsulation part (3) and the second filling part (2), by means of its external joining or coupling means (1 1, 1 1 ', 12) and its means external seal (1 1 "), but without an internal hermetic connection between both parts (2, 3) through the internal means of hermetic connection (13) between the microfluidic connectors (14) of the third part (3) and the fluidic connections (7, 7 ') of the second part (2);
- segunda posición: comprende, además del acoplamiento y conexión externa entre la tercera pieza (3) de encapsulado y la segunda pieza (2) de llenado de la primera posición, también su conexión hermética interna entre los conectores microfluídicos (14) de la tercera pieza (3) y las conexiones fluídicas (7, 7) de la segunda pieza (2), a través de los medios internos de conexión hermética (13).  - second position: it includes, in addition to the coupling and external connection between the third encapsulation part (3) and the second filling part (2) of the first position, its internal hermetic connection between the microfluidic connectors (14) of the third piece (3) and the fluidic connections (7, 7) of the second piece (2), through the internal means of hermetic connection (13).
2.- Dispositivo según la reivindicación anterior, donde la tercera pieza (3) de encapsulado se encuentra conectada a un circuito fluídico (20) o microfluídico. 2. Device according to the preceding claim, wherein the third encapsulation piece (3) is connected to a fluidic (20) or microfluidic circuit.
3. - Dispositivo según cualquiera de las reivindicaciones anteriores, donde los medios externos de cierre hermético (1 1 ") entre la segunda pieza (2) y la tercera pieza (3) comprenden una o más juntas herméticas de anillo. 3. - Device according to any one of the preceding claims, wherein the external sealing means (1 1 ") between the second part (2) and the third part (3) comprise one or more hermetic ring seals.
4. - Dispositivo según cualquiera de las reivindicaciones anteriores, donde los medios externos de unión o acoplamiento (6, 6', 8, 8', 1 1 , 1 1 ', 12) de la primera pieza (1 ) de soporte, de la segunda pieza (2) de llenado y/o de la tercera pieza (3) de encapsulado comprenden sistemas de cierre de tipo clip/agujero. 4. - Device according to any of the preceding claims, wherein the external joining or coupling means (6, 6 ', 8, 8', 1 1, 1 1 ', 12) of the first support piece (1), of the second filling part (2) and / or the third encapsulation part (3) comprise clip / hole closing systems.
5. - Dispositivo según cualquiera de las reivindicaciones anteriores, donde los medios internos de conexión hermética (9, 9', 13) de la segunda pieza (2) de llenado o de la tercera pieza (3) de encapsulado comprenden juntas tóricas. 5. - Device according to any of the preceding claims, wherein the internal means of hermetic connection (9, 9 ', 13) of the second filling part (2) or the third encapsulation part (3) comprise O-rings.
6. - Dispositivo según cualquiera de las reivindicaciones anteriores, donde la primera pieza (1 ) de soporte comprende una ventana (5) configurada para la monitorización de! chip ( 7) de cultivo celular mediante microscopía. 6. - Device according to any of the preceding claims, wherein the first support piece (1) comprises a window (5) configured for the monitoring of! chip (7) of cell culture by microscopy.
7.- Método de encapsulado de sistemas microfluídicos que comprende el uso de un dispositivo según cualquiera de las reivindicaciones anteriores. 7. Method of encapsulation of microfluidic systems comprising the use of a device according to any of the preceding claims.
8.- Método según la reivindicación anterior, que comprende los siguientes pasos: 8. Method according to the preceding claim, comprising the following steps:
a) se aloja un chip (17) equipado con una pluralidad de entradas/salidas (1 8, 1 8') sobre la primera pieza (1 ) de soporte; b) se acopla la segunda pieza (2) de llenado a la primera pieza (1) de soporte, conectando dichas piezas a través de medios externos de unión o acoplamiento (6, 6', 8, 8') de dichas piezas (1 , 2) y proporcionando una conexión hermética entre los canales de entrada/salida (18, 18') del chip (1 ) y los canales fluídicos (7, 7') de la segunda pieza (2), a través de medios internos de conexión hermética (9, 9') dispuestos en dicha segunda pieza (2); a) a chip (17) equipped with a plurality of inputs / outputs (1 8, 1 8 ') is housed on the first support piece (1); b) the second filling part (2) is coupled to the first support part (1), connecting said parts through external means of joining or coupling (6, 6 ', 8, 8') of said parts (1 , 2) and providing a tight connection between the input / output channels (18, 18 ') of the chip (1) and the fluidic channels (7, 7') of the second piece (2), through internal means of hermetic connection (9, 9 ') arranged in said second piece (2);
c) se utiliza un medio de llenado de líquido (19) para el llenado del chip (17) y sus canales de entrada/salida (18, 18'), a través de las conexiones fluídicas (7, 7') de la segunda pieza (2) y se purga de aire tanto el chip (17) como los canales de entrada/salida (18, 18'), hasta saturar de líquido parcial o totalmente los pozos de llenado (10, 10') de la segunda pieza (2); c) a liquid filling means (19) is used for filling the chip (17) and its input / output channels (18, 18 '), through the fluidic connections (7, 7') of the second piece (2) and the chip (17) and the inlet / outlet channels (18, 18 ') are purged of air, until the filling wells (10, 10') of the second piece are partially or completely saturated with liquid (2);
d) opcionaimente, una vez llenado de líquido tanto el chip (17) como los canales de entrada/salida (18, 18') y los pozos de llenado (10, 10') con el medio de llenado (19), se insertan partículas y/o líquido en dicho chip (17); d) optionally, once the chip (17) and the input / output channels (18, 18 ') and the filling wells (10, 10') are filled with liquid with the filling medium (19), they are inserted particles and / or liquid in said chip (17);
e) se conecta la tercera pieza (3) de encapsulado a un circuito fluídico (20) en el que se integra el dispositivo, a través de los conectores microfluídicos (14) de la tercera pieza (3). e) the third encapsulation part (3) is connected to a fluidic circuit (20) in which the device is integrated, through the microfluidic connectors (14) of the third part (3).
f) se conecta la tercera pieza (3) de encapsulado a la segunda pieza (2) de llenado, a través de la aplicación de los medios de cierre hermético externo (11 ") y de medios externos de acoplamiento o unión (11 , 1 1', 12) entre la segunda pieza (2) y la tercera pieza (3), y configurando dichos medios externos de cierre hermético (11 ") y medios externos de acoplamiento (11 , 11 ', 12) en la primera posición de configuración del dispositivo, pero sin aplicar los medios internos de conexión hermética (13) entre las piezas segunda (2) y tercera (3). g) manteniendo el dispositivo en la primera posición de configuración, se bombea líquido a través del circuito fluídico (20), para purgarlo de aire, h) una vez que todo el aire se ha retirado del circuito (20) fluídico o microfluídico, se realiza la conexión hermética interna entre la tercera pieza (3) y la segunda pieza (2), a través de los medios internos de conexión hermética (13), en la segunda posición de configuración del dispositivo, cerrando el circuito fluídico completo de dicho dispositivo y trasladando el líquido que satura los pozos de llenado (10, 10') y, opcionaimente, burbujas de aire residual en el circuito (20), a la cámara de purgado (15). f) the third encapsulation part (3) is connected to the second filling part (2), through the application of the external sealing means (11 ") and external coupling or joining means (11, 1 1 ', 12) between the second part (2) and the third part (3), and configuring said external sealing means (11 ") and external coupling means (11, 11', 12) in the first position of configuration of the device, but without applying the internal means of hermetic connection (13) between the second (2) and third (3) pieces. g) keeping the device in the first configuration position, liquid is pumped through the fluidic circuit (20), to purge it of air, h) once all the air has been removed from the fluidic or microfluidic circuit (20), it makes the internal hermetic connection between the third part (3) and the second part (2), through the internal means of hermetic connection (13), in the second configuration position of the device, closing the complete fluidic circuit of said device and transferring the liquid that saturates the filling wells (10, 10 ') and, optionally, residual air bubbles in the circuit (20), to the purge chamber (15).
PCT/ES2013/000219 2012-10-04 2013-10-03 Device and method for encapsulating microfluidic systems WO2014053678A1 (en)

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