WO2013186408A1 - Cell culture device and method associated with said - Google Patents
Cell culture device and method associated with said Download PDFInfo
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- WO2013186408A1 WO2013186408A1 PCT/ES2013/000141 ES2013000141W WO2013186408A1 WO 2013186408 A1 WO2013186408 A1 WO 2013186408A1 ES 2013000141 W ES2013000141 W ES 2013000141W WO 2013186408 A1 WO2013186408 A1 WO 2013186408A1
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- 238000004113 cell culture Methods 0.000 title claims abstract description 46
- 238000000034 method Methods 0.000 title claims description 18
- 239000000758 substrate Substances 0.000 claims abstract description 24
- 239000000463 material Substances 0.000 claims abstract description 20
- 238000004519 manufacturing process Methods 0.000 claims abstract description 18
- 239000000499 gel Substances 0.000 claims description 38
- 238000000206 photolithography Methods 0.000 claims description 18
- 229910052751 metal Inorganic materials 0.000 claims description 16
- 239000002184 metal Substances 0.000 claims description 16
- 239000012530 fluid Substances 0.000 claims description 13
- 239000000203 mixture Substances 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 8
- 239000001963 growth medium Substances 0.000 claims description 7
- 229920003223 poly(pyromellitimide-1,4-diphenyl ether) Polymers 0.000 claims description 3
- 238000006116 polymerization reaction Methods 0.000 claims description 3
- VYZAMTAEIAYCRO-UHFFFAOYSA-N Chromium Chemical compound [Cr] VYZAMTAEIAYCRO-UHFFFAOYSA-N 0.000 claims description 2
- 229910052782 aluminium Inorganic materials 0.000 claims description 2
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 claims description 2
- 239000013043 chemical agent Substances 0.000 claims description 2
- 210000004748 cultured cell Anatomy 0.000 claims description 2
- 238000005538 encapsulation Methods 0.000 claims description 2
- 238000005304 joining Methods 0.000 claims description 2
- 239000000126 substance Substances 0.000 claims description 2
- 238000004458 analytical method Methods 0.000 abstract description 3
- 239000003814 drug Substances 0.000 abstract description 3
- 229940079593 drug Drugs 0.000 abstract description 3
- 210000004027 cell Anatomy 0.000 description 10
- 238000002474 experimental method Methods 0.000 description 3
- 229920000642 polymer Polymers 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 239000004593 Epoxy Substances 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000011031 large-scale manufacturing process Methods 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 230000004308 accommodation Effects 0.000 description 1
- 238000000429 assembly Methods 0.000 description 1
- 230000000712 assembly Effects 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000000459 effect on growth Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 230000000873 masking effect Effects 0.000 description 1
- 238000007567 mass-production technique Methods 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000004065 semiconductor Substances 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
Classifications
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- 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
- B01L3/502761—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 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
- B01F33/00—Other mixers; Mixing plants; Combinations of mixers
- B01F33/30—Micromixers
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
- B01F35/00—Accessories for mixers; Auxiliary operations or auxiliary devices; Parts or details of general application
- B01F35/80—Forming a predetermined ratio of the substances to be mixed
- B01F35/81—Forming mixtures with changing ratios or gradients
-
- 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
-
- 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
- B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
- B01L2200/0668—Trapping microscopic beads
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/06—Auxiliary integrated devices, integrated components
- B01L2300/069—Absorbents; Gels to retain a fluid
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0809—Geometry, shape and general structure rectangular shaped
- B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
- B01L2300/0864—Configuration of multiple channels and/or chambers in a single devices comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
- B01L2300/0874—Three dimensional network
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
- B01L2300/0883—Serpentine channels
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0887—Laminated structure
Definitions
- the present invention refers to an encapsulable device of the "chip laboratory” type (or, in English, “lab-on-chip”) intended for use in the accommodation and study of cell cultures, as well as a method of manufacture of said device. More specifically, the invention relates to a device oriented to provide a microfluidic culture chamber equipped with a plurality of wells, on which a matrix (or "array") of substrates is deposited, wherein said substrates deposited in each well have preferably different stiffness values.
- the present invention is oriented to satisfy said need.
- the present invention relates to an encapsulable device of the "lab-on-chip" type, intended for use in the study of cell cultures, and preferably composed of a plastic material where they are located different areas or wells for cell culture, at the bottom of which a gelled material with different mechanical properties is deposited in each well.
- Said device obeys the main object of the present invention, which consists in obtaining large-scale analysis systems that allow the study of the response of a certain type of cells, drugs, effect on growth, etc. depending on different values of substrate stiffness, in which the user can perform cell culture studies.
- Said object of the invention is carried out, in a first aspect thereof, by means of a cell culture device characterized in that it comprises:
- a gel generator comprising at least two fluid inlet channels and a mixer connected to said fluid inlet channels, the mixer being configured to create a discrete mixing gradient through a step branching structure, and where the gel generator additionally comprises a plurality of output channels, connected to the mixer in one or more of its branching stages; - a cell culture chamber comprising a plurality of cell culture wells connected to the output channels of the gel generator, so that said gels can be deposited as substrates of the wells, and where the cell culture chamber also comprises , at least one fluid inlet channel and at least one fluid outlet channel, both channels being connected to said cell culture chamber.
- the cell culture chamber is an encapsulable chamber. This facilitates the preparation of the cultures, by using exclusively the channels or microchannels of the device, for the injection of the substrates, cells and culture medium, without the need for additional manual intervention.
- the possibility of encapsulating the device also provides greater robustness to it.
- the device is made as a chip based on a polymerizable material by photolithography.
- a polymerizable material by photolithography.
- the polymerizable manufacturing material of the device comprises an SU-8 epoxy viscous polymer.
- Another object of the present invention relates to a cell culture method comprising the use of a device according to the embodiments described herein, where liquids of different composition are circulated through the inlet channels of the gel generator, so that the gels generated in the exit channels of said gels generator are deposited, by means of the connection with the wells of the cell culture chamber, as substrates of different rigidity of said wells.
- the cultured cells are introduced into the culture chamber, on the substrates of the wells of said chamber.
- a biocompatible liquid and / or a culture medium is circulated through the inlet and outlet channels of the culture chamber.
- Another aspect of the invention relates to a method of manufacturing a device according to the embodiments described herein, which comprises the polymerization by photolithography of polymerizable layers that form a chip by joining it.
- the stiffness values of the substrate matrix of the device are known exactly by the user.
- the stiffness values can be preset by the user by modifying the liquid components that give rise to the gel in the mixer.
- the stiffness gradient produced varies in discrete and preset amounts, unlike a continuous gradient, where stiffness variations are difficult to determine.
- the presence of a flow of biocompatible liquid or a culture medium during cell culture also allows the simulation of shear stress conditions in the cultures, which allows them to be studied, subjected to said tension.
- the main advantage of the device is that it facilitates the massive testing of different substrates, with different stiffness properties, in a single use, automatically and without the need for prior preparation of each well. It is a device that does not currently exist in the market, which causes each research center to invest some time and effort to fill that gap, developing its own systems.
- Figure 1 shows a representation of the elements of the device of the invention, in a preferred embodiment thereof (plant).
- Figure 2 shows a representation of the gel generator structure of the cell culture device, in a preferred embodiment of the invention (plant).
- Figures 3 (a-b) show a representation of the cell culture chamber comprised in the device of the invention, according to a preferred embodiment of the same (3 (a) plant, 3 (b) cross section).
- Figures 4 (a) and 5 (a) show two schematic representations of the different masking stages of the cell culture device according to preferred embodiments of the present invention.
- Figures 4 (b-f) and 5 (b-g) show the plant and profile of the device during the different manufacturing stages according to preferred embodiments of the present invention.
- Figures 6 (ac), 7 (ac) and 8 (ac) show three alternative embodiments of the device of the invention, where general views of each embodiment are provided ( Figures 6a, 7a, 8a), enlarged views of the mixers employed ( Figures 6b, 7b, 8b) and enlarged views of the wells comprised in the culture chamber ( Figures 6c, 7c, 8c).
- Figure 9 shows a top view of a plurality of cell culture devices according to a preferred embodiment of the invention, arranged as a multi-device chip intended for large-scale study of cell cultures.
- Figure 10 shows a schematic representation of the steps of the manufacturing process of a device according to an embodiment of the invention.
- the cell culture device of the present invention comprises:
- a gels generator (1) which is preferably integrated by at least two fluid inlet channels (2, 3), intended to conduct two fluids of different physical-mechanical properties, and a mixer (4) intended for mixing said gels in different proportions, through a plurality of stages, where mixing through said stages provides a plurality of gels of different stiffness, and where the gels generator also comprises outlet channels (5) of said gels;
- a cell culture chamber (6) preferably encapsulable, comprising a plurality of wells (7) for cell culture, where the wells (7) are connected to the outlet channels (5) of the gel generator, of so that these gels (with different stiffness values) can be deposited as substrates of the wells (7).
- the cell culture chamber (6) preferably has a fluid inlet channel (8) and a fluid outlet channel (9), through which it is possible to introduce the culture cells into the wells, as well as circulate a continuous feed flow (culture medium) of the crops studied.
- the mixer (4) of the gel generator (1) preferably comprises a branched tree structure growing in the direction of the outlet channels (5), so that, at each stage of branching, successive mixtures of the two initial gels entering the generator (1) are produced, thus forming a gradient of stiffness in the gels that exit through the exit channels (5), after passing through the mixer (4).
- Said channels are connected, as mentioned above, to the wells (7) of the cell culture chamber (6).
- the mixing ratios that reach the outlet channels (5) can be determined accurately, through the design of the mixer (4 ), for example by varying the number of mixing stages, the number of branches in each stage, the thickness of the mixer channels, etc., which also allows a precise adjustment of the physical properties (for example, the stiffness) of the gel that reaches each well (7), which can be calculated accurately.
- the device and the manufacturing process of the present invention allow the wells (7) to be filled with the different gels that come from the mixer (5), without occupying or obstructing the culture chamber (6).
- the cell culture chamber (6) comprises a plurality of areas / wells (7) to house the culture cells, where the bottoms of said areas / wells (7) are connected to the output channels of the gels generator (1).
- the cell culture chamber (6) comprises a fluid inlet channel (8) and a fluid inlet channel (9), both channels intended for the circulation of the cell culture medium, as well as for the introduction of said cells in the wells (7).
- the manufacture of the cell culture device described herein is carried out by a polymerization process by photolithography, through which different layers are generated that overlap one another to form the elements of the device (gels generator (1 ) and culture chamber (6)).
- photolithographic technology allows the large-scale manufacture of the device, also providing the capacity for serial mounting of a plurality of "lab-on-chip" devices, which is a significant improvement over the state of the art in obtaining of cell culture devices with substrates of varying stiffness and intended for mass production.
- the manufacturing material of the device is preferably an SU-8 epoxy viscous polymer.
- Figures 4 (ag) and 5 (ah) of this document two manufacturing schemes by masks of the device according to Preferred embodiments of the present invention. They represent the following elements:
- FIGS. 4 (b-f) and 5 (b-g) show top and side views of two preferred embodiments of the present invention.
- Both embodiments comprise encapsulated assemblies, in which cell cultures remain isolated from the outside. This makes the preparation of the cultures much easier, by using exclusively the channels or microchannels of the device, for the injection of the substrates, cells and culture medium, without the need for additional manual intervention.
- the possibility of encapsulating the device also provides greater robustness to it.
- Figures 6 (ac), 7 (ac) and 8 (ac) show three alternative embodiments of the device of the invention, where general views of each embodiment are provided ( Figures 6a, 7a, 8a), enlarged views of the mixers employed ( Figures 6b, 7b, 8b) and enlarged views of the wells comprised in the culture chamber ( Figures 6c, 7c, 8c).
- Figure 9 shows a top view of a plurality of cell culture devices according to a preferred embodiment of the invention, arranged as a multi-device chip intended for large-scale study of cell cultures.
- another object of the present invention relates to a method of manufacturing a device according to the above embodiments, in which successive layers of polymerizable material (preferably, said layers comprising an SU-8 polymer) are arranged to form a chip using photolithography techniques.
- successive layers of polymerizable material preferably, said layers comprising an SU-8 polymer
- photolithography techniques preferably, photolithography techniques
- a first SU-8 layer (12) is deposited on the metal layer (10) of the first support surface (11), and defined by photolithography to be used as a "ground” layer of the component mixer (4) of the gels.
- a second SU-8 layer (13) is deposited on the first SU-8 layer (12) of the first support surface (11), and is defined by photolithography to manufacture the channels (5) that are going to be used for the mixture of the components of the gels, and the location of the areas / wells (7) assigned.
- a third layer of SU-8 (14) is deposited on a second support surface (15) comprising a material (16) with low adhesion to the SU-8 (for example, kapton), said third layer of SU being defined -8 (14) by photolithography, to "cover" the structures defined in the first support surface (11), except fluidic entrances and exits to access the channels (2, 3, 5).
- the second support surface (15) faces the first support surface (11), so that the layers of SU-8 (12, 13, 14) comprised on said surfaces are facing and aligned with each other, sticking together. the SU-8 layers of both support surfaces (11, 15) through the application of pressure and temperature.
- a metal layer (17) is deposited on a third support surface (18), preferably of a different metal than the metal layer (10) deposited on the first support surface (11).
- a fourth layer of SU-8 (19) is deposited on the metal layer of the third support surface (18) and is defined through photolithography processes to be used as the "ground” layer of the final device.
- the third support surface (18) faces the first support surface (11), so that the layers of SU-8 (12, 13, 14, 19) deposited on each of said surfaces are facing and aligned with each other, and the SU-8 layers of both surfaces are glued through the application of pressure and temperature.
- a fifth layer of SU-8 (20) is deposited on a fourth support surface (21) comprising a material (22) with low adhesion to SU-8, preferably kapton, and is defined by photolithography processes to be used as the roof layer of the final device.
- a sixth layer of SU-8 (23) is deposited on the fifth layer (20) of the fourth support surface (21), and is defined through photolithography processes to be used as a "culture chamber” layer "of the final device.
- the fourth support surface (21) faces the third support surface (18), so that the layers of SU-8 (12, 13, 14, 19, 20, 23) deposited on each of said surfaces are facing each other, and aligned with each other, and the SU-8 layers of both support surfaces are glued through the application of pressure and temperature.
- the six layers of SU-8 (12, 13, 14, 19, 20, 23) are released from the remaining support surfaces (18, 21), by introducing the wafer formed by said layers into a chemical attacker selective for the metal comprised in the metal layer (17) of the third support surface (18), and by removing the fourth support surface (21) comprising the material (22) with low adhesion to the SU-8.
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Abstract
The invention relates to a capsulatable device to be used in the study of cell cultures, preferably consisting of a plastic material containing various recesses for the cell culture. A gelled material is deposited on the bottom of the recesses, said material having different mechanical properties in each recess. The invention further relates to, preferably, the production of large-scale analysis systems that enable the response of a certain type of cell, drug etc., to be studied according to different substrate rigidity values used for each culture.
Description
DISPOSITIVO DE CULTIVO CELULAR Y MÉTODO ASOCIADO A DICHO DISPOSITIVO CELLULAR CULTURE DEVICE AND METHOD ASSOCIATED WITH SUCH DEVICE
CAMPO DE LA INVENCIÓN FIELD OF THE INVENTION
La presente invención hace referencia a un dispositivo encapsulable de tipo "laboratorio en chip" (o, en inglés, "lab-on-chip") destinado para su uso en el alojamiento y el estudio de cultivos celulares, así como a un método de fabricación de dicho dispositivo. Más concretamente, la invención se refiere a un dispositivo orientado a proporcionar una cámara de cultivo microfluídica equipada con una pluralidad de pocilios, sobre los que se encuentra depositada una matriz (o "array") de sustratos, donde dichos sustratos depositados en cada pocilio presentan, preferentemente, distintos valores de rigidez. ANTECEDENTES DE LA INVENCIÓN The present invention refers to an encapsulable device of the "chip laboratory" type (or, in English, "lab-on-chip") intended for use in the accommodation and study of cell cultures, as well as a method of manufacture of said device. More specifically, the invention relates to a device oriented to provide a microfluidic culture chamber equipped with a plurality of wells, on which a matrix (or "array") of substrates is deposited, wherein said substrates deposited in each well have preferably different stiffness values. BACKGROUND OF THE INVENTION
Actualmente, dentro del campo técnico correspondiente al estudio del comportamiento celular sobre disposiciones de sustratos (definidos como las superficies donde se depositan las muestras biológicas durante su cultivo), es conocido en el estado de la técnica que las propiedades mecánicas de dichos sustratos poseen un importante efecto en el comportamiento y evolución de los cultivos estudiados. Dentro de las propiedades mecánicas que afectan al desarrollo de los cultivos, según lo publicado en estudios de alto impacto científico (en revistas como Science, Cell, etc.), la rigidez de los mismos posee una especial relevancia en su comportamiento. Ello ha motivado, en los grupos de investigación especializada en este campo, una necesidad creciente de obtener muestras de experimentación sobre sustratos de diferente rigidez, lo que ha llevado a la obtención de algunos dispositivos experimentales destinados a tal fin. Dado que dichos dispositivos, hasta la fecha, han de ser desarrollados por cada laboratorio de forma individual, adaptándose a las necesidades concretas de cada experimento, y que dichos dispositivos no pueden aplicarse, por lo general, a más de un uso o experimento, existe hoy en
día una importante carencia tecnológica de producción a gran escala de este tipo de plataformas de cultivo. Currently, within the technical field corresponding to the study of cellular behavior on substrate arrangements (defined as the surfaces where biological samples are deposited during their cultivation), it is known in the state of the art that the mechanical properties of said substrates possess an important effect on the behavior and evolution of the crops studied. Within the mechanical properties that affect the development of crops, as published in studies of high scientific impact (in journals such as Science, Cell, etc.), the rigidity of them has a special relevance in their behavior. This has motivated, in the specialized research groups in this field, a growing need to obtain experimental samples on substrates of different stiffness, which has led to the obtaining of some experimental devices intended for this purpose. Since these devices, to date, have to be developed by each laboratory individually, adapting to the specific needs of each experiment, and that said devices cannot be applied, generally, to more than one use or experiment, there is today in day a major technological lack of large-scale production of this type of cultivation platform.
Es por ello que se plantea, en el estado de la técnica, la necesidad de desarrollar sistemas de análisis a gran escala (o "screening" masivo) que permitan, en un solo uso, hacer el estudio de la respuesta de los cultivos celulares en función de diferentes valores de rigidez del sustrato y realizar una caracterización rápida y sencilla de las células estudiadas, ya sean solas o bajo la influencia de agentes externos (drogas, agentes bioquímicos, etc.). That is why, in the state of the art, there is a need to develop large-scale analysis systems (or "mass screening") that allow, in a single use, to study the response of cell cultures in function of different values of substrate stiffness and perform a quick and simple characterization of the cells studied, either alone or under the influence of external agents (drugs, biochemical agents, etc.).
La presente invención está orientada a satisfacer dicha necesidad. The present invention is oriented to satisfy said need.
DESCRIPCIÓN BREVE DE LA INVENCIÓN BRIEF DESCRIPTION OF THE INVENTION
Tal y como se ha descrito en párrafos precedentes, la presente invención se refiere a un dispositivo encapsulable de tipo "lab-on-chip", destinado para su uso en el estudio de cultivos celulares, y compuesto preferentemente de un material plástico donde se localizan diferentes áreas o pocilios para el cultivo celular, en el fondo de los cuales se deposita un material gelificado con propiedades mecánicas diferentes en cada pocilio. Dicho dispositivo obedece al objeto principal de la presente invención, que consiste en la obtención de sistemas de análisis a gran escala que permitan hacer el estudio de la respuesta de un determinado tipo de células, fármacos, efecto sobre crecimiento, etc. en función de diferentes valores de rigidez del sustrato, en los que el usuario pueda realizar estudios de cultivo celular. As described in the preceding paragraphs, the present invention relates to an encapsulable device of the "lab-on-chip" type, intended for use in the study of cell cultures, and preferably composed of a plastic material where they are located different areas or wells for cell culture, at the bottom of which a gelled material with different mechanical properties is deposited in each well. Said device obeys the main object of the present invention, which consists in obtaining large-scale analysis systems that allow the study of the response of a certain type of cells, drugs, effect on growth, etc. depending on different values of substrate stiffness, in which the user can perform cell culture studies.
El citado objeto de la invención se realiza, en un primer aspecto de la misma, mediante un dispositivo de cultivo celular caracterizado porque comprende: Said object of the invention is carried out, in a first aspect thereof, by means of a cell culture device characterized in that it comprises:
- un generador de geles que comprende, al menos, dos canales de entrada de fluidos y un mezclador conectado a dichos canales de entrada de fluidos, estando el mezclador configurado para crear un gradiente discreto de mezclado a través de una estructura de ramificación por etapas, y donde el generador de geles comprende, adicionalmente, una pluralidad de canales de salida, conectados al mezclador en una o más de sus etapas de ramificación;
- una cámara de cultivo celular que comprende una pluralidad de pocilios de cultivo celular conectados a los canales de salida del generador de geles, de forma que se puedan depositar dichos geles como sustratos de los pocilios, y donde la cámara de cultivo celular comprende, asimismo, al menos un canal de entrada de fluido y, al menos, un canal de salida de fluido, estando ambos canales conectados a dicha cámara de cultivo celular. - a gel generator comprising at least two fluid inlet channels and a mixer connected to said fluid inlet channels, the mixer being configured to create a discrete mixing gradient through a step branching structure, and where the gel generator additionally comprises a plurality of output channels, connected to the mixer in one or more of its branching stages; - a cell culture chamber comprising a plurality of cell culture wells connected to the output channels of the gel generator, so that said gels can be deposited as substrates of the wells, and where the cell culture chamber also comprises , at least one fluid inlet channel and at least one fluid outlet channel, both channels being connected to said cell culture chamber.
En una realización preferente de la presente invención, la cámara de cultivo celular es una cámara encapsulable. Se consigue con ello facilitar la preparación de los cultivos, mediante el uso exclusivamente de los canales o microcanales del dispositivo, para la inyección de los sustratos, células y medio de cultivo, sin la necesidad de intervención manual adicional. La posibilidad de encapsular el dispositivo proporciona, asimismo, mayor robustez al mismo. In a preferred embodiment of the present invention, the cell culture chamber is an encapsulable chamber. This facilitates the preparation of the cultures, by using exclusively the channels or microchannels of the device, for the injection of the substrates, cells and culture medium, without the need for additional manual intervention. The possibility of encapsulating the device also provides greater robustness to it.
En otra realización preferente de la invención, el dispositivo está realizado como un chip basado en un material polimerizable mediante fotolitografía. Se consigue con ello una herramienta implementable a través de las técnicas de producción en masa conocidas en el campo de la fabricación de chips semiconductores, lo que proporciona una forma eficaz para el estudio en masa de cultivos, y su producción con costes competitivos. Preferentemente, el material de fabricación polimerizable del dispositivo comprende un polímero viscoso epoxi SU-8. In another preferred embodiment of the invention, the device is made as a chip based on a polymerizable material by photolithography. This achieves a tool that can be implemented through mass production techniques known in the field of semiconductor chip manufacturing, which provides an effective way to study mass crops, and their production at competitive costs. Preferably, the polymerizable manufacturing material of the device comprises an SU-8 epoxy viscous polymer.
Otro objeto de la presente invención se refiere a un método de cultivo celular que comprende el uso de un dispositivo según las realizaciones descritas en el presente documento, donde se hacen circular líquidos de distinta composición a través de los canales de entrada del generador de geles, de forma que los geles generados en los canales de salida de dicho generador de geles se depositen, por medio de la conexión con los pocilios de la cámara de cultivo celular, como sustratos de diferente rigidez de dichos pocilios. Preferentemente, las células cultivadas son introducidas en la cámara de cultivo, sobre los sustratos de los pocilios de dicha cámara. Another object of the present invention relates to a cell culture method comprising the use of a device according to the embodiments described herein, where liquids of different composition are circulated through the inlet channels of the gel generator, so that the gels generated in the exit channels of said gels generator are deposited, by means of the connection with the wells of the cell culture chamber, as substrates of different rigidity of said wells. Preferably, the cultured cells are introduced into the culture chamber, on the substrates of the wells of said chamber.
En una realización preferente del método de cultivo celular de la invención, se hace circular un líquido biocompatible y/o un medio de cultivo a través de los canales de entrada y salida de la cámara de cultivo.
Otro aspecto de la invención se refiere a un método de fabricación de un dispositivo según las realizaciones aquí descritas, que comprende la polimerización mediante fotolitografía de capas polimerizables que conforman un chip mediante su unión. In a preferred embodiment of the cell culture method of the invention, a biocompatible liquid and / or a culture medium is circulated through the inlet and outlet channels of the culture chamber. Another aspect of the invention relates to a method of manufacturing a device according to the embodiments described herein, which comprises the polymerization by photolithography of polymerizable layers that form a chip by joining it.
Dentro de las ventajas aportadas por el dispositivo de la invención, destacan las siguientes: Among the advantages provided by the device of the invention, the following stand out:
- Los valores de rigidez de la matriz de sustratos del dispositivo son conocidos con exactitud por el usuario. - The stiffness values of the substrate matrix of the device are known exactly by the user.
- Los valores de rigidez pueden ser prefijados por el usuario mediante la modificación de los componentes líquidos que dan lugar al gel en el mezclador. - The stiffness values can be preset by the user by modifying the liquid components that give rise to the gel in the mixer.
- El gradiente de rigidez producido varía en cantidades discretas y prefijadas, a diferencia de un gradiente continuo, donde las variaciones de rigidez son difíciles de determinar. - The stiffness gradient produced varies in discrete and preset amounts, unlike a continuous gradient, where stiffness variations are difficult to determine.
- La presencia de canales de entrada y salida en la cámara de cultivo permite la realización de experimentos a largo plazo (manteniendo dicha entrada y salida como canales de alimentación de un medio de cultivo), sin necesidad de intervención externa del usuario. - The presence of input and output channels in the culture chamber allows for long-term experiments (maintaining said input and output as feeding channels of a culture medium), without the need for external user intervention.
- La presencia de un flujo de líquido biocompatible o un medio de cultivo durante el cultivo celular permite, asimismo, la simulación de condiciones de tensión de cizalladura en los cultivos, lo que permite el estudio de los mismos, sometidos a dicha tensión. La principal ventaja del dispositivo es que facilita el testeo masivo de diferentes sustratos, con propiedades diferentes de rigidez, en un solo uso, de forma automática y sin la necesidad de preparación previa de cada pocilio. Se trata de un dispositivo que no existe actualmente en el mercado, lo que provoca que cada centro de investigación deba invertir cierto tiempo y esfuerzo a cubrir ese vacío, desarrollando sistemas propios. - The presence of a flow of biocompatible liquid or a culture medium during cell culture also allows the simulation of shear stress conditions in the cultures, which allows them to be studied, subjected to said tension. The main advantage of the device is that it facilitates the massive testing of different substrates, with different stiffness properties, in a single use, automatically and without the need for prior preparation of each well. It is a device that does not currently exist in the market, which causes each research center to invest some time and effort to fill that gap, developing its own systems.
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.
DESCRIPCIÓN DE LAS FIGURAS In addition to those already raised, other features and advantages of the invention will be apparent from the following description, as well as from the figures accompanying this document. DESCRIPTION OF THE FIGURES
La Figura 1 muestra una representación de los elementos del dispositivo de la invención, en una realización preferente de la misma (planta). Figure 1 shows a representation of the elements of the device of the invention, in a preferred embodiment thereof (plant).
La Figura 2 muestra una representación de la estructura del generador de geles del dispositivo de cultivo celular, en una realización preferente de la invención (planta). Figure 2 shows a representation of the gel generator structure of the cell culture device, in a preferred embodiment of the invention (plant).
Las Figuras 3(a-b) muestran una representación de la cámara de cultivo celular comprendida en el dispositivo de la invención, según una realización preferente de la misma (3(a) planta, 3(b) sección transversal). Figures 3 (a-b) show a representation of the cell culture chamber comprised in the device of the invention, according to a preferred embodiment of the same (3 (a) plant, 3 (b) cross section).
Las Figuras 4(a) y 5(a) muestran dos representaciones esquemáticas de las diferentes etapas de fabricación por máscaras del dispositivo de cultivo celular según realizaciones preferentes de la presente invención. Figures 4 (a) and 5 (a) show two schematic representations of the different masking stages of the cell culture device according to preferred embodiments of the present invention.
Las Figuras 4(b-f) y 5(b-g) muestran la planta y perfil del dispositivo durante las diferentes etapas de fabricación según realizaciones preferentes de la presente invención. Figures 4 (b-f) and 5 (b-g) show the plant and profile of the device during the different manufacturing stages according to preferred embodiments of the present invention.
Las Figuras 6(a-c), 7(a-c) y 8(a-c) muestran tres realizaciones alternativas del dispositivo de la invención, donde se proporcionan vistas generales de cada realización (Figuras 6a, 7a, 8a), vistas ampliadas de los mezcladores empleados (Figuras 6b, 7b, 8b) y vistas ampliadas de los pocilios comprendidos en la cámara de cultivo (Figuras 6c, 7c, 8c). Figures 6 (ac), 7 (ac) and 8 (ac) show three alternative embodiments of the device of the invention, where general views of each embodiment are provided (Figures 6a, 7a, 8a), enlarged views of the mixers employed ( Figures 6b, 7b, 8b) and enlarged views of the wells comprised in the culture chamber (Figures 6c, 7c, 8c).
La Figura 9 muestra una vista superior de una pluralidad de dispositivos de cultivo celular según una realización preferente de la invención, dispuestos como un chip multi-dispositivo destinado al estudio a gran escala de cultivos celulares. Figure 9 shows a top view of a plurality of cell culture devices according to a preferred embodiment of the invention, arranged as a multi-device chip intended for large-scale study of cell cultures.
La Figura 10 muestra una representación esquemática de las etapas del proceso de fabricación de un dispositivo según una realización de la invención. Figure 10 shows a schematic representation of the steps of the manufacturing process of a device according to an embodiment of the invention.
DESCRIPCIÓN DETALLADA DE LA INVENCIÓN
Tal y como se muestra esquemáticamente en la Figura 1 de este documento, el dispositivo de cultivo celular de la presente invención comprende: DETAILED DESCRIPTION OF THE INVENTION As schematically shown in Figure 1 of this document, the cell culture device of the present invention comprises:
- un generador de geles (1), que está preferentemente integrado por, al menos, dos canales de entrada (2, 3) de fluidos, destinados a conducir dos fluidos de diferentes propiedades físico-mecánicas, y un mezclador (4) destinado a mezclar dichos geles en diferentes proporciones, a través de una pluralidad de etapas, donde la mezcla a través de dichas etapas proporciona una pluralidad de geles de diferente rigidez, y donde el generador de geles comprende, asimismo, canales de salida (5) de dichos geles; - a gels generator (1), which is preferably integrated by at least two fluid inlet channels (2, 3), intended to conduct two fluids of different physical-mechanical properties, and a mixer (4) intended for mixing said gels in different proportions, through a plurality of stages, where mixing through said stages provides a plurality of gels of different stiffness, and where the gels generator also comprises outlet channels (5) of said gels;
- una cámara de cultivo celular (6), preferentemente encapsulable, que comprende una pluralidad de pocilios (7) para el cultivo de células, donde los pocilios (7) están conectados a los canales de salida (5) del generador de geles, de forma que se puedan depositar dichos geles (con distintos valores de rigidez) como sustratos de los pocilios (7). La cámara de cultivo celular (6) presenta, preferentemente, un canal de entrada de fluido (8) y un canal de salida de fluido (9), a través de los cuales es posible introducir las células de cultivo en los pocilios, así como hacer circular un flujo continuo de alimentación (medio de cultivo) de los cultivos estudiados. - a cell culture chamber (6), preferably encapsulable, comprising a plurality of wells (7) for cell culture, where the wells (7) are connected to the outlet channels (5) of the gel generator, of so that these gels (with different stiffness values) can be deposited as substrates of the wells (7). The cell culture chamber (6) preferably has a fluid inlet channel (8) and a fluid outlet channel (9), through which it is possible to introduce the culture cells into the wells, as well as circulate a continuous feed flow (culture medium) of the crops studied.
Adicionalmente, según lo representado en la Figura 2, el mezclador (4) del generador de geles (1) comprende, preferentemente, una estructura ramificada de árbol creciente en dirección a los canales de salida (5), de forma que, en cada etapa de ramificación, se producen mezclados sucesivos de los dos geles iniciales de entrada al generador (1), formándose así un gradiente de rigidez en los geles que salen por los canales de salida (5), tras su paso por el mezclador (4). Dichos canales se conectan, tal y como se ha dicho anteriormente, a los pocilios (7) de la cámara de cultivo celular (6). Mediante las diferentes mezclas de líquidos generadas en el mezclador (4) y la formación de geles en su interior, las proporciones de mezcla que llegan a los canales de salida (5) pueden ser determinadas con exactitud, a través del diseño del mezclador (4), por ejemplo mediante la variación del número de etapas de mezclado, el número de ramas en cada etapa, el grosor de los canales del mezclador, etc.,
lo que permite, asimismo, un ajuste de precisión de las propiedades físicas (por ejemplo, la rigidez) del gel que llega a cada pocilio (7), pudiendo éstas ser calculadas con exactitud. Ventajosamente, el dispositivo y el proceso de fabricación de la presente invención permiten rellenar los pocilios (7) con los diferentes geles que proceden del mezclador (5), sin ocupar ni obstruir la cámara de cultivo (6). Additionally, as depicted in Figure 2, the mixer (4) of the gel generator (1) preferably comprises a branched tree structure growing in the direction of the outlet channels (5), so that, at each stage of branching, successive mixtures of the two initial gels entering the generator (1) are produced, thus forming a gradient of stiffness in the gels that exit through the exit channels (5), after passing through the mixer (4). Said channels are connected, as mentioned above, to the wells (7) of the cell culture chamber (6). Through the different mixtures of liquids generated in the mixer (4) and the formation of gels inside, the mixing ratios that reach the outlet channels (5) can be determined accurately, through the design of the mixer (4 ), for example by varying the number of mixing stages, the number of branches in each stage, the thickness of the mixer channels, etc., which also allows a precise adjustment of the physical properties (for example, the stiffness) of the gel that reaches each well (7), which can be calculated accurately. Advantageously, the device and the manufacturing process of the present invention allow the wells (7) to be filled with the different gels that come from the mixer (5), without occupying or obstructing the culture chamber (6).
Tal y como se muestra en la Figura 3, la cámara de cultivo celular (6) comprende una pluralidad de áreas/pocilios (7) para alojar las células de cultivo, donde los fondos de dichas áreas/pocilios (7) se encuentran conectados a los canales de salida del generador de geles (1). De este modo, previamente a la introducción de las células de cultivo en los pocilios (7), es posible depositar el gradiente de geles generado como sustrato de dichos pocilios (7), obteniendo así una pluralidad de sustratos de diferente rigidez sobre los que se depositarán las células de cultivo. Adicionalmente, según lo descrito anteriormente, la cámara de cultivo celular (6) comprende un canal de entrada de fluido (8) y un canal de entrada de fluido (9), destinados ambos canales para la circulación del medio de cultivo de las células, así como para la introducción de dichas células en los pocilios (7). As shown in Figure 3, the cell culture chamber (6) comprises a plurality of areas / wells (7) to house the culture cells, where the bottoms of said areas / wells (7) are connected to the output channels of the gels generator (1). Thus, prior to the introduction of the culture cells in the wells (7), it is possible to deposit the gradient of gels generated as a substrate of said wells (7), thus obtaining a plurality of substrates of different stiffness on which they will deposit the culture cells. Additionally, as described above, the cell culture chamber (6) comprises a fluid inlet channel (8) and a fluid inlet channel (9), both channels intended for the circulation of the cell culture medium, as well as for the introduction of said cells in the wells (7).
Preferentemente, la fabricación del dispositivo de cultivo celular aquí descrito se lleva a cabo mediante un proceso de polimerización mediante fotolitografía, a través del cual se generan diferentes capas que se superponen unas encima de otras para conformar los elementos del dispositivo (generador de geles (1) y cámara de cultivo (6)). El empleo de tecnología fotolitográfica permite la fabricación a gran escala del dispositivo, proporcionando además la capacidad de montajes en serie de una pluralidad de dispositivos "lab-on-chip", lo que supone una importante mejora frente al estado de la técnica en la obtención de dispositivos de cultivo celular con sustratos de rigidez variable y destinados a su producción masiva. Preferably, the manufacture of the cell culture device described herein is carried out by a polymerization process by photolithography, through which different layers are generated that overlap one another to form the elements of the device (gels generator (1 ) and culture chamber (6)). The use of photolithographic technology allows the large-scale manufacture of the device, also providing the capacity for serial mounting of a plurality of "lab-on-chip" devices, which is a significant improvement over the state of the art in obtaining of cell culture devices with substrates of varying stiffness and intended for mass production.
El material de fabricación del dispositivo es, preferentemente, un polímero viscoso epoxi SU-8. En las Figuras 4(a-g) y 5(a-h) de este documento se muestran dos esquemas de fabricación por máscaras del dispositivo según
realizaciones preferentes de la presente invención. En ellas se representan los siguientes elementos: The manufacturing material of the device is preferably an SU-8 epoxy viscous polymer. In Figures 4 (ag) and 5 (ah) of this document two manufacturing schemes by masks of the device according to Preferred embodiments of the present invention. They represent the following elements:
- Figura 4(a), basada en cinco máscaras: Cubierta superior del dispositivo (C); cámara de cultivo celular (CH); pocilios (W); mezclador (M); y fondo (máscara) inferior del dispositivo (G). - Figure 4 (a), based on five masks: Top cover of the device (C); cell culture chamber (CH); wells (W); mixer (M); and bottom (mask) of the device (G).
- Figura 5(a), basada en seis máscaras: Cubierta (máscara) superior del dispositivo (C); cámara de cultivo celular (CH); mezclador 2 (M2); agujeros (H); mezclador 1 ; y fondo (máscara) inferior del dispositivo (G). - Figure 5 (a), based on six masks: Upper cover (mask) of the device (C); cell culture chamber (CH); mixer 2 (M2); holes (H); mixer 1; and bottom (mask) of the device (G).
Cada una de las Figuras 4(b-f) y 5(b-g) muestran vistas superiores y laterales de dos realizaciones preferentes de la presente invención. Ambas realizaciones comprenden montajes encapsulados, en los cuales los cultivos celulares permanecen aislados del exterior. Ello hace mucho más fácil la preparación de los cultivos, mediante el uso exclusivamente de los canales o microcanales del dispositivo, para la inyección de los sustratos, células y medio de cultivo, sin la necesidad de intervención manual adicional. La posibilidad de encapsular el dispositivo proporciona, asimismo, mayor robustez al mismo. Each of Figures 4 (b-f) and 5 (b-g) show top and side views of two preferred embodiments of the present invention. Both embodiments comprise encapsulated assemblies, in which cell cultures remain isolated from the outside. This makes the preparation of the cultures much easier, by using exclusively the channels or microchannels of the device, for the injection of the substrates, cells and culture medium, without the need for additional manual intervention. The possibility of encapsulating the device also provides greater robustness to it.
Las Figuras 6(a-c), 7(a-c) y 8(a-c) muestran tres realizaciones alternativas del dispositivo de la invención, donde se proporcionan vistas generales de cada realización (Figuras 6a, 7a, 8a), vistas ampliadas de los mezcladores empleados (Figuras 6b, 7b, 8b) y vistas ampliadas de los pocilios comprendidos en la cámara de cultivo (Figuras 6c, 7c, 8c). Figures 6 (ac), 7 (ac) and 8 (ac) show three alternative embodiments of the device of the invention, where general views of each embodiment are provided (Figures 6a, 7a, 8a), enlarged views of the mixers employed ( Figures 6b, 7b, 8b) and enlarged views of the wells comprised in the culture chamber (Figures 6c, 7c, 8c).
Adicionalmente, la Figura 9 muestra una vista superior de una pluralidad de dispositivos de cultivo celular según una realización preferente de la invención, dispuestos como un chip multi-dispositivo destinado al estudio a gran escala de cultivos celulares. Additionally, Figure 9 shows a top view of a plurality of cell culture devices according to a preferred embodiment of the invention, arranged as a multi-device chip intended for large-scale study of cell cultures.
Según lo descrito anteriormente, otro objeto de la presente invención se refiere a un método de fabricación de un dispositivo según las realizaciones anteriores, en el que se disponen capas sucesivas de material polimerizable (preferentemente, comprendiendo dichas capas un polímero SU-8) para conformar un chip mediante técnicas de fotolitografía. En una realización
preferente de dicho método, tal y como se muestra en la Figura 10, se llevan a cabo los siguientes pasos: As described above, another object of the present invention relates to a method of manufacturing a device according to the above embodiments, in which successive layers of polymerizable material (preferably, said layers comprising an SU-8 polymer) are arranged to form a chip using photolithography techniques. In one embodiment Preferred of said method, as shown in Figure 10, the following steps are carried out:
a) Una capa metálica (10), preferentemente de aluminio o cromo, es depositada sobre una primera superficie de soporte (11) (denominada también dicha superficie de soporte como "sustrato", aunque sin confundir con los sustratos de los pocilios/áreas (7) donde se depositan las muestras de cultivo). b) Una primera capa de SU-8 (12) es depositada sobre la capa metálica (10) de la primera superficie de soporte (11), y definida mediante fotolitografía para ser usada como capa "suelo" del mezclador (4) de componentes de los geles. a) A metallic layer (10), preferably of aluminum or chrome, is deposited on a first support surface (11) (also referred to as said support surface as "substrate", although not confused with the substrates of the wells / areas ( 7) where the culture samples are deposited). b) A first SU-8 layer (12) is deposited on the metal layer (10) of the first support surface (11), and defined by photolithography to be used as a "ground" layer of the component mixer (4) of the gels.
c) Una segunda capa de SU-8 (13) se deposita sobre la primera capa de SU-8 (12) de la primera superficie de soporte (11), y es definida mediante fotolitografía para fabricar los canales (5) que van a ser utilizados para la mezcla de los componentes de los geles, y la localización de las áreas/pocilios (7) asignados. c) A second SU-8 layer (13) is deposited on the first SU-8 layer (12) of the first support surface (11), and is defined by photolithography to manufacture the channels (5) that are going to be used for the mixture of the components of the gels, and the location of the areas / wells (7) assigned.
d) Una tercera capa de SU-8 (14) se deposita sobre una segunda superficie de soporte (15) que comprende un material (16) con baja adherencia al SU-8 (por ejemplo, kapton), definiéndose dicha tercera capa de SU-8 (14) mediante fotolitografía, para hacer de "tapa" de las estructuras definidas en la primera superficie de soporte (11), excepto las entradas y salidas fluídicas para poder acceder a los canales (2, 3, 5). d) A third layer of SU-8 (14) is deposited on a second support surface (15) comprising a material (16) with low adhesion to the SU-8 (for example, kapton), said third layer of SU being defined -8 (14) by photolithography, to "cover" the structures defined in the first support surface (11), except fluidic entrances and exits to access the channels (2, 3, 5).
e) La segunda superficie de soporte (15) se enfrenta a la primera superficie de soporte (11), de forma que las capas de SU-8 (12, 13, 14) comprendidas en dichas superficies queden enfrentadas y alineadas entre sí, pegándose las capas de SU-8 de ambas superficies de soporte (11 , 15) a través de la aplicación de presión y temperatura. e) The second support surface (15) faces the first support surface (11), so that the layers of SU-8 (12, 13, 14) comprised on said surfaces are facing and aligned with each other, sticking together. the SU-8 layers of both support surfaces (11, 15) through the application of pressure and temperature.
f) La segunda superficie de soporte (15) es liberada de la primera superficie de soporte (11) a través del material (16) con baja adherencia al SU-8, dejando las capas primera (12), segunda (13) y tercera (14) de SU-8 sobre la primera superficie de soporte (11). f) The second support surface (15) is released from the first support surface (11) through the material (16) with low adhesion to the SU-8, leaving the first (12), second (13) and third layers (14) of SU-8 on the first support surface (11).
g) La oblea formada por la unión de las capas (10, 12, 13, 14) de la primera (11) y la segunda (15) superficie de soporte es introducida en un encapsulado, donde se realizan conexiones fluídicas sobre las entradas y salidas a los canales de SU-8 para introducir los componentes de los geles y, por tanto, para
realizar la mezcla deseada, localizando las diferentes mezclas en las áreas/pocilios (7) asignados. g) The wafer formed by the union of the layers (10, 12, 13, 14) of the first (11) and the second (15) support surface is introduced in an encapsulation, where fluidic connections are made on the entrances and outputs to the SU-8 channels to introduce the components of the gels and, therefore, to make the desired mixture, locating the different mixtures in the assigned areas / wells (7).
h) Se deposita una capa metálica (17) sobre una tercera superficie de soporte (18), preferentemente de un metal diferente al de la capa metálica (10) depositada sobre la primera superficie de soporte (11). h) A metal layer (17) is deposited on a third support surface (18), preferably of a different metal than the metal layer (10) deposited on the first support surface (11).
i) Una cuarta capa de SU-8 (19) se deposita sobre la capa metálica de la tercera superficie de soporte (18) y se define a través de procesos de fotolitografía para ser usada como capa "suelo" del dispositivo final. i) A fourth layer of SU-8 (19) is deposited on the metal layer of the third support surface (18) and is defined through photolithography processes to be used as the "ground" layer of the final device.
j) La tercera superficie de soporte (18) se enfrenta a la primera superficie de soporte (11), de forma que las capas de SU-8 (12, 13, 14, 19) depositadas sobre cada una de dichas superficies queden enfrentadas y alineadas entre sí, y las capas de SU-8 de ambas superficies son pegadas a través de la aplicación de presión y temperatura. j) The third support surface (18) faces the first support surface (11), so that the layers of SU-8 (12, 13, 14, 19) deposited on each of said surfaces are facing and aligned with each other, and the SU-8 layers of both surfaces are glued through the application of pressure and temperature.
k) La oblea formada por las capas de las superficies de soporte primera (11) y tercera (18), pegadas entre sí por medio de las capas de SU-8, es introducida en un agente químico para disolver selectivamente la capa metálica (10) depositada en la primera superficie de soporte (11), de forma que todas las capas de SU-8 (12, 13, 14, 19) quedan adheridas a la tercera superficie de soporte (18); k) The wafer formed by the layers of the first (11) and third (18) support surfaces, glued together by means of the SU-8 layers, is introduced into a chemical agent to selectively dissolve the metal layer (10 ) deposited on the first support surface (11), so that all layers of SU-8 (12, 13, 14, 19) are adhered to the third support surface (18);
I) Se deposita una quinta capa de SU-8 (20) sobre una cuarta superficie de soporte (21) que comprende un material (22) con baja adherencia al SU-8, preferentemente kapton, y se define a través de procesos de fotolitografía para ser usada como capa techo del dispositivo final. I) A fifth layer of SU-8 (20) is deposited on a fourth support surface (21) comprising a material (22) with low adhesion to SU-8, preferably kapton, and is defined by photolithography processes to be used as the roof layer of the final device.
m) Se deposita una sexta capa de SU-8 (23) sobre la quinta capa (20) de la cuarta superficie de soporte (21), y se define a través de procesos de fotolitografía para ser usada como capa de "cámara de cultivo" del dispositivo final. m) A sixth layer of SU-8 (23) is deposited on the fifth layer (20) of the fourth support surface (21), and is defined through photolithography processes to be used as a "culture chamber" layer "of the final device.
n) La cuarta superficie de soporte (21) se enfrenta a la tercera superficie de soporte (18), de forma que las capas de SU-8 (12, 13, 14, 19, 20, 23) depositadas sobre cada una de dichas superficies queden enfrentadas, y alineadas entre sí, y las capas de SU-8 de ambas superficies de soporte son pegadas a través de la aplicación de presión y temperatura.
o) Las seis capas de SU-8 (12, 13, 14, 19, 20, 23) son liberadas de las superficies de soporte restantes (18, 21), mediante la introducción de la oblea formada por dichas capas en un atacante químico selectivo para el metal comprendido en la capa metálica (17) de la tercera superficie de soporte (18), y mediante la retirada de la cuarta superficie de soporte (21) que comprende el material (22) con baja adherencia al SU-8. n) The fourth support surface (21) faces the third support surface (18), so that the layers of SU-8 (12, 13, 14, 19, 20, 23) deposited on each of said surfaces are facing each other, and aligned with each other, and the SU-8 layers of both support surfaces are glued through the application of pressure and temperature. o) The six layers of SU-8 (12, 13, 14, 19, 20, 23) are released from the remaining support surfaces (18, 21), by introducing the wafer formed by said layers into a chemical attacker selective for the metal comprised in the metal layer (17) of the third support surface (18), and by removing the fourth support surface (21) comprising the material (22) with low adhesion to the SU-8.
Una vez descrita la presente invención y algunas de sus realizaciones preferentes, junto con sus principales ventajas sobre el 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 elementos del dispositivo o al método descritos, ni a las realizaciones referidas en los ejemplos de la 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.
Once the present invention and some of its preferred embodiments have been described, together with its main advantages over the state of the art, it should be noted again that its application should not be understood as necessarily limited to a particular configuration of the elements of the device or method described, nor to the embodiments referred to in the examples of the invention, but it is also applicable to other types of configurations and procedures, by means of suitable variations in its elements, provided that said variations do not alter the essence of the invention , as well as the object of it.
Claims
1. - Dispositivo de cultivo celular caracterizado porque comprende: 1. - Cell culture device characterized because it comprises:
- un generador de geles (1) que comprende, al menos, dos canales de entrada (2, 3) de fluidos y un mezclador (4) conectado a dichos canales de entrada (2, - a gel generator (1) that comprises at least two fluid input channels (2, 3) and a mixer (4) connected to said input channels (2, 3).
3) de líquidos, estando el mezclador (4) configurado para crear un gradiente de mezclado a través de una estructura de ramificación por etapas, y donde el generador de geles (1) comprende, adicionalmente, una pluralidad de canales de salida (5), conectados al mezclador en una o más de sus etapas de ramificación; 3) of liquids, the mixer (4) being configured to create a mixing gradient through a staged branching structure, and where the gel generator (1) additionally comprises a plurality of outlet channels (5) , connected to the mixer in one or more of its branch stages;
- una cámara de cultivo celular (6) que comprende una pluralidad de pocilios (7) de cultivo celular conectados a los canales de salida (5) del generador de geles (1), de forma que se puedan depositar dichos geles como sustratos de los pocilios (7), y donde la cámara de cultivo celular (6) comprende, asimismo, al menos un canal de entrada de fluido (8) y, al menos, un canal de salida de fluido (9), estando ambos canales conectados a dicha cámara de cultivo celular (6). - a cell culture chamber (6) that comprises a plurality of cell culture wells (7) connected to the outlet channels (5) of the gel generator (1), so that said gels can be deposited as substrates of the wells (7), and where the cell culture chamber (6) also comprises at least one fluid inlet channel (8) and at least one fluid outlet channel (9), both channels being connected to said cell culture chamber (6).
2. - Dispositivo según la reivindicación anterior, donde la cámara de cultivo celular (6) es una cámara encapsulable. 2. - Device according to the previous claim, where the cell culture chamber (6) is an encapsulable chamber.
3.- Dispositivo de cultivo celular según cualquiera de las reivindicaciones anteriores, caracterizado por estar realizado como un chip basado en un material polimerizable mediante fotolitografía. 3.- Cell culture device according to any of the previous claims, characterized in that it is made as a chip based on a material that can be polymerized by photolithography.
4.- Dispositivo según la reivindicación anterior, donde el material polimerizable comprende SU-8. 4.- Device according to the previous claim, wherein the polymerizable material comprises SU-8.
5.- Método de cultivo celular que comprende el uso de un dispositivo según cualquiera de las reivindicaciones anteriores, y donde se hace circular líquidos de distinta composición a través los canales de entrada (2, 3) del generador de geles (1), de forma que los geles generados en los canales de salida (5) de dicho generador de geles se depositen, por medio de la conexión con los pocilios (7) de la cámara de cultivo celular (6), como sustratos de diferente rigidez de dichos pocilios (7).
5.- Cell culture method that comprises the use of a device according to any of the previous claims, and where liquids of different composition are circulated through the inlet channels (2, 3) of the gel generator (1), of so that the gels generated in the output channels (5) of said gel generator are deposited, by means of the connection with the wells (7) of the cell culture chamber (6), as substrates of different rigidity of said wells. (7).
6. - Método según la reivindicación anterior, que comprende, asimismo, la introducción de las células cultivadas en la cámara de cultivo (6), sobre los sustratos de los pocilios (7) de dicha cámara (6). 6. - Method according to the previous claim, which also comprises the introduction of the cultured cells in the culture chamber (6), on the substrates of the wells (7) of said chamber (6).
7. - Método según cualquiera de las reivindicaciones 5-6, donde se hace circular un líquido biocompatible y/o un medio de cultivo a través de los canales de entrada y salida de la cámara de cultivo. 7. - Method according to any of claims 5-6, wherein a biocompatible liquid and/or a culture medium is circulated through the entry and exit channels of the culture chamber.
8. - Método de fabricación de un dispositivo según cualquiera de las reivindicaciones 1-4, que comprende la polimerización mediante fotolitografía de capas polimerizables que conforman un chip mediante su unión. 8. - Method of manufacturing a device according to any of claims 1-4, which comprises the polymerization by photolithography of polymerizable layers that form a chip by joining them.
9. - Método de fabricación según la reivindicación anterior, que comprende los siguientes pasos: 9. - Manufacturing method according to the previous claim, which includes the following steps:
- una capa metálica - a metallic layer
(10) es depositada sobre una primera superficie de soporte(10) is deposited on a first support surface
(11) ; (eleven) ;
- una primera capa de SU-8 (12) es depositada sobre la capa metálica (10) de la primera superficie de soporte (11), y definida mediante fotolitografía para ser usada como capa suelo del mezclador (4) de componentes de los geles; - a first layer of SU-8 (12) is deposited on the metal layer (10) of the first support surface (11), and defined by photolithography to be used as the ground layer of the mixer (4) of gel components ;
- una segunda capa de SU-8 (13) se deposita sobre la primera capa de SU-8 - a second layer of SU-8 (13) is deposited on the first layer of SU-8
(12) de la primera superficie de soporte (11), y es definida mediante fotolitografía para fabricar los canales que van a ser utilizados para la mezcla de los componentes de los geles, y la localización de las áreas/pocilios (7) asignados; (12) of the first support surface (11), and is defined by photolithography to manufacture the channels that will be used for mixing the components of the gels, and the location of the assigned areas/wells (7);
- una tercera capa de SU-8 (14) se deposita sobre una segunda superficie de soporte (15) que comprende un material (16) con baja adherencia al SU-8, definiéndose dicha tercera capa (14) mediante fotolitografía, para hacer de tapa de las estructuras definidas en la primera superficie de soporte (11), excepto las entradas y salidas fluídicas, para poder acceder a los canales (2, 3, 5); - a third layer of SU-8 (14) is deposited on a second support surface (15) that comprises a material (16) with low adhesion to SU-8, said third layer (14) being defined by photolithography, to make cover of the structures defined in the first support surface (11), except for the fluidic inlets and outlets, to be able to access the channels (2, 3, 5);
- la segunda superficie de soporte (15) se enfrenta a la primera superficie de soporte (11), de forma que las capas de SU-8 (12, 13, 14) comprendidas en dichas superficies queden enfrentadas y alineadas entre sí, pegándose las
capas de SU-8 (12, 13, 14) de ambas superficies (11 , 15) a través de ia aplicación de presión y temperatura; - the second support surface (15) faces the first support surface (11), so that the layers of SU-8 (12, 13, 14) included in said surfaces are facing and aligned with each other, sticking the layers of SU-8 (12, 13, 14) of both surfaces (11, 15) through the application of pressure and temperature;
- la segunda superficie de soporte (15) es liberada de la primera superficie de soporte (11) a través del material (16) con baja adherencia al SU-8, dejando las capas primera (12), segunda (13) y tercera (14) de SU-8 sobre la primera superficie de soporte (11); - the second support surface (15) is released from the first support surface (11) through the material (16) with low adhesion to SU-8, leaving the first (12), second (13) and third layers ( 14) of SU-8 on the first support surface (11);
- la oblea formada por la unión de las capas (10 12, 13, 14) de la primera (11) y la segunda (15) superficie de soporte es introducida en un encapsulado, donde se realizan conexiones fluídicas sobre las entradas y salidas a los canales de SU-8 para introducir los componentes de los geles y, por tanto, para realizar la mezcla deseada, localizando las diferentes mezclas en las áreas/pocilios (7) asignados; - the wafer formed by the union of the layers (10, 12, 13, 14) of the first (11) and the second (15) support surface is introduced into an encapsulation, where fluidic connections are made on the inputs and outputs to the SU-8 channels to introduce the components of the gels and, therefore, to make the desired mixture, locating the different mixtures in the assigned areas/wells (7);
- se deposita una capa metálica (17) sobre una tercera superficie de soporte (18); - a metallic layer (17) is deposited on a third support surface (18);
- una cuarta capa de SU-8 (19) se deposita sobre la capa metálica (17) de la tercera superficie de soporte (18) y se define a través de procesos de fotolitografía para ser usada como capa suelo del dispositivo final; - a fourth layer of SU-8 (19) is deposited on the metal layer (17) of the third support surface (18) and is defined through photolithography processes to be used as the ground layer of the final device;
- la tercera superficie dé soporte (18) se enfrenta a la primera superficie de soporte (11), de forma que las capas de SU-8 (12, 13, 14, 19) depositadas sobre cada una de dichas superficies (11 , 18) queden enfrentadas y alineadas entre sí, y las capas de SU-8 de ambas superficies (12, 13, 14, 19) son pegadas a través de la aplicación de presión y temperatura; - the third support surface (18) faces the first support surface (11), so that the layers of SU-8 (12, 13, 14, 19) deposited on each of said surfaces (11, 18 ) are facing and aligned with each other, and the SU-8 layers of both surfaces (12, 13, 14, 19) are glued through the application of pressure and temperature;
- la oblea formada por las capas de las superficies de soporte primera (11) y tercera (18), pegadas entre sí por medio de las capas de SU-8 (12, 13, 14, 19), es introducida en un agente químico para disolver selectivamente la capa metálica (10) de la primera superficie de soporte (11), de forma que todas las capas de SU-8 (12, 13, 14, 19) quedan adheridas a la tercera superficie de soporte (18); - the wafer formed by the layers of the first (11) and third (18) support surfaces, glued together by means of the SU-8 layers (12, 13, 14, 19), is introduced into a chemical agent to selectively dissolve the metal layer (10) of the first support surface (11), so that all the SU-8 layers (12, 13, 14, 19) are adhered to the third support surface (18);
- se deposita una quinta capa de SU-8 (20) sobre una cuarta superficie de soporte (21) que comprende un material (22) con baja adherencia al SU-8, y se
define a través de procesos de fotolitografía para ser usada como capa techo del dispositivo final; - a fifth layer of SU-8 (20) is deposited on a fourth support surface (21) that comprises a material (22) with low adhesion to SU-8, and defined through photolithography processes to be used as the roof layer of the final device;
- se deposita una sexta capa de SU-8 (23) sobre la quinta capa de SU-8 (20) de la cuarta superficie de soporte (21), y se define a través de procesos de fotolitografía para ser usada como capa de cámara de cultivo del dispositivo final; - a sixth layer of SU-8 (23) is deposited on the fifth layer of SU-8 (20) of the fourth support surface (21), and is defined through photolithography processes to be used as a chamber layer final device cultivation;
- la cuarta superficie de soporte (21) se enfrenta a la tercera superficie de soporte (18), de forma que las capas de SU-8 (12,13, 14, 19, 20, 23) depositadas sobre cada una de dichas superficies queden enfrentadas y alineadas entre sí, y las capas de SU-8 (12, 13, 14, 19, 20, 23) de ambas superficies de soporte son pegadas a través de la aplicación de presión y temperatura; - the fourth support surface (21) faces the third support surface (18), so that the SU-8 layers (12,13, 14, 19, 20, 23) deposited on each of said surfaces are facing and aligned with each other, and the SU-8 layers (12, 13, 14, 19, 20, 23) of both support surfaces are glued through the application of pressure and temperature;
- las seis capas de SU-8 (12, 13, 14, 19, 20, 23) son liberadas de las superficies de soporte restantes (18, 21), mediante la introducción de la oblea formada por dichas capas en un atacante químico selectivo para la capa metálica (17) de la tercera superficie de soporte (18), y mediante la retirada de la cuarta superficie de soporte (21) que comprende el material (22) con baja adherencia al SU-8. - the six SU-8 layers (12, 13, 14, 19, 20, 23) are released from the remaining support surfaces (18, 21), by introducing the wafer formed by said layers into a selective chemical etchant for the metallic layer (17) of the third support surface (18), and by removing the fourth support surface (21) that comprises the material (22) with low adhesion to the SU-8.
10. - Método según la reivindicación anterior, donde el material (16, 22) con baja adherencia a SU-8 comprende kapton. 10. - Method according to the previous claim, where the material (16, 22) with low adhesion to SU-8 comprises kapton.
1. - Método según cualquiera de las reivindicaciones 9-10, donde las capas metálicas (10, 17) comprenden aluminio o cromo. 1. - Method according to any of claims 9-10, where the metal layers (10, 17) comprise aluminum or chrome.
12. - Método según cualquiera de las reivindicaciones 9-12, donde la capa metálica (10) de la primera superficie de soporte (11) comprende un metal diferente al metal de la capa metálica (17) de la tercera superficie de soporte (18). 12. - Method according to any of claims 9-12, wherein the metal layer (10) of the first support surface (11) comprises a different metal than the metal of the metal layer (17) of the third support surface (18). ).
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| ES201230911A ES2438391B1 (en) | 2012-06-12 | 2012-06-12 | CELLULAR CULTURE DEVICE AND METHOD ASSOCIATED WITH SUCH DEVICE |
| ESP201230911 | 2012-06-12 |
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| WO2013186408A1 true WO2013186408A1 (en) | 2013-12-19 |
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| PCT/ES2013/000141 WO2013186408A1 (en) | 2012-06-12 | 2013-06-10 | Cell culture device and method associated with said |
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| ES (1) | ES2438391B1 (en) |
| WO (1) | WO2013186408A1 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018067802A1 (en) * | 2016-10-07 | 2018-04-12 | The Regents Of The University Of Michigan | Systems and methods for high throughput screening |
| CN110732355A (en) * | 2019-09-27 | 2020-01-31 | 东南大学 | micro-mixing micro-fluidic chip |
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|---|---|---|---|---|
| GB2421076A (en) * | 2004-12-07 | 2006-06-14 | Univ Southampton | Identifiable particles and uses thereof |
| WO2010024779A1 (en) * | 2008-08-27 | 2010-03-04 | Agency For Science, Technology And Research | Microfluidic continuous flow device for culturing biological material |
| US20100304306A1 (en) * | 2006-05-22 | 2010-12-02 | Ikerlan Centro De Investigaciones | Flexible micro/nanofluidic devices |
| WO2012040579A2 (en) * | 2010-09-23 | 2012-03-29 | President And Fellows Of Harvard College | Molecular screening across defined stiffness matrices |
-
2012
- 2012-06-12 ES ES201230911A patent/ES2438391B1/en not_active Expired - Fee Related
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| Publication number | Priority date | Publication date | Assignee | Title |
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| GB2421076A (en) * | 2004-12-07 | 2006-06-14 | Univ Southampton | Identifiable particles and uses thereof |
| US20100304306A1 (en) * | 2006-05-22 | 2010-12-02 | Ikerlan Centro De Investigaciones | Flexible micro/nanofluidic devices |
| WO2010024779A1 (en) * | 2008-08-27 | 2010-03-04 | Agency For Science, Technology And Research | Microfluidic continuous flow device for culturing biological material |
| WO2012040579A2 (en) * | 2010-09-23 | 2012-03-29 | President And Fellows Of Harvard College | Molecular screening across defined stiffness matrices |
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| JANG, YH.: "An integrated microfluidic device for two-dimensional combinatorial dilution.", AUTHORMANUSCRIPT AVAILABLE IN PMC THE 22.05.2012 PUBLISHED IN FINAL, vol. 11, no. 19, 7 October 2011 (2011-10-07), pages 3277 - 3286, Retrieved from the Internet <URL:http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3357545/> <DOI: 10.1039/c11c20449a> [retrieved on 20130930] * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018067802A1 (en) * | 2016-10-07 | 2018-04-12 | The Regents Of The University Of Michigan | Systems and methods for high throughput screening |
| CN110732355A (en) * | 2019-09-27 | 2020-01-31 | 东南大学 | micro-mixing micro-fluidic chip |
| CN110732355B (en) * | 2019-09-27 | 2021-09-28 | 东南大学 | Micro-mixing micro-fluidic chip |
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| Publication number | Publication date |
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| ES2438391B1 (en) | 2014-10-22 |
| ES2438391A1 (en) | 2014-01-16 |
| WO2013186408A8 (en) | 2014-02-06 |
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