ES2438391A1 - Cell culture device and method associated with said - Google Patents

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

Cell culture device and method associated with said device

FIELD OF THE INVENTION

The present invention refers to an encapsulable device of the "laboratory chip" (or, in English, "lab-on-chip") intended for use in housing and cell culture study, 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 an array (or "array") of substrates is deposited, wherein said substrates deposited in each well have preferably different stiffness values.

BACKGROUND OF THE INVENTION

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, there is a significant technological lack of large-scale production of this type of cultivation platform.

That is why, in the state of the art, there is a need to develop large-scale analysis systems (or "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.).

The present invention is oriented to satisfy said need.

BRIEF DESCRIPTION OF THE INVENTION

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.

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 wherein 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.

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.

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.

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 output channels of said gel generator are deposited, by means of the connection with the wells of the cell culture chamber, as substrates of different stiffness of said wells. Preferably, the cultured cells are introduced into the culture chamber, on the substrates of the wells of said chamber.

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.

Among the advantages provided by the device of the invention, the following stand out:

-

 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 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.

-

 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.

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

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.

DETAILED DESCRIPTION OF THE INVENTION

As schematically shown in Figure 1 of this document, 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 to mix 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 output 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, 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.

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). These 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 exactly. 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).

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).

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 represents 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. In Figures 4 (a-g) and 5 (a-h) of this document two masking schemes of the device according to preferred embodiments of the present invention are shown. They represent the following elements:

-

 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).

-

 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).

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.

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).

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.

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 a preferred embodiment of said method, as shown in Figure 10, the following steps are carried out:

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) 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 mixing the components of the gels, and the location of the assigned areas / wells (7). 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 means of photolithography, to act as a “cover” of the structures defined in the first support surface (11), except the fluidic entrances and exits to be able to access the channels (2, 3, 5). 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) 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 (14) layers of SU-8 on the first support surface (11). 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 perform the desired mixture, locating the different mixtures in the assigned areas / wells (7). 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) 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) 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) 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); l) 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) 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) 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.

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 (12)

1.- Cell culture device characterized in that it comprises:

-

 a gels generator (1) comprising at least two fluid inlet channels (2, 3) and a mixer

(4) connected to said liquid inlet channels (2, 3), the mixer (4) being configured to create a mixing gradient through a stage branching structure, and where the gels generator (1) comprises , additionally, a plurality of output channels (5), connected to the mixer in one or more of its branching stages;

-

 a cell culture chamber (6) comprising a plurality of cell culture wells (7) connected to the outlet channels (5) of the gels 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. Device according to the preceding claim, wherein the cell culture chamber (6) is an encapsulable chamber. 3. Cell culture device according to any of the preceding claims, characterized in that it is made as a chip based on a polymerizable material by photolithography. 4. Device according to the preceding claim, wherein the polymerizable material comprises SU-8. 5. Method of cell culture comprising the use of a device according to any of the preceding claims, and where liquids of different composition are circulated through the inlet channels (2, 3) of the gels generator (1), of such 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 stiffness of said wells (7). 6. Method according to the preceding claim, which also includes the introduction of cultured cells in the culture chamber (6), on the substrates of the wells (7) of said chamber (6). 7. Method according to any of claims 5-6, wherein a biocompatible liquid and / or a culture medium is circulated through the inlet and outlet channels of the culture chamber. 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 it. 9. Manufacturing method according to the preceding claim, comprising the following steps:

-

 a metal layer (10) is deposited on a first support surface (11);

-

 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 a soil layer of the mixer (4) of gels components;

-

 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 to be used for mixing. of the components of the gels, and the location of the assigned areas / wells (7);

-

 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, said third layer (14) being defined by photolithography, to act as a cover of the structures defined in the first support surface (11), except the fluidic entrances and exits, in order 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, the layers sticking together SU-8 (12, 13, 14) of both surfaces (11, 15) through the application of pressure and temperature;

-

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 (14) layers of SU-8 on the first support surface (11);

-

 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 exits to the SU-8 channels to introduce the components of the gels and, therefore, to perform the desired mixture, locating the different mixtures in the assigned areas / wells (7);

-

 a metal layer (17) is deposited on a third support surface (18);

-

 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;

-

 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) they 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;

-

 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 for selectively dissolving the metal layer (10) of the first support surface (11), so that all layers of SU-8 (12, 13, 14, 19) are adhered to the third support surface (18);

-

 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, and is defined through photolithography processes to be used as a layer roof of the final device;

-

 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 camera layer of culture 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 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;

-

 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 selective chemical attacker for 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. 10. Method according to the preceding claim, wherein the material (16, 22) with low adhesion to SU-8 comprises kapton. 11. Method according to any of claims 9-10, wherein the metal layers (10, 17) comprise aluminum or chrome. 12. Method according to any of claims 9-12, wherein the metal layer (10) of the first support surface (11) comprises a metal other than the metal of the metal layer (17) of the third support surface (18 ). FIG. one FIG. 2 FIG. 3 FIG. 4 FIG. 5 Fig. 6a Fig. 6b Fig. 6c Fig. 7a Fig. 7b Fig. 7c Fig. 8a Fig. 8b Fig. 8c FIG. 9 FIG. 10