JP5594658B2 - Substance distribution control method, device, cell culture method, cell differentiation control method - Google Patents

Description

  The present invention relates to a method for controlling a substance distribution in a micro space filled with a liquid, a device used in the method, a cell culture method using the method, and a cell differentiation control method.

Currently, researches on pluripotent stem cells, especially embryonic stem cells and induced pluripotent stem cells, are widely conducted, and the results of such research include regenerative medicine, cell / tissue engineering, drugs Application to screening is expected. However, at present, the differentiation efficiency of pluripotent stem cells is still low at a few percent, and there is a need for improvement.
Recent research has shown that the concentration of microenvironments surrounding cells (for example, the concentration of substances that affect differentiation (eg, humoral factors such as differentiation regulators), cell-cell interactions, interactions with extracellular matrix, It has been found that physical stimuli, etc.) influence the direction of cell differentiation. Therefore, to improve differentiation efficiency, it is considered important to precisely design and control the microenvironment surrounding the cells.

Conventionally, as a cell culture method, a stationary culture method in which a culture vessel such as a culture dish or a culture plate is used and cultured in a culture solution in a stationary / closed environment is generally used. However, in stationary culture, the supply of substances such as nutrient components and other arbitrary liquid factors can be performed only monotonously, and the distribution of substances in the culture environment cannot be precisely controlled.
As another cell culture method, there is a perfusion culture method in which a culture solution containing nutrient components and oxygen is perfused in a culture vessel. In perfusion culture, the distribution of substances in the culture vessel can be controlled over time by adjusting the composition of the culture solution. However, when culture is performed under perfusion, there is an adverse effect due to shear stress. For example, shear stress caused by flow may cause cells attached to the cell container to detach and cells may be lost.
To solve such problems, a method has been proposed in which a compartment is divided into an upper layer and a lower layer with a semipermeable membrane, and cells are cultured in the upper layer while circulating a culture solution in the lower layer. (Patent Document 1, Non-Patent Document 1).
However, in the above method, the substance distribution cannot be spatially controlled, for example, a substance concentration gradient is formed in the culture environment.

On the other hand, it is known that the behavior of the fluid in the micro space is greatly different from the behavior of the fluid in the macro space. For example, when a liquid is circulated in a micro space (micro channel) having a channel structure, the Reynolds number is small and a laminar flow is easily formed.
Recently, studies have been carried out in which a substance concentration gradient is formed by utilizing the phenomenon peculiar to the microspace and applied to cell culture. For example, in Non-Patent Documents 2 to 7, a substance concentration gradient is formed in the micro space using laminar flow. In Non-Patent Documents 8 to 9, a substance concentration gradient is formed in the micro space by utilizing diffusion of substances.
However, when the culture is performed in a laminar flow field, there is an adverse effect due to shear stress as in the case of the perfusion culture. In particular, in the method of forming a substance concentration gradient using laminar flow, it is necessary to increase the flow velocity to some extent in order to maintain the concentration gradient, and thus the influence of shear stress is large. In addition, in the case of pluripotent stem cells, there are reports that the cells are altered or the direction of differentiation is changed by the shear stress (for example, Non-Patent Document 10).
In addition, when the cultured cells form a three-dimensional structure (for example, an embryoid body or a fertilized egg), the laminar flow is disturbed by the three-dimensional structure, and thus it is difficult to precisely control the material distribution in the micro space. It becomes.
Further, in the method using the diffusion of the substance, the substance distribution (concentration) becomes uniform over time due to the diffusion, and therefore the predetermined substance distribution cannot be maintained in the micro space for a long time. Therefore, cell differentiation cannot be precisely controlled.

JP 2006-246720 A

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  The present invention has been made in view of the above circumstances, and a substance distribution control method capable of precisely controlling a substance distribution in a micro space in terms of time and space while reducing a load due to shear stress, An object is to provide a device used in the method, a cell culture method using the method, and a cell differentiation control method.

As a result of intensive studies, the present inventors have arranged a microchannel through a porous film having a predetermined pore size under a microspace, and formed a predetermined substance distribution in the lower microchannel. When the multi-layered flow is circulated, the same material distribution as in the lower micro-space can be formed in the upper micro-space while reducing the load due to the shear stress due to the multi-layer flow, and the material distribution can be maintained over a long period of time. The present invention was completed.
The present invention has the following aspects.
[1] A method for controlling a material distribution in a micro space filled with a liquid,
N types of liquids (n is an integer of 2 or more) having different compositions are joined together as a laminar flow to form a multilayer flow in which n laminar flows are adjacent in the width direction,
A substance distribution control method, characterized in that the multilayer flow is circulated through a microchannel disposed under a microspace through a porous membrane having a pore diameter of 1 μm or more and 20 μm or less .
[2] The substance distribution control method according to [1], wherein the multilayer flow is circulated so that the Reynolds number is less than 2000 in the microchannel.
[3] A device used in the substance distribution control method according to [1] or [2],
Comprising a first substrate and a second substrate each having a microchannel structure on the surface;
The microchannel structure on the surface of the first substrate includes a multi-layer flow distribution portion through which a multi-layer flow circulates, a multi-layer flow forming portion disposed upstream of the multi-layer flow distribution portion, and a downstream side of the multi-layer flow distribution portion. A multi-layer flow outlet disposed in the
The microchannel structure on the surface of the second substrate has a microspace portion having substantially the same pattern as the multilayer flow circulation portion, and a lead-in / inlet portion for introducing or leading out liquid to the microspace portion. ,
The first substrate and the second substrate are laminated so that the surface on which the microchannel structure is provided is on the inside, and the positions of the multilayer flow circulation portion and the micro space portion are matched,
A device in which a porous film having a pore diameter of 1 μm or more and 20 μm or less is sandwiched between the multilayer flow distribution part and the micro space part.
[4] A method for culturing cells in a microspace filled with liquid,
N types of culture solutions having different compositions (n is an integer of 2 or more) are joined together as a laminar flow to form a multilayer flow in which n laminar flows are adjacent in the width direction;
A cell culturing method, wherein the multilayer flow is circulated through a microchannel disposed under a microspace through a porous membrane having a pore diameter of 1 μm or more and 20 μm or less .
[5] The cell culture method according to [4], wherein pre-culture is performed to attach the cells to the micro space side of the porous membrane, and then the circulation of the multilayer flow is started.
[6] The cell culture method according to [4], wherein the multilayer flow is performed in a state where the cells are not attached to the porous membrane.
[7] The cell culture method according to any one of [4] to [6], wherein the cells are pluripotent stem cells.
[8] The cell culture method according to [6], wherein the cells form a three-dimensional structure.
[9] A method for controlling cell differentiation in a microspace filled with liquid,
The n-type (n is an integer of 2 or more) culture solutions having different types or concentrations of substances that control cell differentiation are combined as laminar flows to form a multi-layer flow in which n laminar flows are adjacent in the width direction. And
A method for controlling cell differentiation, characterized in that the multilayer flow is circulated through a microchannel disposed under a microspace through a porous membrane having a pore diameter of 1 μm or more and 20 μm or less .
[10] The cell differentiation control method according to [9], wherein pre-culture is performed to attach the cells to the microspace side of the porous membrane, and then the circulation of the multilayer flow is started.
[11] The cell differentiation control method according to [9], wherein the multilayer flow is circulated in a state where the cells are not attached to the porous membrane.
[12] The cell differentiation control method according to any one of [9] to [11], wherein the cell is a pluripotent stem cell.
[13] The method for controlling cell differentiation according to [11], wherein the cells form a three-dimensional structure.

  According to the present invention, a material distribution control method capable of precisely controlling a material distribution in a micro space both temporally and spatially while reducing a load due to shear stress, a device used in the method, and the method are used. Cell culture methods and cell differentiation control methods can be provided.

1 is a perspective view of a device 1. FIG. 2 is an exploded perspective view of the device 1. FIG. 2 is a top view of the device 1. FIG. FIG. 6 is an explanatory diagram for explaining an embodiment of a substance concentration control method using the device 1. As a result of Test Example 1 (1) (upper layer flow path when porous membrane B (pore diameter 1 μm), porous membrane C (pore diameter 3 μm), porous membrane D (pore diameter 5 μm), and porous membrane E (pore diameter 10 μm) are used ( It is a fluorescence image which shows FITC distribution in a micro space part. It is a fluorescence image which shows a time-dependent change of the FITC distribution in the upper-layer flow path (micro space part) at the time of using the porous film E among the results of Test example 1 (2). It is the fluorescence image and graph which show the FITC distribution in the lower layer flow path (multilayer flow distribution part) at the time of using the porous film E among the results of Test example 1 (2). It is the fluorescence image and graph which show the FITC distribution in the upper-layer flow path (micro space part) at the time of using the porous film E among the results of Test example 1 (2). It is the fluorescence image and graph which show the result (the FITC distribution of each in a lower layer flow path (multilayer flow circulation part) and an upper layer flow path (micro space part)) of the test example 2 ((a) In a multilayer flow circulation part, (b ) Inside the micro space part). Results of Test Example 3 (Amount of liquid fed to the lower layer flow path (multilayer flow circulation portion) and flow rate of beads in each flow path in the lower layer flow path (multilayer flow flow portion) and in the upper layer flow path (micro space portion) FIG. Fluorescence image and graph showing the results of Test Example 4 (fluorescence observation results of SYBR Green incorporated into cells in the upper layer flow path (microspace part)) ((a) before culture, (b) after 12 hours of culture) It is. Among the results of Test Example 5, the fluorescence images and graphs ((a) before culture, (b) after 5 days of culture) showing the results of GFP fluorescence observation in the upper channel (microspace part) before and after 5 days of culture. is there. It is the fluorescence image and graph which show the DAPI fluorescence observation result in the upper-layer flow path (micro space part) 5 days after culture | cultivation among the results of Test Example 5. It is the fluorescence image and graph ((a) fluorescence image, (b) graph) which show the GFP fluorescence observation result in the upper layer flow path (micro space part) 5 days after culture | cultivation among the results of the test example 6. FIG. It is the fluorescence image and graph ((a) fluorescence image, (b) graph) which show the DAPI fluorescence observation result in the upper channel (micro space part) 5 days after culture | cultivation among the results of Test Example 6.

The substance distribution control method of the present invention is a method for controlling substance distribution in a micro space filled with a liquid, wherein n kinds of liquids (n is an integer of 2 or more) having different compositions are joined together as a laminar flow. A multilayer flow in which n laminar flows are adjacent to each other in the width direction, and the multilayer flow is circulated through a microchannel disposed under the microspace via a porous film having a pore diameter of 1 μm or more. To do.
In the multilayer flow, n types of laminar flows having different compositions (substance concentrations) are sequentially stacked in the width direction of the microchannel, and the substance concentration changes in the width direction of the microchannel. When a multilayer flow having such a material distribution is circulated through the microchannel, the substances contained in each of the n kinds of laminar flows pass through the porous film by diffusion and move into the upper microspace. As a result, a region having the same substance concentration as each laminar flow is formed in the micro space above each laminar flow with substantially the same width as each laminar flow. In this way, a material distribution similar to the multilayer flow is formed in the upper microspace.
Conventionally, it has been considered that the concentration distribution of a substance that has passed through a membrane and transferred from a fluid into a liquid that does not flow is uniform over time due to diffusion. However, according to the knowledge of the present inventors, by using a porous film having a pore diameter of 1 μm or more, surprisingly, a material distribution similar to a multilayer flow flowing through the lower microchannel is formed in the upper microspace. At the same time, the material distribution is maintained while the multilayer flow is being circulated.
Therefore, in the present invention, the substance distribution in the micro space can be precisely controlled (in the micro scale level) both in terms of time and space simply by adjusting the composition and flow rate of the n kinds of liquids. Long-term control over time is also possible. This is useful in controlling the culture and differentiation of cells that require a relatively long time. For example, while culturing cells such as pluripotent cells in a microspace, the type of substance that controls the differentiation of the cultured cells (differentiation inducing factor, differentiation inhibitory factor, etc.) or the distribution of concentration in the microspace By controlling the target and / or time, it is possible to precisely control the differentiation of the cells.
In addition, the shear stress generated by the flow of the multilayer flow is reduced by the porous film. For example, the micro space can be a high-level static environment in which the Brownian motion of fine particles existing in the micro space can be observed. Therefore, even in the micro space, it is possible to culture even a cell having a weak adhesive force. Moreover, the influence on the activity and differentiation of a cultured cell which arises by this shear stress can also be suppressed.
Furthermore, even when cells cultured in a microspace form a three-dimensional structure, the flow in the underlying microchannel is not disturbed, so the precision of controlling the material distribution in the microspace is maintained. Is done.

The formation method of the said multilayer flow is not specifically limited, A well-known method can be utilized. For example, n microchannels (hereinafter referred to as laminar flow forming channels) that merge at the downstream end are provided upstream of the microchannels, and liquid is introduced into each laminar flow forming channel. Can be formed. That is, in each laminar flow forming flow path, the introduced liquid forms a laminar flow and merges at the downstream end. As a result, a multilayer flow in which n laminar flows are arranged in the width direction is formed. The multilayer flow is supplied as it is to the downstream micro-channel and circulates in the micro-channel.
n should just be an integer greater than or equal to 2, and an upper limit is not specifically limited. Preferably, the width of each laminar flow constituting the multi-layer flow is set within a range not smaller than 10 μm. The width of each laminar flow is preferably 10 to 1000 μm, and more preferably 50 to 500 μm.

The substance distribution control method of the present invention can be implemented using, for example, the following devices.
Comprising a first substrate and a second substrate each having a microchannel structure on the surface;
The first micro-channel structure on the surface of the first substrate (hereinafter sometimes referred to as a lower layer flow path) includes a multi-layer flow distribution section through which a multi-layer flow circulates and a multi-layer flow disposed upstream of the multi-layer flow distribution section. A forming portion, and a multilayer flow discharge portion disposed on the downstream side of the multilayer flow circulation portion,
The micro-channel structure on the surface of the second substrate (hereinafter sometimes referred to as the upper-layer channel) includes a micro space part having substantially the same pattern as the multilayer flow circulation part, and introduction of liquid into the micro space part or A derivation entry section for derivation,
The first substrate and the second substrate are laminated so that the surface on which the microchannel structure is provided is on the inside, and the positions of the multilayer flow circulation portion and the micro space portion are matched,
A device in which a porous film having a pore diameter of 1 μm or more is sandwiched between the multilayer flow distribution part and the micro space part.
In the device, a multi-layer flow is formed by introducing n kinds of liquids into the multi-layer flow forming portion of the lower layer flow path, and the multi-layer flow passes through the multi-layer flow circulation portion and passes from the multi-layer flow discharge portion to the outside of the device. It is supposed to be discharged.
In the device, a micro space is formed by the micro space portion of the upper layer flow path and the porous membrane, and a multilayer flow circulation portion is disposed under the micro space via the porous membrane. The micro space part and the multilayer flow circulation part are formed in substantially the same pattern.
Therefore, when n kinds of liquids are introduced into the multilayer flow forming portion of the lower layer flow channel with the liquid filled in the micro space, a multilayer flow is formed, and the multilayer flow circulates in the multilayer flow distribution portion under the micro space. It will be. Since a porous film having a predetermined pore diameter is arranged at the interface between the multilayer flow distribution part and the micro space, the substances in the n kinds of laminar flows constituting the multilayer flow move upward through the porous film. As a result, a material distribution similar to the multilayer flow flowing thereunder is formed in the micro space.
In the device, the multilayer flow circulation part and the micro space part respectively correspond to the micro flow path and the micro space in the material distribution control method of the present invention.

Hereinafter, an embodiment of the device will be described with reference to the drawings.
The device 1 of this embodiment is shown in FIGS. FIG. 1 is a perspective view of the device 1, FIG. 2 is an exploded perspective view of the device 1, and FIG. 3 is a top view of the device 1.
In the device 1, the first substrate 2 having the lower layer flow path 21 formed on the surface and the second substrate 3 having the upper layer flow path 31 formed on the surface form the lower layer flow path 21 and the upper layer flow path 31. A porous body 4 having a pore diameter of 1 μm or more is sandwiched at a predetermined position between the first substrate 2 and the second substrate 3.
In the laminated body, the upper layer channel 31 and the overlapping portion on the lower layer channel 21 are all covered with the porous film 4. Thereby, the liquid flowing through the lower layer channel 21 does not directly flow into the upper layer channel 31.

In the first substrate 2, the lower layer flow path 21 includes a multilayer flow circulation portion 211 (a portion between the positions AA ′ and BB ′ in FIGS. 2 and 3) and the multilayer flow circulation portion 211. A multi-layer flow forming section having three branch flow path sections 212 a to 212 c that merge at the downstream end disposed on the upstream side, and a multi-layer flow discharge section 213 disposed on the downstream side of the multi-layer flow distribution section 211 Is done.
The multi-layer flow forming unit includes branch flow channel portions 212a to 212c, and a communication flow channel portion 212d that connects the junction of the branch flow channel portions 212a to 212c and the upstream end of the multi-layer flow circulation portion 211. In the multilayer flow forming section, when liquids having different compositions are introduced into two or three upstream ends of the branch flow path sections 212a to 212c, each liquid forms a laminar flow and merges into two layers or A three-layer multilayer flow is formed.
The multilayer flow distribution part 211, the branch flow path part 212b, the communication flow path part 212d, and the multilayer flow discharge part 213 are arranged on substantially the same straight line.

In the second substrate 3, the upper layer flow path 31 has a micro space portion 311 having substantially the same pattern as the multilayer flow distribution portion 211 (a portion between the position AA ′ and the position BB ′ in FIGS. 2 and 3). ) And two lead-in / take-in passage portions 312 and 313 for introducing or discharging the liquid to / from the micro space portion 311.
The lead-in / in channel portions 312 and 313 are respectively provided in the longitudinal direction of the micro space portion 311 from both ends in the longitudinal direction of the micro space portion 311 (the upstream end and the downstream end in the flow direction of the multilayer flow in the multilayer flow flow portion 211). Is bent at a certain angle.
The micro space portion 311 is formed in substantially the same pattern as the multilayer flow circulation portion 211. That is, it is formed with the length and width substantially the same as the length (length in the flow direction of the multilayer flow) and the width of the multilayer flow circulation portion 211.

Six through holes 32 to 37 that penetrate the second substrate 3 are formed in the second substrate 3. Among these, the through holes 32 to 34 are provided at positions corresponding to the upstream ends of the branch flow passage sections 212 a to 212 c, and the through holes 35 are provided at positions corresponding to the downstream ends of the multilayer flow discharge section 213. It has been. Thereby, the liquid can be introduced into each of the flow paths 212a to 212c and the liquid (multilayer flow) can be discharged from the multilayer flow discharge portion 213 through the through holes 32 to 37.
The through holes 36 and 37 are provided at positions corresponding to the ends of the lead-in / in passage portions 312 and 313, respectively. As a result, liquid can be introduced into or discharged from the micro space portion 311 through the through holes 36 and 37. When controlling the culture or differentiation of cells in the micro space portion 311, the through holes 36 and 37 can also be used for introducing cells into the micro space portion 311 or taking out cells.

Hereinafter, each of the components of the device 1 described above will be described in more detail.
The multilayer flow circulation part 211 preferably takes into account the physical properties (density and viscosity) and flow rate of the liquid so that the Reynolds number of the liquid (multilayer flow) flowing in the multilayer flow circulation part 211 is less than 2000. Width and height are set. If the Reynolds number is less than 2000, the laminar flow state of the multilayer flow is stable, and the micro flow channel is circulated in a state in which mass transfer between adjacent laminar flows hardly occurs. The Reynolds number is preferably 2000 or less, more preferably 100 or less, and even more preferably 1 or less. The lower limit of the Reynolds number is not particularly limited.
The Reynolds number is conventionally known as one of important indexes for explaining the behavior of a fluid, and is defined by the following equation.
Re = ρ × r × l / μ
[Where Re is the Reynolds number, ρ is the density of the liquid, r is the flow velocity, l is the representative length, and μ is the viscosity of the liquid. ]

The density ρ and viscosity μ of the liquid are density and viscosity, respectively, under temperature conditions during distribution.
About the Reynolds number of the liquid which distribute | circulates the multilayer flow distribution part 211, it calculates | requires in the following procedures. First, the density of n liquids constituting each laminar flow is ρ1, ρ2... Ρn, the viscosity is ρ1, ρ2... Ρn, μ1, μ2,. From the flow rate ratio of each liquid in the circulation part 211, the density ρ ′ and the viscosity μ ′ of the mixed liquid of all the liquids constituting the multilayer flow are calculated. From the density ρ ′ and the viscosity μ ′, the flow velocity and the representative length of the multilayer flow in the multilayer flow circulation portion 211, the Reynolds number of the liquid flowing through the multilayer flow circulation portion 211 is obtained.
As for the flow velocity r (unit: μm / min), the average flow velocity is obtained from the average flow velocity = the flow rate / the cross-sectional area of the flow path.
The representative length l is determined by the cross-sectional area and the perimeter of the location where the flow is most affected when the length is changed. For example, if the cross-sectional shape of the microchannel is rectangular in height h ₁ and the width (depth) w, its characteristic length l is obtained by the following equation.
l = 4 × h ₁ × w / 2 (h ₁ + w)

The Reynolds number varies depending on the physical properties (density and viscosity) of the liquid flowing through the flow path, the flow velocity, and the size (width and height) of the flow path.
Therefore, the width and height of the multilayer flow circulation portion 211 vary depending on the physical properties and flow velocity of the liquid to be circulated, and are not particularly limited, but can be applied to liquids having a wide range of density or viscosity, so that the width ranges from 1 to 100000 μm. Of these, the height is preferably set within a range of 1 to 10,000 μm. As for the width | variety of the multilayer flow distribution part 211, 1-1000 micrometers is more preferable, and 20-1000 micrometers is further more preferable. Further, the height is more preferably 1 to 1000 μm, and further preferably 1 to 500 μm. If the width and height are not more than the upper limit of the above range, the Reynolds number is small, and the stability of the laminar flow is good. When it is at least the lower limit value, there are advantages such that it is easy to form the flow channel structure, it is suitable for application to cell experiments, and the shear stress can be reduced.

  The length of the multilayer flow circulation portion 211 (length in the circulation direction of the multilayer flow) is not particularly limited, and can be set as appropriate according to the purpose. For example, when the length is shortened, the multilayer flow flows from the upstream end to the downstream end of the multilayer flow circulation section 211 in a state where the interface with the adjacent laminar flow is clear (the contrast of the substance concentration between the laminar flows is clear). Therefore, a substance distribution with a clear contrast can be formed over the entire longitudinal direction in the microspace of the upper layer. Further, when the length is increased, mass transfer occurs between the adjacent laminar flows while the multilayer flow circulates in the multilayer flow circulation portion 211. Along with this, the substance distribution formed in the micro space in the upper layer becomes gentler as the concentration gradient becomes closer to the downstream side in the longitudinal direction.

The branch flow channel portions 212a to 212c preferably have physical properties (density and viscosity) of the liquid flowing through each branch flow channel portion so that the Reynolds number of the liquid flowing through the branch flow channel portions 212a to 212c is less than 2000. The width and height are set in consideration of the rate) and the flow velocity. If the Reynolds number is less than 2000, the liquid introduced into the branch flow channel sections 212a to 212c forms a laminar flow, and when each laminar flow joins, it is stable with almost no movement of substances between the laminar flows. Multi-layered flow is formed. The Reynolds number is preferably 2000 or less, more preferably 100 or less, and even more preferably 1 or less. The lower limit of the Reynolds number is not particularly limited.
The width and height of the branch flow channel sections 212a to 212c can be applied to liquids having a wide range of densities or viscosities, and therefore are set within a width range of 0.1 to 100000 μm and a height range of 0.1 to 1000 μm. It is preferable to do. The width of the channels 212a to 212c is more preferably 1 to 1000 μm, and further preferably 20 to 1000 μm. Moreover, 1-1000 micrometers is more preferable and, as for the height of the flow paths 212a-212c, 1-100 micrometers is further more preferable. If the width and height are less than or equal to the upper limit of the above range, the Reynolds number becomes small and a laminar flow is likely to be formed. When it is at least the lower limit value, there are advantages such that the flow channel structure is easily formed, a laminar flow can be formed with a small flow rate, and the amount of reagents used is reduced.

Of the branch channel portions 212a to 212c, the joining angles (θ ₁ and θ _{2 in} FIG. 3) of the branch channel portions 212a and 212c at both ends to the connecting channel portion 212d are preferably _{1 to} 179 degrees, 90 -179 degrees is more preferred. When the merging angle is within the above range, a multilayer flow can be formed satisfactorily. The preferable range of the merging angle is the same when there are two or four or more branch flow passage portions.
Further, the angles formed by the branched flow channel portions 212a to 212c with the adjacent flow channels (the angle formed by the flow channel 212a and the flow channel 212b and the angle formed by the flow channel 212a and the flow channel 212b) are the same. It is preferable that

The communication channel portion 212d is formed to have the same cross-sectional shape in the width direction as the multilayer flow circulation portion 211. As a result, the multilayer flow that has passed through the communication flow path section 212d is smoothly supplied to the multilayer flow distribution section 211.
Moreover, the multilayer flow discharge part 213 is formed in the same cross-sectional shape in the width direction as the multilayer flow circulation part 211. Thereby, the multilayer flow which distribute | circulates the inside of the multilayer flow distribution part 211 is discharged | emitted smoothly.

The material which comprises the 1st board | substrate 2 is not specifically limited, For example, it can select suitably from the well-known materials conventionally used for formation of a microchannel structure according to the objective.
For example, when performing observation in a micro space, a transparent material that transmits visible light is preferable. Examples of the transparent material include silicone rubber such as polydimethylsiloxane (hereinafter referred to as PDMS), acrylic resin, glass, gel, and the like. Moreover, metals, Si, etc. are mentioned other than a transparent material.
In addition, when cell culture or differentiation control is performed in a microspace, it is preferable to use a material that is compatible with cultured cells. As the material, in particular, a material having oxygen permeability is preferable because oxygen in the atmosphere outside the device can be supplied into the micro space. As the material, any known oxygen-permeable material can be used, and examples thereof include a biocompatible oxygen-permeable material used for an oxygen-permeable contact lens. In particular, since the cultured cells in the micro space can be observed from the outside of the device, the material having oxygen permeability is preferably a transparent material.
Specific examples of the biocompatible oxygen permeable material include silicone rubber. In particular, PDMS is preferable because it is biocompatible, has transparency and oxygen permeability, and is an inexpensive material.

As described above, the micro space portion 311 is formed in substantially the same pattern as the multilayer flow circulation portion 211. That is, it is formed with the length and width substantially the same as the length (length in the flow direction of the multilayer flow) and the width of the multilayer flow circulation portion 211.
The height of the micro space portion 311 (width in the direction perpendicular to the surface of the porous membrane 4) is 10,000 μm or less from the viewpoint that the material distribution can be satisfactorily formed in the micro space formed by the micro space portion 311 and the porous membrane 4. Is preferable, and 1000 μm or less is more preferable.
The lower limit of the height is not particularly limited from the viewpoint of controlling the substance distribution, and can be appropriately set according to the purpose. For example, when cell culture or differentiation control is performed in a micro space, the size is set higher than the size of the cultured cells.
The cell size varies depending on the type and adhesion state. For example, the size of a mammalian embryonic stem cell is usually about 10 to 20 μm in major axis and about 5 to 10 μm in minor axis at the time of adhesion, and about 5 to 10 μm when detached and suspended by trypsin or the like. It is a sphere. The size of a fertilized egg is usually about 130 μm for humans, about 80 μm for mice, and about 120 to 130 μm for cows and pigs. An embryoid body is an artificially produced cell mass whose size can be changed by various techniques and is not particularly limited, but is usually about 50 μm to 1 mm.

The lead-in / in passage portions 312 and 313 are formed at the same height as the micro space portion 311.
The lead-in / in channel portions 312 and 313 are respectively provided in the longitudinal direction of the micro space portion 311 from both ends in the longitudinal direction of the micro space portion 311 (the upstream end and the downstream end in the flow direction of the multilayer flow in the multilayer flow flow portion 211). Is bent at a certain angle.
The angle (θ _{3 in} FIG. ₃ ) formed by the lead-in / in passage portion 312 with the longitudinal direction of the micro space portion 311 and the angle (θ _{4 in} FIG. 3) formed by the lead-in / in passage portion 313 with the longitudinal direction of the micro space portion 311 are as follows. Respectively, 0 to 179 degrees is preferable, and 0 to 90 degrees is more preferable. When these angles are within the above range, cells can be uniformly seeded in the micro space portion 311 when cell culture or differentiation control is performed in the micro space portion 311.

  The material which comprises the 2nd board | substrate 3 is not specifically limited, For example, the thing similar to what was mentioned as a material which comprises said 1st board | substrate 2 is mentioned, A transparent material is especially preferable. However, even when cell culture or differentiation control is performed in the micro space, the above-described biocompatibility and oxygen permeability are not necessarily required for the second substrate 3.

The porous film 4 needs to have a pore diameter of 1 μm or more in order to form a substance distribution in the micro space 311. Here, the “porous membrane” is a solid membrane having a large number of pores penetrating the membrane. The pore diameter of the porous membrane (pore diameter) can be measured with an electron microscope (SEM), a laser microscope or the like.
The pore diameter of the porous membrane 4 is preferably 1 μm or more, more preferably 5 μm or more, and further preferably 10 μm or more. The larger the pore size, the more easily the substance distribution of the multi-layer flow flowing through the micro flow path is reflected in the micro space, and the substance distribution with a clear contrast is easily formed. The upper limit of the pore diameter is preferably 50 μm or less, and more preferably 20 μm or less, from the viewpoint of reducing the load on the micro space due to the shear stress generated by the multilayer flow.
When cell culture or differentiation control is performed in a microspace, it is preferable to further consider the size of the cultured cells. That is, if the pores of the porous membrane 4 are too large, cultured cells may infiltrate and flow out of the microchannel. Therefore, in this case, it is preferable to use a porous membrane 4 having a pore size smaller than the size of the cultured cells. The pore diameter of the porous membrane 4 is preferably 100% or less, more preferably 50% or less of the size of the cultured cells. Here, the size of the cultured cell means the diameter of a single cell and can be measured by microscopic observation.
For example, the size of pluripotent stem cells such as embryonic stem cells (hereinafter referred to as ES cells) and induced pluripotent stem cells (hereinafter referred to as iPS cells) is generally about 10 to 20 μm. Therefore, when the cultured cells are embryonic stem cells, the pore diameter of the porous membrane 4 is preferably 15 μm or less, and more preferably 10 μm or less.
The size of a three-dimensional structure such as an embryoid body or a fertilized egg is generally about 50 μm to 1 mm. Therefore, when cultured cells form a three-dimensional structure, the pore diameter of the porous membrane 4 is preferably 50 μm or less, and more preferably 10 μm or less.

The porosity of the porous film 4 is preferably 1 to 15%, more preferably 5 to 10%. If it is above the lower limit of the range, a substance distribution is likely to be formed in the microspace, and if it is below the upper limit, the influence of the flow velocity of the lower layer channel 21 into the upper layer channel 31 can be sufficiently reduced.
The material of the porous film 4 is not particularly limited, and may be the same as that conventionally used as the material of the porous film. Specific examples include polystyrene, polycarbonate, polyester, polyethylene, polytetrafluoroethylene (hereinafter referred to as PTFE), and the like.
Further, the surface of the porous membrane 4 may be subjected to a surface treatment for the purpose of improving cell adhesion.
The thickness of the porous film 4 is preferably 100 μm or less, more preferably 20 μm or less, from the viewpoint of substance permeability.
As the porous membrane 4, a commercial product sold by Whatman or the like can be used.

In the device 1, the first substrate 2 and the second substrate 3 are arranged so that the surfaces on which the lower layer flow paths 21 and 31 are formed are inward so that the positions of the multilayer flow circulation portion 211 and the micro space portion 311 coincide. It is possible to fabricate the film by attaching the film to each other and sandwiching the porous film 4 therebetween.
Each of the first substrate 2 and the second substrate 3 can be manufactured by using a known microfabrication method such as a method conventionally used for manufacturing a microdevice having a channel structure such as a microreactor. Examples of the fine processing method include a lithography method, an etching method, cutting, and injection molding.
If an example of the manufacturing method using the lithography method is given, the 1st board | substrate 2 can be produced by performing the following processes (1)-(5).
(1) First, a photoresist is applied on a substrate by spin coating to form a photoresist layer.
(2) The photoresist layer is patterned by exposing and developing the photoresist layer through a mask having a pattern corresponding to the lower layer flow path 21.
(3) A prepolymer layer of UV curable or thermosetting polymer is applied on the patterned photoresist layer (template) to form a prepolymer layer.
(4) The prepolymer layer is cured by irradiating or heating UV to form a polymer layer.
(5) The polymer layer is peeled off.
On the surface of the polymer layer obtained in this manner, a pattern in which the pattern of the photoresist layer is inverted (for example, a space pattern when the pattern of the photoresist layer is a line pattern), which is the same height as the thickness of the photoresist layer ( A lower depth channel 21 having a depth) is formed. This polymer layer can be used as the first substrate 2.
The second substrate 3 was obtained in the same manner as in the above (1) to (5) except that a mask having a pattern corresponding to the upper layer flow path 31 was used as a mask. It can be produced by opening 32 to 37.

Next, an embodiment of a material distribution control method in the micro space 311 of the device 1 will be described with reference to FIG. FIG. 4 illustrates the device 1 in a state in which two laminar flows L ₁ and L ₂ are adjacent to each other in the width direction of the multilayer flow circulation unit 211 (two-layer flow) are circulating in the multilayer flow circulation unit 211. It is a partial longitudinal cross-sectional view of position AA '.
In the present embodiment, a two-layer flow is formed by introducing liquids L ₁ and L ₂ into the branch flow channel portions 212 a and 212 c in a state where the branch flow channel portion 212 b is sealed, and then flows into the multilayer flow flow portion 211. I am letting. That is, when the liquids L ₁ and L ₂ are introduced from the through holes 32 and 34 into the branch flow channel portions 212a and 212c, laminar flows of the respective liquids are formed in the branch flow channel portions 212a and 212c, and the flow channels 212a and 212c. A multi-layer flow (two-layer flow) in which two laminar flows are aligned in the width direction is formed. The two-layer flow is supplied to the multilayer flow circulation portion 211 through the communication flow path portion 212d, flows through the multilayer flow circulation portion 211, and passes through the through hole 35 from the downstream end of the multilayer flow discharge portion 213. Discharged outside.
When the two-layer flow is circulated through the multilayer flow distribution unit 211 as described above, a material distribution similar to the two-layer flow is formed in the micro space portion 311 in the upper layer of the multilayer flow distribution unit 211.
By simply adjusting the composition and flow rate of the liquids L ₁ and L ₂ , the substance distribution in the micro space 311 can be precisely controlled both temporally and spatially. For example, when a liquid containing the substance A at the concentration a ₁ is used as the liquid L _{1 and} a liquid containing the substance A at the concentration a ₂ (a ₁ <a ₂ ) is used as the liquid L ₂ , the substance is contained in the micro space portion 311. A concentration gradient occurs. That is, the upper portion of the laminar flow of the liquid L ₁ region (low concentration region) which contains the relatively low concentration of substance A, and the upper portion of the laminar flow of the liquid L ₂ is a relatively high concentration of substance A It becomes a contained region (high concentration region), and a concentration gradient of the substance A is generated in the width direction in the micro space portion 311.
When one or both of the concentrations a ₁ and a ₂ are changed, the concentration gradient can be changed.
Further, when the widths w ₁ and w ₂ of the laminar flows L ₁ and L ₂ are changed, the width of the high concentration region and the width of the low concentration region in the micro space portion 311 also change.
The widths w ₁ and w ₂ of the laminar flows L ₁ and L ₂ can be adjusted by adjusting the ratio of the flow rates of the liquids L ₁ and L ₂ . For example, when the ratio of the flow rate of the liquid L ₁ is increased, the width w ₁ of the laminar flow L ₁ is increased. The flow rate of each liquid can be controlled using a known liquid supply device such as a syringe pump, an electroosmotic flow pump, or other mechanically operated pump.
In addition, by changing the types of substances blended in the liquids L ₁ and L ₂ , combining a plurality of kinds of substances, changing the ratio of the plurality of kinds of substances, and the like, a complicated substance distribution can be generated in the micro space portion 311. Can be formed.
In addition, by changing the composition of the liquids L ₁ and L ₂ over time, the substance distribution in the micro space 311 can be changed over time. On the other hand, when the composition of the liquids L ₁ and L ₂ is not changed, the original substance distribution can be maintained as it is.

In the present invention, as described above, it is preferable to circulate the two-layer flow in the multilayer flow circulation portion 211 so that the Reynolds number is less than 2000.
As described above, the Reynolds number can be adjusted not only by the size (width and height) of the multilayer flow circulation portion 211 but also by the physical properties (density ρ, viscosity μ) of the liquid and the flow velocity r. In particular, the flow rate r can be adjusted by a simple method of changing the flow rates of the liquids L ₁ and L ₂ introduced into the branch flow channel portions 212a and 212c, respectively.

The present invention is not limited to the above embodiment.
For example, in the above-described embodiment, the first substrate 2 of the device 1 is provided with three branch flow path portions (flow paths for forming each laminar flow forming a multi-layer flow) that merge with the communication flow path portion 212d. However, the number of branch flow path portions may be two or four or more. The upper limit of the number of branch flow path portions is not particularly limited, and may be set as appropriate according to the number (n) of liquids to be used. Considering the operability of the device, 5 or less is preferable.
Moreover, although the example where the communication flow path part 212d, the multilayer flow circulation part 211, and the multilayer flow discharge part 213 are formed on the same straight line has been shown, the present invention is not limited to this. For example. At least one of the communication flow path part 212d, the multilayer flow circulation part 211, and the multilayer flow discharge part 213 may be formed in a curved shape.
Moreover, although the example where the cross-sectional shape of the width direction of the communication flow path part 212d, the multilayer flow distribution part 211, and the multilayer flow discharge part 213 was the same was shown, this invention is not limited to this, The cross-sectional shape of each flow path May be different. However, from the viewpoint of the stability of the laminar flow, the same is preferable.
Further, in the second substrate 3, the lead-in / inway portions 312 and 313 are provided at both ends in the longitudinal direction of the micro space portion 311, respectively, but only one of the lead-in / inlet passage portions 312 and 313 may be provided. .
Moreover, although the example which forms 2 laminar flow and controls substance concentration distribution was shown in FIG. 4, this invention is not limited to this. For example, when a liquid is introduced into all the branch flow path portions 212a to 212c, a three-layer flow can be formed. In this case, it is possible to control the material distribution more complicated than in the case of the two-layer flow. For example, in the multi-layer flow distribution part 211, a high concentration region-low concentration region-high concentration region concentration gradient material distribution, low concentration region-high concentration region-low concentration region concentration gradient material distribution, substance A-containing region- A substance distribution such as a substance B-containing region-substance C-containing region can be formed.

The liquid used in the material distribution control method of the present invention is not particularly limited as long as it can form a laminar flow, and can be appropriately set according to the purpose.
For example, by using a culture solution as the liquid, the substance distribution control method of the present invention can be used for cell culture.
That is, when a cell to be cultured in a micro space (hereinafter referred to as a cultured cell) is introduced and a multi-layer flow composed of a plurality of culture fluids is circulated through the micro channel, each laminar flow is configured. The nutrient components and oxygen contained in the culture solution to be supplied are supplied to the cultured cells in the upper microspace through the porous membrane. In addition, the waste discharged from the cultured cells in the micro space diffuses into the multilayer flow through the porous membrane and is discharged.
At this time, by adjusting the composition (type and concentration of nutrient components, oxygen concentration, etc.) of a plurality of culture solutions constituting the multilayer flow, the substance distribution in the micro space is surrounded by the cultured cells in the actual living body. It can be similar to the material distribution in the environment. Therefore, according to the present invention, cells can be cultured in an environment closer to the living body than in the past. For example, in a living body, a plurality of cells are present in different environments although they are adjacent to each other (for example, liver cells and pancreatic cells). According to the present invention, a similar environment is formed in a micro space. it can.
Further, as described above, since the influence of the shear stress generated by the multi-layer flow on the micro space is reduced by the porous film, even cells having weak adhesive force can be cultured in the micro space. In addition, there is no influence of shear stress on the activity of cultured cells.
Furthermore, even when cells cultured in the microspace form a three-dimensional structure, the flow in the underlying microchannel is not disturbed, so that the precision of substance distribution control in the microspace is improved. Maintained.

Examples of cultured cells cultured in a micro space include cells derived from mammals such as humans, mice, cows, and pigs.
In particular, embryonic stem cells such as ES cells and iPS cells are preferred because they are useful in various fields such as life science, regenerative medicine, cell / tissue engineering, and developmental engineering. Also preferred are cells that form three-dimensional structures such as embryoid bodies and fertilized eggs.
The present invention can be used for culturing not only these undifferentiated cells but also various organ cells such as hepatocytes and small intestinal cells, plant cells, Escherichia coli, yeast and the like.
For example, when the device is used, the cultured cells can be introduced into the microspace by using a lead-in passage portion for introducing or leading the liquid to or from the microspace portion.

The composition of the culture solution to be used and other culture conditions (culture temperature, culture time, etc.) can be appropriately set according to the cultured cells.
In particular, when a culture solution having a different type or concentration of a substance that controls cell differentiation (hereinafter referred to as differentiation control factor) is used as the culture solution, differentiation of the cultured cell can be controlled. For example, by culturing cells such as pluripotent cells in a microspace, by changing the type of differentiation control factor (for example, differentiation inducing factor or differentiation inhibitory factor) and concentration of the cell cultured in the microspace, The degree of cell differentiation within the microspace can be locally varied. At this time, as described above, since the influence of the shear stress caused by the multilayer flow on the microspace is reduced by the porous film, the influence of the shear stress on the differentiation of the cells does not occur.
The differentiation regulator may be appropriately selected from known differentiation regulators according to the type of cultured cells.

When cell culture or differentiation control is performed as described above, preliminary culture may be performed in the micro space before the multilayer flow is circulated in the micro flow channel.
In particular, when cell culture or differentiation control is performed in a state in which cultured cells are attached to the porous membrane, the cells are attached to the microspace side of the porous membrane by performing pre-culture, and then the multilayer It is preferable to start the flow of the stream.
At this time, if the cultured cells are not adherent to the porous membrane, a matrix solution serving as a scaffold to which the cells adhere is introduced into the microspace before the preculture, and the matrix Is preferably adhered to the surface of the porous membrane. Examples of the matrix include gelatin, fibronectin, collagen, gel and the like.
In the case where the cultured cells have adhesiveness to the porous membrane, the cultured cells may be introduced into the microspace as it is together with the culture solution for pre-culture.
In the case of a cell forming a three-dimensional structure such as an embryoid body or a fertilized egg, culture or differentiation is usually controlled without being attached to the porous membrane. Thus, when culture | cultivation or differentiation control is performed in a state where cultured cells are not attached to the porous membrane, preliminary culture may or may not be performed.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to the examples.
In the test examples described later, fluorescence observation and fluorescence intensity measurement were performed using a phase contrast microscope, a differential interference microscope, and a confocal microscope.

<Production Example 1>
A device having the configuration shown in FIGS. 1 to 3 was manufactured by the following procedure.
First, a silicon substrate was prepared, and a photoresist (trade name “SU-8 2100”; manufactured by Microchem) was applied onto the substrate by spin coating, and baked to form a photoresist layer. Next, exposure and development were performed through the mask of the lower layer flow path 21 pattern, and the mask pattern was transferred to the photoresist layer. On the photoresist layer (mold), uncured PDMS was applied to form a polymer layer and cured by UV irradiation. After curing, the polymer layer is peeled off to form the first substrate 2 (outer diameter: major axis 50 mm × minor axis 20 mm × thickness 5000 μm, multilayer flow distribution part 211: width 1 mm × height 200 μm × length 10000 μm, branch channel part 212a to 212c: width 500 μm × height 200 μm, merging angle θ ₁ of the branch channel portion 212a: 135 degrees, merging angle θ ₂ of the branch channel portion 212c: 135 degrees).
The same operation was performed except that the mask was changed to the pattern of the upper layer flow path 31, and through-holes 32 to 37 were opened in the peeled polymer layer to form the second substrate 3 (outer diameter: major axis 50 mm × minor axis 50 mm × thickness). Length 5000 μm, micro space portion 311: width 1 mm × height 200 μm × length 15000 μm, lead-in passage portions 312, 313: width 1 mm × height 200 μm, angle θ ₃ between lead-in passage portion 312 and micro space portion 311: 60 degrees, and the angle θ ₄ between the lead-in / out path part 313 and the micro space part 311 was 60 degrees).

Separately, as the porous film 4, the following five types (all are "track etch membrane" manufactured by whatman (polycarbonate porous film having a porosity of less than 15%)) are prepared, and SiO ₂ is sputtered on each porous film by sputtering. Coated.
Porous membrane A: porous membrane having a pore diameter of 0.4 μm.
Porous membrane B: A porous membrane having a pore diameter of 1 μm.
Porous membrane C: A porous membrane having a pore diameter of 3 μm.
Porous membrane D: A porous membrane having a pore diameter of 5 μm.
Porous membrane E: A porous membrane having a pore diameter of 10 μm.
After the first substrate 2, the second substrate 3, and the porous film are subjected to O ₂ plasma treatment, the first substrate 2 and the second substrate 3 are placed with the surface on which the lower flow paths 21 and 31 are formed facing inward. Then, the multilayer flow distribution part 211 and the micro space part 311 are arranged so that the positions thereof coincide with each other, and the device is obtained by bonding with the porous film 4 interposed therebetween.

<Production Example 2>
Of the pattern of the lower layer flow path 21, the same as in Production Example 1 except that the multilayer flow distribution section 211 is changed to 150 μm width × 150 μm height × 10000 μm length, and the branch flow path sections 212 a to 212 c are changed to 150 μm width × 150 μm height. Thus, the first substrate 2 was obtained.
In addition, in the pattern of the upper layer flow path 31, the micro space portion 311 is changed to the width of 150 μm × height 150 μm × length 15000 μm, and the lead-in / inlet passage portions 312, 313 are changed to width 150 μm × height 150 μm. A second substrate 3 was obtained in the same procedure.
A device was obtained in the same manner as in Production Example 1 except that the obtained first and second substrates and the porous film E were used.

<Test Example 1>
(1)
Using the five types of devices manufactured in Production Example 1 and having different pore diameters of the porous membrane 4, the influence of the pore size of the porous membrane 4 on the formation of the substance distribution in the micro space portion 311 was evaluated by the following procedure. .
Using a syringe pump, water and a 25 μM aqueous solution of fluorescein isothiocyanate (FITC) as a fluorescent substance were fed from the through holes 32 and 34 at a flow rate of 2 μL / min, respectively. At this time, the flow rates of the liquid in the branch flow channel section 212a, the branch flow channel section 212c, and the multilayer flow circulation section 211 are about 20000 μm / min, about 20000 μm / min, and about 20000 μm / min, respectively, and the Reynolds number is about 0, respectively. 0.095, about 0.095, and about 0.11.
After 180 s from the start of feeding of a 25 μM aqueous solution of water and FITC, fluorescence observation and fluorescence intensity measurement were performed in the upper layer flow path 31 (microspace part 311), and the FITC distribution in each flow path was confirmed.
When the porous film B is used as the porous film B (pore diameter: 1 μm), porous film C (pore diameter: 3 μm), porous film D (pore diameter: 5 μm), and porous film E (pore diameter: 10 μm), the upper layer channel 31 at the time of 180 seconds has elapsed. The fluorescence images are shown in FIG.
As shown in these results, in the example using the porous membranes B to E, a FITC distribution was formed in the micro space portion 311 of the upper layer flow path 31. On the other hand, in the example using the porous film A having a pore diameter of 0.4 μm, the FITC did not diffuse into the micro space portion 311.

(2)
The following evaluation was performed using the device using the porous film E among the devices manufactured in Production Example 1.
First, a 25 μM aqueous solution of FITC was introduced from the through hole 34 to fill the inside of the device (in the upper layer channel 31 and the lower layer channel 21).
Next, using a syringe pump, water and a 25 μM aqueous solution of FITC were fed from the through holes 32 and 34 at a flow rate of 2 μL / min, respectively.
In the lower layer flow path 21 (multi-layer flow circulation section 211) at regular intervals (when 0 s, 30 s, 60 s, 90 s, 120 s, 150 s, 180 s, and 30 min have elapsed) from the start of feeding of a 25 μM aqueous solution of water and FITC. And the fluorescence observation in the upper layer flow path 31 (micro space part 311) and the measurement of the fluorescence intensity were performed, and FITC distribution in each flow path was confirmed.
FIG. 6 shows fluorescence images in the upper flow path 31 at the time when 0 s, 30 s, 60 s, 90 s, 120 s, 150 s, and 180 s have elapsed.
Further, from the measurement results of the fluorescence intensity in the lower layer flow path 21 and the upper layer flow path 31, a graph was created in which the vertical axis represents the fluorescence intensity at the time when 30 minutes elapsed and the horizontal axis represents the position in the width direction of the flow path. The graphs are shown in FIGS. 7 and 8 together with fluorescent images. FIG. 7 is a fluorescent image and graph in the lower layer flow path 21, and FIG. 8 is a fluorescent image and graph in the upper layer flow path 31.
The “position in the width direction of the flow path” indicates a distance (μm) in the width direction when the left end position of the flow path is 0 when the vertical cross section of the flow path is viewed from the downstream side.
As shown in these results, when the upper layer flow path 31 is filled with the FITC aqueous solution and then a two-layer flow of water and the FITC aqueous solution is circulated through the lower layer flow path 21, the FITC in the micro space portion 311 of the upper layer flow path 31 is obtained. The distribution changed from a uniform state to one corresponding to the FITC distribution in the lower layer flow path 21. Further, the FITC distribution was maintained as it was.

<Test Example 2>
Among the devices manufactured in Production Example 1, using a device using the porous membrane E, water was fed from the through hole 33 at a flow rate of 2 μL / min, and a 25 μM FITC aqueous solution was flowed from the through holes 32 and 34 at a flow rate of 2 μL. The same operation as (2) of Test Example 1 was performed except that the solution was fed at / min. The results are shown in FIG. FIG. 9A is a fluorescent image and a graph in the multilayer flow distribution part 211, and FIG. 9B is a fluorescent image and a graph in the micro space part 311.
As shown in the results, a three-layer flow is formed in the multi-layer flow circulation part 211 of the lower layer flow path 21, and the FITC distribution in the multi-layer flow circulation part 211 is accommodated in the upper micro space part 311. A FITC distribution was formed.

<Test Example 3>
Among the devices manufactured in Production Example 1, using the device using the porous membrane E, the flow through the lower layer flow channel 21 (multilayer flow distribution unit 211) is changed to the upper layer flow channel 31 (microspace portion) by the following procedure. 311) was evaluated.
The upper layer flow path 31 of the device was filled with water blended with fluorescent beads (“Fluoresbrite Carbonylate YG microspheres” manufactured by Polysciences, Inc.).
Next, by using a syringe pump, water mixed with fluorescent beads (“Fluoresbrite Carboxylate YG microspheres” manufactured by Polysciences, Inc.) is changed from the through hole 33 (0.2, 0.4, 1.0, 2.0 or 4.0 μL / min), and the lower layer channel 21 was circulated.
Images of fluorescent beads in the multilayer flow distribution part 211 of the lower layer flow path 21 and the micro space part 311 of the upper layer flow path 31 are taken with a phase contrast microscope with a camera every predetermined time from the start of liquid feeding, From the image, the average flow velocity (μm / min) of the fluorescent beads in the lower layer channel 21 and the upper layer channel 31 (hereinafter collectively referred to as beads in the channel) was obtained. The result is shown in the graph of FIG. The horizontal axis of the graph is the flow rate (μL / min) sent to the lower layer flow path 21, and the vertical axis is the average flow rate (μm / min) of the beads in the flow path.
As shown in the results, the average flow rate of the beads in the upper layer flow channel 31 is much smaller than the average flow rate of the beads in the lower flow channel 21, and in particular, the flow rate of the lower flow channel 21 is 0.5 μL. In the case of / min or less, it was 0. From the results, it was confirmed that in the upper layer flow path 31, the influence of the flow of the lower layer flow path 21 is significantly reduced by the porous film.

<Test Example 4>
Among the devices manufactured in Production Example 2, using a device using porous membrane E, HepG2 cells (human hepatoma-derived cell line; obtained from RIKEN cell bank) are cultured according to the following procedure. went.
The same device as used in Test Example 4 was sterilized by irradiation with UV for 30 minutes, and then the upper channel 31 was filled with a fibronectin solution (25 μg / mL) and allowed to stand for 1 hour.
Next, the inside of the device was washed with PBS and a culture solution. As the culture solution, FBS-containing Knock out DMEM was used.
Next, in order to adhere the cells, the upper flow path 31 was filled with a cell suspension (1.0 × 10 ⁷ cells / mL) and allowed to stand overnight in an incubator (temperature: 37 ° C.).
Next, SYBR Green (manufactured by Takara Bio Inc.), which is a fluorescent substance, was added to the culture solution to prepare a SYBR Green (+) culture solution. The SYBR Green (+) culture solution is sent from the through-hole 34, and the culture solution not added with SYBR Green (SYBR Green (-) culture solution) is sent from the through-hole 32 at a flow rate of 2 μL / min using a syringe pump. The liquid (perfusion) was started and culture (culture temperature: 37 ° C.) was performed.

After 12 hours from the start of the culture, fluorescence observation and measurement of fluorescence intensity of SYBR Green taken into the cells in the upper flow path (micro space part) were performed. From the measurement results, a graph was created with the vertical axis representing the fluorescence intensity of SYBR Green and the horizontal axis representing the position in the width direction of the flow path. The graph is shown in FIG. 11 together with the fluorescence image. FIG. 11A shows the state before culturing, and FIG. 11B shows the state after 12 hours of culturing.
As shown in the results, the cells in the region corresponding to the portion where the laminar flow of the SYBR Green (+) culture solution circulated in the lower layer multi-layer flow circulation portion 211 had an increased fluorescence intensity as compared to before perfusion. . From the results, it was confirmed that the cells in the region were exposed to SYBR Green and took up SYBR Green. On the other hand, it was confirmed that the cells in the region corresponding to the portion through which the laminar flow of the SYBR Green (-) culture solution circulated had hardly changed the fluorescence intensity and was hardly exposed to SYBR Green.
As described above, according to the present invention, the substance distribution in the micro space can be spatially controlled, and the local substance exposure can be performed on the cells existing in the same micro space.

<Test Example 5>
Of the devices manufactured in Production Example 1, using a device using porous membrane E, mouse iPS cells (iPS-MEF-Ng-20D-17 cell line; obtained from Kyoto University) by the following procedure. Was cultured. In the iPS-MEF-Ng-20D-17 cell line, green fluorescent protein (GFP) is expressed as a reporter gene of the Nanog gene that is an undifferentiated marker, and it is judged that cells that are fluorescently colored are in an undifferentiated state. it can.
After sterilizing the device in the same manner as in Test Example 4, the upper flow path 31 was filled with a fibronectin solution (concentration 25 μg / mL) and allowed to stand in an incubator (temperature: 37 ° C.) for 1 hour.
Next, the inside of the device was filled with the culture solution. As the culture solution, FBS-containing Knock out DMEM was used.
Next, in order to adhere the cells, a cell suspension (1.0 × 10 ⁶ cells / mL) was introduced into the upper flow path 31 and allowed to stand overnight in an incubator (temperature: 37 ° C.).
Next, an LIF-containing culture solution (undifferentiation maintenance culture solution) in which leukemia inhibitory factor (LIF), which is an undifferentiation maintenance factor of mouse iPS cells, is added to the culture solution, and differentiation of mouse iPS cells into the culture solution. An RA-containing culture solution (differentiation induction culture solution) containing retinoic acid (RA) as an inducer was prepared. Using a syringe pump, the LIF-containing culture solution is sent from the through-hole 34 and the RA-containing culture solution is sent from the through-hole 32 at a flow rate of 2 μL / min to start culture (culture temperature: 37 ° C.). went.

Before culturing and 5 days after the start of culturing, in the micro space 311 of the upper channel 31, fluorescence by GFP expression (GFP fluorescence) was observed and the fluorescence intensity was measured. The graph which took the position of the width direction of a channel was created. GFP fluorescence images and graphs before culture and after 5 days of culture are shown in FIG. 12, respectively. FIG. 12A shows the state before culturing, and FIG. 12B shows the state after 5 days of culturing.
Further, after 5 days of culture, the cells in the micro space 311 of the upper flow path 31 were stained with 4 ′, 6-diamidino-2-phenylindole dihydrochloride (DAPI) to observe fluorescence and measure fluorescence intensity. The graph was created with the vertical axis representing the fluorescence intensity of DAPI and the horizontal axis representing the position in the width direction of the flow path. The fluorescence image and graph are shown in FIG. Since the nuclei of living cells are stained by this DAPI staining, the living cells in the upper channel 31 can be confirmed by measuring DAPI fluorescence. From the results, it was confirmed that the viable cell density in the upper flow path 31 was almost constant after 5 days of culture.
From these results, the cells in the region corresponding to the portion where the laminar flow of the RA-containing culture fluid circulated are induced to differentiate, while the cells in the region corresponding to the portion where the laminar flow of the LIF-containing culture fluid circulated are undifferentiated. It was confirmed that the state of was maintained.
As described above, according to the present invention, it is possible to spatially control the substance distribution in the micro space and to perform local undifferentiation maintenance and differentiation induction on the pluripotent stem cells existing in the same micro space. Is possible.
In addition, when the same operation as described above was performed except that the cell suspension (1.0 × 10 ⁶ cells / mL) was introduced into the lower layer channel 21, the cells in the lower layer channel 21 were removed after 5 days of culture. Most were dead (confirmed by propidium iodide (PI) staining (dead cell staining)). This is thought to be due to the influence of shear stress in the flow.

<Test Example 6>
The same operation as in Test Example 5 except that the LIF-containing culture solution was fed from the through-hole 33 at a flow rate of 2 μL / min, and the RA-containing culture solution was fed from the through-holes 32 and 34 at a flow rate of 2 μL / min. Went.
The results are shown in FIGS. FIG. 14 is a fluorescence image and a graph ((a) fluorescence image, (b) graph) showing a GFP fluorescence observation result in the upper flow path 31 (microspace part 311) after 5 days of culture, and FIG. It is the fluorescence image and graph ((a) fluorescence image, (b) graph) which show the DAPI fluorescence observation result in the upper layer flow path 31 (micro space part 311) after five days.
From these results, differentiation was induced in the cells in the region corresponding to the portion where the laminar flow of the RA-containing culture solution (laminar flow at both ends of the three layers) circulated, while the laminar flow of the LIF-containing culture solution (three layers) It was confirmed that the cells in the region corresponding to the portion where the central laminar flow) was maintained in an undifferentiated state.

  In recent years, culture research on cell tissues including pluripotent stem cells has been actively conducted as an application to regenerative medicine and life sciences, but no practical differentiation induction method has been established. In particular, in a static and uniform culture environment that can be handled by conventional experimental methods, differentiation induction cannot be precisely controlled in space and time. The present invention provides a technique for precisely controlling the microenvironment of the cell tissue for such a problem, and is useful in the field of regenerative medicine and the pharmacokinetic / toxicity test field using animal cells.

  DESCRIPTION OF SYMBOLS 1 ... Device, 2 ... 1st board | substrate, 3 ... 2nd board | substrate, 4 ... Porous film, 21 ... Lower layer flow path, 31 ... Upper layer flow path, 32-37 ... Through-hole, 211 ... Multilayer flow distribution part, 212a -212c ... Branch channel part, 212d ... Communication channel part, 213 ... Multilayer flow discharge part, 311 ... Micro space part, 312-313 ... Lead-in / out part

Claims (13)

A method for controlling material distribution in a microspace filled with liquid,
N types of liquids (n is an integer of 2 or more) having different compositions are joined together as a laminar flow to form a multilayer flow in which n laminar flows are adjacent in the width direction,
A substance distribution control method, characterized in that the multilayer flow is circulated through a microchannel disposed under a microspace through a porous membrane having a pore diameter of 1 μm or more and 20 μm or less .   The material distribution control method according to claim 1, wherein the multilayer flow is circulated so that the Reynolds number is less than 2000 in the microchannel. A device used in the material distribution control method according to claim 1 or 2,
Comprising a first substrate and a second substrate each having a microchannel structure on the surface;
The microchannel structure on the surface of the first substrate includes a multi-layer flow distribution portion through which a multi-layer flow circulates, a multi-layer flow forming portion disposed upstream of the multi-layer flow distribution portion, and a downstream side of the multi-layer flow distribution portion. A multi-layer flow outlet disposed in the
The microchannel structure on the surface of the second substrate has a microspace portion having substantially the same pattern as the multilayer flow circulation portion, and a lead-in / inlet portion for introducing or leading out liquid to the microspace portion. ,
The first substrate and the second substrate are laminated so that the surface on which the microchannel structure is provided is on the inside, and the positions of the multilayer flow circulation portion and the micro space portion are matched,
A device in which a porous film having a pore diameter of 1 μm or more and 20 μm or less is sandwiched between the multilayer flow distribution part and the micro space part. A method of culturing cells in a microspace filled with liquid,
N types of culture solutions having different compositions (n is an integer of 2 or more) are joined together as a laminar flow to form a multilayer flow in which n laminar flows are adjacent in the width direction;
A cell culturing method, wherein the multilayer flow is circulated through a microchannel disposed under a microspace through a porous membrane having a pore diameter of 1 μm or more and 20 μm or less .   The cell culture method according to claim 4, wherein pre-culture is performed to attach the cells to the micro space side of the porous membrane, and then the circulation of the multilayer flow is started.   The cell culture method according to claim 4, wherein the multilayer flow is performed in a state where the cells are not attached to the porous membrane.   The cell culture method according to any one of claims 4 to 6, wherein the cell is a pluripotent stem cell.   The cell culture method according to claim 6, wherein the cells form a three-dimensional structure. A method for controlling cell differentiation in a microspace filled with liquid,
The n-type (n is an integer of 2 or more) culture solutions having different types or concentrations of substances that control cell differentiation are combined as laminar flows to form a multi-layer flow in which n laminar flows are adjacent in the width direction. And
A method for controlling cell differentiation, characterized in that the multilayer flow is circulated through a microchannel disposed under a microspace through a porous membrane having a pore diameter of 1 μm or more and 20 μm or less .   The cell differentiation control method according to claim 9, wherein pre-culture is performed to attach the cells to the microspace side of the porous membrane, and then the circulation of the multilayer flow is started.   The cell differentiation control method according to claim 9, wherein the multilayer flow is circulated in a state where the cells are not attached to the porous membrane.   The cell differentiation control method according to any one of claims 9 to 11, wherein the cell is a pluripotent stem cell.   The cell differentiation control method according to claim 11, wherein the cells form a three-dimensional structure.