JP4219158B2 - Cell culture cell - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a cell for cell culture effective when handling cells, and particularly to a cell for cell culture suitable for use in biotechnology research using a microchip.
[0002]
[Prior art]
Research using a wide range of cells such as drug discovery, food, crop development and genetic engineering has been conducted. Conventionally, cells are cultured in a culture place or the like placed in a petri dish or the like and used for various experiments.
[0003]
When elucidating the functions of cells, it is necessary to examine reactions caused by many drugs and introduced substances. Therefore, there is a demand for a method capable of obtaining results with a large number of types and a small amount of samples at a time for a specific reaction efficiently.
[0004]
In recent years, in the field of analytical chemistry, research on μTAS (Micro Total Analysis Systems) has become active, and it is expected to use a microchip to achieve high-speed analysis, sample reduction, and solvent saving. In the reaction in the micro space on the microchip, there is a possibility that the reaction efficiency can be improved as compared with the reaction using the conventional chemical operation.
[0005]
[Problems to be solved by the invention]
When culturing cells, it is necessary to control the environment surrounding the cells. In particular, it is important to replace the minimum necessary solution for cells and to keep the solution state stable.
[0006]
However, in the microchip, since the space for processing the cell solution is normally a closed space, it is difficult to control the concentration of carbon dioxide in the culture solution by balancing with the outside air.
[0007]
There is also a method of controlling the carbon dioxide concentration in the culture solution by continuing to feed the fresh culture solution. However, particularly in the case of adherent cells, the flow of the solution becomes stress or the cell produces Cell culture cannot be performed successfully due to the fact that substances necessary for growth are washed away.
The object of the present invention is to enable cell culture in a microchip by simple means.
[0008]
[Means for Solving the Problems]
In the present invention, by disposing a porous material that allows gas to pass but not liquid, a structure that can maintain the carbon dioxide gas concentration in the solution by equilibrium with the outside air even in the microchip is realized.
[0009]
That is, the cell culturing cell of the present invention temporarily holds cells by allowing a liquid to flow and a cross-sectional area connected to the flow path and perpendicular to the flow direction of the flow path to be larger than the flow path. The cell culture chamber is provided inside the substrate, and at least a part of the wall surface existing between the cell culture chamber and the external space is made of a porous material and is in contact with the outside air.
[0010]
In general, a microchip is manufactured by bonding substrates having flow paths and the like formed on a bonding surface. At this time, since liquid-tightness is required, materials such as silicon (Si), glass, and plastic are usually used. These materials are usually liquid-tight and air-tight at the same time, and the cell culture chamber formed in the chip generally has a structure in which no liquid / gas exchange occurs except for the solution flow from the flow path. It is. In the cell culture cell structure of the present invention, for example, the material at the upper part of the culture chamber is made of porous glass, so that the exchange of the solution occurs only from the flow path, but the exchange of the gas takes place between the outside air in the culture chamber. realizable.
[0011]
DETAILED DESCRIPTION OF THE INVENTION
The substrate constituting the cell for cell culture can be formed by adhering or joining a plurality of substrates, and the plurality of substrates form a flow path and a cell culture chamber. In order to form the flow path and the cell culture chamber in the substrate, it is easiest to bond or bond a plurality of substrates in this way.
[0012]
In one form in which the flow path and the cell culture chamber are formed by a plurality of substrates, the flow path is formed by one or both of the opposing surfaces of the two substrates, and the cell culture chamber is connected to the flow path and penetrates through one of the substrates. It is formed as a through hole, and the opening of the through hole is sealed with a porous third substrate as the porous material.
[0013]
In that case, the porous third substrate for sealing the opening of the through-hole constituting the cell culture chamber is provided only in the opening region on the substrate on which the through-hole serving as the culture chamber is formed. Can be. In this case, it is only necessary to bond or bond the porous substrate only to the opening region, and the manufacture is easy.
[0014]
In addition, the porous third substrate that seals the opening of the through-hole constituting the cell culture chamber can have substantially the same shape as the substrate on which the through-hole serving as the culture chamber is formed. In this case, the surface of the substrate on which the through-hole serving as the culture chamber is formed becomes flat and easy to handle.
[0015]
In another form in which the flow channel and the cell culture chamber are formed by a plurality of substrates, the flow channel is formed by one or both of the opposing surfaces of the two substrates, and the cell culture chamber is connected to the flow channel and penetrates through one of the substrates. It is formed as a through hole, and the opening is sealed by fitting a porous third substrate, which is a porous material, into the opening of the through hole. In this case, it can be set as the cell culture cell which joined the porous substrate more reliably.
[0016]
Still another embodiment in which the flow path and the cell culture chamber are formed by a plurality of substrates is composed of two substrates, and the flow path and the cell culture chamber are formed as a groove having a bottom on the surface of one of the substrates. The other substrate provided on the surface side of the substrate is a porous substrate as a porous material. In this case, the concentration of carbon dioxide in the solution can be maintained not only in the cell culture chamber but also in the flow path by equilibrium with the outside air.
[0017]
Porous glass or porous resin can be used as the porous material. As the porous glass, for example, Vycor # 7930 (Corning product: hole diameter of several nm and thickness of 0.5 mm) can be used. As the porous resin, for example, polytetrafluoroethylene having a hole diameter of about 100 nm and a thickness of about 1 mm can be used.
[0018]
The cell culture cell of the present invention includes a cell having a plurality of channels connected to the cell culture chamber, and at least one of the channels is provided with a microvalve for controlling the flow of liquid. According to such a cell culture cell, various treatments are possible.
[0019]
【Example】
FIG. 1 shows an embodiment of the cell culture cell of the present invention. (A) to (C) respectively represent three substrates constituting the cell culture cell, and (D) shows the assembled cell culture cell in a direction perpendicular to the flow path at the cell culture chamber position. Sectional drawing of the cut | disconnected state, (E) is sectional drawing of the state which cut | disconnected the cell for cell culture assembled along the flow path.
[0020]
Reference numerals 1 and 2 denote glass substrates, for example, quartz substrates. The thickness of the glass substrate 1 is about 0.2 to 1.0 mm, and the thickness of the glass substrate 2 is about 0.1 to 1.0 mm.
On one side of the glass substrate 1, a minute channel groove 6 used as a liquid sample channel having a width and depth of several hundred μm or less, and a cell culture having a diameter of about 1 mm located at the center of the channel groove 6 A through hole 5 for the chamber and through holes 3 and 4 for sample introduction and discharge located at both ends of the flow channel groove 6 are formed.
[0021]
On the other hand, the glass substrate 2 is an unprocessed substrate.
The surface of the glass substrate 1 on which the flow channel grooves 6 are formed and one surface of the glass substrate 2 are brought into close contact with each other, and are joined in a liquid-tight manner by means such as joining with a hydrofluoric acid solution. A channel groove 6 and a cell culture chamber 5 are formed.
[0022]
A porous glass plate 7 processed into a larger circle than the opening of the cell culture chamber 5 is disposed on the glass substrate 1 so as to cover the opening of the cell culture chamber 5, and bonded with an adhesive 8. Vycor # 7930 (Corning product) is used as the porous glass plate 7. Thereby, the cell for cell culture of one Example is comprised.
[0023]
FIG. 2 shows another embodiment. The glass substrates 1 and 2 and the channel groove 6 formed in the glass substrate 1, the through hole 5 for the cell culture chamber, and the through holes 3 and 4 for introducing and discharging the sample are the same as those in the embodiment of FIG. It is.
[0024]
In this embodiment, a porous glass substrate 10 having the same shape as the glass substrate 1 is provided so as to cover the glass substrate 1. The same porous glass substrate 10 as that of the embodiment of FIG. 1 is used. Through holes are formed in the porous glass substrate 10 at positions corresponding to the through holes 3 and 4 of the glass substrate 1.
[0025]
The glass substrates 1 and 2 and the porous glass substrate 10 are brought into close contact with each other as shown in FIG.
[0026]
FIG. 3 shows still another embodiment. (A) is a top view, (B) is sectional drawing of the state cut | disconnected along the flow path.
11 and 12 are glass substrates, for example, quartz substrates having a thickness of about 0.2 to 1.0 mm.
[0027]
On one side of the glass substrate 12, a minute channel groove 16 used as a channel for a liquid sample having a width and depth of several hundreds μm or less is formed.
[0028]
The glass substrate 11 has a through-hole 15 for a cell culture chamber having a diameter of about 1 mm at a position corresponding to the center of the flow channel 16 and a sample introduction and discharge at positions corresponding to both ends of the flow channel 16. Through-holes 13 and 14 are formed. The through-hole 15 for the cell culture chamber is formed in a shape that expands in a tapered shape so that the diameter is small on the side facing the glass substrate 12 and the diameter is large on the opposite side. A porous glass plate 17 is fitted into the through-hole 15 from the larger diameter side, and is adhered by an adhesive 18 to seal the opening on the larger diameter side of the through-hole 15. A space for cell culture chambers is formed on the smaller diameter side. As the porous glass 17, Vycor # 7930 (a product of Corning) is used.
[0029]
The surface of the glass substrate 12 on which the flow channel grooves 16 are formed and the surface of the glass substrate 11 with the small diameter of the through hole 15 are brought into close contact with each other, and are liquid-tight by means such as bonding with a hydrofluoric acid solution. A cell culture cell is formed by joining.
[0030]
A porous glass 7 processed into a larger circle than the opening of the cell culture chamber 5 is disposed on the glass substrate 1 so as to cover the opening of the cell culture chamber 5, and bonded with an adhesive 8. Thereby, the cell for cell culture of one Example is comprised.
[0031]
FIG. 4 shows still another embodiment.
Reference numeral 22 denotes a glass substrate, for example, a quartz substrate having a thickness of about 0.2 to 1.0 mm. On one side of the glass substrate 22, a minute channel groove 26 used as a liquid sample channel having a width and depth of several hundred μm or less is formed, and the diameter is about 1 mm at the center of the channel groove 26. Thus, a recess 25 for the cell culture chamber having a depth deeper than that of the flow channel groove 26 is formed.
[0032]
The substrate 21 is a porous glass substrate, and the outer shape is the same as that of the glass substrate 22. In the porous glass substrate 21, through holes 23 and 24 for sample introduction and discharge are formed in a tapered shape at positions corresponding to both ends of the flow channel groove 26. As the porous glass substrate 21, Vycor # 7930 (product of Corning) is used.
[0033]
The surface of the glass substrate 22 in which the flow channel grooves 26 are formed and the surface of the porous glass substrate 11 with the small diameters of the through holes 23 and 24 are brought into close contact with each other, for example, means such as bonding with a hydrofluoric acid solution. A cell culture cell is formed by liquid-tight joining.
[0034]
FIG. 5 shows still another embodiment. In the embodiment of FIG. 1, a plurality of flow paths on the cell introduction side are provided.
The glass substrate 31 corresponds to the glass substrate 1 in FIG. 1, but one flow channel leading to the cell culture chamber is a flow channel for introducing cells or reagents, and is branched into two, symbols 36a, It is shown as 36b. Through holes 37a and 37b serving as inlets are formed at the tip positions of the introduction flow paths 36a and 36b. The introduction flow paths 36a and 36b are provided with microvalves 36a and 36b for controlling the flow of the liquid to be interrupted. As the microvalves 36a and 36b, those driven using piezoelectric elements, those driven by electrodes, those driven by air from a compressor placed outside, those using fluid expansion or phase change due to heating Any known one can be used. The other channel groove connected to the cell culture chamber is a cell introduction channel 36, and a through-hole 34 for cell discharge is formed at the tip position.
[0035]
Reference numeral 37 denotes a porous glass substrate that seals the opening of the through hole of the glass substrate 31 serving as a cell culture chamber. Although not shown in the drawing, a glass substrate corresponding to the glass substrate 2 of FIG. 1 is bonded to the lower side of the glass substrate 31.
[0036]
In the cell culture cell of FIG. 5, for example, a cell solution is introduced from one inlet 33a and a reagent is introduced from the other inlet 33b, so that the cells and the reagent are introduced from separate inlets and mixed in the cell culture chamber. It can be used to react.
[0037]
In the above embodiment, the porous material is not limited to glass, and the same effect can be obtained even when polytetrafluoroethylene or other plastic materials are used.
The substrate material is not limited to quartz glass, and various materials such as other glass, Si, ceramic, plastic, and the like can be used. For example, when a plastic material is used, integral molding is also possible.
[0038]
【The invention's effect】
According to the cell culture cell of the present invention, stable cell culture can be achieved with a simple structure in which at least a part of the wall surface existing between the cell culture chamber and the external space is made of a porous material and is in contact with the outside air. It becomes possible.
Since the structure of the present invention is a very simple structure and can be introduced into various manufacturing processes, it can be applied to a complicated microchip incorporating a valve or the like.
By using a microchip, there are many merits such as a small amount of sample, high speed measurement, and simultaneous measurement of multiple samples.
[Brief description of the drawings]
FIG. 1 is a diagram showing a cell culture cell according to one embodiment, wherein (A) to (C) are plan views respectively showing three substrates constituting the cell culture cell, and (D) is assembled. Sectional drawing of the state which cut | disconnected the cell for cell culture which cut | disconnected the direction perpendicular | vertical to a flow path in the cell culture room position, (E) is sectional drawing of the state which cut | disconnected the assembled cell culture cell along a flow path .
FIG. 2 is a cross-sectional view showing a cell culture cell according to another embodiment cut along a flow path.
FIGS. 3A and 3B are diagrams showing a cell culturing cell according to still another embodiment, wherein FIG. 3A is a plan view and FIG. 3B is a cross-sectional view taken along a flow path.
FIG. 4 is a cross-sectional view showing a cell culturing cell of still another embodiment cut along a flow path.
FIG. 5 is a plan view showing a cell culturing cell of still another example.
[Explanation of symbols]
1, 2, 11, 12, 22, 31 Glass substrate 3, 4, 13, 14, 23, 24, 33a, 33b, 34 Through hole 6, 16, 26, 36, 36a, 36b Channel grooves 5, 15, 25 Cell culture chamber 7, 10, 17, 21, 37 Porous glass plate 8, 18 Adhesive

Claims (9)

It is configured by adhering or joining a plurality of substrates, and a flow path capable of flowing a liquid, and a cross-sectional area in a direction perpendicular to the flow direction of the flow path connected to the flow path is larger than the flow path to temporarily store the cells. and holding for cell culture chamber a cell culture cell provided therein,
The flow path is formed on the opposing surface of two of the substrates,
The cell culture cell, wherein the cell culture chamber is formed to have an opening communicating with an external space, and the opening is sealed with a porous material . The cell culture chamber is formed as a through-hole penetrating one of the two substrates forming the flow path, and the opening of the through-hole becomes the opening, and the porous material as the porous material The cell culture cell according to claim 1 , which is sealed by a third substrate. The cell for cell culture according to claim 2 , wherein the porous third substrate for sealing the opening of the through hole is provided only in the opening region on the one substrate. The cell for cell culture according to claim 2 , wherein the porous third substrate for sealing the opening of the through hole has substantially the same shape as the one substrate. The cell culture chamber is formed as a through hole penetrating one of the two substrates forming the flow path, and the opening of the through hole becomes the opening, and the porous material is formed in the opening. The cell culture cell according to claim 1 , wherein the opening is sealed by fitting a porous third substrate. It consists of two substrates, and the flow path and the cell culture chamber are formed as a groove having a bottom on the surface of one substrate, and the other substrate provided on the surface side of the substrate is the porous material. The cell for cell culture according to claim 1 , wherein an opening of the cell culture chamber is sealed with a porous material by being a porous substrate. Cell culture cells according to any one of claims 1 to 6 using a porous glass as the porous material. Cell culture cells according to any one of claims 1 to 6 using a porous resin as the porous material. The cell culture medium according to any one of claims 1 to 8 , wherein a plurality of the flow paths connected to the cell culture chamber are provided, and at least one of the flow paths is provided with a microvalve for controlling a liquid flow. cell.

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