WO2010125665A1

WO2010125665A1 - Reactor

Info

Publication number: WO2010125665A1
Application number: PCT/JP2009/058457
Authority: WO
Inventors: 陽一石川
Original assignee: エイブル株式会社
Priority date: 2009-04-30
Filing date: 2009-04-30
Publication date: 2010-11-04

Abstract

Provided is a reactor that performs environmental regulation of, for example, the pH or dissolved oxygen concentration of a contained liquid that is accommodated therein by supplying a gas from a gas source into the interior from the bottom. A communicating tube that passes through the bottom and the contained liquid and opens above the liquid surface is provided inside the reactor, and the gas is supplied into the space above the liquid surface via the communicating tube. This makes it possible to dissolve the gas in the contained liquid gradually while preventing generation of gas bubbles or foam. Moreover, the range of fluctuation in the pH or dissolved oxygen concentration of the contained liquid is reduced, and the environmental regulation thereof is made smooth.

Description

reactor

The present invention relates to a reactor used for, for example, examination of culture conditions for cells and tissues, and more specifically, a gas used for controlling environmental conditions of a content liquid, for example, pH and dissolved oxygen concentration, as bubbles / bubbles. The present invention relates to a reactor that can be dissolved in a content liquid while preventing occurrence of the above, and a microplate provided with a plurality of such reactors in an aligned state.

A microplate provided with a plurality of reactors as described above (in the microplate, the reactor is also referred to as a well and the microplate is also referred to as a cassette), for example, examination of reaction conditions such as biopolymers, chemical substances, Widely used in various biological and biochemical tests such as examination of culture conditions for cells, tissues, etc. of microorganisms, insects, animals and plants, drug discovery screening, cytotoxicity tests, pharmacokinetic tests, safety evaluation of compounds, etc. There are various types of reactors such as 6, 24, 96, for example.

In various reactions, cultures, tests, etc. using the microplate as described above, various environmental conditions (for example, pH, dissolved oxygen concentration (DO), temperature, etc.) of the contents of each reactor are controlled. In this case, carbon dioxide, ammonia gas, nitrogen gas or the like is used for pH control, and oxygen gas (or air), nitrogen gas or the like is used for dissolved oxygen concentration control.
The various sensors used for these controls, the control gas supply vents, etc. are related equipment such as external detection / control mechanisms (external) means the use of reactors or microplates. In general, it is convenient to be disposed at the bottom of the reactor.

As such a reactor, for example, “CELLERATOR” (trade name manufactured by MicroReactor Technologies Inc.) is available (for example, see Non-Patent Document 1 and Patent Document 1).

FIG. 3 is a partial sectional view for explanation showing the structure of the reactor. The reactor 51 shown in the figure has a container shape made of a transparent material, and a pH sensor 53 (for example, , A fluorescence measurement system pH sensor) and a dissolved oxygen concentration sensor 54 (for example, a fluorescence measurement system dissolved oxygen concentration sensor) are provided in a recess formed in a state of being separated from each other with the center point of the bottom 52 interposed therebetween. In addition, a vent hole 56 penetrating vertically is provided at the center of the bottom portion 52, and a vent filter such as a membrane is attached to the vent hole 56.

Then, the environmental control of the pH of the content liquid 61 and the dissolved oxygen concentration when this reactor 51 is used is performed, for example, in the following manner.
Below the

sensors

53 and 54 on the bottom 52 of the reactor 51, an excitation light source and fluorescence of a fluorometer (microplate reader in the case of a microplate) as an example of an external detection / control mechanism so as to be opposed to each other The light receiving unit is positioned.

In this fluorometer, when light is incident (projected) on each

sensor

53, 54 from an LED (light emitting diode) or the like of the light source, fluorescence is emitted from each

sensor

53, 54. Enters a detector such as a photodiode, and an output signal from the detector enters a signal processing device (computer) where data processing and device control are performed.
The pH and dissolved oxygen concentration at this time can be obtained by measuring fluorescence intensity, decay time, and the like.

Then, the pH of the content liquid 61 and the dissolved oxygen concentration are controlled based on the output signal by the above-described signal processing device (computer) in which the gas 55 whose supply gas type, flow rate, supply time, etc. are controlled is the bottom 52. It is performed by being supplied from the vent hole 56.
The gas 55 supplied in this way is dissolved in the content liquid 61 by stirring the reactor (in the case of a microplate, the whole) by an external stirring means, and the pH and dissolved oxygen concentration of the inner solution are reduced. Will be adjusted and controlled.

Further, the

heat conductors

59 and 60 that can come into contact with the external thermistor 57 and the heater 58 are separated from each other across the center point of the outer surface of the bottom 52, and also from the pH sensor 53 and the dissolved oxygen concentration sensor 54. The temperature of the content liquid 61 can be controlled in substantially the same manner as described above.
Reference numeral 62 in the drawing is an external adapter that communicates with an external supply gas source (not shown), contacts the bottom 52 in an airtight state, and is connected to the vent 55.

US Patent Application Publication No. 2005/0176155

However, in the reactor 51 that supplies the gas 55 from the vent 56 of the bottom 52 as described above, the gas 55 is supplied as bubbles from the upper surface of the vent filter of the vent 56, and the bubbles are stirred in the content liquid. May not increase, but rather increase, causing the following problems.

That is, protein or cells adhere to the bubbles / bubbles and normal contact with the liquid is prevented, the contents liquid flows out of the reactor together with bubbles / bubbles, especially in the case of animal cell culture.・ Cells may be damaged due to the share stress when bubbles are destroyed. Furthermore, when serum is used as the medium, if there are bubbles / bubbles, the bubbles will become noticeable with time, and it may be difficult to continue the culture.
From such a viewpoint, it is strongly required to prevent the generation of bubbles and bubbles in the content liquid.

In addition, from the above, a method of suppressing the generation of bubbles / bubbles by adding an antifoaming agent to the content liquid, or a method of eliminating bubbles / bubbles may be adopted, but also in this method, Problems still remain such as inhibition of culture by an antifoaming agent and, in some cases, separation and removal of the antifoaming agent after culture.

Furthermore, in the reactor having the structure as described above, since the amount of the liquid content is small, the amount of change in pH and dissolved oxygen concentration in one aeration operation is large, so the fluctuation range of pH and dissolved oxygen concentration is large. (Hunting), it becomes difficult to control these. In order to avoid this, even if the gas supply amount is reduced (pressure etc. are reduced), the gas will form larger bubbles on the upper surface of the ventilation filter, and these bubbles will be in the ventilation filter. The above problem cannot be solved because the liquid is intermittently detached from the upper surface and released.

The present invention has been made in view of the problems of the prior art, and the object of the present invention is a reactor in which gas is supplied from the bottom, while preventing the generation of bubbles and bubbles. And a reactor capable of dissolving the supplied gas in the content liquid and enabling smooth control by reducing the fluctuation range of pH and dissolved oxygen concentration in the control, and a plurality of such reactors. It is to provide a microplate.

As a result of intensive studies to achieve the above object, the present inventor established a vent pipe that penetrates the bottom of the reactor and the content liquid, and supplies gas from the bottom of the reactor to the space above the content liquid level. The present inventors have found that the above problems can be solved by achieving gas-liquid contact at the interface with the liquid, and have completed the present invention.

That is, the reactor of the present invention has a bottomed cylindrical shape with an opening that can be sealed by a ventilation member, and supplies the gas from the supply gas source to the inside from the bottom, and the contents stored inside A reactor for controlling the environment of the liquid, comprising a vent pipe that penetrates the bottom of the reactor and the content liquid, opens on the liquid level, and supplies the gas to the space above the liquid level. To do.

In a preferred embodiment of the reactor of the present invention, a vent filter is provided inside the vent tube. In another preferred embodiment of the reactor of the present invention, the environmental control target of the content liquid is pH. In another preferred embodiment of the reactor of the present invention, a pH sensor and / or a dissolved oxygen concentration sensor are provided on the inner surface of the bottom.

Further, the microplate of the present invention is characterized in that a plurality of the reactors are arranged in an aligned state via a support member.

According to the present invention, since the bottom of the reactor and the vent pipe penetrating the content liquid are provided therein, by supplying gas from the bottom of the reactor to the space above the content liquid level, the liquid and Gas-liquid contact occurs at the interface of the gas, and the supplied gas can be dissolved in the content liquid while preventing the generation of bubbles and bubbles, and the fluctuation of the pH and dissolved oxygen concentration in the control becomes small and smooth. Control becomes possible.

FIG. 2 is a longitudinal sectional view (a) showing an embodiment of the reactor of the present invention, and a horizontal sectional view (b) taken along line AA in FIG. 1 (a). It is the partial longitudinal cross-sectional view (a) and partial plan view which show the structural example of the microplate of this invention. It is a fragmentary sectional view for explanation which shows the structure of the conventional reactor.

Hereinafter, the reactor and microplate of the present invention will be described in more detail.

As described above, the reactor and microplate of the present invention are used for various reactions, cultures, tests, etc., especially for examination of optimization of culture conditions such as pH, dissolved oxygen concentration, temperature, medium type and concentration, etc. Preferably used.
In this case, the environmental control of the content liquid (reaction liquid, culture liquid, test liquid, etc.) by gas supply includes, for example, control of pH, dissolved oxygen concentration, etc. alone or in combination. As described above, the gas used for these controls includes, for example, carbon dioxide, ammonia gas, nitrogen gas, etc. for pH control, and oxygen gas (or air), nitrogen gas for dissolved oxygen concentration control. Etc.

The reactor of the present invention has a bottomed cylindrical shape, and its opening is normally used as a breathable member, for example, a spongy synthetic resin (eg, polyurethane resin, silicone resin, etc.) stopper or cap, membrane ( Sheet) or the like.
The shape of the reactor is not particularly limited, such as a cylindrical shape, a rectangular tube shape, a flask shape, or a conical shape. Typically, a reactor having a circular shape with a horizontal section having the same or different diameters at the top and bottom is preferably used. be able to.

Further, examples of the bottom shape of the reactor include a flat bottom, a round bottom, and a V bottom. When various control sensors are provided on the bottom, a flat bottom is preferable.
The volume of the reactor is not particularly limited and is selected and determined according to the purpose. For example, the reactor volume is 100 to 20,000 μl.

A concave portion is formed at an appropriate position on the inner surface of the bottom of the reactor, and as described above, a sensor required for environmental control, such as a pH sensor or a dissolved oxygen concentration sensor, is provided.
In addition, the concave portion at an appropriate position on the outer surface of the bottom is appropriately provided with a heat conductor or the like that comes into contact with an external thermistor or heater as necessary.
In addition, a vertical baffle plate (baffle plate) can be provided on the inner surface of the reactor, thereby changing the state of stirring and shear stress as appropriate.

The material of the reactor is not particularly limited, and commonly used materials such as synthetic resins such as polystyrene, polyethylene, polypropylene, polyacrylonitrile, and polyethylene terephthalate, glass such as borosilicate glass and quartz glass, etc. Can be mentioned.
When information from a pH sensor or a dissolved oxygen sensor provided at the bottom of the reactor is optically read from below the bottom, a transparent material is used for the bottom.

The reactor of the present invention controls the environment of the content liquid, particularly control of pH and dissolved oxygen concentration, and the most characteristic feature is that the bottom of the reactor and the content liquid stored in the reactor are moved up and down ( By establishing a vent pipe that penetrates in the axial direction) and opens in the space above the content liquid level, the gas that is controlled and supplied to this space is allowed to flow at the interface with the content liquid by stirring the content liquid. It is in a point where it is gradually dissolved in the content liquid by gas-liquid contact.

It should be noted that the structure characterized by the present invention other than the installation of a vent pipe inside the reactor, for example, a pH sensor provided at the bottom of the reactor, a dissolved oxygen concentration sensor, and a mechanism relating to external detection and control of information from these sensors As for (detection / control mechanism, for example, a fluorometer, etc.) and the like, it goes without saying that well-known ones and methods can be applied, and description thereof will be omitted.

The lower end of the vent pipe can be provided at an appropriate position on the bottom of the reactor, but is preferably near the center.
As the material of the vent pipe, the same material as that of the main body portion of the reactor can be used. In this case, the bottom portion of the reactor and the vent pipe can be integrally formed. Further, a vent pipe made of a flexible synthetic resin or the like may be used, and the reactor bottom and the vent pipe at this time are connected and fixed by an appropriate method such as adhesion.
The length (height) of the vent pipe is preferably set such that the content liquid does not flow from the upper end opening during stirring. Further, the thickness of the vent pipe is not particularly limited as long as it is sufficient to supply a necessary amount of gas to the space on the content liquid surface.

The lower end opening of the vent pipe (the outer opening at the bottom of the reactor) is detachable and airtight to an external supply gas source controlled by an external detection / control mechanism, such as a cylinder filled with the gas to be supplied. Connected to and communicated with.

The control of the pH and dissolved oxygen concentration of the content liquid stored in the reactor is performed by the same method as described in the conventional example.
That is, information of each sensor is detected by an external detection / control mechanism, for example, an excitation light source and a fluorescence light receiving unit of a fluorometer, which is positioned below each sensor at the bottom of the reactor and is opposed to each sensor, Each output signal is processed by a signal processing device (computer), and the type of gas (ammonia gas, carbon dioxide gas, oxygen (or air), nitrogen gas) from the external supply gas source, flow rate, supply time, etc. Is controlled.

The gas whose kind, supply amount, and the like are controlled in this way is supplied to the space above the liquid level from the upper end portion that passes through the pipe from the lower end opening of the vent pipe and opens on the content liquid level.
This gas supply is usually performed while stirring the content liquid by a known external stirring means, for example, a shaking device that stirs up and down, reciprocating or rotationally shaking or vibrating, etc. By the gas-liquid contact at the interface with the content liquid by such stirring, the content liquid is gradually dissolved.

As a result, the occurrence of bubbles and bubbles in the content liquid at the time of gas supply to the content liquid as in the past is prevented, and the amount of change in pH and dissolved oxygen concentration is also small, so the fluctuation range of the target numerical value is These controls become smoother and smoother.
In addition, about supply of the gas to the said reactor, what kind of methods, such as continuous and intermittent, may be sufficient, for example. Further, the stirring of the content liquid may be any method such as continuous or intermittent.

In addition, when a gas is supplied into the content liquid of the reactor, the generation of bubbles and bubbles promotes evaporation of the content liquid, resulting in a decrease in the liquid volume. It is also possible to prevent a decrease in the amount of liquid due to proper evaporation.
Further, the upper end opening of the vent pipe is positioned at the upper part of the space, and from there, a gas that is higher than the temperature of the content liquid by the heating means (for example, a gas having a temperature 2 to 10 ° C higher than the temperature of the content liquid) If it supplies to, the convection of the gas in space is prevented, As a result, the reduction | decrease in the amount of contents liquid by evaporation is prevented similarly to the above.

The reactor of the present invention can also be used for controlling dissolved carbon dioxide gas.
In addition, as a measuring method of pH and dissolved oxygen concentration of the content liquid during control, in addition to the method of measuring with a pH sensor or a dissolved oxygen concentration sensor provided on the inner surface of the bottom of the reactor as described in the above known example, For example, other known methods such as measuring by inserting a pH sensor or a dissolved oxygen concentration sensor into the content liquid from above the reactor can also be employed.

And in the reactor of this invention, a ventilation filter can also be provided in the inside of the said ventilation pipe.
This ventilation filter is effective in preventing foreign matter (including microorganisms) from entering the contents from the external gas supply source side and intrusion of the contents liquid into the ventilation pipe. The position where such a ventilation filter is provided may be an appropriate position inside the ventilation pipe, but for the purpose described above, it is considered more effective to be near the upper end opening of the ventilation pipe.
In addition, as a material of the said ventilation filter, a well-known thing (filter medium containing a film | membrane etc.) can be used, For example, porous urethane etc. can be mentioned.

The microplate of the present invention is provided with a plurality of reactors arranged in a line through support members, and is also referred to as a cassette as described above.
The alignment state is not particularly limited, but typically the whole is rectangular, and is arranged in a ratio of 2: 3 in the width direction and the longitudinal direction in the number (for example, In the case of 24, 4 × 6 rows, etc.).

The supporting method by the festival support member in which each reactor is arranged may follow a known method. For example, the reactors are horizontally connected to each other by a flat support member at an appropriate height from the opening to the bottom, and a skirt is provided below the support member between the reactors on the outer periphery of the microplate. For example, a method may be used in which the lower end of the skirt is positioned below the bottom of the reactor.

As the support member, the same type of material as that of the reactor can be suitably used as the support member, and the microplate can be manufactured by integrally molding an appropriate number of reactors in the same manner as a known microplate.
In addition, as another support method, it is possible to employ a method in which the reactors are connected and fixed to each other by adhesion or the like on the outer wall of the reactor. In such a case, the adjacent reactor functions as a support member. Become.
When the microplate is used, the lower end of the skirt is fixed in a state of being placed on a fixing member of an external related device.

The microplate of the present invention having such a structure can be effectively used as a cassette capable of performing reactions, cultures, tests, and the like under a plurality of conditions simultaneously in the same manner as a conventional microplate. .

Hereinafter, although the present invention will be described in more detail based on examples with reference to the drawings, it goes without saying that the present invention is not limited to these examples.

Example 1
1A and 1B are a longitudinal sectional view and a horizontal sectional view, respectively, showing an embodiment of the reactor of the present invention.
In the figure, a reactor 1 has a bottomed cylindrical shape, and a side wall 2 and a bottom portion (flat bottom) 3 are integrally molded. In this example, the reactor 1 is made of transparent polystyrene, and the content is as a whole. 10 ml. In this example, the vent pipe 4 having a length of about 7/8 of the reactor 1 is erected in a state of penetrating through the center of the bottom portion 3 in the vertical direction (axial direction).

The lower end of the vent pipe 4 opens toward the outer surface of the bottom portion 3 to form a lower end opening 4a (opening of the bottom portion 3). In the space 6 on the liquid surface 5a of the content liquid 5 (5 ml) stored up to half, it opens as an upper end opening 4b.
In this example, the vent pipe 4 is formed integrally with the bottom 3, and a vent filter 7 made of closed-cell foamed urethane is provided at the upper end opening 4 b of the vent pipe 4. Further, in this embodiment, a vent member 8 made of sponge-like silicon resin is attached to the upper end opening of the reactor 1, and the upper end opening of the reactor 1 is sealed.

Further, an external adapter or the like (not shown) is detachably and airtightly connected to the lower end opening 4a of the vent pipe 3 so as to communicate with an external supply gas source. In this example, the supply gas source is controlled by a fluorometer as an external detection / control mechanism (not shown).

Recesses are formed in the bottom 3 of the reactor 1 at positions spaced apart from each other across the center point on the inner surface thereof, and in each of these recesses, a fluorescence measurement method pH sensor 9 and a fluorescence measurement method dissolved oxygen are respectively provided. A density sensor 10 is provided.
Further, although not shown in the figure, a recess is formed at a position spaced apart from each other across the center point of the outer surface of the bottom 3 and also away from the pH sensor 9 and the dissolved oxygen concentration sensor 10. Thermal conductors that are in contact with the external thermistor and the heater are respectively disposed therein, and thereby the temperature of the content liquid 5 can be controlled via an external control mechanism or the like.

In addition, the usage example of the said reactor 1 is demonstrated in Example 2 as a usage example of the microplate provided with two or more of the reactors 1. FIG.

(Example 2)
FIGS. 2A and 2B are a partial longitudinal sectional view and a partial plan view, respectively, showing an embodiment of the microplate of the present invention, and the microplate includes a plurality of reactors 1 shown in Embodiment 1. These are arranged in an aligned state.

The microplate 20 shown in FIGS. 2 (a) and 2 (b) has a plurality of reactors 1 (pH sensor 9, dissolved oxygen sensor 10 and ventilation filter 7 are omitted) shown in FIG. In this example, it consists of 24 reactors (4 × 6 rows).
The plurality of reactors 1 are supported by a transparent polystyrene support member 21 made of the same material as that of the reactor 1 at an appropriate height position on the side wall 2, and the support member 21 is disposed below the outer periphery of the reactor 1. A skirt 22 is formed so as to extend.

The length of the skirt 22 is set so that its lower end is below the bottom 3 of the reactor 1, and the microplate 20 is placed (set) on an external support (not shown) at this lower end, It will be fixed.
The support member 21 is manufactured by being molded integrally with the side wall 2 of the reactor 1 like a known microplate.

Below, the usage example of the said microplate 20 is demonstrated.
Each reactor 1 constituting the microplate 20 is filled with the content liquid 5 seeded with animal cells, and the opening is sealed with the aeration member 8, and then the culture is started while stirring by a stirring means (not shown). Is done.
During the culture period, environmental control of the content liquid 5 of each reactor 1, for example, control of pH and dissolved oxygen concentration is performed as follows.

Below the bottom 2 of each reactor 1 in the microplate 20, an excitation light source and a fluorescence light receiving unit of a fluorimeter as a detection / control mechanism are positioned relative to the

sensors

9, 10 so that the contents can be obtained. Information from the

sensors

9 and 10 regarding the liquid 5 is detected.
These output signals are processed by a signal processing device (computer), and the type of gas (ammonia gas, carbon dioxide gas, oxygen (or air), nitrogen gas), flow rate, supply time, etc. supplied from an external supply gas source It is adjusted and controlled according to the control target.

The gas 11 controlled according to the target passes through the pipe from the lower end opening 4a of the vent pipe 4, and is supplied from the upper end opening 4b to the space 6 on the content liquid level 5a. The supplied gas 11 is brought into gas-liquid contact at the interface between the content liquid 5 stirred by an external stirring means (not shown) and its liquid surface 5a, thereby preventing generation of bubbles and bubbles, and It is gradually dissolved.
At this time, since the supplied gas 11 is gradually dissolved in the content liquid 5 as described above, the amount of change in pH and dissolved oxygen concentration is small, so that these values in the control do not fluctuate greatly. During the culture, it is smoothly controlled within a desired value range.

Further, the reactor 1 of the present invention can prevent a decrease in the amount of the content liquid 5 due to evaporation.
Furthermore, if the upper end opening 4b of the vent pipe 4 is positioned in the upper part of the space 6 and the gas 11 higher than the temperature of the content liquid 5 is supplied to the space 6, the convection of the gas 11 in the space 6 is prevented. Further, a decrease due to evaporation of the content liquid 5 is also prevented.

Further, the microplate 20 of the present invention can be effectively used as a cassette that can perform various reactions, cultures, tests, and the like under a plurality of conditions in parallel.

DESCRIPTION OF SYMBOLS 1 Reactor 3 Bottom part 4 Vent pipe 5 Content liquid 5a Liquid level 6 Space 7 Vent filter 8 Vent member 9 pH sensor 10 Dissolved oxygen concentration sensor 11 Gas 20 Microplate

Claims

This is a reactor that has a bottomed cylinder with an opening that can be sealed by a ventilation member, supplies the gas from the supply gas source to the inside from the bottom, and controls the environment of the content liquid stored inside. There,
A reactor comprising a vent pipe that penetrates the bottom of the reactor and the content liquid, opens on the liquid level, and supplies the gas to a space above the liquid level.
The reactor according to claim 1, wherein a ventilation filter is provided inside the ventilation pipe.
The reactor according to claim 1 or 2, wherein the target of environmental control of the content liquid is pH and / or dissolved oxygen concentration.
The reactor according to claim 3, further comprising a pH sensor and / or a dissolved oxygen concentration sensor inside the bottom.
A microplate comprising a plurality of reactors according to any one of claims 1 to 4 arranged in an aligned state via a support member.