JP2011120548A5 - - Google Patents

Description

Apparatus and method for promoting cell change by incorporating a plurality of substances into cells

The present invention relates to an apparatus and a method for causing a cell to cause a change effective for a person who uses the cell by causing the cell to incorporate a plurality of substances that trigger the cell change .

The above description is somewhat vague, but if "effective change ... " is replaced with "promotion of cell culture", "promotion of cell proliferation" or "promotion of iPS conversion" of the target cell, its meaning Will be clear.

On the other hand, it is said that so-called “microbubbles” have different properties from ordinary bubbles. This "microbubble" has a low concentration type: there is a distribution peak around 30 μm in diameter, the bubble concentration is about several hundreds / mL, and the appearance is a slightly cloudy water, and a high concentration Type: There is a peak of bubble distribution around 10 μm, the number of bubbles is more than several thousand / mL , and there are things that look like milk.

The composition used by the present invention contains these “microbubbles”, and these bubbles stimulate the cell membrane and promote substance uptake (endocytosis) at the cell membrane. As a result, (1) cells time reduction takes to culture, or (2) normal promoting cell iPS reduction, ie, iPS cells (induced pluripotent stem cells) acquire yield improvement, (3) other, mainly effective on industrial biotechnology The effect is obtained.

Here, as an empirical difference between the above two types of “microbubbles”: the low concentration type (30 μm) and the high concentration type (10 μm), in general, the various effects given by the high concentration type (10 μm) are lower. It is much larger than that of the concentration type (30 μm).

  Therefore, the same applies to the implementation of the present invention, and it is better to implement the high concentration type (10 μm).

  A technique for generating high-concentration type (10 μm) microbubbles is described in Patent Document 6, for example. On the other hand, Patent Document 7 and Patent Document 8 describe low-concentration type (30 μm) or lower (millimeter-scale) type technologies having a larger bubble diameter.

In Patent Document 7 and Patent Document 8, although the description of “cell culture using microbubbles”, which is the same concept as the present invention, is scattered, the bubble diameter is much larger than that of the present invention, such as endocytosis. It does not detract from the patentability of the present invention from the two points that the term does not clearly grasp the phenomenon of cellular uptake .

  Cell culture has countless embodiments in experimental research and practical cell mass production. If several examples are carefully selected, there are culturing skin cells for iPS cell research, which will be described later, and culturing yeast having various and large practical uses such as miso, soy sauce, sake, and the like.

  In such cell culture, it is a necessary condition to allow cells to rapidly and rapidly incorporate a large amount of molecules required for energy acquisition such as nutrients and oxygen. Here, the cellular uptake of cells will be described by the term “endocytosis”.

  Commercially available culture solutions are individually commercialized for each target cell group. These are compositions obtained by screening those in which endocytosis frequently and rapidly occurs in the cell membrane of the target cells from many trials and errors.

  However, endocytotic conditions vary greatly from cell to cell. Commercial cultures that are blended in a least common multiple for each rough target group will require fine tuning for each individual target cell. This tuning naturally includes optimization of the environmental conditions as the liquid phase of the culture solution such as temperature, pressure, hydrogen ion concentration, biological / chemical oxygen demand, etc., which are usually performed.

  However, tuning aimed at optimizing the culture medium itself has been rarely performed because there is no appropriate methodology (technology) and cost effectiveness is unknown.

  In particular, since there are an infinite number of actual cells to be cultured, there was a considerable discrepancy between the commercially available culture solution and the ideal culture environment (including nutrients). Nevertheless, at best, only the optimization of the environmental conditions as the liquid phase of the culture broth has been carried out.

  For example, even if it is speculated that a certain yeast is slightly more preferred than normal culture conditions, that is, it is preferable to use oxygen at a high concentration, the equipment for increasing the oxygen pressure is expensive, and a simple low (millimeter scale) ) Type oxygen bubbling did not provide the desired effect because the oxygen concentration was uneven; some were poorly oxygenated and some were more oxygen rich than necessary.

  Such deviation from the ideal cell culture environment is a major bottleneck in experimental research that requires rapidity, and the head loss time of bio-system experiments spent on cell culture remains large. The present invention provides a suitable methodology to solve this problem.

On the other hand, since no have fertilized importance in reproduction medical ethical without problems iPS cells (induced pluripotent stem cells) course. Experimental attempts have been made to obtain iPS cells using normal cells such as skin and other proliferating cells and other cells. (As mentioned above, there is still a need for large-scale and high-speed culture growth of skin cells and the like.)
Regarding acquisition of iPS cells, the so-called “Yamanaka iPS technology” disclosed in Patent Documents 1 to 5 is disclosed. This technique uses four genes (Oct3 / 4, Sox2, Klf4, c-Myc) for normal cell iPS conversion. These take the name of the discoverer and are also called “Yamanaka factors”.

  However, since the gene for iPS conversion is usually incorporated into the chromosomal DNA of the cell, there is a risk that such a chromosome will spring out into the body and cause other cells to become cancerous.

  In order to solve the problem, Non-Patent Document 1 and Non-Patent Document 2 were announced. The former is the result of the group of Sheng Ding et al. Of the Scripps Research Institute that has been attracting attention in the study of iPS cell establishment using synthetic compounds that are not genes, i.e., iPS cells have been established without using genes.

  In other words, it is a safe and efficient “iPS-based protein” technology that does not use genes, and this technology uses the protein (recombinant protein) encoded by the Yamanaka factor gene at the heart of the idea. is there.

  The authors of Non-Patent Document 1 named the technology of protein-induced pluripotent stem cells (piPS cells).

  However, this “piPS cell technology” also has another problem that such a protein is not easily taken into the nucleus.

  From the current public information, the Yamanaka factor gene has one iPS cell per 100 cells, and the piPS cell technique has one iPS cell per 1000 cells. Therefore, at present, the gene method of “Yamanaka iPS Technology” is 10: 1, which is superior to the protein iPS conversion method (piPS method).

Nonetheless, the Yamanaka iPS gene method has the fatal drawback of the above-mentioned canceration, so the protein uptake into the nucleus can be improved from 1 to 1000 by using the protein iPS (piPS method). In this case, the piPS method is extremely advantageous in practical use. Therefore, there is a need for a technique that promotes the uptake of a plurality of proteins into target cells.

  The present inventor who has recognized such problems has applied for the inventions of Patent Document 9 to Patent Document 12. These control the gas phase environment as the liquid phase of the culture solution with microbubbles, and the gas phase environment that is characteristic of individual cells, which has not been possible in the past, or external substances that are characteristic of individual cells Innovative technology to control the growth of single cells such as yeast and the production of ethanol, etc., which could not be achieved by controlling the gas phase environment or the gas phase environment that facilitates the uptake of endothelium It is.

  Among the inventions of Patent Document 9 to Patent Document 12, the inventions of Patent Document 10 to Patent Document 12 are inventions as devices and control systems, and are not directly related to the present invention. On the other hand, Patent Document 9 describes a concept similar to the present invention.

  However, in Patent Document 9, for example, the invention is not sufficiently conscious of the fact that there are a plurality (four) of the Yamanaka factor and the recombinant proteins derived therefrom.

In order to improve the image, in FIG. 1, the target cell C that generally expects a desired cell change is allowed to incorporate four substances (F1 to F4) required to promote the change of the target cell itself, and the desired cell change. The schematic diagram of obtaining CX which made | formed was shown.
As a specific example of FIG. 2, four recombinant proteins (FY1 to FY4) induced by each of the four Yamanaka factors required to induce and promote iPS cell transformation are obtained from skin cells CY that are expected to become iPS cells. The schematic diagram of obtaining CZ which was made into iPS cell by being embedded is shown.

  In particular, it is known that a recombinant protein derived from Yamanaka factor "Oct3 / 4" has a relatively large molecular weight and is difficult to be taken into cells (see Non-patent Documents 1 and 2).

The present invention proposes a practical technique that corresponds to the need that promotes cellular changes by Maseru taking a plurality of substances into cells as in the example above.
Republished 07-69666 “Nuclear Reprogramming Factor” Kyoto University JP 2009-165478 A "Method for producing induced pluripotent stem cells" Kyoto University JP 2009-165479 A “Use of a nuclear reprogramming factor to produce induced pluripotent stem cells” Kyoto University JP 2009-165480 A “Induced pluripotent stem cells and production method thereof” Kyoto University JP 2009-165481 A “Method for producing somatic cells from induced pluripotent stem cells” Kyoto University Japanese Patent No. 3620797, “Microbubble generator”, IPMS Japanese Patent No. 2762372 “Microbubble Generator” Komatsu Ltd. Japanese Patent Publication No. 5-60353 “Culture method and apparatus by aeration in liquid” Hitachi, Ltd. Japanese Patent Application No. 2009-234683 “A composition containing microbubbles for promoting cell change, and a method for promoting cell change using a composition containing microbubbles” Japanese Patent Application No. 2009-243940 “Composition of microbubbles and microbubble generator” Japanese Patent Application No. 2009-249900 “Apparatus for generating microbubbles in liquid” Japanese Patent Application No. 2009-265832 “Apparatus and method for generating heat by generating microbubbles in liquid” Hongyan Zhou et. al `` Generation of iPS-Cell Using Recombinant Proteins '' Cell Stem Cell Volume 4, Issue 5, 381-384,23 April 2009 Dohoon Kim et. al `` Generation of Human Induced PluripotentStem Cells by Direct Delivery of Reprogramming Proteins '' CellStem Cell Volume 4, Issue 6, 472-476, 28 May 2009

Case object of the present invention is to propose a practical technique for a substance that trigger cellular changes that promote cellular changes by be incorporated into the cells, corresponding to the requirement that, among other things, a plurality trigger substance The technology corresponding to is provided.

The present invention, by developing the invention of Patent Document 9 by the same inventor, since the more substances that promote cell change by Maseru uptake in cells, addresses the need of a more practical method apparatus For example, there is a high molecular weight such as “recombinant protein” derived from Yamanaka factor “Oct3 / 4”, and it is difficult to take it into cells. I took it.

In other words, for these difficult-to-uptake substances, special microbubble gas phase components and concentration conditions depending on the difficulty of taking up the substance, the addition of substances that assist the uptake of the substance to be taken in, and the difficulty of taking in A special ( periodic ) time period is provided to give the target cells environmental conditions such as special temperature, pressure, hydrogen ion concentration, and biological / chemical oxygen demands.

Substances that are easy to take up are time periods different from those that are difficult to take up. Thus, for example, the strategy of extracting only the cell group that has incorporated the recombinant protein derived from Yamanaka factor "Oct3 / 4" and allowing them to incorporate the easy-to-uptake substance, or first The strategy is to allow the incorporation of a recombinant protein derived from “Oct3 / 4”, which is difficult to take in, in a time period different from that of a substance that is difficult to take in after taking in a substance that is easy to take up.

Here, among the individual uptake promoting conditions to a plurality of capture agents, the relationship of components and the concentration of the intake 引気body to be sucked into the microbubbles may be previously experimentally determined. At that time, incorporation meat have substance and uptake easy and material cells fast capturing the substance, i.e. it Shiwakere in like "uptake rate".

That (claim 7), respectively, and a separate capture more substances that Maseru uptake into cells, the relationship between the gaseous components and concentrations the gas body suction means for sucking determined experimentally in advance, a plurality of substances causing Tokoma the uptake easy order or, with sequentially feeding subjected to liquid bath in uptake difficult order to fit such substances individual supply, based on the suction gas component and concentration was determined the experimentally relationship It is preferable to adopt a method that promotes cell change by incorporating a plurality of substances into cells by controlling them .

  In addition, for example, the three types of recombinant proteins derived from genes other than Yamanaka Factor “Oct3 / 4” have relatively low molecular weights, so that they can be incorporated under almost the same conditions without significant changes. May be represented by an average value or the like, and the three types may be taken in at once, which is more practical.

That was determined in the step of determining experimentally, when the concentration each individual suction gas component is similar, when controlling the gas component and concentration, component and the representative of the similar components The suction gas component and the concentration may be controlled to a concentration representing a similar concentration.

Next, an apparatus for carrying out this method will be described. First, FIG. 3, which is a schematic diagram for helping understanding, shows the target values of the suction gas component and concentration according to claim 1 of the present invention, and the target values of control of environmental temperature, pressure, etc. according to claim 3 in time series ( horizontal direction). axis is a schematic diagram showing a time chart of). The auxiliary substance supply amount and concentration target values of claim 2 are shown in FIG.

The apparatus of the present invention comprises a vortex pump and a liquid tank (18) to which a gas suction means, a liquid suction means, and a liquid discharge means are connected, and sucks gas by the gas suction means. However, the liquid suction means sucks the liquid in the liquid tank and discharges the microbubbles containing the gas sucked by the gas suction means from the liquid discharge means into the liquid tank to contain the microbubbles. the liquid composition to produce a liquid bath, by giving it to the cells, a plurality of substances to a device for promoting cellular changes Mase uptake into cells, the microbubble is at least a particle size of 30μm or less Means (12) for controlling the component and concentration of the suction gas upstream of the gas suction unit, and means for controlling the component and concentration of the suction gas. System for issuing target commands Output means of the target command (12A), comprising a decision and the control target of the suction gas component and concentration, based on the gaseous components and concentrations of Effects on the uptake of Maseru uptake into cells into a plurality of substances individual cells The control target of each gas component and concentration of each of the plurality of substances , and the command of the control target is sequentially output individually over time according to each of the plurality of affected substances. This is a device configured as described above.

The apparatus of the present invention ( see claim 2 and FIG. 6 ) supplies a biological substance or a chemical substance for assisting individual uptake of each of a plurality of substances to be taken up by cells to the liquid tank , and auxiliary substances supply and density control means for controlling the concentration, and supplies the auxiliary substances, and, to means for controlling the concentration of the auxiliary substances, a control target to issue a control target command supply amount and concentration of the auxiliary substances output means of the command, to combine the control target supply amount and concentration of the auxiliary substances, based on the influence of the feed rate and the concentration of auxiliary substances given to incorporation into a plurality of substances individual cells that incorporated into cells It is the control target of the supply amount and the concentration of the individual auxiliary substances determined for each of the plurality of substances , and the command of the control target is sequentially and individually over time according to each of the plurality of affected substances. Output to It is preferable to have a configuration such as
The method (Claim 8), a plurality of substances each individual capture that incorporated in the cells, the relationship between the components and the concentrations of biological or chemical substances to aid uptake, determined experimentally in advance, collected in accordance with the plurality of substances each supply to written, the biological material or chemicals that assist in uptake, the added to the liquid bath on the basis of the experimentally determined relationship, it is preferred to carry out the.

The apparatus of the invention (Claim 3) is a means (18A) for controlling the environment of the liquid tank for assisting the individual uptake of a plurality of substances to be taken into the cells, and a means for controlling the environment of the liquid tank. the output means of the control target command issuing control target command environment to assist uptake (30), and combine the control target of the environment of the liquid tank, Maseru uptake into cells to multiple substances individual cells was determined based on the environmental effects of the liquid bath given to capture, each of the plurality of materials is a control target of the specific environment, and also, a command of the control target, the plurality of materials each receiving the influence Accordingly, it is preferable to have a configuration in which the signals are output individually and sequentially over time.
The method (Claim 9), a plurality of material discrete uptake that incorporated in the cells, the relationship between the environmental liquids tank, determined experimentally in advance, a plurality of substances each supply to incorporated together, the environment of the liquid bath, the controlled based on the experimentally determined relationship, preferably implement.

More specifically (claims 4 and 10 ), the components and concentration of the suction gas controlled by the gas component and concentration control means of claims 1 and 7 are oxygen (O2) and / or nitrogen (N2). ), And / or components of carbon dioxide (CO2) and their concentrations.

Similarly, specifically (claims 5 and 11 ), the biological substance or chemical substance for assisting the uptake of a plurality of substances according to claims 2 and 8 is a substance present in the natural growth environment of the target cell, and / or It is a substance that contributes to a biological reaction in the target cell and / or a substance produced in vivo.

  Examples of substances present in the natural growth environment of target cells, substances contributing to biological reactions in target cells, and substances produced in vivo include genes, enzymes, coenzymes, coenzymes, and recombinants biosynthesized by genes. Examples include proteins, amino acids, peptides, sugar chains, and the like, and hormones, pheromones, NGF (nerve cell growth factor), and the like.

Also, specifically (claims 6 and 12 ), the environment of the liquid tank controlled to assist the uptake of a plurality of substances according to claims 3 and 9 is the temperature and / or pressure of the liquid composition. , and / or hydrogen ion concentration, and / or biological or chemical oxygen demand, an environmental condition that.

  Here, it supplements with reference to FIG. 5 which is a schematic diagram for helping an understanding with FIG. FIG. 5 is a schematic diagram of an apparatus in the image of mass automatic processing in units of 1000 cells.

Here, Q1 is an apparatus block having the configuration of FIG. 4, and is a supply source of a liquid composition (cell culture solution) containing microbubbles as described in claim 1. It shows that the environment such as temperature described in claims 3 and 9 is also controlled by the Q3 portion .

Q2 is a supply means for individually supplying a plurality of substances 2 to be taken into cells, for example, F1 to F4.

  Q4 is one in which the supply portion of oxygen (O2) and / or nitrogen (N2) and / or carbon dioxide (CO2) described in claim 4 is surrounded by a one-dot chain line in Q1. . Q5 is an automatic culture vessel having a plurality of target cell culture cells.

In Figure 5, according to claim 1 "according to a plurality of substances each sequentially outputted individually with time", according to claim 7 "is supplied to the sequential liquid bath a plurality of substances, such substance individual in accordance with the supply, to ... control "is shown by a white arrow.
Target cells are cultured in Q5 . The plurality of Q5 are the same and have different time periods. Transfer the liquid composition means R such as an automatic pipettor to transfer the liquid composition 3 containing microbubbles from a liquid tank 18 to Q5 contains microbubbles from a liquid tank 18 Q5, material uptake at each time periodic de Is promoted.

The present invention causes incorporated a wide variety of substances into a plurality cells can Rukoto cause a desired change in the cell. As a result, (1) further mass-acceleration of cell culture and cell growth, and (2) improving the nuclear uptake rate of a plurality of such recombinant proteins in, for example, the “piPS cell technology”, that is, the “protein iPS conversion” method. Practical mass acquisition (mass production) of iPS cells becomes possible.

The schematic diagram of obtaining CX which made the target cell C take in four substances required to promote a change, and made the desired cell change. Schematic diagram of obtaining by incorporated four recombinant proteins was induced in four Yamanaka factors required to induce promotes iPS cells into skin cells CY None of iPS cells of CZ. The schematic diagram which showed the output of the command of the control target of the suction | inhalation gas component and density | concentration (Claim 1) , environmental temperature, pressure, etc. (Claim 3) of this invention in time series. 3 (a) is relatively time variation of a line segment indicating the control target of the air body component concentration of 12 to control the length, FIG. 3 (b), the control target of the liquid bath environment 18A controls It is time change. It is an example figure of the apparatus structure except the means of Q2 in the apparatus of this invention. See FIG. 5 for the relationship with the claims. The structural example figure of the apparatus of this invention. For the meaning of Q1 to Q5, refer to the description of the reference numerals. According to claim 1, “in accordance with each of a plurality of substances, it is output individually and sequentially over time”, and according to claim 7, “a plurality of substances are sequentially supplied to the liquid tank, and in accordance with the supply of each such substance. ,... Control "is indicated by white arrows. Target cells are cultured in Q5. The plurality of Q5 are the same and have different time periods. The liquid composition R contains infinitesimal bubbles transferred from the liquid tank 18 to Q5, material uptake at each time period is accelerated. The control target commands for the auxiliary substances of the present invention (Claim 2) are individually and sequentially Explanatory drawing of being output to. FIG. 6 (a) shows a biological material that assists in the uptake of F1 or Change in the relative value of the supply target and concentration control target of chemical substance over time, Fig. 6 (b) shows F2 uptake Change of relative value of supply target and concentration control target of biological substance or chemical substance It is.

2 Cell change promoting substances (a plurality of substances that trigger cell changes), generally representing examples of F1 to F4.
3 Microbubble-containing cell culture composition mixed with cell change promoting substance 2 4 Gas suction means 10 Microbubble generator 11 Multiple gas supply means (one of them)
12 A means for controlling the component and concentration of the suction gas 12A A gas component / concentration control target command output means for issuing a control target command for the gas component and concentration to the means 12 for controlling the component and concentration of the suction gas.
13 tip portion 15 1 6 tip portion of the liquid discharge means of a liquid suction means of means 14 1 6 to supply the suction air 16 which is controlled component and density (discharge nozzles)
16 A housing containing a vortex pump to which gas suction means, liquid suction means, and liquid discharge means are connected 17 Turbidity sensor 18 for measuring turbidity by transmitted light or scattered light measurement method Liquid tank (cell culture medium container)
18A Environment control means 30 such as temperature, pressure, hydrogen ion concentration, biological / chemical oxygen demand of liquid tank (cell culture medium container) 30 Environment control target command to means 18A for controlling the environment of liquid tank Liquid tank environment control target command output means 31 A plurality of substances The data on the influence of gas components and concentration on the uptake of individual cells (claim 1) , or a plurality of substances determined experimentally in advance and a separate capture, data of the relationship between the gas component and concentration the gas suction means for sucking (claim 7), gives in response to 30 requests database
32 Data on the effect of the environment of the liquid tank on the uptake of cells into individual cells (Claim 2) , or the individual uptake of each of a plurality of substances determined experimentally in advance and the environment of the liquid tank A database C which provides the data of the relationship with (Claim 9 ) upon request of 30 C Cell nucleus CX of target cell CC C or CY before desired cell change Target cell CY iPS cellization with desired cell change Skin cell CZ before being treated First of a plurality of substances that promote changes in iPS cellized skin cells F1 cells ( first substances that trigger cell changes)
Second of multiple substances that promote F2 cell changes ( second substances that trigger cell changes)
Third of a plurality of substances that promote F3 cell change ( third substance that triggers cell change)
The fourth of a plurality of substances that promote F4 cell changes ( fourth substance that triggers cell changes)
FY1 iPS cells of the mountains factor Oct3 / 4 recombinant protein FY4 iPS cells induced by the induced recombinant protein FY3 iPS cells of the mountains factor Klf4 in the mountains factor Sox2 of the induced recombinant protein FY2 iPS cell of at changes in recombinant protein P0 suction gaseous components and concentrations derived by Yamanaka factor c-Myc of reduction, the change of the liquid bath temperature, pressure, and hydrogen ion concentration-biological to chemical oxygen demand is, the target cell so as not to give a biological shock, the control command gaseous component-concentrations uptake cheap gas component and its concentration of the first substance F1 of the time period P1 plurality of material required to make a rough sea et kana change is outputted This time period is the gas component / concentration. The environmental condition of the liquid tank is determined in the same manner, and the first substance F1 is taken in and controlled to a low environmental condition. P2 The time period when the gas component and the concentration control command are output, and the gas component / concentration control command is output for the gas component and the concentration of the second substance F2 out of the plurality of substances. The environmental condition of the liquid tank is determined in the same manner, and the second substance F2 is taken in and controlled to a low environmental condition. Q1 An apparatus block having the configuration shown in FIG. 4, which is a supply source of a liquid composition containing microbubbles as defined in claim 1.
Q2 Supply means for individually supplying a plurality of substances 2 to be taken up by cells.
Q3 Of Q1, an environment control portion such as temperature described in claim 3 is surrounded by a dotted line.
Q4 Of Q1, the oxygen (O2), nitrogen (N2), and carbon dioxide (CO2) supply portion described in claim 4 is surrounded by a one-dot chain line.
Q5 Automatic culture container with culture cells for multiple target cells
Means such as an automatic pipetter for transferring the liquid composition 3 containing R microbubbles from the liquid tank 18 to Q5

Claims (12)

Comprises a gas suction means and the liquid suction means and liquid discharge means vortex flow pump and a liquid tank which is connected, while sucking the gas in the gas suction means, said by sucking the liquid in the liquid tank by the liquid suction means By the liquid composition containing the microbubbles generated by discharging the microbubbles containing the gas sucked by the gas suction means from the liquid discharge means into the liquid in the liquid tank,
A plurality of substances A and Mase capture the cellular machinery to promote cell change,
The microbubbles include at least microbubbles having a particle size of 30 μm or less,
Means for controlling the composition and concentration of the suction gas upstream of the gas suction means; and
A means for controlling the component and concentration of the suction gas, and a control target command output means for outputting a control target command for the suction gas component and concentration;
The control target for the aspirated gas component and concentration is determined based on the influence of the gas component and the concentration on the uptake of each of the plurality of materials to be taken into the cells, and the individual gas components and the concentrations of each of the plurality of materials. Control objectives, and also
Command of the control target, according to a plurality of substances each receiving the impact, in which are successively individually output over time, device that promotes cellular changes by Maseru taking a plurality of substances to cells. The apparatus of claim 1.
The biological or chemical plurality of substances Maseru uptake in the cells to aid the individual capture is supplied to the liquid tank, and auxiliary substances supply and density control means for controlling the concentration of the auxiliary substances and,
The auxiliary material supply, and a means for controlling the concentration of the auxiliary substances, and combines the output means, the control target command issuing control target command supply amount and concentration of the auxiliary substances,
The control target supply amount and concentration of the auxiliary material, the supply amount of auxiliary substances applied to the incorporation of Maseru uptake into cells into a plurality of substances individual cells and was determined based on the concentration of the impacts, individually plurality of substances Is the control target for the supply and concentration of auxiliary substances , and also
Command of the control target, according to a plurality of substances each receiving the impact, in which are successively individually output with time, device. The apparatus of claim 1 or claim 2 ,
Means for controlling the environment of the liquid tank to assist the individual uptake of each of the plurality of substances to be taken up by the cells; and
Combined with the means for controlling the environment of the liquid tank, the output means of the control target command for issuing the control target command of the environment to assist the intake,
Control target of the environment of the liquid tank, cells were determined based on the environmental effects of the liquid tank to provide for the acquisition Maseru incorporation into a plurality of substances individual cells, control of a plurality of substances each individual environment Goal, and again
Command of the control target, according to a plurality of substances each receiving the impact, in which are successively individually output with time, device. The component and concentration of the suction gas of claim 1 are
An apparatus that is a component of oxygen (O2) and / or nitrogen (N2) and / or carbon dioxide (CO2) and their concentrations. The biological material or chemical substance for assisting uptake according to claim 2,
A device that is a substance that exists in the natural growth environment of a target cell and / or a substance that contributes to a biological reaction in the target cell and / or a substance produced in vivo. The controlled liquid bath environment of claim 3 is
A device which is the temperature of the liquid in the liquid tank and / or the pressure of the liquid and / or the hydrogen ion concentration of the liquid and / or the biological or chemical oxygen requirement of the liquid . Using the apparatus of claim 1, a plurality of substances to a method of promoting cell changes Mase uptake into cells,
Respectively separate capture more substances that Maseru uptake into cells, the gas suction means the relationship between the gas carrying component and the concentration determined experimentally in advance for sucking,
Preparative more substances uptake is easy order to written or supplies to sequentially liquids tank in uptake difficult order to fit such substances each supply, the suction gaseous components and concentrations, before Symbol experimental by controlled based on the determined relationship, a method of promoting cytopathic reduction by a plurality of material was incorporated into the cells. The method of claim 7, wherein
Individual uptake of multiple substances to be taken up by cells and biological substances or Determines experimentally the relationship between the chemical composition and concentration in advance,
Biological or chemical substances that assist in the intake of multiple substances to be taken up according to the individual supply In a liquid bath based on the experimentally determined relationship. The method of claim 7, wherein
The relationship between the individual uptake of multiple substances to be taken into cells and the environment of the liquid tank Determined experimentally,
The environment of the liquid tank was determined experimentally to suit the individual supply of the multiple substances to be taken up. A method of controlling based on relationships. The component and concentration of the suction gas according to claim 7 are:
A method of oxygen (O 2) and / or nitrogen (N 2) and / or carbon dioxide (CO 2) components and their concentrations . The biological substance or chemical substance for assisting uptake according to claim 8,
Substances existing in the natural habitat of the target cells, and / or, preferred Azukasuru agents to a biological response of a subject in a cell, and / or, an in vivo production materials, methods. The controlled liquid bath environment of claim 9 is
A method of the temperature of the liquid in the liquid bath and / or the pressure of the liquid and / or the hydrogen ion concentration of the liquid and / or the biological or chemical oxygen requirement of the liquid .