JP2009089698A - Cell culture method and cell culture apparatus - Google Patents

Abstract

When a solution is added in cell culture, a concentration change in a local region in a culture tank is quickly resolved.
When cell culture is performed using a liquid medium, a solution to be added to the liquid medium is spatially divided and added. Examples of the solution include an alkaline solution, an acidic solution, or a medium solution containing at least one of amino acids, vitamins, minerals, and proteins.
[Selection] Figure 1

Description

  The present invention relates to a cell culturing method and a cell culturing apparatus that are applied when culturing cells that produce a substance that is a main raw material such as pharmaceuticals.

  Drugs including antibody drugs contain substances produced by cells as the main component. Since such a substance is secreted and produced by animal cells, for example, it can be obtained by culturing animal cells and separating and purifying the target substance secreted in the culture solution.

  The culture method in cell culture is a batch culture method in which nothing is added until the end of the culture after inoculation of the seed cells, a fed-batch culture method in which a restriction substrate is added during the culture but the culture solution is not withdrawn until the end of the culture, It can be classified into three types: continuous culture method that continuously adds and removes the same amount of medium. For industrial mass culture, batch culture is currently used more frequently than the ease of validation.

  In the production method of useful substances using cells, the cost of a medium used in large quantities is high, and it is difficult to provide useful substances at low cost. Therefore, a more efficient culture method is desired in the method for producing useful substances using cells. In the current mainstream batch culture method, the nutrient source in the medium decreases with the growth of the cells, and harmful metabolites such as lactic acid and ammonia accumulate, so that the cell growth period cannot be maintained for a long time. The yield is low.

  In order to solve this problem, a fed-batch culture method has been studied in recent years. In this method, a method for devising a fed-batch culture and a fed-batch method has been proposed. Specifically, the energy source is efficiently used by utilizing glucose, which is one of energy sources, and mainly from lactic acid metabolism to complete oxidation by TCA cycle. Thereby, accumulation of harmful metabolites can be prevented and cell culture can be achieved at high density. In addition, the fed-batch culture method has an advantage that the equipment and know-how of the existing microorganism culture device used in the batch culture method can be applied.

  Patent Document 1 discloses a method of adding a sterilization additive to a fermentation culture tank in a discontinuous mist form. According to the culture method disclosed in Patent Document 1, bubbles on the surface of the medium are efficiently defoamed by adding the sterilizing additive in a discontinuous mist. Patent Document 2 discloses a culture apparatus that continuously adds a carbon source feed solution. Furthermore, Patent Document 3 discloses an apparatus for supplying a hydrophobic solvent having a high specific gravity so that a plurality of droplets drift in a culture solution in a culture tank.

Special table 2003-505102 gazette JP-A-5-76346 Japanese Patent Laid-Open No. 11-187868

  By the way, in any culture method as described above, various solutions may be added to the medium during the culture period. For example, an alkaline solution or acidic solution for controlling the pH in the medium, a feed medium in fed-batch culture, or the like is added to the medium at a desired timing. When these various solutions are added to the medium, a region where the solution exists at a high concentration is formed, but the region becomes a severe environment for cells to be cultured. For example, the addition of a high-concentration alkaline solution creates a region with a high pH, where cells may be killed or damaged. In addition, since the feed medium is prepared at a high concentration, when a feed medium is added in fed-batch culture, a high nutrient concentration region is formed. Can not.

  That is, cell growth may be affected by adding various solutions in cell culture. For example, when various substances are produced using cells, there is a problem that productivity is lowered due to this. Then, in view of the above-mentioned situation, the present invention has an object to provide a culture method and a culture apparatus in which a concentration change in a local region in a culture tank can be quickly eliminated when a solution is added in cell culture.

  The cell culture method according to the present invention that achieves the above-described object is characterized in that when cell culture is performed using a liquid medium, a solution to be added to the liquid medium is spatially divided and added. It is. In particular, as the above solution, a solution that reduces the productivity of the product as the concentration becomes high, a solution that kills or damages the cell as the concentration becomes high, a solution that has a property of worsening rotation of energy metabolism in the cell, Examples thereof include a solution having a property of causing denaturation of a culture solution component as the concentration is increased, or a solution having a property of generating a by-product as the concentration is increased. More specifically, examples of the solution include an alkaline solution, an acidic solution, or a medium solution containing at least one of amino acids, vitamins, minerals, and proteins. In the cell culture method according to the present invention, as an example, the solution is added to the medium by a spray nozzle. As another example, the solution is added to the culture medium as a plurality of droplets.

  Furthermore, the cell culture device according to the present invention that has achieved the above-described object is a culture vessel that performs cell culture using a liquid medium, and the solution is spatially divided and added to the culture medium in the culture vessel. Solution adding means. The cell culture device according to the present invention includes, for example, a solution that decreases the productivity of the produced substance as the concentration increases, a solution that kills or damages the cells as the concentration increases, and rotation of energy metabolism in the cell. It is possible to add a solution having a property of degrading the concentration, a solution having a property of denaturing the culture medium components as the concentration becomes high, or a solution having a property of generating a by-product as the concentration is increased to the medium. . More specifically, examples of the solution include an alkaline solution, an acidic solution, or a medium solution containing at least one of amino acids, vitamins, minerals, and proteins. In the cell culture device according to the present invention, the solution supply means preferably includes at least one spray nozzle. In the cell culture apparatus according to the present invention, it is preferable that the solution supply means includes at least one nozzle for adding the solution to the culture medium as a plurality of droplets.

  According to the cell culture method according to the present invention, the survival rate, growth state, and substance productivity of cells to be cultured can be well maintained even in a culture system in which various solutions are added to the medium. In addition, according to the cell culture device of the present invention, even when a culture method in which various solutions are added to the medium is applied, the survival rate, growth state, and substance productivity of the cells to be cultured are well maintained. can do. Therefore, according to the cell culture method and the cell culture apparatus according to the present invention, for example, when a substance is produced in a cell to be cultured, the productivity of the substance can be improved.

  Hereinafter, a cell culture method and a cell culture apparatus according to the present invention will be described in detail with reference to the drawings. The cell culturing method and the cell culturing apparatus according to the present invention can be applied when culturing cells that produce substances that are main raw materials such as pharmaceuticals. In the present invention, the substance to be produced is not limited in any way, and examples thereof include proteins such as antibodies and enzymes, physiologically active substances such as low molecular compounds and high molecular compounds. Further, the cells to be cultured are not limited in any way, and examples include animal cells, plant cells, insect cells, bacteria, yeasts, fungi, and algae. In particular, the cell culture method according to the present invention preferably targets animal cells that produce proteins such as antibodies and enzymes. In addition, the cell culture method and the cell culture apparatus according to the present invention are not limited to those for the purpose of producing substances as described above, and are also applied when cells are cultured to obtain a large amount of cells. be able to.

  In the cell culture method and the cell culture apparatus according to the present invention, an initial medium in which the amino acid concentration and / or the carbon source concentration is adjusted to a predetermined value is prepared. Examples of amino acids include 20 kinds of amino acids constituting natural proteins, cystine, hydroxylysine, hydroxyproline, thyroxine, O-phosphoserine, desmosine and the like. Although it does not specifically limit as a carbon source, The carbon source generally used for cell culture can be mentioned. Examples thereof include saccharides such as glucose, lactose, galactose, raffinose, mannose, cellobiose, arabinose, xylose, sorbitol, fructose, sucrose, and maltose. Moreover, as a carbon source, you may use alcohol other than saccharides.

  As an example of the cell culture apparatus, as shown in FIG. 1, an inoculation port 1 for inoculating cells, a DO electrode 2, a pH electrode 3 and a resistance temperature detector 4 for measuring a state in a culture solution, The heater 5 for adjusting the temperature of the culture solution, the stirring blade 6 for stirring the culture solution, the sampling port 7 for sampling the culture solution, and the alkali addition pipe for adding the solution during the culture 8, feed medium addition pipe 9, air inlet 10 for gas exchange, exhaust outlet 11, sparger 12 for venting in the liquid, and defoaming for removing bubbles generated on the liquid surface by venting An electrode 13 and a liquid outlet 14 for collecting the culture solution at the end of the culture are provided. The cell culture apparatus also includes a temperature-controlled water inlet 15 for sterilizing the culture tank 20, a temperature-controlled water outlet 16, a CIP (stationary cleaning) port 17 for cleaning, and a spray ball 18. Although not shown, the cell culture device has a viewing window and illumination for visually observing the state of the cells 19 being cultured.

  Further, around the culture tank 20 in the cell culture apparatus, as shown in FIG. 2, a medium preparation tank 21 for preparing a medium, a medium supply tank 22 storing a feed medium for supplementing medium components, A recording device 23 for recording data obtained by monitoring the situation, an analysis device 24 for judging the culture state from the monitoring, a control device 25 for controlling the culture operation from the analysis result, a pure water production device 26 for use in washing or sterilization, A pure steam (PS) generator 27 and a WFI (Water for Injection) generator 28 are installed. The medium preparation tank 21 and the culture tank 20 are connected via a filter 29.

  In particular, the cell culture device has a solution addition means for adding a solution to the medium. In the cell culture apparatus shown in FIGS. 1 and 2, the solution addition means is the alkali addition pipe 8, the feed medium addition pipe 9, and the medium supply tank 22. The solution addition means records the pH value of the medium measured with the pH electrode 3 with the recording device 23, and when this pH value falls below the threshold value, a predetermined amount of alkaline solution is supplied from the alkali addition pipe 8 by the control device 25. The structure which adds may be sufficient. Further, the solution addition means may be configured to add a predetermined amount of feed medium from the feed medium addition pipe 9 by the control device 25 based on a preset feed medium addition timing.

  In particular, in the cell culture device according to the present invention, the solution adding means spatially divides and adds the solution to be added. Here, the solution to be added is not particularly limited, and examples thereof include various solutions added to the medium during cell culture. As a solution to which the present invention is applied, it is desirable to add it in a plurality of spatially divided solutions, which have properties that reduce the productivity of the product substance as the concentration in the medium increases, and as the concentration increases. Solutions that have the property of killing or damaging cells, solutions that slow down the turnover of intracellular energy metabolism, solutions that have the property of causing degeneration of culture fluid components as the concentration increases, or high concentrations As a result, a solution having a property of generating a by-product can be mentioned. More specifically, examples of the solution include an alkaline solution and an acidic solution for adjusting the pH of the medium. These alkaline solutions and acidic solutions, when in contact with cells in a high concentration state, act to reduce the productivity of the produced substance, act to kill or damage the cells, and denature the culture solution components. There is a risk that it will occur. Examples of the solution include a medium solution for supplementing medium components. The medium solution means a solution having a composition containing at least one of amino acids, vitamins, minerals, and proteins. This medium solution acts to deteriorate the turnover of energy metabolism in the cell when it comes into contact with the cell at a high concentration, acts to denature the culture medium components as the concentration increases, or increases the concentration. As it becomes, there exists a possibility of having the property to produce | generate a by-product.

  In order to spatially divide and add the solution to be added, for example, a configuration in which a plurality of openings at the tip of the pipe are provided or a configuration in which the tip of the pipe is meshed can be given. . When a predetermined amount of solution is added in a spatially divided manner, each droplet has a smaller diameter and a larger specific surface area in contact with the medium than when an equal amount of solution is added all at once. As a result, it is dispersed in a short time and becomes uniform. For this reason, when a predetermined amount of solution is spatially divided and added, a region where the solution exists at a high concentration can be reduced, and a uniform concentration state can be achieved in a shorter time.

  Further, in order to add the solution to be added in a spatially divided manner, as shown in FIG. 3, a solution addition path 31 in which the tip of the pipe facing the culture medium is branched into a plurality is used. Also good. In this solution addition path 31, the tip of the pipe may have a configuration having a plurality of openings or a mesh configuration.

  Furthermore, in order to add the solution to be added in a spatially divided manner, for example, the solution may be in the form of a mist. In order to add the medium to be added in the form of a mist, for example, as shown in FIG. 4, a configuration using a spray nozzle can be applied. The spray nozzle is composed of, for example, a high-pressure air chamber 32 of 0.5 to 10 kPa and a solution chamber 33 to which an additive solution is supplied. By supplying the solution to the solution chamber 33 and supplying high-pressure air to the high-pressure air chamber 32, the solution discharged from the tip can be made into a mist. A solution can be supplied from the solution tank T to the solution chamber 33 using the pump device 35. The supply amount of the solution can be controlled by using a strain gauge pressure gauge 34. In practice, the addition control can be performed by sending a signal when the addition is necessary and controlling the strain gauge pressure gauge.

  Furthermore, in order to add the medium to be added in the form of a mist, for example, as shown in FIG. 5, a bubble jet type nozzle can be used. In the bubble jet type nozzle, a diaphragm 37 having a piezo element 36 disposed on the upper surface, a supply hole 38 for supplying a solution, and a nozzle member having a nozzle hole for discharging the solution supplied to the supply hole 38 are integrated. It is configured as follows. In the bubble jet type nozzle, the solution entering from the supply hole 38 enters the space between the diaphragm 37 and the nozzle member. When a voltage is applied to the piezo element 36, the diaphragm 37 is curved, the internal space is narrowed, and the solution 40 is ejected from the nozzle hole 39. Note that the addition control can be performed by sending a signal when the solution needs to be added and applying a voltage to the piezo element.

  In addition, when the spray nozzle as shown in FIG. 4 or the bubble jet type nozzle as shown in FIG. 5 is used, the cell culture apparatus has a system that can reliably sterilize the spray nozzle and the bubble jet type nozzle. It is preferable to provide. Specifically, a pure steam (PS) generator 27 is connected via a valve 53 to a middle portion of a pipe 51 for supplying a solution to be added to a spray nozzle or a bubble jet type nozzle. A steam pipe 52 for supplying pure steam from the generator 27 into the culture tank is provided, and a drain trap 54 is provided at the bottom of the culture tank. By comprising as mentioned above, even if it is a case where a spray nozzle or a bubble jet system nozzle is used, it can sterilize reliably. Specifically, regarding the sterilization of the nozzle, the valve 53 is opened and, for example, pure steam at 121 ° C. is allowed to flow through the pipe 51 to sterilize the pipe 51 and remove a liquid pool formed in the pipe 51. At the same time, for example, 121 ° C. pure steam is caused to flow through the pipe 52 to raise the temperature in the culture tank to 100 ° C. or higher. Thereby, the liquid pool in a nozzle can be removed reliably. At this time, although a liquid pool is generated at the bottom in the culture tank, it can be removed by opening the drain trap 54. By the above operation, all of the pipe 51, the culture tank, and the nozzle can be sterilized. The sterilization method is not limited to this method, and other sterilization methods such as dry heat sterilization can also be applied.

  According to the cell culture apparatus configured as described above, cell culture can be performed without adversely affecting cell growth and substance production by cells as follows. Before culturing, the inside of the culture tank and the piping are sterilized with steam at 121 ° C. for 20 minutes or longer as described above. By using the pure steam from the PS generator 27, it is possible to sterilize each instrument such as a pH meter, such as all pipes and culture tanks involved in the culture. A medium is prepared using WFI in the medium preparation tank 21, and fed to the culture tank through a filter 29 having a 0.25 μm pore. The temperature of the culture medium in the culture tank is controlled to 37 ° C. by flowing temperature-controlled water through the heater 5, and a mixed gas in which air, nitrogen, oxygen, and carbon dioxide are appropriately mixed while being stirred by the stirring blade 6 is supplied to the air inlet. 10 to adjust the pH and DO (dissolved oxygen concentration) of the medium to values suitable for culture. Thereafter, the seed-cultured cells were aseptically introduced from the inoculation port 1.

  After the start of culture, the culture solution is sampled from the sampling port 7 in a timely manner in order to confirm and control the culture state. As for sampling, for sterilization, first, pure steam at 121 ° C. is allowed to flow through the pipe for 20 minutes or more and dried by blowing sterilized air. You may repeat this process in multiple times as needed. Thereafter, the temperature of the pipe is cooled, the valve is opened, the culture solution is collected from the sampling port 7, and when the target amount is secured, the valve is closed and the pipe is sterilized through the sterilization step. The collected culture solution can be analyzed using an analyzer suitable for the component to be examined.

  If the exchange of gas on the liquid level is not in time for the control during the culture, or before that, the mixed gas is vented from the liquid level to the liquid level using the sparger 12. Can do.

  In particular, according to the cell culture device according to the present invention, when the pH of the medium is more acidic than the set value as cells grow, the pH of the medium can be adjusted by adding an alkaline solution. . At this time, the amount of the alkaline solution added to the medium is calculated according to the pH value of the target medium. The alkaline solution whose amount of addition is determined is spatially divided into a plurality and added to the medium. Thereby, since the area | region where a high concentration alkaline solution exists in high concentration can be made small, the damage and death to a cell can be prevented. Further, the added alkaline solution can be uniformly dispersed in a shorter time, and the pH of the whole medium can be adjusted in a shorter time.

  In the cell culture device according to the present invention, a feed medium for supplementing medium components is added at a predetermined timing. The addition of the feed medium is performed by analyzing the components of the sampled culture medium during the cultivation, determining the nutrient amount consumed by the cells from the results, and calculating the amount of the feed medium necessary for the next feeding. The feed medium whose amount is determined is spatially divided into a plurality and added to the medium. Thereby, since it can prevent that a feed culture medium becomes high concentration locally in a culture medium, the energy metabolic efficiency in the cell in culture | cultivation can be maintained, and production | generation of a by-product etc. can be prevented. As a result, inhibition of cell growth and substance production by the cells can be prevented.

EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, the technical scope of this invention is not limited to a following example.
[Example 1] Effect of dividing the added solution (experimental method)
In this example, mouse mouse hybridoma CRL-1606 cells (purchased from the American Type Culture Collection) were used. These cells are floating cells that secrete anti-fibronectin antibodies. For the culture, a medium in which Fetal bovine serum (FBS) was added to Iscove's Modified Dulbecco's Medium (IMDM medium) to a concentration of 5% was used. Hereinafter referred to as serum medium.

Cell preparation is shown. Cells cultured in serum medium are sedimented with a centrifuge (800 rpm, 5 minutes), the supernatant (culture medium) is removed, serum medium prepared to pH 6.0 with 2 mol / l hydrochloric acid is added, and the cell number density is increased. After adjusting to 0.8 × 10 ⁶ cells / ml, 10 ml was added to a round culture dish having a diameter of 9 cm. The addition method of an alkaline solution is shown. In order to return the pH to neutrality, when one drop of 0.5 ml of 2% NaOH is added to the center of the culture dish in the configuration as shown in FIG. When adding 2 drops of 0.25 ml of 2% NaOH (total 0.5 ml) in the configuration as shown, 3 drops of 0.17 ml of 2% NaOH in the configuration as shown in FIG. In the case of adding 5 ml), the culture was carried out in four ways: when 0.5 ml of 2% NaOH was sprayed and added in the configuration shown in FIG. The evaluation was performed based on the cell viability before and after the addition of NaOH. For the cell viability, BioProfile 100Plus (Beckman Coulter) was used using trypan blue staining.

  The results are shown in FIG. When NaOH was added from one place, the survival rate was greatly reduced to 68% before addition, whereas when NaOH was added separately from two places, it became 80% and 82%, respectively. When added in the form of 85%, cell damage by the alkaline solution was improved. From the above results, it was clarified that the effects of damage and death on cells in culture can be greatly reduced by adding an alkaline solution to adjust the pH in the medium in multiple spatial divisions. .

It is a schematic block diagram which shows typically the culture tank in the cell culture apparatus to which this invention is applied. It is a schematic block diagram which shows typically the peripheral apparatus of the culture tank in the cell culture apparatus to which this invention is applied. It is a schematic block diagram which shows typically the branch type piping in the cell culture apparatus to which this invention is applied. It is a schematic block diagram which shows typically the addition system using a nozzle. It is a schematic block diagram which shows typically the addition system using a bubble jet. It is a figure which shows the addition method of NaOH in an Example. When (a) is added all at once to the center of the culture dish, (b) is added simultaneously from two parts of the culture dish, (c) is added simultaneously from three parts of the culture dish, (d ) In the case of adding in a mist form by spraying, each X in the figure shown on the right indicates the position in the culture dish to which NaOH was added. It is the figure which showed the change of the survival rate by the difference in the addition method of NaOH in an Example.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Inoculation port, 2 ... DO electrode, 3 ... pH electrode, 4 ... Resistance temperature detector, 5 ... Heater, 6 ... Stirrer blade, 7 ... Sampling port, 8 ... Alkaline addition piping, 9 ... Feed culture medium addition piping, 10 DESCRIPTION OF SYMBOLS ... Air inlet, 11 ... Exhaust port, 12 ... Sparger, 13 ... Defoaming electrode, 14 ... Liquid outlet, 15 ... Temperature control water inlet, 16 ... Temperature control water outlet, 17 ... CIP (stationary washing) port, 18 ... Spray ball, 19 ... cell, 20 ... culture tank, 21 ... medium preparation tank, 22 ... medium supply tank, 23 ... recording device, 24 ... analyzer, 25 ... control device, 26 ... pure water production device, 27 ... pure steam (PS) generator, 28 ... WFI generator, 29 ... filter, 30 ... pump, 31 ... branch pipe, 32 ... high pressure air chamber, 33 ... alkali solution chamber, 34 ... strain gauge pressure gauge, 35 ... pump unit 36 ... Piezo element 37 ... Diaphragm 38 ... Kyuana, 39 ... nozzle hole 40 ... droplets, 51 ... alkaline solution addition pipe, 52 ... piping steam, 53 ... drain valve, 54 ... drain trap

Claims (10)

  A method for culturing a cell, characterized in that when cell culture is performed using a liquid medium, a solution to be added to the liquid medium is spatially divided and added.   The above solutions are solutions that reduce the productivity of the product as the concentration increases, solutions that kill or damage the cells as the concentration increases, solutions that deteriorate the rotation of intracellular energy metabolism, and concentrations that increase. 2. The cell culture method according to claim 1, wherein the cell culture method is a solution having a property of causing denaturation of a culture solution component, or a solution having a property of generating a byproduct as the concentration becomes high.   The cell culture method according to claim 1, wherein the solution is an alkaline solution, an acidic solution, or a medium solution containing at least one of amino acids, vitamins, minerals, and proteins.   The cell culture method according to claim 1, wherein the solution is added to the medium by a spray nozzle.   The cell culture method according to claim 1, wherein the solution is added to the medium as a plurality of droplets. A culture vessel for culturing cells using a liquid medium;
A cell culturing apparatus comprising solution adding means for spatially dividing and adding a solution to the medium in the culture tank.   The above solutions are solutions that reduce the productivity of the product as the concentration increases, solutions that kill or damage the cells as the concentration increases, solutions that deteriorate the rotation of intracellular energy metabolism, and concentrations that increase. 7. The cell culture device according to claim 6, wherein the cell culture device is a solution having a property of causing denaturation of a culture solution component, or a solution having a property of generating a byproduct as the concentration becomes high.   The cell culture device according to claim 6, wherein the solution is an alkaline solution, an acidic solution, or a medium solution containing at least one of amino acids, vitamins, minerals, and proteins.   The cell culture apparatus according to claim 6, wherein the solution supply means includes at least one spray nozzle.   The cell culture device according to claim 6, wherein the solution supply means includes at least one or more nozzles for adding the solution to the culture medium as a plurality of droplets.

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