JPH0813268B2 - Cell culture method and device - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method and apparatus for culturing animal cells, plant cells, microorganisms and the like.

[Conventional technology]

During operation of the culture device, the internal conditions of the culture device continue to change due to growth of cultured cells, consumption of nutrients, accumulation of waste components, etc. Normally, the growth of cells and accumulation of products end at a certain period. . On the other hand, by supplying a fresh medium into the culture tank and extracting the medium at the same flow rate, the state in the culture tank can be always kept constant,
Continuous culture is possible. Biochemical engineering fundamentals (1986
Year) 380 to 383 (Biochemical Engineering Fu
There is a method called chemostat as discussed in ndamentals (1986) P380-383). In this method, fresh medium is continuously supplied to the culture device, and a mixed solution of medium and cells at the same flow rate as that is withdrawn from the culture device.The number of cells growing in the culture device and the outflow from the outlet. By balancing the number of cells, the cell density in the culture device can be kept constant. This relationship is expressed by the following equation.

D = F / V (2) where x: cell density (cells / l) t: time (h) D: exchange rate (h ^-1 ) μ: cell growth rate (h ^-1 ) F: medium supply And withdrawal flow rate (l / h) V: Incubator capacity (l) If the cell density x in the incubator in the formula (1) is constant, that is, dx / dt = 0, the following equation holds.

D = μ (3) That is, the exchange rate D and the growth rate μ must be equal. In other words, the dilution rate is determined by the growth rate of the cells to be cultured, and a higher dilution rate cannot be obtained. For example, in the case of animal cells, the growth rate μ is 0.01 to 0.02 (h ^-1 ), and the same exchange rate D
In the case of culturing at 1, the culture medium can supply only a little less than half of the total volume of the culture apparatus per day, and the cell density that can be maintained is about 1 × 10 ⁹ (cells / l) at most.

On the other hand, various attempts have been made to prevent the outflow of cells from the culture device and replace the culture medium in the culture device at a rate higher than the cell growth rate. In order to prevent the outflow of cells from this culture device, a means for separating the cells from the culture medium is required, and there are methods such as gravity sedimentation type, centrifugation type, and excess type. 1 × 10 by using these methods
A cell density of ¹⁰ (cells / l) or more has been obtained.

[Problems to be Solved by the Invention]

In the culture using this cell separation device, since the exchange rate D of the formula (1) is excluded, dx / dt> 0 is always maintained, and the cell density should be infinitely large. Not at all, even if it is large, at most 1 × 10 ¹¹ (cells /
up to l). This is because cells are inhibited by some factor or the number of cells that die is increased, and usually, starvation due to lack of nutritional components and inhibition due to accumulation of waste components are common. Depending on the type of cell or product, the starved state may secrete more product, but on the contrary, the metabolic pathway of the cell may change in an unfavorable direction or the enzyme caused by the decomposition of dead cells. The spillage into the medium may adversely affect the recovery of the product. In order to prevent this adverse effect, it is necessary to maintain the medium replacement rate α (1 / h · cell) per cell at a certain value or more, which is expressed by the following equation.

α = D / x = α ₀ (4) However, as described above, the exchange rate D is finite with respect to the cell density x that is going to continue increasing indefinitely, and therefore, at some point, the equation (4) must be used. There was no means to stop the increase in cell density x before that.

The present invention is a cell culture method capable of obtaining high productivity by maintaining the cell density x constant in a state where the condition of the formula (4) is satisfied and the inhibition to cells does not occur at a given exchange rate D. And to provide a device.

[Means for solving the problem]

In order to achieve the above object, cells are taken out of the culture device at a certain ratio so that the cell density does not increase more than the intended value. The amount of cells to be extracted is not something that is uniquely determined by the exchange rate D as in chemostat, but must be one that can be arbitrarily controlled. For this purpose, the culture device (tank) is provided with separate outlets for the medium from the cell separation device and outlets for the mixture of medium and cells, and the ratio of the amount of withdrawal from both outlets is controlled. Is.

[Action]

When the withdrawal flow rate from the cell separation device is F ₁ (l / h) and the direct withdrawal flow rate from the culture device is F ₂ (l / h), the following relationship is established in the culture device.

D = (F ₁ + F ₂ ) / V …… (6) η ＝ F ₂ / (F ₁ + F ₂ ) …… (7) In the equations (5) to (7), the case where F ₁ = 0 becomes a chemostat. And F ₂ = 0 corresponds to the case where only the conventional cell separation device is used. Here, when the exchange rate D and the medium exchange rate per cell α ₀ are given, the target cell density x ₀ can be obtained by the equation (4). If the extraction flow distribution ratio η is set so that η <μ / D when the actual cell density x is smaller than x ₀ , dx / dt> 0 as shown in equation (5), and the cell density increases. To do. On the contrary, if the cell density x is larger than x _{0, then} η> μ / D so that dx / dt <0 and the cell density decreases. Here, since the extraction flow distribution ratio η can be controlled independently of the exchange rate D and the cell density x, x = x ₀
Can be easily controlled.

〔Example〕

An embodiment of the present invention will be described below with reference to FIG. In the figure, a pump 3 is applied to cells being cultured in the culture device 1.
To supply fresh medium. Liquid level is upper limit level sensor 6
Then, the supply of the fresh medium is stopped, and only the medium is extracted by the pump 4 via the cell separation device 2, and at the same time, the mixed solution of the cells and the medium is extracted by the pump 5. At this time, if the flow rate of the pump 5 is set to be sufficiently higher than that of the pump 4, most of the liquid extracted from the culture device 1 is from the pump 5. When the liquid level drops below the extraction nozzle 8 of the pump 5, only the gas in the gas phase is extracted from the pump 5. The liquid level drops only by the medium withdrawn from the pump 4. When the liquid level reaches the lower limit level sensor 7, the pump 4 and the pump 5 are stopped and the pump 3 is started to restart the supply of the fresh medium.

According to this embodiment, the extraction amount distribution ratio η can be changed depending on the height of the extraction nozzle 8 of the pump 5.

η = h ² / (h ₁ + h ₂ ) (8) Next, another embodiment of the present invention will be described with reference to FIG.

In the figure, a constant flow rate F ₀ (l /
Supply fresh medium by pump 3 in h). By keeping the pump 5 constant at a flow rate F ₂ ′ (l / h) which is sufficiently higher than that of the pump 3, only the amount of the mixed liquid of the cells and the medium, which is higher than that of the extraction nozzle 8 of the pump 5, can be obtained. Pull out. The medium withdrawal flow rate F ₁ (l / h) from the pump 4 is calculated by the following equation, and the controller 9 outputs a speed command signal to the pump 4.

F ₁ = (1−η) F ₀ …… (9) The extraction volume distribution ratio η at this time is set so that η = μ / D, but the cell density x measured by the cell densitometer 10 to reduce the deviation by'll increased or decreased according to the deviation from the control target value x _0, it can be brought close to the control target value x _0.

〔The invention's effect〕

According to the present invention, the medium exchange rate α per cell can be kept constant by changing the withdrawal distribution ratio η, so that the cell state can be maintained at any value such as the growth phase or stationary phase. The productivity can be improved and the reproducibility of the culture can be improved.

[Brief description of drawings]

FIG. 1 is an explanatory view of a culture device according to one embodiment of the present invention, and FIG. 2 is an explanatory view of a culture device according to another embodiment of the present invention. 1 ... Culture device, 2 ... Cell separation device, 3 ... Pump,
4 ... Pump, 5 ... Pump, 6 ... Upper level sensor, 7 ... Lower level sensor, 8 ... Extraction nozzle, 9 ...
… Control device, 10 …… Cell densitometer

Claims (5)

[Claims] 1. A cell culture method for culturing cells in a liquid, comprising means (A) for extracting a cell-free medium from the culture tank and means (B) for extracting a mixture of cells and the medium, A cell culturing method, characterized in that a cell density in a culturing device is controlled by changing a flow distribution ratio of a flow extracted by two means (A) and (B). 2. A means for extracting the medium containing no cells,
The cell culture method according to claim 1, which is any one of gravity sedimentation, centrifugation, and filtration. 3. The cell culture method according to claim 1, wherein the flow distribution ratio is determined so as to satisfy the equation η = μ / D. Where η = F ₂ / (F ₁ + F ₂ ) D = (F ₁ + F ₂ ) V where η: withdrawal flow distribution ratio μ: cell growth rate (h ^-1 ) D: exchange rate (h ^-1 ) F ₁ : Flow rate of withdrawing only the medium through the cell separator (l / h) F ₂ : Direct withdrawal rate of cell and medium mixture from the incubator (l / h) V: Capacity of the incubator (l) 4. An animal cell or plant provided with an outlet (C) for withdrawing a cell-free medium through a cell separation device and an outlet (D) for withdrawing a mixed solution of cells and medium. cell,
A cell culture device for culturing microorganisms and the like in a liquid, comprising a setting means for arbitrarily setting a flow rate ratio of the outlet (C) and the outlet (D). 5. The setting means controls the flow rate using a measuring means for measuring the cell density in the culture tank and a deviation between the cell density measured by the measuring means and the control target cell density. 5. The cell culture device according to claim 4, further comprising: