JPH04126072A - Cell culture and apparatus therefor - Google Patents

Abstract

PURPOSE:To readily control the cell density in a culturing unit and to improve productivity by equipping a device for taking out a cell-free culture medium from a culture tank and a device for taking out a mixture solution of cells and a culture medium and changing the ratio between the quantities taken out by both the units. CONSTITUTION:Animal cells, plant cells or cells of microorganisms, etc., are cultured in a culturing unit 1 and a fresh culture medium is supplied to the cells during culture using a pump 3. A cell-free culture medium is taken out through a cell separator unit 2 (e.g. hydrophobic filtration membrane) using a pump 4 and a mixture solution of cells and a culture medium is simultaneously taken out using a pump 5. During the above-mentioned operation, the ratio of the quantity taken out by the pump 4 to that by the pump 5 is controlled using a controller 9 so as to control the cell density in the culturing unit 1. The cell density in the culturing unit 1 can be kept to a prescribed value thereby and a high productivity is realized therefor.

Description

Detailed Description of the Invention [Industrial Application Field 1] The present invention relates to a method and apparatus for culturing animal cells, plant cells, microorganisms, etc. 6 [Prior Art] In the operation of a culture apparatus, the growth of cultured cells, nutritional components, etc. The internal conditions of the culture apparatus continue to change due to consumption of cells, accumulation of waste components, etc.1 Usually, cell proliferation and product accumulation come to an end after a certain period of time. On the other hand, by supplying a fresh medium into the culture tank and withdrawing the medium at the same flow rate, the condition inside the culture tank can be kept constant at all times, making continuous culture possible. This continuous culture method is based on Biochemical Engineering Fundamentals (D98
6th year) pages 380 to 383 (Biochemical
al Engineering Fundamen
tals (D986) P2S5-383).
There is a method called t). In this method, fresh medium is continuously supplied to the culture device, and the same flow rate of a mixed solution of culture medium and cells is extracted from the culture device. By balancing the number of cells, the cell density within the culture device can be kept constant. This relationship is expressed by the following equation.

D=F/V −−−−−−−−−−−−−−−−
--(2) where χ: cell density (cells/l2) t: time (h) D, exchange $ (h-") μ, cell proliferation rate (h-') F: medium supply and withdrawal rate (I2/h)V: Culture device capacity (I2) In equation (D), if the cell density χ in the culture device is constant, that is, dχ/dt=0, then the following relationship holds true.

D=μ−−−−−−−−−−−−−−−−−−−−
--(3) That is, the exchange rate and the proliferation rate U must be equal. In other words, the dilution rate is determined by the proliferation rate of the cells to be cultured, and a higher dilution rate cannot be obtained.For example, in the case of animal cells, the proliferation rate μ is 0.01~ 002(h-“),
If you culture at the same exchange rate, you will be able to supply a little less than half of the total capacity of the culture device in one day, and you will be able to maintain cells! The degree is at most l x 10' (
cells/ff).

In response, various attempts have been made to prevent cells from flowing out of the culture device and to replace the medium in the culture device at a rate higher than the cell proliferation rate. In order to prevent the cells from flowing out of the culture device, a means for separating the cells and the medium is required, and methods include gravity sedimentation, centrifugation, and filtration. By using these methods, lXl0”
It has become possible to obtain cell densities of (cells/42) or higher.

[Problem to be solved by the invention 1] In culture using this cell separation device, since the exchange rate of formula (D) is excluded, dχ/dt>O always holds, and the cell density becomes infinitely large. It should be, but in reality it doesn't happen, and even if it's big, it's at most l X I O"
(cells/ff).

This is because cells are inhibited by some factor or the number of cells that die increases. Depending on the type of product, more products may be secreted during this state of starvation, but on the other hand, the metabolic pathway of the cell may change in an unfavorable direction, or the production of enzymes due to the decomposition of dead cells may occur. Spillage into the culture medium may adversely affect product recovery. In order to prevent this negative effect, the medium exchange rate per cell α (D/h-
cell) must be maintained at a value above a certain amount,
This is expressed by the following formula.

α=D/χ=α. −−−−−−−−−−−−−(4)
However, as mentioned above, the exchange rate is finite for the cell density χ, which continues to increase indefinitely, so at some point, 6. In this case, it becomes impossible to satisfy formula (4),
There was no way to stop the increase in cell density χ before that point.

In the present invention, at a given exchange rate, the cell density χ
An object of the present invention is to provide a cell culture method and device that can obtain high productivity by maintaining constant cell culture.

[Means for solving the problem] In order to achieve the above purpose, cells are cultured at a certain rate so that the cell density does not increase beyond the target value! ! It is designed so that it can be extracted outside the device. The amount of cells to be extracted is not uniquely determined by the exchange rate like in a chemostat (but must be able to be controlled arbitrarily; for this purpose, the cell separation device is connected to the culture device (tank). A medium outlet for the medium and a outlet for the mixed solution of the culture medium and cells are provided separately, and the ratio of the amounts withdrawn from the outlets of both methods is controlled.

[Function] The extraction speed from the cell separation device is set to F, [! /h), and when the direct extraction rate from the culture device is F, (β/h),
Culture! The following relationship holds within the device.

dχ m= (U-ηD)χ −−−−−−−−−−(5) t D= (F, +F,)/V −−−−−−−−(6
) η=F2/(F, +F,) −−−−−−(7) In equations (5) to (7), the case where F', = 0 is the case of a chemostat, and F, = O is Conventional cell fraction ilI
This corresponds to the case where only the device is used. Here, the exchange rate and the medium exchange rate per cell α. When is given, the target cell density χ0 is determined by equation (4). The actual cell density χ is χ. If the extraction flow rate distribution ratio η is set so that η is smaller than μ/D, dχ/dt>O as shown in equation (5), and the cell density increases. Also, conversely, the cell density χ is χ. When it is larger than , if η>μ/D, dχ/dt<0 and the cell density decreases.

Here, the extraction flow rate distribution ratio η can be controlled independently of the exchange rate and cell density χ, so χ=χ. The sound can be easily controlled.

[Example] Hereinafter, in Figure 0, an example of the present invention will be explained with reference to Figure 1, in which a fresh medium is supplied by a pump 3 to cells being cultured in a culture M device 1. When the liquid level reaches the upper limit level sensor 6, the supply of fresh medium is stopped, and the pump 4 extracts only the medium via the cell separation device 2. At the same time, the pump 5 extracts the mixture of cells and medium. If the flow rate of pump 5 is set sufficiently larger than that of pump 4,
Most of what is extracted from the culture device 1 comes from the pump 5. When the liquid level falls below the extraction nozzle 8 of the pump 5, only the gas in the gas phase is extracted from the pump 5, and the liquid level is lowered only by the culture medium extracted from the pump 4. When the liquid level reaches the lower limit level sensor 7, the pumps 4 and 5 are stopped, and the pump 3 is started to restart the supply of fresh culture 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”/ (h1+h2) −−−−−
-(8) Next, another embodiment of the present invention will be described with reference to FIG.

In the figure, a constant flow rate F is used to maintain a predetermined exchange rate for the cells being cultured in the culture device L. (R/
h) Supply fresh medium by pump 3. By keeping the flow IF2' (I2/h) of pump 5 constant, which is sufficiently larger than that of pump 3, the mixture of cells and culture medium can be removed by the amount that the liquid level is higher than the extraction nozzle 8 of pump 5. put out. The rate of withdrawal of the medium from the pump 4 Fl (p/h
) is calculated by the following equation, and the control device 9 outputs a speed command signal to the pump 4.

F 1= (D-n)Fo ------------------------(9
) At this time, the extraction amount distribution ratio η is set so that η=μ/D, and the control target value χ of the cell heatness χ measured by the cell density meter 10. By increasing or decreasing the deviation in accordance with the deviation, the deviation is made smaller and the control target value χ is increased. can be approached.

〔Effect of the invention〕

According to the present invention, the medium exchange rate α per cell can be kept constant by changing the extraction volume distribution ratio q.
The state of the cells can be maintained at any value such as the growth phase or the stationary phase, thereby improving productivity and culturing reproducibility.

[Brief explanation of drawings]

FIG. 1 is an explanatory diagram of a culturing apparatus according to one embodiment of the present invention, and FIG. 2 is an explanatory diagram of a culturing apparatus according to another embodiment of the present invention. 1-----One culture device, 2-----One cell separation device, 3-------Pump, 4-------Pump, 5-
------- Pump, 6 ------- One upper limit level sensor, 7--- Lower limit level sensor, 8--- One extraction nozzle, 9--- One controller, 10---One cell area

Claims (1)

[Scope of Claims] 1. A cell culture method in which cells are cultured in liquid, comprising means (A) for extracting a medium containing no cells from a culture tank and means (B) for extracting a mixed solution of cells and medium. provision, said means (A)
A cell culture method characterized by controlling the cell density within the culture device by changing the extraction ratio of (B). 2. The cell culture method according to claim 1, wherein the means for extracting the cell-free medium is a desired method among gravity sedimentation, centrifugation, or filtration. 3. The cell culture method according to claim 1, wherein the means for extracting the cell-free medium is a hydrophobic filtration membrane. 4. The cell culture method according to claim 1, claim 2, or claim 3, wherein the cells are animal cells, plant cells, or microorganisms. 5. Animal cells, plant cells, and microorganisms provided with an extraction port (C) for extracting a medium without cells through a cell separation device and an extraction port (D) for extracting a mixed solution of cells and culture medium. 1. A cell culture device for submerged culture of a cell, etc., characterized in that the cell culture device is provided with means for arbitrarily setting a flow rate ratio between the extraction port (C) and the extraction port (D). 6. The means for setting the flow rate ratio between the extraction port (C) and the extraction port (D) is such that the extraction port (D) is provided near the liquid surface of the culture tank, and the mixture of cells and culture medium is supplied from the extraction port (D). 6. The cell culture device according to claim 5, wherein the cell culture device is configured such that the gas in the gas phase portion of the culture tank is alternately extracted and controlled. 7. The cell culture apparatus according to claim 5, wherein the means for setting the flow rate ratio between the outlet (C) and the outlet (D) uses a variable flow rate pump. 8. The cell culture device according to claim 5, wherein the means for setting the flow rate ratio between the extraction port (C) and the extraction port (D) uses a throttle valve. 9. The means for setting the flow rate ratio between the outlet (C) and the outlet (D) is configured by opening and closing a valve.
6. The method of operating a cell culture apparatus according to claim 5, comprising: D) controlling respective opening and closing times. 10. The means for setting the flow rate ratio of the extraction port (C) and the extraction port (D) measures the cell density in the culture tank, and adjusts the flow rate using the deviation between the measured cell density and the control target cell density. 6. The method of operating a cell culture apparatus according to claim 5, wherein the ratio is controlled. 11. The method of operating a cell culture apparatus according to claim 10, wherein the cell density detection means is a turbidity meter that measures transmitted light, scattered light, etc. 12. The method of operating a cell culture apparatus according to claim 10, wherein the cell density detection means is a device that processes a cell image obtained by a television camera. 13. Claim 1 or Claim 2, characterized in that the proportion of the flow rate of the mixture of cells and culture medium directly extracted from the culture tank in the total extraction amount is determined by the following formula:
, or claim 3, or instructional claim 4, or claim 10
Cell culture method described. η=μ/D However, η=F_2/(F_1+F_2) D=(F_1+F_2)/V Here, η: Extraction flow rate distribution ratio μ: Cell proliferation rate (h^-^1) D: Exchange rate (h^-^ 1) F_1: Extraction flow rate of only the medium via the cell separation device (
(l/h) F_2: Direct removal rate of cells and medium mixture from the culture device (l/h) V: Culture device capacity (l)