JPH0644863B2 - Method for setting up high-density continuous culture of cells - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of application] The present invention, in order to carry out high-density continuous culture of various cells, simultaneously supplies fresh medium and filters the culture solution, and at the same time, achieves cell density up to a target cell density. A startup method for increasing the density.

[Conventional technology]

Attempts to continuously culture cells at high density using a culture device that combines a culture tank with an ultrafiltration membrane or a microfiltration membrane have already been attempted at the laboratory level (Biotechnology an
d Bioengineering Vol.28, No.4, p.523-p.53
3, 1986, RIKEN Symposium "Separate Reactor" Proceedings p. 9, 1986 and JP-A-61-88872). The conventional culture method of this kind is that the culture solution is filtered with the maximum capacity of the filtration membrane until the target cell density is reached.
A high-density continuous culture of cells is set up by replenishing a fresh medium in an amount equal to the filtration amount using a signal from a liquid level sensor or the like. That is, the filtration rate of the culture solution decreases with the passage of time due to clogging of the membrane, fouling of the membrane surface, etc., so the method of starting up the method in which the supply rate of the fresh medium also decreases with the passage of time Has been adopted.

[Problems to be solved by the invention]

The conventional start-up method considers only the condition that the maximum filtration capacity of the filtration membrane is realized by ignoring the growth characteristics, substrate consumption characteristics, and metabolite production characteristics of cells in the culture device, and the filtration rate of the culture solution is considered. Since the supply rate of the fresh medium was controlled, the cell density was low, the consumption rate of the substrate by the cells in the culture device, the production rate of the metabolites were low, and the supply rate of the fresh medium and the culture solution at the initial stage of start-up. The filtration rate is high, conversely, the cell density is high, the consumption rate of the substrate and the production rate of the metabolites are high, and the supply rate of the fresh medium and the filtration rate are low at the final stage of startup, which is an irrational operation method. Was there. That is, in the initial stage of start-up, the supplied substrate is not fully consumed in the culture device and is discharged together with the culture filtrate to the outside of the device to reduce the utilization efficiency, or to perform filtration at an unnecessarily large rate. There was a problem that the membrane was clogged earlier. In addition, at the final stage of start-up, it becomes impossible to supply a substrate commensurate with the amount of consumption of the substrate of the cell, and metabolites that inhibit the growth of the cell accumulate in the device, resulting in a decrease in the cell growth rate. There were drawbacks such as.

[Means for solving problems]

In order to solve the above-mentioned problems, the present invention arranges a sensor for directly measuring the cell density online in a culture device to monitor a change over time in the cell density, and the cell density in a culture solution reaches a target value. The fresh medium supply pump is automatically controlled to increase the supply rate of the fresh medium according to the increase of the cell density until the time of reaching, and the discharge rate of the culture filtrate is the signal of the culture liquid level sensor arranged in the culture device. Based on the above, the feed rate of the fresh medium is kept almost equal, and after the cell density reaches the target value, the culture medium is continuously discharged from the cell culture device by the culture medium discharge pump and switched to high-density continuous culture. It is characterized by

[Action]

In the present invention, a sensor for measuring the cell density online is arranged in the culture device to monitor the time-dependent change of the cell density, and the cell density in the culture solution is adjusted to the increase of the cell density until the time when the cell density reaches a target value. By increasing the supply rate of the fresh medium and the discharge rate of the culture filtrate in the same manner, it is possible to supply an appropriate amount of the substrate and remove the growth-inhibiting metabolites during the entire start-up.

〔The invention's effect〕

According to the present invention, an appropriate amount of substrate necessary for cell growth is supplied and the growth-inhibiting metabolite can be removed throughout the entire period of startup, so that the utilization efficiency of the supplied substrate is increased and the metabolite is increased. It is possible to prevent the growth rate of cells from slowing down due to the accumulation of, and not to perform filtration at an unnecessarily large rate,
There is an effect that the filter membrane is less likely to be clogged, and as a result, it is possible to improve productivity by improving the start-up period and improve the cell yield per substrate used.

〔Example〕

FIG. 1 shows a cell culture device used for starting a high-density continuous culture of lactic acid bacteria, which will be described later, as an embodiment of the present invention.
The cell density in the culture tank (1) is measured online by the cell density sensor (5) and transmitted to the computer (3). The computer calculates the supply rate of the fresh medium from this cell density by the method described below, and controls the flow rate of the fresh medium supply pump (7). The fresh medium is supplied from the fresh medium tank (2) to the culture tank via the fresh medium supply line (12). Further, the culture solution in the culture tank is supplied from the culture solution supply line (15) by the culture solution supply pump (8) to the culture solution filtration module (4) through the culture solution supply flow rate control valve (18) to return the culture solution. The circulation flow control valve (19) and the culture fluid return line (16) are used to return to the culture tank. The two filtration modules are connected by a culture fluid circulation line (17), and the culture fluid is constantly circulated in the closed circuit by a culture fluid circulation pump (9). The culture fluid flow rate in the filtration module is adjusted by the culture fluid circulation flow control valve (20).
A liquid level sensor (6) detects an increase in the liquid level in the culture medium tank due to the supply of fresh medium, and the signal is used to discharge the culture filtrate solenoid valve (1
By opening 1), the culture solution is filtered and discharged from the culture filtrate discharge line (13). In this way, the supply rate of the fresh medium and the discharge rate of the culture filtrate are increased according to the increase of the cell density, and when the cell density in the culture device reaches the target value, the culture medium discharge pump (10) is turned on. It is possible to switch to high-density continuous culture by operating the medium and continuously withdrawing the medium from the medium discharge line.

An example of starting high-density continuous culture of lactic acid bacteria will be described below.

Glucose 40 g /, corn steep liquor 150 g /, K ₂ HPO ₄ 1 g /, KH in a 30-volume culture tank.
Lactobacillus pre-cultured with a medium consisting of ₂ PO ₄ 1 g /, MnSO ₄ 0.06 g /
Inoculate 150 ml of s casei seeds at 35 ° C ± 0.5
Batch culture was performed while the temperature and pH were controlled at constant values within the range of ℃ and pH 6.5 ± 0.3.

After 16 hours of culturing, the L-lactic acid concentration reached 25 g /, and beyond that, the growth rate of cells gradually decreased. From this time, while supplying the fresh medium by the start-up method specified in the present invention, The culture solution was filtered to increase the cell density.
The fresh medium supplied was glucose 25 g /, corn steep liquor 100 g /, K ₂ HPO ₄ 1 g /,
The composition of KH ₂ PO ₄ 1 g / and MnSO ₄ 0.06 g / was used. The membrane used in the experiment was a CarboSep SV7 module (zirconia membrane, molecular weight cut-off 15,000, membrane area 0.16, manufactured by Sumitomo Kikai Envirotech Co., Ltd.).
m ² , operating temperature <150 ° C., operating pH 0 to 14) 2. The cell density sensor is a laser turbidimeter (light source He-Ne laser 5 mW, wavelength 632.8n manufactured by RIKEN Institute of Technology.
m, optical path length 2 mm, detector photomultiplier).

The method of calculating the medium supply rate from the online measurement result of the cell density will be described below. When lactic acid bacteria were cultivated while supplying a fresh medium, the concentration of glucose, which is a growth limiting substrate, was 0.
At around g /, a linear relationship as shown in FIG. 2 is obtained between the glucose specific consumption rate ν and the specific growth rate μ. Such a relationship is experimentally obtained in advance and stored in the computer. Further, the specific growth rate μ _c is calculated from the cell density X _c measured online according to the equation (1), and more preferably, the specific growth rate μ _c is removed by the method of JP-A-59-11390 to eliminate the influence of noise. And calculate the fourth
From the relationship in the figure, the corresponding glucose specific consumption rate ν _c
Ask for.

μ _c = (ΔX / Δt) / X _c (1) where ΔX is the increase in cell density per unit time, and Δt is the unit time.

The mass balance equation for glucose S in this culture device is given by equation (2).

ds / dt = (Fin Sf−Fout S) / V−νX (2) where Fin and Sf are the supply rate of the fresh medium, the glucose concentration in the fresh medium, and Fout is the discharge rate of the culture filtrate, V
Represents the amount of culture solution. Now, the start-up method of the present invention is characterized by keeping the supply rate of the fresh medium and the discharge rate of the culture filtrate equal, and further, in this example, in order to improve the utilization efficiency of the supplied medium, The glucose concentration, that is, the glucose concentration S in the culture solution is set to 0.
Since it was adjusted to around g /, (3),
The relationship of equation (4) is established.

Fin = Fout (3) SO, ds / dtO (4) Substituting the relationships of these equations (3) and (4) into equation (2) and solving for Fin yields equation (5).

Fin = νXV / Sf (5) The culture solution volume V in the equation (5), the glucose concentration Sf in the fresh medium are constant values determined by the culture conditions, and the glucose specific consumption rate ν is the target specific growth rate μ _c Constant value ν _c corresponding to
Further, by substituting the cell density obtained from the signal transmitted from the cell density sensor for the cell density X, the supply rate Fin of the fresh medium corresponding to the increase of the cell density can be obtained.

(Example 1) FIG. 3 shows changes with time in cell density, glucose concentration, L-lactic acid concentration, supply rate of fresh medium and integrated supply amount in the course of culture. Immediately after the 16th hour, the L-lactic acid concentration could be maintained at 25 g / or less, and the glucose concentration could be maintained at almost 0 g / after 20 hours. As a result, the cell density was 40 g-dry-cell / the target value at 30 hours after 14 hours from the start-up.
By reaching the stomach and continuously discharging the culture solution at 2.54 / h at that time, the growth rate and the discharge rate of the cells in the culture device are almost balanced, and the cell density is 40 g-dry.
-Cell / could be maintained for continuous culture. The specific growth rate μ of the cells during the start-up period is 0.169 (h ^-1 ).
I was able to keep it.

The amount of fresh medium supplied during the startup period was 123
Met.

(Example 2) FIG. 4 is an example in the case of adopting a start-up method such that the supply rate of the fresh medium was constant, and the supply rate of the fresh medium was 5.06 / h. Other culture conditions are
This is similar to the case of the first embodiment. In this case as well, the L-lactic acid concentration and the glucose concentration decreased immediately after the start-up at the 16th hour, and after the 20th hour, the L-lactic acid concentration could be maintained at 25 g / or less and the glucose concentration at 0 g /. . However, the target cell density was 40g-dry-ce.
It took 32 hours to reach ll /, and the amount of fresh medium supplied during the start-up period was 162.

Thus, when the fresh medium was supplied at a constant rate,
As the cell density increases, the glucose specific consumption rate ν decreases, and as a result, the cell specific growth rate μ and the cell-to-glucose yield Y _{x / s} decrease, as is clear from the relationship in FIG. Compared to the first embodiment, the startup period is 2,
3 times, the amount of fresh medium used was 1.3 times.

As is clear from the above two examples, in order to maintain a high cell growth rate to shorten the start-up period and to reduce the amount of fresh medium used, simply maintain the L-lactic acid concentration low. Or maintaining the glucose concentration low is not sufficient, and Example 1 according to the start-up method of the present invention was used.
As described above, by increasing the supply rate of the fresh medium and the discharge rate of the culture filtrate in accordance with the increase of the cell density, it is possible to shorten the start-up period and the amount of the fresh medium used.

[Brief description of drawings]

FIG. 1 is a block diagram of a high-density cell culture device used for carrying out the present invention, FIG. 2 is a relationship diagram of glucose specific consumption rate, specific growth rate of cells, and glucose yield, and FIG. Time-course diagram of culture when fresh medium was supplied according to increase in cell density, and FIG. 4 is time-course diagram when fresh medium was supplied at a constant rate. 1 ... Culture tank, 2 ... Fresh medium tank, 3 ... Computer, 4 ... Culture fluid filtration module, 5 ... Cell density sensor, 6 ... Liquid level sensor, 7 ... Fresh medium supply pump, 11 ... Solenoid valve for discharging culture filtrate.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Hiroshi Misawa 1-1-19 Higashishimbashi, Minato-ku, Tokyo Yakult Honsha Co., Ltd. (72) Inventor Tsuneo Terajima 1-1-1 Higashishimbashi, Minato-ku, Tokyo No. 19 Yakult Honsha Co., Ltd. (72) Inventor Takahiko Makudai 1-1-19 Higashishimbashi, Minato-ku, Tokyo Yakult Honsha Co., Ltd. (56) References Japanese Patent Publication Sho 60-15307 (JP, B2) Japanese Patent Publication Sho 54-11392 (JP, B2) Biotechnology and Beoengineering (28) P. 523-533 (1986)

Claims (1)

[Claims] 1. A sensor for directly online measuring the cell density in a culture device for high-density continuous culture of cells in a cell culture device comprising a fresh medium supply port, a culture filtrate discharge port, and a culture solution discharge port. To monitor the change over time in cell density and increase the supply rate of fresh medium by automatically controlling the fresh medium supply pump according to the increase in cell density until the cell density in the culture reaches the target value. And the discharge rate of the culture filtrate is controlled based on the signal of the culture liquid level sensor placed in the culture device to keep it almost equal to the supply rate of the fresh medium, and after the cell density reaches the target value, the culture liquid is reached. A method for starting a high-density continuous culture of cells, which comprises continuously discharging a culture solution from a cell culture device by a discharge pump to switch to a high-density continuous culture.