JPH06209761A - Cell culture system - Google Patents

Abstract

PURPOSE:To provide a cell culture system designed to produce physiologically active substances at a lower cost by perfusion culture, i.e., conducting a high- density culture with the culture environment maintained stably for a long period of time. CONSTITUTION:In a culture system where a culture tank 1 is continuously fed with fresh culture fluid along with discharging the culture fluid accumulated with cell metabolites out of the tank 1 and recovering the culture fluid, the inside of the culture tank 1 is equipped with a sedimentation separation tube 4 to continuously separate the supernatant of the culture fluid from the microcarrier and the cells adhered thereto; the opening 4a of said tube 4, through which culture fluid is introduced, is directed to the downstream side of the circulating flow due to culture fluid agitation, and the tube 4 has at least one bent part 4b.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a cell culture device having a continuous cell separation device for carrying out perfusion culture, and more specifically, to adhere adherent animal cells on microcarriers in a cell culture tank. In the system for culturing in, the sedimentation separation tube type cells installed in the culture tank to continuously separate the microcarriers and the cells adhered to the microcarriers and the culture supernatant during the perfusion culture. The present invention relates to a cell culture device equipped with a separation device.

[0002]

2. Description of the Related Art In recent years, as a result of technological development in the field of biotechnology, various physiologically active substances, which have been very difficult to obtain conventionally, have been produced and utilized through the culture of animal cells. There is. However, the production of physiologically active substances by cell culture generally has the drawbacks of low productivity and high cost.

Perfusion culture is intended to realize high-density culture by maintaining a stable cell culture environment, improve productivity, and solve cost problems. Various efforts have been made towards this.

As a method of continuously separating cells and a culture solution in perfusion culture, in addition to sedimentation, a method of using a continuous centrifugal separator, a method of installing a spin filter in a tank, a UF membrane or an MF membrane. A method using is proposed. The method is roughly classified into a method of obtaining a clarified liquid by performing separation in a tank, and a method of guiding a culture solution to a separation device outside the culture tank for separation and returning the cell concentrate to the culture tank.

In the case of a separation method in which separation is performed outside the culture tank (for example, centrifugation, separation with a membrane or a filter, etc.), the cells are exfoliated from the microcarriers and deadly to the cells due to the influence of the shearing force during liquid feeding. There is a danger of causing specific damage. Further, in the separation using a membrane or a filter, it is necessary to periodically replace the filter element due to clogging or to eliminate clogging by backwashing, which causes a problem that the operation and equipment become more complicated.

When a spin filter is installed in a culture tank for separation, the equipment becomes complicated and
If there is no choice but to eliminate the clogging of the filter by backwashing, and if the clogging cannot be removed, it becomes impossible to continue the perfusion culture.

On the other hand, in the sedimentation separation, since the microcarriers and the cells attached to the microcarriers and the culture medium are separated by the difference in specific gravity between them, the damage to the cells is small. Further, the sedimentation separation has an advantage that the structure of the separation equipment is simple and clogging is less likely to occur.

So far, in perfusion culture of adherent cells, a settling zone is provided by a sedimentation separation tube,
Methods have been used to facilitate the separation of culture medium from microcarriers and cells attached to microcarriers (Reference M. Butler et.al .: High Yields from Microca
rrier Cultures by Medium Perfusion: J. Cell Sci.6
1, 351-363, 1983).

The sedimentation separation tube disclosed in the same document is installed perpendicularly to the gas-liquid interface on the culture solution, and the portion for conducting the culture solution is opened at the bottom of the sedimentation separation tube. In the separation tube provided perpendicularly to the gas-liquid interface, the cells and the culture solution are separated due to the difference in specific gravity.

However, in the separation tube having this structure, in order to separate the culture solution from the microcarriers and the cells attached to the microcarriers, there is a place where the inside of the settling zone of the sedimentation separation tube should be in a state where there is almost no disturbance of the culture solution. , The turbulent state due to the flow of the culture solution bulk due to the stirring operation partially or mostly affects the settling zone inside the sedimentation separation tube. Due to this, the efficiency of separation due to the difference in specific gravity between the microcarrier and the culture medium is significantly impaired, and the microcarrier and the cells attached to the microcarrier are retained in the culture tank, and a clear culture medium that does not contain the microcarrier. Would be extremely difficult to obtain.

Further, in the separation tube having such a structure,
It is possible to reduce the cross-sectional area of the opening of the sedimentation separation tube in order to prevent the disturbance of the culture solution bulk in the sedimentation separation tube, but the microcarriers that have already entered the sedimentation separation tube accumulate, There is a high possibility that it will not be possible to return to the culture solution bulk, and as a result, the microcarriers will be blocked in the sedimentation separation tube and will flow out to the culture solution collection line. In addition, when microcarriers are deposited in the sedimentation separation tube, the culture environment of cells attached to the microcarriers is compared to when the microcarriers are present in the culture medium, because of the local dissolved oxygen and other nutrient sources. Depletion of wastewater and accumulation of waste products are expected, which is an obstacle to high-density culture.

[0012]

In view of the above-mentioned circumstances, an object of the present invention is to reduce the cost of physiologically active substances by performing high-density culture while maintaining the culture environment stably for a long period in perfusion culture. It is to develop a cultured cell device for production.

[0013]

Means for Solving the Problems The inventors of the present invention have conducted extensive studies in order to realize high-density culture. As a result, in perfusion culture, microcarriers and cells attached to the microcarriers were continuously treated with a culture supernatant. The present invention has invented a cell culture device having a sedimentation separation tube capable of forming a clear settling zone by rapidly attenuating turbulence of a culture solution due to stirring during sedimentation separation.

That is, the present invention provides a system in which cells are adhered onto a microcarrier and agitated and cultivated in a cell culture tank, while continuously supplying fresh culture medium to the cell culture tank. In a cell culture device for discharging and recovering the culture solution in which the metabolites of the above are accumulated outside the cell culture tank, a settling separation tube for continuously separating microcarriers and cells attached to the microcarriers and the culture solution supernatant is provided. Installed in a cell culture tank, the sedimentation separation tube forms a flow path for allowing a culture solution to pass while precipitating microcarriers and cells attached to the microcarriers in the cell culture tank,
The opening for introducing the culture solution in the flow channel is characterized in that it is directed in the downstream direction of the circulation flow due to the stirring of the culture solution, and at least one portion of the flow channel is bent.

The stirring culture is usually carried out by rotating a stirring blade provided in a culture tank to suspend and mix a culture solution containing microcarriers and cells.

Nutrient sources constituting the culture solution include salts, sugars,
Vitamins, amino acids and serum components are usually used.

In the cell culture device of the present invention, various cells such as animal cells, plant cells, microbial cells, cells produced by artificial manipulations such as hybridomas can be cultured. In particular, the present invention is adherent. Suitable for culturing animal cells.

[0018] Metabolites of cultured cells contain useful substances such as physiologically active substances, and by separating and purifying them, they can be used as pharmaceuticals and the like.

The sedimentation / separation tube separates the microcarriers and the cells attached to the microcarriers from the culture solution, retains the microcarriers and the cells attached to the microcarriers in the culture tank, and the culture solution supernatant, that is, the supernatant. It is installed in the culture tank to collect only

The sedimentation separation tube has one end opened in the culture solution in the culture tank, and the other end is piped to a culture solution supernatant recovery tank or container via a nozzle of the culture tank and a liquid feed pump. Alternatively, it can be connected with a tube or the like. When the culture solution is extracted at a predetermined flow rate by the liquid feed pump, the culture solution is gently introduced into the sedimentation separation tube. The microcarriers and the cells attached to the microcarriers are separated by sedimentation due to the difference in specific gravity from the culture supernatant, and only the culture supernatant is selectively sent from the sedimentation separation tube to the culture supernatant collection tank or container. .

In the present invention, the circulation flow by stirring means the flow of the whole culture solution which is generated in the rotating direction of the stirring blade, for example, by rotating the stirring blade, and the downstream of the circulation flow means the circulation flow in the culture solution. At the point of existence, it indicates the direction in which the circulating flow flows. In the device of the present invention, the opening of the sedimentation separation tube for introducing the culture solution is opened toward the downstream side.

The apparatus of the present invention will be described in more detail below. FIG. 1 is a schematic diagram showing an example of a cell culture device according to the present invention. As shown in FIG. 1 (a), the cell culture device according to the present invention has a culture tank 1 for performing perfusion culture, and a sedimentation separation tube 4 is provided in the culture tank 1.
Is installed.

The upper part of the sedimentation separation pipe 4 is connected to the recovery pipe 6 through the discharge port 4c for discharging the culture solution supernatant to the outside of the culture tank 1. The culture solution supernatant is collected in the container 8a by the pump 7a through the collection pipe 6.

On the other hand, the lower part of the sedimentation separation tube 4 is connected to the culture solution 5 through an opening 4a in the culture solution 5.

A stirring blade 2 driven by a motor 12 is provided in the culture tank 1. By rotating the stirring blade 2, the microcarriers in the culture solution 5 and the cells attached to the microcarriers are uniformly stirred. It

Further, in this apparatus, the fresh medium contained in the container 8b is supplied from the pipe 9 into the culture tank 1 via the pump 7b. Further, in the culture tank 1, a gas supply pipe 13 for supplying a mixed gas containing oxygen as a main component and an exhaust pipe 1 for discharging the mixed gas from the culture tank 1 to the outside of the system.
0 is provided, and a temperature control jacket 11 is provided outside the culture tank 1.

Further, in the sedimentation separation tube 4, the culture solution 5
The part that is present therein has a bend 4b.
A portion of the settling separation tube 4 extending from the bent portion 4b toward the outlet 4c connected to the recovery pipe 6 is provided at a substantially vertical angle or nearly vertical to the liquid surface of the culture solution 5, while the bent portion 4b is provided. The opening 4a is bent from the direction perpendicular to the liquid surface of the culture medium 5 toward the liquid surface.

With such a bent portion 4b, as shown in FIG.
As shown in, the opening 4a of the sedimentation separation tube 4 is directed to the downstream direction with respect to the circulation flow of the culture solution (indicated by arrow B) in the rotation direction of the stirring blade 2 (indicated by arrow A).

It is desirable that the opening 4a be mounted at a position where it is difficult to bring the fluidized state by stirring.
In the radial direction of, the position closer to the stirring rotary shaft 3 is more preferable, and particularly within 3/4 of the radius of the culture tank 1 with the stirring rotary shaft 3 as the center. Further, in the height direction of the culture tank 1, the opening 4a is located at a position closer to the liquid surface of the culture liquid 5, particularly at a position that is 1/2 or more of the distance from the upper end of the stirring blade 2 to the liquid surface of the culture liquid 5.
Is preferably provided.

Further, as shown in FIG. 2, for example, the direction toward the downstream side of the opening is the streamline 20 due to the circulation flow.
Can be set approximately equal to the tangential direction of (shown by arrow C), but if the opening of the separation tube does not face the circulation flow,
The openings may be oriented in a direction deviating from the tangential direction (for example, indicated by arrows D and E).

It is desirable that the opening 4a has a structure in which it is difficult to bring the fluidized state by stirring into the sedimentation separation tube 4.
Further, it is desirable that the microcarriers that have flowed into the settling separation tube 4 are less likely to deposit inside. As such a structure, as shown in FIG. 3, it is preferable that the vicinity of the opening is inclined with respect to the liquid surface of the culture solution.

Further, as shown in FIG. 3 (a), the tube walls facing the opening are formed substantially parallel to each other, or, as shown in FIG. 3 (b).
By adopting a trumpet shape that does not easily generate turbulence or vortex near the opening as described above, it is possible to minimize the carry-in of the flow state of the bulk culture solution.

If the microcarriers do not deposit in the sedimentation separation tube, the inner tube inner diameter may be gradually enlarged with respect to the inner diameter of the opening as shown in FIG. 3 (c). Further, as shown in FIG. 3 (d), one having a separation plate 31 in the opening, and one having a plurality of openings in order to obtain the cross-sectional area required for sedimentation separation as shown in FIG. 3 (e). Etc. can also be used.

The inclination in the vicinity of the opening is preferably an angle along the streamline of the stirring circulation flow, but the elevation angle α of the opening relative to the liquid surface of the culture solution as shown in FIG. 4 is within the range of 5 to 80 °. More preferably. If the elevation angle is less than 5 °, microcarriers and the like that have flowed into the sedimentation separation tube are likely to accumulate,
On the other hand, when the angle exceeds 80 °, the effect of attenuating the disturbance of the culture solution due to stirring decreases.

The cross-sectional shape of the opening is generally circular, but may be elliptical, track field-shaped, rectangular, or the like.

In the sedimentation separation tube shown in FIG. 3, the culture solution supernatant is discharged from the upper end of the sedimentation separation tube. For example, as shown in FIG. 5, the culture solution is discharged from the side of the sedimentation separation tube. It may be a structure for discharging.

By the sedimentation separation tube concretely shown above, a flow path for allowing the culture solution to pass through while allowing the microcarriers and the cells attached to the microcarriers to settle in the culture tank is formed. The opening for introducing the culture solution in this flow channel is directed toward the downstream direction of the circulation flow due to the stirring of the culture solution, and at least one portion of the flow channel is bent.

[0038]

[Function] By forming one or more bent parts in the flow path formed by the sedimentation separation tube, the turbulent flow brought in from the bulk of the culture solution can be rapidly attenuated, and the liquid from the bent part to the upper part can be attenuated. A settling zone without any disturbance can be formed.

Further, since the opening of the sedimentation separation tube is directed to the downstream direction of the circulation flow due to stirring, the circulation flow is prevented from directly entering the sedimentation separation pipe, and the influence of the flow state of the culture solution bulk is affected. To prevent it from being applied inside the sedimentation separation tube.

The greater the number of bent portions, the more completely the liquid turbulence can be attenuated. However, since the internal volume of the culture tank is limited, it is more realistic that the number of bent portions be within three. FIG. 6 shows an example of a compaction settling separation tube. As shown in FIG. 6, forming a flow path by a sedimentation separation tube in a spiral shape is also included in the technical scope of providing one or more bent portions.

The cross-sectional area of the settling zone is designed so that the liquid removal linear velocity of the culture medium does not exceed the terminal velocity of particles of the microcarrier to be separated.

The length of the settling zone is affected by the strength of turbulence of the culture solution due to stirring, the structure of the opening, the number of bent portions, the shape, etc.
The range of 01 to 100 times, particularly 0.1 to 10 times is preferable.

[0043]

EXAMPLES Next, the present invention will be described in more detail by way of examples.

PB in a spinner flask of 1.5 L capacity
Microcarrier 9 swollen in S1.5L and PBS 9
g (CultiSpher-GL / Hyclone Lab.) was added and the microcarrier concentration was adjusted to 6 g / L. Mix gently (5
(0 rpm) to make the microcarriers uniformly dispersed in the flask. A settling separation tube whose shape is shown in FIGS. 3 (a), 3 (b) and 3 (c) is made of glass, and each is set in a PBS solution in a spinner flask so that the opening of the settling separation tube is electrically connected. . The other end of the tube was connected to the liquid inlet nozzle of the spinner flask so that the liquid could be returned to the spinner flask via a peristaltic pump by connecting a tube to the liquid outlet on the top of the settling separation tube.

The PBS supply rate and the drainage rate were 3.6.
ml / min (perfusion speed: PR = 3.
The rotation speed of the peristaltic pump was set so as to be 5). The pump was operated for about 3 hours while adjusting the temperature of the spinner flask to 37 ° C., and the flow state of the PBS solution containing the microcarriers in the sedimentation separation tube was visually confirmed.

The opening of the sedimentation separation tube is opened in the downstream direction of the stirring circulation flow, and the opening is inclined with respect to the liquid surface of the culture solution,
Moreover, when the separation tube of FIG. 3 (a) in which the tube was provided with one bent portion was used, a relatively violent flow state including microcarriers was observed on the culture tank side from the bent portion in the sedimentation separation tube.
A clear settling zone without any microcarriers was formed on the outlet side of the culture tank from the bent portion, and it was confirmed that the sedimentation separation tube according to the present invention has sufficient microcarrier separation performance. In addition, microcarriers were hardly deposited on the inclined portions of the bent portions.
Further, as a result of similarly testing the settling separation tubes having the shapes shown in FIGS. 3 (b) and 3 (c), the same results as in FIG. 3 (a) were obtained.

Next, an actual perfusion culture experiment was carried out using the sedimentation separation tube which gave good results in the above test, and the separation performance of the microcarriers in the actual culture and the cells attached to the microcarriers and the cells Proliferation was examined.

Recombinant CHO cells capable of producing human t-PA (tissue plasminogen activator) were set to 1.5 L, which is a specification capable of carrying out perfusion culture by installing the continuous cell separation device of the present invention. It culture | cultivated using the volume culture tank. The sedimentation separation tube used had the shape shown in FIG.

The continuous supply of fresh medium and the continuous recovery of the culture supernatant are carried out by a peristaltic pump at a predetermined medium supply rate and culture solution recovery rate (perfusion rate = PR). The pump rotation speed was set. For medium, ITES supplemented with Far East Pharmaceuticals e-
The RDF medium was MultiSpher-GL manufactured by Hyclone at a concentration of 6 g / L as a microcarrier.

The culture temperature was 37 ° C. and the stirring speed was 35 r.
pm. The dissolved oxygen concentration of the culture solution was monitored by a DO sensor inserted in the tank in the culture solution, and oxygen was indirectly supplied via a silicon tube so as to prevent oxygen deficiency.

The cell density at the time of seeding was 1.2 × 10 ⁵ cells /
It was ml. At the start of culturing (day 0 to 4), the medium was not exchanged, and the procedure of gradually increasing the perfusion speed from day 4 was taken (day 4 to 6: PR = 0.
5, days 6-8: P.I. R. = 1.0, after day 8 P.I. R. = 1.5) Perfusion culture was performed.

The results of the culture are shown in Table 1. Very good results were obtained with respect to cell proliferation, and 1 × 1 was obtained on the 13th day.
0 was reached ⁷ cells / ml density. Even after that, the same level of cell density could be maintained for 2 weeks or more. During this period, no microcarriers were found to be mixed in the culture medium supernatant collection container, which revealed that the sedimentation separation tube according to the present invention sufficiently exhibited the intended function. In addition, the accumulation of microcarriers at the inclined bent portion did not occur during the main culture period.

The above sedimentation / separation tube is changed depending on the culture conditions such as the cells to be actually cultured and the microcarriers used. The cell culture apparatus of the present invention has the above-described specific shape of the sedimentation / separation tube. However, the present invention is not limited to the example using.

[0054]

[Table 1]

[0055]

[Comparative Example] In the same manner as in the example, a spinner flask having a capacity of 1.5 L was charged with 1.5 L of PBS and 9 g of microcarrier swollen with PBS (CultiSpher-GL / Hyclone Lab.).
Was added to adjust the microcarrier concentration to 6 g / L. The mixture was gently stirred (50 rpm) to make the microcarriers evenly dispersed in the flask.

A settling separation tube of which the shape is shown in FIG. 7 was made of glass, and each was made into PBS in a spinner flask.
It was installed so that the opening of the sedimentation separation tube would be in communication with the liquid. A tube was connected to the liquid outlet on the upper part of the sedimentation separation tube, and the other end of the tube was connected to the liquid inlet nozzle of the spinner flask so that the extracted liquid returned to the spinner flask via a peristaltic pump.

PBS feed rate and drain rate were 3.6
ml / min (perfusion speed: PR = 3.
The rotation speed of the peristaltic pump was set so as to be 5). The pump was operated for about 3 hours while adjusting the temperature of the spinner flask to 37 ° C., and the flow state of the PBS solution containing the microcarriers in the sedimentation separation tube was visually confirmed.

The opening of the settling separation tube does not open in the downstream direction of the stirring circulation flow, and there is no bent portion in the tube.
In, a relatively violent flow state containing microcarriers was observed throughout the sedimentation separation tube, and a clear settling zone was not formed. Although the turbulent velocity of the liquid tends to gradually decrease toward the upper part of the sedimentation separation tube, many microcarriers exist in the liquid extraction tube of the spinner flask, and it functions as a continuous separation device for microcarriers. I couldn't.

The opening of the settling separation tube opens in the downstream direction of the stirring circulation flow, but there is no bend in the tube.
In (b), a slightly clear settling zone could be formed in the sedimentation separation tube, but liquid turbulence could not be sufficiently attenuated, and irregular winding of microcarriers was observed. The amount of microcarriers in the liquid extraction tube of the spinner flask was reduced as compared with the case of using FIG. 7A, but it was not possible to completely separate them.

[0060]

EFFECTS OF THE INVENTION In the cell culture device of the present invention, since the opening of the sedimentation separation tube installed inside the culture tank is opened toward the downstream direction of the stirring circulation flow, the flow state of the culture liquid bulk can be improved. The effect is less likely to be exerted inside the sedimentation separation tube. Further, by forming at least one bent portion in the flow path formed by the sedimentation separation tube, the disturbance of the culture medium containing the microcarriers that has entered the sedimentation separation tube can be rapidly attenuated.

By these two-step actions, it becomes possible to separate the microcarriers and the cells attached to the microcarriers from the culture supernatant, and the culture is substantially free of microcarriers and cells attached to the microcarriers. A liquid supernatant can be obtained.

Since the device of the present invention is not a separation device such as a filter which inevitably causes clogging but a simple tubular structure, it is particularly advantageous in perfusion culture in which high cell density and a long culture period are long. Therefore, the physiologically active substance and the like can be efficiently produced.

[Brief description of drawings]

FIG. 1 is a schematic view showing an example of a cell culture device according to the present invention.

FIG. 2 is a schematic view showing a direction in which an opening of a sedimentation separation tube is provided in the present invention.

FIG. 3 is a schematic view showing a specific example of a sedimentation separation tube used in the cell culture device of the present invention.

FIG. 4 is a schematic view showing a sedimentation separation tube in which an opening portion is inclined at a predetermined angle α with respect to a culture liquid surface in the present invention.

FIG. 5 is a schematic view showing an example of a sedimentation separation tube for discharging a culture solution from the side portion of the tube in the present invention.

FIG. 6 is a schematic view showing an example of a sedimentation separation tube in which a culture fluid channel has a spiral shape in the present invention.

FIG. 7 is a schematic view of a sedimentation separation tube used as a comparative example.

[Explanation of symbols]

1. Culture tank 2. Stirrer 3. Agitation rotary shaft 4. Settling Separation Tube 4a. Opening 4b. Bent portion 4c. Discharge port 5. Culture solution 6. Recovery pipe 7a. Pump 7b. Pump 8a. Container 8b.
Container 9. Piping 10. Exhaust pipe 11. Temperature control jacket 12. Motor 13. Gas supply pipe 20. Streamline 31. Separation plate

Claims (1)

[Claims] 1. In a system in which cells are adhered onto a microcarrier and stirred and cultured in a cell culture tank, a metabolite of the cells is metabolized while continuously supplying a fresh culture solution to the cell culture tank. In a cell culture device for discharging the accumulated culture solution to the outside of the cell culture tank and collecting the cell culture tank, a settling separation tube for continuously separating microcarriers and cells attached to the microcarriers and culture solution supernatant is provided in the cell culture tank. And a channel for allowing the culture solution to pass through while allowing the microcarriers and the cells attached to the microcarriers to settle in the cell culture tank is formed by the sedimentation separation tube, and an opening for introducing the culture solution in the flow channel The part is directed in the downstream direction of the circulation flow due to stirring of the culture solution, and at least one part of the flow path is bent.
Cell culture device.