JPH0734736B2 - Incubator - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Field of Industrial Application The present invention relates to a culture device. More specifically, it relates to a culture device suitable for high-density and continuous culture.

(B) Conventional technology When a large amount of conventional animal cells are to be cultured, in order to efficiently culture them with a small device, a medium containing a large amount of waste products is continuously, rapidly and reliably recovered from the culture solution. Therefore, a culture device equipped with a culture medium exchange means is used. Particularly in the case of floating animal cells, a culture device in which a culture solution exchange means is attached to a stirring-type culture tank is often used (Sugino Yukio, "Cell Culture Technology," Kodansha, 1985, p146-150. ). The culture medium exchange means separates cells and medium from each other such as a cell settling tube provided in the culture tank, a filtration means provided in the culture tank or a culture solution circulation path attached to the culture tank, and the like. This means a combination of means for discharging the separated medium to the outside of the culture tank and means for supplying the fresh medium to the culture tank.

(C) Problem to be Solved by the Invention When cells are continuously cultured, dead cells solidify to form a cell mass, which is more likely to sediment than a living cell. Therefore, when culturing with a culturing device having a cell settling tube, the above-mentioned cell clusters are less likely to be discharged to the outside of the culture tank than living cells, so that the cell clusters are gradually accumulated in the culture tank, which is an unfavorable environment for the cells. Becomes
Further, when continuously culturing in a culture device having a filtration means in a culture solution circulation path attached to the culture tank or attached to the culture tank, clogging of the filtration means becomes faster due to the cell mass, and the medium Cannot be replaced sufficiently.

The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a culture device having a unit capable of removing cell clusters from a culture medium and configured to perform stable continuous culture.

(D) Means for Solving the Problems Thus, according to the present invention, a culture system for circulating or storing a culture solution containing cells and the like to increase the number of cells and the like, and a liquid medium in the culture system are continuously A liquid-replenishment / separation means that can be exchanged with a fresh medium is provided in the culture system, and a bypass flow path that can temporarily bypass the culture solution to the outside of the system is provided in the culture system, and cells in the culture solution are provided in the bypass flow path. A tubular body having a built-in filter that does not allow the passage of cell masses and the like, but the washing solution supply means is connected to the downstream side of the tubular body divided by the filter, and the upstream side of the tubular body is connected. There is provided a culturing device in which a cell mass removing part configured by connecting a drainage line to a drain is inserted.

The present invention is characterized in that it is a culture device configured so as to be able to efficiently remove cell aggregates generated in the culture medium as needed without stopping the culture operation.

The culturing apparatus of the present invention is suitable for continuous and high-density culturing, and can also be applied to a bioreactor.

In the culture device of the present invention, a culture system in which a culture solution containing cells or the like is circulated or stored to grow cells or the like, and the culture solution can be circulated or stored, and a stirring means, a liquid level sensor, and a pH meter. It means a culture tank known in the art, which is equipped with necessary members for isoculturing as necessary, or a culture tank having a function equivalent thereto. Further, as the liquid-supplying / separating means provided in the culture apparatus of the present invention, known ones, for example, a cell settling tube provided in the culture tank, a culture solution circulation passage provided in the culture tank or attached to the culture tank It is possible to use, as it is, a means for separating cells and a liquid medium and discharging the separated medium to the outside of the culture tank, such as a provided filtration part, and a fresh medium supplying means.

The culture solution bypass flow path provided in the culture system in the culture apparatus of the present invention is configured so that the temporary culture solution can be bypassed to the outside of the culture system. The culture medium can be circulated between the two. This bypass flow path may be provided directly in the culture tank, or may be provided in the separation means of the liquid-supply / separation means. In the latter case, when the separation means side is composed of a culture solution circulation passage attached to the culture tank having a filtration portion, the bypass flow passage is provided in a stage before the filtration portion of the culture fluid circulation passage. Also, the flow path switching means between the bypass flow path and the culture solution circulation path is set. At this time, the liquid sending means set in the bypass flow path may be the same as the liquid sending means set in the culture solution circulation path. For an example of this configuration, the description of the examples described later is referred to.

A cell mass removing means is provided in the culture medium bypass flow path.
The cell clump removing means has a tubular body having liquid inlets and liquid outlets that can be connected to the bypass passage on opposite end surfaces, a filter provided over a cross section inside the tubular body, It is mainly composed of a cleaning liquid supply means which is divided by a filter and is connected to a downstream side of the tubular body by a pipe line, and a drainage line which is connected to an upstream side of the tubular body and is connected to a drain. As the filter, a mesh-like filter is selected so that cells and the like in the culture medium can be transmitted but cell aggregates and the like cannot be transmitted. About 0.5 to 2 mm is suitable for the pore size of this mesh.
The material of the tubular body is preferably one that allows the inside of the tubular body to be seen through from the outside, and glass or transparent resin is suitable for this, while stainless steel or synthetic resin is selected as the material of the filter, and synthetic resin For this, nylon is preferred. It is preferable that the cleaning liquid supply means connected to the downstream side of the tubular body by a pipe line is connected to a cleaning liquid storage tank via a liquid feed pump. On the other hand, a flow path opening / closing valve is provided in the discharge conduit connected to the upstream side of the tubular body.

In the device of the present invention, the timing of the transfer of the culture solution to the bypass flow path may be manually performed based on the state of generation of cell clumps in the culture tank based on visual observation, and at predetermined time intervals. The switching operation may be controlled by a program preset so as to be mechanically operable. Also,
The timing of supplying the washing solution to the cell clump removing means inserted in the bypass channel may be manually performed according to the accumulation state of the cell clumps in the tubular body based on visual observation, and the machine may be operated at predetermined intervals. The switching operation may be controlled by a preset program so that the switching operation can be performed automatically. Further, as another configuration related to this timing, a pressure gauge is attached to each of the upstream flow passage and the downstream flow passage of the filter built in the removal unit, and the clogging state of the filter is automatically adjusted by the pressure fluctuations of both. It is also possible to adopt a configuration in which it is performed by sensing.

(E) Action According to the present invention, the culture solution containing the proliferating cells, the cell mass, the liquid medium and the like from the culture system and bypassed to the outside of the culture system,
In the filter of the cell mass removal section provided in the middle of the bypass flow path, only the cell slugs are separated and then returned to the culture system. When the separated cell droplets in the filter accumulate to some extent, the transfer of the culture solution from the culture system to the bypass flow path is stopped, and the cleaning solution is supplied to the downstream side in the removal section and further the cleaning solution passes through the filter. And flows back to the upstream side, and is discharged to the drain through the discharge pipe together with the cell mass accumulated in the upstream portion.

Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited thereto.

(F) Embodiment FIG. 1 is a structural explanatory view of an embodiment of the culture apparatus of the present invention. In the figure, the culture device (1) includes a culture tank (2), a stirring means (3), a fresh medium supply means (4), a culture medium filtration means (5), a cell clump removal means (6), and a control section (7). ) Is mainly composed of.

The culture tank (2) is composed of a culture solution storage tank and a lid capable of detachably sealing the culture solution storage tank. A liquid level sensor (21), a stirring blade (31), a pH electrode (not shown), a DO sensor (not shown), and pipes (described later) are set at predetermined positions on the lid. In addition, a temperature controller (not shown) is attached to this culture tank so that the temperature of the culture solution can be kept constant at a predetermined temperature.

The stirring means (3) is composed of the stirring blade (31) and a rotation drive unit (not shown) for rotating the stirring blade (31).

The fresh medium supply means (4) is opened in the medium tank (41) for storing the fresh medium and in the vicinity of the bottom of the tank, and is connected to the lid of the culture tank (2) via the peristaltic pump (42).
It is composed of a medium supply pipe (a) that opens to the upper space in the culture tank.

The culture solution filtration means (5) is attached to the lid by opening in the vicinity of the bottom of the culture tank, and is connected to the upper end of the filtration section (53) through the peristaltic pump (51) and the solenoid valve (52) in this order. pumping conduit is road connected (b ₁₎ and,該Ro over section (53) return line to be set again opened in the upper space in the culture tank is connected to the lid from the lower end (b ₂₎ And a recovery pipe line (b ₃ ) connected to the recovery tank (55) from the lower part of the side surface of the filtration part (53) through the peristaltic pump (54). The pumping-out conduit (b ₁ ) and the returning conduit (b ₂ ) form a culture solution circulation path (b). Above filter (5
3) includes a filter pipe having a cylindrical filter surface and set coaxially with the flow passage, and an outer cylindrical container for temporarily storing the permeated liquid from the inside of the filter pipe outside the filter pipe. A cross-flow filter device composed of and is used. As the filter pore diameter of the above-mentioned filter surface, a pore diameter (for example, about 0.2 to 10 μm) that does not permeate cells or the like but permeates dissolved components in the culture medium is used.

The cell clump removing means (6) is connected to the upstream side of the solenoid valve (52) of the pumping-out pipeline (b ₁ ) of the culture solution filtration means (5) by a solenoid valve (61) and a solenoid valve ( 62) through the pumping line (b ₁ ) again in this order, and a bypass line (c) connected to the downstream side of the solenoid valve (52) of the pipe line (b ₁ ) and the bypass line (c). A cell clump removal unit (63), a wash solution supply conduit (d ₁ ) and a cell clump removal unit connected to the downstream side of the cell clump removal unit (63) from the wash solution tank (64) via a peristaltic pump (65). The cell clump removing section (68) is composed of a recovery conduit (d ₂ ) connected to the washing liquid recovery tank (67) via the solenoid valve (66) from the upstream side of (63).

An example of the structure of the cell mass removal unit (63) is shown in FIG. As shown in the figure, the cell mass removing unit (63) has a conduit (e) connectable to the bypass conduit (c) on one end surface and an edge portion whose other end is expanded outward. A mesh filter (9) consisting of a pair of open cylindrical container bodies (8) and having their opening end face peripheral edges opposed to each other is provided with an O-ring (10) (1) from both sides of the mesh filter (9).
The tightly sandwiched state is held by the retaining bands (12) straddled along the outer peripheries of the pair of opposed opening peripheral edges while being closely sandwiched via 1). In the cell mass removal unit (63) configured as described above, the cylindrical container body (hereinafter referred to as the liquid introduction side portion (a)) and the bypass conduit (c) connected to the upstream side of the bypass conduit (c). Cylindrical container body connected to the downstream side (hereinafter referred to as liquid discharge side portion (b))
Communication holes (81) (82) are set in each. Of these, the conduit (d ₁ ) of the cleaning liquid backflow means (68) is connected to the communication hole (82) of the liquid discharge side (b), and the communication hole (81) of the one liquid introduction side (a) is connected. Is connected to the conduit (d ₂ ) of the washing liquid backflow means (68). The mesh filter (9) is made of stainless steel having a mesh of 2 mm, and the cylindrical container body (8), that is, the liquid introduction side portion (a) and the liquid discharge side portion (b) are made of glass.

The control section (7) includes a liquid level sensor (21), a stirring means (3),
pH electrode, DO sensor, solenoid valve (52), (61), (62),
(66), Peristaltic pumps (42), (51), (54), (6
5) each of which is electrically connected and has a program capable of maintaining them at preset set values or driving them at preset timings, and is configured to instruct each of the above parts to operate according to this program. There is.

The operation of the culture device (1) configured as above will be described.

First, when conducting normal continuous culture, the solenoid valve (52) is opened and the solenoid valves (61) (62) and (66) are closed. In this state, when the peristaltic pumps (51) and (54) are driven at preset speeds, respectively, the culture solution in the culture tank (2) is delivered at a predetermined amount, and the culture tank (2) → pipe line (b) ₁ ) → filtration section (53) → pipe (b ₂ ) → culture tank (2). By this operation, the cells and the like in the culture solution are returned to the culture tank (2) without being filtered in the filtration section (53) set on the circulation path, while the dissolved components in the culture solution are permeated and collected. It is recovered in the recovery tank (55) through the pipeline (b ₃ ). Due to this circulation operation, cells and the like are proliferated in the culture medium, and waste products and the like that are generated by the proliferation and dissolved in the culture medium are removed from the filtration part (53), and the liquid level in the culture tank is lowered. When the liquid level is detected by the liquid level sensor (21), the control section (7) operates the peristaltic pump (42) for a predetermined time based on the output from the sensor (21). It is driven at a speed, which replenishes a predetermined amount of fresh medium. In this way, the culture / proliferation is continuously performed.

When the above operating state progresses to some extent, cell clumps start to form in the culture solution, but when this is noticeable, switching based on visual observation or output of a timer (setting from the start of culture until the elapse of a predetermined time) set in the control unit The solenoid valves (61) and (62) are opened by switching based on the one-way solenoid valve (52).
Close. In this state, the culture fluid pumped up by the peristaltic pump (51) is the solenoid valve (52) of the conduit (b ₁ ).
From the upstream part of the outlet to the bypass line (c) and the solenoid valve (61)
→ flowed into the downstream portion of the re conduit bypassing the order of cell mass removal unit (63) → solenoid valve (62) (b ₁₎ solenoid valve (52), are the as circulation below. By this bypass circulation, the cell mass in the culture medium is separated into the liquid introduction part side (a) in the cell mass removal unit (63), while the cells to be proliferated are reticulated in the unit (63) ( 9) and returned to the culture tank (2). As the bypassed circulation progresses, the separated cell mass begins to accumulate on the liquid introduction part side (a) of the cell mass removal unit (63) based on visual observation or within the liquid introduction part side (a) and liquid discharge. When the solenoid valve (52) is opened and the solenoid valves (61) and (63) are closed by a command from the control unit based on the comparative pressure value of each pressure sensor that is set to measure the pressure in side (b). At the same time, the solenoid valve (66) is opened to drive the peristaltic pump (65). By this operation, the culture solution circulates again in the culture solution circulation path as described above, and at the same time, the washing solution tank (6) is added to the cell mass removal unit (63).
4) The washing solution (in this case, fresh medium or isotonic buffer solution equivalent to the medium tank (41)) is connected to the communication hole (82) → liquid discharge side (b) → mesh filter (9) → liquid introduction side. (B) →
It flows backward in the order of the communication hole (81) and is collected in the cleaning liquid collection tank (67) through the solenoid valve (66). The cell mass accumulated in the reticulated filter (9) and the liquid introduction side (a) by this washing liquid backflow operation is removed from these by the washing liquid flow and discharged into the recovery tank (67), and the cell mass is cultured system. Will be removed to the outside. After that, the peristaltic pump (65) is stopped and the solenoid valve (66) is closed by the visual judgment or the automatic operation after a lapse of a certain time to terminate the washing liquid backflow operation, and the normal continuous culture state is again established.

Therefore, by repeating the above operation, the filter section (53)
It is possible to carry out stable continuous culture for a long period of time with less clogging at.

(G) Effect of the Invention According to the present invention, it is possible to efficiently remove cell clumps generated in the culture medium, and to reduce the number of inconvenient states such as clogging at the filtration part of the culture medium circulation passage attached to the culture tank. Since the liquid can be kept in a preferable state for culture, stable cell culture can be continuously performed for a long time.

[Brief description of drawings]

FIG. 1 is a structural explanatory view of an embodiment of the culture apparatus of the present invention,
FIG. 2 is a structural explanatory view of an example of a cell mass removal unit used in the culture apparatus of FIG. (2) ... culture tank, (3) ... stirring means, (4) ... fresh medium supplying means, (5) ... culture solution filtering means, (6) ... cell mass removing means, (7) ...... Control part, (8) ...... Cylindrical container body, (9) ...... Mesh filter, (10) (11) …… O ring, (12) …… Stop band, (21) …… Liquid level sensor , (31) …… stirring blade, (41) …… medium tank, (42) (51) (54) (65) …… perista pump, (52) (61) (62) (66) …… solenoid valve, (53) …… Filter part, (55) …… Recovery tank, (63) …… Cell clump removal unit, (64) …… Wash solution tank, (67) …… Wash solution recovery tank, (68) …… Wash solution reflux means, (a) ...... medium supply line, (b ₁₎ ...... pumping conduit, (b ₂₎ ...... return line, (c) ...... bypass conduit.

Claims (1)

[Claims] 1. A culture system in which a culture medium containing cells and the like is circulated or stored to grow cells and the like, and a liquid medium in the culture system that can continuously replace a fresh medium-supplementation / separation The above culture system is provided with a bypass flow path that allows the culture solution to be temporarily bypassed to the outside of the system, and the cells and the like in the culture solution can pass through the bypass flow path, but the cell mass and the like cannot pass through. It is composed of a tubular body with a built-in filter, the cleaning liquid supply means is connected to the downstream side of the tubular body divided by the filter, and the drainage line to the drain is connected to the upstream side of the tubular body. A culture device in which a cell mass removal unit is inserted.