JPH01206988A - Culture apparatus - Google Patents

Abstract

PURPOSE:To provide the title apparatus so designed that a bypass channel for temporarily bypassing a culture solution externally is provided and fitted with a cylinder with a special filter included, thereby efficiently taking out cell aggregates consisting mainly of dead cells from said solution. CONSTITUTION:In a culture apparatus designed to perform culture by making cells float on a liquid culture medium, a culture tank 2 is connected to a fresh culture medium feed means 4 and provided with a circulation system comprising a drawing pipe b1 for culture solution, a specific pump 51, a valve 52, a filter 53 and a feedback pipe b2. The filter 53 is fitted with a filter impermeable for cells to return them to the tank 2, and the dissolved components in the culture solution are put into a withdrawal vessel 55 either singly or together with said solution. Said circulation system is provided with a bypass channel C having such a structure that a valve 61, a filter 63 and another valve 62 are connected in this order from either side of the valve 52 through a pipe channel. The filter 63 is fitted with a filter impermeable for cell aggregates, and when cell aggregates are accumulated to some extent in its upstream side, the valves 61, 62 are closed to subject a washing liquid to back-flow fro a vessel 64 to the filter 63, thus collecting said aggregates together with the washing liquid into a vessel 67.

Description

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

(B) Conventional technology Conventionally, when attempting to culture animal cells in large quantities, in order to efficiently culture them in a small device, it is necessary to continuously, rapidly, and reliably collect the medium containing many waste products from the culture solution. Therefore, a culture device equipped with a means for exchanging the culture medium is used.

Especially in the case of planktonic animal cells, use a stirred culture medium! ! A culture device with a culture medium exchange means attached to the tank is often used (Yukio Sugino, ed., RM culture technology, Kodansha,
1985, p146-150). The above-mentioned culture solution exchange means include a cell sedimentation tube provided in the culture tank, a port passage means provided in the culture tank or a culture solution circulation path attached to the culture tank, etc. This refers to a combination of means for discharging the separated culture medium out of the culture tank and means for supplying fresh culture medium to the culture tank.

(c) Problems to be Solved by the Invention When cells are cultured continuously, dead cells eventually solidify and form cell clusters, but these cell clusters tend to settle more easily than living cells. Therefore, when culturing is carried out in a culture device with a cell sedimentation tube, the above-mentioned cell clusters are more likely to be cultured than living cells. I
Since it is difficult to discharge the cells to the outside of the tank, cell clumps gradually accumulate in the culture tank, creating an unfavorable environment for the cells. Furthermore, when culturing is carried out continuously in a culture device having a passage means in a culture tank or a culture solution circulation path attached to a culture tank, the passage means is quickly clogged by the cell mass, and the culture medium is replacement cannot be carried out adequately.

The present invention has been made in view of the above situation, and it is an object of the present invention to provide a culture device that is configured to have a means for removing cell clusters from the culture solution and to perform stable continuous culture.

(d) Means for Solving the Problems Thus, according to the present invention, there is provided a culture system in which a culture solution containing cells, etc. is circulated or stored to grow cells, etc., and a liquid medium in the culture system is continuously maintained. The culture system is equipped with a liquid replenishment/separation means that can be exchanged with a fresh medium, and the culture system is provided with a detour flow path through which the culture medium can be temporarily bypassed outside the system, and the detour flow path is provided with a means for replenishing and separating the culture medium. A cleaning liquid supply means is connected to the downstream side of the cylindrical body divided by the filter, and a cleaning liquid supply means is connected to the upstream side of the cylindrical body. There should be provided a culture device in which a cell mass removal section is inserted on the side of the cell clump, which is configured by connecting a discharge pipe to a drain.

The present invention is characterized in that it is a culture apparatus configured to be able to efficiently remove cell clusters generated in a culture solution as needed without stopping the culture operation.

The culture device of the present invention is suitable for continuous and high-density culture, and can also be applied to bioreactors.

In the culture apparatus of the present invention, a culture system that circulates or stores a culture solution containing cells, etc. to grow cells, etc. is a culture system that can circulate or store the culture solution, and includes a stirring means, a liquid level sensor, and a pt. - A culture flF known in the field that is equipped with necessary parts for culture such as an i-meter! Or means a culture tank with equivalent functions. In addition, as the liquid replenishment/separation means included in the culture apparatus of the present invention, known ones may be used, such as a cell sedimentation tube provided in the culture tank, and a culture liquid circulation path attached to the culture tank or culture tank. Separate the cells from the liquid medium using a mouth opening, etc., and culture this separated medium! ! means for discharging outside the tank;
A means for supplying a fresh culture medium can be used as is.

In the culture device of the present invention, the culture solution bypass flow path provided in the culture system is configured to temporarily bypass the culture solution to outside the culture system, and is configured to temporarily bypass the culture solution to the inside and outside of the culture system through the liquid feeding means. The structure is such that the culture solution can be circulated between the This detour channel may be provided directly in the culture tank, or may be provided in the separation means of the liquid replenishment/separation means. In the latter case, when the separation means side is configured with a culture solution circulation path attached to the culture tank with an opening section, the detour channel is provided before the opening section of the culture solution circulation path. and a flow path switching means between the detour flow path and the culture solution circulation path is set. Note that at this time, the liquid feeding means set in the detour channel may be the same as the liquid feeding means set in the culture solution circulation path. For an example of this configuration, refer to the description of the embodiment described later.

A cell mass removing means is provided in the culture solution detour channel.

This cell mass removal means includes a cylindrical body having a liquid inlet port and a liquid discharge port on opposite end faces, which can be connected to the bypass flow path, and a filter provided over a cross section inside the cylindrical body. It mainly consists of a cleaning liquid supply means connected to the downstream side of the cylindrical body divided by the filter, and a discharge conduit to a drain connected to the downstream side of the cylindrical body. The filter is selected to have a mesh shape that allows it to pass through cells and the like in the culture medium but not through cell clusters and the like. A suitable pore size for this mesh is approximately 0.5 to 2 mI+1. The material of the cylindrical body is preferably one that allows the inside to be seen through from the outside of the cylindrical body, and glass or transparent resin is suitable for this, while the material of the filter is selected from stainless steel, synthetic resin, and synthetic resin. Nylon is preferred as the resin. It is preferable that the cleaning liquid supply means connected to the downstream side of the cylindrical body by a pipeline is constituted by a pipeline connected to the cleaning liquid storage tank via a liquid feeding pump.

On the other hand, a flow path opening/closing valve is provided in the discharge pipe connected to the upstream side of the cylindrical body.

In the apparatus of the present invention, the timing of transferring the culture medium to the detour channel may be manually based on the state of cell mass formation in the culture tank based on visual observation, or may be carried out at predetermined intervals. The switching operation may be controlled by a preset program that can be mechanically operated. Furthermore, the timing of supplying the cleaning liquid to the cell mass removal means inserted in the detour channel may be performed manually depending on the state of accumulation of cell mass inside the cylindrical body based on visual observation, and the cleaning solution may be supplied at predetermined intervals. The switching operation may be controlled by a preset program that can be mechanically operated. Furthermore, as another configuration regarding this timing, a pressure gauge is attached to each of the upstream flow path and the downstream flow path of the filter built in the removal section, and the clogging state of the filter is automatically detected by pressure fluctuations in both. It may also be configured to perform this by sensing.

(E) Effect According to the present invention, the culture solution containing proliferating cells, cell masses, liquid medium, etc. and bypassed from the culture system to the outside of the culture system is
After only the cell aggregates are filtered through the filter of the cell aggregate removal section provided in the middle of the detour channel, they are returned to the culture system. When the cell arms harvested in the filter accumulate to a certain extent, the transfer of the culture solution from the culture system to the detour channel is stopped, and a washing solution is supplied to the downstream side of this removal section, and this washing solution is further passed through the filter. The cell mass is then flowed back upstream, and is discharged to the drain through the discharge pipe along with the cell mass accumulated in this upstream section.

The present invention will be described in detail below with reference to Examples, but the present invention is not limited thereby.

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

The culture tank (2) is composed of a culture solution storage tank and a lid that can removably seal the tank. A liquid level sensor (21), a stirring blade (31), a pH1 pole (not shown), a Do sensor (not shown), and each pipe line described later are set at predetermined positions on the lid.

This cultivation again! A temperature controller (not shown) is attached to the 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) that rotates the stirring blade (31).

The fresh medium supply means (4) opens near the bottom of a medium tank (41) that stores fresh medium, and is connected to the lid of the culture 111 (2) via a peristaltic pump (42), and Culture medium supply pipe opening into the upper space in the tank (
It consists of a).

The culture liquid passage means (5) opens near the bottom of the culture tank and is attached to the lid, and connects a pipe to the upper end of the mouth passage part (53) through a peristaltic pump (51) and a solenoid valve (52) in this order. The pumping pipe (b,) connected to the pipe and the mouth part (5)
3) A return pipe (b,) which is connected to the lid again from the lower end and opened to the upper space in the culture tank, and a peristaltic pump (54) from the lower side of the mouth part (53). A recovery pipe (b) is connected to the recovery tank (55).
3) and (Y). The above pumping pipe (bυ
The culture solution circulation path (b) is constituted by the return pipe (b,) and the return pipe (b,). The mouth passage part (53) includes a filter pipe having a cylindrical filter surface and set coaxially with the flow path, and a permeate from inside the filter pipe is temporarily stored on the outside of this filter pipe. A cross-flow type gating device is used, which consists of an outer cylindrical container. The filter pore size of the filter surface used is a pore size (for example, about 0.2 to 10 μm) that does not allow cells to pass through, but allows dissolved components in the culture solution to pass through.

The cell mass removing means (6) is connected to the upstream side of the solenoid valve (52) of the pumping conduit (bl) of the culture liquid filtration means (5), and includes II solenoid valve 61) and II&! Valve (62)
In this order, pump out again through the solenoid valve (b,) of the pipe (b,).
52), and a cell mass removal unit (6) inserted into this detour pipe (e).
3) and the peristaltic pump (6) from the cleaning liquid tank (64).
5) to the washing liquid supply pipe (DL) connected to the downstream side of the cell lump removal unit (63) and the washing liquid from the upstream side of the cell lump removal unit (63) via the solenoid valve (66). Recovery pipe (67) connected to recovery tank (67)
d2) and a cell mass removing section (68).

An example of the configuration of the cell mass removal unit (63) is shown in FIG. 2. As shown in the figure, this cell mass removal unit (63) has a conduit (e) connectable to the detour conduit (c) on one end surface, and the other end has an outwardly expanded edge. Consisting of a pair of opened cylindrical container bodies (8), these openings d
A mesh filter (9) having a predetermined mesh between the edges of the end faces facing each other is inserted into an O-ring (10) from both sides.
(11), and is held by a retaining band (12) that extends along the outer periphery of the pair of opposing opening peripheries. Cell mass removal unit (63
), 1, a cylindrical container body (hereinafter referred to as liquid introduction side part (a)) connected to the upstream side of the detour pipe (c) and a detour pipe (
Communication holes (81) and (82) are set in each of the cylindrical container bodies (hereinafter referred to as liquid discharge side parts (b)) connected to the downstream side of c). Of these, the communication hole (82) on the liquid discharge side (b)
) is connected to the pipe line (dυ) of the cleaning liquid backflow means (68), and the communication hole (81) of the negative liquid introduction side (a) is connected to the pipe line (d,) of the cleaning liquid backflow means (68). The mesh filter (9) is made of stainless steel with a mesh of 2m+++, and the cylindrical container body (8), that is, the liquid introduction side (a) and the liquid discharge side (b) are made of glass. belongs to.

The control section (7) includes a liquid level sensor (21) and a stirring means (3).
, pH electrode, DO sensor, solenoid valve (52), (61),
(62).

(66), peristaltic pump (42), (51), (54
) and (65), and has a program that can maintain these at preset values or drive them at preset timing, and instructs each of the above parts to operate according to this program. It is configured.

The operation of the culture apparatus (1) configured as above will be explained.

First, when performing 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 respective preset speeds, the culture medium II tank (
The culture solution in 2) is fed at a predetermined amount, and the culture +! (2)
→It is circulated through the pipe (b,) - the port (53) - the pipe (b,) - the culture tank (2). Through this operation, cells in the culture solution are returned to the culture 1 (2) without being passed through the mouth passage part (53) set on the circulation path, while dissolved components in the culture solution are permeated and collected. It is collected into the collection tank (55) through the pipe (b,). Due to this circulation operation, cells, etc. are multiplied in the culture medium, and waste products, etc., generated along with this multiplication and dissolved in the culture medium are removed from the mouth passage (53), and the liquid level in the culture tank is lowered. However, when this liquid level is detected by the liquid level sensor (21), the control unit (
7) drives the peristaltic pump (42) at a predetermined speed for a predetermined time based on the output from this sensor (21), thereby replenishing a predetermined amount of fresh culture medium. Cultivation and proliferation are performed continuously in this manner.

When the above operating state progresses to a certain extent, cell clusters will begin to form in the culture solution, but when this becomes noticeable, the switch should be made based on visual inspection, or the output of a timer (set from the start of culture until after a predetermined time has elapsed) set in the control unit. By switching based on
52) Close. In this state, the peristaltic pump (5
The culture solution pumped up by 1) flows out from the upstream part of the solenoid valve (52) of the conduit (bl) to the detour conduit (c), and is passed through the solenoid valve (61) - cell mass removal unit (63) - solenoid valve. The solenoid valve (5) of the pipe (bl) is detoured again in the order of (62).
2) and then circulated as described above. Due to this detour circulation, cell aggregates in the culture solution are transferred to the liquid introduction part side (a) in the cell aggregate removal unit (63), while cells to be proliferated are transferred to the reticular filter (a) in the unit (63). 9) and returned to the 1% culture medium (2).

As the detour circulation progresses, the cell mass removal unit) (6
3) When the cell mass collected on the liquid inlet side (a) begins to accumulate, the pressure in the liquid inlet side (a) and the liquid outlet side (b) can be measured based on visual observation or the pressure inside the liquid inlet side (a) and the liquid outlet side (b). Based on the comparative pressure value of each pressure sensor that has been set, the solenoid valve (52) is opened by a command from the control unit and the solenoid valves (61) and (63) are opened.
) and at the same time open the solenoid valve (66) to drive the peristaltic pump (65). By this operation, the culture solution is circulated through the culture solution circulation path again as in the beginning, and at the same time, the cell mass removal unit (63) is transferred from the washing solution tank (64) to the washing solution (in this case, a fresh medium equivalent to the medium tank (41) or the like). (buffer solution) from the communication hole (82) to the liquid discharge side (
(b) - The liquid flows backward in the order of the mesh filter (9) -> the liquid introduction side part (a) -> the communication hole (81), and is collected in the cleaning liquid recovery tank (67) via the electromagnetic valve (66). Due to this washing liquid backflow operation, the cell clumps accumulated in the mesh filter (9) and the liquid introduction side part (a) are removed from them by this washing liquid flow and discharged into the recovery tank (67), and the cell clumps are removed from the culture system. It will be removed outside. Thereafter, by visual judgment or automatic operation after a certain period of time has elapsed, the peristaltic pump (65) is stopped and the solenoid valve (66) is closed to complete the washing liquid backflow operation and return to the normal continuous culture state.

Therefore, by repeating the above operation, the opening part (53
) with little clogging, allowing for stable and continuous culture over a long period of time.

(G) Effects of the Invention According to the present invention, cell clusters generated in the culture solution can be efficiently removed, and inconvenient conditions such as clogging at the opening of the culture solution circulation path attached to the culture tank can be minimized. Since the liquid can be maintained in a state suitable for culture, stable cell culture can be carried out continuously over a long period of time.

[Brief explanation of the drawing]

FIG. 1 is an explanatory diagram of the configuration of an embodiment of the culture device of the present invention;
FIG. 2 is an explanatory diagram of the configuration of an example of a cell mass removal unit used in the culture apparatus of FIG. 1. (2)・・・・・・Culture tank, (3)・・・・・・
Stirring means, (4) Fresh medium supply means, (5
)・・・・・・Culture solution passing means, (6)・・・Cell mass removal means, (7)・・・Control unit, (
8)...Cylindrical container body, (9)...Mesh filter, (loXtt)...0 ring,
(12)...Stopping band, (21)...Liquid level sensor, (3[)...
... Stirring blade, (41) ... Culture medium tank, (42X51X54) (65) ... Peristaltic pump, (52) (6106206B) ... Solenoid valve, (53) ...・・・・・・Last part of the mouth," (55)...
...Recovery tank, (63) ...Cell mass removal unit, (64) ...Washing liquid tank, (67) ...Washing liquid recovery tank, (68)・
... Washing liquid backflow means, (a) ... Culture medium supply pipe line, (b+) ... Pumping pipe line, (b, ).
...Return pipe line, (c)...Detour pipe line. Figure 1 Figure 2

Claims (1)

[Scope of Claims] 1. A culture system in which cells, etc. are grown by circulating or storing a culture medium containing cells, etc., and a liquid medium in which the liquid medium in the culture system can be continuously replaced with fresh medium. The culture system is provided with a replenishment/separation means, and the culture system is provided with a bypass flow path that allows the culture solution to temporarily bypass the system, and the bypass flow path is provided with a bypass flow path that allows cells, etc. in the culture solution to pass through, but not cell clusters, etc. A cleaning liquid supply means is connected to the downstream side of the cylindrical body divided by the filter, and a discharge conduit to the drain is connected to the upstream side of the cylindrical body. A culture device in which a cell mass removal section is inserted.