JP6423942B1 - Microcarrier stirring culture device - Google Patents

Abstract

A stirring culture apparatus using a microcarrier that eliminates the need for backwashing a filtration membrane separately.
A microcarrier agitation culture apparatus according to the present invention includes a culture container that contains a microcarrier, a culture, and a medium, and one culture zone and the other zone that are provided in the culture container. A filtration membrane that does not pass through the microcarrier but passes through the culture and the medium, a drive shaft that is provided in the culture vessel and reciprocates through the filtration membrane, and the one One stirring blade fixed to the drive shaft portion in the zone, the other stirring blade fixed to the drive shaft portion in the other zone, and the contents in the one zone are stored in the culture vessel. It is characterized by comprising discharge means for discharging to the outside and supply means for supplying contents such as a culture, a culture medium and a microcarrier into the other zone.
[Selection] Figure 1

Description

  The present invention particularly relates to a stirring culture apparatus for culturing cells and the like using microcarriers.

  In a culture vessel, iPS cells, animals and plants using a microparticle carrier having a culture surface that allows cells to adhere to and grow inside the culture vessel, for example, about 100 to 300 μm in size. There is a culture apparatus for efficiently culturing cultures such as cells and cells.

  In order to extract a culture such as cells cultured in the culture container to the outside of the culture container, conventionally, the culture container is provided with a discharge pipe and a discharge pump, and the microcarrier is provided in the discharge pipe. Using a culture apparatus provided with a filtration membrane (filter) that does not pass through the culture medium such as cells and culture medium such as a culture solution, and negative pressure is applied to the inside of the discharge pipe by the discharge pump (or culture) Pressurize the inside of the vessel), introduce the contents in the culture vessel into the discharge tube, and draw only the culture and medium other than the microcarrier out of the culture vessel by the filtration membrane. Yes.

  For example, Patent Literature 1 discloses a culture apparatus in which a filtration membrane is provided on a discharge tube of a culture container.

Japanese Unexamined Patent Publication No. Hei 8-172956

  However, in the conventional culture apparatus, a separate operation for backwashing (backflow) of the discharge pipe is periodically required to prevent clogging of the filtration membrane provided in the discharge pipe.

  The present invention eliminates the above-mentioned drawbacks.

  In order to achieve the above object, a microcarrier stirring culture apparatus of the present invention comprises a culture container containing a microcarrier, a culture, and a medium, and one zone inside the culture container provided in the culture container. A filtration membrane that is divided into the other zone, does not pass through the microcarrier, and passes through the culture and the culture medium; and a drive shaft that is provided in the culture vessel and reciprocates through the filtration membrane; One stirring blade fixed to the drive shaft portion in the one zone, the other stirring blade fixed to the drive shaft portion in the other zone, and the contents in the one zone. And a discharge means for discharging the culture container and a supply means for supplying contents such as a culture, a culture medium and a microcarrier into the other zone.

  The microcarrier agitation culture apparatus of the present invention includes a culture container in which a microcarrier, a culture, and a medium are contained, and the culture container provided in the culture container is divided into a lower zone and an upper zone, A filtration membrane that does not pass through the microcarrier and allows the culture and the medium to pass through, a drive shaft that is provided in the culture vessel and reciprocates vertically through the filtration membrane, and in the lower zone A lower wing fixed to the drive shaft portion, an upper wing fixed to the drive shaft portion in the upper zone, and a discharge means for discharging the contents in the lower zone to the outside of the culture vessel, The upper zone includes supply means for supplying contents such as a culture, a medium, and a microcarrier.

  According to the present invention, only cells and the like can be extracted, and a separate backwash operation for the filtration membrane can be made unnecessary.

It is a vertical front view of the stirring culture apparatus of Example 1 of this invention.

  The example of the form for carrying out the present invention is shown below.

  A first embodiment of the present invention will be described with reference to FIG.

  Reference numeral 1 denotes a microcarrier agitation culture apparatus of the present invention. Reference numeral 2 denotes, for example, a vertical culture container of the agitation culture apparatus 1. The culture container 2 includes, for example, iPS cells, animal and plant cells, It contains a culture such as microbial cells, a medium such as a culture solution, and a fine particle carrier (microcarrier 3) having a culture surface on which cells or the like can adhere and grow on the surface or inside.

  Reference numeral 4 denotes a filtration membrane (filter), and the filtration membrane 4 is partitioned horizontally on the inner peripheral surface of the middle portion in the vertical direction of the culture vessel 2, for example, horizontally. By being fixed, the lower side zone 5 (lower space part) and the upper side upper zone 6 (upper space part) are formed in the culture vessel 2 by the filtration membrane 4.

  The pore size (mesh size) of the filtration membrane 4 is set to a size that allows passage of cultures such as cells and culture medium but does not allow the microcarriers 4 to be used, that is, the microcarrier diameter. .

  Moreover, the said filtration membrane 4 may be inclined if the said culture vessel 2 is partitioned up and down other than providing horizontally, for example. In other words, it may be partitioned in the reciprocating direction of the drive shaft.

  7 indicates a drive shaft, and the drive shaft 7 passes through the opening 8 at the center of the ceiling portion 2a of the culture vessel 2 and passes through the through hole 4a formed in the filtration membrane 4, It is suspended in the culture vessel 2 and inserted.

  The drive shaft 7 is supported by a support portion 9 such as a drive shaft seal portion or a thrust bearing portion provided in the opening portion 8 so as to be movable up and down. It is connected to a reciprocating drive device 10 provided above the portion 2 a and is moved up and down by driving the reciprocating drive device 10.

  The drive shaft 7 is provided so as to be slidable up and down in the through hole 4a. Between the through hole 4a and the stirring shaft 7, the microcarrier 4 contained in the stirring container 2 is provided. It is formed at intervals that cannot pass.

  Reference numeral 11 denotes a lower wing. The lower wing 11 is formed, for example, in a circular flat plate shape, and the surface is orthogonal to the drive shaft portion, for example, the lower end, located in the lower zone 5 in the culture vessel 2. (Crossed) and fixed.

  The lower wing 11 may be provided in two or more stages in addition to the first stage on the drive shaft 7, and the position where the lower wing 11 is fixed to the drive shaft 7 is such that the drive shaft 7 moves up and down. Even if it moves, if it does not hit the filtration membrane 4, it may be the middle part instead of the lower end, and there is no particular limitation.

  In addition to the circular shape, the lower blade 11 has various shapes including a three-dimensional shape such as an elliptical shape, an oval shape, a rectangular shape, a cross shape, and a conical shape. Further, a part or all of the stirring blades may be bent at an arbitrary position.

  Reference numeral 12 denotes an upper blade, and the upper blade 12 is formed in, for example, a circular flat plate shape, and the drive shaft portion located in the upper zone 6 in the culture vessel 2, for example, the upper portion of the drive shaft 7 The surface is fixed orthogonally (crossed) to the part.

  The upper blade 12 may be provided in two or more stages in addition to the first stage on the drive shaft 7, and the position where the upper blade 12 is fixed to the drive shaft 7 is such that the drive shaft 7 moves up and down. Even if it moves, if it does not hit the filtration membrane 4, it may be an intermediate portion instead of the upper portion, and there is no particular limitation.

  In addition to the circular shape, the upper blade 12 has various shapes including a three-dimensional shape such as an elliptical shape, an oval shape, a rectangular shape, a cross shape, and a conical shape. Further, a part or all of the stirring blades may be bent at an arbitrary position.

  The lower blade 11 and the upper blade 12 include various shapes and structures. In the blades 11 and 12 of the present embodiment, the blade surfaces of the blades 11 and 12 are arranged on the drive shaft 4. The drive shaft is fixed so as to be perpendicular to the axial direction, but at an angle with respect to the vertical surface so that all or part of the blade surfaces of the blades 11 and 12 are directed in an arbitrary direction. 4, and all or part of the wings 11, 12 are angled around the axis of the drive shaft 4 (that is, around the axis with respect to a line extending radially outward from the drive shaft 4). Including those fixed to the drive shaft 4 by being inclined in the direction).

  Reference numeral 13 denotes a supply pipe as a supply means for supplying contents such as a culture, a culture medium, and a microcarrier into the upper zone 6, and the supply pipe 13 includes, for example, a ceiling portion 2 a of the culture vessel 2. It penetrates and the front-end | tip part is provided so that it may be located in the said upper zone 6 in this culture container 2. As shown in FIG.

  The supply pipe 13 has an on-off valve (not shown) and a supply pipe-side pump section for supplying a culture medium or culture containing microcarriers into the upper zone 6 of the culture vessel 2 ( (Not shown), and if necessary, the on-off valve is opened, and microcarriers are introduced into the upper zone 6 of the culture vessel 2 through the supply pipe 13 by the supply pipe side pump unit. A medium or culture containing it is supplied.

  Note that only the medium excluding the microcarriers may be supplied from the supply tube 13 into the culture vessel 2.

  14 shows a discharge pipe as discharge means for discharging the contents in the lower zone 5 to the outside of the culture container, and the discharge pipe 14 penetrates the ceiling portion 2a of the culture container 2, for example, The tip of the filtration membrane 4 is provided so as to be located in the lower zone 5 in the culture vessel 2.

  The discharge pipe 14 includes an on-off valve (not shown) and a discharge pipe side pump section (not shown) for discharging the contents in the lower zone 5 of the culture vessel 2 to the outside of the culture vessel. When necessary, the on-off valve is opened, and the contents in the lower zone 5 of the culture vessel 2 are transferred to the outside of the culture vessel through the discharge tube 14 by the discharge pipe side pump unit. Come out.

  Next, the operation and effect of the stirring culture apparatus using the microcarrier of the present invention will be described.

  In the agitating culture apparatus 1 of the present invention, first, the opening / closing valve of the supply pipe 13 is opened, and the supply pipe side pump unit causes the microcarriers, cells and medium to be transferred from the supply pipe 13 into the culture vessel 2. The contents (the material to be stirred) containing the contents of the culture vessel 2 are accommodated to a desired height (a desired amount) of the culture vessel 2.

  Thereby, since cultures and media such as cells can pass through the filtration membrane 4, they are contained in the lower zone 5 and the upper zone 6, and only the microcarrier 4 is contained in the filtration membrane. Since it does not pass through 4, it can be contained only in the upper zone 5.

  Then, the on / off valve of the supply pipe 13 is closed, the pump on the supply pipe side is stopped, and the drive shaft 7 is reciprocated up and down by the reciprocating drive device 10, whereby the lower blade 11 and the upper blade 12 reciprocates in the vertical direction, and the contents (stirred objects) in the lower zone 5 and the upper zone 6 are stirred and cultured.

  At this time, since the microcarrier 3 does not pass through the filtration membrane 4, the microcarrier 3 is stirred only in the upper zone 6, and the culture and medium such as the cells can pass through the filtration membrane 4. Agitation occurs in the upper zone 5 and the lower zone 6.

  Even if the filtration membrane 4 is clogged by the microcarrier 3, the upward movement of the upper blade and the lower blade causes an upward flow in the filtration membrane 4, and the filtration membrane 4 The microcarrier 3 attached to the surface is separated, and clogging is suppressed.

  That is, the inside of the culture vessel 2 is agitated by the lower blade 11 and the upper blade 12, and at the same time, the filtration membrane 4 has a backwashing action, and clogging is suppressed.

  When the contents in the lower zone 5 are not withdrawn, the discharge pipe side pump is stopped and the on-off valve of the discharge pipe 14 is closed.

  Then, after the passage of a desired time, the on-off valve of the discharge pipe 14 is opened and the discharge pipe side pump unit is driven to discharge the contents in the lower zone 5 so that only the cells and the medium are discharged. Can be extracted.

  When the contents in the lower zone 5 are extracted, the reciprocating drive device 10 may be stopped.

  Further, simultaneously with the discharge of the contents in the culture vessel 2, a medium, a culture, or a microcarrier may be supplied from the supply unit 13 into the culture vessel 2.

  According to the present invention, cells excluding microcarriers can be extracted out of the culture vessel.

  In addition, the reciprocating motion of the lower blade and the upper blade allows the stirring and the backwashing of the filtration membrane to be performed at the same time, without performing a separate backwashing operation to prevent clogging of the filtration membrane as in the prior art. It will be good.

  In the above-described embodiment, an example in which the drive shaft is vertically suspended and the drive shaft 7 is moved in the up and down direction has been shown. However, for example, the drive shaft 4 is disposed horizontally and moved in the lateral direction. You may make it move to an arbitrary direction and to reciprocate in this arbitrary direction.

  When the drive shaft moves in the lateral direction, the filtration membrane fixed in the culture vessel is fixed so as to partition the culture vessel into the left zone and the right zone in the lateral direction, and the drive When the shaft moves in an arbitrary direction, the filtration membrane fixed in the culture vessel is fixed so as to partition the culture vessel into one zone and the other zone in that direction.

  For example, one stirring blade such as a lower blade or a left blade is fixed to a drive shaft portion positioned in the one zone, and the other stirring blade such as an upper blade or a right blade is connected to the other blade. It comes to be fixed to the drive shaft part located in the zone.

  The agitation culture apparatus of the present invention is used for a culture apparatus related to medical products, foods, and the like.

DESCRIPTION OF SYMBOLS 1 Stirring culture apparatus 2 Culture container 2a Ceiling part 3 Microcarrier 4 Filtration membrane 4a Through-hole 5 Lower zone 6 Upper zone 7 Drive shaft 8 Opening part 9 Support part 10 Reciprocating drive apparatus 11 Lower stage blade 12 Upper stage blade 13 Supply pipe 14 Discharge pipe

Claims (2)

A culture vessel in which a microcarrier, a culture and a medium are contained;
A filtration membrane provided in the culture vessel, which partitions the inside of the culture vessel into one zone and the other zone, does not pass through the microcarrier, and allows the culture and the medium to pass through;
A drive shaft provided in the culture vessel and reciprocating through the filtration membrane;
One stirring blade fixed to the drive shaft portion in the one zone;
The other stirring blade fixed to the drive shaft portion in the other zone;
Discharging means for discharging the contents in the one zone out of the culture vessel;
A microcarrier agitation culture apparatus comprising supply means for supplying contents such as a culture, a culture medium and a microcarrier into the other zone. A culture vessel in which a microcarrier, a culture and a medium are contained;
A filtration membrane that is provided in the culture vessel and divides the inside of the culture vessel into a lower zone and an upper zone, does not pass through the microcarrier, and passes the culture and the medium;
A drive shaft provided in the culture vessel and reciprocating vertically through the filtration membrane;
A lower blade fixed to the drive shaft portion in the lower zone;
An upper blade fixed to the drive shaft portion in the upper zone;
Discharging means for discharging the contents in the lower zone out of the culture vessel;
A microcarrier agitation culture apparatus comprising supply means for supplying contents such as a culture, a culture medium, and a microcarrier into the upper zone.

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