WO2016157326A1

WO2016157326A1 - Division/detachment member, culture vessel, and device

Info

Publication number: WO2016157326A1
Application number: PCT/JP2015/059731
Authority: WO
Inventors: 広斌周; 鈴木　大介; 絵里乃松本; 洸斉藤
Original assignee: 株式会社日立製作所
Priority date: 2015-03-27
Filing date: 2015-03-27
Publication date: 2016-10-06

Abstract

Provided is a cell colony division/detachment member which makes it possible to detach uniform cell colonies. A division/detachment member is constructed, which is equipped with: a division section 7 which can divide a cell colony 2 into pieces each having a proper size on a culture surface 1 on which the cell colony 2 is cultured, prior to the detachment of the cell colony 2 from the culture surface 1; an enzyme introduction section 6 through which a detaching enzyme 4 capable of detaching cells is introduced into division gaps formed after the division by the division section 7; and a detachment section 8 which can detach the divided cell colonies completely. A cell culture device is so configured that a cell colony can be divided/detached using the member. Therefore, the cell culture device enables the culturing of cells with high efficiency and with less damage to the cells.

Description

Dividing / peeling member, culture vessel, and apparatus

The present invention relates to a technique for dividing / separating cell colonies in a cell culture apparatus.

Induced pluripotent stem cells (i nduced P luripotent S tem cell : iPS cells) due to their pluripotency can unlimited growth, application to regenerative medicine and drug screening is expected. Although iPS cells can be cultured in a suspension culture, planar culture in which the cells are adhered to feeder cells or various polymers is generally used. This expansion culture that expands the number of iPS cells requires repeated subculture. At that time, in order to culture a large colony, it is necessary to peel the cultured iPS cell colony from the adhesive surface and pulverize it to a size suitable for the next cell seeding.

Therefore, in general, the entire cultured iPS cell colony is immersed in a peeling enzyme, the colony is lifted from the outer periphery, and then the colony is peeled off from the culture surface and pulverized using a cell scraper. Here, if the seeded cell colonies created are too large, iPS cells tend to differentiate, while if the seeded cell colonies are too small, they tend to be unable to proliferate. It is important to grind to an appropriate size of about 200 to 300 μm.

Patent Document 1 proposes a method of exfoliating colonies with an exfoliating enzyme and then pulverizing them to a seeding size with a cutter. Patent Document 2 proposes a method of dividing a cell cluster of pluripotent stem cells through a microgrid. Patent Document 3 proposes a method of fragmenting cell colonies using a laser cutting pattern.

WO2007 / 148098 Special table 2010-526530 gazette Special table 2012-514981 gazette

However, in the method described in Patent Document 1, it is difficult to control the detachment enzyme treatment time, and there is a problem that damage to cells due to long-time treatment is large. In addition, the method described in Patent Document 2 has a problem that when it is used for culturing a large amount of pluripotent stem cells, clogging of a microgrid or the like occurs and appropriate and efficient passage cannot be realized. Furthermore, in the method described in Patent Document 3, since laser light is used to provide a laser cutting pattern on the culture surface, there is a problem that the colony dividing operation becomes complicated.

An object of the present invention is to provide a cell colony dividing / separating member, a culture container, and an apparatus that solve the above-described problems, reduce cell damage, and allow cell colonies to be efficiently cultured. There is.

In order to achieve the above object, in the present invention, a divided part for dividing a cultured cell colony on the culture surface, an enzyme introducing part for introducing a cell detachment enzyme into the divided gap after division, and a divided cell There is provided a splitting / peeling member configured to include a peeling part for peeling a colony from a culture surface.

In order to achieve the above object, the present invention comprises a culture surface for culturing a cell colony and a dividing / peeling member for dividing / peeling the cell colony, and the splitting / peeling member is a cultured cell. A culture container having a structure including a dividing part for dividing a colony on the culture surface, an enzyme introducing part for introducing a cell detachment enzyme into the divided gap after division, and a peeling part for peeling the divided cell colony from the culture surface. provide.

Furthermore, in order to achieve the above object, in the present invention, a culture container having a culture surface for culturing cell colonies and a dividing / separating member for dividing / separating cell colonies, a cell detachment enzyme feeding pump, a cell And a control unit that controls the detachment enzyme feeding pump, and the dividing / peeling member is an enzyme that divides the cultured cell colony on the culture surface and an enzyme that introduces the cell detachment enzyme into the divided gap after the division Provided is a culture apparatus having an introduction part and a peeling part for peeling a divided cell colony from a culture surface.

According to the present invention, uniform cell colonies can be efficiently detached.

It is a schematic diagram which shows the Example of the cell colony division | segmentation and peeling method which concerns on Example 1. FIG. 6 is a plan view showing an application process of a cell colony dividing / peeling method according to Example 1. FIG. It is a figure which shows one structural example of the peeling part of the cell colony division | segmentation / peeling member which concerns on Example 1. FIG. It is a figure which shows an example of the front-end | tip part of the division part of the cell colony division | segmentation / peeling member which concerns on Example 1. FIG. FIG. 6 is a diagram showing a configuration example of a cell colony dividing / peeling member according to Example 2. 6 is a diagram illustrating another configuration example of the cell colony dividing / peeling member according to Embodiment 2. FIG. 6 is a diagram illustrating an example of a schematic configuration of a culture container according to Example 3. FIG. 6 is a diagram illustrating an example of a schematic configuration of a culture container according to Example 3. FIG. FIG. 4 is a diagram illustrating a schematic configuration example of a culture apparatus according to Example 3. FIG. 10 is a flowchart showing an example of subculture processing by the culture device according to Example 3.

Hereinafter, various embodiments of the present invention will be described with reference to the drawings. In the drawings corresponding to each embodiment, the same components are given the same number.

An example of the cell colony dividing / detaching method according to Example 1 will be described with reference to FIGS. The cell colony dividing / peeling method of this example divides the cell colony before peeling the cell colony from the culture surface, introduces a cell peeling enzyme into the gap after division, and then peels the cell colony from the culture surface This is an example.

FIG. 1 is a schematic view showing an embodiment of the cell colony dividing / detaching method of the present embodiment, and FIG. 2 is a plan view showing an application process of the cell colony dividing / detaching method of the present embodiment. As shown in FIG. 1, the cell colony dividing / peeling method of the present embodiment is applied to the cell colony 2 cultured on the cell culture surface 1. FIG. 2A shows a state in which a cell colony is divided into two and a divided gap 3 is formed. FIG. 2B shows a state in which the cell detachment enzyme 4 is introduced at the division interval 3. FIG. 2C shows the state of the divided / detached cell colony 5 having a desired size after the introduction of the release enzyme 4.

As shown in FIG. 1, the dividing / peeling member of this example is composed of a peeling enzyme introduction part 6, a plow-shaped dividing part 7, and a cell scraper-shaped peeling part 8. The exfoliation enzyme introduction part 6 has an exfoliation enzyme introduction part inlet 6A for introducing / derived the cell exfoliation enzyme 4 and an exfoliation enzyme introduction part outlet 6B, and the dividing part 7 and the exfoliation part 8 are located in the vicinity of the exfoliation enzyme introduction part outlet 6B. is set up. The peeling enzyme 4 introduced from the peeling enzyme introduction part inlet 6A of the peeling enzyme introduction part 6 is introduced into the divided gap 3 of the divided cell colony 2 on the cell culture surface 1 from the peeling enzyme introduction part outlet 6B.

That is, in the cell colony dividing / peeling method of the present embodiment, the cell colony 2 is cultured on the cell culture surface 1, and after the cell colony 2 becomes a predetermined size, the dividing portion of the dividing / peeling member of FIG. 7 is moved at a predetermined interval on the cell culture surface 1 to form a division gap 3 in the cell colony 2 as shown in FIG. At the same time, the cell detachment enzyme 4 is injected into the separation gap 3 from the detachment enzyme introduction part outlet 6B through the detachment enzyme introduction part 6 as shown in FIG. After injection of the detachment enzyme 4, the detachment part 8 of the detachment / separation member detaches, and a detachment / detachment cell colony 5 is generated as shown in FIG.

If the cell colony dividing / detaching method of the present example is used, the time required for the release enzyme treatment can be shortened, resulting in less damage to the cells by the release enzyme. Further, since the cell colony 2 is divided into a desired size of the next seeding size before detachment, the cell colony crushing operation after detachment becomes unnecessary, and the operation time at the time of cell colony passage can be shortened.

FIG. 3 shows an example of the peeling part 8 of the cell colony dividing / peeling member of this example. In the same figure, since parts other than the culture medium 9 have the same configuration as that of the dividing / peeling member in FIG. 1, the description thereof is omitted here. Peeling of the cell colony of this example can be performed in a shorter time because the cell colony after the culture is divided and peeled off, but in order to further shorten, the peeling part 8 of the cell colony dividing / peeling member having a cell scraper shape is used. Is used. That is, the cell scraper-shaped exfoliation part 8 can exfoliate the cell colony from the culture surface after the exfoliation enzyme-treated cell colony 2 has been lifted from the outer periphery in the same manner as the cell scraper.

FIG. 4 is a diagram showing an example of a tip portion of the dividing portion 7 of the cell colony dividing / peeling member having a plow shape used in the cell colony dividing / peeling method of the present embodiment. If the plow-shaped division part 7 provided with

such tip parts

7A and 7B is used, the cell colony 2 can be divided smoothly.

For the cell colony dividing / peeling member of this example, it is desirable to use a material that can withstand high humidity and sterilization, such as resin. Thereby, it can be adapted to sterilization such as γ-ray, and can be applied to physics and chemistry use and regenerative medicine use.

According to Example 1 described in detail above, uniform cell colonies can be efficiently detached by dividing a cell colony into an appropriate size and then introducing a release enzyme into the dividing gap. In this embodiment, the shape of the cell culture surface is not specifically described. However, the cell colony dividing / peeling method and the dividing / peeling member of this embodiment can be applied to each shape of the culture surface.

As Example 2, an example of a dividing / peeling member suitable for a large culture area and capable of dividing and peeling a large number of cell colonies into a predetermined size at the same time will be described.

FIG. 5 is a schematic diagram showing a multiple split / peel member that integrates a plurality of split / peel members according to the second embodiment. In the figure, portions other than the communication unit 10 are configured to include a plurality of configurations similar to those in the first embodiment, and thus description thereof is omitted here. As shown in FIG. 5, the detachment enzyme introduction part 6 of the dividing / separation member includes one detachment enzyme introduction part inlet 6 </ b> A for introducing cell detachment enzyme, a plurality of detachment enzyme introduction part outlets 6 </ b> B, and a detachment enzyme introduction part inlet. The communication part 10 which connects 6A and several peeling enzyme introduction part exit 6B is provided.

That is, the dividing / peeling member of the present embodiment has the multiple structure of the first embodiment by the communication portion 10. And, since the peeling enzyme 4 can be introduced from one peeling enzyme introduction part inlet 6A and can be led out from the plurality of peeling enzyme introduction part outlets 6B through the communication part 10, a large number of cell colonies can be simultaneously formed to a predetermined size. It becomes possible to divide and peel.

FIG. 6 is a view showing a hollow grid type dividing / peeling member, which is a modification of the dividing / peeling member of Example 2. In (A) and (B) of the figure, the portions other than the hollow grid 11 corresponding to the communication portion 10 and the leg portions 11A of the hollow grid are the same as those in the first embodiment. As shown in FIGS. 6A and 6B, the detachment enzyme introduction part 6 of the dividing / peeling member includes one detachment enzyme introduction part inlet 6A for introducing a cell detachment enzyme and a plurality of detachment enzyme introduction part outlets. The hollow grid 11 which connects 6B, the peeling enzyme introduction part inlet 6A, and the some peeling enzyme introduction part outlet 6B is provided.

As shown in (A) and (B) of the figure, the cell colony 2 is divided into a predetermined size by a dividing part attached to the leg part 11A of the hollow grid, and the peeling enzyme 4 is further separated from the leg part 11A of the hollow grid 11. The cell colonies can be detached from the culture surface in a shorter time by injecting into the dividing gap.

According to the present embodiment, it is possible to simultaneously divide and detach a large number of cell colonies into a predetermined size using a dividing / separating member having a shape matched to the cell culture surface.

The third embodiment is an embodiment of the cell colony dividing / peeling method, the culture container and the culture apparatus using the splitting / peeling member of each embodiment described above.
7A and 7B are diagrams showing a schematic configuration example of the culture vessel of Example 3. FIG. The culture vessel 12 of FIGS. 7A and 7B includes a culture surface 12A of the culture vessel corresponding to the cell culture surface 1, a lid 12B of the culture vessel, a medium inlet 13, a medium outlet 14, a mixed gas inlet 15, a mixed gas outlet 16, and A culture vessel inner magnet 17 and a culture vessel outer magnet 18 are provided as a mechanism for moving the separation / separation member. This embodiment is an implementation of a culture container that performs culture medium exchange to perform culture medium discharge / injection to the culture container 12, mixed gas exchange to perform mixed gas discharge / injection, cultures cell colonies, and further divides and detaches cell colonies. It is an example.

As shown in FIGS. 7A and 7B, the splitting / peeling member has the same configuration as that of the second embodiment, and a split plow portion corresponding to the split portion and a peeler scraper corresponding to the split portion. Department. These switching operations by the culture vessel inner magnet 17 and the culture vessel outer magnet 18 will be briefly described.

As shown in FIG. 7A, the center of the culture vessel external magnet 18 is an axis (indicated by a dotted line), and the culture vessel external magnet 18 is rotated 180 degrees under the control of a control terminal constituting a control unit described later. By rotating the internal magnet 17 by 180 degrees, it is possible to switch between the split plow portion and the peel scraper portion of the multiple split / peel member. Further, as shown in FIG. 7B, the culture vessel external magnet 18 is moved in the direction of the arrow (shown in white), and the culture vessel internal magnet 17 is moved in the same direction, whereby the culture surface 12A is divided. The plow part and the peeling scraper part can be moved.

FIG. 8 is a diagram showing a schematic configuration example of the culture apparatus according to the present embodiment. The culture apparatus shown in FIG. 8 is installed in an incubator 28 and is connected to a culture medium inlet 13 through a flow path, a liquid feed pump 19, a liquid supply pump 19 and a culture medium bottle 19 </ b> A connected through a flow path, a culture medium outlet 14 and a flow path. The drainage pump 20 connected in the above, the drainage bottle 20A connected to the drainage pump 20 by the flow path, the mixed gas controller 21 connected to the mixed gas inlet 15 by the flow path, the exhaust gas filter 22, the separation enzyme feed A pump 23, a peeling enzyme bottle 23A connected to the peeling enzyme feeding pump 23 through a flow path, a moving mechanism 24 of the dividing / peeling member, a microscope 25, a moving mechanism 26 of the microscope 25, a temperature control unit 27, and a control signal line 29 And the control terminal 30 constituting the control unit.

The control terminal 30 is configured by a personal computer (PC) having a computer configuration including a normal central processing unit (CPU), a storage unit, an input / output interface unit, and the like, and the CPU executes control software. By this control terminal 30, the medium feeding pump 19, the drainage pump 20, the mixed gas controller 21, the peeling enzyme feeding pump 23, the dividing / peeling member moving mechanism 24, the microscope 25, the microscope moving mechanism 26, the temperature The control unit 27 is controlled through the control signal line 29. A cell image is input from the microscope 25 and transmitted to the server in the control terminal 30 or via the network, and image processing is executed by various image processing software.

FIG. 9 is a flowchart showing an example of subculture processing by the culture apparatus according to the present embodiment. In the figure, when the culture process is started under the control of the control terminal 30, iPS cell colonies are seeded in the culture vessel (ST1). Thereafter, conditions such as temperature and humidity are adjusted, and the cells are cultured (ST2). Subsequently, when a predetermined medium replacement time is reached, the old medium in the culture container is discharged from the culture container 12 (ST3). Thereafter, a new medium is poured into the culture vessel 12 (ST4). ST3 and ST4 are repeated until the number of cells reaches a predetermined time (ST5).

When the number of cells reaches a predetermined time (Yes), the medium is discharged from the culture vessel (ST6). Subsequently, the cell colony cultured in the dividing unit 7 is divided into an appropriate size, and a peeling enzyme is introduced (ST7). Further, the cell colonies are detached at the separation unit 8 (ST8), and the divided / detached cell colonies 5 after the separation / detachment are collected (ST9). ST1 to ST9 are repeated until the predetermined number of cells is reached (ST10). When the predetermined number of cells is reached (Yes), the culture is terminated.

That is, as apparent from the flowchart of FIG. 9, in the cell colony dividing / detaching method of the present example, the divided / detached cell colonies obtained in the removing step are used for subculture. At this time, when the culture time of the cell colonies on the culture surface reaches a predetermined time, or when the number of cells of the cell colonies cultured on the culture surface reaches a predetermined number, the dividing step is performed. In addition, when the number of the divided / detached cell colonies peeled off by the peeling process does not reach a predetermined number, the divided / separated cell colonies are seeded in a culture vessel and repeated subculture, and the divided When the number of exfoliated cell colonies reaches a predetermined number, the subculture of the cell colonies is terminated.

According to the culture apparatus of this example, by dividing a cell colony into an appropriate size and then introducing a detaching enzyme into the dividing gap, it becomes possible to efficiently detach a uniform cell colony. Subculture can be performed. Moreover, the culture apparatus of the present embodiment has mechanisms such as automatic cell seeding, automatic medium exchange, automatic passage, and automatic observation, and can maintain and manage more uniform culture quality.

Although various embodiments of the present invention have been described above, the present invention is not limited to the above-described embodiments unless the characteristics of the present invention are impaired, and can be considered within the scope of the technical idea of the present invention. Various other modifications are included. For example, the above-described embodiments have been described in detail for better understanding of the present invention, and are not necessarily limited to those having all the configurations described. Further, a part of the configuration of one embodiment can be replaced with the configuration of another embodiment, and the configuration of another embodiment can be added to the configuration of one embodiment. Further, it is possible to add, delete, and replace other configurations for a part of the configuration of each embodiment.

Furthermore, although each of the above-described configurations, functions, control terminals, etc. has been described with respect to an example of creating a program executed by a CPU that realizes some or all of them, some or all of them are, for example, integrated circuits. It goes without saying that it may be realized by hardware by designing or the like.

DESCRIPTION OF SYMBOLS 1 Culture surface 2 Cell colony 3 Dividing gap 4 Stripping enzyme 5 Splitting / peeling cell colony 6 Stripping enzyme introduction part 6A Stripping enzyme introduction part inlet 6B Stripping enzyme introduction part outlet 7 Division part 8 Stripping part 9 Medium 10 Communication part 11 Hollow grid 11A Hollow grid legs 12 Culture vessel 12A Culture vessel culture surface 12B Culture vessel lid 13 Medium inlet 14 Medium outlet 15 Mixed gas inlet 16 Mixed gas outlet 17 Culture vessel internal magnet 18 Culture vessel external magnet 19 Feed pump 19A Medium bottle 20 Wastewater pump 20A Wastewater bottle 21 Mixed gas controller 22 Exhaust gas filter 23 Peeling enzyme feed pump 23A Peeling enzyme bottle 24 Dividing / peeling member moving mechanism 25 Microscope 26 Microscope moving mechanism 27 Temperature control unit 28 Incubator 29 Control signal Line 30 control terminal

Claims

A dividing part for dividing the cultured cell colony on the culture surface;
An enzyme introduction part for introducing a cell detachment enzyme into the divided gap after the division;
A separation part for separating the divided cell colonies from the culture surface,
A dividing / peeling member characterized by that.
The splitting / peeling member according to claim 1,
The dividing / peeling member, wherein the dividing portion has a plow shape, and the peeling portion has a cell scraper shape.
The splitting / peeling member according to claim 1,
The enzyme introduction part has an enzyme introduction part inlet and an enzyme introduction part outlet for introducing / derived the cell detachment enzyme,
The split part and the peeling part are installed in the vicinity of the enzyme introduction part outlet,
A dividing / peeling member characterized by that.
The splitting / peeling member according to claim 1,
Further comprising a communication portion for communicating the plurality of the dividing / peeling members;
A dividing / peeling member characterized by that.
The splitting / peeling member according to claim 1,
Further comprising a hollow grid communicating the plurality of the dividing / peeling members.
A dividing / peeling member characterized by that.
A culture surface for culturing the cell colony;
A dividing / peeling member for dividing / peeling the cell colony,
The dividing / peeling member includes a dividing unit that divides the cultured cell colony on the culture surface, an enzyme introduction unit that introduces a cell peeling enzyme into the divided gap after the division, and the divided cell colony A peeling portion that peels from the culture surface,
A culture vessel characterized by that.
The culture container according to claim 6,
The enzyme introduction part of the splitting / separating member has an enzyme introduction part inlet and an enzyme introduction part outlet for introducing / derived the cell peeling enzyme,
The split part and the peeling part are installed in the vicinity of the enzyme introduction part outlet,
A culture vessel characterized by that.
The culture container according to claim 6,
The enzyme introduction part of the splitting / separation member includes an enzyme introduction part inlet for introducing the cell detachment enzyme, a plurality of enzyme introduction part outlets, and a communication that communicates the enzyme introduction part inlet and the plurality of enzyme introduction part outlets. Comprising a part,
A culture vessel characterized by that.
The culture container according to claim 6,
The enzyme introduction part of the splitting / separating member includes an enzyme introduction part inlet for introducing the cell detachment enzyme, a plurality of enzyme introduction part outlets, and a hollow communicating the enzyme introduction part inlet and the plurality of enzyme introduction part outlets. With a grid,
A culture vessel characterized by that.
The culture container according to claim 6,
A moving mechanism for moving the dividing / peeling member on the culture surface;
A culture vessel characterized by that.
A culture vessel having a culture surface for culturing the cell colony and a dividing / peeling member for dividing / peeling the cell colony, a cell detachment enzyme feeding pump, and a controller for controlling the cell detachment enzyme feeding pump, With
The dividing / peeling member includes a dividing unit that divides the cultured cell colony on the culture surface, an enzyme introduction unit that introduces a cell peeling enzyme into the divided gap after the division, and the divided cell colony A peeling portion that peels from the culture surface,
A culture apparatus characterized by that.
The culture apparatus according to claim 11,
The controller is
When the culture time of the cell colonies on the culture surface reaches a predetermined time, or when the number of cells of the cell colonies cultured on the culture surface reaches a predetermined number, the dividing / peeling member Control to perform splitting and peeling,
A culture apparatus characterized by that.
A culture device according to claim 12,
The control unit controls the cell detachment enzyme pump to control introduction of the cell detachment enzyme into the enzyme introduction unit;
A culture measure characterized by that.
A culture device according to claim 12,
A moving mechanism for moving the dividing / peeling member on the culture surface;
The controller is
By controlling the moving mechanism, control to carry out the division and detachment of the cell colony by the division and detachment member,
A culture apparatus characterized by that.
The culture apparatus according to claim 14, wherein
The controller is
By switching the moving mechanism, the split part and the peel part of the split / peel member are switched,
A culture apparatus characterized by that.