Culture system for pluripotent stem cells and method for subculturing pluripotent stem cells

• US 9,738,861 B2
• Filed: 02/20/2014
• Issued: 08/22/2017
• Est. Priority Date: 03/06/2013
• Status: Active Grant

• Current Assignee: Kyoto University, Nipro Corporation
• Original Assignee: Kyoto University, Nipro Corporation
• Inventors: Nakatsuji, Norio, Yoshikawa, Yoshihiro, Takeuchi, Masakatsu, Tateyama, Daiki
• Primary Examiner(s): Prakash, Gautam
• Application Number:
  US14/771,358
• Publication Number:
  US 20160060588A1
• Time in Patent Office: 1,279 Days
• CPC Class Codes: C12M 23/14 Bags
• CPC Class Codes: C12M 23/58 Reaction vessels connected in series or in parallel combinations of bioreactors with other apparatus, C12M43/00
• CPC Class Codes: C12M 25/02 Membranes; Filters filters or filtration in general B01D24/00-B01D41/00
• CPC Class Codes: C12M 29/00 Means for introduction, extraction or recirculation of materials, e.g. pumps pumps per se F04B
• CPC Class Codes: C12M 29/04 Filters; Permeable or porous membranes or plates, e.g. dialysis
• CPC Class Codes: C12M 41/00 Means for regulation, monitoring, measurement or control, e.g. flow regulation controlling or regulating chemical, physical or physicochemical processes B01J19/0006; heating or cooling apparatus for laboratory use B01L7/00; electro optical investigation of individual particles, flow cytometers G01N15/14; automatic analysis G01N35/00; controlling or regulating in general G06N

Abstract

A culture system has a culture bag for suspending and culturing pluripotent stem cells in a culture medium; a waste liquid container for storing used culture medium that is connected to the culture bag; a fresh medium container for storing a fresh culture medium that is connected to the culture bag; three-way stopcocks for switching flow channels from the culture bag to the waste liquid container or the fresh culture medium container, etc.; a trap portion for trapping the pluripotent stem cells in the culture medium in the flow toward the waste liquid container side; and a filter portion for subculturing that is formed in fourth and fifth flow channels in parallel to a first flow channel. The first flow channel connects the trap part to the culture bag, and has a mesh by which a cell mass of the pluripotent stem cells can be divided. A pump is used to pump the liquid in the individual flow channels.

Claims

1. A culture system for pluripotent stem cells comprising:

a culture container for suspending and culturing pluripotent stem cells in a culture medium;

a waste liquid container which is connected to the culture container by flow channels through which liquid can flow and which stores a used culture medium flowing out of the culture container;

a fresh culture medium container which is connected to the culture container by flow channels through which liquid can flow and which stores a fresh culture medium to be supplied to the culture container;

first switching portions which switch the flow channels from the culture container to the waste liquid container or the fresh culture medium container;

a trap portion which is provided in the flow channel on a side of the culture container with respect to the first switching portions and which traps the pluripotent stem cells in the culture medium in flow to a side of the waste liquid container;

a subculturing filter portion which is provided in flow channels provided in parallel to a flow channel connecting the trap portion and the culture container between the trap portion and the culture container and which has a mesh capable of dividing a cell mass of the pluripotent stem cells; and

second switching portions which switch flow channels from the trap portion to the culture container or the subculturing filter portion.

2. The culture system for pluripotent stem cells according to claim 1, further comprising:

a backwashing container which is provided in a flow channel connecting the culture container and the subculturing filter portion in such a manner as to allow passage of liquid and which stores a backwashing culture medium to be supplied to the subculturing filter portion.

3. The culture system for pluripotent stem cells according to claim 1, further comprising:

a control portion for controlling a flow rate of liquid in each of the flow channels, wherein

the control portion sets a flow rate per unit square cm of liquid flowing through the mesh of the subculturing filter portion to 95 mL or more per minute.

4. The culture system for pluripotent stem cells according to claim 1, further comprising:

a pump for letting liquid flow through each of the flow channels; and

a control portion which controls drive of the pump, wherein

the control portion drives the pump at a fixed flow rate of 90 mL or more per minute at least in flow from the trap portion to the subculturing filter portion.

5. The culture system for pluripotent stem cells according to claim 1, wherein the pluripotent stem cells are ES cells or iPS cells.

6. The culture system for pluripotent stem cells according to claim 1, wherein the pluripotent stem cells are human-derived pluripotent stem cells.

7. A method for subculturing pluripotent stem cells comprising:

a culturing step of culturing suspending and culturing a cell mass of pluripotent stem cells;

a first dividing step of letting the cell mass of the cultured pluripotent stem cells flow into a mesh, which is provided in a flow channel through which a culture medium can flow, in one direction together with a culture medium to thereby divide the cell mass, and then accommodating the divided cell masses in a culture container;

a backwashing step of closing a flow channel to the culture container, and then letting the culture medium flow into the mesh in an opposite direction to the direction in the dividing process to remove a cell mass clogging the mesh; and

a second dividing step of opening the flow channel to the culture container, letting the removed cell mass flow in one direction together with the culture medium to thereby divide the cell mass, and then accommodating the divided cell masses in the culture container.

8. The method for subculturing pluripotent stem cells according to claim 7, wherein the pluripotent stem cells are ES cells or iPS cells.

9. The method for subculturing pluripotent stem cells according to claim 7, wherein the pluripotent stem cells are human-derived pluripotent stem cells.