Method for determining undifferentiated state of pluripotent stem cells by culture medium analysis

Patent metadata

Field	Value
Publication number	US-10704073-B2
Application number	US-201515514610-A
Country	US
Kind code	B2
Filing date	Sep 29, 2015
Priority date	Sep 29, 2014
Publication date	Jul 7, 2020
Grant date	Jul 7, 2020

Abstract

There is provided a method for easily determining an undifferentiated state of pluripotent stem cells without relying on the judgment of a skilled technician. The method includes: a step of evaluating an undifferentiated state of pluripotent stem cells based on a time-dependent change in a variation value of an extracellular metabolite contained in a culture medium in which the pluripotent stem cells are cultured, wherein the extracellular metabolite is at least one selected from a group consisting of L-glutamic acid, L-alanine and ammonia.

First claim

What is claimed is:

1. A method for automatically determining an undifferentiated state of pluripotent stem cells in order to subculture the pluripotent stem cells, comprising:
automatically determining, using a computer, whether pluripotent stem cells are in an undifferentiated state or not based on a time-dependent change in a variation value of an extracellular metabolite contained in a culture medium in which the pluripotent stem cells are cultured, in order to remove or maintain the pluripotent stem cells based on determination results in the automatically determining whether the pluripotent stem cells are in the undifferentiated state or not, wherein the pluripotent stem cells are removed when the pluripotent stem cells are determined as not being in the undifferentiated state and the pluripotent stem cells are maintained when the pluripotent stem cells are determined as being in the undifferentiated state, in order to perform the automated subculture of the pluripotent stem cells,
wherein the extracellular metabolite is at least one selected from a group consisting of L-glutamic acid, L-alanine and ammonia, and
wherein a type of the culture medium is a culture medium configured to maintain the undifferentiated state of the pluripotent stem cells, and
wherein the variation value is found as a ratio of a variation amount of the L-glutamic acid, the L-alanine or the ammonia to a variation amount of L-lactic acid which is a further extracellular metabolite contained in the culture medium.

2. The method of claim 1 , wherein the culture medium is a culture medium used until a subsequent medium replacement is carried out from a previous medium replacement.

3. The method of claim 2 , wherein a time period between culture medium replacements is 24 to 48 hours.

4. The method of claim 1 , wherein the variation value is a variation value of at least one selected from a group consisting of L-glutamic acid, L-alanine and ammonia, which is available until the subsequent medium replacement after the culture medium is replaced.

5. The method of claim 1 , wherein the time-dependent change in the variation value is found as an increasing or decreasing trend of a variation value obtained at each medium replacement time.

6. The method of claim 1 , wherein if the variation value shows a decreasing trend as compared with a variation value in a culture medium obtained at an immediately-previous medium replacement time, it is evaluated that the pluripotent stem cells are undifferentiated cells.

7. The method of claim 6 , wherein if the variation value continuously shows the decreasing trend, it is evaluated that the pluripotent stem cells are undifferentiated cells.

8. The method of claim 1 , wherein if the variation value shows a trend other than the decreasing trend as compared with a variation value in a culture medium obtained at the immediately-previous medium replacement time, it is evaluated that the pluripotent stem cells are differentiation-started cells.

9. The method of claim 1 , further comprising:
a step of evaluating a quality of a colony of the pluripotent stem cells based on a differential-filter-processed image of an image of a colony formed by the pluripotent stem cells.

Assignees

• Tokyo Electron Ltd

Inventors

• Hatabayashi Kunitada
• Tomita Kentaro
• Gomi Shinichi
• Kurakazu Tomoaki
• Oshima Yasuhiro
• Kagawa Kenichi
• Ozaki Shigenori

Classifications

• C12M41/36 Primary

  of biomass, e.g. colony counters or by turbidity measurements (electrooptical investigation of individual particles G01N15/14, flow cytometers G01N15/1404) · CPC title

• C12N2500/32

  Amino acids · CPC title

• C12M41/46

  of cellular or enzymatic activity or functionality, e.g. cell viability · CPC title

• C12N5/0696

  Artificially induced pluripotent stem cells, e.g. iPS · CPC title

• G01N33/5073

  Stem cells · CPC title