Method and device for identifying multipotent stem cell colony, and method and device for automatically culturing multipotent stem cells

• US 8,977,031 B2
• Filed: 06/22/2011
• Issued: 03/10/2015
• Est. Priority Date: 06/25/2010
• Status: Expired due to Fees

• Current Assignee: Kawasaki Jukogyo Kabushiki Kaisha (Kawasaki Heavy Industries Limited)
• Original Assignee: Kawasaki Jukogyo Kabushiki Kaisha (Kawasaki Heavy Industries Limited)
• Inventors: Sakurai, Takashi, Ohji, Osamu, Nakashima, Katsumi, Watakabe, Keizo
• Primary Examiner(s): CHANG, JON CARLTON
• Application Number:
  US13/805,767
• Publication Number:
  US 20130130228A1
• Time in Patent Office: 1,357 Days
• Field of Search: None
• US Class Current: 382/133
• CPC Class Codes: C12M 41/46 of cellular or enzymatic activity or functionality, e.g. cell viability

Abstract

A method and device achieves identification on whether a colony on a cultivation container is differentiated or undifferentiated on an occasion of cultivation of multipotent stem cell, and a method and device achieves a rapid culture of undifferentiated multipotent stem cells. Undifferentiated colony containing only undifferentiated multipotent stem cells is identified from other types of colonies by means of the circularity C of the colony in an image-processed taken image of the inside of cultivation container of a multipotent stem cell. More specifically, when a first threshold of the circularity of the colony is C1, and a second predetermined threshold is C2 (C1<C2), a colony satisfying C2≦C is determined as the undifferentiated colony, a colony satisfying C1≦C<C2 is determined as an undefined colony, and a colony satisfying C<C1 is determined as the differentiated colony.

Claims

1. A method for identifying a colony comprising identifying an undifferentiated colony and an undefined colony based on an image taken inside of an incubator of multipotent stem cells, wherein, when a predetermined first threshold of a circularity of the colony is C1 and a second threshold of the circularity of the colony is C2 (C1<

C2), a circularity C of the colony obtained from the image is obtained by calculation, and

a colony satisfying

C2≦

C

is determined as the undifferentiated colony containing only undifferentiated multipotent stem cells, and

a colony satisfying

C1≦

C<

C2

is determined as an undefined colony that is potentially the undifferentiated colony.

2. The method for identifying a colony according to claim 1, further comprising determining that a colony satisfying

C<

C1

is a differentiated colony containing multipotent stem cells.

3. The method for identifying a colony according to claim 2, wherein the undifferentiated colony, the undefined colony, and the differentiated colony are identified based on a luminance B of the colony in the image, in addition to the circularity.

4. The method for identifying a colony according to claim 3, wherein, when the undifferentiated colony that is identified based on the circularity C is (A), the undefined colony that is identified based on the circularity C is (B), the differentiated colony that is identified based on the circularity C is (C), a first predetermined threshold for the luminance of the colony is B1, a second predetermined threshold for the luminance of the colony is B2, a third predetermined threshold for the luminance of the colony is B3, and a fourth predetermined threshold for the luminance of the colony is B4 (B1<

B2<

B3<

B4), a colony satisfying B2≦

B≦

B3 for all luminances within a colony contour is determined as an undifferentiated colony (a), a colony satisfying B<

B1 for some of the luminances within the colony contour is determined as a multi-layered colony (d), a colony satisfying B4<

B for some of the luminances within the colony contour is determined as a differentiated colony (c), and a colony other than the undifferentiated colony (a), the multi-layered colony (d) and the differentiated colony (c) is determined as an undefined colony (b), then

a colony satisfying

(A) and (a)

is determined as the undifferentiated colony,

a colony satisfying

(A) and (b), or satisfying (B) and (a), or satisfying (B) and (b)

is determined as the undefined colony,

a colony satisfying

(d)

is determined as the multi-layered colony containing multipotent stem cells that are stacked to form multiple layers, and

a colony satisfying

(c) or (C), and (a) or (C), and (b)

is determined as the differentiated colony.

5. The method for identifying a colony according to claim 1, further comprising acquiring a fine image for the undefined colony, and identifying the undefined colony as the undifferentiated colony when a cell within the undefined colony in the fine image is smaller than a predetermined size.

6. The method for identifying a colony according to claim 1, wherein the image is subjected to image processing to accentuate a contour of the image.

7. A method for automatically culturing multipotent stem cells, comprising:

for identifying an undifferentiated colony and a colony other than the undifferentiated colony by the method for identifying a colony according to claim 1;

acquiring positional information of the undifferentiated colony and the colony other than the undifferentiated colony;

supplying a cell dissociation agent in the cultivation container;

dissociating the undifferentiated colony based on the positional information; and

recovering the undifferentiated colony obtained by the dissociation of the undifferentiated colony.

8. A method for automatically culturing multipotent stem cells, comprising:

identifying a colony other than an undifferentiated colony by the method for identifying a colony according to claim 1;

acquiring positional information of the colony other than the undifferentiated colony;

supplying a cell dissociation agent in the cultivation container;

dissociating the colony other than the undifferentiated colony based on the positional information;

removing the multipotent stems cell obtained by the dissociation of the colony other than the undifferentiated colony; and

dissociating the undifferentiated colony to recover the undifferentiated colony multipotent stem cells.

9. A method for automatically culturing multipotent stem cells, comprising:

identifying an undifferentiated colony by the method for identifying a colony according to claim 1;

acquiring a number of the undifferentiated colonies and the number of colonies other than the undifferentiated colonies; and

dissociating and recovering all colonies by supplying a cell dissociation agent in the cultivation container, when a relation;

(number of undifferentiated colonies)/(total number of colonies)≧

(predetermined threshold) is satisfied.

10. The method for automatically culturing multipotent stem cells according to claim 9, further comprising:

acquiring a fine image for undefined colonies and adding a number of colonies that are determined as the undifferentiated colonies based on the fine image to the number of the undifferentiated colonies to obtain a new number of the undifferentiated colonies, when a relation;

(number of undifferentiated colonies)/(total number of colonies)<

(predetermined threshold)

is satisfied;

supplying a cell dissociation agent to the cultivation container to dissociate and recover all colonies, when a relation;

(new number of undifferentiated colonies)/(total number of colonies)≧

(predetermined threshold) is satisfied; and

selectively dissociating and recovering the undifferentiated colonies and the undefined colonies determined as the undifferentiated colonies based on the fine image, when a relation;

(new number of undifferentiated colonies)/(total number of colonies)<

(predetermined threshold) is satisfied.

11. A device for identifying colonies, comprising:

an image acquisition means for acquiring an image taken inside of an incubator of multipotent stem cells; and

an identification means for identifying an differentiated colony and an undifferentiated colony based on a circularity C of the colony obtained from the image,

wherein, when a predetermined first threshold of the circularity of the colony is C1, and a second predetermined threshold of the circularity of the colony is C2 (C1<

C2), the identification means determines that;

a colony satisfying

C2≦

C

is the undifferentiated colony containing only undifferentiated multipotent stem cells,

a colony satisfying

C1≦

C<

C2

is an undefined colony that is potentially an undifferentiated colony, and

a colony satisfying

C<

C1

is the differentiated colony containing multipotent stem cells.

12. The device for identifying colonies according to claim 11, wherein the identification means identifies the undifferentiated colony, the undefined colony, and the undefined colony based on a luminance B of the colony in the image, in addition to the circularity.

13. The device for identifying colonies according to claim 12, wherein when the undifferentiated colony that is identified based on the circularity C is (A), the undefined colony that is identified based on the circularity C is (B), the differentiated colony that is identified based on the circularity C is (C), a first predetermined threshold for the luminance of the colony is B1, a second predetermined threshold for the luminance of the colony is B2, a third predetermined threshold for the luminance of the colony is B3, and a fourth predetermined threshold for the luminance of the colony is B4 (B1<

B2<

B3<

B4), the identification means determines a colony satisfying B2≦

B≦

B3 for all luminances within a colony contour as an undifferentiated colony (a), a colony satisfying B<

B1 for some of the luminances within the colony contour as a multi-layered colony (d), a colony satisfying B4<

B for some of the luminances within the colony contour as a differentiated colony (c), and a colony other than the undifferentiated colony (a), the multi-layered colony (d) and the differentiated colony (c) as an undefined colony (b), then,

the identification means determines that a colony satisfying

(A) and (a)

is the undifferentiated colony,

a colony satisfying

(A) and (b), or satisfying (B) and (a), or satisfying (B) and (b)

is the undefined colony,

a colony satisfying

(d)

is the multi-layered colony containing multipotent stem cells that are stacked to form multiple layers, and

a colony satisfying

(c) or (C), and (a) or (C), and (b)

is the differentiated colony.

14. The device for identifying colonies according to claim 11, further comprising a fine image acquisition means for acquiring a fine image for the undefined colony, the identification means identifies that the undefined colony is the undifferentiated colony when a cell in the undefined colony in the fine image is smaller than a predetermined size.

15. The device for identifying colonies according to claim 11, wherein the image is subjected to image processing to accentuate a contour of the image.

16. A device for automatically culturing multipotent stem cells, comprising:

a device for identifying colonies according to claim 11;

a dissociation agent supply means for supplying cell dissociation agent in the cultivation container; and

a pipetting device for dissociating the undifferentiated colony based on positional information of the respective colonies and recovering the undifferentiated colony acquired by the dissociation of the undifferentiated colony.

17. A device for automatically culturing multipotent stem cells, comprising:

a device for identifying colonies according to claim 11;

a positional information acquisition means for acquiring positional information of a colony other than the undifferentiated colony;

a dissociation agent supply means for supplying a cell dissociation agent in the cultivation container; and

a pipetting device for dissociating a colony other than the undifferentiated colony based on positional information acquired in the positional information acquisition means, removing the multipotent stem cells acquired by the dissociation of the colony other than the undifferentiated colony, and further dissociating the undifferentiated colony to recover the undifferentiated colony.

18. A device for automatically culturing multipotent stem cells, comprising:

a device for identifying colonies according to claim 11;

a colony number acquisition means for acquiring the number of the undifferentiated colonies and the number of colonies other than the undifferentiated colonies; and

a pipetting device for supplying a cell dissociation agent to the cultivation container to dissociate and recover all colonies, when a relation;

(number of undifferentiated colonies)/(total number of colonies)≧

(predetermined threshold) is satisfied.

19. The device for automatically culturing multipotent stem cells according to claim 18, further comprising a fine image acquisition means for further acquiring a fine image for each of the undefined colonies when a relation:

(number of undifferentiated colonies)/(total number of colonies)<

(predetermined threshold) is satisfied,

wherein the colony number acquisition means adds the number of colonies that are determined as the undifferentiated colonies based on the fine image to the number of the undifferentiated colonies to obtain new number of the undifferentiated colonies, and

wherein the pipetting device supplies a cell dissociation agent to the cultivation container to dissociate and recover all colonies, when a relation;

(new number of undifferentiated colonies)/(total number of colonies)≧

(predetermined threshold) is satisfied, and

wherein the pipetting device selectively dissociates and recovers the undifferentiated colonies and the undefined colonies determined as the undifferentiated colonies based on the fine image, when a relation;

(new number of undifferentiated colonies)/(total number of colonies)<

(predetermined threshold) is satisfied.