Method for producing layered cell sheet and layered cell sheet produced by the same

Patent metadata

Field	Value
Publication number	US-2020140807-A1
Application number	US-201816627156-A
Country	US
Kind code	A1
Filing date	Jun 28, 2018
Priority date	Jun 29, 2017
Publication date	May 7, 2020
Grant date	—

Abstract

The present invention provides a method for swiftly producing a layered cell sheet that is non-invasively obtained and is utilizable for transplantation, etc., the method including (1) a step of applying a centrifugal force to a first cell sheet on a temperature-responsive culture surface for a predetermined time in a temperature range from a lower critical solution temperature of the temperature-responsive culture surface to 45° C., (2) a step of further placing a second cell sheet on the first cell sheet, and (3) a step of applying a centrifugal force to the first cell sheet and the second cell sheet on the temperature-responsive culture surface for a predetermined time in the temperature range from the lower critical solution temperature to 45° C.; and also provides a layered cell sheet obtained by the method.

First claim

1 . A method for producing a layered cell sheet on a temperature-responsive culture surface, the method comprising:
(1) a step of placing a first cell sheet on the temperature-responsive culture surface applying a centrifugal force to the first cell sheet for a predetermined time in a temperature range from a lower critical solution temperature of the temperature-responsive culture surface to 45° C., (2) a step of further placing a second cell sheet on the first cell sheet on the temperature-responsive culture surface, and (3) a step of applying a centrifugal force to the first cell sheet and the second cell sheet on the temperature-responsive culture surface for a predetermined time in the temperature range from the lower critical solution temperature to 45° C.

2 . The method according to claim 1 , further comprising:
(4) a step of repeating the steps (2) and (3) an arbitrary number of times.

3 . The method according to claim 1 , wherein the temperature-responsive culture surface is at least partially coated with poly(N-isopropylacrylamide).

4 . The method according to claim 1 , wherein the lower critical solution temperature is 32° C.

5 . The method according to claim 1 , wherein the temperature range is from 34 to 39° C.

6 . The method according to claim 1 , wherein the predetermined time is from 1 to 10 minutes.

7 . The method according to claim 1 , wherein the centrifugal force is from 25×g to 150×g.

8 . The method according to claim 1 , wherein a time until reaching the centrifugal force is from 15 to 60 seconds.

9 . The method according to claim 1 , wherein a time until ceasing the centrifugal force is from 15 to 60 seconds.

10 . The method according to claim 1 , wherein the first cell sheet and/or the second cell sheet contains one type of cells or two or more types of cells selected from the group consisting of cardiomyocytes, hepatocytes, fibroblasts, myoblasts, pancreatic cells, renal cells, vascular endothelial cells, and epithelial cells.

11 . The method according to claim 1 , further comprising:
(5) a step of releasing a layered cell sheet from the temperature-responsive culture surface by exposing the temperature-responsive culture surface to a temperature less than the lower critical solution temperature.

12 . A layered cell sheet obtained by the method according to claim 1 .

13 . The method according to claim 1 , wherein the predetermined time of the step (3) is shorter than the predetermined time of the step (1).

Assignees

• Nihon Kohden Corp
• Univ Tokyo Womens Medical

Inventors

• Kagawa Yuki
• Shimizu Tatsuya
• Haraguchi Yuji
• Kubo Hirotsugu

Classifications

• A61L27/3895

  using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions · CPC title

• C12N2533/30

  Synthetic polymers · CPC title

• C12N5/0658 Primary

  Skeletal muscle cells, e.g. myocytes, myotubes, myoblasts · CPC title

• C12N2523/00

  Culture process characterised by temperature · CPC title

• C12N5/0062 Primary

  General methods for three-dimensional culture · CPC title