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US-12616776-B2 - Three-dimensional tissue body, method for producing same, and formation agent for three-dimensional tissue body

US12616776B2US 12616776 B2US12616776 B2US 12616776B2US-12616776-B2

Abstract

A method may comprises bringing cells suspended in an aqueous medium into contact with a plurality of fragmented collagen pieces and, after the cells brought into contact with the plurality of fragmented collagen pieces and the plurality of fragmented collagen pieces are concentrated, culturing the cells brought into contact with the fragmented collagen pieces, with the plurality of fragmented collagen pieces, to form a three-dimensional tissue.

Inventors

  • Michiya Matsusaki
  • Shinji Irie
  • Shiro Kitano

Assignees

  • TOPPAN PRINTING CO., LTD.
  • OSAKA UNIVERSITY

Dates

Publication Date
20260505
Application Date
20211015
Priority Date
20170131

Claims (17)

  1. 1 . A method, comprising: bringing cells into contact with a plurality of fragmented collagen pieces derived from exogenous collagen which is exogenous to the cells in an aqueous medium, the bringing of the cells including adding a dispersion of the plurality of fragmented collagen pieces to the aqueous medium containing the cells, or adding the cells to the aqueous medium containing a dispersion of the plurality of fragmented collagen pieces, resulting in a suspension of the cells and the plurality of fragmented collagen pieces in the aqueous medium; and culturing the cells with the plurality of fragmented collagen pieces, pieces to form a three-dimensional tissue, the culturing including standing the suspension of the cells and the plurality of fragmented collagen pieces to allow sedimentation, resulting in the plurality of fragmented collagen pieces being uniformly distributed among the cells, three-dimensionally throughout the three-dimensional tissue.
  2. 2 . The method according to claim 1 , wherein the plurality of fragmented collagen pieces have an average length of from 100 nm to 200 μm.
  3. 3 . The method according to claim 1 , wherein the plurality of fragmented collagen pieces have an average diameter of from 50 nm to 30 μm.
  4. 4 . The method according to claim 1 , wherein the cells comprise a collagen-producing cell.
  5. 5 . The method according to claim 1 , wherein the cells comprise one or more types of cells selected from a group of cells consisting of vascular endothelial cells, cancer cells, cardiomyocytes, smooth muscle cells, and epithelial cells.
  6. 6 . The method according to claim 1 , wherein a mass ratio between the plurality of fragmented collagen pieces and the cells is 900:1 to 9:1.
  7. 7 . The method according to claim 1 , wherein collagen in the three-dimensional tissue comprises 10 weight percent (wt %) to 90 wt % based on weight of the three-dimensional tissue.
  8. 8 . The method according to claim 1 , wherein a thickness of the three-dimensional tissue is 10 μm or thicker.
  9. 9 . The method according to claim 1 , further comprising: treating the three-dimensional tissue with a trypsin treatment at a trypsin concentration of 0.25%, a temperature of 37° C., and a pH of 7.4 for a reaction time of 15 min.
  10. 10 . The method according to claim 9 , wherein a residual percentage of the three-dimensional tissue after the trypsin treatment is 70% or more, the residual percentage of the three-dimensional tissue is calculated from a mass of the three-dimensional tissue before and after the trypsin treatment.
  11. 11 . A method comprising: bringing cells into contact with a plurality of fragmented collagen pieces in an aqueous medium, the bringing of the cells including adding a dispersion of the plurality of fragmented collagen pieces to the aqueous medium containing the cells, or adding the cells to the aqueous medium containing a dispersion of the plurality of fragmented collagen pieces, resulting in a suspension of the cells and the plurality of fragmented collagen pieces in the aqueous medium; culturing the cells with the plurality of fragmented collagen pieces to form a three-dimensional tissue, the culturing including standing the suspension of the cells and the plurality of fragmented collagen pieces to allow sedimentation, resulting in the plurality of fragmented collagen pieces being uniformly distributed among the cells, three-dimensionally throughout the three-dimensional tissue.
  12. 12 . The method according to claim 11 , wherein the plurality of fragmented collagen pieces are exogenous collagen pieces derived from collagen which is exogenous to the cells in the aqueous medium.
  13. 13 . The method according to claim 11 , wherein the plurality of fragmented collagen pieces are concentrated by the sedimentation.
  14. 14 . The method according to claim 11 , wherein the plurality of fragmented collagen pieces have an average length of from 100 nm to 200 μm.
  15. 15 . The method according to claim 11 , wherein the plurality of fragmented collagen pieces have an average diameter of from 50 nm to 30 μm.
  16. 16 . The method according to claim 11 , wherein the cells comprise a collagen-producing cell.
  17. 17 . The method according to claim 11 , wherein a mass ratio between the fragmented collagen pieces and the cells is 900:1 to 9:1.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation application of U.S. patent application Ser. No. 16/480,114, filed on Jul. 23, 2019, which is a U.S. National Stage Application under 35 U.S.C. § 371 of PCT International Application No. PCT/JP2018/003249, filed on Jan. 31, 2018, which claims the foreign priority benefit under 35 U.S.C. § 119 of Japanese Patent Application No. 2017-169834, filed Sep. 4, 2017, and Japanese Patent Application No. 2017-015958, filed on Jan. 31, 2017, both in the Japanese Patent Office. The entire contents of the U.S. patent application Ser. No. 16/480,114, PCT International Application No. PCT/JP2018/003249, Japanese Patent Application No. 2017-169834, and Japanese Patent Application No. 2017-015958 are hereby incorporated by reference. TECHNICAL FIELD The present invention relates to a three-dimensional tissue, a method for producing the same, and a formation agent for the three-dimensional tissue. BACKGROUND ART Recently, techniques for ex vivo construction of a cell-based three-dimensional tissue have been developed. Proposed are, for instance, a method for producing a three-dimensional tissue by culturing cells coated such that the whole surface of each cultured cell is coated with an adhesion membrane (Patent Literature 1); and a method for producing a three-dimensional tissue by seeding cells on a scaffold made of a material such as poly-lactic acid (Non Patent Literature 1). In addition, the present inventors have proposed before a method for producing a three-dimensional tissue, comprising the step of three-dimensionally arranging cells coated with a collagen-containing film to form a three-dimensional tissue (Patent Literature 2); and a method for producing a three-dimensional tissue, comprising the step of forming a coated cell having a coating film on the cell surface and the step of three-dimensionally arranging the coated cell, wherein forming the coated cell includes immersing a cell in a liquid containing a coating film component and separating the immersed cell and the liquid containing the coating film component with a liquid permeable membrane (Patent Literature 3). Such three-dimensional tissue bodies should be used as graft materials and alternatives for experimental animals, etc. CITATION LIST Patent Literature Patent Literature 1: Japanese Unexamined Patent Publication No. 2012-115254Patent Literature 2: International Publication No. WO 2015/072164Patent Literature 3: International Publication No. WO 2016/027853 Non Patent Literature Non Patent Literature 1: Nature Biotechnology, 2005, Vol. 23, NO. 7, p. 879-884 SUMMARY OF INVENTION Technical Problem The concentration of collagen such as fibrillar collagen in biological tissues is about 20 to 30 wt %. Accordingly, when a three-dimensional tissue is applied as a graft material, an alternative for an experimental animal, etc., it is preferable to prepare a three-dimensional tissue in which collagen is included at a concentration close to that of a biological tissue of interest. However, previously developed three-dimensional tissue bodies are limited to those having a very high cell density due to the restriction of their production method, etc. Thus, three-dimensional tissue bodies in which the concentration of collagen is close to that of a biological tissue have previously been unknown. The present invention has been made in light of the above situations. The purpose of the present invention is to provide a three-dimensional tissue in which the concentration of collagen is close to that of a biological tissue, a method for producing the same, and a formation agent capable of being used for production of the three-dimensional tissue. Solution to Problem The present inventors have conducted intensive research and, as a result, have found that the above problem can be solved by the following items of the present invention. [1] A three-dimensional tissue comprising cells and collagen including endogenous collagen, wherein at least a portion of the cells is adhered to the collagen, and content of the collagen is from 10 wt % to 90 wt % based on the three-dimensional tissue. [2] The three-dimensional tissue according to [1], wherein the cells comprise a collagen-producing cell. [3] The three-dimensional tissue according to [1] or [2], wherein a residual percentage of the three-dimensional tissue after trypsin treatment at a trypsin concentration of 0.25%, a temperature of 37° C., and a pH of 7.4 for a reaction time of 15 min is 70% or more. [4] The three-dimensional tissue according to any one of [1] to [3], wherein a thickness thereof is 10 μm or longer. [5] The three-dimensional tissue according to any one of [1] to [4], wherein the cells comprise one or more types of cells selected from the group consisting of vascular endothelial cells, cancer cells, cardiomyocytes, smooth muscle cells, and epithelial cells. [6] The three-dimensional tissue according to any one of [1] to [5], wher