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BR-112021015968-B1 - METHOD FOR PRODUCING THREE-DIMENSIONAL MUSCLE TISSUE

BR112021015968B1BR 112021015968 B1BR112021015968 B1BR 112021015968B1BR-112021015968-B1

Abstract

THREE-DIMENSIONAL MUSCLE TISSUE AND METHOD OF PRODUCING THE SAME. The present invention relates to a method of producing a three-dimensional muscle tissue including the steps of: preparing a first approximately rectangular cell module containing skeletal myoblasts in a hydrogel and having a plurality of approximately rectangular holes parallel to each other, and a second approximately rectangular cell module containing skeletal myoblasts in a hydrogel and having a plurality of approximately rectangular holes parallel to each other in positions different from those of the first cell module in a vertical direction; and alternately stacking the first prepared cell module and the second prepared cell module to obtain a stack; subjecting the skeletal myoblasts contained in the obtained stack to proliferation culture; and inducing the proliferated skeletal myoblasts to differentiate within the myotubes.

Inventors

  • Shoji Takeuchi
  • Yuya MORIMOTO
  • Ai Shima
  • MAI FURUHASHI

Assignees

  • THE UNIVERSITY OF TOKYO
  • NISSIN FOODS HOLDINGS CO., LTD

Dates

Publication Date
20260310
Application Date
20200122
Priority Date
20190304

Claims (10)

  1. 1. A method for producing a three-dimensional muscle tissue, characterized by comprising the steps of: preparing a first approximately rectangular cell module containing skeletal myoblasts in a hydrogel, and having a plurality of approximately rectangular holes, parallel to each other, and a second approximately rectangular cell module containing skeletal myoblasts in a hydrogel, and having a plurality of approximately rectangular holes, parallel to each other, at least part of the holes of the second cell module being in different positions from those of the holes of the first cell module in a vertical direction; alternately stacking the first prepared cell module and the second prepared cell module to obtain a stack; and inducing the skeletal myoblasts to differentiate within the myotubes, wherein the stacking is carried out in such a way that the holes of the first cell module do not overlap at least part of the holes of the second cell module immediately above and immediately below.
  2. 2. Production method according to claim 1, characterized in that the hydrogels each contain collagen at 0.3 mg/mL or more.
  3. 3. Production method according to claim 1 or 2, characterized in that the hydrogels each contain ascorbic acid or a salt thereof, or a derivative thereof, from about 10 μM to 1000 μM.
  4. 4. Production method, according to any one of claims 1 to 3, characterized in that the holes of the first cell module and the second cell module each have a width of about 200 to 2000 μm in a short-side direction.
  5. 5. Production method, according to any one of claims 1 to 4, characterized in that the distance between adjacent holes of each of the first cell module and the second cell module is 200 to 2000 μm in a short-side direction.
  6. 6. Production method, according to any one of claims 1 to 5, characterized in that the first cell module and the second cell module each have a size measuring 3 mm or more in a short-side direction and 9 mm or more in a long-side direction.
  7. 7. Production method, according to any one of claims 1 to 6, characterized in that the total number of first cell modules and second cell modules to be stacked is equal to 6 plus.
  8. 8. Production method according to any one of claims 1 to 7, characterized in that, when stacked, both ends of each of the first cell module and the second cell module in a long side direction are immobilized.
  9. 9. Production method according to any one of claims 1 to 8, characterized in that the production method is a production method for an edible three-dimensional muscle tissue.
  10. 10. Production method according to any one of claims 1 to 9, characterized in that the skeletal myoblasts are bovine skeletal myoblasts.

Description

Technical field [0001] The present invention basically relates to a production method for a three-dimensional muscle tissue. Antecedent Technique [0002] Along with population growth and income growth in emerging countries, there is an increasing demand for meat. Meanwhile, increasing the amount of meat supply is difficult due to problems related to rising grain prices for livestock feed and securing land for raising livestock, and thus, the development of meat analogues is expected. [0003] Cultured meat (three-dimensional muscle tissue) is produced by forming a tissue through the use of skeletal muscle cells proliferated by culture. Cultured meat can be produced in a laboratory, and thus can be produced independently of climate change. Additionally, cultured meat has a reduced environmental impact due to the low level of greenhouse gas emissions compared to conventional animal farming. Citation List Patent Literature PTL 1: JP 2018-000194 A PTL 2: JP 6427836 B2 Summary of the Invention Technical Problem [0004] In order to produce meat with a steak-like texture from cells, a mature three-dimensional muscle tissue needs to be constructed. In the field of regenerative medicine, there are reports on the construction of three-dimensional muscle tissues using human and rodent musculoskeletal cells (Patent Literature 1 and 2). However, specialized conditions for edible cultured meat still need to be sufficiently investigated. [0005] One objective of the present invention is to provide a method for producing a three-dimensional muscle tissue particularly suitable for food use. Solution to the Problem [0006] The inventors of the present invention have carried out extensive investigations with a view to the aforementioned objective, and as a result, have discovered that the objective can be achieved by a production method, including the steps of: preparing a first approximately rectangular cell module containing skeletal myoblasts in a hydrogel, and having a plurality of approximately rectangular holes parallel to each other, and a second approximately rectangular cell module containing skeletal myoblasts in a hydrogel, and having a plurality of approximately rectangular holes, parallel to each other, in positions different from those of the first cell module in a vertical direction; alternately stacking the first prepared cell module and the second prepared cell module to obtain a stack; subjecting the skeletal myoblasts contained in the obtained stack to proliferation culture; and inducing the proliferated skeletal myoblasts to differentiate within the myotubes. The present invention has been completed by carrying out further investigations based on such discoveries. [0007] The present invention encompasses the following aspects. [0008] Item 1. A production method for a three-dimensional muscle tissue including the steps of: preparing a first approximately rectangular cell module containing skeletal myoblasts in a hydrogel, and having a plurality of approximately rectangular holes, parallel to each other, and a second approximately rectangular cell module containing skeletal myoblasts in a hydrogel, and having a plurality of approximately rectangular holes, parallel to each other, at least some of the holes of the second cell module being in different positions from those of the holes of the first cell module in a vertical direction; alternately stacking the first prepared cell module and the second prepared cell module to obtain a stack; and inducing the skeletal myoblasts to differentiate within the myotubes. [0009] Item 2. The production method according to item 1, in which the hydrogels each contain collagen at 0.3 mg/mL. [0010] Item 3. The production method, according to item 1 or 2, in which the hydrogels each contain ascorbic acid or a salt thereof, or a derivative thereof, from 10 μM to 1000 μM. [0011] Item 4. The production method, according to any one of items 1 to 3, in which the holes of the first cell module and the second cell module each have a width of 200 μm to 2000 μm in a short-side direction. [0012] Item 5. The production method, according to any one of items 1 to 4, in which a distance between the adjacent holes of each of the first cell module and the second cell module is 200 μm to 2000 μm in a short-side direction. [0013] Item 6. The production method, according to any one of items 1 to 5, in which the first cell module and the second cell module each have a size measuring 3 mm or more in a short-side direction and 9 mm or more in a long-side direction. [0014] Item 7. The production method, according to any of items 1 to 6, in which the total number of first cell modules and second cell modules to be stacked is 6 or more. [0015] Item 8. The production method, according to any one of items 1 to 7, in which, in the stack, both ends of each of the first cell module and the second cell module, in a long direction, are immobilized. [0016] Item 9. The production method, according to any one of ite