JP2024018683A - Method for producing three-dimensional muscle tissue - Google Patents
Method for producing three-dimensional muscle tissue Download PDFInfo
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- JP2024018683A JP2024018683A JP2022122160A JP2022122160A JP2024018683A JP 2024018683 A JP2024018683 A JP 2024018683A JP 2022122160 A JP2022122160 A JP 2022122160A JP 2022122160 A JP2022122160 A JP 2022122160A JP 2024018683 A JP2024018683 A JP 2024018683A
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Abstract
Description
本発明は、三次元筋組織の製造方法、及び三次元筋組織を製造するための培養容器に関する。 The present invention relates to a method for producing three-dimensional muscle tissue and a culture vessel for producing three-dimensional muscle tissue.
世界的な人口増加、新興国での経済発展や食生活の多様化に伴って、世界的に食肉需要が加速している。しかしながら、家畜の生産には飼育コストもかかり、家畜生産量は無限ではないため、やがて食肉の供給が追いつかなくなることが予想されている。
そこで、食肉の安定的供給の解決策の１つとして、代替肉の開発研究がなされてきている。
Demand for meat is accelerating worldwide due to global population growth, economic development in emerging countries, and diversification of dietary habits. However, the production of livestock requires breeding costs, and the amount of livestock produced is not infinite, so it is predicted that the supply of meat will eventually not be able to keep up.
Therefore, as one solution to ensuring a stable supply of meat, research is being conducted to develop alternative meats.
家畜から生産される食肉の代替肉と一口に言ってもいろいろな種類があり、肉もどき、植物肉、培養ミンチ肉、培養ステーキ肉等に分類される。このうち、培養ミンチ肉と培養ステーキ肉は培養肉と呼ばれるが、培養ミンチ肉がバラバラの筋細胞の集合体であり食肉の食感が劣るのに対し、培養ステーキ肉は筋組織の立体構造を再現したものであり食肉の食感を感じることが可能な代替肉である。 There are various types of meat substitutes for meat produced from livestock, and they are classified into meat imitations, plant meats, cultured minced meat, cultured steak meat, etc. Among these, cultured minced meat and cultured steak meat are called cultured meat, but cultured minced meat is an aggregate of disjointed muscle cells and has an inferior texture, whereas cultured steak meat has a three-dimensional structure of muscle tissue. It is a meat substitute that has been reproduced and allows you to feel the texture of meat.
ここで、筋組織の立体構造を再現するには、すなわち、三次元筋組織を生体外で構築するためには、三次元的に細胞を培養することが必要である。しかし、三次元的に細胞を培養するのに乗り越えるべき技術的課題も多い。 Here, in order to reproduce the three-dimensional structure of muscle tissue, that is, to construct three-dimensional muscle tissue in vitro, it is necessary to culture cells three-dimensionally. However, there are many technical challenges that must be overcome in order to culture cells three-dimensionally.
引用文献１には、特に食用に適した三次元筋組織の製造方法として、ハイドロゲルに骨格筋芽細胞を含んでおり、互いに平行な略長方形の穴を複数有する略長方形の第１の細胞モジュール、及び、ハイドロゲルに骨格筋芽細胞を含んでおり、垂直方向において前記第１の細胞モジュールとは異なる位置に互いに平行な略長方形の穴を複数有する略長方形の第２の細胞モジュールを作成する工程、得られた前記第１の細胞モジュールと前記第２の細胞モジュールとを交互に積層し積層体を得る工程、得られた積層体に含まれる骨格筋芽細胞を増殖培養する工程、及び増殖した骨格筋芽細胞を筋管へと分化誘導する工程を含む、三次元筋組織の製造方法が開示されている。 Cited document 1 describes a method for producing a three-dimensional muscle tissue particularly suitable for human consumption, in which a hydrogel contains skeletal myoblasts and a substantially rectangular first cell module has a plurality of substantially rectangular holes parallel to each other. , and creating a substantially rectangular second cell module containing skeletal myoblasts in the hydrogel and having a plurality of substantially rectangular holes parallel to each other at positions different from the first cell module in the vertical direction. step, a step of alternately stacking the obtained first cell module and the second cell module to obtain a laminate, a step of proliferating and culturing skeletal myoblasts contained in the obtained laminate, and proliferation. A method for producing three-dimensional muscle tissue is disclosed, which includes a step of inducing differentiation of skeletal myoblasts into myotubes.
引用文献１に記載の技術により、特に食用に適した三次元筋組織、より具体的には、生体由来の筋肉と同様にサルコメア構造を有し、食用に供した際に畜産された肉に近い食感が期待できる三次元筋組織の製造方法が提供される。
一方で、食用に適した十分な大きさを有する三次元筋組織を簡便に製造することに関し、更なる改善が必要であった。
本発明は、食用に適した十分な大きさを有する三次元筋組織を簡便に製造することができる三次元筋組織の製造方法及び三次元筋組織を製造するための培養容器を提供することを課題とする。
The technology described in Cited Document 1 produces three-dimensional muscle tissue that is especially suitable for human consumption, and more specifically, has a sarcomere structure similar to that of living body-derived muscle, and is similar to livestock meat when used for human consumption. A method for producing three-dimensional muscle tissue with a promising texture is provided.
On the other hand, further improvements are needed regarding the simple production of three-dimensional muscle tissue that is large enough to be eaten.
The present invention aims to provide a method for producing three-dimensional muscle tissue that can easily produce three-dimensional muscle tissue having a sufficient size suitable for human consumption, and a culture container for producing three-dimensional muscle tissue. Take it as a challenge.
本発明者は、上記課題を鑑みて鋭意検討を重ねた結果、容器端部に向かい合う一対のアンカー部と、前記一対のアンカー部間にわたって複数の凸部が配列してなる培養容器を使用することにより上記の課題を解決できることを見いだした。本発明は、このような発見にさらに検討を重ねて完成したものである。 As a result of intensive studies in view of the above-mentioned problems, the present inventors have discovered the use of a culture container comprising a pair of anchor parts facing each other at the ends of the container, and a plurality of convex parts arranged between the pair of anchor parts. We have found that the above problems can be solved by The present invention has been completed through further study of these discoveries.
本発明は、以下の態様を包含する。
〔１〕 下記の工程（Ａ）及び（Ｂ）を含む三次元筋組織の製造方法：
（Ａ）筋芽細胞を含むハイドロゲルを培養容器へ供給する工程であって、
前記培養容器は、容器端部に向かい合う一対のアンカー部と、前記一対のアンカー部間にわたって複数の凸部が配列してなる培養容器である工程、並びに、
（Ｂ）前記培養容器中で、筋芽細胞を筋管へと分化誘導する工程。
〔２〕 前記培養容器において、前記凸部の長手方向の長さ（Ｘ）が１ｃｍ以上、かつ、前記凸部の短手方向の長さ（Ｙ）が１ｃｍ以上である、上記〔１〕に記載の三次元筋組織の製造方法。
〔３〕 前記培養容器において、前記凸部の長手方向の長さ（Ｘ）に対する前記凸部の短手方向の長さ（Ｙ）の比〔（Ｙ）／（Ｘ）〕が０．７以上である、上記〔１〕又は〔２〕に記載の三次元筋組織の製造方法。
〔４〕 前記凸部が、高さ５０μｍ～１ｍｍである、上記〔１〕～〔３〕のいずれか１項に記載の三次元筋組織の製造方法。
〔５〕 前記凸部の互いの間隔が、１０μｍ～１ｍｍである、上記〔１〕～〔４〕のいずれか１項に記載の三次元筋組織の製造方法。
〔６〕 上記〔１〕～〔５〕のいずれか１項に記載の製造方法により得られる三次元筋組織。
〔７〕 三次元筋組織を製造するための培養容器であって、容器端部に向かい合う一対のアンカー部と、前記一対のアンカー部間にわたって複数の凸部が配列してなる、培養容器。
The present invention includes the following aspects.
[1] A method for producing three-dimensional muscle tissue including the following steps (A) and (B):
(A) A step of supplying a hydrogel containing myoblasts to a culture container,
a step in which the culture container is a culture container including a pair of anchor portions facing each other at ends of the container, and a plurality of convex portions arranged between the pair of anchor portions, and
(B) A step of inducing differentiation of myoblasts into myotubes in the culture vessel.
[2] In the above [1], in the culture container, the length (X) of the protrusion in the longitudinal direction is 1 cm or more, and the length (Y) of the protrusion in the transverse direction is 1 cm or more. The described method for producing three-dimensional muscle tissue.
[3] In the culture container, the ratio [(Y)/(X)] of the length (Y) of the convex part in the transverse direction to the length (X) of the convex part in the longitudinal direction is 0.7 or more. The method for producing three-dimensional muscle tissue according to [1] or [2] above.
[4] The method for producing three-dimensional muscle tissue according to any one of [1] to [3] above, wherein the convex portion has a height of 50 μm to 1 mm.
[5] The method for producing three-dimensional muscle tissue according to any one of [1] to [4] above, wherein the distance between the convex portions is 10 μm to 1 mm.
[6] Three-dimensional muscle tissue obtained by the production method according to any one of [1] to [5] above.
[7] A culture container for producing three-dimensional muscle tissue, comprising a pair of anchor parts facing each other at the ends of the container, and a plurality of convex parts arranged between the pair of anchor parts.
本発明により、特に食用に適した三次元筋組織の製造方法が提供される。本発明の製造方法により製造される三次元筋組織は、生体由来の筋肉と同様に、サルコメア構造を有する。また、食用に適した十分な大きさを有する三次元筋組織を製造することができる。そのため、本発明の製造方法により製造された三次元筋組織は、食用に供した際に畜産された肉に近い食感が期待できる。 The present invention provides a method for producing three-dimensional muscle tissue that is particularly suitable for human consumption. The three-dimensional muscle tissue produced by the production method of the present invention has a sarcomere structure, similar to muscles of biological origin. Moreover, three-dimensional muscle tissue having a sufficient size suitable for consumption can be produced. Therefore, the three-dimensional muscle tissue produced by the production method of the present invention can be expected to have a texture similar to that of livestock meat when used for human consumption.
［三次元筋組織の製造方法］
本発明の三次元筋組織の製造方法は、下記の工程（Ａ）及び（Ｂ）を含む：
（Ａ）筋芽細胞を含むハイドロゲルを培養容器へ供給する工程であって、
前記培養容器は、容器端部に向かい合う一対のアンカー部と、前記一対のアンカー部間にわたって複数の凸部が配列してなる培養容器である工程、並びに、
（Ｂ）前記培養容器中で、筋芽細胞を筋管へと分化誘導する工程。
[Method for manufacturing three-dimensional muscle tissue]
The method for producing three-dimensional muscle tissue of the present invention includes the following steps (A) and (B):
(A) A step of supplying a hydrogel containing myoblasts to a culture container,
a step in which the culture container is a culture container including a pair of anchor portions facing each other at ends of the container, and a plurality of convex portions arranged between the pair of anchor portions, and
(B) A step of inducing differentiation of myoblasts into myotubes in the culture vessel.
＜三次元筋組織＞
本発明において、三次元筋組織とは、生体に由来せず、人工的に製造された筋肉を主に意味する。本発明の三次元筋組織は、筋細胞から構成される。筋細胞は、好ましくは収縮能を有する横紋筋細胞であり、具体的には骨格筋細胞又は心筋細胞である。筋細胞は、その前駆体である筋芽細胞が多核化した筋管又は筋線維の形態である。
<Three-dimensional muscle tissue>
In the present invention, three-dimensional muscle tissue mainly refers to muscles that are not derived from a living body but are artificially manufactured. The three-dimensional muscle tissue of the present invention is composed of muscle cells. The muscle cells are preferably striated muscle cells having contractile ability, specifically skeletal muscle cells or cardiac muscle cells. Myocytes are myoblast precursors in the form of multinucleated myotubes or myofibers.
一般に、筋線維は、筋肉を構成するタンパク質であるアクチンの線維（アクチンフィラメント）及び筋肉を構成するタンパク質であるミオシンの線維（ミオシンフィラメント）から構成される筋原線維を構成単位とする。さらに、筋原線維は複数のサルコメア構造が長軸方向に連なった構造を有している。サルコメアにおけるアクチン及びミオシンの相互作用（滑り込み）に基づき、筋肉の収縮及び弛緩が発生することが知られている。 In general, muscle fibers have myofibrils as constituent units, which are composed of actin filaments, which are proteins that make up muscles, and myosin filaments, which are proteins that make up muscles. Furthermore, myofibrils have a structure in which a plurality of sarcomere structures are connected in the longitudinal direction. It is known that muscle contraction and relaxation occur based on the interaction (sliding) of actin and myosin in sarcomeres.
本発明の三次元筋組織は、サルコメア構造を有する。ただし、サルコメア構造における滑り込みが生じるか否かは問わない。 The three-dimensional muscle tissue of the present invention has a sarcomere structure. However, it does not matter whether or not sliding occurs in the sarcomere structure.
三次元筋組織は、サルコメア構造を有するか否かは、公知の手法により評価することができる。例えば、サルコメア構造のＺ膜を構成するタンパク質であるｓａｒｃｏｍｅｒｉｃ α－ａｃｔｉｎｉｎ（ＳＡＡ）が存在することをＳＡＡの免疫染色により評価し、ＳＡＡ免疫染色が陽性かつＳＡＡが規則的な縞状に分布している場合にサルコメア構造を有すると判定することができる。 Whether or not a three-dimensional muscle tissue has a sarcomere structure can be evaluated by a known method. For example, the presence of sarcomeric α-actinin (SAA), a protein that constitutes the Z membrane of the sarcomere structure, was evaluated by immunostaining for SAA, and the SAA immunostaining was positive and SAA was distributed in a regular stripe pattern. If there is a sarcomere structure, it can be determined that it has a sarcomere structure.
本発明の三次元筋組織は、好ましくは食用の三次元筋組織である。例えば食用の三次元筋組織である場合、筋組織を構成する筋細胞は、好ましくは骨格筋細胞である。食用の三次元筋組織は、「培養肉」、「人工食肉」等と換言することができる。 The three-dimensional muscle tissue of the present invention is preferably an edible three-dimensional muscle tissue. For example, in the case of edible three-dimensional muscle tissue, the muscle cells constituting the muscle tissue are preferably skeletal muscle cells. Edible three-dimensional muscle tissue can be referred to as "cultured meat", "artificial meat", etc.
＜工程（Ａ）＞
工程（Ａ）は、筋芽細胞を含むハイドロゲルを培養容器へ供給する工程であって、
前記培養容器は、底部に複数の互いに平行な略長方形の凸部、及び前記凸部の長手方向の両端にアンカー部を備えた培養容器である。
<Process (A)>
Step (A) is a step of supplying a hydrogel containing myoblasts to a culture container,
The culture container has a plurality of substantially rectangular convex portions parallel to each other on the bottom, and anchor portions at both longitudinal ends of the convex portions.
〔筋芽細胞を含むハイドロゲル〕
（筋芽細胞）
筋芽細胞は、公知の手法により調製することができる。例えば、生体由来の筋組織を分解酵素（例えば、コラゲナーゼ）の処理を施して得られる初代筋芽細胞を使用することができる。
例えば、三次元組織を構成する筋細胞が骨格筋細胞であるとき、筋芽細胞は骨格筋芽細胞である。
なお、初代筋芽細胞から結合組織などの不純物を除去するためにフィルター処理を施すことができる。一方で、筋芽細胞以外の細胞を完全に除去することは必須ではなく、筋芽細胞は、筋芽細胞以外の細胞を含む混合状態で使用することができる。
[Hydrogel containing myoblasts]
(myoblast)
Myoblasts can be prepared by known techniques. For example, primary myoblasts obtained by treating muscle tissue derived from a living body with a degrading enzyme (eg, collagenase) can be used.
For example, when the muscle cells constituting the three-dimensional tissue are skeletal muscle cells, the myoblasts are skeletal myoblasts.
Note that filter processing can be performed to remove impurities such as connective tissue from primary myoblasts. On the other hand, it is not essential to completely remove cells other than myoblasts, and myoblasts can be used in a mixed state containing cells other than myoblasts.
また、筋芽細胞は、ＥＳ細胞、ｉＰＳ細胞のような万能性を有する幹細胞や筋芽細胞へ分化する能力を有する体性幹細胞から分化誘導した細胞を用いることもできる。 Further, as myoblasts, cells induced to differentiate from stem cells having pluripotency such as ES cells and iPS cells, or somatic stem cells having the ability to differentiate into myoblasts can also be used.
筋芽細胞は、ほ乳類動物、鳥類動物、は虫類動物、両生類動物、魚類動物等の脊椎動物に由来する。ほ乳類動物としては、サル、ウシ、ウマ、ブタ、ヒツジ、ヤギ、イヌ、ネコ、モルモット、ラット、マウス等の非ヒトほ乳類動物が挙げられる。鳥類動物としては、ダチョウ、ニワトリ、カモ、スズメ等が挙げられる。は虫類動物としては、ヘビ、ワニ、トカゲ、カメ等が挙げられる。両生類動物としては、カエル、イモリ、サンショウウオ等が挙げられる。魚類動物としては、サケ、マグロ、サメ、タイ、コイ等が挙げられる。三次元筋組織を食用とする場合、筋芽細胞はウシ、ブタ、ヒツジ、ヤギ、ウマ等の畜産のために飼育されるほ乳類動物に由来することが好ましく、ウシ由来であることがより好ましい。 Myoblasts are derived from vertebrates such as mammals, birds, reptiles, amphibians, and fish. Examples of mammals include non-human mammals such as monkeys, cows, horses, pigs, sheep, goats, dogs, cats, guinea pigs, rats, and mice. Examples of avian animals include ostriches, chickens, ducks, and sparrows. Examples of reptile animals include snakes, crocodiles, lizards, and turtles. Amphibians include frogs, newts, salamanders, and the like. Examples of fish animals include salmon, tuna, shark, sea bream, and carp. When the three-dimensional muscle tissue is used as food, the myoblasts are preferably derived from mammals raised for livestock such as cows, pigs, sheep, goats, and horses, and more preferably from cows.
筋芽細胞は、相同組み替え法、ＣＲＩＳＰＲ／Ｃａｓ９法等のゲノム編集の手法等により遺伝子改変をされた筋芽細胞または遺伝子改変されていない筋芽細胞を用いることができる。三次元筋組織を食用とする場合の一つの態様においては、安全性及び消費者の嗜好の観点から、筋芽細胞は遺伝子改変されていない筋芽細胞を用いることが好ましい。 As myoblasts, myoblasts that have been genetically modified by homologous recombination, CRISPR/Cas9, or other genome editing methods, or myoblasts that have not been genetically modified can be used. In one embodiment when the three-dimensional muscle tissue is used as food, it is preferable to use myoblasts that have not been genetically modified from the viewpoint of safety and consumer preference.
（ハイドロゲル）
ハイドロゲルは、三次元筋組織の培養時の足場材として機能する。
ハイドロゲルの好ましい態様の１つとしては、フィブリン、フィブロネクチン、ラミニン、コラーゲン（例えば、Ｉ型、ＩＩ型、ＩＩＩ型、Ｖ型、ＸＩ型など）、寒天、アガロース、グリコサミノグリカン、ヒアルロン酸、プロテオグリカンなどを等の細胞外基底膜マトリックスを構成する成分のゲルを使用することができる。ハイドロゲルとして市販品を使用することもできる例えば、「マトリゲル」の商品名で販売されるマウスＥＨＳ腫瘍抽出物（ＩＶ型コラーゲン、ラミニン、ヘパラン硫酸プロテオグリカンなどを含む）に基づく成分を使用することができる。
(hydrogel)
The hydrogel functions as a scaffolding material during the culture of three-dimensional muscle tissue.
One of the preferred embodiments of the hydrogel includes fibrin, fibronectin, laminin, collagen (for example, type I, type II, type III, type V, type XI, etc.), agar, agarose, glycosaminoglycan, hyaluronic acid, A gel of components constituting the extracellular basement membrane matrix, such as proteoglycans, can be used. Commercial products can also be used as hydrogels. For example, components based on mouse EHS tumor extract (containing type IV collagen, laminin, heparan sulfate proteoglycans, etc.) sold under the trade name "Matrigel" can be used. can.
なお、本明細書において「コラーゲン」は、未変性のコラーゲン及び変性したコラーゲンを包含する。変性したコラーゲンとしては、ゼラチンが例示される。 Note that in this specification, "collagen" includes undenatured collagen and denatured collagen. An example of denatured collagen is gelatin.
ハイドロゲルの別の好ましい態様の１つとして、血液由来血漿及び凝固剤を含むゲルが挙げられる。
血液の由来動物は、入手容易性の観点から、ウシ、ブタ、ヒツジ、ヤギ、ウマ等の畜産のために飼育されるほ乳類動物に由来であり、より好ましくはウシ由来である。
由来動物は分娩前の胎児であっても、又は、分娩後の成体のいずれであってもよい。由来動物の日齢、月齢、年齢は限定されない。成体である場合、雌性個体から分娩された後の、幼い個体、若い個体、成熟した個体、老いた個体のいずれであってもよい。
好ましい態様において由来動物は成体のウシであり、中でも市場からの入手のしやすさ及び屠畜解体される量から、より好ましくは成熟したウシである。
成牛の血液は、食肉市場や卸売市場において屠畜解体される牛から検体として採取する方法や、その他の方法により得られる。
Another preferred embodiment of the hydrogel includes a gel containing blood-derived plasma and a coagulant.
From the viewpoint of availability, the animal from which the blood is derived is derived from a mammalian animal raised for livestock production such as a cow, pig, sheep, goat, or horse, and is more preferably derived from a cow.
The animal of origin may be a fetus before parturition or an adult after parturition. The age in days, months, and age of the source animal is not limited. In the case of an adult, it may be a young individual, a young individual, a mature individual, or an old individual after delivery from a female individual.
In a preferred embodiment, the source animal is an adult cow, and more preferably an adult cow from the viewpoint of easy availability on the market and the amount to be slaughtered and slaughtered.
Blood from adult cows can be obtained by collecting specimens from slaughtered cows at meat and wholesale markets, or by other methods.
血液は、流通や保管時の凝固を防ぐため、抗凝固剤を添加してから配送することがある。抗凝固剤が添加された血液は、遠心分離により、赤血球と、フィブリノーゲンを含む血漿に分離される。ここで、血液に抗凝固剤が添加されている場合は、赤血球も、凝固成分となるフィブリノーゲンを含む血漿も、凝固及びゲル化しない。 To prevent blood from clotting during distribution and storage, anticoagulants may be added to blood before delivery. Blood to which an anticoagulant has been added is separated into red blood cells and plasma containing fibrinogen by centrifugation. Here, if an anticoagulant is added to the blood, neither red blood cells nor plasma containing fibrinogen, which is a coagulation component, will coagulate or gel.
遠心分離により得られたフィブリノーゲンを含む血液由来血漿に、凝固剤を添加すると、血漿はゲル化する。
なお、本態様のハイドロゲルは、血漿を含むため、細胞培養時の栄養分たる細胞培養成分の補給機能も期待される。
When a coagulant is added to blood-derived plasma containing fibrinogen obtained by centrifugation, the plasma gels.
In addition, since the hydrogel of this embodiment contains plasma, it is also expected to have a function of replenishing cell culture components, which are nutrients during cell culture.
また、上記フィブリノーゲンを含む成牛血液由来血漿は、多血小板血漿（ＰＲＰ血漿；Ｐｌａｔｅｌｅｔ－ｒｉｃｈ Ｐｌａｓｍａ）であってもよい。多血小板血漿は、血漿を遠心分離して調製した、血小板に富む血漿濃縮物であり、血小板の他、可溶性タンパク質や成長因子が濃縮されている。 Further, the adult bovine blood-derived plasma containing fibrinogen may be platelet-rich plasma (PRP plasma). Platelet-rich plasma is a platelet-rich plasma concentrate prepared by centrifuging plasma, and is enriched with platelets as well as soluble proteins and growth factors.
多血小板血漿は、抗凝固剤が添加された血液の遠心分離、二重遠心分離、選択的濾過等で回収することができる。具体的には赤血球を除いた血漿をさらに遠心分離して回収してもよいし、赤血球も含まれる血液では血漿層のうち赤血球との境界に濃縮した層を回収してもよい。 Platelet-rich plasma can be collected by centrifugation, double centrifugation, selective filtration, etc. of blood to which an anticoagulant has been added. Specifically, plasma from which red blood cells have been removed may be further centrifuged and collected, or in the case of blood that also contains red blood cells, a layer of the plasma layer concentrated at the boundary with red blood cells may be collected.
多血小板血漿を用いた場合は、血漿全体を用いた場合に比べ、ゲル化が速やかに進む傾向にある。このため、多血小板血漿を用いた細胞培養用ゲルでは、細胞の沈殿が起こりにくく、細胞をより均一に分散させることができる。 When platelet-rich plasma is used, gelation tends to proceed more quickly than when the whole plasma is used. Therefore, in cell culture gels using platelet-rich plasma, cell precipitation is less likely to occur, and cells can be more uniformly dispersed.
抗凝固剤は、血液の凝固を防ぐものであれば限定されないが、クエン酸ナトリウム、ＥＤＴＡ（エチレンジアミン四酢酸）、フッ化ナトリウム等の、血液の凝固に不可欠なカルシウムイオンと結合するものが例示される。
クエン酸ナトリウムの場合、血液及び抗凝固剤全量に対し、２体積％～４体積％、好ましくは、３体積％前後となるように添加することがより望ましい。
Anticoagulants are not limited as long as they prevent blood coagulation, but examples include those that bind calcium ions essential for blood coagulation, such as sodium citrate, EDTA (ethylenediaminetetraacetic acid), and sodium fluoride. Ru.
In the case of sodium citrate, it is more desirable to add it in an amount of 2% to 4% by volume, preferably around 3% by volume, based on the total amount of blood and anticoagulant.
凝固剤は、抗凝固剤を含む血液の血漿をゲル化できるものであれば限定されないが、塩化カルシウム、ＤＭＥＭ（Ｄｕｌｂｅｃｃｏ’ｓ Ｍｏｄｉｆｉｅｄ Ｅａｇｌｅ’ｓ Ｍｅｄｉｕｍ）等が例示され、カルシウムイオンを含有するものがより好ましい。 The coagulant is not limited as long as it can gel blood plasma containing an anticoagulant, but examples include calcium chloride, DMEM (Dulbecco's Modified Eagle's Medium), and those containing calcium ions. More preferred.
凝固剤が塩化カルシウムの場合、血漿及び凝固剤全量に対し、カルシウムイオン濃度として５ｍＭ～７０ｍＭ、より好ましくは１０ｍＭ～６５ｍＭ、さらに好ましくは１０ｍＭ～６０ｍＭとなるように添加することができる。
なお、本明細書において。「ＸＸ～ＹＹ」は、「ＸＸ以上ＹＹ以下」を意味する。
When the coagulant is calcium chloride, it can be added to the total amount of plasma and coagulant so that the calcium ion concentration is 5mM to 70mM, more preferably 10mM to 65mM, and even more preferably 10mM to 60mM.
Note that in this specification. "XX to YY" means "more than or equal to XX and less than or equal to YY."
ＤＭＥＭには、Ｌ－アルギニン、Ｌ－シスチン、Ｌ－ヒスチジン、Ｌ－イソロイシン、Ｌ－ロイシン、Ｌ－リシン、Ｌ－フェニルアラニン、Ｌ－トレオニン、Ｌ－トリプトファン、Ｌ－チロシン、Ｌ－バリン、塩化カルシウム、塩化カリウム、硫酸マグネシウム、ナトリウム塩化物、リン酸二水素ナトリウム、Ｄ－グルコース、葉酸、ニコチンアミド、リボフラビン、ビタミンＢ１２、コリン、イノシトール、パントテン酸、ピリドキサールリン酸、チアミン、鉄等が含まれる。
凝固剤がＤＭＥＭの場合、ＤＭＥＭの量に対し、血液由来血漿の量を、０．１体積％以上３５体積％以下、好ましくは０．１体積％以上３０体積％以下、より好ましくは０．２体積％以上３０体積％以下となるように添加することができる。
DMEM contains L-arginine, L-cystine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-phenylalanine, L-threonine, L-tryptophan, L-tyrosine, L-valine, and calcium chloride. , potassium chloride, magnesium sulfate, sodium chloride, sodium dihydrogen phosphate, D-glucose, folic acid, nicotinamide, riboflavin, vitamin B12, choline, inositol, pantothenic acid, pyridoxal phosphate, thiamine, iron, and the like.
When the coagulant is DMEM, the amount of blood-derived plasma is 0.1 volume% or more and 35 volume% or less, preferably 0.1 volume% or more and 30 volume% or less, and more preferably 0.2 volume%, based on the amount of DMEM. It can be added in an amount of at least 30% by volume.
ハイドロゲルは、さらに培地成分を含んでもよい。
培地成分としては、ＤＭＥＭ（例えば、ＧＩＢＣＯ社製等）や、ＥＭＥＭ（例えば、ＧＩＢＣＯ社製）、ＭＥＭＡＬＰＨＡ（例えば、ＧＩＢＣＯ社製）、ＲＰＭＩ－１６４０（Ｒｏｓｗｅｌｌ Ｐａｒｋ Ｍｅｍｏｒｉａｌ Ｉｎｓｔｉｔｕｔｅ １６４０培地；ＧＩＢＣＯ社製）等が挙げられる。
さらに培地成分には、通常培地で使用する添加剤成分を適宜配合することができる。添加剤成分としては、抗生物質の他、ビタミン類、核酸、アミノ酸、無機塩、糖、ポリアミン、炭水化物、タンパク質、脂肪酸、脂質、ｐＨ調整剤、亜鉛、銅、セレン等が例示される。
The hydrogel may further include a medium component.
Medium components include DMEM (for example, manufactured by GIBCO), EMEM (for example, manufactured by GIBCO), MEMALPHA (for example, manufactured by GIBCO), RPMI-1640 (Roswell Park Memorial Institute 1640 medium; manufactured by GIBCO), etc. can be mentioned.
Furthermore, additive components commonly used in culture media can be appropriately blended into the culture medium components. Examples of additive components include antibiotics, vitamins, nucleic acids, amino acids, inorganic salts, sugars, polyamines, carbohydrates, proteins, fatty acids, lipids, pH adjusters, zinc, copper, selenium, and the like.
ハイドロゲルにおける筋芽細胞は、例えば細胞密度が約１．０×１０６個／ｍｌ以上、好ましくは約１．０×１０７個／ｍｌ～約１．０×１０８個／ｍｌ、さらに好ましくは５．０×１０７個／ｍｌ～約１．０×１０８個／ｍｌであることが好ましい。 The myoblasts in the hydrogel have a cell density of, for example, about 1.0 x 10 6 cells/ml or more, preferably about 1.0 x 10 7 cells/ml to about 1.0 x 10 8 cells/ml, more preferably is preferably 5.0×10 7 cells/ml to about 1.0×10 8 cells/ml.
（培養容器）
本発明で使用する培養容器は、三次元筋組織を製造するための培養容器であって、容器端部に向かい合う一対のアンカー部と、前記一対のアンカー部間にわたって複数の凸部が配列してなる培養容器である。
該凸部を備えることで、培養時に筋管及び筋線維の束の配向が効率的に形成されると考えられる。
(Culture container)
The culture container used in the present invention is a culture container for producing three-dimensional muscle tissue, and includes a pair of anchor portions facing each other at the ends of the container, and a plurality of convex portions arranged between the pair of anchor portions. This is a culture container.
It is thought that by providing the convex portion, the orientation of myotubes and myofiber bundles is efficiently formed during culture.
本発明を実施するための好適な実施形態について、図面を用いて説明する。
図１は、本実施形態に係る培養容器１００の構成例の斜視図を示す。図２は、培養容器１００の断面図を示す。
Preferred embodiments for carrying out the present invention will be described with reference to the drawings.
FIG. 1 shows a perspective view of a configuration example of a culture container 100 according to the present embodiment. FIG. 2 shows a cross-sectional view of the culture container 100.
培養容器１００を構成する素材は限定されない。例えば、培養容器１００を構成する素材は熱可塑性樹脂又は熱硬化性樹脂である。熱可塑性樹脂としては、ポリプロピレン等のポリオレフィン系樹脂；ポリエチレンテレフタレート等のポリエステル系樹脂；アクリル系樹脂；熱可塑性エラストマー：ポリジメチルシロキサン（ＰＤＭＳ）等のシリコーン系樹脂等が挙げられる。
また、培養容器１００を構成する素材は、好ましくは細胞に対して非接着性の材質又は非接着性となる表面処理が施されている素材である。非接着性となる表面処理としては、パリレンコーティングが挙げられる。
培養容器１００を構成する素材は、透明又は不透明のいずれであってもよい。観察の容易性の観点から、好ましくは透明である。
培養容器１００は、例えば３Ｄプリンターを用いて製造することができる。
The material constituting the culture container 100 is not limited. For example, the material constituting the culture container 100 is a thermoplastic resin or a thermosetting resin. Examples of the thermoplastic resin include polyolefin resins such as polypropylene; polyester resins such as polyethylene terephthalate; acrylic resins; thermoplastic elastomers: silicone resins such as polydimethylsiloxane (PDMS), and the like.
Further, the material constituting the culture container 100 is preferably a material that is non-adhesive to cells or a material that has been surface-treated to become non-adhesive. Non-adhesive surface treatments include parylene coatings.
The material constituting the culture container 100 may be transparent or opaque. From the viewpoint of ease of observation, it is preferably transparent.
The culture container 100 can be manufactured using, for example, a 3D printer.
培養容器は好ましくは四角形である。この場合、前記凸部の長手方向の長さ（Ｘ）が好ましくは１ｃｍ以上、より好ましくは１．５ｃｍ以上、かつ、前記凸部の短手方向の長さ（Ｙ）が好ましくは１ｃｍ以上、より好ましくは２ｃｍ以上である。凸部の長手方向とは、後述する一対のアンカー部が向かい合う方向であり、凸部の短手方向とは凸部が配列される方向である。
また、前記凸部の長手方向の長さ（Ｘ）に対する前記凸部の短手方向の長さ（Ｙ）の比〔（Ｙ）／（Ｘ）〕は、好ましくは０．７以上である。〔（Ｙ）／（Ｘ）〕は、１以上、１．５以上、２以上とすることもできる。上限は特に限定されないが、例えば４以下である。
The culture vessel is preferably rectangular. In this case, the length (X) of the protrusion in the longitudinal direction is preferably 1 cm or more, more preferably 1.5 cm or more, and the length (Y) of the protrusion in the lateral direction is preferably 1 cm or more, More preferably, it is 2 cm or more. The longitudinal direction of the convex portion is the direction in which a pair of anchor portions, which will be described later, face each other, and the lateral direction of the convex portion is the direction in which the convex portions are arranged.
Further, the ratio [(Y)/(X)] of the length (Y) of the convex portion in the transverse direction to the length (X) of the convex portion in the longitudinal direction is preferably 0.7 or more. [(Y)/(X)] can also be 1 or more, 1.5 or more, or 2 or more. The upper limit is not particularly limited, but is, for example, 4 or less.
培養容器１００は、後述する一対のアンカー部２０間にわたって複数の凸部が配列してなる。
アンカー部の形状は限定されない。例えば、培養容器の上部からの平面視の形状が略長方形である。
The culture container 100 has a plurality of convex portions arranged between a pair of anchor portions 20, which will be described later.
The shape of the anchor part is not limited. For example, the culture container has a substantially rectangular shape when viewed from above.
複数の凸部が配列する態様は限定されない。好ましくは、複数の凸部は底部に互いに平行となるように設けられている。
凸部１０は、好ましくは高さ５０μｍ～１ｍｍ、より好ましくは１００μｍ～３００μｍである。
また、前記凸部の互いの間隔が、１０μｍ～５ｍｍ、より好ましくは５０μｍ～２．５ｍｍ、更に好ましくは１００μｍ～１ｍｍである。
The manner in which the plurality of convex portions are arranged is not limited. Preferably, the plurality of convex portions are provided on the bottom so as to be parallel to each other.
The height of the convex portion 10 is preferably 50 μm to 1 mm, more preferably 100 μm to 300 μm.
Further, the distance between the convex portions is 10 μm to 5 mm, more preferably 50 μm to 2.5 mm, and even more preferably 100 μm to 1 mm.
本発明で使用する培養容器は、容器端部に向かい合う一対のアンカー部を備える。アンカー部は、ハイドロゲル及び製造される三次元筋組織を固定する手段であれば、特に限定されない。例えば、接着力を有する成分（例えば、フィブリン等のハイドロゲル）を用いて固定する部材が挙げられる。
図１に示す好ましい態様である培養容器１００は、容器端部のそれぞれに複数の円柱状からなるのアンカー部２０が向かい合っている。円柱状のアンカー部２０は、直径が１００μｍ～２ｍｍ程度、高さが３ｍｍ～１０ｍｍ程度である。
The culture container used in the present invention includes a pair of anchor portions facing each other at the ends of the container. The anchor part is not particularly limited as long as it is a means for fixing the hydrogel and the manufactured three-dimensional muscle tissue. For example, a member that is fixed using a component having adhesive strength (for example, hydrogel such as fibrin) can be mentioned.
In the preferred embodiment of the culture container 100 shown in FIG. 1, a plurality of cylindrical anchor portions 20 face each other at each end of the container. The cylindrical anchor portion 20 has a diameter of approximately 100 μm to 2 mm and a height of approximately 3 mm to 10 mm.
図４～９は、他の実施形態に係る培養容器２００、培養容器３００、及び培養容器４００の構成例を斜視図、並びに、平面図、正面図、右側面図及び底面図により示す。 4 to 9 show configuration examples of a culture container 200, a culture container 300, and a culture container 400 according to other embodiments using a perspective view, a top view, a front view, a right side view, and a bottom view.
（ハイドロゲルの供給）
筋芽細胞を含むハイドロゲルは、上記培養容器に供給される。好ましくは、培養容器を底面とし、側面に型枠を設けて、筋芽細胞を含むハイドロゲルを供給する。
供給する筋芽細胞を含むハイドロゲルの量は適宜設定することができる。例えば、ハイドロゲルの厚さが好ましくは１ｍｍ～１ｃｍ、より好ましくは２～５ｍｍとなる量とすることができる。
(Hydrogel supply)
A hydrogel containing myoblasts is supplied to the culture vessel. Preferably, a culture container is used as the bottom surface, a mold is provided on the side surface, and the hydrogel containing myoblasts is supplied.
The amount of hydrogel containing myoblasts to be supplied can be set appropriately. For example, the amount may be such that the thickness of the hydrogel is preferably 1 mm to 1 cm, more preferably 2 to 5 mm.
培養容器に供給されたハイドロゲルは、加温し固化することができる。加温の温度範囲は、好ましくは３７℃程度である。また、加温時間は、ゲル化の進み具合で調整されるが、５分～６０分程度が例示され、より好ましくは約１０分程度である。 The hydrogel supplied to the culture container can be heated and solidified. The temperature range for heating is preferably about 37°C. Further, the heating time is adjusted depending on the progress of gelation, and is exemplified to be about 5 minutes to 60 minutes, and more preferably about 10 minutes.
また、筋芽細胞を含むハイドロゲルは、増殖培養に供し、含まれる筋芽細胞を細胞増殖させることができる。
例えば、筋芽細胞がハイドロゲルに十分量（例えば、１．０×１０８個／ｍ以上）含まれる場合、増殖培養を行うことなく次の分化誘導の工程を行うことができる。例えば、筋芽細胞を増殖させる必要がある場合は、増殖培養を行った後に次の分化誘導の工程を行うことができる。
Furthermore, the hydrogel containing myoblasts can be subjected to proliferation culture to allow the myoblasts contained therein to proliferate.
For example, if the hydrogel contains a sufficient amount of myoblasts (for example, 1.0×10 8 cells/m or more), the next step of inducing differentiation can be performed without performing proliferation culture. For example, if it is necessary to proliferate myoblasts, the next step of inducing differentiation can be performed after proliferation culture.
上記培養は、例えば上記の増殖培養用の培地中で、当業者に公知の手法で行なうことができる。好適な培養を行なう手法として、約３７℃程度および二酸化炭素濃度約５～１０％（ｖ／ｖ）程度の条件下で培養する手法が例示されるが、これに限定されるものではない。上記条件での培養は、例えば公知のＣＯ２インキュベータを用いて行なうことができる。 The above-mentioned culture can be carried out, for example, in the above-mentioned growth culture medium by a method known to those skilled in the art. An example of a suitable culture method is a method of culturing under conditions of about 37° C. and a carbon dioxide concentration of about 5 to 10% (v/v), but is not limited thereto. Cultivation under the above conditions can be performed using, for example, a known CO 2 incubator.
増殖培養用の培地としては、ＤＭＥＭ（Ｄｕｌｂｅｃｃｏ’ｓ Ｍｏｄｉｆｉｅｄ Ｅａｇｌｅ’ｓ Ｍｅｄｉｕｍ）、ＥＭＥＭ（Ｅａｇｌｅ’ｓ ｍｉｎｉｍａｌ ｅｓｓｅｎｔｉａｌ ｍｅｄｉｕｍ）、αＭＥＭ（ａｌｐｈａ Ｍｏｄｉｆｉｅｄ Ｍｉｎｉｍｕｍ Ｅｓｓｅｎｔｉａｌ Ｍｅｄｉｕｍ）などの通常の液体培地に、血清成分（例えば、ウマ血清（Ｈｏｒｓｅ Ｓｅｒｕｍ）ウシ胎児血清（Ｆｅｔａｌ Ｂｏｖｉｎｅ Ｓｅｒｕｍ（ＦＢＳ））、ヒト血清（Ｈｕｍａｎ Ｓｅｒｕｍ）など）、成長因子等の成分；ペニシリン、ストレプトマイシン等の抗生物質を添加した培地を使用することができる。 Examples of growth culture media include DMEM (Dulbecco's Modified Eagle's Medium), EMEM (Eagle's minimal essential medium), and αMEM (alpha Modified Minimum Essenti). Serum components (e.g. Components such as horse serum (Horse Serum, Fetal Bovine Serum (FBS), Human Serum, etc.), growth factors; A medium supplemented with antibiotics such as penicillin and streptomycin may be used. can.
増殖培養用の培地に血清成分を添加する場合、血清成分としてはウシ胎児血清を用いることができる。血清成分の濃度は１０％（ｖ／ｖ）程度とすることができる。 When a serum component is added to a growth culture medium, fetal bovine serum can be used as the serum component. The concentration of serum components can be about 10% (v/v).
培養期間は、例えば、１日～２週間程度とすることができる。 The culture period can be, for example, about 1 day to 2 weeks.
必要に応じて、培地交換を行うことができる。培養条件は、常法に準じることができる。 Culture medium can be replaced if necessary. Culture conditions can be based on conventional methods.
＜工程（Ｂ）＞
工程（Ｂ）は、前記培養容器中で、筋芽細胞を筋管へと分化誘導する工程である。
当該工程により、筋芽細胞は周囲の細胞と細胞融合により多核化し、筋管が形成される。筋管はさらに成熟することで筋線維を形成する。
<Process (B)>
Step (B) is a step of inducing differentiation of myoblasts into myotubes in the culture vessel.
Through this step, myoblasts become multinucleated by cell fusion with surrounding cells, and myotubes are formed. Myotubes further mature to form muscle fibers.
上記培養は、例えば分化誘導用（多核化用培地）の培地中で、当業者に公知の手法で行なうことができる。好適な培養を行なう手法として、約３７℃程度および二酸化炭素濃度約５～１０％（ｖ／ｖ）程度の条件下で培養する手法が例示されるが、これに限定されるものではない。上記条件での培養は、例えば公知のＣＯ２インキュベータを用いて行なうことができる。 The above culture can be performed, for example, in a medium for inducing differentiation (medium for multinucleation) by a method known to those skilled in the art. An example of a suitable culture method is a method of culturing under conditions of about 37° C. and a carbon dioxide concentration of about 5 to 10% (v/v), but is not limited thereto. Cultivation under the above conditions can be performed using, for example, a known CO 2 incubator.
筋芽細胞は栄養分が少なくなると，周囲の細胞を巻きこみ多核化を開始することが知られている。そのため、筋管への分化誘導は、前記の増殖培養よりも栄養分が少ない培地を用いて行うことができる It is known that when myoblasts become depleted of nutrients, they engulf surrounding cells and begin to become multinucleated. Therefore, differentiation into myotubes can be induced using a medium containing fewer nutrients than the aforementioned proliferation culture.
かくして三次元筋組織が製造される。
本発明は、本発明の製造方法により得られる三次元筋組織にも関する。
A three-dimensional muscle tissue is thus produced.
The present invention also relates to a three-dimensional muscle tissue obtained by the production method of the present invention.
次に実施例により本発明を更に具体的に説明する。しかし下記の実施例は本発明の範囲を限定するものではない。 Next, the present invention will be explained in more detail with reference to Examples. However, the following examples are not intended to limit the scope of the invention.
実施例１：ウシ筋芽細胞を用いた収縮可能な大型筋組織の作製
ウシ筋芽細胞を下記に示す組成のハイドロゲルに包埋し、両端を固定し培養することにより長さ７ｍｍの筋組織を作製した。ハイドロゲル組成および細胞密度を変化させ、筋組織の形成条件並びに成熟条件を検討した。
Example 1: Preparation of large contractile muscle tissue using bovine myoblasts Bovine myoblasts were embedded in a hydrogel with the composition shown below, and both ends were fixed and cultured to produce a muscle tissue with a length of 7 mm. was created. We investigated the formation and maturation conditions of muscle tissue by changing the hydrogel composition and cell density.
（培養容器１００の作製）
３Ｄプリンター（ＢＬＵＬＥ Ｉｎｃ社製、Ｆｏｒｍｌａｂ３Ｂ）を用いて図１０に示す培養容器１００を作成した。培養容器１００は凸部の長手方向の長さ（Ｘ）が４ｃｍ、凸部の短手方向の長さ（Ｙ）が３ｃｍであった。筋細胞の配向を促すための凸部１０は高さが２００μｍ及び幅５μｍであり、アンカー部と２ｍｍ離し、長手方向の長さが３ｃｍであった。
アンカー部２０は、直径０．５ｍｍ、高さ５ｍｍの円柱状であり、中心点１ｍｍ間隔で設けた。
作成した培養容器の表面を、パリレン蒸着装置（Ｓｐｅｃｉａｌｉｔｙ Ｃｏａｔｉｎｇ Ｓｙｓｔｅｍ社製、ラボコーターＰＤＳ２０１０）を用いて表面をパリレンコーティングした。次いで、オゾン滅菌装置を用いて培養容器を滅菌後、細胞接着を促すためアンカー部２０にフィブロネクチンコーティングを施した。
(Preparation of culture container 100)
A culture container 100 shown in FIG. 10 was created using a 3D printer (Formlab 3B, manufactured by BLULE Inc.). In the culture container 100, the length (X) of the protrusion in the longitudinal direction was 4 cm, and the length (Y) of the protrusion in the transverse direction was 3 cm. The convex portion 10 for promoting the orientation of muscle cells had a height of 200 μm and a width of 5 μm, was separated from the anchor portion by 2 mm, and had a longitudinal length of 3 cm.
The anchor portions 20 had a cylindrical shape with a diameter of 0.5 mm and a height of 5 mm, and were provided at intervals of 1 mm between centers.
The surface of the created culture container was coated with parylene using a parylene vapor deposition device (Lab coater PDS2010, manufactured by Specialty Coating Systems). Next, after sterilizing the culture container using an ozone sterilizer, the anchor portion 20 was coated with fibronectin to promote cell adhesion.
（対照培養容器の作製）
凸部を設けない以外は培養容器１００と同様にして、対照培養容器を作成した。
(Preparation of control culture container)
A control culture container was created in the same manner as culture container 100 except that no convex portion was provided.
（ハイドロゲル組成（１））
フィブリノーゲン（２５ｍｇ／ｍＬ） ４８０μＬ（終濃度４ｍｇ／ｍＬ）（Ｓｉｇｍａ社製、Ｆ８６３０）
マトリゲル ６００μＬ（終濃度２０％）（Ｃｏｒｎｉｎｇ社製、３５６２３１）
Ｔｈｒｏｍｂｉｎ（２００Ｕｎｉｔ／ｍＬ） ３０μＬ（終濃度２Ｕｎｉｔ／ｍＬ）（Ｓｉｇｍａ社製、Ｔ４６４８－１０ＫＵ）
ＤＭＥＭ １８９０μＬ（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ社製、１１９６５１１８）
(Hydrogel composition (1))
Fibrinogen (25mg/mL) 480μL (final concentration 4mg/mL) (Sigma, F8630)
Matrigel 600 μL (final concentration 20%) (Corning, 356231)
Thrombin (200 Unit/mL) 30 μL (final concentration 2 Unit/mL) (manufactured by Sigma, T4648-10KU)
DMEM 1890 μL (manufactured by Thermo Fisher Scientific, 11965118)
（ハイドロゲルの作製）
筋芽細胞３．０×１０７ｃｅｌｌｓを、１．０×１０７ｃｅｌｌｓ／ｍＬとなるようにハイドロゲルと混合し、細胞を包埋したハイドロゲルを作製した。培養容器１００又は対照培養容器を底面とし、側面に型枠を設けて、そこに得られた細胞を包埋したハイドロゲルを流し込んだ。３７℃、３０分間、ＣＯ２インキュベータ内で静置し、ハイドロゲルを固化した。
(Preparation of hydrogel)
3.0×10 7 cells of myoblasts were mixed with the hydrogel at 1.0×10 7 cells/mL to prepare a hydrogel in which the cells were embedded. The culture container 100 or the control culture container was used as the bottom, a mold was provided on the side, and the obtained hydrogel in which cells were embedded was poured into the mold. The hydrogel was left standing in a CO 2 incubator at 37° C. for 30 minutes to solidify the hydrogel.
（培養）
次いで、増殖用培地（１０％ＦＢＳ ＤＭＥＭ＋１％Ｐｅｎｉｃｉｌｌｉｎ／Ｓｔｒｅｐｔｏｍｙｃｉｎ）を添加し、加えて３７℃、５％ＣＯ２で２日間培養した。
その後、培地を分化用培地（２％ＨＳ ＤＭＥＭ＋１％Ｐｅｎｉｃｉｌｌｉｎ／Ｓｔｒｅｐｔｏｍｙｃｉｎ＋１００μＭ）に交換し、さらに５日間培養を継続し、三次元筋組織を得た。培地は隔日で交換した。
培養の時間経過に伴い、三次元筋組織は凸部の短手方向に収縮（シュリンク）した。
(culture)
Next, a growth medium (10% FBS DMEM + 1% Penicillin/Streptomycin) was added and cultured for 2 days at 37° C. and 5% CO 2 .
Thereafter, the medium was replaced with a differentiation medium (2% HS DMEM + 1% Penicillin/Streptomycin + 100 μM), and culture was continued for an additional 5 days to obtain three-dimensional muscle tissue. The medium was changed every other day.
As the culture progressed over time, the three-dimensional muscle tissue contracted (shrinked) in the short direction of the convex part.
（電気刺激）
培養５日目及び７日目に得られた三次元筋組織に対して、電気刺激培養システムＣ－Ｐａｃｅ（ＩｏｎＯｐｔｉｃ社製）を用いて、下記条件の電気刺激を与えた：頻度：１Ｈｚ、強さ：０．３Ｖ／ｍｍ、持続時間：４０ｍｓ。顕微鏡観察により、電気刺激に応答した収縮運動の有無を観察した。
培養容器１００を用いて得た三次元筋組織において、明確な収縮運動が観察された。一方、対照培養容器を用いて得た三次元筋組織においては、収縮運動は観察できなかった。
(electrical stimulation)
Electrical stimulation was applied to the three-dimensional muscle tissue obtained on the 5th and 7th day of culture using the electrical stimulation culture system C-Pace (manufactured by IonOptic) under the following conditions: frequency: 1 Hz, strong Power: 0.3V/mm, Duration: 40ms. The presence or absence of contraction movement in response to electrical stimulation was observed by microscopic observation.
Clear contraction movement was observed in the three-dimensional muscle tissue obtained using the culture vessel 100. On the other hand, no contraction movement could be observed in the three-dimensional muscle tissue obtained using the control culture vessel.
（ＳＡＡ免疫染色）
７日間経過後、得られた三次元筋組織を４％ＰＦＡで固定し、Ｓａｒｃｏｍｅｒｉｃ α－ａｃｔｉｎｉｎ （ＳＡＡ）の免疫染色及びＨｏｅｃｈｓｔ３３３４２を用いた細胞核染色を行った。
顕微鏡観察の結果を図１１に示す。
培養容器１００を用いて得た三次元筋組織（左図）では、ＳＡＡで染色される筋管領域が多数観察され、また筋管領域は互いに配向していた。一方、対照培養容器を用いて得た三次元筋組織（右図）では、観察される筋管領域は少なく、かつ、配向性が乏しかった。
(SAA immunostaining)
After 7 days, the obtained three-dimensional muscle tissue was fixed with 4% PFA, and immunostaining for Sarcomeric α-actinin (SAA) and cell nucleus staining using Hoechst 33342 were performed.
The results of microscopic observation are shown in FIG.
In the three-dimensional muscle tissue (left figure) obtained using the culture vessel 100, many myotube regions stained with SAA were observed, and the myotube regions were oriented with respect to each other. On the other hand, in the three-dimensional muscle tissue obtained using the control culture vessel (right figure), there were few myotube regions observed and poor orientation.
以上の結果から、培養容器の凸構造によって、三次元筋組織中の筋芽細胞から筋管への細胞分化及び筋管の配向が促されることが示唆された。 The above results suggested that the convex structure of the culture container promoted cell differentiation from myoblasts to myotubes in three-dimensional muscle tissue and the orientation of myotubes.
実施例２：培養筋組織の大きさ検討
（培養容器２００、培養容器３００、培養容器４００の作製）
実施例１における培養容器１００と同様にして、凸部の長手方向の長さ（Ｘ）：凸部の短手方向の長さ（Ｙ）が、４ｃｍ：３ｃｍである培養容器２００；４ｃｍ：８ｃｍである培養容器３００、１０ｃｍ：１０ｃｍである培養容器４００を作製した。
Example 2: Examination of the size of cultured muscle tissue (fabrication of culture vessels 200, 300, and 400)
In the same manner as the culture container 100 in Example 1, the culture container 200 has a length of the convex part in the longitudinal direction (X): a length of the convex part in the transverse direction (Y) of 4 cm: 3 cm; 4 cm: 8 cm. A culture vessel 300 with a diameter of 10 cm and a culture vessel 400 with a diameter of 10 cm were produced.
（ハイドロゲルの作製及び培養）
実施例１と同様にしてハイドロゲルを作製した。
得られた細胞を包埋したハイドロゲルを培養容器２００には３ｍＬ、培養容器３００には８ｍＬ、培養容器４００には２４ｍＬそれぞれ流し込み、３７℃、３０分間、ＣＯ２インキュベータ内で静置し、ハイドロゲルを固化した。
実施例１と同様の方法で、７日間培養し、三次元筋組織を得た。培養の時間経過に伴い、三次元筋組織は凸部の短手方向に収縮（シュリンク）した。
ハイドロゲルから三次元筋組織への培養中、凸部の短手方向の長さを経時的に測定し、収縮の程度（シュリンク量）を観察した。結果を図１２に示す。
７日間培養後、培養容器２００及び培養容器３００を用いた三次元筋組織ではシュリンク量は約２ｃｍであり、培養容器４００を用いた三次元筋組織ではシュリンク量は約５ｃｍであった。
このことから、三次元筋組織のシュリンク量は、培養容器の凸部の短手方向の長さの約２分の１であることが示唆され、シュリンク量を推定した大きさの培養容器を使用することで、任意の大きさの三次元筋組織の作製が可能であることが明らかになった。
(Preparation and culture of hydrogel)
A hydrogel was produced in the same manner as in Example 1.
3 mL of the resulting cell-embedded hydrogel was poured into the culture container 200, 8 mL into the culture container 300, and 24 mL into the culture container 400, and left standing in a CO 2 incubator at 37° C. for 30 minutes. The gel was solidified.
The cells were cultured for 7 days in the same manner as in Example 1 to obtain three-dimensional muscle tissue. As the culture progressed over time, the three-dimensional muscle tissue contracted (shrinked) in the short direction of the convex part.
During the culture of three-dimensional muscle tissue from the hydrogel, the length of the convex portion in the transverse direction was measured over time to observe the degree of contraction (shrink amount). The results are shown in FIG.
After culturing for 7 days, the shrinkage amount of the three-dimensional muscle tissue using culture container 200 and culture container 300 was about 2 cm, and the shrinkage amount of the three-dimensional muscle tissue using culture container 400 was about 5 cm.
This suggests that the amount of shrinkage of three-dimensional muscle tissue is approximately half the length of the convex part of the culture container in the transverse direction, and a culture container of the size estimated for the amount of shrinkage was used. By doing this, it became clear that it was possible to create three-dimensional muscle tissue of any size.
実施例３：食用ブタ血液を用いたハイドロゲルの作製
（培養容器５００の作製）
実施例１における培養容器１００と同様にして、凸部の長手方向の長さ（Ｘ）：凸部の短手方向の長さ（Ｙ）が、８ｃｍ：１２ｃｍである培養容器５００を作製した。
Example 3: Production of hydrogel using edible pig blood (production of culture container 500)
In the same manner as the culture container 100 in Example 1, a culture container 500 was produced in which the length of the protrusion in the longitudinal direction (X): the length of the protrusion in the transverse direction (Y) was 8 cm:12 cm.
（ハイドロゲル組成（２））
フィブリノーゲン（２５ｍｇ／ｍＬ） ４０００μＬ（終濃度４ｍｇ／ｍＬ）（Ｓｉｇｍａ社製、Ｆ８６３０）
食用ブタ血液 ５０００μＬ（終濃度２０％）
Ｔｈｒｏｍｂｉｎ（２００Ｕｎｉｔ／ｍＬ） ２５０μＬ（終濃度２Ｕｎｉｔ／ｍＬ）（Ｓｉｇｍａ社製、Ｔ４６４８－１０ＫＵ）
ＤＭＥＭ １５７５０μＬ（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ社製、１１９６５１１８）
(Hydrogel composition (2))
Fibrinogen (25mg/mL) 4000μL (final concentration 4mg/mL) (Sigma, F8630)
Edible pig blood 5000μL (final concentration 20%)
Thrombin (200 Unit/mL) 250 μL (final concentration 2 Unit/mL) (manufactured by Sigma, T4648-10KU)
DMEM 15750 μL (manufactured by Thermo Fisher Scientific, 11965118)
（ハイドロゲルの作製及び培養）
実施例１におけるハイドロゲル組成において、マトリゲルに替えて食用ブタ血液を用いた上記組成によりハイドロゲルを作製した。
筋芽細胞２．５×１０８ｃｅｌｌｓを、１．０×１０７ｃｅｌｌｓ／ｍＬとなるようにハイドロゲルと混合し、細胞を包埋したハイドロゲルを作製した。培養容器５００又は対照培養容器を底面とし、側面に型枠を設けて、そこに得られた細胞を包埋したハイドロゲルを流し込んだ。３７℃、３０分間、ＣＯ２インキュベータ内で静置し、ハイドロゲルを固化した。
次いで、培養期間を１４日間とする以外は実施例１と同様の方法で、１４日間培養し、三次元筋組織を得た。
(Preparation and culture of hydrogel)
In the hydrogel composition in Example 1, a hydrogel was produced using the above composition using edible pig blood instead of Matrigel.
2.5×10 8 myoblast cells were mixed with the hydrogel at a concentration of 1.0×10 7 cells/mL to produce a hydrogel in which the cells were embedded. The culture container 500 or the control culture container was used as the bottom, a mold was provided on the side, and the obtained hydrogel in which cells were embedded was poured into the mold. The hydrogel was left standing in a CO 2 incubator at 37° C. for 30 minutes to solidify the hydrogel.
Next, the cells were cultured for 14 days in the same manner as in Example 1 except that the culture period was changed to 14 days to obtain a three-dimensional muscle tissue.
培養７日目で実施例１と同様の方法で電気刺激を与えて、顕微鏡観察により、電気刺激に応答した収縮運動の有無を観察した。その結果、収縮運動が観察された。
１４日間培養により、約８ｃｍ×８ｃｍの三次元筋組織を得た。培養の時間経過に伴い、三次元筋組織は凸部の短手方向に約４ｃｍ収縮（シュリンク）した。
以上の結果より、マトリゲルを食用ブタ血液に変更しても収縮可能な組織が作製可能であること、組織のシュリンク量はマトリゲルと同等であることが明らかとなった。
On the 7th day of culture, electrical stimulation was applied in the same manner as in Example 1, and the presence or absence of contraction movement in response to the electrical stimulation was observed by microscopic observation. As a result, contraction movement was observed.
A three-dimensional muscle tissue of approximately 8 cm x 8 cm was obtained by culturing for 14 days. As the culture time progressed, the three-dimensional muscle tissue shrank by about 4 cm in the transverse direction of the convex portion.
The above results revealed that it is possible to create a contractible tissue even when Matrigel is replaced with edible pig blood, and that the amount of shrinkage of the tissue is equivalent to that of Matrigel.
実施例４：凸部の高さ検討
（培養容器の作製）
凸部１０の高さ以外は実施例１における培養容器１００と同様にして、凸部１０の高さが１００μｍである培養容器６００、凸部１０の高さが３００μｍである培養容器７００、及び凸部１０の高さが５００μｍである培養容器８００を作製した。
（ハイドロゲルの作製及び培養）
実施例１と同様にしてハイドロゲルを作製及び培養をした。
Example 4: Examination of height of convex portion (fabrication of culture container)
A culture container 600 in which the height of the projection 10 is 100 μm, a culture container 700 in which the height of the projection 10 is 300 μm, and a culture container 700 in which the height of the projection 10 is 300 μm are prepared in the same manner as the culture container 100 in Example 1 except for the height of the projection 10. A culture container 800 in which the height of the portion 10 is 500 μm was produced.
(Preparation and culture of hydrogel)
A hydrogel was prepared and cultured in the same manner as in Example 1.
（電気刺激）
培養１４日目に実施例３と同様にして電気刺激を行い、電気刺激に応答した三次元筋組織の収縮運動を顕微鏡にて観察及び動画撮影した。撮影した動画に基づき、収縮に伴う三次元筋組織の移動量（収縮量）の経時変化をグラフ化した。
結果を図１３に示す。
(electrical stimulation)
On the 14th day of culture, electrical stimulation was performed in the same manner as in Example 3, and the contraction movement of the three-dimensional muscle tissue in response to the electrical stimulation was observed using a microscope and videotaped. Based on the video footage, we created a graph of changes over time in the amount of movement (contraction amount) of three-dimensional muscle tissue associated with contraction.
The results are shown in FIG.
いずれの場合においても、三次元筋組織の収縮運動が観察された。中でも、凸部１０の高さが１００μｍである場合に移動量（収縮量）が特に大きかった。 In both cases, contraction movements of three-dimensional muscle tissue were observed. Among these, the amount of movement (amount of contraction) was particularly large when the height of the convex portion 10 was 100 μm.
１００ 培養容器
１０ 凸部
２０ アンカー部
２００ 培養容器
３００ 培養容器
４００ 培養容器
100 Culture container 10 Convex portion 20 Anchor portion 200 Culture container 300 Culture container 400 Culture container
Claims (7)
（Ａ）筋芽細胞を含むハイドロゲルを培養容器へ供給する工程であって、
前記培養容器は、容器端部に向かい合う一対のアンカー部と、前記一対のアンカー部間にわたって複数の凸部が配列してなる培養容器である工程、並びに、
（Ｂ）前記培養容器中で、筋芽細胞を筋管へと分化誘導する工程。 A method for producing three-dimensional muscle tissue including the following steps (A) and (B):
(A) A step of supplying a hydrogel containing myoblasts to a culture container,
a step in which the culture container is a culture container including a pair of anchor portions facing each other at ends of the container, and a plurality of convex portions arranged between the pair of anchor portions, and
(B) A step of inducing differentiation of myoblasts into myotubes in the culture vessel.
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