JP2020202814A - Method and kit of culturing three-dimensional cellular tissue - Google Patents
Method and kit of culturing three-dimensional cellular tissue Download PDFInfo
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- JP2020202814A JP2020202814A JP2019113681A JP2019113681A JP2020202814A JP 2020202814 A JP2020202814 A JP 2020202814A JP 2019113681 A JP2019113681 A JP 2019113681A JP 2019113681 A JP2019113681 A JP 2019113681A JP 2020202814 A JP2020202814 A JP 2020202814A
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Abstract
Description
本発明は、立体的細胞組織の培養方法及びキットに関する。 The present invention relates to a method and a kit for culturing a three-dimensional cell tissue.
近年、再生医療はもとより、生体に近い環境が求められる薬剤のアッセイ系において、平板上で成育させた細胞よりも立体的に組織化させた三次元細胞組織を使用することの優位性が示されており、生体外で細胞の三次元組織を構築するための様々な技術が開発されている。例えば、細胞が付着できない表面基板上で細胞塊を形成させる方法や液滴中で細胞塊を形成させる方法、透過性膜上に細胞を集積させる方法等が開発されている。このような細胞の組織化を維持するためには、生体自身が産生するコラーゲンなどの細胞外マトリックス（ＥＣＭ）が細胞間の結合や足場形成に必要である。そのため、人為的に細胞組織を構築する際、ＥＣＭを外部から添加することが検討されてきた。特許文献１には、酵素処理等により単離した細胞を、代表的なＥＣＭであって細胞保護作用を有する水溶性高分子であるコラーゲン等と接触させた後、三次元集合体として培養する方法が開示されている。この方法によれば、培養中に細胞周辺で水溶性高分子のゲルが形成される。特許文献２には、温度応答性の樹脂であるｐｏｌｙ（Ｎ−ｉｓｏｐｒｏｐｙｌａｃｒｙｌａｍｉｄｅ）（ＰＩＰＡＡｍ）を表面に固定化した培養皿を用いて細胞シートを作製し、作製した細胞シートを積層することで三次元組織を構築する方法が開示されている。 In recent years, the superiority of using three-dimensionally organized three-dimensional cell tissue over cells grown on a flat plate has been shown in the assay system of drugs that require an environment close to that of a living body as well as regenerative medicine. Various techniques have been developed for constructing three-dimensional tissues of cells in vitro. For example, a method of forming a cell mass on a surface substrate to which cells cannot adhere, a method of forming a cell mass in a droplet, a method of accumulating cells on a permeable membrane, and the like have been developed. In order to maintain such cell organization, extracellular matrix (ECM) such as collagen produced by the living body itself is required for cell-cell binding and scaffold formation. Therefore, it has been studied to add ECM from the outside when artificially constructing a cell tissue. Patent Document 1 describes a method in which cells isolated by enzyme treatment or the like are brought into contact with collagen or the like, which is a typical ECM and a water-soluble polymer having a cytoprotective effect, and then cultured as a three-dimensional aggregate. Is disclosed. According to this method, a water-soluble polymer gel is formed around the cells during culturing. In Patent Document 2, a cell sheet is prepared using a culture dish in which poly (N-isotropylacrylamide) (PIPAAm), which is a temperature-responsive resin, is immobilized on the surface, and the prepared cell sheets are laminated to form a three-dimensional structure. How to build an organization is disclosed.
ところで、発明者らは、これまでに、細胞と、カチオン性物質、細胞外マトリックス成分及び高分子電解質と混合し、培養することにより、立体的細胞組織を作製する方法の研究を行ってきた（例えば、特許文献３を参照）。 By the way, the inventors have been studying a method for producing a three-dimensional cell tissue by mixing and culturing cells with a cationic substance, an extracellular matrix component and a polymer electrolyte (by the way). For example, see Patent Document 3).
発明者らは、特許文献３に記載の方法により、立体的細胞組織を作製し、継続して培養すると、次第に立体的細胞組織の厚みが薄くなることを見出した。このような立体的細胞組織は、組織としての機能が低下していると考えられ、生体内の組織の機能を模倣としているとは言えない場合がある。また、薬剤のアッセイ系にも適していない場合がある。 The inventors have found that when a three-dimensional cell tissue is prepared by the method described in Patent Document 3 and continuously cultured, the thickness of the three-dimensional cell tissue gradually decreases. Such a three-dimensional cell tissue is considered to have a reduced function as a tissue, and may not be said to imitate the function of a tissue in a living body. It may also not be suitable for drug assay systems.
そこで、本発明の解決すべき課題は、立体的細胞組織を培養する方法であって、従来の培養方法において見られた培養期間中の細胞組織の体積の減少を改善する方法を提供することである。 Therefore, the problem to be solved of the present invention is a method for culturing a three-dimensional cell tissue, and by providing a method for improving the decrease in the volume of the cell tissue during the culturing period observed in the conventional culturing method. is there.
［１］立体的細胞組織をＦｉｂｒｏｂｌａｓｔ ｇｒｏｗｔｈ ｆａｃｔｏｒを含む培地中で培養する工程を含む、前記立体的細胞組織の培養方法。
［２］前記立体的細胞組織が、細胞をカチオン性物質、細胞外マトリックス成分及び高分子電解質と混合して混合物を得る工程と、得られた前記混合物中の前記細胞を培養して前記立体的細胞組織を得る工程と、含む方法により製造されたものである、［１］に記載の立体的細胞組織の培養方法。
［３］前記細胞外マトリックス成分は、コラーゲン、ラミニン、フィブロネクチン、ビトロネクチン、エラスチン、テネイシン、エンタクチン、フィブリリン、プロテオグリカン、ならびにそれらの組み合わせからなる群から選択され、前記高分子電解質は、グリコサミノグリカン、デキストラン硫酸、ラムナン硫酸、フコイダン、カラギナン、ポリスチレンスルホン酸、ポリアクリルアミド−２−メチルプロパンスルホン酸、ポリアクリル酸、ならびにそれらの組み合わせからなる群から選択される、［２］に記載の立体的細胞組織の培養方法。
［４］前記混合物中の前記細胞外マトリックス成分の濃度が、０．０１ｍｇ／ｍＬ以上１．０ｍｇ／ｍＬ未満であり、前記混合物中の前記高分子電解質の濃度が、０．０１ｍｇ／ｍＬ以上１．０ｍｇ／ｍＬ未満である、［２］又は［３］に記載の立体的細胞組織の培養方法。
［５］前記細胞を培養して前記立体的細胞組織を得る工程が、前記細胞を、Ｆｉｂｒｏｂｌａｓｔ ｇｒｏｗｔｈ ｆａｃｔｏｒを含まない培地で培養することにより行われる、［２］〜［４］のいずれかに記載の立体的細胞組織の培養方法。
［６］前記Ｆｉｂｒｏｂｌａｓｔ ｇｒｏｗｔｈ ｆａｃｔｏｒを含む培地を７２時間に１回以上交換する、［１］〜［５］のいずれかに記載の立体的細胞組織の培養方法。
［７］前記Ｆｉｂｒｏｂｌａｓｔ ｇｒｏｗｔｈ ｆａｃｔｏｒがＦＧＦ２である、［１］〜［６］のいずれかに記載の立体的細胞組織の培養方法。
［８］前記Ｆｉｂｒｏｂｌａｓｔ ｇｒｏｗｔｈ ｆａｃｔｏｒを含む培地中の前記Ｆｉｂｒｏｂｌａｓｔ ｇｒｏｗｔｈ ｆａｃｔｏｒの濃度が、１００ｐｇ／ｍＬ以上である、［１］〜［７］のいずれかに記載の立体的細胞組織の培養方法。
［９］前記立体的細胞組織中の生細胞の数が、１．５×１０６個以上である、［１］〜［８］のいずれかに記載の立体的細胞組織の培養方法。
［１０］前記立体的細胞組織中の前記生細胞の密度が、４．０×１０６個／ｃｍ２以上である、請求項［１］〜［９］のいずれかに記載の立体的細胞組織の培養方法。
［１１］前記立体的細胞組織の厚みが６０μｍ以上である、［１］〜［１０］のいずれかに記載の立体的細胞組織の培養方法。
［１２］［１］〜［９］のいずれかに記載の立体的細胞組織の培養方法により製造される、シート状の立体的細胞組織。
［１３］［１］〜［１１］のいずれかに記載の立体的細胞組織の培養方法に用いられ、Ｆｉｂｒｏｂｌａｓｔ ｇｒｏｗｔｈ ｆａｃｔｏｒを含む、立体的細胞組織の培養用キット。
[1] The method for culturing a three-dimensional cell tissue, which comprises a step of culturing the three-dimensional cell tissue in a medium containing Fibroblast growth factor.
[2] The three-dimensional cell tissue is a step of mixing cells with a cationic substance, an extracellular matrix component, and a polymer electrolyte to obtain a mixture, and culturing the cells in the obtained mixture to obtain the three-dimensional cells. The method for culturing a three-dimensional cell tissue according to [1], which is produced by a step of obtaining a cell tissue and a method including the cell tissue.
[3] The extracellular matrix component is selected from the group consisting of collagen, laminin, fibronectin, vitronectin, elastin, tenesin, entactin, fibrilline, proteoglycan, and combinations thereof, and the polymer electrolyte is glycosaminoglycan, The steric cell tissue according to [2], which is selected from the group consisting of dextran sulfate, ramnan sulfate, fucoidan, caraginan, polystyrene sulfonic acid, polyacrylamide-2-methylpropanesulfonic acid, polyacrylic acid, and combinations thereof. Cultivation method.
[4] The concentration of the extracellular matrix component in the mixture is 0.01 mg / mL or more and less than 1.0 mg / mL, and the concentration of the polymer electrolyte in the mixture is 0.01 mg / mL or more and 1 The method for culturing a three-dimensional cell tissue according to [2] or [3], which is less than 0.0 mg / mL.
[5] The step of culturing the cells to obtain the three-dimensional cell tissue is carried out by culturing the cells in a medium containing no Fibroblast growth factor, according to any one of [2] to [4]. Method of culturing three-dimensional cell tissue.
[6] The method for culturing a three-dimensional cell tissue according to any one of [1] to [5], wherein the medium containing the Fibroblast growth factor is exchanged at least once every 72 hours.
[7] The method for culturing a three-dimensional cell tissue according to any one of [1] to [6], wherein the Fibroblast growth factor is FGF2.
[8] The method for culturing a three-dimensional cell tissue according to any one of [1] to [7], wherein the concentration of the Fibroblast growth factor in the medium containing the Fibroblast growth factor is 100 pg / mL or more.
[9] The number of viable cells of said three-dimensional cell tissue is at 1.5 × 10 6 or more, [1] to a method of culturing three-dimensional tissue according to any one of [8].
[10] The three-dimensional cell tissue according to any one of claims [1] to [9], wherein the density of the living cells in the three-dimensional cell tissue is 4.0 × 10 6 cells / cm 2 or more. Cultivation method.
[11] The method for culturing a three-dimensional cell tissue according to any one of [1] to [10], wherein the thickness of the three-dimensional cell tissue is 60 μm or more.
[12] A sheet-shaped three-dimensional cell tissue produced by the method for culturing a three-dimensional cell tissue according to any one of [1] to [9].
[13] A kit for culturing a three-dimensional cell tissue, which is used in the method for culturing a three-dimensional cell tissue according to any one of [1] to [11] and includes a Fibroblast growth factor.
本発明によれば、従来技術と比較して、培養期間中の立体的細胞組織の体積の減少を有意に抑制し改善することができる。 According to the present invention, it is possible to significantly suppress and improve the decrease in the volume of the three-dimensional cell tissue during the culture period as compared with the prior art.
本発明の適用の対象となる立体的細胞組織の形態に特に制限は無く、例えば、コラーゲンなどの天然生体高分子や合成高分子によって構成されたスキャフォール内で細胞を培養して形成した立体的細胞組織や、細胞凝集体（スフェロイド）、シート状の細胞構造体、などに適用し得る。特に、２以上の細胞層からなる積層構造等を有する立体的細胞組織（例えば、シート状の細胞構造体）の場合には、細胞組織の減少によって組織が痩せ細り、当初構成していた層構成が崩れるおそれがあるが、本発明を適用することによりそのリスクを抑えることが可能であることから、より効果的に採用し得るものである。 The morphology of the three-dimensional cell tissue to which the present invention is applied is not particularly limited, and for example, a three-dimensional cell formed by culturing cells in a scaffold composed of a natural biopolymer such as collagen or a synthetic polymer. It can be applied to cell tissues, cell aggregates (spheroids), sheet-like cell structures, and the like. In particular, in the case of a three-dimensional cell tissue having a laminated structure consisting of two or more cell layers (for example, a sheet-like cell structure), the tissue becomes thin due to the decrease in the cell tissue, and the layer structure initially formed. However, since it is possible to suppress the risk by applying the present invention, it can be adopted more effectively.
また、本明細書において用いる場合、「立体的細胞組織」とは、少なくとも一種類の細胞を含む立体的な集合体を意味する。本発明によって構築される立体的細胞組織には、皮膚、毛髪、骨、軟骨、歯、角膜、血管、リンパ管、心臓、肝臓、膵臓、神経、食道などの生体組織、ならびに固形癌モデル（例えば、胃癌、食道癌、大腸癌、結腸癌、直腸癌、膵臓癌、乳癌、卵巣癌、前立腺癌、腎細胞癌、肝癌など）が挙げられるが、これらに限定されない。 Further, as used herein, the term "three-dimensional cell tissue" means a three-dimensional aggregate containing at least one type of cell. The three-dimensional cell tissues constructed by the present invention include biological tissues such as skin, hair, bone, cartilage, teeth, cornea, blood vessels, lymphatic vessels, heart, liver, pancreatic cancer, nerves, and esophageal cancer, as well as solid cancer models (for example, solid cancer models). , Gastric cancer, esophageal cancer, colon cancer, colon cancer, rectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, renal cell cancer, liver cancer, etc.), but is not limited thereto.
シート状の細胞構造体としては、例えば、細胞を単層培養した後に培養容器から剥離し、他の単層培養細胞に積層していくことで形成したものや、下記に記載する立体的細胞組織の製造方法によって形成したものに適用することができる。 Examples of the sheet-shaped cell structure include those formed by culturing cells in a single layer, peeling them from a culture vessel, and stacking them on other single-layer cultured cells, or the three-dimensional cell tissue described below. It can be applied to those formed by the manufacturing method of.
［培養方法］
１実施形態において、本発明は、立体的細胞組織をＦｉｂｒｏｂｌａｓｔ ｇｒｏｗｔｈ ｆａｃｔｏｒを含む培地中で培養する工程を含む、前記立体的細胞組織の培養方法を提供する。
[Culture method]
In one embodiment, the present invention provides a method for culturing a three-dimensional cell tissue, which comprises the step of culturing the three-dimensional cell tissue in a medium containing Fibroblast growth factor.
実施例において後述するように、立体的細胞組織を、Ｆｉｂｒｏｂｌａｓｔ ｇｒｏｗｔｈ ｆａｃｔｏｒ（以下、ＦＧＦと呼ぶ場合がある）を含まない培地で培養すると、培養期間の経過とともに立体的細胞組織の体積が減少し、立体的細胞組織に含まれる生細胞数が減少することがある。厚みに対して、厚み方向とは垂直な方向の長さが長い組織の場合には、立体的細胞組織の体積の減少は、厚みの減少として観察されてもよい。本明細書において、立体的細胞組織の厚みとは組織の自重方向の長さである。自重方向とは重力のかかる方向である。 As will be described later in Examples, when the three-dimensional cell tissue is cultured in a medium containing no Fibroblast growth factor (hereinafter, may be referred to as FGF), the volume of the three-dimensional cell tissue decreases with the lapse of the culture period. The number of viable cells contained in the steric cell tissue may decrease. In the case of a tissue having a long length in a direction perpendicular to the thickness direction with respect to the thickness, a decrease in the volume of the three-dimensional cell tissue may be observed as a decrease in thickness. In the present specification, the thickness of the three-dimensional cell tissue is the length of the tissue in the direction of its own weight. The direction of its own weight is the direction in which gravity is applied.
理論に拘泥するものではないが、立体的細胞組織においては、培地に含まれる栄養素等が組織の内部にまで十分に浸透せず、その結果、組織の内部の細胞が壊死している、又は、組織の内部において細胞の増殖が抑制されていると考えられる。組織の内部の細胞が壊死している、又は、組織の内部において細胞の増殖が抑制されている結果、立体的細胞組織の厚みが減少する可能性がある。立体的細胞組織の厚みが減少すると、細胞の活性が低下し、立体的細胞組織としての機能が低下、消失すると考えられる。 Although not bound by theory, in a three-dimensional cell tissue, nutrients contained in the medium do not sufficiently penetrate into the tissue, and as a result, the cells inside the tissue are necrotic or necrotic. It is considered that cell proliferation is suppressed inside the tissue. As a result of cell necrosis inside the tissue or suppression of cell proliferation inside the tissue, the thickness of the three-dimensional cell tissue may decrease. It is considered that when the thickness of the three-dimensional cell tissue decreases, the activity of the cell decreases, and the function as the three-dimensional cell tissue decreases or disappears.
実施例において後述するように、立体的細胞組織を、ＦＧＦを含む培地で培養すると、立体的細胞組織の厚みが減少することが抑制され、立体的細胞組織に含まれる生細胞数が減少することが抑制される。 As will be described later in Examples, when the three-dimensional cell tissue is cultured in a medium containing FGF, the decrease in the thickness of the three-dimensional cell tissue is suppressed, and the number of viable cells contained in the three-dimensional cell tissue is reduced. Is suppressed.
立体的細胞組織は、細胞をカチオン性物質、細胞外マトリックス成分及び高分子電解質と混合して混合物を得る工程と、得られた前記混合物中の前記細胞を培養して前記立体的細胞組織を得る工程と、含む方法により製造されたものであってもよい。 The three-dimensional cell tissue is obtained by mixing cells with a cationic substance, an extracellular matrix component and a polymer electrolyte to obtain a mixture, and culturing the cells in the obtained mixture to obtain the three-dimensional cell tissue. It may be manufactured by a process and a method including.
また、立体的細胞組織は、
（Ａ）細胞をカチオン性物質、細胞外マトリックス成分及び高分子電解質と混合して混合物を得る工程と、
（Ｂ）得られた混合物から細胞を集め、基材上に細胞集合体を形成する工程と、
（Ｃ）細胞を培養して立体的細胞組織を得る工程と、
を含む方法によって製造されたものであってもよい。
In addition, the three-dimensional cell tissue
(A) A step of mixing cells with a cationic substance, an extracellular matrix component, and a polymer electrolyte to obtain a mixture, and
(B) A step of collecting cells from the obtained mixture to form a cell aggregate on a substrate, and
(C) A step of culturing cells to obtain a three-dimensional cell tissue,
It may be manufactured by a method including.
上述した、工程（Ａ）、工程（Ｂ）、工程（Ｃ）においては、細胞をＦＧＦに接触させなくてもよいし、ＦＧＦに接触させてもよい。細胞をＦＧＦに接触させない場合であっても、立体的細胞組織を得ることができる。 In the steps (A), (B), and (C) described above, the cells may not be brought into contact with FGF, or may be brought into contact with FGF. Three-dimensional cell tissue can be obtained even when cells are not brought into contact with FGF.
前記立体的細胞組織の製造方法の工程（Ａ）において、細胞をカチオン性物質、細胞外マトリックス成分及び高分子電解質と混合し、この細胞混合物から細胞集合体を形成することにより、内部に大きな空隙が少ない立体的細胞組織を得ることができる。また、得られた立体的細胞組織は、比較的安定であるため、少なくとも数日間の培養が可能であり、かつ培地交換時にも組織が崩壊し難い。 In the step (A) of the method for producing a three-dimensional cell tissue, cells are mixed with a cationic substance, an extracellular matrix component, and a polymer electrolyte, and a cell aggregate is formed from this cell mixture to form a large void inside. It is possible to obtain a three-dimensional cell tissue with less. Moreover, since the obtained three-dimensional cell tissue is relatively stable, it can be cultured for at least several days, and the tissue is unlikely to collapse even when the medium is exchanged.
また、前記立体的細胞組織の製造方法の工程（Ａ）の後に、（Ａ’−１）得られた混合物から液体部分を除去し、細胞集合体を得る工程、および（Ａ’−２）細胞集合体を溶液に懸濁する工程、を含み、かつ工程（Ｂ）に代えて、（Ｂ’）得られた懸濁液から細胞を沈殿し、基材上に細胞の沈殿体を形成する工程を含んでもよい。上述の工程（Ａ）〜（Ｃ）を実施することで所望の組織体を得ることができるが、工程（Ａ）の後に（Ａ’−１）および（Ａ’−２）を実施し、工程（Ｂ）に代えて工程（Ｂ’）を実施することで、より均質な組織体を得ることができる。 Further, after the step (A) of the method for producing a three-dimensional cell tissue, a step (A'-1) of removing a liquid portion from the obtained mixture to obtain a cell aggregate, and (A'-2) cells. A step of suspending the aggregate in a solution, and instead of step (B), (B'), a step of precipitating cells from the obtained suspension to form a cell precipitate on a substrate. May include. A desired structure can be obtained by carrying out the above-mentioned steps (A) to (C), but (A'-1) and (A'-2) are carried out after the step (A), and the steps are carried out. By carrying out the step (B') instead of (B), a more homogeneous structure can be obtained.
前記立体的細胞組織の製造方法における細胞とカチオン性物質、高分子電解質、および細胞外マトリックス成分との混合は、ディッシュ、チューブ、フラスコ、ボトル、プレートなどの適当な容器中で行われてもよく、工程（Ｂ）において用いられる基材上で行われてもよい。工程（Ａ’−２）における懸濁もまた、ディッシュ、チューブ、フラスコ、ボトル、プレートなどの適当な容器中で行われてもよく、工程（Ｂ’）において用いられる基材上で行われてもよい。 Mixing of cells with cationic substances, polymeric electrolytes, and extracellular matrix components in the method for producing steric cell tissue may be carried out in a suitable container such as a dish, tube, flask, bottle, or plate. , May be performed on the substrate used in step (B). Suspension in step (A'-2) may also be done in a suitable container such as a dish, tube, flask, bottle, plate, etc., and is done on the substrate used in step (B'). May be good.
本明細書において用いる場合、「細胞集合体」とは細胞の集団を意味する。細胞集合体には、遠心分離やろ過などによって得られる細胞の沈殿体も含まれる。ある実施形態では、細胞集合体はスラリー状の粘稠体である。「スラリー状の粘稠体」とは、ゲル様の細胞集合体を指す（例えば、非特許文献２を参照）。 As used herein, "cell assembly" means a population of cells. The cell aggregate also includes a cell precipitate obtained by centrifugation, filtration, or the like. In certain embodiments, the cell aggregate is a slurry-like viscous body. The “slurry-like viscous body” refers to a gel-like cell aggregate (see, for example, Non-Patent Document 2).
前記立体的細胞組織の製造方法におけるカチオン性物質としては、細胞の生育および細胞集合体の形成に悪影響を及ぼさない限り、任意の正電荷を有する物質を用いることができる。カチオン性物質には、トリス−塩酸緩衝液、トリス−マレイン酸緩衝液、ビス−トリス−緩衝液、およびＨＥＰＥＳなどのカチオン性緩衝液、ならびにエタノールアミン、ジエタノールアミン、トリエタノールアミン、ポリビニルアミン、ポリアリルアミン、ポリリシン、ポリヒスチジン、およびポリアルギニンが挙げられるが、これらに限定されない。好ましい実施形態では、本発明で用いられるカチオン性物質はカチオン性緩衝液である。より好ましい実施形態では、本発明で用いられるカチオン性物質はトリス−塩酸緩衝液である。 As the cationic substance in the method for producing a three-dimensional cell tissue, a substance having an arbitrary positive charge can be used as long as it does not adversely affect the growth of cells and the formation of cell aggregates. Cationic substances include cationic buffers such as Tris-hydrogen buffer, Tris-maleic acid buffer, Bis-Tris-buffer, and HEPES, as well as ethanolamine, diethanolamine, triethanolamine, polyvinylamine, polyallylamine. , Polylysine, polyhistidine, and polyarginine, but are not limited thereto. In a preferred embodiment, the cationic material used in the present invention is a cationic buffer. In a more preferred embodiment, the cationic material used in the present invention is Tris-hydrochloric acid buffer.
前記立体的細胞組織の製造方法におけるカチオン性物質の濃度は、細胞の生育および細胞集合体の形成に悪影響を及ぼさない限り、特に限定されない。本実施形態で用いられるカチオン性物質の濃度は１０〜１００ｍＭであることが好ましい。例えば、本実施形態で用いられるカチオン性物質の濃度は、２０〜９０ｍＭ、３０〜８０ｍＭ、４０〜７０ｍＭ、４５〜６０ｍＭであることが好ましい。本実施形態で用いられるカチオン性物質の濃度は５０ｍＭであることがより好ましい。 The concentration of the cationic substance in the method for producing a three-dimensional cell tissue is not particularly limited as long as it does not adversely affect the growth of cells and the formation of cell aggregates. The concentration of the cationic substance used in this embodiment is preferably 10 to 100 mM. For example, the concentration of the cationic substance used in this embodiment is preferably 20 to 90 mM, 30 to 80 mM, 40 to 70 mM, and 45 to 60 mM. The concentration of the cationic substance used in this embodiment is more preferably 50 mM.
前記立体的細胞組織の製造方法におけるカチオン性物質としてカチオン性緩衝液が用いられる場合、カチオン性緩衝液のｐＨは、細胞の生育および細胞集合体の形成に悪影響を及ぼさない限り、特に限定されない。本実施形態で用いられるカチオン性緩衝液のｐＨは６．０〜８．０であることが好ましい。例えば、本実施形態で用いられるカチオン性緩衝液のｐＨは、７．０、７．１、７．２、７．３、７．４、７．５、７．６、７．７、７．８、７．９、または８．０である。本実施形態で用いられるカチオン性緩衝液のｐＨは７．２〜７．６であることがより好ましい。本実施形態で用いられるカチオン性緩衝液のｐＨは７．４であることがさらに好ましい。 When a cationic buffer solution is used as the cationic substance in the method for producing a three-dimensional cell tissue, the pH of the cationic buffer solution is not particularly limited as long as it does not adversely affect the growth of cells and the formation of cell aggregates. The pH of the cationic buffer used in this embodiment is preferably 6.0 to 8.0. For example, the pH of the cationic buffer used in this embodiment is 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7. It is 8, 7.9, or 8.0. The pH of the cationic buffer used in this embodiment is more preferably 7.2 to 7.6. The pH of the cationic buffer used in this embodiment is more preferably 7.4.
本明細書において、「高分子電解質」とは、高分子鎖中に解離可能な官能基を有する高分子を意味する。本実施形態で用いられる高分子電解質としては、細胞の生育および細胞集合体の形成に悪影響を及ぼさない限り、任意の高分子電解質を用いることができる。高分子電解質には、ヘパリンや、コンドロイチン硫酸（例えば、コンドロイチン４−硫酸、コンドロイチン６−硫酸）、ヘパラン硫酸、デルマタン硫酸、ケラタン硫酸、ヒアルロン酸等のグリコサミノグリカン；デキストラン硫酸や、ラムナン硫酸、フコイダンや、カラギナン、ポリスチレンスルホン酸、およびポリアクリルアミド−２−メチルプロパンスルホン酸、ポリアクリル酸等が挙げられるが、これらに限定されない。これらの高分子電解質は、単独で用いてもよく、組み合わせて用いてもよい。本実施形態で用いられる高分子電解質はグリコサミノグリカンであることが好ましい。また、本実施形態で用いられる高分子電解質はヘパリンまたはデキストラン硫酸、コンドロイチン硫酸、またはデルマタン硫酸であることがより好ましい。本実施形態で用いられる高分子電解質はヘパリンであることがさらに好ましい。細胞の生育および細胞集合体の形成に悪影響を及ぼさない限り、上述の高分子電解質の誘導体を用いてもよい。 As used herein, the term "polymer electrolyte" means a polymer having a dissociable functional group in the polymer chain. As the polymer electrolyte used in the present embodiment, any polymer electrolyte can be used as long as it does not adversely affect the growth of cells and the formation of cell aggregates. High molecular weight electrolytes include heparin, chondroitin sulfate (for example, chondroitin 4-sulfate, chondroitin 6-sulfate), heparan sulfate, dermatan sulfate, keratane sulfate, hyaluronic acid and other glycosaminoglycans; Examples thereof include, but are not limited to, fucoidan, caraginan, polystyrene sulfonic acid, polyacrylamide-2-methylpropane sulfonic acid, and polyacrylic acid. These polymer electrolytes may be used alone or in combination. The polymer electrolyte used in this embodiment is preferably glycosaminoglycan. Further, the polymer electrolyte used in the present embodiment is more preferably heparin or dextran sulfate, chondroitin sulfate, or dermatan sulfate. It is more preferable that the polymer electrolyte used in this embodiment is heparin. Derivatives of the above-mentioned polymer electrolytes may be used as long as they do not adversely affect the growth of cells and the formation of cell aggregates.
前記立体的細胞組織の製造方法における高分子電解質の濃度は、細胞の生育および細胞集合体の形成に悪影響を及ぼさない限り、特に限定されない。本実施形態で用いられる高分子電解質の濃度は０ｍｇ／ｍＬ超１．０ｍｇ／ｍＬ未満であることが好ましい。本実施形態で用いられる高分子電解質の濃度は０．０１ｍｇ／ｍＬ以上１．０ｍｇ／ｍＬ以下がより好ましく、０．０２５ｍｇ／ｍＬ以上０．１ｍｇ／ｍＬ以下がさらに好ましい。例えば、０．０１、０．０２５、０．０５、０．０７５、または０．１ｍｇ／ｍＬである。本実施形態で用いられる高分子電解質の濃度は０．０５ｍｇ／ｍＬ以上０．１ｍｇ／ｍＬ以下であることがさらに好ましい。本実施形態で用いられる高分子電解質の濃度は０．０５ｍｇ／ｍＬであることがさらに好ましい。本実施形態において、高分子電解質を適切な溶媒に溶解して用いてもよい。溶媒の例としては、水および緩衝液が挙げられるが、これらに限定されない。上述のカチオン性物質としてカチオン性緩衝液が用いられる場合、高分子電解質をカチオン性緩衝液に溶解して用いてもよい。 The concentration of the polymer electrolyte in the method for producing a three-dimensional cell tissue is not particularly limited as long as it does not adversely affect the growth of cells and the formation of cell aggregates. The concentration of the polymer electrolyte used in this embodiment is preferably more than 0 mg / mL and less than 1.0 mg / mL. The concentration of the polymer electrolyte used in the present embodiment is more preferably 0.01 mg / mL or more and 1.0 mg / mL or less, and further preferably 0.025 mg / mL or more and 0.1 mg / mL or less. For example, 0.01, 0.025, 0.05, 0.075, or 0.1 mg / mL. The concentration of the polymer electrolyte used in the present embodiment is more preferably 0.05 mg / mL or more and 0.1 mg / mL or less. The concentration of the polymer electrolyte used in this embodiment is more preferably 0.05 mg / mL. In the present embodiment, the polymer electrolyte may be dissolved in an appropriate solvent and used. Examples of solvents include, but are not limited to, water and buffers. When a cationic buffer solution is used as the above-mentioned cationic substance, the polymer electrolyte may be dissolved in the cationic buffer solution and used.
前記立体的細胞組織の製造方法における細胞外マトリックス成分としては、細胞の生育および細胞集合体の形成に悪影響を及ぼさない限り、細胞外マトリックス（ＥＣＭ）を構成する任意の成分を用いることができる。細胞外マトリックス成分には、コラーゲンや、ラミニン、フィブロネクチン、ビトロネクチン、エラスチン、テネイシン、エンタクチン、フィブリリン、およびプロテオグリカン等が挙げられるが、これらに限定されない。これらの細胞外マトリックス成分は、単独で用いてもよく、組み合わせて用いてもよい。プロテオグリカンには、コンドロイチン硫酸プロテオグリカン、ヘパラン硫酸プロテオグリカン、ケラタン硫酸プロテオグリカン、デルマタン硫酸プロテオグリカンが挙げられるが、これらに限定されない。本実施形態で用いられる細胞外マトリックス成分はコラーゲン、ラミニン、フィブロネクチンであり、中でもコラーゲンであることが好ましい。細胞の生育および細胞集合体の形成に悪影響を及ぼさない限り、上述の細胞外マトリックス成分の改変体およびバリアントを用いてもよい。 As the extracellular matrix component in the method for producing a three-dimensional cell tissue, any component constituting the extracellular matrix (ECM) can be used as long as it does not adversely affect the growth of cells and the formation of cell aggregates. The extracellular matrix component includes, but is not limited to, collagen, laminin, fibronectin, vitronectin, elastin, tenascin, entactin, fibrillin, proteoglycan and the like. These extracellular matrix components may be used alone or in combination. Examples of proteoglycans include, but are not limited to, chondroitin sulfate proteoglycan, heparan sulfate proteoglycan, keratan sulfate proteoglycan, and dermatan sulfate proteoglycan. The extracellular matrix component used in this embodiment is collagen, laminin, fibronectin, and collagen is preferable. Variants and variants of the extracellular matrix components described above may be used as long as they do not adversely affect cell growth and cell assembly formation.
細胞外マトリックス成分の濃度は、細胞の生育および細胞集合体の形成に悪影響を及ぼさない限り、特に限定されない。本実施形態で用いられる細胞外マトリックス成分の濃度は０ｍｇ／ｍＬ超１．０ｍｇ／ｍＬ未満であることが好ましい。本実施形態で用いられる細胞外マトリックス成分の濃度は０．０１ｍｇ／ｍＬ以上１．０ｍｇ／ｍＬ以下がより好ましく、０．０２５ｍｇ／ｍＬ以上０．１ｍｇ／ｍＬ以下であることがさらに好ましく、例えば、０．０１、０．０２５、０．０５、０．０７５、または０．１ｍｇ／ｍＬである。本実施形態で用いられる細胞外マトリックス成分の濃度は０．０５ｍｇ／ｍＬ以上０．１ｍｇ／ｍＬ以下であることがさらに好ましい。本実施形態で用いられる細胞外マトリックス成分の濃度は０．０５ｍｇ／ｍＬであることがさらに好ましい。本実施形態において、細胞外マトリックス成分を適切な溶媒に溶解して用いてもよい。溶媒の例としては、水、緩衝液、酢酸などが挙げられるが、これらに限定されない。本実施形態では、細胞外マトリックス成分は緩衝液または酢酸に溶解されることが好ましい。 The concentration of the extracellular matrix component is not particularly limited as long as it does not adversely affect the growth of cells and the formation of cell aggregates. The concentration of the extracellular matrix component used in this embodiment is preferably more than 0 mg / mL and less than 1.0 mg / mL. The concentration of the extracellular matrix component used in the present embodiment is more preferably 0.01 mg / mL or more and 1.0 mg / mL or less, further preferably 0.025 mg / mL or more and 0.1 mg / mL or less, for example. It is 0.01, 0.025, 0.05, 0.075, or 0.1 mg / mL. The concentration of the extracellular matrix component used in the present embodiment is more preferably 0.05 mg / mL or more and 0.1 mg / mL or less. The concentration of the extracellular matrix component used in this embodiment is more preferably 0.05 mg / mL. In the present embodiment, the extracellular matrix component may be dissolved in an appropriate solvent and used. Examples of the solvent include, but are not limited to, water, buffer, acetic acid and the like. In this embodiment, the extracellular matrix component is preferably dissolved in buffer or acetic acid.
前記立体的細胞組織の製造方法における高分子電解質と細胞外マトリックス成分との配合比は１：２〜２：１であることが好ましい。本実施形態で用いられる高分子電解質と細胞外マトリックス成分との配合比は１：１．５〜１．５：１であることがより好ましい。本実施形態で用いられる高分子電解質と細胞外マトリックス成分との配合比は１：１であることがさらに好ましい。 The compounding ratio of the polymer electrolyte and the extracellular matrix component in the method for producing a three-dimensional cell tissue is preferably 1: 2 to 2: 1. The compounding ratio of the polymer electrolyte used in the present embodiment and the extracellular matrix component is more preferably 1: 1.5 to 1.5: 1. It is more preferable that the compounding ratio of the polymer electrolyte used in the present embodiment and the extracellular matrix component is 1: 1.
前記立体的細胞組織の製造方法に用いられる細胞は特に限定されないが、例えば、ヒトや、サル、イヌ、ネコ、ウサギ、ブタ、ウシ、マウス、ラット等の動物に由来する細胞である。細胞の由来部位も特に限定されず、骨や、筋肉、内臓、神経、脳、骨、皮膚、血液等などに由来する体細胞であってもよく、生殖細胞であってもよい。さらに、本実施形態に係る方法に用いられる細胞は、誘導多能性幹細胞細胞（ｉＰＳ細胞）、胚性幹細胞（ＥＳ細胞）であってもよい。あるいは、初代培養細胞や、継代培養細胞、および細胞株細胞などの培養細胞であってもよい。一種類の細胞を用いてもよいし、複数種類の細胞を用いてもよい。本実施形態に係る方法に用いられる細胞には、例えば、神経細胞や、樹状細胞、免疫細胞、血管内皮細胞、リンパ管内皮細胞、線維芽細胞、肝癌細胞等の癌細胞、上皮細胞、心筋細胞、肝細胞、膵島細胞、組織幹細胞、平滑筋細胞等が挙げられるが、これらに限定されない。
本実施形態の立体的細胞組織は、生体内の組織の機能を模倣する組織であるため、前記立体的細胞組織の製造方法に用いられる細胞は、不死化された細胞以外の細胞であることが好ましい。
The cells used in the method for producing a three-dimensional cell tissue are not particularly limited, and are, for example, cells derived from animals such as humans, monkeys, dogs, cats, rabbits, pigs, cows, mice, and rats. The origin of the cell is not particularly limited, and may be a somatic cell derived from bone, muscle, internal organs, nerve, brain, bone, skin, blood, etc., or a germ cell. Furthermore, the cells used in the method according to the present embodiment may be induced pluripotent stem cell cells (iPS cells) or embryonic stem cells (ES cells). Alternatively, it may be a cultured cell such as a primary cultured cell, a subcultured cell, and a cell line cell. One type of cell may be used, or a plurality of types of cells may be used. The cells used in the method according to the present embodiment include, for example, nerve cells, dendritic cells, immune cells, vascular endothelial cells, lymphatic endothelial cells, fibroblasts, hepatoma cells and other cancer cells, epithelial cells, and myocardium. Examples include, but are not limited to, cells, hepatocytes, pancreatic islet cells, tissue stem cells, smooth muscle cells and the like.
Since the three-dimensional cell tissue of the present embodiment is a tissue that mimics the function of the tissue in the living body, the cell used in the method for producing the three-dimensional cell tissue may be a cell other than an immortalized cell. preferable.
前記立体的細胞組織の製造方法により得られる立体的細胞組織は、一種類の細胞を含んでいてもよく、複数種類の細胞を含んでいてもよい。本実施形態では、立体的細胞組織に含まれる細胞は、神経細胞、樹状細胞、免疫細胞、血管内皮細胞、リンパ管内皮細胞、線維芽細胞、癌細胞、上皮細胞、心筋細胞、肝細胞、膵島細胞、組織幹細胞、および平滑筋細胞からなる群から選択される。また、本実施形態に係る方法により得られる立体的細胞組織は、脈管構造を有していてもよい。「脈管構造」とは、生体組織における血管網やリンパ管網のような、ネットワーク状の構造を指す。 The three-dimensional cell tissue obtained by the method for producing a three-dimensional cell tissue may contain one type of cell or may contain a plurality of types of cells. In the present embodiment, the cells contained in the three-dimensional cell tissue include nerve cells, dendritic cells, immune cells, vascular endothelial cells, lymphatic endothelial cells, fibroblasts, cancer cells, epithelial cells, myocardial cells, and hepatocytes. It is selected from the group consisting of pancreatic islet cells, tissue stem cells, and smooth muscle cells. In addition, the three-dimensional cell tissue obtained by the method according to the present embodiment may have a vascular structure. "Vascular structure" refers to a network-like structure such as a vascular network or a lymphatic network in a living tissue.
前記立体的細胞組織の製造方法では、工程（Ａ’−１）における液体部分を除去する手段として、当業者に公知の手法を用いることができる。例えば、遠心分離やろ過によって、液体部分を除去してもよい。遠心分離の条件は、細胞の生育および細胞集合体の形成に悪影響を及ぼさない限り、特に限定されない。例えば、混合物の入ったマイクロチューブを室温、４００×ｇで１分間の遠心分離に供して液体部分と細胞集合体とを分離することによって、液体部分を除去する。あるいは、自然沈降によって細胞を集めた後、液体部分を除去してもよい。 In the method for producing a three-dimensional cell tissue, a method known to those skilled in the art can be used as a means for removing the liquid portion in the step (A'-1). For example, the liquid portion may be removed by centrifugation or filtration. The conditions for centrifugation are not particularly limited as long as they do not adversely affect the growth of cells and the formation of cell aggregates. For example, the liquid portion is removed by subjecting the microtube containing the mixture to centrifugation at 400 xg for 1 minute at room temperature to separate the liquid portion from the cell aggregate. Alternatively, the liquid portion may be removed after the cells have been collected by spontaneous sedimentation.
前記立体的細胞組織の製造方法における工程（Ａ’−２）において用いられる溶液は、細胞の生育および細胞集合体の形成に悪影響を及ぼさない限り、特に限定されない。例えば、使用される細胞に適した細胞培養培地または緩衝液が用いられる。 The solution used in the step (A'-2) in the method for producing a three-dimensional cell tissue is not particularly limited as long as it does not adversely affect the growth of cells and the formation of cell aggregates. For example, a cell culture medium or buffer suitable for the cells used is used.
前記立体的細胞組織の製造方法の工程（Ｂ）または（Ｂ’）において用いられる基材には、細胞の培養に用いるための培養容器が挙げられる。培養容器は、細胞や微生物の培養に通常用いられている素材、形状を有する容器であってよい。培養容器の素材としては、ガラスや、ステンレス、プラスチックなどが挙げられるが、これらに限定されない。培養容器としては、ディッシュや、チューブ、フラスコ、ボトル、プレートなどが挙げられるが、これらに限定されない。基材は、例えば、液体中の細胞を通過させず、液体を通すことが可能な材料である。基材は、透過膜であることが好ましい。かかる透過膜を有する容器としては、Ｔｒａｎｓｗｅｌｌ（登録商標）インサート、Ｎｅｔｗｅｌｌ（登録商標）インサート、Ｆａｌｃｏｎ（登録商標）セルカルチャーインサート、Ｍｉｌｌｉｃｅｌｌ（登録商標）セルカルチャーインサートなどのセルカルチャーインサートが挙げられるが、これらに限定されない。 Examples of the base material used in the step (B) or (B') of the method for producing a three-dimensional cell tissue include a culture vessel for use in culturing cells. The culture container may be a container having a material and shape usually used for culturing cells and microorganisms. Examples of the material of the culture container include, but are not limited to, glass, stainless steel, and plastic. Examples of the culture vessel include, but are not limited to, dishes, tubes, flasks, bottles, plates, and the like. The base material is, for example, a material capable of passing a liquid without passing cells in the liquid. The base material is preferably a permeable membrane. Examples of the container having such a permeable membrane include cell culture inserts such as Transwell (registered trademark) insert, Netwell (registered trademark) insert, Falcon (registered trademark) cell culture insert, and Millicell (registered trademark) cell culture insert. Not limited to these.
前記立体的細胞組織の製造方法では、工程（Ｂ）または（Ｂ’）における細胞を集める手段として、当業者に公知の手法を用いることができる。例えば、遠心分離、磁性分離、またはろ過によって、細胞を集めてもよい。遠心分離の条件は、細胞の生育に悪影響を及ぼさない限り、特に限定されない。例えば、混合物または懸濁液をセルカルチャーインサートに播種し、１０℃、４００×ｇで１分間の遠心分離に供することで、細胞を集める。あるいは、自然沈降によって細胞を集めてもよい。工程（Ｂ’）では、例えば遠心分離やろ過によって懸濁液から液体部分を除去することで、基材上に細胞の沈殿体を形成してもよい。あるいは、自然沈降によって基材上に細胞の沈殿体を形成してもよい。工程（Ｂ）における細胞集合体または工程（Ｂ’）における細胞の沈殿体は層状であってもよい。 In the method for producing a three-dimensional cell tissue, a method known to those skilled in the art can be used as a means for collecting cells in the step (B) or (B'). Cells may be collected, for example, by centrifugation, magnetic separation, or filtration. The conditions for centrifugation are not particularly limited as long as they do not adversely affect the growth of cells. For example, cells are collected by seeding the mixture or suspension into cell culture inserts and subjecting them to centrifugation at 400 xg for 1 minute at 10 ° C. Alternatively, cells may be collected by spontaneous sedimentation. In step (B'), cell precipitates may be formed on the substrate by removing the liquid portion from the suspension, for example by centrifugation or filtration. Alternatively, a cell precipitate may be formed on the substrate by natural precipitation. The cell aggregate in step (B) or the precipitate of cells in step (B') may be layered.
前記立体的細胞組織の製造方法では、構築された立体的細胞組織の変形（例えば、組織の収縮、組織末端の剥離等）を抑制するための物質が用いられる。このような物質としては、選択的ＲＯＣＫ（Ｒｈｏ−ａｓｓｏｃｉａｔｅｄ ｃｏｉｌｅｄ−ｃｏｉｌ ｆｏｒｍｉｎｇ ｋｉｎａｓｅ／Ｒｈｏ結合キナーゼ）阻害剤であるＹ−２７６３２が挙げられるが、これに限定されない。本実施形態では、当該物質の存在下で工程（Ｃ）を行う。当該物質の存在下で細胞集合体を培養することにより構築された立体的細胞組織の収縮が抑制される。その結果、構築された組織がセルカルチャーインサート等の基材から剥離することが抑制される。例えば、工程（Ａ）において、このような物質をさらに混合してもよい。あるいは、工程（Ａ’−２）において用いる溶液に、このような物質をさらに添加してもよい。あるいは、工程（Ｃ）で細胞を培養する際に、このような物質を培地に添加してもよい。 In the method for producing a three-dimensional cell tissue, a substance for suppressing deformation of the constructed three-dimensional cell tissue (for example, tissue contraction, exfoliation of tissue ends, etc.) is used. Such substances include, but are not limited to, Y-27632, which is a selective ROCK (Rho-associated coiled-coil forming kinase) inhibitor. In this embodiment, step (C) is performed in the presence of the substance. The contraction of the three-dimensional cell tissue constructed by culturing the cell aggregate in the presence of the substance is suppressed. As a result, the constructed structure is suppressed from peeling from the substrate such as the cell culture insert. For example, in step (A), such substances may be further mixed. Alternatively, such a substance may be further added to the solution used in the step (A'-2). Alternatively, such a substance may be added to the medium when the cells are cultured in the step (C).
前記立体的細胞組織の製造方法の工程（Ｃ）において、細胞の培養は、培養される細胞に適した培養条件下で行うことができる。当業者は、細胞の種類や所望の機能に応じて適切な培地を選択することができる。細胞培養培地としては特に限定されないが、Ｄ−ＭＥＭ、Ｅ−ＭＥＭ、ＭＥＭα、ＲＰＭＩ−１６４０、Ｍｃｃｏｙ‘５ａ、Ｈａｍ’ｓ Ｆ−１２等や、これらにＣＳ（ウシ血清）、ＦＢＳ（ウシ胎児血清）、ＨＢＳ（ウマ胎児血清）等の血清を１〜２０容量％程度になるように添加した培地が挙げられる。培養環境の温度や大気組成等の諸条件もまた、当業者が容易に定めうる。 In the step (C) of the method for producing a three-dimensional cell tissue, the cells can be cultured under culture conditions suitable for the cells to be cultured. One of ordinary skill in the art can select an appropriate medium according to the cell type and desired function. The cell culture medium is not particularly limited, but D-MEM, E-MEM, MEMα, RPMI-1640, Mccoy'5a, Ham's F-12, etc., and CS (fetal bovine serum), FBS (fetal bovine serum), etc. ), HBS (fetal bovine serum) and other culture media added in an amount of about 1 to 20% by volume. Conditions such as the temperature of the culture environment and the atmospheric composition can also be easily determined by those skilled in the art.
本発明の一態様に係る立体的細胞組織の培養方法は、培地中にＦｉｂｒｏｂｌａｓｔ ｇｒｏｗｔｈ ｆａｃｔｏｒ（ＦＧＦ）を含有する。培地としては、立体的細胞組織の製造方法の工程（Ｃ）で使用したものと同様に当業者が適宜定め得る。ＦＧＦは血管新生、創傷治癒、胚発生に関係する成長因子群の総称であり、広範囲の細胞の増殖や分化の過程において重要な役割を果たしていると考えられている。ＦＧＦの種類としては特に限定されないが、例えば、ＦＧＦ１（ａｃｉｄ ＦＧＦ）、ＦＧＦ２（ｂａｓｉｃ ＦＧＦ）、ＦＧＦ３、ＦＧＦ４、ＦＧＦ５、ＦＧＦ６、ＦＧＦ７、ＦＧＦ８、ＦＧＦ９、ＦＧＦ１０、ＦＧＦ１１、ＦＧＦ１２、ＦＧＦ１３、ＦＧＦ１４、ＦＧＦ１６，ＦＧＦ１７，ＦＧＦ１８、ＦＧＦ１９，ＦＧＦ２０、ＦＧＦ２１、ＦＧＦ２２、ＦＧＦ２３など、ヒト由来のＦＧＦが上げられる。実施例において後述するように、ＦＧＦはＦＧＦ２であることが好ましい。培地中に含有されるＦＧＦの濃度については、立体的細胞組織の体積の減少が抑制される機能が発揮される程度に含まれていれば特に制限されない。一般的に、生理条件下におけるＦＧＦの半減期は短く、ＦＧＦ２においては約８時間程度といわれているため（非特許文献１）、生理条件に近い培養条件で培養を行う場合、好ましくは、１００ｐｇ／ｍＬ以上、より好ましくは０．６ｎｇ／ｍＬ以上、更に好ましくは５ｎｇ／ｍＬ以上、もしくは１５ｎｇ／ｍＬ以上含まれることが好ましい。但し、培養温度や培地交換頻度などの諸条件もまた、当業者が容易に定め得る。例えば、ＦＧＦを最低限必要な量よりも多めに含有した培地によって立体的細胞組織の培養を行えば、培養期間中に含有されるＦＧＦの一部が消費・分解されたとしても機能を維持することが可能であり、例えば、２日に１度、３日に１度、４日に１度などのように、培地交換の頻度を少なくすることが可能である。培地交換の頻度を少なくすることができれば、薬剤等の効果などを立体的細胞組織によって検証する際に、培地に含有される薬剤等がより長期間維持されるため、立体的細胞組織好適に利用することができる。実施例において後述するように、立体的細胞組織の培養において、ＦＧＦを含む培地を、７２時間に１回以上交換してもよい。ＦＧＦをその機能が十分に発揮できる程度に含有する培地によって立体的細胞組織の培養を行えば、ＦＧＦを過剰に添加する必要がなくなり、また、培養期間中の培地内のＦＧＦ濃度を一定範囲に保ちやすくなるため、ＦＧＦ濃度が影響を及ぼす可能性があるアッセイを並行して行う場合などに好適に利用される。 The method for culturing a three-dimensional cell tissue according to one aspect of the present invention contains Fibroblast growth factor (FGF) in a medium. The medium can be appropriately determined by those skilled in the art in the same manner as that used in step (C) of the method for producing a three-dimensional cell tissue. FGF is a general term for growth factors involved in angiogenesis, wound healing, and embryogenesis, and is considered to play an important role in the process of proliferation and differentiation of a wide range of cells. The type of FGF is not particularly limited, but for example, FGF1 (acid FGF), FGF2 (basic FGF), FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF16, Human-derived FGFs such as FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23 can be raised. As will be described later in the examples, the FGF is preferably FGF2. The concentration of FGF contained in the medium is not particularly limited as long as it is contained to such an extent that the function of suppressing the decrease in the volume of the three-dimensional cell tissue is exhibited. Generally, the half-life of FGF is short under physiological conditions, and it is said that FGF2 has about 8 hours (Non-Patent Document 1). Therefore, when culturing under culturing conditions close to physiological conditions, 100 pg is preferable. It is preferably contained in an amount of / mL or more, more preferably 0.6 ng / mL or more, still more preferably 5 ng / mL or more, or 15 ng / mL or more. However, various conditions such as culture temperature and medium exchange frequency can also be easily determined by those skilled in the art. For example, if the three-dimensional cell tissue is cultured in a medium containing a larger amount of FGF than the minimum required amount, the function is maintained even if a part of the FGF contained during the culture period is consumed and decomposed. It is possible to reduce the frequency of medium exchange, for example, once every two days, once every three days, once every four days, and so on. If the frequency of medium exchange can be reduced, the drug contained in the medium can be maintained for a longer period of time when the effect of the drug or the like is verified by the three-dimensional cell tissue, so that the three-dimensional cell tissue is preferably used. can do. As will be described later in the examples, the medium containing FGF may be changed at least once every 72 hours in culturing the three-dimensional cell tissue. If the three-dimensional cell tissue is cultured in a medium containing FGF to the extent that its function can be fully exerted, it is not necessary to add an excessive amount of FGF, and the FGF concentration in the medium during the culture period is kept within a certain range. Since it is easy to maintain, it is preferably used when performing an assay in which the FGF concentration may affect it in parallel.
立体的細胞組織中の細胞の数は、立体的細胞組織を製造する際に播種された細胞数によって決められてもよい。また、立体的細胞組織中の細胞の数は、生細胞の数であってもよい。立体的細胞組織中の生細胞の数は、１．５×１０６個以上であってもよい。 The number of cells in the three-dimensional cell tissue may be determined by the number of cells seeded during the production of the three-dimensional cell tissue. Further, the number of cells in the three-dimensional cell tissue may be the number of living cells. The number of viable cells in the three-dimensional cell tissue may be of 1.5 × 10 6 or more.
実施例において後述するように、底面積が０．３３ｃｍ２であるセルカルチャーインサートにおいて、立体的細胞組織を、ＦＧＦ２を含むＦＢＳ含有ＤＭＥＭで培養した場合、立体的細胞組織の生細胞の数を１．５×１０６個以上とすることができる。すなわち、ウェル内の立体的細胞組織は４．０×１０６個／ｃｍ２以上であるということもできる。 As will be described later in Examples, when the three-dimensional cell tissue is cultured in FBS-containing DMEM containing FGF2 in the cell culture insert having a bottom area of 0.33 cm 2 , the number of living cells in the three-dimensional cell tissue is 1. .5 × 10 It can be 6 or more. That is, it can be said that the three-dimensional cell tissue in the well is 4.0 × 10 6 cells / cm 2 or more.
前記立体的細胞組織の製造方法の工程（Ａ）〜（Ｂ）または（Ａ）〜（Ｂ’）を繰り返すことで、細胞集合体または細胞の沈殿体を積層することが可能である。これにより、複数の層を有する立体的細胞組織を構築することができる。この場合、複数種類の細胞を用いて、異なる種類の細胞によって構成される立体的細胞組織を構築してもよい。前記立体的細胞組織の製造方法によれば、構築される立体的細胞組織の厚さは約５〜約３００、約４００、あるいは約５００μｍである。好ましくは、６０μｍ以上、より好ましくは２００μｍ以上、さらに好ましくは２５０μｍ以上である。本発明の上記方法の工程（Ａ）〜（Ｂ）または（Ａ）〜（Ｂ’）を繰り返すことにより、例えば、厚さが１５０〜５００μｍと厚みがあるにもかかわらず、内部に空隙の少ない立体的細胞組織が得られる。本実施形態では、構築される立体的細胞組織における細胞層の数は１〜約１００層であることが好ましく、約１０〜約１００層であることがより好ましい。細胞層とは、立体的細胞組織の厚み方向の断面の切片画像において、細胞核を認識できる倍率、つまり、染色した切片の厚みの全体が視野に入る倍率で観察した際に、細胞の自重、高さ方向に対して細胞核が重ならない時に別の層と定義する。なお、その際、自重と垂直（横）方向の視野は、少なくとも２００μｍ以上は確保する。なお、本実施形態においては、１００〜２００倍の倍率で立体的細胞組織の厚み方向の断面の切片画像を観察する。 By repeating the steps (A) to (B) or (A) to (B') of the method for producing a three-dimensional cell tissue, it is possible to stack cell aggregates or cell precipitates. This makes it possible to construct a three-dimensional cell tissue having a plurality of layers. In this case, a plurality of types of cells may be used to construct a three-dimensional cell tissue composed of different types of cells. According to the method for producing a three-dimensional cell tissue, the thickness of the three-dimensional cell tissue to be constructed is about 5 to about 300, about 400, or about 500 μm. It is preferably 60 μm or more, more preferably 200 μm or more, still more preferably 250 μm or more. By repeating the steps (A) to (B) or (A) to (B') of the above method of the present invention, for example, although the thickness is as thick as 150 to 500 μm, there are few voids inside. A three-dimensional cell tissue is obtained. In the present embodiment, the number of cell layers in the three-dimensional cell tissue to be constructed is preferably 1 to about 100 layers, and more preferably about 10 to about 100 layers. The cell layer is the weight and height of the cells when observed in a section image of a cross section in the thickness direction of the three-dimensional cell tissue at a magnification at which the cell nucleus can be recognized, that is, at a magnification at which the entire thickness of the stained section can be seen. It is defined as another layer when the cell nuclei do not overlap in the vertical direction. At that time, the field of view in the direction perpendicular to the own weight (horizontal) should be at least 200 μm or more. In this embodiment, a section image of a cross section of a three-dimensional cell tissue in the thickness direction is observed at a magnification of 100 to 200 times.
前記立体的細胞組織の製造方法の工程（Ａ）〜（Ｂ）または（Ａ）〜（Ｂ’）を繰り返すことで得られた立体的細胞組織は、従来の細胞の積層方法により得られた立体的細胞組織と比較して、単位厚み当たりの細胞層の数が少ない。前記立体的細胞組織の製造方法において、構築される立体的細胞組織は、生体外で得られる。また、前記立体的細胞組織の製造方法に係る立体的細胞組織は、細胞と、細胞外マトリックス成分とを含有し、厚み１０μｍ当たりの細胞層の数が、２．８層以下であり、好ましくは２．５層以下であり、より好ましくは２．２層以下である。前記立体的細胞組織の製造方法において、得られた立体的細胞組織において、厚みが最大となる位置（最大地点）を含む領域における厚み方向１００μｍ、幅方向５０μｍの面積あたりの細胞数が５個以上７０個以下であることが好ましく、１０個以上６０個以下であることがより好ましく、１５個以上５０個以下であることがさらに好ましい。
本実施形態においては、組織の厚み方向の断面からなる切片を用いて、細胞数を以下のように測定することができる。当該切片において、所定の大きさ以上の空隙がない領域のうち、立体的細胞組織の厚みが最大になる位置（最大地点）を決定する。厚みの最大値付近を含む幅５０μｍの短冊状（組織の天面から底面までを含む）の領域を測定領域として、当該測定領域中に含まれる細胞核の数を計数する。当該測定領域には空隙を含まないようにする。なお、空隙の所定の大きさとは、最大径５０μｍ以上をいう。空隙の最大径とは空隙が矩形の場合は長辺、球形の場合は直径、楕円形の場合は長径、不定形の場合は略楕円に近似した場合の長径をいう。なお、ＨＥ染色した切片上で空隙は染色されない。但し、厚みの最大値を含む領域の天面が凸形状の場合、組織が基材から剥離している場合もある。このような場合はその最大値近傍でかつ天面が比較的平坦な領域を測定領域とする。この場合、厚みの最大値付近を含む幅方向１００μm〜６５０μmの領域を測定領域とする。
計数した当該測定領域に少なくとも一部分が含まれている細胞核は、測定領域に含まれ
る細胞核として計数する。計数した細胞数から、厚み方向１００μｍ、幅方向５０μｍの
面積あたりの前記細胞数を算出する。
The three-dimensional cell tissue obtained by repeating the steps (A) to (B) or (A) to (B') of the method for producing a three-dimensional cell tissue is a three-dimensional obtained by a conventional cell stacking method. The number of cell layers per unit thickness is smaller than that of target cell tissue. In the method for producing a three-dimensional cell tissue, the three-dimensional cell tissue constructed is obtained in vitro. The three-dimensional cell tissue according to the method for producing a three-dimensional cell tissue contains cells and an extracellular matrix component, and the number of cell layers per 10 μm thickness is 2.8 or less, preferably 2.8 layers or less. It is 2.5 layers or less, more preferably 2.2 layers or less. In the method for producing a three-dimensional cell tissue, the number of cells per area of 100 μm in the thickness direction and 50 μm in the width direction in the region including the position (maximum point) where the thickness is maximum is 5 or more in the obtained three-dimensional cell tissue. The number is preferably 70 or less, more preferably 10 or more and 60 or less, and further preferably 15 or more and 50 or less.
In the present embodiment, the number of cells can be measured as follows using a section having a cross section in the thickness direction of the tissue. In the section, the position (maximum point) at which the thickness of the three-dimensional cell tissue is maximized is determined in the region where there is no void of a predetermined size or larger. The number of cell nuclei contained in the measurement region is counted with a strip-shaped region (including the top surface to the bottom surface of the tissue) having a width of 50 μm including the vicinity of the maximum thickness as the measurement region. The measurement area should not contain voids. The predetermined size of the void means a maximum diameter of 50 μm or more. The maximum diameter of the void means the long side when the void is rectangular, the diameter when it is spherical, the major axis when it is elliptical, and the major axis when it is close to an ellipse when it is amorphous. The voids are not stained on the HE-stained section. However, when the top surface of the region including the maximum thickness is convex, the structure may be peeled off from the base material. In such a case, the region near the maximum value and the top surface is relatively flat is defined as the measurement region. In this case, the measurement region is a region of 100 μm to 650 μm in the width direction including the vicinity of the maximum thickness.
Cell nuclei containing at least a part of the counted measurement region are counted as cell nuclei contained in the measurement region. From the counted number of cells, the number of cells per area of 100 μm in the thickness direction and 50 μm in the width direction is calculated.
［立体的細胞組織］
１実施形態において、本発明は、上述した立体的細胞組織の培養方法により製造される、立体的細胞組織を提供する。立体的細胞組織の形状は特に限定されず、シート状であってもよく、塊状であってもよく、球状であってもよい。
[Three-dimensional cell tissue]
In one embodiment, the present invention provides a three-dimensional cell tissue produced by the method for culturing a three-dimensional cell tissue described above. The shape of the three-dimensional cell tissue is not particularly limited, and may be sheet-like, lump-like, or spherical.
本実施形態のシート状の立体的細胞組織と、例えば、他の方法によって製造された三次元化した組織とは、組織の構造、組織を構成する細胞の活性等に相違が存在する可能性がある。しかしながら、そのような相違が存在するか否かは定かではなく、また、そのような相違を特定して、組織を構成する細胞の活性等により本実施形態のシート状の立体的細胞組織を特定するためには、著しく多くの試行錯誤を重ねることが必要であり、実質的に不可能である。したがって、本実施形態のシート状の立体的細胞組織は、上述した製造方法により製造されたことにより特定することが実際的であるといえる。 There may be differences in the structure of the tissue, the activity of the cells constituting the tissue, etc. between the sheet-shaped three-dimensional cell tissue of the present embodiment and the three-dimensional tissue produced by another method, for example. is there. However, it is not clear whether or not such a difference exists, and the sheet-like three-dimensional cell tissue of the present embodiment is specified by identifying such a difference and by the activity of cells constituting the tissue or the like. In order to do so, it is necessary to repeat a great deal of trial and error, which is practically impossible. Therefore, it can be said that it is practical to specify the sheet-shaped three-dimensional cell tissue of the present embodiment by being produced by the above-mentioned production method.
［キット］
１実施形態において、本発明は、上述した立体的細胞組織の培養方法に用いられ、Ｆｉｂｒｏｂｌａｓｔ ｇｒｏｗｔｈ ｆａｃｔｏｒを含む、立体的細胞組織の培養用キットを提供する。
[kit]
In one embodiment, the present invention is used in the method for culturing three-dimensional cell tissue described above, and provides a kit for culturing three-dimensional cell tissue, which comprises Fibroblast growth factor.
例えば、本実施形態のキットに付属した、Ｆｉｂｒｏｂｌａｓｔ ｇｒｏｗｔｈ ｆａｃｔｏｒと、細胞とを接触させることにより、立体的細胞組織を容易に製造することができる。 For example, a three-dimensional cell tissue can be easily produced by contacting cells with the Fibroblast growth factor attached to the kit of the present embodiment.
ＦＧＦは、水溶液中で分解されやすいため、本実施形態のキットに付属したＦＧＦは、粉末であることが好ましい。 Since FGF is easily decomposed in an aqueous solution, the FGF attached to the kit of this embodiment is preferably a powder.
ＦＧＦとしては、培養方法において上述したものであってもよく、好ましくは、ＦＧＦ２である。 The FGF may be the one described above in the culture method, and is preferably FGF2.
本実施形態のキットは、培地を更に含んでもよい。培地としては、例えば、培養方法において上述したものを用いることができる。 The kit of this embodiment may further include a medium. As the medium, for example, the medium described above in the culture method can be used.
本実施形態のキットは、血清を更に含んでもよい。上述の基礎培地には、血清を添加することが好ましい。培地に添加する血清としては、例えば、ウシ血清、ウシ胎児血清、ウマ血清、ヒト血清等が挙げられる。 The kit of this embodiment may further contain serum. It is preferable to add serum to the above-mentioned basal medium. Examples of the serum added to the medium include bovine serum, fetal bovine serum, horse serum, human serum and the like.
本実施形態のキットは、培養容器を更に含んでもよい。培養容器としては、培養方法において上述したものを用いることができる。培養容器としては、ディッシュや、チューブ、フラスコ、ボトル、プレートなどが挙げられるが、これらに限定されない。培養容器は、は透過膜を有するものであってもよい。 The kit of this embodiment may further include a culture vessel. As the culture container, the one described above in the culture method can be used. Examples of the culture vessel include, but are not limited to, dishes, tubes, flasks, bottles, plates, and the like. The culture vessel may have a permeable membrane.
本実施形態のキットは、カチオン性物質、細胞外マトリックス成分又は高分子電解質を更に含んでもよい。カチオン性物質、細胞外マトリックス成分、高分子電解質としては、培養方法において上述したものが挙げられる。 The kit of this embodiment may further include a cationic substance, an extracellular matrix component or a polymer electrolyte. Examples of the cationic substance, extracellular matrix component, and polymer electrolyte include those described above in the culture method.
本実施形態のキットは、立体的細胞組織の培養方法を行うことを指示する説明書を含んでもよい。説明書に従って、高品質の立体的細胞組織を再現性良く培養することができる。 The kit of this embodiment may include instructions instructing the method of culturing three-dimensional cell tissue. High-quality three-dimensional cell tissues can be cultured with good reproducibility according to the instructions.
以下に実施例を示して本発明をより詳細かつ具体的に説明するが、実施例は本発明の範
囲を限定するものではない。以下の実施例において、特に説明がない限り、コラーゲンとしてコラーゲンＩを用いた。
Hereinafter, the present invention will be described in more detail and concretely with reference to Examples, but the Examples do not limit the scope of the present invention. In the following examples, collagen I was used as collagen unless otherwise specified.
［実験例１］ＦＧＦを含有する培養培地による立体的細胞組織の培養１
＜立体的細胞組織の構築および培養＞
（サンプルＡ）
２．０×１０６細胞の正常ヒト皮膚線維芽細胞（ＮＨＤＦ）および１．０×１０４細胞のＲＦＰ導入ヒト臍帯静脈内皮細胞（ＨＵＶＥＣ）を、１５０μＬの０．２ｍｇ／ｍＬ ヘパリン／５０ｍＭ トリス−塩酸緩衝液（ｐＨ７．４）と、１５０μＬの０．２ｍｇ／ｍＬ コラーゲン／５ｍＭ 酢酸溶液（ｐＨ３．７）溶液との等量混合液に懸濁した。得られた混合物を室温、１０００×ｇで１分間、遠心し、粘稠体を得た。得られた粘稠体を１０％ＦＢＳ含有ＤＭＥＭに懸濁した。得られた懸濁液の全量を２４ｗｅｌｌ セルカルチャーインサート（底面積０．３３ｃｍ２）内に播種し、室温、４００×ｇ（重力加速度）で１分間、遠心した。これにより、セルカルチャーインサート上に細胞集合体を形成した。次いで、１０％ＦＢＳ含有ＤＭＥＭをセルカルチャーインサートの内側および外側の合計液量が２ｍＬ超となる様に添加し、ＣＯ２インキュベーター（３７℃、５％ＣＯ２）にて培養した（この培養を開始した時点を培養開始時点とする）。培養開始時点から２４時間培養後、５ｎｇ／ｍＬのＦＧＦ２を含む１０％ＦＢＳ含有ＤＭＥＭに培地交換した。以降、培養開始時点から、４８時間後、１２０時間後、１４４時間後、１６８時間後に、同様にして５ｎｇ／ｍＬのＦＧＦ２を含む１０％ＦＢＳ含有ＤＭＥＭによる培地交換を行った。培養終了後、立体的細胞組織の底面積が変化するような外観上の変化（ウェルカルチャーインサートからの剥がれ等）は観察されなかった。
[Experimental Example 1] Culturing a three-dimensional cell tissue in a culture medium containing FGF 1
<Construction and culture of three-dimensional cell tissue>
(Sample A)
2.0 × 10 6 cells of normal human skin fibroblasts (NHDF) and 1.0 × 10 4 cells of RFP-introduced human umbilical vein endothelial cells (HUVEC) in 150 μL of 0.2 mg / mL heparin / 50 mM Tris- Suspended in an equal volume mixture of 150 μL of 0.2 mg / mL collagen / 5 mM acetic acid solution (pH 3.7) solution of hydrochloric acid buffer (pH 7.4). The obtained mixture was centrifuged at 1000 × g for 1 minute at room temperature to obtain a viscous body. The obtained viscous material was suspended in DMEM containing 10% FBS. The entire amount of the obtained suspension was seeded in a 24-well cell culture insert (bottom area 0.33 cm 2 ) and centrifuged at room temperature, 400 × g (gravitational acceleration) for 1 minute. As a result, a cell aggregate was formed on the cell culture insert. Next, DMEM containing 10% FBS was added so that the total amount of liquid inside and outside the cell culture insert was more than 2 mL, and the cells were cultured in a CO 2 incubator (37 ° C., 5% CO 2 ) (this culture was started). The time when the culture is started is defined as the start time of the culture). After culturing for 24 hours from the start of culturing, the medium was replaced with DMEM containing 10% FBS containing 5 ng / mL FGF2. Subsequently, 48 hours, 120 hours, 144 hours, and 168 hours after the start of the culture, the medium was exchanged with DMEM containing 10% FBS containing 5 ng / mL FGF2 in the same manner. After the completion of the culture, no change in appearance (peeling from the well culture insert, etc.) such as a change in the bottom area of the three-dimensional cell tissue was observed.
＜立体的細胞組織の免疫組織染色＞
培養開始時点から１９２時間後の時点での立体的細胞組織をホルマリン固定し、パラフィン包埋切片を作製した。パラフィン包埋切片の作製は公知の方法に従った。作製した切片について、抗ＣＤ３１抗体による組織免疫染色を行った。組織免疫染色は公知の方法に従った。
<Immunohistochemical staining of three-dimensional cell tissue>
The three-dimensional cell tissue at 192 hours after the start of culture was fixed with formalin to prepare paraffin-embedded sections. Preparation of paraffin-embedded sections was performed according to a known method. The prepared sections were subjected to tissue immunostaining with an anti-CD31 antibody. Tissue immunostaining followed a known method.
＜立体的細胞組織の分散液の作成＞
培養開始時点から１９２時間後の時点での立体的細胞組織について、当該セルカルチャーインサート内の培地を除いた後、ＰＢＳを適量添加し、その後、液体成分を十分に除去した。この洗浄操作を繰り返し３回実施した。次いで、当該トランズウェルセルカルチャーインサートに０．２５％トリプシン‐ＥＤＴＡ溶液（Ｉｎｖｉｔｒｏｇｅｎ社製、）を２００μＬ添加し、ＣＯ２インキュベータ（３７℃，５％ＣＯ２）で１５分間インキュベートした。その後、溶液全量を回収用１．５ｍＬチューブに移した。次いで、当該トランズウェルセルカルチャーインサートに０．２５％トリプシン‐ＥＤＴＡ溶液（Ｉｎｖｉｔｒｏｇｅｎ社製、）を２００μＬ添加し、ＣＯ２インキュベータ（３７℃，５％ＣＯ２）で５分間インキュベートし、その後同様にして溶液全量を回収用１．５ｍＬチューブに移した。更に、トランズウェルセルカルチャーインサートにＰＢＳを２００μＬ添加し、攪拌した後、その溶液全量を回収した。回収溶液全量に対し、ＤＭＥＭ４００μＬを加え、合計１ｍＬの分散液を得た。
<Creation of a dispersion of three-dimensional cell tissue>
For the three-dimensional cell tissue at 192 hours after the start of culture, the medium in the cell culture insert was removed, an appropriate amount of PBS was added, and then the liquid component was sufficiently removed. This cleaning operation was repeated 3 times. Then, 200 μL of a 0.25% trypsin-EDTA solution (manufactured by Invitrogen) was added to the Transwell cell culture insert, and the mixture was incubated in a CO 2 incubator (37 ° C., 5% CO 2 ) for 15 minutes. The entire solution was then transferred to a recovery 1.5 mL tube. Then, 200 μL of 0.25% trypsin-EDTA solution (manufactured by Invitrogen) was added to the Transwell cell culture insert, and the mixture was incubated in a CO 2 incubator (37 ° C., 5% CO 2 ) for 5 minutes, and then similarly. The entire solution was transferred to a 1.5 mL recovery tube. Further, 200 μL of PBS was added to the Transwell cell culture insert, and after stirring, the entire amount of the solution was recovered. DMEM 400 μL was added to the total amount of the recovered solution to obtain a total of 1 mL of the dispersion.
＜分散液中の生細胞数測定＞
得られた立体的細胞組織の分散液をトリパンブルー溶液に浸漬させてトリパンブルー染色した後、ＲＦＰの蛍光を発しておりかつトリパンブルー染色されていない細胞を、生きているがん細胞として計数した。細胞の計数は、セルカウンター「ＣｏｕｎｔｅｓｓＩＩ」（ライフテクノロジーズ社製）を使用して行った。
<Measurement of the number of living cells in the dispersion>
After immersing the obtained three-dimensional cell tissue dispersion in a trypan blue solution and staining it with trypan blue, cells emitting RFP fluorescence and not stained with trypan blue were counted as living cancer cells. .. Cell counting was performed using a cell counter "Countess II" (manufactured by Life Technologies).
（サンプルＢ）
サンプルＡと同様にして作成した立体的細胞組織について、サンプルＡと同様に１０％ＦＢＳ含有ＤＭＥＭで２４時間培養した後、１５ｎｇ／ｍＬのＦＧＦ２を含む１０％ＦＢＳ含有ＤＭＥＭに培地交換した。以降、培養開始時点から換算して、４８時間後、１２０時間後、１４４時間後、１６８時間後に、同様にして１５ｎｇ／ｍＬのＦＧＦ２を含む１０％ＦＢＳ含有ＤＭＥＭによる培地交換を行った。培養した立体的細胞組織について、サンプルＡと同様にして、免疫組織染色および生細胞数測定を実施した。培養終了後、立体的細胞組織の底面積が変化するような外観上の変化（ウェルカルチャーインサートからの剥がれ等）は観察されなかった。
（サンプルＣ）
サンプルＡと同様にして作成した立体的細胞組織について、サンプルＡと同様に１０％ＦＢＳ含有ＤＭＥＭで２４時間培養した後、ＦＧＦを加えない１０％ＦＢＳ含有ＤＭＥＭに培地交換した。以降、培養開始時点から換算して、４８時間後、１２０時間後、１４４時間後、１６８時間後に、同様にしてＦＧＦを加えない１０％ＦＢＳ含有ＤＭＥＭによる培地交換を行った。培養した立体的細胞組織について、サンプルＡと同様にして、免疫組織染色および生細胞数測定を実施した。培養終了後、立体的細胞組織の底面積が変化するような外観上の変化（ウェルカルチャーインサートからの剥がれ等）は観察されなかった。
(Sample B)
The three-dimensional cell tissue prepared in the same manner as in Sample A was cultured in DMEM containing 10% FBS in the same manner as in Sample A for 24 hours, and then the medium was replaced with DMEM containing 10% FBS containing 15 ng / mL FGF2. After that, the medium was exchanged with DMEM containing 15 ng / mL of FGF2 in the same manner 48 hours, 120 hours, 144 hours, and 168 hours after the start of the culture. For the cultured three-dimensional cell tissue, immunohistochemical staining and viable cell number measurement were carried out in the same manner as in Sample A. After the completion of the culture, no change in appearance (peeling from the well culture insert, etc.) such as a change in the bottom area of the three-dimensional cell tissue was observed.
(Sample C)
The three-dimensional cell tissue prepared in the same manner as in Sample A was cultured in DMEM containing 10% FBS in the same manner as in Sample A for 24 hours, and then the medium was exchanged in DMEM containing 10% FBS without adding FGF. After that, the medium was exchanged with DMEM containing 10% FBS without adding FGF in the same manner after 48 hours, 120 hours, 144 hours, and 168 hours after the start of culturing. For the cultured three-dimensional cell tissue, immunohistochemical staining and viable cell number measurement were carried out in the same manner as in Sample A. After the completion of the culture, no change in appearance (peeling from the well culture insert, etc.) such as a change in the bottom area of the three-dimensional cell tissue was observed.
サンプルＡ、Ｂ、Ｃの生細胞数測定の結果を表１及び図１に示す。尚、図中の「初日」は、各サンプルと同タイミングで作成した立体的細胞組織体について培養開始時点から２４時間後の時点で分散液を作成し、生細胞数測定を行った際の値である。図１に示した通り、ＦＧＦを含む培地で培養したサンプルＡ、ＢはサンプルＣと比較して、生細胞数の経時減少が有意に抑制できていることが分かった。 The results of measuring the number of viable cells of samples A, B, and C are shown in Table 1 and FIG. The "first day" in the figure is a value when a dispersion was prepared 24 hours after the start of culturing for the three-dimensional cell tissue prepared at the same timing as each sample and the number of living cells was measured. Is. As shown in FIG. 1, it was found that the samples A and B cultured in the medium containing FGF could significantly suppress the decrease in the number of living cells with time as compared with the sample C.
サンプルＡ、Ｂ、Ｃのそれぞれの免疫組織染色の顕微鏡観察図を図２Ａ〜Ｃに記載する。また、組織染色過程の影響で極端な組織の収縮や形状の崩れが見られる両端部を除いた任意の３点の拡大図も併せて図２Ａ〜Ｃに記載する。更に、図２Ａ〜Ｃの各拡大図において点線で表示した領域の長さを表２に示す。図２Ａ〜Ｃおよび表２の結果から、サンプルＣに比べて、サンプルＡ、Ｂの組織の厚みは優位に厚く、その結果組織内部の管腔(組織内部で縁が黒く染色されている部分)が全体的により大きく形成できていることが分かった。また、ＦＢＳ含有ＤＭＥＭを、ＦＧＦ２を含むＦＢＳ含有ＤＭＥＭに交換することにより、組織の厚みを維持できることが明らかになった。 Microscopic observations of immunohistochemical staining of samples A, B, and C are shown in FIGS. 2A to 2C. In addition, enlarged views of any three points excluding both ends where extreme tissue contraction and shape collapse are observed due to the influence of the tissue staining process are also shown in FIGS. 2A to 2C. Further, Table 2 shows the length of the area indicated by the dotted line in each enlarged view of FIGS. 2A to 2C. From the results of FIGS. 2A to 2C and Table 2, the tissue thickness of samples A and B was significantly thicker than that of sample C, and as a result, the cavity inside the tissue (the part where the edge was stained black inside the tissue). Was found to be able to form larger overall. Further, it was revealed that the tissue thickness can be maintained by exchanging FBS-containing DMEM with FBS-containing DMEM containing FGF2.
［実験例２］ＦＧＦを含有する培養培地による立体的細胞組織の培養２
＜立体的細胞組織の構築および培養＞
（サンプルＡ）
２．０×１０６細胞の正常ヒト皮膚線維芽細胞（ＮＨＤＦ）および１．０×１０４細胞のＲＦＰ導入ヒト臍帯静脈内皮細胞（ＨＵＶＥＣ）を、１５０μＬの０．２ｍｇ／ｍＬ ヘパリン／５０ｍＭ トリス−塩酸緩衝液（ｐＨ７．４）と、１５０μＬの０．２ｍｇ／ｍＬ コラーゲン／５ｍＭ 酢酸溶液（ｐＨ３．７）溶液との等量混合液に懸濁した。得られた混合物を室温、１０００×ｇで１分間、遠心し、粘稠体を得た。得られた粘稠体を１０％ＦＢＳ含有ＤＭＥＭに懸濁した。得られた懸濁液の全量を２４ｗｅｌｌ セルカルチャーインサート内に播種し、室温、４００×ｇ（重力加速度）で１分間、遠心した。これにより、セルカルチャーインサート上に細胞層を形成した。次いで、１０％ＦＢＳ含有ＤＭＥＭをセルカルチャーインサートの内側および外側の合計液量が２ｍL超となる様に添加し、ＣＯ２インキュベーター（３７℃、５％ＣＯ２）にて培養した（この培養を開始した時点を培養開始時点とする）。培養開始時点から２４時間培養後、５ｎｇ／ｍＬのＦＧＦ２を含む１０％ＦＢＳ含有ＤＭＥＭに培地交換した。以降、培養開始時点から換算して、４８時間後、７２時間後、９６時間後、１２０時間後、１４４時間後、１６８時間後に、同様にして５ｎｇ／ｍＬのＦＧＦ２を含む１０％ＦＢＳ含有ＤＭＥＭによる培地交換を行った。培養終了後、立体的細胞組織の底面積が変化するような外観上の変化（ウェルカルチャーインサートからの剥がれ等）は観察されなかった。
[Experimental Example 2] Culturing a three-dimensional cell tissue in a culture medium containing FGF 2
<Construction and culture of three-dimensional cell tissue>
(Sample A)
2.0 × 10 6 cells of normal human skin fibroblasts (NHDF) and 1.0 × 10 4 cells of RFP-introduced human umbilical vein endothelial cells (HUVEC) in 150 μL of 0.2 mg / mL heparin / 50 mM Tris- Suspended in an equal volume mixture of 150 μL of 0.2 mg / mL collagen / 5 mM acetic acid solution (pH 3.7) solution of hydrochloric acid buffer (pH 7.4). The obtained mixture was centrifuged at 1000 × g for 1 minute at room temperature to obtain a viscous body. The obtained viscous material was suspended in DMEM containing 10% FBS. The entire volume of the resulting suspension was seeded in a 24-well cell culture insert and centrifuged at room temperature, 400 xg (gravitational acceleration) for 1 minute. As a result, a cell layer was formed on the cell culture insert. Next, DMEM containing 10% FBS was added so that the total amount of liquid inside and outside the cell culture insert was more than 2 mL, and the cells were cultured in a CO 2 incubator (37 ° C., 5% CO 2 ) (this culture was started). The time when the culture is started is defined as the start time of the culture). After culturing for 24 hours from the start of culturing, the medium was replaced with DMEM containing 10% FBS containing 5 ng / mL FGF2. After that, 48 hours, 72 hours, 96 hours, 120 hours, 144 hours, and 168 hours after conversion from the start of culturing, similarly by DMEM containing 5 ng / mL of FGF2 and containing 10% FBS. The medium was exchanged. After the completion of the culture, no change in appearance (peeling from the well culture insert, etc.) such as a change in the bottom area of the three-dimensional cell tissue was observed.
＜立体的細胞組織の分散液の作成、および生細胞数の測定＞
培養開始時点から１９２時間後の時点での立体的細胞組織について、実施例１と同様にして、合計１ｍＬの分散液を得て、当該分散液をトリパンブルー溶液に浸漬させてトリパンブルー染色した後、ＲＦＰの蛍光を発しておりかつトリパンブルー染色されていない細胞を、生きているがん細胞として計数した。細胞の計数は、セルカウンター「ＣｏｕｎｔｅｓｓII」（ライフテクノロジーズ社製）を使用して行った。
<Preparation of dispersion of three-dimensional cell tissue and measurement of viable cell number>
For the three-dimensional cell tissue at the time point 192 hours after the start of culturing, a total of 1 mL of a dispersion was obtained in the same manner as in Example 1, and the dispersion was immersed in a trypan blue solution and stained with trypan blue. , RFP-fluorescent and unstained with trypan blue were counted as living cancer cells. Cell counting was performed using a cell counter "Countess II" (manufactured by Life Technologies).
（サンプルＢ）
サンプルＡと同様にして作成した立体的細胞組織について、サンプルＡと同様に１０％ＦＢＳ含有ＤＭＥＭで２４時間培養した後、５ｎｇ／ｍＬのＦＧＦ２を含む１０％ＦＢＳ含有ＤＭＥＭに培地交換した。以降、培養開始時点から換算して、７２時間後、１２０時間後、１６８時間後に、同様にして５ｎｇ／ｍＬのＦＧＦ２を含む１０％ＦＢＳ含有ＤＭＥＭによる培地交換を行った。培養した立体的細胞組織について、サンプルＡと同様にして、生細胞数測定を実施した。培養終了後、立体的細胞組織の底面積が変化するような外観上の変化（ウェルカルチャーインサートからの剥がれ等）は観察されなかった。
(Sample B)
The three-dimensional cell tissue prepared in the same manner as in Sample A was cultured in DMEM containing 10% FBS in the same manner as in Sample A for 24 hours, and then the medium was replaced with DMEM containing 10% FBS containing 5 ng / mL FGF2. After that, 72 hours, 120 hours, and 168 hours after the start of culturing, the medium was exchanged with DMEM containing 5 ng / mL of FGF2 in the same manner. For the cultured three-dimensional cell tissue, the number of viable cells was measured in the same manner as in Sample A. After the completion of the culture, no change in appearance (peeling from the well culture insert, etc.) such as a change in the bottom area of the three-dimensional cell tissue was observed.
（サンプルＣ）
サンプルＡと同様にして作成した立体的細胞組織について、サンプルＡと同様に１０％ＦＢＳ含有ＤＭＥＭで２４時間培養した後、５ｎｇ／ｍＬのＦＧＦ２を含む１０％ＦＢＳ含有ＤＭＥＭに培地交換した。以降、培養開始時点から換算して、９６時間後、１６８時間後に、同様にして５ｎｇ／ｍＬのＦＧＦ２を含む１０％ＦＢＳ含有ＤＭＥＭによる培地交換を行った。培養した立体的細胞組織について、サンプルＡと同様にして、生細胞数測定を実施した。培養終了後、立体的細胞組織の底面積が変化するような外観上の変化（ウェルカルチャーインサートからの剥がれ等）は観察されなかった。
(Sample C)
The three-dimensional cell tissue prepared in the same manner as in Sample A was cultured in DMEM containing 10% FBS in the same manner as in Sample A for 24 hours, and then the medium was replaced with DMEM containing 10% FBS containing 5 ng / mL FGF2. Then, 96 hours and 168 hours after the start of culturing, the medium was exchanged with DMEM containing 5 ng / mL FGF2 in the same manner. For the cultured three-dimensional cell tissue, the number of viable cells was measured in the same manner as in Sample A. After the completion of the culture, no change in appearance (peeling from the well culture insert, etc.) such as a change in the bottom area of the three-dimensional cell tissue was observed.
（サンプルＤ）
サンプルＡと同様にして作成した立体的細胞組織について、サンプルＡと同様に１０％ＦＢＳ含有ＤＭＥＭで２４時間培養した後、ＦＧＦを加えない１０％ＦＢＳ含有ＤＭＥＭに培地交換した。以降、培養開始時点から換算して、４８時間後、７２時間後、９６時間後、１２０時間後、１４４時間後、１６８時間後に、同様にしてＦＧＦを加えない１０％ＦＢＳ含有ＤＭＥＭによる培地交換を行った。培養した立体的細胞組織について、サンプルＡと同様にして、生細胞数測定を実施した。培養終了後、立体的細胞組織の底面積が変化するような外観上の変化（ウェルカルチャーインサートからの剥がれ等）は観察されなかった。
(Sample D)
The three-dimensional cell tissue prepared in the same manner as in Sample A was cultured in DMEM containing 10% FBS in the same manner as in Sample A for 24 hours, and then the medium was exchanged in DMEM containing 10% FBS without adding FGF. After that, after 48 hours, 72 hours, 96 hours, 120 hours, 144 hours, and 168 hours, the medium was replaced with DMEM containing 10% FBS without adding FGF in the same manner from the start of culturing. went. For the cultured three-dimensional cell tissue, the number of viable cells was measured in the same manner as in Sample A. After the completion of the culture, no change in appearance (peeling from the well culture insert, etc.) such as a change in the bottom area of the three-dimensional cell tissue was observed.
サンプルＡ、Ｂ、Ｃ、Ｄの生細胞数測定の結果を表３及び図３に示す。尚、表３中及び図３中の「初日」は、各サンプルと同タイミングで作成した立体的細胞組織体について培養開始時点から２４時間後の時点で分散液を作成し、生細胞数測定を行った際の値である。表３及び図３に示した通り、ＦＧＦ２を加えた培地で培養したサンプルＡ、Ｂ、ＣはサンプルＤと比較して、生細胞数の経時減少が有意に抑制できていることが分かった。更に、７２時間に一度の培地交換を行ったサンプルＣは、サンプルＡ、Ｂと比較しても遜色なく、サンプルＤに対して有意に細胞数の減少を抑制できていたことから、培地交換後、４８時間経過後に培地内に残留しているＦＧＦ量であっても、立体的細胞組織の体積の減少を抑制する効果が発揮されていることが分かった。生理条件下におけるＦＧＦ２の半減期が約８時間程度であることを考慮すると、培養開始時点のＦＧＦ濃度が５ｎｇ／ｍＬだったとすれば４８時間後には約１００ｐｇ／ｍＬ程度となっていることから、少なくとも１００ｐｇ／ｍＬ超のＦＧＦ２を含む培地であれば、立体的細胞組織の体積の減少を抑制する効果が発揮されることが示された。 The results of measuring the number of viable cells of samples A, B, C and D are shown in Table 3 and FIG. On the "first day" in Table 3 and FIG. 3, a dispersion was prepared 24 hours after the start of culturing for the three-dimensional cell tissue prepared at the same timing as each sample, and the number of living cells was measured. It is the value when it went. As shown in Table 3 and FIG. 3, it was found that the samples A, B, and C cultured in the medium supplemented with FGF2 were able to significantly suppress the decrease in the number of living cells with time as compared with the sample D. Furthermore, Sample C, which had undergone medium exchange once every 72 hours, was comparable to Samples A and B, and was able to significantly suppress the decrease in the number of cells with respect to Sample D. It was found that even the amount of FGF remaining in the medium after 48 hours had the effect of suppressing the decrease in the volume of the three-dimensional cell tissue. Considering that the half-life of FGF2 under physiological conditions is about 8 hours, if the FGF concentration at the start of culture is 5 ng / mL, it will be about 100 pg / mL after 48 hours. It has been shown that a medium containing at least 100 pg / mL of FGF2 exerts an effect of suppressing a decrease in the volume of steric cell tissue.
本発明の方法を用いることで、従来技術と比較してより厚い立体的細胞組織を製造することができる。また、本発明の方法を用いることで、従来技術と比較してより安定的に立体的細胞組織を製造することができる。これにより、これまで安定的に構築することが困難であった厚さの立体的細胞組織を安定的に製造することが可能になる。 By using the method of the present invention, a thicker three-dimensional cell tissue can be produced as compared with the prior art. In addition, by using the method of the present invention, a three-dimensional cell tissue can be produced more stably as compared with the prior art. This makes it possible to stably produce a three-dimensional cell tissue having a thickness that has been difficult to construct stably.
Claims (13)
細胞をカチオン性物質、細胞外マトリックス成分及び高分子電解質と混合して混合物を得る工程と、
得られた前記混合物中の前記細胞を培養して前記立体的細胞組織を得る工程と、含む方法により製造されたものである、請求項１に記載の立体的細胞組織の培養方法。 The three-dimensional cell tissue
A step of mixing cells with a cationic substance, extracellular matrix components and a polymer electrolyte to obtain a mixture, and
The method for culturing a three-dimensional cell tissue according to claim 1, which is produced by a step of culturing the cells in the obtained mixture to obtain the three-dimensional cell tissue and a method including the method.
前記高分子電解質は、グリコサミノグリカン、デキストラン硫酸、ラムナン硫酸、フコイダン、カラギナン、ポリスチレンスルホン酸、ポリアクリルアミド−２−メチルプロパンスルホン酸、ポリアクリル酸、ならびにそれらの組み合わせからなる群から選択される、請求項２に記載の立体的細胞組織の培養方法。 The extracellular matrix component is selected from the group consisting of collagen, laminin, fibronectin, vitronectin, elastin, tenascin, entactin, fibrillin, proteoglycan, and combinations thereof.
The polymer electrolyte is selected from the group consisting of glycosaminoglycan, dextran sulfate, ramnan sulfate, fucoidan, caraginan, polystyrene sulfonic acid, polyacrylamide-2-methylpropane sulfonic acid, polyacrylic acid, and combinations thereof. The method for culturing a three-dimensional cell tissue according to claim 2.
前記混合物中の前記高分子電解質の濃度が、０．０１ｍｇ／ｍＬ以上１．０ｍｇ／ｍＬ未満である、請求項２又は３に記載の立体的細胞組織の培養方法。 The concentration of the extracellular matrix component in the mixture is 0.01 mg / mL or more and less than 1.0 mg / mL.
The method for culturing a three-dimensional cell tissue according to claim 2 or 3, wherein the concentration of the polymer electrolyte in the mixture is 0.01 mg / mL or more and less than 1.0 mg / mL.
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