JP2020141599A - Cellular tissue production method and cellular tissue production set - Google Patents
Cellular tissue production method and cellular tissue production set Download PDFInfo
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本発明は細胞組織を作成する方法および該方法に適した細胞組織作製セットに関する。
The present invention relates to a method for producing a cell tissue and a cell tissue preparation set suitable for the method.
近年、ＲＦＩＤタグなどの微細な回路を印刷（塗布）方式で描画して形成するプリンテッドエレクトロニクス技術が急速に発展してきている。微細な回路のパターンや電極パターンを形成する方式としては、インクジェット方式、ディスペンサ方式などが一般的であるが、塗布ピンを用いた方式も広範囲の粘度の材料を用いて微細な塗布が可能な点で、注目されている。 In recent years, printed electronics technology for drawing and forming minute circuits such as RFID tags by a printing (coating) method has been rapidly developed. As a method for forming a fine circuit pattern or an electrode pattern, an inkjet method, a dispenser method, etc. are generally used, but a method using a coating pin also allows fine coating using a material having a wide range of viscosities. So, it is attracting attention.
このようなプリンテッドエレクトロニクス技術は、回路や電極の形成への応用のみでなく、細胞組織細胞の作成技術として応用するバイオプリンティング技術として研究されている（例えば、特許文献１）。 Such printed electronics technology is being studied as a bioprinting technology applied not only to the formation of circuits and electrodes but also as a technology for producing cell tissue cells (for example, Patent Document 1).
本出願人らは、プリンティング技術の１つとして、塗布ピンを用いる技術を有しており、塗布ピン技術をバイオプリンティング技術へ応用し、生体外で細胞の三次元組織を構築する技術を研究開発している（特願２０１７−２１３０９５号）。塗布ピンを用いて液体材料の微細な塗布を行なう方法としては、例えば、特許文献２に開示されている。このような塗布ユニットは、微細パターンの欠陥を修正することを目的とするもので、広範囲な粘度の塗布液を用いて微細な塗布を行なうことが可能である。塗布動作の際、塗布液を保持する塗布液容器の底部に形成された貫通孔から、１本の塗布ピンを突出させる。塗布ピンは、先端に付着している塗布液を被塗布物 に接触させて塗布を行なう。 The applicants have a technology using a coating pin as one of the printing technologies, and apply the coating pin technology to the bioprinting technology to research and develop a technology for constructing a three-dimensional tissue of cells in vitro. (Japanese Patent Application No. 2017-21309). For example, Patent Document 2 discloses a method for finely coating a liquid material using a coating pin. Such a coating unit is intended to correct defects in a fine pattern, and can perform fine coating using a coating liquid having a wide range of viscosities. During the coating operation, one coating pin is projected from a through hole formed in the bottom of the coating liquid container that holds the coating liquid. The coating pin is applied by bringing the coating liquid adhering to the tip into contact with the object to be coated.
上記塗布ピンを応用して、ｉＰＳ由来心筋細胞をフィブリノーゲン溶液に分散させて塗布した後、トロンビン溶液に浸浸することでゲル化による組織固定を塗布方法が行われている。該方法においては、フィブリノーゲンを心筋細胞を塗布する際のゲル化剤として用いるにあたり、１液目に心筋細胞とフィブリノーゲンを分散させた塗布剤を塗布し、２液目にゲル化開始剤として塗布剤を覆うようにトロンビンを添加することで塗布を行っている。しかしながら、塗布工程を実行する前に、１液目内部の心筋細胞とフィブリノーゲン溶液に、意図しないゲル化が発生する現象が確認された。そのため、塗布液容器内部でゲル化した心筋細胞塊が目詰まりを起こす場合や、ゲル化した心筋細胞が塗布液容器に残り塗布できない場合があるという不具合が発生した。 By applying the above coating pin, iPS-derived cardiomyocytes are dispersed in a fibrinogen solution and coated, and then immersed in a thrombin solution to fix the tissue by gelation. In this method, when fibrinogen is used as a gelling agent when applying cardiomyocytes, a coating agent in which cardiomyocytes and fibrinogen are dispersed is applied to the first solution, and a coating agent is applied to the second solution as a gelling initiator. The coating is performed by adding thrombin so as to cover the above. However, before performing the coating process, it was confirmed that the cardiomyocytes inside the first solution and the fibrinogen solution developed an unintended gelation. Therefore, there is a problem that the gelled cardiomyocyte mass inside the coating liquid container may be clogged, or the gelled cardiomyocytes may remain in the coating liquid container and cannot be applied.
本発明は、上記事情に鑑みなされたもので、上記のようなバイオプリンティング技術において、塗布工程を実行する前の塗布液容器内部でゲル化、それに伴う目詰まり等の不具合が生じないバイオプリンティング技術を提供する目的で行われた。
驚いたことに、上記目的を達成できるバイオプリンティング技術を用いれば、細胞の生存率を維持した上で、高密度かつ保形性の高い細胞組織を作成できることが見出された。
本発明は、塗布液容器内部でゲル化、それに伴う目詰まり等の不具合が生じず、しかも、細胞の生存率を維持した上で、高密度かつ保形性の高い細胞組織を作製できる細胞の組織作製方法を提供するものである。
The present invention has been made in view of the above circumstances, and in the above-mentioned bioprinting technique, a bioprinting technique that does not cause problems such as gelation inside the coating liquid container before executing the coating step and clogging associated therewith. Was done for the purpose of providing.
Surprisingly, it was found that using bioprinting technology that can achieve the above objectives, it is possible to produce a cell tissue having high density and high shape retention while maintaining cell viability.
INDUSTRIAL APPLICABILITY According to the present invention, a cell tissue capable of producing a high-density and highly shape-retaining cell tissue without causing problems such as gelation inside the coating liquid container and clogging associated therewith, and maintaining the cell viability. It provides a method for creating a tissue.
すなわち、本発明は、第１の塗布液を塗布対象物に塗布する第１塗布工程、および
前記第１塗布工程で塗布された第１の塗布液に、第２の塗布液を重ねて塗布し、第１溶液の塗布液の流動状態を維持しつつ、第１の塗布液と第２の塗布液の界面をゲル化する第２塗布工程、
を含むことを特徴とする細胞組織の作製方法を提供するものである。
That is, in the present invention, the first coating liquid is applied to the object to be coated, and the second coating liquid is superposed on the first coating liquid applied in the first coating step. A second coating step of gelling the interface between the first coating liquid and the second coating liquid while maintaining the fluid state of the coating liquid of the first solution.
The present invention provides a method for producing a cell tissue, which is characterized by containing.
本発明の方法に従えば、塗布ピン等のバイオプリンティング技術における塗布工程において、塗布工程実行前の塗布液容器内部でゲル化、それに伴う目詰まり等の不具合が生じない。
また、本発明の方法に従えば、細胞の生存率を維持した上で、高密度かつ保形性の高い細胞組織を作成できる。
According to the method of the present invention, in the coating process in the bioprinting technique of the coating pin or the like, problems such as gelation inside the coating liquid container before the execution of the coating process and the accompanying clogging do not occur.
Further, according to the method of the present invention, it is possible to prepare a cell tissue having high density and high shape retention while maintaining the cell viability.
本発明の第１の側面は、細胞とゲル化開始剤を含む第１の塗布液を塗布対象物に塗布する第１塗布工程、および
前記第１塗布工程で塗布された第１の塗布液に、ゲル化剤を含む第２の塗布液を重ねて塗布しゲル化反応を開始させる第２塗布工程、
を含むことを特徴とする細胞の組織作製方法に関する。
本発明の第２の側面は、細胞とゲル化開始剤を含む第１の塗布液を含む第１の容器、およびゲル化剤を含む第２の塗布液を含む第２の容器を含む、細胞組織作製セットに関する。
The first aspect of the present invention is a first coating step of applying a first coating solution containing cells and a gelation initiator to an object to be coated, and a first coating solution applied in the first coating step. , A second coating step of superimposing a second coating solution containing a gelling agent to initiate a gelation reaction,
The present invention relates to a method for producing a tissue of a cell, which comprises.
A second aspect of the invention comprises cells, including a first container containing a first coating solution containing cells and a gelation initiator, and a second container containing a second coating solution containing a gelling agent. Regarding tissue preparation set.
まず、本発明の第１の側面である、
細胞とゲル化開始剤を含む第１の塗布液を塗布対象物に塗布する第１塗布工程、および
前記第１塗布工程で塗布された第１の塗布液に、ゲル化剤を含む第２の塗布液を重ねて塗布しゲル化反応を開始させる第２塗布工程、
を含むことを特徴とする細胞の組織作製方法について説明する。
First, the first aspect of the present invention,
A first coating step of applying a first coating solution containing cells and a gelation initiator to an object to be coated, and a second coating solution containing a gelling agent in the first coating solution applied in the first coating step. The second coating step, in which the coating liquids are layered and the gelation reaction is started.
A method for producing a tissue of cells, which is characterized by containing
第１塗布工程
本発明の方法が適用できる細胞は、特に限定されるものではないが、iPS細胞由来の心筋細胞、正常ヒト心臓線維芽細胞、正常ヒト線維芽細胞、ヒト血管内皮細胞、HePG2細胞等の細胞である。２種以上混合して使用してもよい。本発明は、これらの中でも、ｉＰＳ細胞由来の心筋細胞、正常ヒト線維芽細胞、HepG2細胞、特に、ｉＰＳ細胞由来の心筋細胞に適用することが好ましい。ｉＰＳ細胞由来の心筋細胞を使用する場合、より生体の心臓の形態に近づけ、正常ヒト心臓線維芽細胞から発される因子によりiPS細胞由来の心筋細胞を成熟化させる理由で、通常、正常ヒト心臓線維芽細胞と混合して使用される。細胞としては、未修飾の細胞、またはタンパク質、糖鎖、核酸等で修飾された細胞、例えばフィブロネクチン、ゼラチン、コラーゲン、ラミニン、エラスチン、マトリゲス等の既に知られているコーティング剤、コーティング方法でコーティングされた細胞を用いることができる。
First application step The cells to which the method of the present invention can be applied are not particularly limited, but are iPS cell-derived cardiomyocytes, normal human cardiac fibroblasts, normal human fibroblasts, human vascular endothelial cells, and HePG2 cells. Etc. cells. Two or more kinds may be mixed and used. Among these, the present invention is preferably applied to iPS cell-derived cardiomyocytes, normal human fibroblasts, HepG2 cells, and particularly iPS cell-derived cardiomyocytes. When iPS cell-derived cardiomyocytes are used, they are usually closer to the morphology of the living heart and are usually normal human hearts because of the maturation of iPS cell-derived cardiomyocytes by factors emanating from normal human heart fibroblasts. Used in combination with fibroblasts. As cells, unmodified cells or cells modified with proteins, sugar chains, nucleic acids, etc., for example, coated with already known coating agents and coating methods such as fibronectin, gelatin, collagen, laminin, elastin, and matriges. Can be used.
第１の塗布液に含ませるゲル化開始剤としては、トロンビン、塩化カルシウム、アルコール類、例えば、エチルアルコール、グリセリン等）を使用すればよい。これらのゲル化開始剤は、通常、第２溶液に含ませるゲル化剤の種類に応じて使い分けられている。これらの使い分けは、当業者に公知であり、そのような公知の組合せで使用すればよい。例えば、ゲル化開始剤とゲル化剤の使用の組合せ（ゲル化開始剤：ゲル化剤）としては、トロンビン：フィブリノーゲン（ゲル化開始剤：ゲル化剤）、塩化カルシウム：アルギン酸ナトリウム（ゲル化開始剤：ゲル化剤）、塩化カルシウム：カラギーナン（ゲル化開始剤：ゲル化剤）、アルコール類：タマリンドシードガム（ゲル化開始剤：ゲル化剤）等の組合せ使用が挙げられる。
細胞として、ｉＰＳ細胞由来の心筋細胞を使用する場合は、トロンビン：フィブリノーゲン（ゲル化開始剤：ゲル化剤）の組合せ使用が好ましい。
As the gelation initiator contained in the first coating liquid, thrombin, calcium chloride, alcohols, for example, ethyl alcohol, glycerin, etc. may be used. These gelation initiators are usually used properly according to the type of gelling agent contained in the second solution. The proper use of these is known to those skilled in the art, and such known combinations may be used. For example, as a combination of use of a gelling initiator and a gelling agent (gelling initiator: gelling agent), thrombin: fibrinogen (gelling initiator: gelling agent), calcium chloride: sodium alginate (gelling initiation). Agent: gelling agent), calcium chloride: carrageenan (gelling initiator: gelling agent), alcohols: tamarind seed gum (gelling initiator: gelling agent) and the like.
When iPS cell-derived cardiomyocytes are used as cells, it is preferable to use a combination of thrombin: fibrinogen (gelling initiator: gelling agent).
第１の塗布液は、上記細胞とゲル化開始剤が、水性溶液に含まれるものである。 In the first coating solution, the cells and the gelation initiator are contained in an aqueous solution.
水性溶液としては、水、各種の生理食塩水、例えば、リン酸緩衝生理食塩水、トリス緩衝生理食塩水等を使用するようにすればよい。他にも、細胞培養液として使用される培地、例えばDulbecco’s Modified Eagle Medium等を使用することができる。これらの水溶液は、通常、細胞の種類に応じて使い分けられている。これらの使い分けは、当業者に公知であり、そのような公知の組合せで使用すればよい。例えば、ｉＰＳ細胞由来の心筋細胞であれば、リン酸緩衝生理食塩水（PBS)、Dulbecco’s Modified Eagle Medium、各社専用培地、特に、リン酸緩衝生理食塩水を使用することが好ましい。 As the aqueous solution, water or various physiological salines, for example, phosphate buffered saline, Tris buffered saline or the like may be used. In addition, a medium used as a cell culture medium, such as Dulbecco's Modified Eagle Medium, can be used. These aqueous solutions are usually used properly according to the type of cells. The proper use of these is known to those skilled in the art, and such known combinations may be used. For example, in the case of cardiomyocytes derived from iPS cells, it is preferable to use phosphate buffered saline (PBS), Dulbecco's Modified Eagle Medium, a medium dedicated to each company, and in particular, phosphate buffered saline.
上記水性液に含ませる細胞の量は、特に、限定されるものではないが、1ｘ１０5個／ｍＬ〜1ｘ１０9個／ｍＬ、好ましくは、1ｘ１０6個／ｍＬ〜1ｘ１０8個／ｍＬ、より好ましくは、1ｘ１０7個／ｍＬ〜1ｘ１０8個／ｍＬ程度の濃度で含有させるようにすればよい。多すぎると、溶液の調整が困難であり、少なすぎると、塗布量によっては細胞が含まれないものとなる。 The amount of cells contained in the aqueous solution is not particularly limited, but is 1x10 5 cells / mL to 1x10 9 cells / mL, preferably 1x10 6 cells / mL to 1x10 8 cells / mL, more preferably. May be contained at a concentration of about 1x10 7 pieces / mL to 1x10 8 pieces / mL. If it is too large, it will be difficult to prepare the solution, and if it is too small, cells will not be contained depending on the application amount.
ゲル化開始剤の含有量は、特に限定されるものではなく、通常、ゲル化の速さを考慮し、適宜その含有量を設定するようにすればよい。その量が、少なすぎると、ゲル化の速さが遅くなり、1液目の保形性が低下する。例えば、トロンビンを用いる場合、その濃度は、1 ｕｎｉｔ／ｍＬ〜１０００ ｕｎｉｔ／ｍＬ、好ましくは１０ ｕｎｉｔ／ｍＬ〜１０００ ｕｎｉｔ／ｍＬ、より好ましくは、１００ ｕｎｉｔ／ｍＬ〜８００ ｕｎｉｔ／ｍＬ程度の濃度で含有させるようにすればよい。 The content of the gelation initiator is not particularly limited, and usually, the content thereof may be appropriately set in consideration of the speed of gelation. If the amount is too small, the gelation speed will be slowed down and the shape retention of the first liquid will be reduced. For example, when thrombin is used, its concentration is about 1 unit / mL to 1000 unit / mL, preferably 10 unit / mL to 1000 unit / mL, and more preferably 100 unit / mL to 800 unit / mL. It may be contained.
第１の塗布液には、細胞が安定して接着・増殖できる環境を与えるために、フィブロネクチン、ゼラチン、コラーゲン、ラミニン、エラスチン、マトリゲル等の細胞外マトリックス成分、線維芽細胞増殖因子や血小板由来成長因子等の細胞増殖因子、その他、血管内皮細胞やリンパ管内皮細胞、各種幹細胞等の添加剤を含ませてもよい。 In order to provide an environment in which cells can stably adhere and proliferate, the first coating solution contains extracellular matrix components such as fibronectin, gelatin, collagen, laminin, elastin, and matrigel, fibroblast growth factor, and platelet-derived growth. In addition to cell growth factors such as factors, additives such as vascular endothelial cells, lymphatic endothelial cells, and various stem cells may be included.
第１塗布溶液には、添加剤としてさらに増粘剤を添加してもよい。増粘剤としては、増粘多糖類、例えば、ヒアルロン酸ナトリウム、アルギン酸ナトリウムを使用することができる。 A thickener may be further added as an additive to the first coating solution. As the thickener, thickening polysaccharides such as sodium hyaluronate and sodium alginate can be used.
増粘剤は、第１の塗布溶液に保形性を付与するために含ませるものであり、特に、増粘多糖類は、保形性の高い三次元細胞組織の作成に効果がある。 The thickener is included in the first coating solution to impart shape retention, and in particular, the thickening polysaccharide is effective in producing a three-dimensional cell tissue having high shape retention.
増粘剤を含ませる場合、その量は、第1の塗布液の保形性、使用する塗布装置の可能な粘度範囲、コスト、取り扱い性等を考慮し適宜設定するようにすればよい。例えば、塗布ピン法では１ｍＰａ・ｓ〜１×１０５ｍＰａ・ｓ、好ましくは、３×１０３ｍＰａ・ｓ〜１×１０５ｍＰａ・ｓ、より好ましくは、１×１０４ｍＰａ・ｓ〜５×１０４ｍＰａ・ｓの粘度になるように増粘剤を含ませるようにすればよい。その粘度が低すぎると第１の塗布溶液の保形性が低下する。なお、塗布液の粘度は、回転粘度法により25℃で測定された値を用いている。 When the thickener is included, the amount thereof may be appropriately set in consideration of the shape retention of the first coating liquid, the possible viscosity range of the coating apparatus to be used, the cost, the handleability, and the like. For example, in the coating pin method, 1 mPa · s to 1 x 10 5 mPa · s, preferably 3 x 10 3 mPa · s to 1 x 10 5 mPa · s, more preferably 1 x 10 4 mPa · s to 5. A thickener may be added so as to have a viscosity of × 10 4 mPa · s. If the viscosity is too low, the shape retention of the first coating solution will decrease. The viscosity of the coating liquid uses the value measured at 25 ° C. by the rotational viscosity method.
細胞外マトリックスは、細胞の成長を促し、細胞と細胞、あるいは細胞と基質の接着を促すために含ませるものである。細胞外マトリックスを含ませる場合、その量は特に制限はない。作製したい組織に合わせてその種類、濃度を指定するようにすればよい。 The extracellular matrix is included to promote cell growth and promote cell-to-cell or cell-to-matrix adhesion. When the extracellular matrix is included, the amount is not particularly limited. The type and concentration may be specified according to the tissue to be produced.
本発明の第１塗布工程においては、第１の塗布液を塗布対象物に塗布する。 In the first coating step of the present invention, the first coating liquid is applied to the object to be coated.
使用される塗布対象物は、特に限定されず、細胞増殖のために通常使用されている、基板、プレート、例えば、６から３８４のような多ウェルプレート、ディッシュ、シャーレ、セルデスク（商標、住友ベークライト）等を使用することができる。 The application object used is not particularly limited, and substrates, plates, multi-well plates such as 6 to 384, dishes, petri dishes, cell desks (trademark, Sumitomo Bakelite), which are usually used for cell proliferation, are used. ) Etc. can be used.
塗布方法は、特に限定されるものではなく、例えば、公知のバイオプリンティング技術、例えば、インクジェット方式、ディスペンサ方式、レーザーアシスト方式等を使用することができる。 The coating method is not particularly limited, and for example, known bioprinting techniques such as an inkjet method, a dispenser method, and a laser assist method can be used.
溶液の粘度が高い場合は、塗布ピン法が好適に使用できる。塗布ピン法を用いて溶液、液体の塗布を行う方法については、例えば、特許第４８０２０２７号公報明細書（第０００１段〜第００５０段）、図面（図１〜図５）、および特願２０１７−２１３０９５号明細書（第０００１段〜第００８２段）、図面（図１〜図１５）に開示、記載されている。それらの明細書、図面に開示、記載されている全内容を本願明細書の記載内容の一部としてここに引用する。 When the viscosity of the solution is high, the coating pin method can be preferably used. Regarding the method of applying a solution or a liquid using the coating pin method, for example, Japanese Patent No. 4802027 (stages 0001 to 0050), drawings (FIGS. 1 to 5), and Japanese Patent Application No. 2017- It is disclosed and described in the specification No. 213095 (stages 0001 to 802) and drawings (FIGS. 1 to 15). The entire contents disclosed and described in those specifications and drawings are cited herein as part of the contents described in the present specification.
塗布ピン法は、広範囲な粘度の塗布液を用いて微細な塗布を行なうことが可能である。塗布動作の際、塗布液を保持する塗布液容器の底部に形成された貫通孔から、１本の塗布ピンを突出させる。塗布ピン法は、先端に付着している塗布液を被塗布物に接触させて塗布を行なうものである。 In the coating pin method, fine coating can be performed using a coating liquid having a wide range of viscosities. During the coating operation, one coating pin is projected from a through hole formed in the bottom of the coating liquid container that holds the coating liquid. In the coating pin method, the coating liquid adhering to the tip is brought into contact with the object to be coated to perform coating.
塗布ピン法においては、先端に極微量な塗布液を付着させた針（塗布針）を対象物（基板等）に接触させることにより、数ｐＬ（ピコリットル）から数100μL(マイクロリットル)の塗布量となる液滴を高い配置精度、例えば±１５μｍ以下、好ましくは±３μｍ以下の配置精度で塗布することができる。また、塗布液の粘度としては、１×１０５ｍＰａ・ｓまでの材料を塗布することが可能であり、高粘度の細胞分散液の塗布が可能となる。このように、塗布ピン法によれば、高粘度の細胞分散液を対象物（基板等）に対して所定の位置に精密塗布することが可能である。特に、粘度として、3×１０3ｍＰａ・ｓ程度以上の塗布溶液を使用することにより、塗布液を立体的に、詳しくは、立体的ドーム状に塗布することができ、第２塗布工程までの間にその立体的形状を保つことができる。本発明においてはこの特性を「保形性」という。 In the coating pin method, a needle (coating needle) having a very small amount of coating liquid attached to the tip is brought into contact with an object (such as a substrate) to coat several pL (picolitre) to several hundred μL (microliter). A large amount of droplets can be applied with high placement accuracy, for example, ± 15 μm or less, preferably ± 3 μm or less. Further, as the viscosity of the coating liquid, it is possible to apply a material up to 1 × 10 5 mPa · s, and it is possible to apply a highly viscous cell dispersion liquid. As described above, according to the coating pin method, it is possible to precisely coat a highly viscous cell dispersion liquid at a predetermined position on an object (such as a substrate). In particular, by using a coating solution having a viscosity of about 3 × 10 3 mPa · s or more, the coating solution can be coated three-dimensionally, more specifically, in a three-dimensional dome shape, up to the second coating step. The three-dimensional shape can be maintained in between. In the present invention, this property is referred to as "shape retention".
塗布量は、所望の量に応じて塗布方法の条件を適宜設定すればよい。塗布ピン法においては、塗布ピンの直径、塗布回数等により調整可能である。 As for the coating amount, the conditions of the coating method may be appropriately set according to the desired amount. In the coating pin method, the diameter of the coating pin, the number of coatings, and the like can be adjusted.
より具体的には、塗布ピン法は、
第１の塗布液を所定量貯留する塗布液溜りを有する塗布液容器と、
前記第１の塗布液が貯留された前記塗布液溜りを貫通可能な塗布針と、を含む塗布ユニットを備えた微細塗布装置を用い、
前記塗布液溜りに充填された前記第１の塗布液に前記塗布針の先端を浸漬させる待機工程、
前記塗布針の先端が前記塗布液溜りを貫通して、前記第１の塗布液が付着された前記塗布針の先端を下降させる下降工程、
前記第１の塗布液が付着された前記塗布針の先端を塗布対象物に接触させて、前記第１の塗布液を前記塗布対象物に塗布して液滴スポットを形成する塗布工程、および
前記塗布針の先端を上昇させて、前記塗布針の先端を前記塗布液溜りに収容する収容工程、
を含んで実行される。
More specifically, the coating pin method
A coating liquid container having a coating liquid reservoir for storing a predetermined amount of the first coating liquid, and
Using a fine coating device including a coating unit including a coating needle capable of penetrating the coating liquid pool in which the first coating liquid is stored,
A standby step of immersing the tip of the coating needle in the first coating liquid filled in the coating liquid pool.
A lowering step in which the tip of the coating needle penetrates the coating liquid pool and the tip of the coating needle to which the first coating liquid is attached is lowered.
A coating step in which the tip of the coating needle to which the first coating liquid is attached is brought into contact with the coating object, and the first coating liquid is applied to the coating object to form a liquid drop spot, and the above. A storage step in which the tip of the coating needle is raised and the tip of the coating needle is accommodated in the coating liquid pool.
Is executed including.
塗布ピン法以外のバイオプリンティング技術を使用する場合においても、塗布条件を適宜設定することにより塗布ピンと同様の塗布物が得られるように適宜塗布条件を調整して塗布を行えばよい。 Even when a bioprinting technique other than the coating pin method is used, the coating conditions may be appropriately adjusted so that a coating material similar to that of the coating pin can be obtained by appropriately setting the coating conditions.
第２塗布工程
第２塗布工程において使用される第２の塗布液は、ゲル化剤が、水性溶液に含まれるものである。
Second coating step The second coating liquid used in the second coating step contains a gelling agent in an aqueous solution.
第２の塗布液に含ませるゲル化剤としては、フィブリノーゲン、アルギン酸ナトリウム等を使用すればよい。これらのゲル化剤は、通常、第１溶液に含ませるゲル化開始剤の種類に応じて使い分けられている。これらの使い分け、組合わせは、上記第１溶液において説明した。 As the gelling agent to be contained in the second coating liquid, fibrinogen, sodium alginate or the like may be used. These gelling agents are usually used properly according to the type of gelation initiator contained in the first solution. The proper use and combination of these have been described in the first solution.
第２の塗布液に含ませるゲル化剤として、ゲル化開始剤なしで硬化するゲル化剤を使用してもよい。このようなゲル化剤を使用する場合は、第１の塗布液にゲル化開始剤を含ませる必要はない。このようなゲル化剤として、コラーゲン、ゼラチン、寒天（アガロース）等が挙げられる。例えば、コラーゲンは加熱することによりゲル化し、ゼラチンは冷却することによりゲル化する。このようなゲル化剤を含有する第２の塗布液を使用した場合は、第２の塗布液を塗布した後、コラーゲンを含む場合は加熱操作が、ゼラチンを含む場合は冷却操作を行いゲル化反応する必要がある。 As the gelling agent to be contained in the second coating liquid, a gelling agent that cures without a gelation initiator may be used. When such a gelling agent is used, it is not necessary to include the gelation initiator in the first coating liquid. Examples of such a gelling agent include collagen, gelatin, agar (agarose) and the like. For example, collagen gels when heated and gelatin gels when cooled. When a second coating solution containing such a gelling agent is used, after applying the second coating solution, a heating operation is performed when collagen is contained, and a cooling operation is performed when gelatin is contained to gel. You need to react.
第２塗布液に含まれる水性溶液は、上記した第１の塗布液で使用する同様の水性溶液を使用することができ、第１塗布液で使用されている同一の水性溶液でもよいし、異なる種類の水性溶液を使用してもよい。 As the aqueous solution contained in the second coating liquid, the same aqueous solution used in the first coating liquid described above can be used, and the same aqueous solution used in the first coating liquid may be used, or different. Any type of aqueous solution may be used.
上記水性液に含ませるゲル化剤の量は、特に限定されるものではなく、ゲル化の速さ、ゲル化剤の濃度、ゲルの硬さ等を考慮し、適宜その含有量を設定するようにすればよい。少なすぎると、ゲル化の速さが遅くなる。例えば、フィブリノーゲンを用いる場合、その濃度は０．１ｍｇ／ｍＬ〜１００ｍｇ／ｍＬ、好ましくは、１ｍｇ／ｍＬ〜８０ｍｇ／ｍＬ、より好ましくは、１ｍｇ／ｍＬ〜３０ｍｇ／ｍＬの濃度になるようにすればよい。
第２の塗布液には、増粘剤等の成分（さらなる添加剤）が含まれてもよい。
The amount of the gelling agent contained in the aqueous solution is not particularly limited, and the content thereof should be appropriately set in consideration of the speed of gelation, the concentration of the gelling agent, the hardness of the gel, and the like. It should be. If it is too small, the gelation speed will be slow. For example, when fibrinogen is used, its concentration may be 0.1 mg / mL to 100 mg / mL, preferably 1 mg / mL to 80 mg / mL, and more preferably 1 mg / mL to 30 mg / mL. Good.
The second coating liquid may contain a component (additional additive) such as a thickener.
増粘剤は、塗布済の第１の塗布液の保形の観点から含ませるもので、ヒアルロン酸ナトリウム、アルギン酸ナトリウム等を使用することができる。増粘剤を含ませる場合、その量は、塗布済の第1の塗布液の保形性、使用する塗布装置の可能な粘度範囲、コスト、取り扱い性等を考慮し適宜設定するようにすればよい。例えば、塗布ピン法では、５×１０２ｍＰａ・ｓ〜１×１０５ｍＰａ・ｓ、好ましくは、５×１０２ｍＰａ・ｓ〜１×１０４ｍＰａ・ｓ、より好ましくは、５×１０２ｍＰａ・ｓ〜５×１０４ｍＰａ・ｓの粘度になるように増粘剤を含ませるようにすればよい。その量が少なく、粘度が低すぎると、塗布済の第１の塗布液の保形性が保てなくなる。なお、塗布液の粘度は、回転粘度法により25℃で測定された値を用いている。 The thickener is included from the viewpoint of shape retention of the first coated liquid, and sodium hyaluronate, sodium alginate and the like can be used. When a thickener is included, the amount thereof should be appropriately set in consideration of the shape retention of the first coated liquid, the possible viscosity range of the coating device to be used, the cost, the handleability, and the like. Good. For example, in the coating pin method, 5 × 10 2 mPa · s to 1 × 10 5 mPa · s, preferably 5 × 10 2 mPa · s to 1 × 10 4 mPa · s, more preferably 5 × 10 2. A thickener may be added so as to have a viscosity of mPa · s to 5 × 10 4 mPa · s. If the amount is too small and the viscosity is too low, the shape-retaining property of the first coated liquid that has been applied cannot be maintained. The viscosity of the coating liquid uses the value measured at 25 ° C. by the rotational viscosity method.
第２の塗布液は、第１塗布工程で塗布された第１の塗布液に、第２の塗布液を重ねて塗布する。「重ねて」とは、「第１塗布工程で塗布された第１の溶液の塗布対象物（基板等）と接している面以外の面を覆うように」という意味である。 The second coating liquid is formed by superimposing the second coating liquid on the first coating liquid applied in the first coating step. "Overlapping" means "covering a surface other than the surface in contact with the object to be coated (such as a substrate) of the first solution applied in the first coating step".
上記のように、第２の塗布液を、第１の塗布液に重ねて塗布することにより、塗布された第１の溶液と第２の溶液との界面がゲル化する。この時、このゲル化した界面の下に存在する第１の塗布液の内部はゲル化せず、第１の溶液の溶液状態（粘度）を保っている。そのため、第１の塗布溶液内部の細胞は、重力により沈降沈殿し、塗布溶液下部に凝集、堆積、積層する。 As described above, by applying the second coating solution on top of the first coating solution, the interface between the applied first solution and the second solution is gelled. At this time, the inside of the first coating liquid existing under the gelled interface does not gel, and the solution state (viscosity) of the first solution is maintained. Therefore, the cells inside the first coating solution settle and settle due to gravity, and aggregate, deposit, and stack under the coating solution.
第１塗布工程終了から、第２の塗布工程開始までの時間間隔は、できる限り短いことが望ましい。その、時間間隔が長すぎると、第１の塗布液が乾燥し、含有される細胞が死滅するという問題が生じる。 It is desirable that the time interval from the end of the first coating process to the start of the second coating process is as short as possible. If the time interval is too long, there arises a problem that the first coating liquid dries and the contained cells die.
第２の塗布液の塗布量は、第１の塗布液の塗布量、ゲル化の速さ等を考慮して、第２の塗布液が第１の塗布液に重ねて塗布されるように適宜設定される。 The coating amount of the second coating liquid is appropriately adjusted so that the second coating liquid is overlaid on the first coating liquid in consideration of the coating amount of the first coating liquid, the speed of gelation, and the like. Set.
塗布方法は、特に限定されるものではなく、例えば、手作業による塗布、例えば、注射器、スポイト等による液滴塗布、公知のバイオプリンティング技術、例えば、塗布ピン法、インクジェット法、ディスペンサ法等を使用することができる。 The coating method is not particularly limited, and for example, manual coating, for example, droplet coating with a syringe, a dropper, or the like, a known bioprinting technique, for example, a coating pin method, an inkjet method, a dispenser method, or the like is used. can do.
第２の溶液を塗布後、第１の溶液と第２の溶液の接触面（界面）のみがゲル化し、ゲル化した界面の下に存在する第１の溶液内部は硬化開始剤が液体として存在し、第１の溶液の溶液状態（粘度）を保っており、時間の経過に従って、細胞が沈降沈殿し、溶液下部に凝集、堆積、積層することにより、複雑な製造工程を介さず高密度かつ保形性の高い三次元細胞組織が形成される。
塗布直後の細胞組織の外周部である第１の溶液と第２の溶液の接触面がゲル化し、第１溶液の内部は液体の状態で保持されるため、細胞組織の乾燥も抑制できる。
After applying the second solution, only the contact surface (interface) between the first solution and the second solution gels, and the curing initiator exists as a liquid inside the first solution existing under the gelled interface. However, the solution state (viscosity) of the first solution is maintained, and as time passes, cells settle and settle, and by aggregating, accumulating, and laminating under the solution, high density and high density are achieved without going through a complicated manufacturing process. A three-dimensional cell tissue with high shape retention is formed.
Since the contact surface between the first solution and the second solution, which is the outer peripheral portion of the cell tissue immediately after application, is gelled and the inside of the first solution is kept in a liquid state, drying of the cell tissue can be suppressed.
細胞の培養は、第１塗布工程および第２塗布工程で塗布された塗布物を培地中に浸しておこなう。このとき、塗布対象物（基板）と共に培地に浸してもよい。培地としては、細胞との関係で、通常使用される培地を使用すればよく、例えば、細胞にｉＰＳ細胞由来心筋細胞を含む場合は、Dulbecco’s Modified Eagle Medium、各社市販専用培地が使用される。 The cells are cultured by immersing the coating material applied in the first coating step and the second coating step in the medium. At this time, it may be immersed in the medium together with the object to be coated (substrate). As the medium, a medium usually used may be used in relation to the cells. For example, when the cells contain iPS cell-derived cardiomyocytes, Dulbecco's Modified Eagle Medium, a medium commercially available from each company is used.
図１に、本発明で実施した細胞の組織作製方法の概略、塗布後の細胞の三次元集積の形態を模式図で表した。
細胞とゲル化開始剤とを懸濁含有する第１の塗布液を用意し、該第１の塗布液を塗布容器に充填する。塗布ピンにより基板に第１の塗布液を塗布する（１液目）。次に、ゲル化剤を含む第２の塗布液（２液目）を、１液目を覆うように塗布する。１液目と２液目の接触面から外周までがゲル化した後、培地を添加する。１液目の第１の塗布液内部は、流動性が保たれた液体状態が保持されてドーム構造となっているため、時間経過とともに細胞が沈降沈殿し、塗布溶液下部に凝集、堆積、積層する。その結果、高密度の三次元細胞組織を作成することができる。
本発明において、「高密度」とは、細胞間にフィブリンゲル等が存在せず、細胞が集積している状態を意味し、細胞密度として数値的に表すと、１×１０８個／ｍＬ〜１×１０9個／ｍＬ程度の密度を有する場合を意味している。
FIG. 1 is a schematic diagram showing the outline of the cell tissue preparation method carried out in the present invention and the morphology of three-dimensional accumulation of cells after application.
A first coating solution containing the cells and the gelation initiator in suspension is prepared, and the first coating solution is filled in the coating container. The first coating liquid is applied to the substrate by the coating pin (first liquid). Next, the second coating liquid (second liquid) containing the gelling agent is applied so as to cover the first liquid. After gelation from the contact surface of the first liquid and the second liquid to the outer circumference, the medium is added. Since the inside of the first coating liquid of the first liquid is maintained in a liquid state in which fluidity is maintained and has a dome structure, cells settle and settle over time, and aggregate, deposit, and stack under the coating solution. To do. As a result, a high-density three-dimensional cell tissue can be created.
In the present invention, "high density" means a state in which no fibrin gel or the like exists between cells and cells are accumulated, and when expressed numerically as cell density, 1 × 10 8 cells / mL ~. This means that the cells have a density of about 1 × 10 9 cells / mL.
本発明の第２の側面は、少なくとも、細胞とゲル化開始剤を含む第１の塗布液を含む第１の容器、およびゲル化剤を含む第２の塗布液を含む第２の容器を含む、細胞組織作製セットに関する。
該セットは、上記した本発明の細胞の組織作製方法への使用に適している。
上記細胞組織作製セットに規定する第１の塗布液および第２の塗布液は、上記した本発明の細胞の組織作製方法で説明した第１の塗布液および第２の塗布液と同義である。
A second aspect of the invention includes at least a first container containing a first coating solution containing cells and a gelation initiator, and a second container containing a second coating solution containing a gelling agent. , Concerning the cell tissue production set.
The set is suitable for use in the above-mentioned method for producing a tissue of cells of the present invention.
The first coating solution and the second coating solution specified in the cell tissue preparation set are synonymous with the first coating solution and the second coating solution described in the above-described method for producing cell tissue of the present invention.
以下、具体的に実施例を用いて本発明を説明するが、本発明はそれらの実施例に限定的に解釈されるべきでなく、本発明の概念に接した当業者が想到し、実施可能であると観念するであろうあらゆる技術的思想、その具体的態様が本発明に含まれるものとして理解されるべきものである Hereinafter, the present invention will be specifically described with reference to Examples, but the present invention should not be construed as being limited to those Examples, and can be conceived and implemented by those skilled in the art who are familiar with the concept of the present invention. Any technical idea that would be considered to be, and its specific aspects, should be understood as being included in the present invention.
実施例１
（１）塗布方法
（１−１）第１の溶液の塗布
本実施例で使用した第１の溶液の塗布装置（塗布ピン法）を図２、塗布機構を図３に示す。
ＸＹＺステージに支持された塗布機構４は、カム４３の動作により、可動部４６と塗布ピン２０がＺ方向に往復動作を行い、塗布液容器２１に貫通可能に支持された塗布ピン２４が同様のＺ方向往復動作を行う。この動作により、針表面に付着した塗布材料容器２１内の塗布材が対象物に塗布される。
Example 1
(1) Coating method (1-1) Coating of the first solution FIG. 2 shows the coating device (coating pin method) for the first solution used in this embodiment, and FIG. 3 shows the coating mechanism.
In the coating mechanism 4 supported by the XYZ stage, the movable portion 46 and the coating pin 20 reciprocate in the Z direction by the operation of the cam 43, and the coating pin 24 supported so as to penetrate the coating liquid container 21 is the same. Reciprocating in the Z direction is performed. By this operation, the coating material in the coating material container 21 adhering to the needle surface is applied to the object.
制御部は、モニタ９、制御用コンピュータ１０、操作パネル８からなる。操作パネルから塗布速度指令値を入力し、制御用コンピュータ１０の記憶装置に保管する。塗布動作時には塗布速度指令値を記憶装置から読みだして塗布機構の制御プログラムに送る。制御プログラムは塗布速度指令値に基づいて塗布機構のモータ４１の回転速度を決定し、所定の速度で塗布ピンを上下動させて塗布動作を行う。制御用コンピュータが上位の制御システム（図示せず）と通信している場合には、塗布速度指令値を上位の制御システムから受け取ってもよい。また、塗布液の種類に応じたパラメータを記憶部に保管し、指定された塗布液の種類と塗布量または塗布寸法に応じて塗布速度指令値を算出してもよい。 The control unit includes a monitor 9, a control computer 10, and an operation panel 8. The coating speed command value is input from the operation panel and stored in the storage device of the control computer 10. At the time of coating operation, the coating speed command value is read from the storage device and sent to the control program of the coating mechanism. The control program determines the rotation speed of the motor 41 of the coating mechanism based on the coating speed command value, and moves the coating pin up and down at a predetermined speed to perform the coating operation. When the control computer is communicating with a higher control system (not shown), the coating speed command value may be received from the higher control system. Further, the parameters corresponding to the type of the coating liquid may be stored in the storage unit, and the coating speed command value may be calculated according to the specified type and coating amount of the coating liquid or the coating dimension.
塗布ピンは塗布材料容器の底面に設けられた先端穴から突出して塗布動作を行う。塗布ピン先端が塗布材料容器の底面から突き出す時に、塗布ピン先端部に塗布材料が付着して容器外に出る。この時、表面張力によって塗布材料が上方に引き上げられ、塗布ピン先端にはほぼ一定量の塗布材料が残される。残された塗布材料を塗布対象に転写することで、再現性のある塗布を実現できる。 The coating pin projects from the tip hole provided on the bottom surface of the coating material container to perform the coating operation. When the tip of the coating pin protrudes from the bottom surface of the coating material container, the coating material adheres to the tip of the coating pin and goes out of the container. At this time, the coating material is pulled upward by the surface tension, and a substantially constant amount of the coating material is left at the tip of the coating pin. By transferring the remaining coating material to the coating target, reproducible coating can be realized.
（１−２）第２の溶液の塗布
第２の溶液の塗布は、マイクロピペットを用いて手動で滴下することにより行った。
(1-2) Application of the second solution The application of the second solution was carried out by manually dropping the solution using a micropipette.
（２）塗布条件
・ピン径： φ１０００μｍ
・ピン形状： ストレート
・塗布回数： １０回
・第１の塗布液の塗布量： 数１００ nＬ/10回
・第２の塗布液の塗布量： １５ μＬ/1回
・塗布容器内待機時間： １０２０ ｍｓ
・ピン降下後待機時間： ０ｍｓ
・上昇時間： ５μｍ／１回
・２液目添加までの所要時間： ５ｓ以内
・培地添加までの所要時間： ４個作成後まとめて、マイクロピペットにて手動で添加
(2) Coating conditions ・ Pin diameter: φ1000 μm
・ Pin shape: Straight ・ Number of coatings: 10 times ・ Coating amount of the first coating liquid: Several 100 nL / 10 times ・ Coating amount of the second coating liquid: 15 μL / time ・ Standby time in the coating container: 1020 ms
・ Wait time after pin descent: 0ms
・ Rise time: 5 μm / once ・ Time required to add the second liquid: Within 5 s ・ Time required to add the medium: After making 4 pieces, add them manually with a micropipette
（３）塗布液
（３−１）第１の塗布液（１液目）
・ｉＰＳ細胞由来心筋細胞： ヒト心臓線維芽細胞 ＝ ３：１
・細胞数： ８ｘ１０７／ｍＬ
・ヒアルロン酸ナトリウム： １３ｍｇ／ｍＬ
・トロンビン： ８００ｕｎｉｔ／ｍＬ
・ＰＢＳ
・粘度： １.５×１０4ｍＰａ・ｓ）
（３−２）第２の塗布液（２液目）
・添加量： １５μＬ
・ヒアルロン酸ナトリウム： ０．５ ｍｇ／ｍＬ
・フィブリノーゲン： １０ ｍｇ／ｍＬ
・ＰＢＳ
・粘度： 1×１０3ｍＰａ・ｓ
(3) Coating liquid (3-1) First coating liquid (first liquid)
-IPS cell-derived cardiomyocytes: human cardiac fibroblasts = 3: 1
- number of cells: 8x10 7 / mL
・ Sodium hyaluronate: 13 mg / mL
・ Thrombin: 800 units / mL
・ PBS
・ Viscosity: 1.5 × 10 4 mPa ・ s)
(3-2) Second coating liquid (second liquid)
・ Addition amount: 15 μL
・ Sodium hyaluronate: 0.5 mg / mL
-Fibrinogen: 10 mg / mL
・ PBS
・ Viscosity: 1 × 10 3 mPa ・ s
（４）結果考察
（４−１）作製方法
上記塗布条件で繰返し塗布を行い、１液目容器内の心筋細胞とゲル化開始剤のトロンビンを残らず塗布できることを確認した。また塗布終了後、容器内部を目視したところ、ゲルの発生は確認されなかった。
(4) Consideration of results (4-1) Production method Repeated application under the above application conditions, and it was confirmed that all the cardiomyocytes in the first liquid container and thrombin, which is a gelling initiator, could be applied. Moreover, when the inside of the container was visually inspected after the application was completed, no gel generation was confirmed.
上記の条件にて96ウェルプレート内の40ウェルに塗布を行った結果の塗布状態を、プレート上方から撮影した写真を、図４(倍率２倍）に示す。図４に示されているように、多数回塗布しても、概ね細胞数にばらつきはなかった。 FIG. 4 (2x magnification) shows a photograph taken from above the plate showing the coating state as a result of applying the coating to 40 wells in the 96-well plate under the above conditions. As shown in FIG. 4, there was almost no variation in the number of cells even when applied many times.
（４−２）組織観察
（４−２−１）密度
塗布１時間後に得られた細胞組織を、位相差顕微鏡を用いて顕微鏡観察した。該顕微鏡観察は、塗布液（図１の右下に描いたゲルドームと記載された状態）を上から観察したものである。その写真を図５に示す。
図５からわかるように、細胞と細胞との間に細胞間マトリックスがほとんど存在せず、高密度の三次元細胞組織が作製されたことが解る。密度は３.５×１０８個／ｍＬであった。
なお、培養は、温度 ３７℃、ＣＯ２ ５％の環境下、７日間行った。
(4-2) Tissue Observation (4-2-1) The cell tissue obtained 1 hour after the density application was observed under a microscope using a phase contrast microscope. The microscopic observation is a top view of the coating liquid (state described as the gel dome drawn in the lower right of FIG. 1). The photograph is shown in FIG.
As can be seen from FIG. 5, it can be seen that there is almost no extracellular matrix between cells, and a high-density three-dimensional cell tissue is produced. The density was 3.5 × 10 8 pieces / mL.
Incidentally, the culture, the temperature 37 ° C., CO 2 under 5% environment were performed 7 days.
（４−２−２）厚さ
培養後に得られた細胞組織の厚さを、細胞核および細胞質をヘマトキシリン・エオジン（ＨＥ）染色により測定した。ヘマトキシリン・エオジン（ＨＥ）染色測定結果を図６に示す。
図６より、この組織の厚さは、５０μｍ程度の厚さであった。
(4-2-2) Thickness The thickness of the cell tissue obtained after culturing was measured by staining the cell nucleus and cytoplasm with hematoxylin and eosin (HE). The measurement results of hematoxylin and eosin (HE) staining are shown in FIG.
From FIG. 6, the thickness of this structure was about 50 μm.
（４−２−３）細胞組織
細胞核、アクチン、トロポニン染色を行った。
その蛍光染色の結果を図７に示す。
この写真は、本来カラー写真であり、細胞核が青で、アクチンが赤で、心筋トロポニンＴが緑で写し出されている。図７より、心筋細胞に特徴的な心筋トロポニンＴの横紋構造の発現を確認した。また拍動数は成人の心臓に近い50〜60拍動/分であった。
(4-2-3) Cell tissue Cell nuclei, actin, and troponin were stained.
The result of the fluorescent staining is shown in FIG.
This photograph is originally a color photograph, and the cell nucleus is blue, actin is red, and myocardial troponin T is projected in green. From FIG. 7, the expression of the striated structure of myocardial troponin T, which is characteristic of cardiomyocytes, was confirmed. The number of beats was 50 to 60 beats / minute, which is close to that of the adult heart.
（４−３）考察
１液目に心筋細胞とゲル化開始剤のトロンビンを分散させた塗布液を塗布し、２液目にゲル化剤であるフィブリノーゲンを含む塗布液を、１液目の塗布液を覆うように添加する塗布方法により、高密度かつ保形性の高い三次元細胞組織を、複雑な製造工程を介さず、細胞生存率を維持し、簡易に作成することができた。
(4-3) Discussion A coating solution in which cardiomyocytes and thrombin, a gelling initiator, are dispersed is applied to the first solution, and a coating solution containing fibrinogen, which is a gelling agent, is applied to the second solution. By the coating method of adding so as to cover the liquid, a high-density and highly shape-retaining three-dimensional cell tissue could be easily prepared while maintaining the cell viability without going through a complicated manufacturing process.
比較例１
（１）実施例１で使用した同じ塗布装置を使用して、下記塗布条件で、塗布液を塗布した。
比較例においては、ゲル化剤（フィブリノーゲン）が第１の溶液に、ゲル化開始剤（トロンビン）が第２の溶液に入っている点が、実施例１と大きく異なる点である。
Comparative Example 1
(1) Using the same coating apparatus used in Example 1, the coating liquid was applied under the following coating conditions.
In the comparative example, the gelling agent (fibrinogen) is contained in the first solution and the gelation initiator (thrombin) is contained in the second solution, which is a major difference from Example 1.
（２）塗布条件
・ピン径： φ１０００μｍ
・ピン形状： ストレート
・塗布回数： １０回
・第１の塗布液の塗布量： 数nＬ/10回
・第２の塗布液の塗布量： １５ μＬ/1回
・塗布容器内待機時間： １０２０ ｍｓ
・ピン降下後待機時間： ０ｍｓ
・上昇時間： ５μｍ／１回
・２液目添加までの所要時間： ５ｓ以内
・培地添加までの所要時間： ４個作成後まとめて、マイクロピペットにて手動で添加
(2) Coating conditions・ Pin diameter: φ1000 μm
・ Pin shape: Straight ・ Number of coatings: 10 times ・ Coating amount of the first coating liquid: several nL / 10 times ・ Coating amount of the second coating liquid: 15 μL / time ・ Standby time in the coating container: 1020 ms
・ Wait time after pin descent: 0ms
・ Rise time: 5 μm / once ・ Time required to add the second liquid: Within 5 s ・ Time required to add the medium: After making 4 pieces, add them manually with a micropipette
（３）塗布液
（３−１）第１の塗布液（１液目）
・ｉＰＳ細胞由来心筋細胞： ヒト心臓線維芽細胞 ＝ ３：１
・細胞数： ８ｘ１０７／ｍＬ
・ヒアルロン酸ナトリウム： １３ｍｇ／ｍＬ
・フィブリノーゲン： １０ ｍｇ／ｍＬ
・ＰＢＳ
・粘度：１.５×１０4ｍＰａ・ｓ）
（３−２）第２の塗布液（２液目）
・添加量 １５μＬ
・ヒアルロン酸ナトリウム ０．５ ｍｇ／ｍＬ
・トロンビン： ８００ｕｎｉｔ／ｍＬ
・ＰＢＳ
・粘度： １×１０3ｍＰａ・ｓ
(3) Coating liquid (3-1) First coating liquid (first liquid)
-IPS cell-derived cardiomyocytes: human cardiac fibroblasts = 3: 1
- number of cells: 8x10 7 / mL
・ Sodium hyaluronate: 13 mg / mL
-Fibrinogen: 10 mg / mL
・ PBS
・ Viscosity: 1.5 × 10 4 mPa ・ s)
(3-2) Second coating liquid (second liquid)
・ Addition amount 15 μL
・ Sodium hyaluronate 0.5 mg / mL
・ Thrombin: 800 units / mL
・ PBS
・ Viscosity: 1 × 10 3 mPa ・ s
（４）結果考察
（４−１）組織観察
（４−１−１）密度
塗布直後に得られた細胞組織を、位相差顕微鏡を用いて顕微鏡観察を行った。その、写真を図９に示す。
図９においては、略球状の細胞と細胞の間に、細胞間マトリックスが存在していることが解る。塗布直後の細胞が沈殿する前の状態において、図５と比較すると図９では細胞密度が低いことが分かる。これは、１液目にゲル化剤を混合することにより細胞は疎の状態で固定化されてしまい、沈殿が起こらないためと考えている。
(4) Consideration of results (4-1) Tissue observation (4-1-1) Density The cell tissue obtained immediately after application was observed under a microscope using a phase-contrast microscope. The photograph is shown in FIG.
In FIG. 9, it can be seen that an extracellular matrix exists between the substantially spherical cells. It can be seen that the cell density in FIG. 9 is lower than that in FIG. 5 in the state immediately after the application before the cells settle. It is considered that this is because the cells are immobilized in a sparse state by mixing the gelling agent in the first liquid, and precipitation does not occur.
（４−２）作製方法
１液目に心筋細胞とフィブリノーゲンを分散させた場合、１５分経過した後、塗布容器内に心筋細胞を包埋したゲル状物質を確認した(図８）。
(4-2) Preparation method When cardiomyocytes and fibrinogen were dispersed in the first solution, a gel-like substance in which cardiomyocytes were embedded in a coating container was confirmed after 15 minutes had passed (FIG. 8).
図８(a)は、貫通可能に支持された塗布ピンと塗布容器を示しており、図８（b）は、塗布容器貫通部の周囲に形成された細胞包埋フィブリルゲルを示している。図８（c)は、塗布容器から取り出した細胞包埋フィブリルゲルを、図８(d)は、図８（c）の拡大図を示している。
本比較例の作製方法においては、塗布容器内に細胞が残留し、塗布液内の細胞数のバラツキが発生した。
FIG. 8A shows a penetratingly supported coating pin and coating container, and FIG. 8B shows a cell-embedded fibril gel formed around the penetrating portion of the coating container. FIG. 8 (c) shows the cell-embedded fibril gel taken out from the coating container, and FIG. 8 (d) shows an enlarged view of FIG. 8 (c).
In the production method of this comparative example, cells remained in the coating container, and the number of cells in the coating solution varied.
（４−３）考察
１液目に心筋細胞とフィブリノーゲンを分散させた塗布液を塗布し、２液目にゲル化開始剤として１液目塗布液を覆うようにトロンビンを含む塗布液を塗布する本比較例においては、心筋細胞を包埋する形でフィブリノーゲンがゲル化するため、心筋細胞がゲル内で疎に分散し、高密度かつ保形性の高い三次元心筋細胞組織を、細胞生存率を維持し、簡易に作成することが困難となる不具合が発生した。
また、１液目に心筋細胞とフィブリノーゲンを分散させた塗布液を、繰り返し塗布を繰り返すか、塗布容器内に塗布液を長時間保持ししていると、容器内で第１溶液のゲル化が生じ、そのため、塗布液容器内部でゲル化した心筋細胞塊が目詰まりを起こす場合や、ゲル化した心筋細胞が塗布液容器に残り塗布できない場合があるという不具合が発生した。
(4-3) Discussion A coating solution containing cardiomyocytes and fibrinogen is applied to the first solution, and a coating solution containing thrombin is applied to the second solution as a gelling initiator so as to cover the first solution. In this comparative example, since fibrinogen gels in the form of embedding cardiomyocytes, the cardiomyocytes are sparsely dispersed in the gel, and a high-density and highly shape-retaining three-dimensional cardiomyocyte tissue is obtained with a cell viability. There was a problem that it became difficult to maintain and easily create.
In addition, if the coating solution in which cardiomyocytes and fibrinogen are dispersed in the first solution is repeatedly applied or the coating solution is held in the application container for a long time, the first solution gels in the container. As a result, there is a problem that the gelled cardiomyocyte mass inside the coating liquid container may be clogged, or the gelled cardiomyocytes may remain in the coating liquid container and cannot be applied.
以上開示事項から、本発明の第１の側面に係る発明のより具体的な態様として、例えば、下記のものが提供される。
（１）第１の塗布液を塗布対象物に塗布する第１塗布工程、および
前記第１塗布工程で塗布された第１の塗布液に、第２の塗布液を重ねて塗布し、第１溶液の塗布液の流動状態を維持しつつ、第１の塗布液と第２の塗布液の界面をゲル化する第２塗布工程、
を含むことを特徴とする細胞の組織作製方法。
（２）第１の塗布溶液が細胞およびゲル化開始剤を含む、上記（１）に記載の細胞組織の作製方法。
（３）第２の塗布溶液がゲル化剤を含む、上記（１）または上記（２）に記載の細胞組織の作製方法。
（４）細胞とゲル化開始剤を含む第１の塗布液を塗布対象物に塗布する第１塗布工程、および
前記第１塗布工程で塗布された第１の塗布液に、ゲル化剤を含む第２の塗布液を重ねて塗布し、第１の塗布液と第２の塗布液の界面でゲル化反応を開始させる第２塗布工程、
を含むことを特徴とする細胞組織の作製方法。
（５）第１の塗布液および第２の塗布液が塗布された塗布対象物を培地中に浸浸し、細胞を培養する工程を含む、上記（１）〜上記（４）いずれかに記載の細胞組織の作製方法。
（６）第１の塗布溶液が増粘多糖類を含む、上記（１）〜（５）いずれかに記載の細胞組織の作製方法。
（７）第２の塗布溶液が増粘多糖類を含む、上記（１）〜（６）いずれかに記載の細胞組織の作製方法。
（８）ゲル化開始剤がトロンビンである、上記（２）〜（７）いずれかに記載の細胞組織の作製方法。
（９）ゲル化剤がフィブリノーゲン、コラーゲン、ゼラチンまたはそれらの２種以上の混合物である、上記（３）〜（８）いずれかに記載の細胞組織の作製方法。
（１０）増粘多糖類が、ヒアルロン酸ナトリウム、アルギン酸ナトリウムまたはそれらの混合物であり、上記（６）〜（９）いずれかに記載の細胞組織の作製方法。
（１１）細胞が心筋細胞である、上記（２）〜（１０）いずれかに記載の細胞組織の作製方法。
From the above disclosure items, for example, the following is provided as a more specific aspect of the invention according to the first aspect of the present invention.
(1) A second coating liquid is superposed on the first coating liquid applied in the first coating step of applying the first coating liquid to the object to be coated and the first coating liquid, and the first coating liquid is applied. A second coating step of gelling the interface between the first coating liquid and the second coating liquid while maintaining the fluid state of the coating liquid of the solution.
A method for producing a tissue of a cell, which comprises.
(2) The method for producing a cell tissue according to (1) above, wherein the first coating solution contains cells and a gelation initiator.
(3) The method for producing a cell tissue according to (1) or (2) above, wherein the second coating solution contains a gelling agent.
(4) The gelling agent is contained in the first coating step of applying the first coating solution containing the cells and the gelation initiator to the object to be coated, and the first coating solution applied in the first coating step. A second coating step in which the second coating liquid is applied in layers and the gelation reaction is started at the interface between the first coating liquid and the second coating liquid.
A method for producing a cell tissue, which comprises.
(5) The above-mentioned (1) to (4), which comprises a step of immersing a first coating solution and a coating object coated with the second coating solution in a medium and culturing cells. Method for producing cell tissue.
(6) The method for producing a cell tissue according to any one of (1) to (5) above, wherein the first coating solution contains a thickening polysaccharide.
(7) The method for producing a cell tissue according to any one of (1) to (6) above, wherein the second coating solution contains a thickening polysaccharide.
(8) The method for producing a cell tissue according to any one of (2) to (7) above, wherein the gelation initiator is thrombin.
(9) The method for producing a cell tissue according to any one of (3) to (8) above, wherein the gelling agent is fibrinogen, collagen, gelatin or a mixture of two or more thereof.
(10) The method for producing a cell tissue according to any one of (6) to (9) above, wherein the thickening polysaccharide is sodium hyaluronate, sodium alginate or a mixture thereof.
(11) The method for producing a cell tissue according to any one of (2) to (10) above, wherein the cell is a cardiomyocyte.
また、本発明の第２の側面に係る発明のより具体的な態様として、例えば、下記のものが提供される。
（１２）少なくとも、細胞とゲル化開始剤を含む第１の塗布液を含む第１の容器、およびゲル化剤を含む第２の塗布液を含む第２の容器を含む、細胞組織形成セット。
（１３）第１の塗布溶液が増粘多糖類を含む、上記（１２）に記載の細胞組織形成セット。
（１４）第２の塗布溶液が増粘多糖類を含む、上記（１２）〜（１３）いずれかに記載の細胞組織形成セット。
（１５）ゲル化開始剤がトロンビンである、上記（１２）〜（１４）いずれかに記載の細胞組織形成セット。
（１６）ゲル化剤がフィブリノーゲン、コラーゲン、ゼラチンまたはそれらの２種以上の混合物である、上記（１２）〜（１５）いずれかに記載の細胞組織形成セット。
（１７）増粘多糖類が、ヒアルロン酸ナトリウム、アルギン酸ナトリウムまたはそれらの混合物であり、上記（１３）〜（１６）いずれかに記載の細胞組織形成セット。
（１８）細胞が心筋細胞である、上記（１２）〜（１７）いずれかに記載の細胞組織形成セット。
（１９）上記（１）〜上記（１１）いずれかに記載の細胞組織の作製方法により作製された細胞組織。
（２０）第１塗布工程を塗布ピン法を用いて行う、上記（１）〜（１１）いずれかに記載の細胞組織の作製方法。
Further, as a more specific aspect of the invention according to the second aspect of the present invention, for example, the following is provided.
(12) A cell tissue formation set comprising at least a first container containing a first coating solution containing cells and a gelation initiator, and a second container containing a second coating solution containing a gelling agent.
(13) The cell tissue formation set according to (12) above, wherein the first coating solution contains a thickening polysaccharide.
(14) The cell tissue formation set according to any one of (12) to (13) above, wherein the second coating solution contains a thickening polysaccharide.
(15) The cell tissue formation set according to any one of (12) to (14) above, wherein the gelation initiator is thrombin.
(16) The cell tissue formation set according to any one of (12) to (15) above, wherein the gelling agent is fibrinogen, collagen, gelatin or a mixture of two or more thereof.
(17) The cell tissue formation set according to any one of (13) to (16) above, wherein the thickening polysaccharide is sodium hyaluronate, sodium alginate or a mixture thereof.
(18) The cell tissue formation set according to any one of (12) to (17) above, wherein the cells are cardiomyocytes.
(19) A cell tissue prepared by the method for producing a cell tissue according to any one of (1) to (11) above.
(20) The method for producing a cell tissue according to any one of (1) to (11) above, wherein the first coating step is performed using a coating pin method.
Claims (20)
前記第１塗布工程で塗布された第１の塗布液に、第２の塗布液を重ねて塗布し、第１溶液の塗布液の流動状態を維持しつつ、第１の塗布液と第２の塗布液の界面をゲル化する第２塗布工程、
を含むことを特徴とする細胞組織の作製方法。 The second coating solution is overlaid on the first coating step of applying the first coating solution to the object to be coated and the first coating solution applied in the first coating step, and the first solution is applied. A second coating step of gelling the interface between the first coating liquid and the second coating liquid while maintaining the fluid state of the liquid.
A method for producing a cell tissue, which comprises.
前記第１塗布工程で塗布された第１の塗布液に、ゲル化剤を含む第２の塗布液を重ねて塗布し、第１の塗布液と第２の塗布液の界面でゲル化反応を開始させる第２塗布工程、
を含むことを特徴とする細胞組織の作製方法。 A first coating step of applying a first coating solution containing cells and a gelation initiator to an object to be coated, and a second coating solution containing a gelling agent in the first coating solution applied in the first coating step. A second coating step in which the coating liquids are applied in layers and the gelation reaction is started at the interface between the first coating liquid and the second coating liquid.
A method for producing a cell tissue, which comprises.
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| CN202080018860.1A CN113474092B (en) | 2019-03-06 | 2020-03-03 | Method for producing cell tissue, cell tissue production kit, and culture container containing cell tissue produced by the production method |
| EP20765757.8A EP3936238A4 (en) | 2019-03-06 | 2020-03-03 | Cell tissue production method, cell tissue production set, and cultivation container containing cell tissue produced by said production method |
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