JP2022055039A - Cell transplant material, manufacturing method thereof, and method of use thereof - Google Patents
Cell transplant material, manufacturing method thereof, and method of use thereof Download PDFInfo
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Abstract
Description
本発明は、再生医療などの医学、創薬等の分野に有用な細胞移植材、その製造方法及び使用方法に関する。 The present invention relates to a cell transplant material useful in the fields of medicine such as regenerative medicine, drug discovery, etc., and a method for producing and using the same.
再生医療における一つの手法として細胞移植が注目されている。しかしながら、細胞を直接注入して移植した場合は、移植部位における移植細胞の生着率が低いという問題があった。そこで、特許文献１では、脂肪細胞含有細胞シートを移植材料として用いることを提案している。特許文献２では、細胞シートと該細胞シートの片面または両面に接着する特定のゼラチンを含有する支持体層からなる積層体を提案している。
Cell transplantation is attracting attention as a method in regenerative medicine. However, when the cells are directly injected and transplanted, there is a problem that the engraftment rate of the transplanted cells at the transplant site is low. Therefore,
しかし、特許文献１に記載のような移植材料の場合、細胞シートが脆弱であり、取扱性に劣っていた。また、特許文献１に記載のような移植材料や特許文献２に記載の積層体の場合、移植部位における再生効果をさらに改良することが求められている。
However, in the case of the transplant material as described in
本発明は、前記従来の問題を解決するため、取扱性が良好であり、移植部位における移植細胞の生着率及び再生効果が高い細胞移植材、その製造方法及び使用方法を提供する。 In order to solve the above-mentioned conventional problems, the present invention provides a cell transplant material having good handleability and a high engraftment rate and regeneration effect of transplanted cells at a transplant site, and a method for producing and using the same.
本発明は、１以上の実施形態において、第一の細胞シート、第二の細胞シート、第一の生体適合長繊維不織布、及び第二の生体適合長繊維不織布を含み、第一の生体適合長繊維不織布及び第二の生体適合長繊維不織布は、いずれも、生体適合性ポリマーを主成分とする生体適合長繊維不織布であり、第一の生体適合長繊維不織布は第一の細胞シートの一方の表面に接着しており、第二の生体適合長繊維不織布は第二の細胞シートの一方の表面に接着しており、第一の細胞シートの他方の表面と第二の細胞シートの他方の表面が接着していることを特徴とする細胞移植材に関する。 The present invention comprises, in one or more embodiments, a first cell sheet, a second cell sheet, a first biocompatible long fiber non-woven fabric, and a second biocompatible long fiber non-woven fabric, the first biocompatible length. Both the fibrous nonwoven fabric and the second biocompatible nonwoven fabric are biocompatible nonwoven fabrics containing a biocompatible polymer as a main component, and the first biocompatible nonwoven fabric is one of the first cell sheets. Adhering to the surface, the second biocompatible long fiber non-woven fabric is attached to one surface of the second cell sheet, the other surface of the first cell sheet and the other surface of the second cell sheet. The present invention relates to a cell transplant material characterized by being adhered to the fabric.
本発明は、１以上の実施形態において、前記細胞移植材の製造方法であって、第一の細胞シート及び第二の細胞シートを準備する工程と、第一の細胞シートの一方の表面に第一の生体適合長繊維不織布層を積層して第一の積層体を得る工程と、第二の細胞シートの一方の表面に第二の生体適合長繊維不織布層を積層して第二の積層体を得る工程と、第一の細胞シートの他方の表面と第二の細胞シートの他方の表面が接するように第一の積層体と第二の積層体を積層し、細胞移植材を得る工程を含む細胞移植材の製造方法に関する。 The present invention is the method for producing the cell transplant material according to one or more embodiments, wherein the first cell sheet and the second cell sheet are prepared, and the first cell sheet is formed on one surface of the first cell sheet. A step of laminating one biocompatible long fiber non-woven fabric layer to obtain a first laminated body and a second laminating body by laminating a second biocompatible long fiber non-woven fabric layer on one surface of a second cell sheet. And a step of laminating the first laminate and the second laminate so that the other surface of the first cell sheet and the other surface of the second cell sheet are in contact with each other to obtain a cell transplant material. The present invention relates to a method for producing a cell transplant material containing.
本発明は、１以上の実施形態において、細胞移植材を生体（ただし、人体を除く。）内の所定部位に移植する工程を含む細胞移植材の使用方法に関する。 The present invention relates to a method of using a cell transplant material, which comprises a step of transplanting a cell transplant material to a predetermined site in a living body (excluding a human body) in one or more embodiments.
本発明は、取扱性が良好であり、移植部位における移植細胞の生着率及び再生効果が高い細胞移植材を提供することができる。
本発明の製造方法によれば、取扱性が良好であり、移植部位における移植細胞の生着率及び再生効果が高い細胞移植材を得ることができる。
本発明の細胞移植材を用いることで、取扱性良く細胞移植材を移植でき、移植部位における移植細胞の生着率及び再生効果を高めることができる。
INDUSTRIAL APPLICABILITY The present invention can provide a cell transplantation material which is easy to handle and has a high engraftment rate and regeneration effect of transplanted cells at a transplantation site.
According to the production method of the present invention, it is possible to obtain a cell transplant material which is easy to handle and has a high engraftment rate and regeneration effect of transplanted cells at the transplantation site.
By using the cell transplant material of the present invention, the cell transplant material can be transplanted with good handleability, and the engraftment rate and the regeneration effect of the transplanted cells at the transplant site can be enhanced.
本発明の発明者らは、上述した問題を解決するため、検討を重ねた。その結果、生体適合性ポリマーを主成分とする第一及び第二の生体適合長繊維不織布を第一の細胞シートの一方の表面及び第二の細胞シートの一方の表面にそれぞれ接着させるとともに、第一の細胞シートの他方の表面と第二の他方の表面を接着させて細胞移植材にすることで、該細胞移植材の取扱性が良好になるとともに、移植部位における移植細胞の生着率及び再生効果が高まることを見出した。
特に、生体適合性ポリマーとしてゼラチンを用い、間葉系幹細胞を含む骨髄由来細胞や脂肪由来細胞等で細胞シートを構成することで、膀胱組織へ移植した場合、取扱性が良好になるとともに、膀胱組織における移植細胞の生着率及び平滑筋細胞への分化が促進され、下部尿路障害治療用細胞移植材として好適に使用し得ることを見出した。
The inventors of the present invention have repeated studies in order to solve the above-mentioned problems. As a result, the first and second biocompatible long fiber nonwoven fabrics containing the biocompatible polymer as a main component are adhered to one surface of the first cell sheet and one surface of the second cell sheet, respectively, and at the same time, the second By adhering the other surface of one cell sheet to the other surface of the second to form a cell transplant material, the handleability of the cell transplant material is improved, and the engraftment rate of the transplanted cells at the transplant site and the engraftment rate of the transplanted cells are improved. We found that the reproduction effect was enhanced.
In particular, by using gelatin as a biocompatible polymer and forming a cell sheet with bone marrow-derived cells including mesenchymal stem cells, adipose-derived cells, etc., when transplanted into bladder tissue, handleability is improved and the bladder is bladder. It has been found that the engraftment rate of transplanted cells in tissues and the differentiation into smooth muscle cells are promoted, and that they can be suitably used as a cell transplant material for treating lower urinary tract disorders.
＜生体適合長繊維不織布＞
本発明の１以上の実施形態において、生体適合長繊維不織布は、生体適合性ポリマーを主成分とする。本発明の１以上の実施形態において、主成分とは、生体適合性ポリマーを９０質量％以上含むことを意味する。他の成分は、必要に応じて、架橋剤、薬剤、可塑剤、他の添加剤等であってもよい。本発明の１以上の実施形態において、生体適合長繊維不織布は、実質的に１００質量％の生体適合性ポリマーで構成されてもよい。なお、本発明の１以上の実施形態の細胞移植材において、生体適合長繊維不織布は、膨潤した状態である。本発明の１以上の実施形態において、「膨潤」とは、水、緩衝液又は液体培地からなる群から選ばれる一つ以上の液体で飽和状態まで膨潤することを意味する。前記飽和状態とは、液体が最大限に含まれた状態であり、液体の含有量が一定限度にとどまりそれ以上増えない状態を意味する。
<Biocompatible long fiber non-woven fabric>
In one or more embodiments of the present invention, the biocompatible long fiber nonwoven fabric contains a biocompatible polymer as a main component. In one or more embodiments of the invention, the principal component means containing 90% by weight or more of the biocompatible polymer. The other component may be a cross-linking agent, a drug, a plasticizer, another additive, or the like, if necessary. In one or more embodiments of the invention, the biocompatible nonwoven fabric may be composed of substantially 100% by weight of the biocompatible polymer. In the cell transplant material of one or more embodiments of the present invention, the biocompatible long fiber non-woven fabric is in a swollen state. In one or more embodiments of the invention, "swelling" means swelling to saturation with one or more liquids selected from the group consisting of water, buffer or liquid medium. The saturated state means a state in which the liquid is contained to the maximum, and the content of the liquid stays at a certain limit and does not increase any more.
前記生体適合性ポリマーとしては、特に限定されないが、例えば、天然高分子や合成高分子を用いることができる。天然高分子としては、例えばタンパク質や多糖類が挙げられる。タンパク質としては、例えばコラーゲン、フィブロネクチン、フィブリノーゲン、ラミニン、フィブリン等が挙げられる。多糖類としては、例えばキトサン、アルギン酸カルシウム、ヘパラン硫酸、コンドロイチン硫酸、ヒアルロン酸、ヘパリン、 デンプン、ジェランガム、アガロース、グァーガム、キサンタンガム、カラギーナン、ペクチン、ローカストビーンガム、タマリンドガム、ダイユータンガム等の天然高分子を用いてもよく、カルボキシメチルセルロース等の天然高分子の誘導体を用いてもよい。合成高分子としては、例えば、ポリエチレングリコールポロエチレングリコール、ポリエチレンテレフタレート、ポリビニルアルコール、熱可塑性エラストマー、ポリプロピレン、ポリエチレン、ポリスチレン、ポリメタクリル酸メチル、ポリカーボネート、ポリジメチルシロキサン、シクロオレフィンポリマー、アモルファスフッ素樹脂等の非吸収性の合成高分子や、ポリ乳酸、ポリグルコール酸、ポリカプロラクトン、ポリジオキサノン等の生体吸収性高分子等が挙げられる。前記生体適合性ポリマーは、１種を用いてもよく、２種以上を用いてもよい。 The biocompatible polymer is not particularly limited, and for example, a natural polymer or a synthetic polymer can be used. Examples of natural polymers include proteins and polysaccharides. Examples of the protein include collagen, fibronectin, fibrinogen, laminin, fibrin and the like. Examples of polysaccharides include natural high polymers such as chitosan, calcium alginate, heparan sulfate, chondroitin sulfate, hyaluronic acid, heparin, starch, gellan gum, agarose, guar gum, xanthan gum, carrageenan, pectin, locust bean gum, tamarind gum, and dieutan gum. A molecule may be used, or a derivative of a natural polymer such as carboxymethyl cellulose may be used. Examples of the synthetic polymer include polyethylene glycol poloethylene glycol, polyethylene terephthalate, polyvinyl alcohol, thermoplastic elastomer, polypropylene, polyethylene, polystyrene, polymethylmethacrylate, polycarbonate, polydimethylsiloxane, cycloolefin polymer, and amorphous fluororesin. Examples thereof include non-absorbable synthetic polymers and bioabsorbable polymers such as polylactic acid, polyglucoric acid, polycaprolactone and polydioxanone. The biocompatible polymer may be used alone or in combination of two or more.
前記生体適合性ポリマーは、溶液紡糸が可能で、繊維形成後にゲル化し、架橋させることができ、形態安定性の高い不織布に形成できる観点から、水溶性高分子であることが好ましく、ゼラチンであることがより好ましい。ゼラチン長繊維不織布は、安全性が高く、生体吸収性に優れるゼラチンを主成分とすることから、細胞移植材に好適に用いることができる。特に、間葉系幹細胞を含む骨髄由来細胞や脂肪由来細胞等で構成された細胞シートと併用した場合、ゼラチン長繊維不織布と細胞シートの相乗作用が発生やすく、移植部位における目的細胞への分化が高まる。 The biocompatible polymer is preferably a water-soluble polymer, preferably gelatin, from the viewpoint that it can be solution-spun, gelled and crosslinked after fiber formation, and can be formed into a non-woven fabric having high morphological stability. Is more preferable. Since the gelatin long fiber non-woven fabric contains gelatin as a main component, which is highly safe and has excellent bioabsorbability, it can be suitably used as a cell transplant material. In particular, when used in combination with a cell sheet composed of bone marrow-derived cells including mesenchymal stem cells, adipose-derived cells, etc., synergistic action between the gelatin long fiber non-woven fabric and the cell sheet is likely to occur, and differentiation into target cells at the transplantation site is likely to occur. It will increase.
前記生体適合長繊維不織布を構成するゼラチン長繊維等の生体適合長繊維は、平均繊維径が２μｍ以上４００μｍ以下であることが好ましく、より好ましくは５μｍ以上３００μｍ以下であり、さらに好ましくは７μｍ以上２５０μｍ以下であり、特に好ましくは１０μｍ以上１５０μｍ以下である。生体適合長繊維の平均繊維径が上記範囲内であると、細胞が生体適合長繊維不織布に侵入しやすく、移植箇所における移植細胞の生着率がより高まる。本発明の１以上の実施形態において、「平均繊維径」は、膨潤後の生体適合長繊維不織布から任意に選択した５０本の繊維の直径を測定してその平均値を算出することで求めることができる。 The biocompatible long fibers such as gelatin long fibers constituting the biocompatible long fiber nonwoven fabric preferably have an average fiber diameter of 2 μm or more and 400 μm or less, more preferably 5 μm or more and 300 μm or less, and further preferably 7 μm or more and 250 μm. It is less than or equal to, and particularly preferably 10 μm or more and 150 μm or less. When the average fiber diameter of the biocompatible long fibers is within the above range, the cells easily invade the biocompatible long fiber non-woven fabric, and the engraftment rate of the transplanted cells at the transplant site is further increased. In one or more embodiments of the present invention, the "average fiber diameter" is determined by measuring the diameters of 50 fibers arbitrarily selected from the swelled biocompatible long fiber non-woven fabric and calculating the average value thereof. Can be done.
前記生体適合長繊維不織布を構成するゼラチン長繊維等の生体適合長繊維は、繊維交点が部分的に溶着していることが好ましい。この部分的溶着は、特に限定されないが、例えば、後述するように、生体適合長繊維不織布の製造時に圧力流体によって吹き飛ばされた完全に固化していない状態のゼラチン長繊維等の生体適合長繊維を堆積することで発現させることができる。この部分的溶着により、生体適合長繊維不織布はブリッジ構造となり、所望の形に成形しやすく、かつ成形安定性も高いものとなる。また、繊維交点が部分的に溶着していることにより、生体適合長繊維不織布は水に濡れてもへたらない。また、生体適合長繊維不織布において、繊維交点は一部が溶着してもよく、繊維交点の全部が溶着してもよい。 It is preferable that the biocompatible long fibers such as gelatin long fibers constituting the biocompatible long fiber non-woven fabric are partially welded at the fiber intersections. This partial welding is not particularly limited, but for example, as described later, biocompatible long fibers such as gelatin long fibers in a state in which they are not completely solidified and blown off by a pressure fluid during the production of the biocompatible long fiber nonwoven fabric are used. It can be expressed by depositing. By this partial welding, the biocompatible long fiber non-woven fabric has a bridge structure, which is easy to mold into a desired shape and has high molding stability. Further, since the fiber intersections are partially welded, the biocompatible long fiber non-woven fabric does not become worn even when it gets wet with water. Further, in the biocompatible long fiber non-woven fabric, a part of the fiber intersections may be welded, or all the fiber intersections may be welded.
前記生体適合長繊維不織布は、特に限定されないが、例えば、取扱性、細胞シートとの接着性、及び移植後の細胞シートとの相互作用を高める観点から、厚みが０．１ｍｍ以上３．０ｍｍ以下であることが好ましく、０．２ｍｍ以上２．５ｍｍ以下であることがより好ましく、０．３ｍｍ以上２．０ｍｍ以下であることがさらに好ましい。 The biocompatible long fiber non-woven fabric is not particularly limited, but has a thickness of 0.1 mm or more and 3.0 mm or less from the viewpoint of enhancing handleability, adhesion to the cell sheet, and interaction with the cell sheet after transplantation. It is more preferably 0.2 mm or more and 2.5 mm or less, and further preferably 0.3 mm or more and 2.0 mm or less.
前記生体適合長繊維不織布は、特に限定されないが、例えば、取扱性、細胞シートの接着性、及び移植後の細胞シートとの相互作用を高める観点から、１．０ｋＰａの圧縮応力時の圧縮変形率（以下において、単に「圧縮変形率」とも記す。）が１％以上４０％以下であることが好ましく、５％以上３５％以下であることがより好ましく、１０％以上３０％以下であることがさらに好ましい。本明細書において、圧縮変形率は、水で飽和状態まで膨潤した後の積層体において、無荷重の時の厚さを（Ｈ１）とし、１．０ｋＰａの圧縮応力時の厚さを（Ｈ２）とした場合、下記式で算出したものである。圧縮試験は、後述のとおりに行う。
圧縮変形率（％）＝１００－｛（Ｈ２／Ｈ１）×１００｝
The biocompatible long fiber non-woven fabric is not particularly limited, but for example, from the viewpoint of enhancing the handleability, the adhesiveness of the cell sheet, and the interaction with the cell sheet after transplantation, the compressive deformation rate under a compressive stress of 1.0 kPa. (Hereinafter, also simply referred to as "compression deformation rate") is preferably 1% or more and 40% or less, more preferably 5% or more and 35% or less, and 10% or more and 30% or less. More preferred. In the present specification, the compressive deformation rate is defined as (H1) the thickness under no load and (H2) the thickness under compressive stress of 1.0 kPa in the laminated body after being swollen to a saturated state with water. If, it is calculated by the following formula. The compression test is performed as described later.
Compression deformation rate (%) = 100-{(H2 / H1) x 100}
本発明の１以上の実施形態において、生体適合長繊維不織布は、細胞接着因子、細胞誘導因子、細胞増殖因子、細胞に栄養やエネルギ－を与える物質、細胞の機能を抑制又は亢進する物質等でコーティングされてもよい。細胞接着因子としては、特に限定されないが、例えば、フィブロネクチン等が挙げられる。細胞接着因子で生体適合長繊維不織布をコーティングすることで、細胞シートとの接着が強固になる。細胞に栄養やエネルギ－を与える物質としては、特に限定されないが、例えば、ＡＴＰ、ピルビン酸、グルタミン等が挙げられる。また、本発明の１以上の実施形態において、生体適合長繊維不織布を細胞誘導因子、細胞増殖因子等の生理活性物質を含む溶液に浸して、これらの成分を含ませてもよい。移植後に、生体適合長繊維不織布から、これらの生理活性物質が徐々に放出されることで、目的細胞への分化等を促進することができる。 In one or more embodiments of the present invention, the biocompatible long fiber nonwoven fabric is a cell adhesion factor, a cell inducing factor, a cell growth factor, a substance that gives nutrients and energy to cells, a substance that suppresses or enhances cell function, and the like. It may be coated. The cell adhesion factor is not particularly limited, and examples thereof include fibronectin and the like. By coating the biocompatible long fiber non-woven fabric with cell adhesion factor, the adhesion with the cell sheet is strengthened. The substance that gives nutrition and energy to the cells is not particularly limited, and examples thereof include ATP, pyruvic acid, and glutamine. Further, in one or more embodiments of the present invention, the biocompatible long fiber nonwoven fabric may be immersed in a solution containing a physiologically active substance such as a cell inducing factor or a cell growth factor to contain these components. After transplantation, these bioactive substances are gradually released from the biocompatible long fiber non-woven fabric, so that differentiation into target cells and the like can be promoted.
本発明の１以上の実施形態において、生体適合長繊維不織布は、特に限定されないが、夾雑物の発生を抑制し、製品汚染を防ぐ観点から、ゼラチン等の生体適合性ポリマーを含む紡糸液をノズル吐出口から空気中に押し出し、前記ノズル吐出口の後方に位置し、前記ノズル吐出口とは非接触状態の流体噴射口から前方に向けて圧力流体を噴射し、前記押し出された紡糸液を前記圧力流体に随伴させて繊維形成させ、得られた生体適合長繊維を集積させて不織布とすることで作製することが好ましい。 In one or more embodiments of the present invention, the biocompatible long fiber non-woven fabric is not particularly limited, but from the viewpoint of suppressing the generation of impurities and preventing product contamination, a spinning fluid containing a biocompatible polymer such as gelatin is nozzleed. The pressure fluid is extruded into the air from the discharge port, is located behind the nozzle discharge port, and injects pressure fluid forward from the fluid injection port in a non-contact state with the nozzle discharge port, and the extruded spinning liquid is said to be the same. It is preferable to form fibers by accompany the pressure fluid and to accumulate the obtained biocompatible long fibers to form a non-woven fabric.
本発明の１以上の実施形態において、生体適合長繊維不織布は、架橋されていることが好ましい。これにより形態安定性及び耐水性を高めることができる。架橋は、架橋剤等の化合物を用いた化学架橋であってもよいが、生体安全性の観点から、生体安全性を有する架橋剤を用いる架橋、架橋剤を用いない架橋であることが好ましい。架橋剤を用いない架橋としては、例えば、熱架橋、電子線架橋、γ線等の放射線架橋、紫外線架橋等が挙げられる。電子線照射、γ線等の放射線照射の場合は、滅菌と架橋を同時にすることもできる。簡便に所望の架橋効果を得やすい観点から、熱架橋であることが好ましく、熱脱水架橋であることがより好ましい。熱脱水架橋は、例えば、１００℃以上１６０℃以下で、２４時間以上９６時間以下行ってもよい。また、熱脱水架橋は、例えば、１ｋＰａ以下の真空下で行ってもよい。前記積層体は、架橋する前に乾燥してもよい。乾燥は、室温における風乾でもよく、真空凍結乾燥でもよい。 In one or more embodiments of the present invention, the biocompatible long fiber nonwoven fabric is preferably crosslinked. This makes it possible to improve morphological stability and water resistance. The cross-linking may be a chemical cross-linking using a compound such as a cross-linking agent, but from the viewpoint of biosafety, a cross-linking using a cross-linking agent having bio-safety and a cross-linking without using a cross-linking agent are preferable. Examples of the cross-linking without using a cross-linking agent include thermal cross-linking, electron beam cross-linking, radiation cross-linking such as γ-rays, and ultraviolet cross-linking. In the case of radiation irradiation such as electron beam irradiation and γ-ray, sterilization and cross-linking can be performed at the same time. From the viewpoint of easily obtaining the desired crosslinking effect, thermal crosslinking is preferable, and thermal dehydration crosslinking is more preferable. The thermal dehydration crosslinking may be performed, for example, at 100 ° C. or higher and 160 ° C. or lower for 24 hours or longer and 96 hours or lower. Further, the thermal dehydration crosslinking may be performed under a vacuum of 1 kPa or less, for example. The laminate may be dried before cross-linking. The drying may be air drying at room temperature or vacuum freeze drying.
本発明の１以上の実施形態において、生体適合長繊維不織布は、具体的には、国際公開公報２０１８／２３５７４５号に記載のとおりに作製し、必要に応じて膨潤させて用いることができる。また、ゼラチン長繊維不織布として、例えば、株式会社ニッケ・メディカル製の「Ｇｅｎｏｃｅｌ（登録商標）」シートタイプ等の市販品を適宜に膨潤させて用いてもよい。 In one or more embodiments of the present invention, the biocompatible long fiber nonwoven fabric can be specifically prepared as described in International Publication No. 2018/235745 and can be inflated and used as necessary. Further, as the gelatin long fiber non-woven fabric, for example, a commercially available product such as "Genocel (registered trademark)" sheet type manufactured by Nikke Medical Co., Ltd. may be appropriately swollen and used.
＜細胞シート＞
本発明の１以上の実施形態において、細胞シートは、細胞同士が細胞間結合によりシート状になったものを意味する。細胞同士は、直接接着していてもよく、介在物質を介して互いに接着していてもよい。介在物質としては、細胞同士を接着し得る物質であれば特に限定されないが、例えば、細胞外マトリックス等が挙げられる。介在物質は、特に限定されないが、細胞由来のものであることが好ましい。
<Cell sheet>
In one or more embodiments of the present invention, the cell sheet means that cells are formed into a sheet by cell-cell binding. The cells may be directly adhered to each other or may be adhered to each other via an intervening substance. The intervening substance is not particularly limited as long as it is a substance capable of adhering cells to each other, and examples thereof include an extracellular matrix. The intervening substance is not particularly limited, but is preferably derived from cells.
本発明の１以上の実施形態において、細胞シートを構成する細胞は、浮遊細胞であってもよく、接着性細胞であってもよいが、細胞シートを形成しやすい観点から接着性細胞であることが好ましい。 In one or more embodiments of the present invention, the cells constituting the cell sheet may be floating cells or adhesive cells, but they are adhesive cells from the viewpoint of easily forming a cell sheet. Is preferable.
本発明の１以上の実施形態において、細胞シートを構成する細胞は、特に限定されず、動物由来の細胞を好適に用いることができる。動物としては、哺乳類であることが好ましく、ヒトであることがより好ましい。ヒト以外の哺乳類動物としては、例えば、サル、チンパンジー等の霊長類、ウシ、ヒツジ、ヤギ、ブタ等の有蹄類等が挙げられる。前記細胞は、生体組織や器官から直接採収した初代培養した初代細胞、継代させた継代細胞、及び細胞株等のいずれであってもよい。 In one or more embodiments of the present invention, the cells constituting the cell sheet are not particularly limited, and animal-derived cells can be preferably used. As the animal, a mammal is preferable, and a human is more preferable. Examples of mammals other than humans include primates such as monkeys and chimpanzees, and ungulates such as cows, sheep, goats, and pigs. The cell may be any of a primary cultured cell directly collected from a living tissue or an organ, a subcultured subculture cell, a cell line, or the like.
本発明の１以上の実施形態において、細胞としては、特に限定されないが、例えば、体細胞、幹細胞、前駆細胞、生殖細胞、免疫細胞等が挙げられる。 In one or more embodiments of the present invention, the cells are not particularly limited, and examples thereof include somatic cells, stem cells, progenitor cells, germ cells, immune cells, and the like.
体細胞は、生体を構成する体細胞や体細胞から派生したがん細胞を含む。生体を構成する体細胞としては、特に限定されず、例えば、線維芽細胞、筋細胞、内皮細胞、骨芽細胞、内皮細胞、膀胱細胞、肺細胞、骨細胞、神経細胞、肝細胞、軟骨細胞、上皮細胞、中皮細胞等が挙げられる。癌細胞としては、特に限定されず、例えば、乳癌細胞、腎癌細胞、前立腺癌細胞、肺癌細胞、肝癌細胞、子宮頸癌細胞、食道上皮癌、膵癌、大腸癌、膀胱癌等が挙げられる。 Somatic cells include somatic cells that make up the living body and cancer cells derived from somatic cells. The somatic cells constituting the living body are not particularly limited, and are, for example, fibroblasts, muscle cells, endothelial cells, osteoblasts, endothelial cells, bladder cells, lung cells, osteoocytes, nerve cells, hepatocytes, and cartilage cells. , Epithelial cells, mesophageal cells and the like. The cancer cells are not particularly limited, and examples thereof include breast cancer cells, renal cancer cells, prostate cancer cells, lung cancer cells, liver cancer cells, cervical cancer cells, esophageal epithelial cancer, pancreatic cancer, colon cancer, and bladder cancer.
幹細胞は、様々な特殊化した細胞型へ分化する可能性がある細胞である。幹細胞としては、特に限定されず、例えば、胚性幹細胞（ＥＳ細胞）、胚性癌腫細胞（ＥＣ）、胚性生殖幹細胞（ＥＧ）、人工多能性幹細胞（ｉＰＳ細胞）、成体幹細胞、胚盤胞由来幹細胞、生殖隆起由来幹細胞、奇形腫由来幹細胞、オンコスタチン非依存性幹細胞（ＯＩＳＣ）、骨髄由来間葉系幹細胞、脂肪由来間葉系幹細胞、羊膜由来間葉系幹細胞、皮膚由来間葉系幹細胞、骨膜由来間葉系幹細胞等が挙げられる。 Stem cells are cells that can differentiate into a variety of specialized cell types. The stem cells are not particularly limited, and are, for example, embryonic stem cells (ES cells), embryonic cancer tumor cells (EC), embryonic germ stem cells (EG), artificial pluripotent stem cells (iPS cells), adult stem cells, and scutellum. Spore-derived stem cells, reproductive ridge-derived stem cells, malformation-derived stem cells, oncostatin-independent stem cells (OISC), bone marrow-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, sheep membrane-derived mesenchymal stem cells, skin-derived mesenchymal stem cells Examples thereof include stem cells and bone membrane-derived mesenchymal stem cells.
前駆細胞は、前記幹細胞から発生し生体を構成する最終分化細胞へ分化することができる細胞である。生殖細胞としては、例えば、精子、精細胞、卵子、卵細胞等が挙げられる。免疫細胞としては、特に限定されないが、例えば、マクロファージ、リンパ球、樹状細胞等が挙げられる。 Progenitor cells are cells that can develop from the stem cells and differentiate into the final differentiated cells constituting the living body. Examples of germ cells include sperm, sperm cells, eggs, and egg cells. The immune cell is not particularly limited, and examples thereof include macrophages, lymphocytes, and dendritic cells.
上述した細胞は、１種を単独で用いてもよく、目的等に応じて２種以上を併用してもよい。例えば、下部尿路障害治療用細胞移植材として好適に用いる観点から、前記細胞は、骨髄由来細胞及び脂肪由来細胞からなる群から選ばれる１以上であることが好ましく、間葉系幹細胞を含むことがより好ましい。 As the above-mentioned cells, one type may be used alone, or two or more types may be used in combination depending on the purpose and the like. For example, from the viewpoint of being suitably used as a cell transplant material for treating lower urinary tract disorders, the cells are preferably one or more selected from the group consisting of bone marrow-derived cells and adipose-derived cells, and include mesenchymal stem cells. Is more preferable.
移植後の免疫拒絶反応を抑制する観点から、細胞は、同種同系あるいは自己由来の幹細胞及び前駆細胞からなる群から選ばれる一種以上であることが好ましく、同種同系あるいは自己由来の幹細胞であることがより好ましく、同種同系あるいは自己由来の間葉系幹細胞であることがさらに好ましい。間葉系幹細胞としては、特に由来組織、臓器に限定されず、例えば、骨髄由来間葉系幹細胞、脂肪組織由来間葉系幹細胞、羊膜由来間葉系幹細胞、皮膚由来間葉系幹細胞、骨膜由来間葉系幹細胞等が挙げられる。 From the viewpoint of suppressing the immune rejection reaction after transplantation, the cell is preferably one or more selected from the group consisting of allogeneic or autologous stem cells and precursor cells, and is preferably allogeneic or autologous stem cell. More preferably, allogeneic or autologous mesenchymal stem cells are even more preferred. The mesenchymal stem cells are not particularly limited to derived tissues and organs, and are, for example, bone marrow-derived mesenchymal stem cells, adipose tissue-derived mesenchymal stem cells, sheep membrane-derived mesenchymal stem cells, skin-derived mesenchymal stem cells, and bone membrane-derived mesenchymal stem cells. Mesenchymal stem cells and the like can be mentioned.
＜細胞移植材＞
本発明の１以上の実施形態において、細胞移植材は、第一の細胞シート、第二の細胞シート、第一の生体適合長繊維不織布、及び第二の生体適合長繊維不織布を含み、第一の生体適合長繊維不織布は第一の細胞シートの一方の表面に接着しており、第二の生体適合長繊維不織布は第二の細胞シートの一方の表面に接着しており、第一の細胞シートの他方の表面と第二の細胞シートの他方の表面が接着している。本発明の１以上の実施形態の細胞移植材において、第一の生体適合長繊維不織布、及び第二の生体適合長繊維不織布のそれぞれは、細胞移植材の両表面のそれぞれに配置され、第一の生体適合長繊維不織布、及び第二の生体適合長繊維不織布の間に細胞シートが配置されていることになる。これにより、細胞移植材の取扱性が良好になるとともに、移植部位における細胞移植材の生着率も高くなる。また、細胞として幹細胞や前駆細胞等の未分化細胞を用いた場合、目的細胞への分化も促進される。
<Cell transplant material>
In one or more embodiments of the invention, the cell transplant material comprises a first cell sheet, a second cell sheet, a first biocompatible nonwoven fabric, and a second biocompatible nonwoven fabric, the first. The biocompatible long fiber non-woven fabric is adhered to one surface of the first cell sheet, and the second biocompatible long fiber non-woven fabric is adhered to one surface of the second cell sheet. The other surface of the sheet and the other surface of the second cell sheet are adhered. In the cell transplant material of one or more embodiments of the present invention, each of the first biocompatible long fiber non-woven fabric and the second biocompatible long fiber non-woven fabric is arranged on both surfaces of the cell transplant material, and the first The cell sheet is arranged between the biocompatible long fiber non-woven fabric and the second biocompatible long fiber non-woven fabric. As a result, the handleability of the cell transplant material is improved, and the engraftment rate of the cell transplant material at the transplant site is also increased. In addition, when undifferentiated cells such as stem cells and progenitor cells are used as cells, differentiation into target cells is also promoted.
第一の生体適合長繊維不織布は第一の細胞シートの一方の表面に部分的に接着してもよく、第一の細胞シートの一方の表面の全体に接着してもよい。また、第二の生体適合長繊維不織布は第二の細胞シートの一方の表面に部分的に接着してもよく、第一の細胞シートの一方の表面の全体に接着してもよい。また、第一の細胞シートと第二の細胞シートは、部分的に接着してもよく、全体的に接着してもよい。 The first biocompatible long fiber nonwoven fabric may be partially adhered to one surface of the first cell sheet or may be adhered to the entire surface of one surface of the first cell sheet. Further, the second biocompatible long fiber nonwoven fabric may be partially adhered to one surface of the second cell sheet, or may be adhered to the entire surface of one surface of the first cell sheet. Further, the first cell sheet and the second cell sheet may be partially adhered or may be totally adhered to each other.
第一の生体適合長繊維不織布及び第二の生体適合長繊維不織布としては、上述した生体適合長繊維不織布を適宜用いることができる。第一の生体適合長繊維不織布及び第二の生体適合長繊維不織布は、同じ生体適合性ポリマーで構成されたものであってもよく、異なる生体適合性ポリマーで構成されたものであってもよい。また、第一の生体適合長繊維不織布及び第二の生体適合長繊維不織布は、厚みや生体適合長繊維の平均繊維径等が同じものであってもよく、異なるものであってもよい。 As the first biocompatible long fiber non-woven fabric and the second biocompatible long fiber non-woven fabric, the above-mentioned biocompatible long fiber non-woven fabric can be appropriately used. The first biocompatible long fiber non-woven fabric and the second biocompatible long fiber non-woven fabric may be made of the same biocompatible polymer or may be made of different biocompatible polymers. .. Further, the first biocompatible long fiber non-woven fabric and the second biocompatible long fiber non-woven fabric may have the same thickness, the average fiber diameter of the biocompatible long fibers, or the like, or may be different.
第一の細胞シート及び第二の細胞シートとしては、上述した細胞シートを適宜用いることができる。第一の細胞シート及び第二の細胞シートは、同じ種類の細胞で構成された細胞シートであってもよく、異なる種類の細胞で構成された細胞シートであってもよい。また、第一の細胞シート及び第二の細胞シートは、それぞれ、１種類の細胞で構成された細胞シートであってもよく、２種類以上の細胞で構成された細胞シートであってもよい。 As the first cell sheet and the second cell sheet, the above-mentioned cell sheet can be appropriately used. The first cell sheet and the second cell sheet may be a cell sheet composed of cells of the same type, or may be a cell sheet composed of cells of different types. Further, the first cell sheet and the second cell sheet may be cell sheets composed of one type of cells, respectively, or may be cell sheets composed of two or more types of cells.
本発明の１以上の実施形態において、細胞移植材は、第一の細胞シート及び第二の細胞シートに加えて、必要に応じて他の細胞シートをさらに含んでもよく、前記他の細胞シートは、第一の細胞シートと第二の細胞シートの間に配置されており、第一の細胞シートの他方の表面と第二の他方の表面は他の細胞シートを介して接着されてもよい。他の細胞シートは、１つであってもよく、２つ以上であってもよい。 In one or more embodiments of the present invention, the cell transplant material may further contain another cell sheet, if necessary, in addition to the first cell sheet and the second cell sheet, wherein the other cell sheet is , Which is located between the first cell sheet and the second cell sheet, the other surface of the first cell sheet and the other surface of the second may be adhered via the other cell sheet. The other cell sheets may be one or more.
本発明の１以上の実施形態において、細胞移植材は、第一の生体適合長繊維不織布、及び第二の生体適合長繊維不織布に加えて、必要に応じて他の生体適合長繊維不織布をさらに含んでもよく、前記他の生体適合長繊維不織布は、細胞シートの間に配置されていてもよい。 In one or more embodiments of the present invention, the cell transplant material includes a first biocompatible long fiber non-woven fabric and a second biocompatible long fiber non-woven fabric, and if necessary, another biocompatible long fiber non-woven fabric. The other biocompatible long fiber nonwoven fabric may be included and may be disposed between the cell sheets.
本発明の１以上の実施形態の細胞移植材において、生体適合長繊維不織布と細胞シートは直接接着してもよく、介在物質、例えば細胞外マトリックス等を介して接着してもよい。
また、本発明の１以上の実施形態の細胞移植材において、細胞シートと細胞シートは直接接着してもよく、介在物質、例えば細胞外マトリックス等を介して接着してもよい。
In the cell transplant material of one or more embodiments of the present invention, the biocompatible long fiber non-woven fabric and the cell sheet may be directly adhered or may be adhered via an intervening substance such as an extracellular matrix.
Further, in the cell transplant material of one or more embodiments of the present invention, the cell sheet and the cell sheet may be directly adhered to each other, or may be adhered to each other via an intervening substance such as an extracellular matrix.
＜細胞移植材の製造方法＞
本発明の１以上の実施形態において、特に限定されないが、細胞移植材の作製は、下記のような工程を含む。
（１）細胞シートを準備する工程；
（２）細胞シートと生体適合長繊維不織布層を積層して積層体を得る工程；
（３）２つの積層体を積層し、細胞移植材を得る工程。
<Manufacturing method of cell transplant material>
In one or more embodiments of the present invention, the production of the cell transplant material includes, but is not limited to, the following steps.
(1) Step of preparing cell sheet;
(2) A step of laminating a cell sheet and a biocompatible long fiber non-woven fabric layer to obtain a laminate;
(3) A step of laminating two laminates to obtain a cell transplant material.
本発明の１以上の実施形態において、第一の細胞シート及び第二の細胞シート等の細胞シートは、例えば、細胞を公知の培養方法により接着培養することで得ることができる。培養皿等の培養容器からの細胞シートの剥離は、公知の方法で行うことができる。培養皿等の培養容器については、特に限定されないが、例えば、細胞シートを剥離しやすい培養容器を用いることが好ましい。このような培養容器としては、例えば、株式会社セルシード製の温度応答性培養基材等の市販品を用いてもよい。 In one or more embodiments of the present invention, cell sheets such as the first cell sheet and the second cell sheet can be obtained, for example, by adhering and culturing cells by a known culturing method. The cell sheet can be peeled off from a culture container such as a culture dish by a known method. The culture container such as a culture dish is not particularly limited, but for example, it is preferable to use a culture container in which the cell sheet can be easily peeled off. As such a culture vessel, for example, a commercially available product such as a temperature-responsive culture substrate manufactured by CellSeed Co., Ltd. may be used.
細胞培養時には、培地としては、特に限定されず、細胞の種類に応じて、細胞の生存増殖に必要な成分を含むものを適宜用いることができる。前記培地は、血清、抗生物質及び成長因子等を含んでもよい。血清は、例えば、ウシ血清、ウシ胎児血清、ウマ血清、ヒト血清等を適宜用いることができる。抗生物質は、ペニシリン、ストレプトマイシン、ゲンタマイシン、アンフォテリシン、アンピシリン、ミノマイシン、カナマイシン等を適宜用いることができる。成長因子は、細胞増殖因子、分化誘導因子、細胞接着因子等を適宜用いることができる。 At the time of cell culture, the medium is not particularly limited, and a medium containing components necessary for cell survival and proliferation can be appropriately used depending on the type of cell. The medium may contain serum, antibiotics, growth factors and the like. As the serum, for example, bovine serum, fetal bovine serum, horse serum, human serum and the like can be appropriately used. As the antibiotic, penicillin, streptomycin, gentamicin, amphotericin, ampicillin, minocycline, kanamycin and the like can be appropriately used. As the growth factor, a cell proliferation factor, a differentiation inducing factor, a cell adhesion factor and the like can be appropriately used.
細胞培養時の播種する細胞数は、細胞の種類などに応じて適宜決めればよいが、例えば、培養容器の接着面積に対して、０．１０ｘ１０５Ｃｅｌｌｓ／ｃｍ２以上０．６０ｘ１０５Ｃｅｌｌｓ／ｃｍ２以下であってもよく、０．２０ｘ１０５Ｃｅｌｌｓ／ｃｍ２以上０．５０ｘ１０５Ｃｅｌｌｓ／ｃｍ２以下であってもよく、０．３５ｘ１０５Ｃｅｌｌｓ／ｃｍ２以上０．４５ｘ１０５Ｃｅｌｌｓ／ｃｍ２以下であることがさらに好ましい。 The number of cells to be seeded at the time of cell culture may be appropriately determined according to the type of cells and the like. For example, 0.10x10 5 Cells / cm 2 or more and 0.60x10 5 Cells / cm with respect to the adhesion area of the culture vessel. It may be 2 or less, 0.20x10 5 Cells / cm 2 or more and 0.50x10 5 Cells / cm 2 or less, 0.35x10 5 Cells / cm 2 or more and 0.45x10 5 Cells / cm 2 or less. Is more preferable.
細胞培養は、例えば、２７℃以上４０℃以下で行ってもよく、３１℃以上３７℃以下であってもよい。二酸化炭素は、２％以上１０％以下の範囲であってもよい。 The cell culture may be carried out, for example, at 27 ° C. or higher and 40 ° C. or lower, or 31 ° C. or higher and 37 ° C. or lower. Carbon dioxide may be in the range of 2% or more and 10% or less.
培養時間は、細胞種類、細胞数等に応じて適宜決めればよいが、例えば、２～８日継続して培養してもよく、３～７日継続して行ってもよく、４～６日継続して行ってもよい。培地は、２～４日毎に交換してもよい。 The culturing time may be appropriately determined according to the cell type, the number of cells, etc., but for example, the culturing may be continued for 2 to 8 days, continuously for 3 to 7 days, or 4 to 6 days. You may continue to do so. The medium may be changed every 2-4 days.
培養容器から剥離した細胞シートの一方の表面上に、培地がない状態で、培地で膨潤させたゼラチン長繊維不織布等の生体適合長繊維不織布を積層し、例えば、室温（２０℃以上２５℃以下）で約２分以上１０分以下静置することで、ゼラチン長繊維不織布等の生体適合長繊維不織布と細胞シートを接着させて、積層体を得ることができる。 A biocompatible long fiber non-woven fabric such as a gelatin long fiber non-woven fabric swollen with a medium is laminated on one surface of the cell sheet peeled from the culture container in the absence of a medium, and for example, at room temperature (20 ° C or higher and 25 ° C or lower). ) For about 2 minutes or more and 10 minutes or less, the biocompatible long fiber non-woven fabric such as gelatin long fiber non-woven fabric and the cell sheet can be adhered to obtain a laminated body.
得られた第一の細胞シートと第一の生体適合長繊維不織布の積層体、及び第二の細胞シートと第二の生体適合長繊維不織布の積層体を、第一の細胞シートの第一の生体適合長繊維不織布と接していない表面と第二の細胞シートの第二の生体適合長繊維不織布と接していない表面が接するように積層し、例えば、培地がない状態で、２％以上１０％以下の二酸化炭素の雰囲気下、２７℃以上４０℃以下の温度で約６０分以上１２０分以下培養することで、第一の細胞シートと第二の細胞シートを接着させて、細胞移植材を得ることができる。 The obtained laminated body of the first cell sheet and the first biocompatible long fiber non-woven fabric, and the laminated body of the second cell sheet and the second biocompatible long fiber non-woven fabric were used as the first of the first cell sheets. Laminated so that the surface not in contact with the biocompatible long fiber non-woven fabric and the surface not in contact with the second biocompatible long fiber non-woven fabric of the second cell sheet are in contact with each other. By culturing at a temperature of 27 ° C. or higher and 40 ° C. or lower for about 60 minutes or more and 120 minutes or less in the following atmosphere of carbon dioxide, the first cell sheet and the second cell sheet are adhered to obtain a cell transplant material. be able to.
＜細胞移植材の使用方法＞
本発明の１以上の実施形態において、細胞移植材を生体内の所定部位に移植することができる。細胞移植材の移植方法は特に限定されず、生体内の患部に直接付着させてもよく、生体内で使用可能な接着剤で接合してもよく、生体内で使用可能な縫合材で縫合してもよい。縫合する際に、細胞移植材の一方又は両方の表面に生体適合性材料で構成された補強材を配置した後に縫合してもよい。本発明の１以上の実施形態において、細胞移植材は、細胞種類等に応じて、所定の条件下で培養した後に、生体内の所定部位に移植してもよい。
<How to use cell transplant material>
In one or more embodiments of the present invention, the cell transplant material can be transplanted to a predetermined site in a living body. The method of transplanting the cell transplant material is not particularly limited, and the cell transplant material may be directly attached to the affected area in the living body, may be bonded with an adhesive that can be used in the living body, or sutured with a suturing material that can be used in the living body. May be. At the time of suturing, a reinforcing material composed of a biocompatible material may be placed on one or both surfaces of the cell transplant material and then sutured. In one or more embodiments of the present invention, the cell transplant material may be cultured under predetermined conditions according to the cell type and the like, and then transplanted to a predetermined site in the living body.
本発明の１以上の実施形態において、細胞シートが骨髄由来細胞や脂肪由来細胞で構成されている場合、膀胱拡大術や下部尿路障害治療用細胞移植材として好適に用いることができる。 In one or more embodiments of the present invention, when the cell sheet is composed of bone marrow-derived cells or adipose-derived cells, it can be suitably used as a cell transplant material for bladder enlargement surgery or treatment of lower urinary tract disorders.
以下、実施例を用いてさらに具体的に説明する。なお、本発明は下記の実施例に限定されるものではない。 Hereinafter, a more specific description will be given using examples. The present invention is not limited to the following examples.
測定・評価方法は下記のとおりである。
＜平均繊維径＞
膨潤後のゼラチン長繊維不織布をマイクロスコープ（ＣＫＸ５３、オリンパス社製）で観察し、任意に選択した５０本の繊維を用いて、膨潤後の平均繊維径を測定した。
＜生体適合長繊維不織布の厚み＞
膨潤後のゼラチン長繊維不織布の厚みをミツトヨ社製のデジタルノギスで計測した。
The measurement / evaluation method is as follows.
<Average fiber diameter>
The swelled gelatin long fiber non-woven fabric was observed with a microscope (CKX53, manufactured by Olympus Corporation), and the average fiber diameter after swelling was measured using 50 arbitrarily selected fibers.
<Thickness of biocompatible long fiber non-woven fabric>
The thickness of the gelatin long fiber non-woven fabric after swelling was measured with a digital caliper manufactured by Mitutoyo.
（実施例１）
＜骨髄細胞の採取及び初代培養＞
動物は１０週齢雄ＧＦＰ－ＳＤラットを使用した。麻酔下のラットの左右の大腿骨を摘出した。摘出した大腿骨の両端を切断して一方から注射針２１Ｇ（内径０．５１ｍｍ）を付けたシリンジを刺して、培養培地（１５％牛胎児血清（ＦＢＳ）、及び０．１％ペニシリン－ストレプトマイシン含有ＤＭＥＭ、以下同様）で大腿骨内に存在する細胞を反対側の切断部位へ押し流した。押し流された細胞を遠心管に採取した。遠心分離を１０００ｒｐｍで４分間行った後、上清を除去した。下層に集積した細胞を新鮮培養培地で懸濁して、コラーゲンコートされた１０ｃｍ培養皿（ＩＷＡＫＩ コラーゲンＩコートディッシュ、ＡＧＣテクノグラス製）に播種した。培養皿を５％ＣＯ２、３７℃の条件でインキュベートし、コンフルエントに達するまで、７日から１０日間初代培養を行った。なお、初代培養期間中、全量培地交換を毎日行い、コラーゲンコートした培養皿に接着しなかった細胞（造血幹細胞、血球細胞や死細胞など）を除去した。培養皿に接着・伸展して増殖したコラーゲン接着性を有する細胞を骨髄由来細胞として取り扱った。
＜細胞シートの作製＞
初代培養を経てコンフルエントに達した骨髄由来細胞（Ｐ０）をトリプシン処理して、コラーゲンコート培養皿から回収した。遠心分離を１０００ｒｐｍで４分間行った後、上清を除去して、新鮮培養培地で懸濁した。その細胞懸濁液から細胞カウントを行い、０．５×１０６ｃｅｌｌｓ／ｍＬとなるように細胞懸濁液を新鮮培養培地で調製した。調製した細胞懸濁液２ｍＬ（細胞数１．０×１０６ｃｅｌｌｓ）を６ｃｍの温度応答性培養皿（ＵｐＣｅｌｌ（登録商標）、セルシード社製、接着面積２８．３ｍｍ）に播種した。新鮮培養培地３ｍＬを追加して、５％ＣＯ２、３７℃の条件でインキュベートし、オーバーコンフルエントに達するまで、７日から１０日間培養を行った。このとき培地は２日おきに全量交換した。
＜移植材の作製＞
６ｃｍの温度応答性培養皿で骨髄由来細胞がオーバーコンフルエントになった後、培地を除去して、培養皿の底の温度を２０～２５℃まで低下させた。培養皿を１～２０分静置して、骨髄由来細胞を培養皿から剥がし、シート状に遊離させた。この時の細胞シートの厚さは３０～５０μｍであった。得られた細胞シートの一方の表面上に培地で膨潤させたゼラチン長繊維不織布（Ｇｅｎｏｃｅｌ（登録商標）、シートタイプ、ニッケ・メディカル社製）を積層して、約５分静置し、ゼラチン長繊維不織布と細胞シートを接着させた後、ゼラチン長繊維不織布と細胞シートの積層体を回収した。なお、ゼラチン長繊維不織布は、膨潤時の繊維の平均繊維径は４７～５０μｍ、直径は約１２ｍｍ、厚さは約４５０μｍであった。回収した積層体を２枚、細胞シートの他方の表面同士が重なるように積層した。積層体を１０ｃｍの培養皿に置き、細胞シートが互いに接着するように、培地が無い状態で５％ＣＯ２、３７℃の条件で９０分間インキュベーションし、細胞移植材を得た。
＜膀胱拡大術＞
上記で得られた細胞移植材に、新鮮培地１０～１２ｍＬ添加して、３日間、培地交換なしで培養を行った。培養３日後、移植直前のゼラチン長繊維不織布で挟んだ細胞層の厚さ（第一の細胞シート及び第二の細胞シートの合計厚さ）は、約２００μｍであった。
動物は１０週齢雌ＳＤラットを用いた。麻酔をかけたラットの下腹部正中切開より膀胱を露出させた。膀胱を頂部から膀胱頚部にかけて約２／３（頂部から三角部にかけて）を切開して、膀胱前壁と後壁を開いた。膀胱割断面に上記で得られた細胞移植材を置き、積層体の上にポリグリコール酸の不織布を乗せ１２、３、６、９時方向で縫合した。尿リークがないことを確認して、閉腹した。
(Example 1)
<Bone marrow cell collection and primary culture>
Animals used 10-week-old male GFP-SD rats. The left and right femurs of anesthetized rats were removed. Both ends of the removed femoral bone were cut and a syringe with an injection needle 21G (inner diameter 0.51 mm) was inserted from one side to contain culture medium (15% fetal bovine serum (FBS)) and 0.1% penicillin-streptomycin. DMEM, and so on) flushed the cells present in the femoral bone to the contralateral cutting site. The washed cells were collected in a centrifuge tube. Centrifugation was performed at 1000 rpm for 4 minutes and then the supernatant was removed. The cells accumulated in the lower layer were suspended in fresh culture medium and seeded in a collagen-coated 10 cm culture dish (IWAKI collagen I-coated dish, manufactured by AGC Technoglass). Culture dishes were incubated at 5% CO 2 , 37 ° C. and primary cultures were performed for 7 to 10 days until confluence was reached. During the primary culture period, the total medium was exchanged every day to remove cells (hematopoietic stem cells, blood cells, dead cells, etc.) that did not adhere to the collagen-coated culture dish. Collagen-adherent cells that adhered to and expanded on a culture dish and proliferated were treated as bone marrow-derived cells.
<Preparation of cell sheet>
Bone marrow-derived cells (P0) that reached confluence through primary culture were trypsinized and recovered from collagen-coated culture dishes. Centrifugation was performed at 1000 rpm for 4 minutes, then the supernatant was removed and suspended in fresh culture medium. Cell counts were performed from the cell suspension and the cell suspension was prepared in fresh culture medium to 0.5 × 10 6 cells / mL. 2 mL of the prepared cell suspension (cell number 1.0 × 10 6 cells) was seeded in a 6 cm temperature-responsive culture dish (UpCell®, Cellseed, 28.3 mm adhesion area). 3 mL of fresh culture medium was added, and the cells were incubated at 5% CO 2 , 37 ° C., and cultured for 7 to 10 days until overconfluent was reached. At this time, the whole medium was replaced every two days.
<Preparation of transplant material>
After the bone marrow-derived cells became overconfluent in a 6 cm temperature-responsive culture dish, the medium was removed to reduce the temperature at the bottom of the culture dish to 20-25 ° C. The culture dish was allowed to stand for 1 to 20 minutes, and the bone marrow-derived cells were peeled off from the culture dish and released in the form of a sheet. The thickness of the cell sheet at this time was 30 to 50 μm. A gelatin long fiber non-woven fabric (Genocel (registered trademark), sheet type, manufactured by Nikke Medical Co., Ltd.) swollen with a medium was laminated on one surface of the obtained cell sheet, and allowed to stand for about 5 minutes to form a gelatin length. After adhering the fibrous nonwoven fabric and the cell sheet, a laminate of the gelatin long fiber nonwoven fabric and the cell sheet was recovered. In the gelatin long fiber non-woven fabric, the average fiber diameter at the time of swelling was 47 to 50 μm, the diameter was about 12 mm, and the thickness was about 450 μm. Two of the collected laminates were laminated so that the other surfaces of the cell sheets overlapped with each other. The laminate was placed in a 10 cm culture dish and incubated for 90 minutes at 5% CO 2 and 37 ° C. in the absence of medium so that the cell sheets adhered to each other to obtain a cell transplant material.
<Bladder enlargement>
To the cell transplant material obtained above, 10 to 12 mL of fresh medium was added, and the cells were cultured for 3 days without changing the medium. After 3 days of culturing, the thickness of the cell layer (total thickness of the first cell sheet and the second cell sheet) sandwiched between the gelatin long fiber non-woven fabrics immediately before transplantation was about 200 μm.
As animals, 10-week-old female SD rats were used. The bladder was exposed through a midline incision in the lower abdomen of anesthetized rats. An incision was made in the bladder from the apex to the bladder neck about two-thirds (from the apex to the trigone) to open the anterior and posterior walls of the bladder. The cell transplant material obtained above was placed on the cross section of the bladder, and a non-woven fabric of polyglycolic acid was placed on the laminate and sutured at 12, 3, 6 and 9 o'clock. After confirming that there was no urine leak, the abdomen was closed.
（比較例１）
実施例１で用いたものと同様の培地で膨潤させたゼラチン長繊維不織布を２枚積層して、積層体を得た。
その後、該積層体を１０ｃｍの培養皿に置き、培地が無い状態で５％ＣＯ２、３７℃の条件で９０分間インキュベーションし、移植材を得た。
該移植材を用いた以外は、実施例１と同様にして膀胱拡大術を行った。
(Comparative Example 1)
Two pieces of gelatin long fiber non-woven fabric swollen with the same medium as that used in Example 1 were laminated to obtain a laminated body.
Then, the laminate was placed in a 10 cm culture dish and incubated for 90 minutes under the conditions of 5% CO 2 and 37 ° C. in the absence of a medium to obtain a transplant material.
Bladder enlargement was performed in the same manner as in Example 1 except that the transplant material was used.
（比較例２）
実施例１と同様にしてゼラチン長繊維不織布と細胞シートの積層体を作製した。
その後、該積層体を１０ｃｍの培養皿に置き、培地が無い状態で５％ＣＯ２、３７℃の条件で９０分間インキュベーションし、細胞移植材を得た。
該細胞移植材を用いた以外は、実施例１と同様にして膀胱拡大術を行った。
(Comparative Example 2)
A laminated body of gelatin long fiber non-woven fabric and a cell sheet was prepared in the same manner as in Example 1.
Then, the laminate was placed in a 10 cm culture dish and incubated for 90 minutes under the conditions of 5% CO 2 and 37 ° C. in the absence of a medium to obtain a cell transplant material.
Bladder enlargement was performed in the same manner as in Example 1 except that the cell transplant material was used.
（比較例３）
実施例１と同様にしてゼラチン長繊維不織布と細胞シートの積層体を作製した。次に、該積層体の細胞シートの他方の表面上に実施例１で用いたものと同様の培地で膨潤させたゼラチン長繊維不織布を積層して、約5分静置し、ゼラチン長繊維不織布と細胞シートを接着させ、細胞シートの両表面上にゼラチン長繊維不織布が接着された積層体を得た。
その後、該積層体を１０cmの培養皿に置き、培地が無い状態で５％ＣＯ２、３７℃の条件で９０分間インキュベーションし、細胞移植材を得た。
該細胞移植材を用いた以外は、実施例１と同様にして膀胱拡大術を行った。
(Comparative Example 3)
A laminated body of gelatin long fiber non-woven fabric and a cell sheet was prepared in the same manner as in Example 1. Next, a gelatin long-fiber non-woven fabric swollen with the same medium as that used in Example 1 was laminated on the other surface of the cell sheet of the laminated body, and allowed to stand for about 5 minutes, and the gelatin long-fiber non-woven fabric was allowed to stand. And the cell sheet were adhered to obtain a laminate in which gelatin long fiber non-woven fabric was adhered on both surfaces of the cell sheet.
Then, the laminate was placed in a 10 cm culture dish and incubated for 90 minutes under the conditions of 5% CO 2 and 37 ° C. in the absence of a medium to obtain a cell transplant material.
Bladder enlargement was performed in the same manner as in Example 1 except that the cell transplant material was used.
（比較例４）
実施例１と同様にして得られた細胞シートを膀胱拡大術に用いようとしたところ、細胞シートのみでは柔らかく、移植部位に移送、留置することはできなかった。
(Comparative Example 4)
When the cell sheet obtained in the same manner as in Example 1 was tried to be used for bladder enlargement, the cell sheet alone was soft and could not be transferred to or placed at the transplant site.
（移植材の構造観察）
細胞移植材をパラフィン包埋した。包埋組織を５μｍ程度に薄切して、スライドガラスに乗せ標本とした。組織標本を脱パラフィン処理して、マッソントリクローム染色を実施し、細胞移植材の断面方向を観察した。
(Observation of the structure of the transplant material)
The cell transplant material was embedded in paraffin. The embedded tissue was sliced to about 5 μm and placed on a slide glass to prepare a specimen. The tissue specimen was deparaffinized, Masson's trichrome staining was performed, and the cross-sectional direction of the cell transplant material was observed.
図１は、実施例１の細胞移植材の断面写真である。図２は、同部分的拡大図である。図３は、比較例３の細胞移植材の断面写真である。図１及び図２おいて、スケールバーは、５０μｍを示す。
実施例１の細胞移植材１は、第一の細胞シート２、第二の細胞シート３、第一の生体適合長繊維不織布４、及び第二の生体適合長繊維不織布５を含む。
比較例３の細胞移植材１１は、細胞シート１３と、２枚の生体適合長繊維不織布１２、１４を含む。
２枚の細胞シートを含む実施例１の細胞移植材の場合、図１から分かるように、細胞層部分に２枚の細胞シートを積層したことに由来する細胞シート間空隙６が見られた。また、図２から分かるように、細胞シート間空隙６には２枚の細胞シート２及び３を橋渡しする細胞成分が見られた。この橋渡し成分は細胞シート間以外の空隙では見られず、細胞シート間の特徴的な構造である。
１枚の細胞シートを含む比較例３の細胞移植材１１の場合、図３から分かるように、実施例１の細胞移植材のような細胞シート間空隙は観察されない。
図４は、実施例１において、細胞移植材を所定期間培養し、移植する直前に観察した断面写真である。図４から分かるように、２枚の細胞シートにおいて、細胞の増殖及び遊走が生じており、細胞シート間空隙が観察できない。図４において、スケールバーは、１００μｍを示す。
FIG. 1 is a cross-sectional photograph of the cell transplant material of Example 1. FIG. 2 is a partially enlarged view of the same. FIG. 3 is a cross-sectional photograph of the cell transplant material of Comparative Example 3. In FIGS. 1 and 2, the scale bar indicates 50 μm.
The
The
In the case of the cell transplant material of Example 1 containing two cell sheets, as can be seen from FIG. 1, the
In the case of the
FIG. 4 is a cross-sectional photograph of the cell transplant material observed in Example 1 immediately before transplanting after culturing the cell transplant material for a predetermined period. As can be seen from FIG. 4, in the two cell sheets, cell proliferation and migration occur, and the space between the cell sheets cannot be observed. In FIG. 4, the scale bar indicates 100 μm.
（膀胱再生の肉眼的観察結果）
膀胱拡大術４週間後、再度、動物に麻酔をかけて、正中切開より膀胱を露出した。癒着した膀胱周辺の脂肪組織などの組織を膀胱からできる限り剥離した後、尿道と膣壁の剥離を行い、尿管を切断して膀胱（一部尿道を含む）を摘出した。摘出した膀胱の肉眼的観察を行った。実施例及び比較例の全てにおいて、切開した膀胱は治癒していた。しかし、積層体を移植した部位において、実施例では、比較例よりも厚い組織が再生されていた。
(Macroscopic observation of bladder regeneration)
Four weeks after the bladder enlargement, the animals were anesthetized again to expose the bladder through a midline incision. After as much tissue as possible, such as adipose tissue around the adhered bladder, was detached from the bladder, the urethra and vaginal wall were detached, the ureter was cut, and the bladder (including a part of the urethra) was removed. Macroscopic observation of the removed bladder was performed. In all of the examples and comparative examples, the incised bladder was healed. However, in the example, a thicker tissue than in the comparative example was regenerated at the site where the laminate was transplanted.
（膀胱組織解析方法）
膀胱拡大術４週間後、再度、動物に麻酔をかけて、正中切開より膀胱を露出した。癒着した膀胱周辺の脂肪組織などの組織を膀胱からできる限り剥離した後、尿道と膣壁の剥離を行い、尿管を切断して膀胱（一部尿道を含む）を摘出した。摘出した膀胱を４％パラフォルムアルデヒドに浸漬固定を一晩行った。浸漬固定した膀胱から、膀胱摘出時に剥離できなかった癒着組織をできるだけ剥離して、膀胱頂部から尿道にかけて２分割した。膀胱上皮から間質、排尿筋層が観察できるようにパラフィン包埋した。包埋組織を５μｍ程度に薄切して、スライドガラスに乗せ標本とした。組織標本を脱パラフィン処理して、マッソントリクローム染色を実施した。
染色した標本を１００倍レンズ顕微鏡（ＢＨ－２、オリンパス社製）で観察して、縫合痕を確認する。縫合痕から、頂部にかけて５～１０枚、組織像が連続する様に顕微鏡用デジタルカメラ（ＤＰ２、オリンパス社製）で撮影した。撮影した画像から、排尿筋層（平滑筋細胞で構成される平滑筋層）の厚さを１枚の画像から３、４カ所をデジタルカメラ付属のソフトウェアを用いて測定した。撮影、測定した排尿筋層の厚みの平均値を求めた。
(Bladder tissue analysis method)
Four weeks after the bladder enlargement, the animals were anesthetized again to expose the bladder through a midline incision. After as much tissue as possible, such as adipose tissue around the adhered bladder, was detached from the bladder, the urethra and vaginal wall were detached, the ureter was cut, and the bladder (including a part of the urethra) was removed. The removed bladder was immersed in 4% paraformaldehyde and fixed overnight. Adhesive tissue that could not be detached at the time of bladder removal was detached as much as possible from the bladder that had been immersed and fixed, and divided into two from the top of the bladder to the urethra. Paraffin was embedded so that the stroma and detrusor muscularis could be observed from the bladder epithelium. The embedded tissue was sliced to about 5 μm and placed on a slide glass to prepare a specimen. Tissue specimens were deparaffinized and Masson's trichrome staining was performed.
Observe the stained specimen with a 100x lens microscope (BH-2, manufactured by Olympus) to confirm the suture marks. From the suture marks to the top, 5 to 10 images were taken with a digital camera for microscope (DP2, manufactured by Olympus Corporation) so that the histological images were continuous. From the captured images, the thickness of the detrusor muscle layer (smooth muscle layer composed of smooth muscle cells) was measured from one image at three or four places using software attached to a digital camera. The average value of the thickness of the detrusor muscle layer that was photographed and measured was calculated.
図５～図８のそれぞれに、実施例１、比較例１～３において、膀胱拡大術４週間に摘出した膀胱の組織切片像を示した。図５～図８において、スケールバーは、１００μｍを示す。通常（正常）膀胱組織は、内側（内腔側）から、尿路上皮層、間質層、排尿筋層の３層で形成されている。実施例１、及び比較例１、２、３のいずれにおいても、正常膀胱組織に類似した尿路上皮層、及び間質層の再生（再構築）が確認された。一方、実施例において、排尿筋層は、正常膀胱組織に近い組織像を呈しており、比較例と比べて厚い排尿筋層が再生（再構築）されていた。図５～図８において、排尿筋層部分を両矢印で示した。移植した積層体の種類によって、膀胱の組織切片にみられる筋層の厚さが変化した。下記表１に排尿筋層の厚さを定量化した結果を示した。 In Examples 1 and Comparative Examples 1 to 3, the tissue section images of the bladder excised 4 weeks after the bladder enlargement operation are shown in FIGS. 5 to 8, respectively. In FIGS. 5-8, the scale bar indicates 100 μm. The normal (normal) bladder tissue is formed from the inside (luminal side) by three layers: the urinary tract epithelial layer, the stromal layer, and the detrusor muscular layer. In both Example 1 and Comparative Examples 1, 2 and 3, regeneration (reconstruction) of the urinary tract epithelial layer and the stromal layer similar to normal bladder tissue was confirmed. On the other hand, in the examples, the detrusor muscular layer exhibited a histological image close to that of normal bladder tissue, and the thick detrusor muscular layer was regenerated (reconstructed) as compared with the comparative example. In FIGS. 5 to 8, the detrusor muscular layer portion is indicated by double-headed arrows. Depending on the type of laminate transplanted, the thickness of the muscle layer found in the tissue section of the bladder varied. Table 1 below shows the results of quantifying the thickness of the detrusor muscularis.
表１からわかるように、実施例１の場合は、細胞シート２枚をゼラチン長繊維不織布２枚で挟んだハイブリッド積層体（以下、ハイブリッド型積層細胞シートとも記す。）とすることで、比較例に対し３倍以上の排尿筋層の再生がみられた。細胞シート２枚をゼラチン長繊維不織布２枚で挟んだハイブリッド積層体の場合、移植部位における移植細胞の生着率及び再生効果が高いことが分かる。 As can be seen from Table 1, in the case of Example 1, a hybrid laminated body in which two cell sheets are sandwiched between two gelatin long fiber non-woven fabrics (hereinafter, also referred to as a hybrid type laminated cell sheet) is used as a comparative example. Regeneration of the detrusor muscle layer was observed more than 3 times as much as that. It can be seen that in the case of a hybrid laminate in which two cell sheets are sandwiched between two gelatin long fiber non-woven fabrics, the engraftment rate and the regeneration effect of the transplanted cells at the transplantation site are high.
図９に、実施例１において、膀胱拡大術４週間に摘出した膀胱の組織切片像（スケールバー：１００μｍ）を示した。図９に示されているように、ゼラチン長繊維不織布上に線維芽細胞様細胞が伸展して、その細胞に沿うように平滑筋細胞が遊走した。遊走してきた平滑筋細胞同士が結合して、排尿筋層の一部を形成した。ハイブリッド型積層細胞シートを形成している骨髄由来細胞の平滑筋細胞への分化、及び分化した平滑筋細胞同士の結合による排尿筋層も考えられる。これは、移植材としてハイブリッド型積層細胞シートを用いることで、膀胱組織の再生機序を解明する可能性を示唆した。 FIG. 9 shows a tissue section image (scale bar: 100 μm) of the bladder removed in 4 weeks after the bladder enlargement operation in Example 1. As shown in FIG. 9, fibroblast-like cells were extended on the gelatin long fiber non-woven fabric, and smooth muscle cells migrated along the cells. The migrating smooth muscle cells bound to each other to form a part of the detrusor muscularis. Differentiation of bone marrow-derived cells forming a hybrid laminated cell sheet into smooth muscle cells and urinary muscular layer due to binding between differentiated smooth muscle cells are also conceivable. This suggests the possibility of elucidating the regeneration mechanism of bladder tissue by using a hybrid laminated cell sheet as a transplant material.
（実施例２）
＜脂肪細胞の採取及び初代培養＞
動物は１０週齢雄GFP-SDラット、１０週齢雄または雌SDラット、１０週齢雄または、雌NZWウサギを使用した。麻酔下の動物の下腹部を正中切開して、脂肪組織（０．５～１．０g）を摘出した。摘出した脂肪組織をリン酸緩衝生理食塩水（PBS）にて洗浄した後、０．２％コラゲナーゼ含有DMEMに移して、細かく切断した。その後、３７℃で約１００rpmで旋回させ、明らかな固形の脂肪組織がなくなるまで、６０～９０分旋回コラゲナーゼ酵素処理を行った。酵素処理した細胞含有溶液を遠心管に移した。遠心分離を１０００ｒｐｍで４分間行った後、上清を除去した。下層に集積した細胞を新鮮培養培地で懸濁して、コラーゲンコートされた１０ｃｍ培養皿に播種した。培養皿を５％ＣＯ２、３７℃の条件でインキュベートし、コンフルエントに達するまで、５日から７日間初代培養を行った。なお、初代培養期間中、全量培地交換を毎日行い、コラーゲンコートした培養皿に接着しなかった細胞（血球細胞や死細胞など）を除去した。培養皿に接着・伸展して増殖したコラーゲン接着性を有する細胞を脂肪由来細胞として取り扱った。
＜脂肪細胞の継代培養＞
初代培養を経てコンフルエントに達した脂肪由来細胞（Ｐ０）をトリプシン処理して、コラーゲンコート培養皿から回収した。遠心分離を１０００ｒｐｍで４分間行った後、上清を除去して、新鮮培地で懸濁した。その細胞懸濁液から細胞カウントを行い、０．５×１０６ｃｅｌｌｓ／ｍｌとなるように細胞懸濁液を新鮮培地で調製した。調製した細胞懸濁液２ｍＬ（細胞数１．０×１０６ ｃｅｌｌｓ）を６ｃｍの温度応答性培養皿に播種した。新鮮培地３ｍＬを追加して、５％ＣＯ２、３７℃の条件インキュベートし、オーバーコンフルエントに達するまで、５日から７日間培養を行った。このとき、培地は２日おきに全量交換した。
＜移植材の作製＞
骨髄由来細胞の代わりに、上記で得られた脂肪由来細胞を用いた以外は、実施例１に記載のとおりに移植材を作製した。
(Example 2)
<Fat cell collection and primary culture>
As animals, 10-week-old male GFP-SD rats, 10-week-old male or female SD rats, 10-week-old males or female NZW rabbits were used. A midline incision was made in the lower abdomen of the anesthetized animal and adipose tissue (0.5-1.0 g) was removed. The excised adipose tissue was washed with phosphate buffered saline (PBS), transferred to DMEM containing 0.2% collagenase, and cut into small pieces. Then, it was swirled at 37 ° C. at about 100 rpm and subjected to swirling collagenase enzyme treatment for 60 to 90 minutes until there was no obvious solid adipose tissue. The enzyme-treated cell-containing solution was transferred to a centrifuge tube. Centrifugation was performed at 1000 rpm for 4 minutes and then the supernatant was removed. The cells accumulated in the lower layer were suspended in fresh culture medium and seeded in a collagen-coated 10 cm culture dish. Culture dishes were incubated at 5% CO 2 , 37 ° C. and primary cultures were performed for 5 to 7 days until confluence was reached. During the primary culture period, the total amount of the medium was changed every day to remove cells (blood cells, dead cells, etc.) that did not adhere to the collagen-coated culture dish. Collagen-adhesive cells that adhered to and expanded on a culture dish and proliferated were treated as adipose-derived cells.
<Subculture of adipocytes>
Fat-derived cells (P0) that reached confluence through primary culture were trypsinized and recovered from collagen-coated culture dishes. Centrifugation was performed at 1000 rpm for 4 minutes, then the supernatant was removed and suspended in fresh medium. Cell counts were performed from the cell suspension and the cell suspension was prepared in fresh medium to a concentration of 0.5 × 10 6 cells / ml. 2 mL of the prepared cell suspension (cell number 1.0 × 10 6 cells) was seeded in a 6 cm temperature-responsive culture dish. 3 mL of fresh medium was added, incubated under conditions of 5% CO 2 , 37 ° C., and cultured for 5 to 7 days until overconfluent was reached. At this time, the whole medium was replaced every two days.
<Preparation of transplant material>
A transplant material was prepared as described in Example 1 except that the fat-derived cells obtained above were used instead of the bone marrow-derived cells.
（比較例５）
実施例２と同様にしてゼラチン長繊維不織布と細胞シートの積層体を用いた以外は、比較例３と同様にして移植材を作製した。
(Comparative Example 5)
A transplant material was prepared in the same manner as in Comparative Example 3 except that a laminated body of gelatin long fiber nonwoven fabric and a cell sheet was used in the same manner as in Example 2.
（細胞移植材の構造観察）
実施例１の場合と同様、実施例２及び比較例５で得られた細胞移植材の断面構造をそれぞれ観察し、その結果を図１０及び図１１にそれぞれ示した。図１０及び図１１において、スケールバーは、５０μｍを示す。
図１０に示すように、実施例２の細胞移植材２１は、第一の細胞シート２２、第二の細胞シート２３、第一の生体適合長繊維不織布２４、及び第二の生体適合長繊維不織布２５を含む。
比較例３の細胞移植材３１は、細胞シート３３と、２枚の生体適合長繊維不織布３２、３４を含む。
図１０から分かるように、実施例２の細胞移植材２１の場合、細胞部分に２枚の細胞シート２２、２３を積層したことに由来する細胞シート間空隙２６が見られた。一方、１枚の細胞シートを含む比較例５の細胞移植材３１の場合、図１１から分かるように、実施例２の細胞移植材のような細胞シート間空隙は観察されない。
(Structural observation of cell transplant material)
Similar to the case of Example 1, the cross-sectional structures of the cell transplant materials obtained in Example 2 and Comparative Example 5 were observed, respectively, and the results are shown in FIGS. 10 and 11, respectively. In FIGS. 10 and 11, the scale bar indicates 50 μm.
As shown in FIG. 10, the
The
As can be seen from FIG. 10, in the case of the
実施例２の細胞移植材も、実施例１の細胞移植材のように膀胱拡大術に用いることができる。 The cell transplant material of Example 2 can also be used for bladder enlargement like the cell transplant material of Example 1.
本発明のハイブリッド型積層骨髄由来細胞シートは、移植によって厚い排尿筋層の組織を形成するのに用いることができる。また、本発明の本発明のハイブリッド型積層骨髄由来細胞シートは、組織再生機序への実験モデルとして利用が可能である。加えて、ヒト又はヒト以外の動物の組織の増大、修復又は再生に用いることができる。 The hybrid laminated bone marrow-derived cell sheet of the present invention can be used to form a thick detrusor muscular tissue by transplantation. In addition, the hybrid-type laminated bone marrow-derived cell sheet of the present invention of the present invention can be used as an experimental model for the tissue regeneration mechanism. In addition, it can be used to grow, repair or regenerate human or non-human animal tissues.
１、１１、２１、３１ 細胞移植材
２、３、１３、２２、２３、３３ 細胞シート
４、５、１２、１４、２４、２５、３２、３４ 生体適合長繊維不織布（ゼラチン長繊維不織布）
６、２６ 細胞シート間空隙
1,11,21,31
6,26 gap between cell sheets
Claims (11)
第一の生体適合長繊維不織布及び第二の生体適合長繊維不織布は、いずれも、生体適合性ポリマーを主成分とする生体適合長繊維不織布であり、
第一の生体適合長繊維不織布は第一の細胞シートの一方の表面に接着しており、
第二の生体適合長繊維不織布は第二の細胞シートの一方の表面に接着しており、
第一の細胞シートの他方の表面と第二の細胞シートの他方の表面が接着していることを特徴とする細胞移植材。 Includes a first cell sheet, a second cell sheet, a first biocompatible long fiber non-woven fabric, and a second biocompatible long fiber non-woven fabric.
The first biocompatible long fiber non-woven fabric and the second biocompatible long fiber non-woven fabric are both biocompatible long fiber non-woven fabrics containing a biocompatible polymer as a main component.
The first biocompatible long fiber non-woven fabric is adhered to one surface of the first cell sheet.
The second biocompatible long fiber non-woven fabric is adhered to one surface of the second cell sheet.
A cell transplant material characterized in that the other surface of the first cell sheet and the other surface of the second cell sheet are adhered to each other.
第一の細胞シート及び第二の細胞シートを準備する工程と、
第一の細胞シートの一方の表面に第一の生体適合長繊維不織布層を積層して第一の積層体を得る工程と、
第二の細胞シートの一方の表面に第二の生体適合長繊維不織布層を積層して第二の積層体を得る工程と、
第一の細胞シートの他方の表面と第二の他方の表面が接するように第一の積層体と第二の積層体を積層し、細胞移植材を得る工程を含む細胞移植材の製造方法。 The method for producing a cell transplant material according to any one of claims 1 to 9.
The process of preparing the first cell sheet and the second cell sheet,
A step of laminating a first biocompatible long fiber non-woven fabric layer on one surface of a first cell sheet to obtain a first laminate,
A step of laminating a second biocompatible long fiber non-woven fabric layer on one surface of a second cell sheet to obtain a second laminate,
A method for producing a cell transplant material, which comprises a step of laminating a first laminate and a second laminate so that the other surface of the first cell sheet and the second surface are in contact with each other to obtain a cell transplant material.
細胞移植材を生体（ただし、人体を除く。）内の所定部位に移植する工程を含む細胞移植材の使用方法。 The method for using the cell transplant material according to any one of claims 1 to 9.
A method of using a cell transplant material, which comprises a step of transplanting the cell transplant material to a predetermined site in a living body (excluding the human body).
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