JP2005110537A - Cell culture carrier - Google Patents
Cell culture carrier Download PDFInfo
- Publication number
- JP2005110537A JP2005110537A JP2003346547A JP2003346547A JP2005110537A JP 2005110537 A JP2005110537 A JP 2005110537A JP 2003346547 A JP2003346547 A JP 2003346547A JP 2003346547 A JP2003346547 A JP 2003346547A JP 2005110537 A JP2005110537 A JP 2005110537A
- Authority
- JP
- Japan
- Prior art keywords
- cell
- cell culture
- gel layer
- polyvalent metal
- gel
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Abstract
Description
本発明は、細胞培養の技術に関し、具体的には、細胞培養担体、該細胞培養担体を利用した細胞の培養方法、該培養方法により得られる細胞培養物、細胞シート、該細胞培養物を利用した細胞転写法、細胞重層化法、該重層化方法により得られる重層化細胞に関する。 The present invention relates to a cell culture technique, and specifically, a cell culture carrier, a cell culture method using the cell culture carrier, a cell culture obtained by the culture method, a cell sheet, and the cell culture The present invention relates to a cell transfer method, a cell stratification method, and a stratified cell obtained by the stratification method.
高分子含水ゲルは、生体に類似した構造を持ち、温度、酸性・アルカリ性等の外部条件によって膨張、収縮する性質を有するため、人工筋肉などの人工臓器・組織への応用、内部に薬剤を封入して放出量をコントロールする医療分野への応用のみならず、各種サイトカイン等を含むゲルとして細胞を培養する際に細胞成長の足場としての利用も試みられている。 The polymer hydrogel has a structure similar to that of a living body, and has the property of expanding and contracting depending on external conditions such as temperature, acidity and alkalinity, so it can be applied to artificial organs and tissues such as artificial muscles, and drugs are enclosed inside In addition to application to the medical field of controlling the amount of release, attempts have been made to use it as a scaffold for cell growth when cells are cultured as gels containing various cytokines.
高分子含水ゲルの中でも温度応答性高分子であるN-イソプロピルアクリルアミド（N-isopropylacrylamide）（以下「NIPAM」と称する）は、低温では膨潤して液状であるが、34℃付近で相転移し急激に収縮しゲル化する。従来、培養細胞の階層化を行うに当たり、37℃の温度条件下、ゲル化したNIPAM上で培養した細胞をNIPAMごと別の細胞層に重ね、その後34℃以下に下げることによりNIPAMを液状化させて取り除き、細胞同士を直接重ねるという手法が取られていた（非特許文献１から５参照）。 Among water-containing polymer gels, N-isopropylacrylamide (NIPAM), which is a temperature-responsive polymer, swells and is liquid at low temperatures, but rapidly undergoes a phase transition near 34 ° C. Shrinks into a gel. Conventionally, when layering cultured cells, the cells cultured on gelled NIPAM at 37 ° C are layered on another cell layer together with NIPAM, and then lowered to 34 ° C or lower to liquefy NIPAM. A method of removing cells and directly stacking cells has been taken (see Non-Patent Documents 1 to 5).
NIPAM上で細胞培養を行った場合、通常、細胞は単層状に成長する。この際、隣り合った細胞同士ではコラーゲン等の細胞外マトリックス（Extracellular Matrix）（以下「ECM」と称する）が形成される。そして、細胞が増殖するためには、ECMに接着する必要がある。しかし、細胞の上部及び細胞と基底層であるNIPAM間は他の細胞と接着しておらず、細胞接着に必要なECMは形成されない。
従って、NIPAM上で培養した単層の細胞層同士を重層し、細胞層を34℃以下の条件下でNIPAMを可溶化して取り除き、細胞同士が直接接するように重ねても、上部に重ねた細胞は、増殖するための足場が十分でなく、従って安定した増殖は望むことができなかった。
また、液状化したNIPAMが細胞毒として作用するため細胞の正常な成長を阻害する現象も認められるため、細胞の階層化の手段としては極めて不向きで不安定な技術であった。これまでに各種ゲル上に細胞外マトリックス成分を重層化しゲル化させた培地での細胞培養は成功例がなく、重層後に不要となったゲルを容易に除去できるような培地を使用した培養系確立の試みが続けられていた。
When cell culture is performed on NIPAM, cells usually grow in a monolayer. At this time, an extracellular matrix such as collagen (hereinafter referred to as “ECM”) is formed between adjacent cells. And in order for a cell to proliferate, it is necessary to adhere to ECM. However, the upper part of the cell and between the cell and the basal layer NIPAM are not adhered to other cells, and the ECM necessary for cell adhesion is not formed.
Therefore, the cell layers of monolayers cultured on NIPAM are overlaid, and the cell layer is solubilized and removed under conditions of 34 ° C or lower, and the cells are stacked so that the cells are in direct contact with each other, or stacked on top. The cells did not have enough scaffold to grow, so stable growth could not be desired.
In addition, since liquefied NIPAM acts as a cytotoxin and inhibits the normal growth of cells, it was an extremely unsuitable and unstable technique for cell stratification. To date, there has been no successful cell culture in a medium in which extracellular matrix components are layered and gelled on various gels, and a culture system using a medium that can easily remove unnecessary gels after layering has been established. The attempt was continued.
この問題の解決法として多孔質膜と該多孔質膜上にアルギン酸ゲル層と細胞外マトリックス成分ゲル層又は細胞外マトリックス成分スポンジ層を重層化させたことを特徴とする細胞培養担体が提案されているが（特許文献１）、細胞外マトリックス層が厚く、乾燥膜の状態で細胞培養担体を保管することが不可能であった。また、細胞の重層化操作にも邪魔になり安定に重層化細胞を得られない問題があった。また、この方法ではアルギン酸ゲル層の溶解にEDTA水溶液を用いて細胞培養物を細胞培養担体から剥離しているが、このときの侵襲により細胞がダメージを受ける問題があった。
さらに、該特許に記載されている多孔質膜は透明性がなく、細胞培養中に生きた細胞の育成状況を目視または顕微鏡等により直接観察することができないという問題があった。このため、シャーレ上に置いた細胞培養担体の周りのシャーレ上での細胞の育成状態から細胞担体上の細胞の状況を同等状態であると類推していたが、細胞培養担体の周りのシャーレ上での細胞の育成状態は、細胞担体上の細胞の状態と必ずしも一致せず、細胞の増殖が不足していたり、オーバーコンフルエントであったりする不都合があった。
従って、細胞の育成状態を確実に把握するために、細胞培養担体には通常の細胞培養で用いるシャーレのように、透明な細胞培養担体が望まれていた。
Furthermore, the porous membrane described in the patent has no transparency, and there is a problem that the growth state of living cells during cell culture cannot be observed directly or visually with a microscope. For this reason, the state of the cells on the cell carrier was analogized from the state of cell growth on the petri dish around the cell culture carrier placed on the petri dish. In this case, the cell growth state does not always match the cell state on the cell carrier, and there is a disadvantage that the cell growth is insufficient or overconfluent.
Therefore, in order to reliably grasp the cell growth state, a transparent cell culture carrier, such as a petri dish used in normal cell culture, has been desired as a cell culture carrier.
本発明は、細胞の育成状態を確実に把握でき、かつ培養した細胞層を、該細胞層中の細胞の成長・増殖を阻害することなく剥離できる、透明性のある細胞培養担体の提供を目的とする。 An object of the present invention is to provide a transparent cell culture carrier capable of reliably grasping a cell growth state and capable of peeling a cultured cell layer without inhibiting the growth and proliferation of cells in the cell layer. And
本発明者らは上記の課題を解決すべく鋭意研究を行い、支持体である高分子含水ゲル層、及び細胞培養面側最表面層に細胞接着性ゲル層を有し、該高分子含水ゲル層と該細胞接着性ゲル層との間にアニオン性多糖類及び多価金属イオンを含むゲル層を有する細胞培養担体が、透明性を有し細胞の生育状況の観察を簡便に行え、かつ細胞層の剥離性に優れることを見出した。本発明はこれらの知見を基に完成されたものである。 The present inventors have intensively studied to solve the above problems, and have a polymer hydrogel layer as a support, and a cell adhesive gel layer on the cell culture surface side outermost surface layer, and the polymer hydrogel The cell culture carrier having a gel layer containing an anionic polysaccharide and a polyvalent metal ion between the cell adhesion gel layer and the cell adhesion gel layer is transparent and allows easy observation of the growth state of the cells. It was found that the peelability of the layer was excellent. The present invention has been completed based on these findings.
すなわち、本発明は、支持体である高分子含水ゲル層、及び細胞培養面側最表面層に細胞接着性ゲル層を有し、該高分子含水ゲル層と該細胞接着性ゲル層との間にアニオン性多糖類及び多価金属イオンを含むゲル層を有する細胞培養担体を提供するものである。本発明の好ましい態様によれば、細胞接着性ゲル層とアニオン性多糖類及び多価金属イオンを含むゲル層との間に高分子含水ゲル層を有さない上記の細胞培養坦体；細胞培養面側最表面層とアニオン性多糖類及び多価金属イオンを含むゲル層が隣接している上記いずれかの細胞培養坦体が提供される。本発明のさらに好ましい態様によれば、アニオン性多糖類及び多価金属イオンを含むゲル層がアルギン酸ゲル層である上記のいずれか一項に記載の細胞培養担体；アルギン酸ゲル層の乾膜厚が0.01μm以上50μm以下である上記細胞培養担体；アルギン酸ゲルがアルギン酸カルシウムゲルである上記細胞培養担体が提供される。 That is, the present invention has a polymer hydrous gel layer as a support and a cell adhesive gel layer on the cell culture surface side outermost surface layer, and the polymer hydrous gel layer and the cell adhesive gel layer are interposed between them. The present invention provides a cell culture carrier having a gel layer containing an anionic polysaccharide and a polyvalent metal ion. According to a preferred embodiment of the present invention, the above cell culture carrier having no polymer hydrogel layer between the cell adhesive gel layer and the gel layer containing the anionic polysaccharide and the polyvalent metal ion; Any one of the above cell culture carriers is provided in which the outermost surface layer is adjacent to a gel layer containing an anionic polysaccharide and a polyvalent metal ion. According to a further preferred embodiment of the present invention, the cell culture carrier according to any one of the above, wherein the gel layer containing an anionic polysaccharide and polyvalent metal ions is an alginate gel layer; the dry film thickness of the alginate gel layer is Provided is the above cell culture carrier of 0.01 μm or more and 50 μm or less; and the above cell culture carrier wherein the alginate gel is a calcium alginate gel.
本発明の特に好ましい態様によれば、電子線、γ線、紫外線のいずれかまたは複数を照射することで滅菌された上記いずれかの記載の細胞培養担体；高分子含水ゲルがキトサンゲルである上記いずれかの細胞培養担体；細胞培養面側最表面層が細胞接着性コラーゲンゲル層である上記いずれの細胞培養担体が提供される。 According to a particularly preferred embodiment of the present invention, the cell culture carrier according to any one of the above, which has been sterilized by irradiation with one or more of an electron beam, a γ-ray and an ultraviolet ray; the polymer-containing gel is a chitosan gel Any of the above cell culture carriers; any one of the above cell culture carriers, wherein the cell culture surface side outermost layer is a cell adhesive collagen gel layer.
また、別の観点からは、上記いずれかの細胞培養担体を用いる細胞培養方法が提供される。
さらに別の観点からは、上記の方法により得られる細胞培養物のアニオン性多糖類及び多価金属イオンを含むゲル層を可溶化して得られる細胞シートが提供される。この発明の好ましい態様によれば、アニオン性多糖類及び多価金属イオンを含むゲル層の可溶化を多価金属カチオンが存在しない培地又は多価金属カチオンの総和モル数に対して90モル%以上のキレート剤を添加した培地を用いて行う上記の細胞シートが提供される。
From another viewpoint, a cell culture method using any one of the above cell culture carriers is provided.
From another viewpoint, a cell sheet obtained by solubilizing a gel layer containing an anionic polysaccharide and a polyvalent metal ion of a cell culture obtained by the above method is provided. According to a preferred embodiment of the present invention, the solubilization of the gel layer containing the anionic polysaccharide and the polyvalent metal ion is 90 mol% or more based on the total number of moles of the medium or polyvalent metal cation without the polyvalent metal cation. There is provided the above-described cell sheet performed using a medium supplemented with the above chelating agent.
さらに別の観点からは、上記の方法により得られる細胞培養物のアニオン性多糖類及び多価金属イオンを含むゲル層を多価金属カチオンが存在しない培地又は多価金属カチオンの総和モル数に対して90モル%以上のキレート剤を添加した培地を用いて可溶化処理して細胞シートを得る細胞培養方法；上記の方法により得られる細胞培養物を他の細胞培養担体上でさらに培養する細胞転写法；該細胞転写法によって得られる細胞培養物のアニオン性多糖類及び多価金属イオンを含むゲル層を可溶化処理して得られる重層化細胞；該細胞転写法により得られる細胞培養物を他の培養細胞上でさらに培養する細胞重層化法が提供される。 From another viewpoint, the gel layer containing the anionic polysaccharide and the polyvalent metal ion of the cell culture obtained by the above method can be used with respect to a medium in which no polyvalent metal cation is present or the total number of moles of the polyvalent metal cation. Cell culture method for obtaining a cell sheet by solubilization treatment using a medium to which a chelating agent of 90 mol% or more is added; cell transfer in which the cell culture obtained by the above method is further cultured on another cell culture carrier A layered cell obtained by solubilizing a gel layer containing an anionic polysaccharide and a polyvalent metal ion of a cell culture obtained by the cell transcription method; a cell culture obtained by the cell transcription method There is provided a cell stratification method of further culturing on the cultured cells.
本発明により、細胞シートの剥離性に優れた細胞培養担体が提供される。従って本発明の細胞培養担体を用いて細胞層の重層化を容易に行なうことができる。また、本発明の細胞培養担体は、透明性を有し、細胞培養中の細胞の生きた状態を直接観察できる。 According to the present invention, a cell culture carrier having excellent cell sheet peelability is provided. Therefore, cell layers can be easily layered using the cell culture carrier of the present invention. In addition, the cell culture carrier of the present invention has transparency, and can directly observe the living state of cells during cell culture.
以下、本発明を詳細に説明する。
「細胞培養担体」とは、細胞を培養する際の担体又は支持体となり得るものを意味する。
「アニオン性多糖類及び多価金属イオンを含むゲル層」とはアルギン酸、デキストラン硫酸、カルボキシメチルセルロース、カルボキシメチルデキストラン、ヒアルロン酸などのアニオン性多糖類とこれらのゲル化を引き起こし得る多価金属カチオンを含むゲル層をあらわすが、本発明においてはアルギン酸ゲル層が好ましい。
Hereinafter, the present invention will be described in detail.
“Cell culture carrier” means a carrier or support that can be used to culture cells.
"Gel layer containing anionic polysaccharide and polyvalent metal ion" refers to anionic polysaccharides such as alginic acid, dextran sulfate, carboxymethylcellulose, carboxymethyldextran, and hyaluronic acid and polyvalent metal cations that can cause gelation of these. In the present invention, an alginate gel layer is preferable.
「アルギン酸ゲル」とは、アルギン酸の分子中のカルボン酸基と多価金属イオンとがキレート構造を形成してゲル化したものを意味し、「アルギン酸ゲル層」とは、層状のアルギン酸ゲルを意味する。アルギン酸は、グルクロン酸（Ｇ）とマンヌロン酸（Ｍ）よりなるブロック共重合体であり、Ｍブロックが有するポケット構造に多価金属カチオンが侵入してエッグボックスを形成し、ゲル化すると考えられている。アルギン酸のゲル化を引き起こし得る多価金属カチオンの具体例としては、バリウム（Ba）、鉛（Pb）、銅（Cu）、ストロンチウム（Sr）、カドミウム（Cd）、カルシウム（Ca）、亜鉛（Zn）、ニッケル（Ni）、コバルト（Co）、マンガン（Mn）、鉄（Fe）、マグネシウム（Mg）等の金属イオンを例示でき、これらのうち特に好ましいものとして、カルシウムイオン、マグネシウムイオン、バリウムイオン、ストロンチウムイオンを例示できる。また、「アルギン酸ゲル」はアルギン酸とカチオン残基を有する有機高分子化合物のポリイオンコンプレックスゲルでも良い。ここでいうカチオン残基を有する有機高分子化合物の例としては、ポリリジン、キトサン、ゼラチン、コラーゲンなどの複数のアミノ基を有する化合物が挙げられる。 “Alginic acid gel” means a gel formed by forming a chelate structure between a carboxylic acid group in a molecule of alginic acid and a polyvalent metal ion, and “alginate gel layer” means a layered alginate gel. To do. Alginic acid is a block copolymer composed of glucuronic acid (G) and mannuronic acid (M). It is thought that polyvalent metal cations enter the pocket structure of M block to form an egg box and gel. Yes. Specific examples of polyvalent metal cations that can cause gelation of alginic acid include barium (Ba), lead (Pb), copper (Cu), strontium (Sr), cadmium (Cd), calcium (Ca), zinc (Zn ), Nickel (Ni), cobalt (Co), manganese (Mn), iron (Fe), magnesium (Mg) and other metal ions can be exemplified, and among these, calcium ions, magnesium ions, barium ions are particularly preferable. And strontium ions. The “alginic acid gel” may be a polyion complex gel of an organic polymer compound having alginic acid and a cation residue. Examples of the organic polymer compound having a cation residue mentioned here include compounds having a plurality of amino groups such as polylysine, chitosan, gelatin and collagen.
アルギン酸のゲル化は、常法に従って行なうことができる。アルギン酸のゲル化は、例えばイオン交換を利用して行なうことができる。例えば、アルギン酸ナトリウム水溶液にカルシウムイオンを添加すると速やかにイオン交換が生じ、アルギン酸カルシウムゲルが得られる。より具体的には、0.2〜５％アルギン酸ナトリウム水溶液を、高分子含水ゲル（例えば、キトサン）の膜上に塗布後0.01〜1.0M 塩化カルシウム水溶液中に浸漬して塩化カルシウムをしみ込ませ、20〜30℃で3分〜３時間放置することによりアルギン酸カルシウムゲル層が得られる。このように高分子含水ゲルを用いてアルギン酸のゲル化を行なえば、高分子含水ゲル膜と該高分子含水ゲル膜上に形成されたアルギン酸ゲル層とを含む細胞培養担体を得ることができる。 The gelation of alginic acid can be performed according to a conventional method. The gelation of alginic acid can be performed using, for example, ion exchange. For example, when calcium ions are added to a sodium alginate aqueous solution, ion exchange occurs rapidly, and a calcium alginate gel is obtained. More specifically, a 0.2-5% sodium alginate aqueous solution is applied onto a film of a polymer hydrous gel (for example, chitosan) and then immersed in a 0.01-1.0M calcium chloride aqueous solution to impregnate calcium chloride. A calcium alginate gel layer is obtained by leaving it at 30 ° C. for 3 minutes to 3 hours. When gelation of alginic acid is performed using the polymer hydrogel as described above, a cell culture carrier comprising a polymer hydrogel membrane and an alginate gel layer formed on the polymer hydrogel membrane can be obtained.
アルギン酸ゲル層の乾膜の厚さは0.01μm以上50μm以下であることが好ましく、0.1μm以上10μm以下が好ましく、0.5μm以上5μm以下であることがさらに好ましい。アルギン酸ゲル層の固形分量が少なすぎると充分な膜を形成できず穴が空いてしまい、多すぎると乾燥膜でのカールや割れの発生、培養工程での変形やアルギン酸ゲル溶解工程での溶解不良といった問題が生じる。 The dry film thickness of the alginate gel layer is preferably 0.01 μm or more and 50 μm or less, preferably 0.1 μm or more and 10 μm or less, and more preferably 0.5 μm or more and 5 μm or less. If the amount of solid content in the alginate gel layer is too small, a sufficient film cannot be formed and holes are formed, and if it is too much, curling and cracking occur in the dried film, deformation in the culture process, and poor dissolution in the alginate gel dissolution process. Problems arise.
本明細書において、ゲル層の厚さは充分に乾燥した状態で計測した値であり、電子顕微鏡断面像、膜厚計、エリプソメーター、角度可変XPSなど、好ましくは電子顕微鏡断面像から計測した値である。
アルギン酸は、褐藻類の細胞壁構成多糖又は細胞間充填物質として天然に存在しており、これらを原料として採取可能である。原料褐藻類の具体例としては、ヒバマタ目ダービリア科ダービリア属（例えばD.potatorum）、ヒバマタ目ヒバマタ科アスコフィラム属（例えばA.nodosum）、コンブ目コンブ科コンブ属（例えばマコンブ、ナガコンブ）、コンブ目コンブ科アラメ属（例えばアラメ）、コンブ目コンブ科カジメ属（例えばカジメ、ウロメ）、コンブ目レッソニア科レッソニア属（例えばL.flavikans）の褐藻類を例示できる。また、市販のアルギン酸を使用することもできる。アルギン酸のG/Mの比は特に限定されないが、G/Mの比が大きいほどゲル形成能が大きいので、G/Mの比は大きい方が好ましく、具体的には0.1〜１であるのが好ましく、0.2〜0.5であるのがさらに好ましい。
In this specification, the thickness of the gel layer is a value measured in a sufficiently dry state, such as an electron microscope cross-sectional image, a film thickness meter, an ellipsometer, and an angle variable XPS, preferably a value measured from an electron microscopic cross-sectional image. It is.
Alginic acid exists naturally as a cell wall-constituting polysaccharide or intercellular packing material of brown algae, and these can be collected as raw materials. Specific examples of the raw material brown algae include the genus Davilia (D.potatorum), the genus Ascophyllum (eg A. nodosum), the genus Ascomphila (eg, Macombu, Nagacomb), Examples include brown algae of the genus Alameda (for example, arame), the order of the genus Coleaceae (for example, Kajime, Urome), and the genus Lessoniaceae (for example, L. flavikans). Commercially available alginic acid can also be used. Although the ratio of G / M of alginic acid is not particularly limited, the larger the ratio of G / M, the larger the gel forming ability. Therefore, the larger ratio of G / M is preferable, specifically 0.1 to 1. Preferably, it is 0.2-0.5.
「細胞接着性ゲル層」とは、層状の細胞接着性を有するハイドロゲルを意味し、細胞毒性が無く、通常の培養条件で細胞が付着するゲルであれば天然、合成の化合物いずれでも良いが、好ましくは層状の細胞外マトリックス成分ゲルである。ここで「細胞接着性ゲル層」にキトサンは含まれない。細胞外マトリックスは、一般的には「動物組織中の細胞の外側に存在する安定な生体構造物で、細胞が合成し、細胞外に分泌・蓄積した生体高分子の複雑な会合体」と定義されており（生化学辞典（第３版）p.570,東京化学同人（株））、細胞を物質的に支持する役割や細胞の活性を調節する役割（すなわち細胞外の情報を細胞に伝えその活性に変化を与える役割）等を担っている。「細胞外マトリックス成分」とは、細胞外マトリックスの構成成分を意味し、その具体例としては、コラーゲン、エラスチン、プロテオグリカン、グルコサミノグリカン（ヒアルロン酸、コンドロイチン硫酸、デルマタン硫酸、ヘパラン硫酸、ヘパリン、ケラタン硫酸など）、フィブロネクチン、ラミニン、ビトロネクチン等を例示でき、これらのうち特に好ましいものとして、コラーゲン、アテロコラーゲン、マトリゲル（IV型コラーゲン、ラミニン、ヘパラン硫酸よりなるゲル）、ヒアルロン酸を例示できる。最も好ましいのはコラーゲンである。細胞外マトリックス成分は、常法に従って得ることができる。また、市販の細胞外マトリックス成分を使用してもよい。細胞接着性成分のゲル化は、常法に従って行なうことができる。細胞外マトリックス成分のゲル化の際には、必要に応じてゲル化剤を使用してもよい。例えば、細胞接着性成分がコラーゲンである場合には、0.3〜0.5％コラーゲン水溶液を37℃で10〜20分間インキュベーションすることにより、コラーゲンゲルを得ることができる。コラーゲンゲル層の乾膜の厚さは0.005μm以上5.0μm以下であり、0. 005μm以上1.0μm以下が好ましく、0. 005μm以上0.5μm以下がさらに好ましい。コラーゲンゲル層が厚いと乾燥時に膜に亀裂が発生するばかりか、細胞の転写が著しく困難になる。 “Cell-adhesive gel layer” means a layered cell-adhesive hydrogel, and any natural or synthetic compound may be used as long as it is non-cytotoxic and adheres to cells under normal culture conditions. Preferably, it is a layered extracellular matrix component gel. Here, chitosan is not included in the “cell adhesive gel layer”. The extracellular matrix is generally defined as "a complex body of biopolymers, which are stable biological structures that exist outside the cells in animal tissues and are synthesized and secreted and accumulated outside the cells." (Biochemical Dictionary (Third Edition) p.570, Tokyo Chemical Doujin Co., Ltd.), the role of materially supporting cells and the role of regulating cell activity (ie, transmitting extracellular information to cells) It plays a role in changing its activity). “Extracellular matrix component” means a component of the extracellular matrix, and specific examples thereof include collagen, elastin, proteoglycan, glucosaminoglycan (hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparan sulfate, heparin, Keratan sulfate, etc.), fibronectin, laminin, vitronectin and the like can be exemplified. Among these, collagen, atelocollagen, matrigel (gel made of type IV collagen, laminin, heparan sulfate), and hyaluronic acid can be exemplified. Most preferred is collagen. The extracellular matrix component can be obtained according to a conventional method. Commercially available extracellular matrix components may also be used. Gelation of the cell adhesion component can be performed according to a conventional method. When the extracellular matrix component is gelled, a gelling agent may be used as necessary. For example, when the cell adhesion component is collagen, a collagen gel can be obtained by incubating a 0.3 to 0.5% aqueous collagen solution at 37 ° C. for 10 to 20 minutes. The dry thickness of the collagen gel layer is from 0.005 μm to 5.0 μm, preferably from 0.005 μm to 1.0 μm, and more preferably from 0.005 μm to 0.5 μm. If the collagen gel layer is thick, not only will the film be cracked during drying, but cell transfer will be extremely difficult.
本明細書において「細胞接着性ゲル層」は、培養面側の最表層に位置するが、これは種類の異なる細胞接着性ゲル層が複数積層している態様も含む。また、本発明の細胞培養担体においては「アニオン性多糖類及び多価金属イオンを含むゲル層」と「細胞接着性ゲル層」が隣接しているのが好ましい。この２層の間に中間層が存在すると細胞の培養時に培地成分や酸素の供給が不足して細胞の成長や増殖を阻害する場合や、細胞が生存できなくなる場合がある。さらに、前述の「アニオン性多糖類及び多価金属イオンを含むゲル層」の可溶化処理時にその溶解性に影響する場合がある。また、細胞シートを剥離した際に細胞接着性層以外の層が残存し、重層化における細胞間の相互作用に影響を与える場合がある。 In the present specification, the “cell-adhesive gel layer” is located on the outermost layer on the culture surface side, but this includes an embodiment in which a plurality of different types of cell-adhesive gel layers are laminated. In the cell culture carrier of the present invention, it is preferable that the “gel layer containing an anionic polysaccharide and polyvalent metal ions” and the “cell adhesive gel layer” are adjacent to each other. If an intermediate layer exists between the two layers, the supply of medium components and oxygen may be insufficient at the time of cell culture, which may inhibit cell growth and proliferation, or cells may not be viable. Furthermore, the solubility may be affected during the solubilization treatment of the aforementioned “gel layer containing anionic polysaccharide and polyvalent metal ions”. Further, when the cell sheet is peeled off, a layer other than the cell adhesive layer remains, which may affect the interaction between cells in the layering.
アニオン性多糖類及び多価金属イオンを含むゲル上に細胞接着性ゲル層を形成する際には、アニオン性多糖類及び多価金属イオンを含むゲルと細胞接着性ゲル層とを別々に作製した後、両者を重ねてもよいが、アニオン性多糖類及び多価金属イオンを含むゲル上にと細胞接着性ゲル成分含有水溶液を添加した後、該水溶液をゲル化させるのが好ましい。細胞接着性ゲル層は脱着を行なうのに十分な物理的強度を有していないため、細胞接着性ゲル層を形成させた容器（例えばディッシュ、シャーレ等）から細胞接着性ゲル層を剥離するのは困難だからである。また、極薄層の細胞接着性ゲル層は、アニオン性多糖類及び多価金属イオンを含むゲルを再簿接着性成分の溶液に浸漬すること（浸漬法）や塗布すること（塗布法）、またはキャストすること（キャスト法）で簡便に得ることができるが、本発明の細胞培養担体の作成においては、これらのいずれの方法を用いてもよい。このうちキャスト法が好ましく用いられる。例えばアルギン酸ゲル上にコラーゲンゲル層を形成する場合には、市販の0.3〜0.5％コラーゲン水溶液を必要により適当な濃度に希釈し、上記の方法で作成したアルギン酸カルシウムゲル上にこの溶液をキャストし、乾燥することで得られる。 When the cell adhesive gel layer was formed on the gel containing the anionic polysaccharide and the polyvalent metal ion, the gel containing the anionic polysaccharide and the polyvalent metal ion and the cell adhesive gel layer were prepared separately. Thereafter, the two may be overlapped, but it is preferable that the aqueous solution containing the cell adhesive gel component is added to the gel containing the anionic polysaccharide and the polyvalent metal ion, and then the aqueous solution is gelled. Since the cell-adhesive gel layer does not have sufficient physical strength for desorption, the cell-adhesive gel layer is peeled off from the container (for example, dish, petri dish, etc.) on which the cell-adhesive gel layer is formed. Because it is difficult. Moreover, the cell adhesive gel layer of an ultra-thin layer immerses the gel containing an anionic polysaccharide and a polyvalent metal ion in the solution of a re-adhesive adhesive component (immersion method), or applies (application method), Alternatively, it can be easily obtained by casting (casting method), but any of these methods may be used in preparing the cell culture carrier of the present invention. Of these, the casting method is preferably used. For example, when forming a collagen gel layer on an alginate gel, a commercially available 0.3 to 0.5% collagen aqueous solution is diluted to an appropriate concentration as necessary, and this solution is cast on the calcium alginate gel prepared by the above method, Obtained by drying.
本発明の細胞培養担体はアニオン性多糖類及び多価金属イオンを含むゲル層と細胞接着性ゲル層との間にキトサンゲルなどの高分子含水ゲル層を有さないことが好ましい。アニオン性多糖類及び多価金属イオンを含むゲル層と細胞接着性ゲル層との間に高分子含水ゲル層を有するとアニオン性多糖類及び多価金属イオンを含むゲル層の可溶化による細胞シートの剥離が悪くなり、剥離できなくなる場合がある。 The cell culture carrier of the present invention preferably does not have a polymer hydrous gel layer such as chitosan gel between the gel layer containing an anionic polysaccharide and polyvalent metal ions and the cell adhesive gel layer. Cell sheet by solubilization of gel layer containing anionic polysaccharide and polyvalent metal ion when polymer hydrous gel layer is provided between gel layer containing anionic polysaccharide and polyvalent metal ion and cell adhesive gel layer May become worse and may not be peeled off.
「高分子含水ゲル」とは親水性高分子を意味し、特に水には溶解しないが高分子中に水を含み系全体にわたって2次元的または３次元的な支持構造を有する吸水性高分子を意味する。例えば、「高分子含水ゲル」とは、アルギン酸ゲルは透過させないが、キレート化剤は透過させ得る膜を意味する。「高分子含水ゲル」は、アルギン酸ゲルは透過させないが、キレート化剤は透過させ得る膜である限り特に限定されず、合成高分子であっても、天然高分子、生体高分子であってもよい。さらに本発明に用いられる高分子含水ゲルは透明性を有するものが好ましい。「高分子含水ゲル」の例としては、アクリルアミドゲル、架橋アクリル酸ゲル、寒天、ゼラチン、コラーゲン、デキストラン、キトサン、シリカゲルなどがある。本発明では、キトサンを用いることが好ましい。なお、本発明において高分子含水ゲル上に被覆する被覆層の数（高分子含水ゲル層上の積層の層の数）は９以下が好ましく、６以下がさらに好ましい。 “Polymer-containing gel” means a hydrophilic polymer, especially a water-absorbing polymer that does not dissolve in water but contains water in the polymer and has a two-dimensional or three-dimensional support structure throughout the system. means. For example, “polymer hydrous gel” means a membrane that does not allow permeation of alginic acid gel but allows permeation of chelating agent. “Polymer hydrogel” is not particularly limited as long as it is a membrane that does not allow permeation of alginic acid gel but allows permeation of a chelating agent. It may be a synthetic polymer, a natural polymer, or a biopolymer. Good. Further, the polymer hydrogel used in the present invention preferably has transparency. Examples of “polymer hydrous gel” include acrylamide gel, cross-linked acrylic acid gel, agar, gelatin, collagen, dextran, chitosan, silica gel and the like. In the present invention, it is preferable to use chitosan. In the present invention, the number of coating layers coated on the polymer hydrogel (number of layers on the polymer hydrogel layer) is preferably 9 or less, and more preferably 6 or less.
本発明の細胞培養担体で培養し得る細胞の具体例としては、繊維芽細胞、血管内皮細胞、軟骨細胞、肝細胞、小腸上皮細胞、表皮角化細胞、骨芽細胞、骨髄間葉細胞等を例示でき、好ましいものとしては繊維芽細胞を例示できる。細胞の培養の際には、通常、細胞濃度１〜1.5万cells/mlの培養液（例えば、D-MEM培地、MEM培地、HamF12培地、HamF10培地）を細胞接着性ゲル層上に添加する。細胞の培養条件は、培養する細胞に従って適宜選択し得る。コラーゲンゲル層上で細胞を培養する場合には、通常、コラーゲンゲル層上にコンフルエントな単層の細胞層が形成されるまで行なう。 Specific examples of cells that can be cultured with the cell culture carrier of the present invention include fibroblasts, vascular endothelial cells, chondrocytes, hepatocytes, small intestinal epithelial cells, epidermal keratinocytes, osteoblasts, bone marrow mesenchymal cells and the like. As a preferable example, a fibroblast can be exemplified. When culturing cells, a culture solution (eg, D-MEM medium, MEM medium, HamF12 medium, HamF10 medium) having a cell concentration of 1 to 15,000 cells / ml is usually added onto the cell adhesive gel layer. Cell culture conditions can be appropriately selected according to the cells to be cultured. When culturing cells on a collagen gel layer, it is usually performed until a confluent monolayer cell layer is formed on the collagen gel layer.
本発明の細胞培養担体を用いた細胞の培養は具体的には次のようにして行なうことができる。細胞培養担体をシャーレ等の内部に設置し、シャーレ内に適当な培養液（例えば、D-MEM培地、MEM培地、HamF12培地、HamF10培地）を添加して5分浸漬後培地交換することを3回繰り返したのち12〜24時間放置し、培養液を細胞培養担体中に浸潤させる。シャーレ内の培養液を捨て、細胞培養担体の細胞接着性ゲル層上に細胞を播き、シャーレ内に適当な培養液（例えば、D-MEM培地、MEM培地、HamF12培地、HamF10培地）を添加する。37℃で１〜２時間放置し、細胞接着性ゲル層に保持（接着）させた後、37℃で培養を続ける。培養の際には、必要に応じて培養液を交換してもよい。通常は培養0.5〜２日ごとに培養液を交換する。 Specifically, the cell culture using the cell culture carrier of the present invention can be performed as follows. Place the cell culture carrier inside a petri dish, etc., add an appropriate culture medium (for example, D-MEM medium, MEM medium, HamF12 medium, HamF10 medium) in the petri dish, soak the medium for 5 minutes and replace the medium. After being repeated for 12 to 24 hours, the culture solution is infiltrated into the cell culture carrier. Discard the culture medium in the petri dish, seed the cells on the cell adhesion gel layer of the cell culture carrier, and add an appropriate culture medium (for example, D-MEM medium, MEM medium, HamF12 medium, HamF10 medium) to the petri dish. . After standing at 37 ° C. for 1 to 2 hours and holding (adhering) the cell adhesive gel layer, the culture is continued at 37 ° C. When culturing, the culture solution may be exchanged as necessary. Usually, the culture medium is changed every 0.5 to 2 days of culture.
本発明の細胞培養担体を用いた細胞の培養により得られる細胞培養物は、本発明の細胞培養担体と該細胞培養担体に保持された細胞層とを含む。「細胞培養担体に保持された細胞層」は細胞接着性ゲル層上に形成された細胞層である。 A cell culture obtained by culturing cells using the cell culture carrier of the present invention includes the cell culture carrier of the present invention and a cell layer held on the cell culture carrier. The “cell layer held on the cell culture carrier” is a cell layer formed on the cell adhesive gel layer.
細胞培養物は、アニオン性多糖類及び多価金属イオンを含むゲル層を可溶化処理することにより、培養された細胞層を含む細胞シートとして脱離させることができる。アニオン性多糖類及び多価金属イオンを含むゲル層の可溶化処理は、アニオン性多糖類及び多価金属イオンを含むゲルを構成するカチオン成分を除去することにより実施でき、カチオン種が多価金属イオンの場合はリン酸などの多価金属カチオンと錯形成もしくは難溶性塩を形成するイオンの添加、キレート剤水溶液の使用、倍地中の多価金属イオンの低減、倍地中の多価金属イオンのキレート剤による隠蔽によって実施できる。通常、細胞培養用の培地にはリン酸イオンが多く存在するため、予め多価金属イオンを除去した培地もしくは多価金属カチオンの総和モル数に対して90モル%以上のキレート剤を添加した培地に浸漬することが細胞への侵襲を低減する観点から好ましく用いられる。該キレート剤の濃度は90モル%以上10000モル%以下が好ましく、90モル%以上1000モル%以下がさらに好ましい。 The cell culture can be detached as a cell sheet containing the cultured cell layer by solubilizing the gel layer containing the anionic polysaccharide and the polyvalent metal ion. The solubilization treatment of the gel layer containing the anionic polysaccharide and the polyvalent metal ion can be carried out by removing the cation component constituting the gel containing the anionic polysaccharide and the polyvalent metal ion. In the case of ions, addition of ions that form complex or sparingly soluble salts with polyvalent metal cations such as phosphoric acid, use of chelating agent aqueous solution, reduction of polyvalent metal ions in the medium, polyvalent metal in the medium This can be done by concealing ions with a chelating agent. Usually, since there are many phosphate ions in the medium for cell culture, a medium in which polyvalent metal ions have been removed beforehand or a medium in which a chelating agent of 90 mol% or more is added to the total number of moles of polyvalent metal cations. It is preferably used in view of reducing invasion to cells. The concentration of the chelating agent is preferably from 90 mol% to 10,000 mol%, more preferably from 90 mol% to 1000 mol%.
本発明のキレート剤としては下記化合物が挙げられる。すなわちエチレンジアミンジオルトヒドロキシフェニル酢酸、ジアミノプロパン四酢酸、ニトリロ三酢酸、ヒドロキシエチルエチレンジアミン三酢酸、ジヒドロキシエチルグリシン、エチレンジアミン二酢酸、エチレンジアミン二プロピオン酸、イミノ二酢酸、ジエチレントリアミン五酢酸、ヒドロキシエチルイミノ二酢酸、1,3-ジアミノプロパノール四酢酸、トリエチレンテトラミン六酢酸、トランスシクロヘキサンジアミン四酢酸、エチレンジアミン四酢酸（edta）、グリコールエーテルジアミン四酢酸、O,O'-ビス(2-アミノエチル)エチレングリコール-N,N,N',N'-四酢酸(egta)、エチレンジアミンテトラキスメチレンホスホン酸、ジエチレントリアミンペンタメチレンホスホン酸、ニトリロトリメチレンホスホン酸、１-ヒドロキシエチリデン-1,1-ジホスホン酸、1,1-ジホスホノエタン-2-カルボン酸、2-ホスホノブタン-1,2,4-トリカルボン酸、1-ヒドロキシ-1-ホスホノプロパン-1,3,3-トリカルボン酸、カテコール-3,5-ジスルホン酸、ピロリン酸ナトリウム、テトラポリリン酸ナトリウム、ヘキサメタリン酸ナトリウムが挙げられ、特に好ましくは例えばジエチレントリアミン五酢酸、トリエチレンテトラミン六酢酸、1,3-ジアミノプロパノール四酢酸、グリコールエーテルジアミン四酢酸、ヒドロキシエチルエチレンジアミン三酢酸、2-ホスホノブタン-1,2,4-トリカルボン酸、1,1-ジホスホノエタン-2-カルボン酸、ニトリロトリメチレンホスホン酸、エチレンジアミンテトラホスホン酸、ジエチレントリアミンペンタホスホン酸、1-ヒドロキシプロピリデン−1,1-ジホスホン酸、1-アミノエチリデン-1,1-ジホスホン酸、1-ヒドロキシエチリデン-1,1-ジホスホン酸やこれらの塩がある。これらのうち好ましいものとしてはedta、egta、エチレンジアミンテトラホスホン酸、１-ヒドロキシエチリデン-1,1-ジホスホン酸がある。 Examples of the chelating agent of the present invention include the following compounds. In other words, ethylenediaminediorthydroxyphenylacetic acid, diaminopropanetetraacetic acid, nitrilotriacetic acid, hydroxyethylethylenediaminetriacetic acid, dihydroxyethylglycine, ethylenediaminediacetic acid, ethylenediaminedipropionic acid, iminodiacetic acid, diethylenetriaminepentaacetic acid, hydroxyethyliminodiacetic acid, 1,3-diaminopropanoltetraacetic acid, triethylenetetraminehexaacetic acid, transcyclohexanediaminetetraacetic acid, ethylenediaminetetraacetic acid (edta), glycol etherdiaminetetraacetic acid, O, O'-bis (2-aminoethyl) ethylene glycol-N , N, N ′, N′-tetraacetic acid (egta), ethylenediaminetetrakismethylenephosphonic acid, diethylenetriaminepentamethylenephosphonic acid, nitrilotrimethylenephosphonic acid, 1-hydroxyethylidene-1,1-di Phosphonic acid, 1,1-diphosphonoethane-2-carboxylic acid, 2-phosphonobutane-1,2,4-tricarboxylic acid, 1-hydroxy-1-phosphonopropane-1,3,3-tricarboxylic acid, catechol-3, 5-disulfonic acid, sodium pyrophosphate, sodium tetrapolyphosphate, sodium hexametaphosphate, and particularly preferably, for example, diethylenetriaminepentaacetic acid, triethylenetetraminehexaacetic acid, 1,3-diaminopropanoltetraacetic acid, glycol etherdiaminetetraacetic acid, Hydroxyethylethylenediaminetriacetic acid, 2-phosphonobutane-1,2,4-tricarboxylic acid, 1,1-diphosphonoethane-2-carboxylic acid, nitrilotrimethylenephosphonic acid, ethylenediaminetetraphosphonic acid, diethylenetriaminepentaphosphonic acid, 1-hydroxypropylidene -1,1-diphosphonic acid, 1-aminoethylidene-1,1-di Suhon acid, 1-hydroxyethylidene-1,1-diphosphonic acid and salts thereof. Among these, edta, egta, ethylenediaminetetraphosphonic acid, and 1-hydroxyethylidene-1,1-diphosphonic acid are preferable.
キレート化剤を用いたアニオン性多糖類及び多価金属イオンを含むゲル層の可溶化処理は、高分子含水ゲルからキレート化剤をしみこませて行なうのが好ましい。これによって、高分子含水ゲルとアニオン性多糖類及び多価金属イオンを含むゲル層とを容易に分離することができ、細胞培養物を高分子含水ゲルから容易に脱離させることができる。アニオン性多糖類及び多価金属イオンを含むゲル層の可溶化処理によってアニオン性多糖類及び多価金属イオンを含むゲル層を完全に除去する必要はなく、可溶化されなかったアニオン性多糖類及び多価金属イオンを含むゲル層が残っていてもよいが、アニオン性多糖類及び多価金属イオンを含むゲル層はできるだけ可溶化して除去するのが好ましい。 The solubilization treatment of the gel layer containing the anionic polysaccharide and the polyvalent metal ion using the chelating agent is preferably carried out by soaking the chelating agent from the water-containing polymer gel. Thus, the polymer hydrogel and the gel layer containing the anionic polysaccharide and the polyvalent metal ion can be easily separated, and the cell culture can be easily detached from the polymer hydrogel. It is not necessary to completely remove the gel layer containing the anionic polysaccharide and the polyvalent metal ion by the solubilization treatment of the gel layer containing the anionic polysaccharide and the polyvalent metal ion. Although a gel layer containing a polyvalent metal ion may remain, it is preferable to remove the gel layer containing an anionic polysaccharide and a polyvalent metal ion as much as possible.
本発明の細胞培養担体を用いた細胞の培養により得られる細胞培養物は、細胞層を含んでいるので、細胞層の重層化ならびに転写に使用できる。細胞層の重層化の際には、予め培養した細胞上に荷重をかけた状態で培養したのちアニオン性多糖類及び多価金属イオンを含むゲルを可溶化してもよいし、アニオン性多糖類及び多価金属イオンを含むゲル層を可溶化して得られる細胞シート同士を重層化してもよいし、アニオン性多糖類及び多価金属イオンを含むゲル層を可溶化して得られる細胞シートを別に作製した細胞層に重層化してもよい。重層化する細胞層の細胞の種類は、同一であっても異なっていてもよい。重層化する細胞層の数は特に限定されないが、通常１〜10、好ましくは１〜５、さらに好ましくは１〜３である。また、細胞層の転写の際には、別の細胞培養用基材上に荷重をかけた状態で培養したのちアニオン性多糖類及び多価金属イオンを含むゲルを可溶化してもよいし、アニオン性多糖類及び多価金属イオンを含むゲル層を可溶化して得られる細胞シートを他の媒体に転写してもよい。好ましい重層化ならびに転写方法としては予め培養した細胞上もしくは別の細胞培養基材上に荷重をかけた状態で培養したのちアニオン性多糖類及び多価金属イオンを含むゲルを溶解する方法である。 Since the cell culture obtained by culturing cells using the cell culture carrier of the present invention contains a cell layer, it can be used for layering and transcription of cell layers. When the cell layer is stratified, the gel containing anionic polysaccharide and polyvalent metal ion may be solubilized after culturing under a load on previously cultured cells, or the anionic polysaccharide may be solubilized. And cell sheets obtained by solubilizing gel layers containing polyvalent metal ions may be layered, or cell sheets obtained by solubilizing gel layers containing anionic polysaccharides and polyvalent metal ions. A cell layer prepared separately may be layered. The cell type of the cell layer to be stratified may be the same or different. The number of cell layers to be stratified is not particularly limited, but is usually 1 to 10, preferably 1 to 5, and more preferably 1 to 3. In addition, when the cell layer is transferred, the gel containing the anionic polysaccharide and the polyvalent metal ion may be solubilized after culturing under a load on another cell culture substrate, A cell sheet obtained by solubilizing a gel layer containing an anionic polysaccharide and polyvalent metal ions may be transferred to another medium. A preferred layering and transfer method is a method in which a gel containing an anionic polysaccharide and a polyvalent metal ion is dissolved after culturing the cells on a pre-cultured cell or another cell culture substrate under a load.
荷重をかけた細胞の培養法は細胞が転写される細胞もしくは基材にムラが生じない程度に充分荷重がかけられていればいかなる方法でもよい。ここで、荷重をかける際に細胞が密閉されると窒息をすることから、転写する側もしくは受ける側の少なくとも一方の細胞培養基材が水透過性のゲルや高分子含水ゲルもしくはこれらの組み合わせでできていることが好ましい。また、ムラ無く転写するには細胞面を充分に覆う状態で荷重をかける必要があるが、均一に接触することで酸素の拡散を妨害することとなるため、不織布（ナイロン、ポリエステル、ステンレスなど）等を介して酸素の拡散を妨げないで荷重することが好ましい。
荷重をかけた細胞の培養法の荷重は0.１g/cm2以上50g/ cm2以下であることが好ましく、0.5g/ cm2以上10g/ cm2以下であることがさらに好ましい。荷重をかけた細胞の培養の時間は充分な細胞の転写が実現できればいかようでもよいが4時間以上72時間以下が好ましく、6時間以上48時間以下がさらに好ましい。
Any method may be used for culturing a cell under load as long as the cell or substrate to which the cell is transferred is sufficiently loaded so as not to cause unevenness. Here, when cells are sealed when a load is applied, they suffocate, so at least one cell culture substrate on the transfer side or the receiving side is made of a water-permeable gel, a polymer hydrous gel, or a combination thereof. Preferably it is made. In addition, in order to transfer without unevenness, it is necessary to apply a load in a state where the cell surface is sufficiently covered. However, since uniform contact prevents oxygen diffusion, nonwoven fabric (nylon, polyester, stainless steel, etc.) It is preferable to apply the load without hindering the diffusion of oxygen through the like.
The load of the cell culture method under load is preferably 0.1 g / cm 2 or more and 50 g / cm 2 or less, more preferably 0.5 g / cm 2 or more and 10 g / cm 2 or less. The culture time of the loaded cells may be any time as long as sufficient cell transfer can be realized, but is preferably 4 hours or longer and 72 hours or shorter, more preferably 6 hours or longer and 48 hours or shorter.
本発明の細胞培養担体を調製する際にカルボジイミド類を含んだ調製液を用いて調製することにより密着性が改善できる。カルボジイミド類およびN-ヒドロキシコハクイミドはいかなる層の調製液に添加してもよいが、アニオン性多糖類及び多価金属イオンを含むゲル層調製液もしくは予め高分子含水ゲルに含浸させておくこと、あるいはアニオン性多糖類及び多価金属イオンを含むゲル層塗布後に塩化カルシウムと共溶解した液に浸漬することが好ましい。該カルボジイミド類は水溶性のものが好ましく、例えば1-エチル-3-(3-ジメチルアミノプロピル)-カルボジイミド塩酸塩などが挙げられる。該カルボジイミドの濃度としては0.1mg/l以上200g/l以下が好ましく、1mg/l以上100g/ml以下である。このときN-ヒドロキシコハクイミドを触媒として使用してもよく、濃度としては該カルボジイミドに対して1重量%以上50重量%以下が好ましい。 Adhesion can be improved by preparing a cell culture carrier of the present invention using a preparation solution containing carbodiimides. Carbodiimides and N-hydroxysuccinimide may be added to any layer preparation liquid, but impregnation in a gel layer preparation liquid containing anionic polysaccharide and polyvalent metal ions or a polymer hydrogel in advance. Or it is preferable to immerse in the liquid co-dissolved with calcium chloride after coating the gel layer containing anionic polysaccharide and polyvalent metal ion. The carbodiimide is preferably water-soluble, such as 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride. The concentration of the carbodiimide is preferably 0.1 mg / l or more and 200 g / l or less, and 1 mg / l or more and 100 g / ml or less. At this time, N-hydroxysuccinimide may be used as a catalyst, and the concentration is preferably 1% by weight or more and 50% by weight or less based on the carbodiimide.
本発明の細胞培養担体は、いかなる方法で滅菌されてもよいが、電子線、γ線、X線、紫外線などの放射線による滅菌が好ましく用いられ、電子線、γ線、紫外線がさらに好ましく用いられ、電子線滅菌が特に好ましい。本発明の電子線滅菌の照射線量としては0.1kGy以上65kGy以下が好ましく、1kGy以上40kGy以下が特に好ましい。EOG滅菌などの化学滅菌、高圧蒸気ガス滅菌などの高熱をかける滅菌は細胞接着性ゲル層やアニオン性多糖類及び多価金属イオンを含むゲルを分解するため好ましくない。このように滅菌した細胞培養担体は無菌条件下であれば長期間に渡って室温保管が可能である。
滅菌法は単独もしくは複数種の組み合わせで実施されても良く、同一種の滅菌法を繰り返し使用してもよい。
The cell culture carrier of the present invention may be sterilized by any method, but sterilization by radiation such as electron beam, γ-ray, X-ray, ultraviolet ray is preferably used, and electron beam, γ-ray, ultraviolet ray is more preferably used. Electron beam sterilization is particularly preferred. The irradiation dose for electron beam sterilization according to the present invention is preferably 0.1 kGy or more and 65 kGy or less, particularly preferably 1 kGy or more and 40 kGy or less. Sterilization by applying high heat such as chemical sterilization such as EOG sterilization or high-pressure steam gas sterilization is not preferable because it degrades the cell-adhesive gel layer or gel containing anionic polysaccharide and polyvalent metal ions. The cell culture carrier thus sterilized can be stored at room temperature for a long time under aseptic conditions.
The sterilization method may be carried out alone or in combination of a plurality of types, and the same type of sterilization method may be used repeatedly.
重層化する細胞層として、例えば、血管内皮細胞層、肝細胞層を使用すれば、肝臓の３次元組織構造物を構築できる。この３次元組織構造物は、in vitroにおける薬物の透過性試験へ適用できるとともに、動物実験代替モデルや移植用臓器へ応用できる。重層化した細胞層は、細胞層を構成する細胞の種類に応じた培養条件で培養することができる。培養の際には、例えば、D-MEM培地、MEM培地、HamF12培地、HamF10培地等の培地を使用できる。 If, for example, a vascular endothelial cell layer or a hepatocyte layer is used as the cell layer to be layered, a three-dimensional tissue structure of the liver can be constructed. This three-dimensional tissue structure can be applied to in vitro drug permeability tests, and can also be applied to animal experiment alternative models and transplanted organs. The layered cell layer can be cultured under culture conditions according to the type of cells constituting the cell layer. In the culture, for example, a medium such as D-MEM medium, MEM medium, HamF12 medium, HamF10 medium or the like can be used.
以下、本発明を実施例によりさらに具体的に説明するが、本発明の範囲は下記の実施例に限定されることはない。 Hereinafter, the present invention will be described more specifically with reference to examples. However, the scope of the present invention is not limited to the following examples.
〔実施例１〕細胞培養担体の作製
（１）含水キトサンゲル層の調製
キトサン（和光純薬製CT-100） 20ｇを1重量％の酢酸水溶液1000ｇに徐々に添加して40℃で３時間攪拌して溶解し、富士写真フイルム製ミクロフィルターFG-30でろ過した。
ろ過したキトサンの酢酸水溶液を、あらかじめ用意しておいたポリエチレンテレフタレートフイルム（フイルム厚200ミクロン）上にアプリケータでウェット膜厚300ミクロンとなるように塗布し、40℃にて3時間乾燥した。
乾燥したキトサンゲル層を、10重量％の水酸化ナトリウムのメタノール溶液に60分間浸漬し、引き続きPBS（Dulbecco's Phosphate buffered Saline）溶液に60分間浸漬した。その後、流水下で60分洗浄後、キトサンゲルを得た。こうして得た含水キトサンゲル層をイオン交換水中室温にて保存した。
[Example 1] Preparation of cell culture carrier (1) Preparation of hydrous chitosan gel layer 20 g of chitosan (CT-100 manufactured by Wako Pure Chemical Industries, Ltd.) was gradually added to 1000 g of a 1% by weight acetic acid aqueous solution and stirred at 40 ° C for 3 hours. Then, it was dissolved and filtered with a micro filter FG-30 manufactured by Fuji Photo Film.
The filtered aqueous solution of chitosan in acetic acid was applied to a polyethylene terephthalate film (film thickness 200 microns) prepared in advance so as to have a wet film thickness of 300 microns with an applicator and dried at 40 ° C. for 3 hours.
The dried chitosan gel layer was immersed in a methanol solution of 10% by weight sodium hydroxide for 60 minutes, and then immersed in a PBS (Dulbecco's Phosphate buffered Saline) solution for 60 minutes. Then, after washing for 60 minutes under running water, chitosan gel was obtained. The hydrous chitosan gel layer thus obtained was stored in ion-exchanged water at room temperature.
（２）アルギン酸カルシウム層の形成
含水キトサンゲル層/アルギン酸カルシウム層積層膜の調製
（１）で得た含水キトサンゲル膜を、0.1Mの塩化カルシウム水溶液に5分浸漬したのちスポンジローラーで水分を拭い、２重量%のアルギン酸ナトリウム水溶液を100ml/m2の厚さで塗布した。この塗布物を0.4Mの塩化カルシウムと10mg/lのWSCを含む水溶液に60分浸漬したのち、流水で30分洗浄することで含水キトサンゲル/アルギン酸カルシウム積層ａ−２を得た。アルギン酸ゲル層乾膜の厚さは電子顕微鏡断面像から計測すると1.0μmであった。
(2) Formation of calcium alginate layer Preparation of water-containing chitosan gel layer / calcium alginate layer laminated film The water-containing chitosan gel film obtained in (1) was immersed in 0.1M calcium chloride aqueous solution for 5 minutes and then wiped with a sponge roller. A 2% by weight aqueous sodium alginate solution was applied at a thickness of 100 ml / m 2 . This coated material was immersed in an aqueous solution containing 0.4 M calcium chloride and 10 mg / l WSC for 60 minutes, and then washed with running water for 30 minutes to obtain a hydrous chitosan gel / calcium alginate laminate a-2. The thickness of the alginic acid gel layer dry film was 1.0 μm as measured from an electron microscope cross-sectional image.
また、アルギン酸ナトリウム水溶液の塗布膜厚を50、250、500ml/ m2 に変更して同様のサンプルａ−１、ａ−３、ａ−４を作成した。 Further, similar samples a-1, a-3, and a-4 were prepared by changing the coating film thickness of the sodium alginate aqueous solution to 50, 250, and 500 ml / m 2 .
（３）コラーゲンゲル層修飾(極薄層コラーゲンゲル層修飾)
（２）で得た乾燥させていない含水キトサンゲル／アルギン酸カルシウム積層膜の上にCellmatrix I-C（新田ゼラチン製）の15倍希釈水溶液をキャストしたのち、乾燥することで極薄コラーゲンゲル層修飾膜を得た(上記ａ−１、ａ−２、ａ−３、及びａ−４に対してそれぞれＡ−１、Ａ−２、Ａ−３、及びＡ−４を得た)。Ａ−２において、コラーゲンゲル層およびアルギン酸ゲル層の厚さの合計は電子顕微鏡写真から1.2μmであり、アルギン酸ゲル層の厚さ1.0μmとの差からコラーゲン層の厚さは0.2μmであった。
また、キトサンの種類をＣＴ-500、ＣＴ-1000（いずれも和光純薬製）に変更しても同様のサンプルが得られた。
＜比較例用細胞培養担体の作製＞
比較例として、下記サンプルを作成した。
（４）高分子含水ゲルの代わりに、含水しない高分子シートを用いたサンプルＢ
上記（１）でキトサンの代わりにナイロンミクロフィルター使用した以外は、（１）と同様にサンプルＢを作成した。
（５）アルギン酸ゲル層を有しないサンプルＣ
上記（２）でアルギン酸を塗布しない以外は、（２）と同様にサンプルＣを作成した。
（６）コラーゲンゲル層とアルギン酸ゲル層との間にキトサン層が存在するサンプルＤ
（２）で得た試料（ａ−２）を上記のろ過したキトサンの酢酸水溶液に1時間浸漬、流水で1時間洗浄後３５℃、３０％ＲＨで乾燥し、上記（３）の工程に用いてサンプルＤを作製した。
(3) Collagen gel layer modification (Ultra-thin collagen gel layer modification)
After casting a 15-fold diluted aqueous solution of Cellmatrix IC (manufactured by Nitta Gelatin) on the non-dried hydrous chitosan gel / calcium alginate laminated film obtained in (2) and drying it, an ultrathin collagen gel layer modified film (A-1, A-2, A-3, and A-4 were obtained for a-1, a-2, a-3, and a-4, respectively). In A-2, the total thickness of the collagen gel layer and the alginate gel layer was 1.2 μm from the electron micrograph, and the thickness of the collagen layer was 0.2 μm from the difference from the thickness of the alginate gel layer of 1.0 μm. .
Similar samples were obtained even when the type of chitosan was changed to CT-500 or CT-1000 (both manufactured by Wako Pure Chemical Industries, Ltd.).
<Preparation of cell culture carrier for comparative example>
The following sample was created as a comparative example.
(4) Sample B using a polymer sheet that does not contain water instead of the polymer hydrous gel
Sample B was prepared in the same manner as (1) except that a nylon microfilter was used instead of chitosan in (1) above.
(5) Sample C not having an alginate gel layer
Sample C was prepared in the same manner as (2) except that alginic acid was not applied in (2) above.
(6) Sample D in which a chitosan layer is present between the collagen gel layer and the alginate gel layer
The sample (a-2) obtained in (2) was immersed in the above filtered chitosan acetic acid aqueous solution for 1 hour, washed with running water for 1 hour, dried at 35 ° C. and 30% RH, and used in the above step (3). Sample D was prepared.
〔実施例２〕滅菌
実施例１の膜をUV滅菌1、2、3時間、電子線滅菌20、40、60、80、100kGyの6種類の滅菌を施したところ、いずれも菌が確認されなかった。このとき、滅菌処理を施していないサンプルからは7200個/m2の菌が確認された。
[Example 2] Sterilization When the membrane of Example 1 was sterilized by UV sterilization for 1, 2, and 3 hours, electron beam sterilization 20, 40, 60, 80, and 100 kGy, none of the bacteria were confirmed. It was. At this time, 7200 cells / m 2 of bacteria were confirmed from the sample that had not been sterilized.
〔実施例３〕細胞培養担体を用いた細胞の培養
次のようにして細胞培養担体を用いた細胞の培養を行なった。
（１）使用細胞
・CHL（チャイニーズハムスター肺由来細胞）
（２）使用培地
・Eagle最小培地、10%牛胎児血清
（３）細胞培養担体
実施例１で作製した細胞培養担体をコラーゲンゲル層を上向にポリスチレン製細胞培養用シャーレの底面に置いたもの、およびポリスチレン製細胞培養用シャーレのみの比較例をUV滅菌もしくは電子線滅菌を施したのち、培地を添加して5分浸漬後培地交換することを3回繰り返したのち二晩放置し、培地を細胞培養担体中に浸潤させた。
（４）細胞の播種
予め培養しておいた細胞をトリプシン処理で回収し、細胞濃度を40000cell/mlに調製した。セル及びシャーレ内の培地を捨てた後、この細胞液を細胞数8000cell/cm2となるようにシャーレ内に播種し培地を添加した。
（５）培養
CO2インキュベーターを用いて37℃で３日間培養した。
（６）結果
本発明の実施例１で作製した細胞培養担体をポリスチレン製細胞培養用シャーレの底面に置いたものは、透明性を有することから細胞培養中の細胞の生育状態が詳細に観察でき、細胞接着性や毒性に問題はなく、ポリスチレン製細胞培養用シャーレのみのサンプルとほぼ同様の状態であった。
また、本発明の細胞培養担体のサンプルにおいては、細胞培養中に高分子含水ゲルからの細胞シートの剥離はなく、問題はなかった。一方、比較例のサンプルＢでは、ナイロンミクロフィルターが不透明であることから細胞培養中の生きた細胞の育成状態が観察できなかった。
細胞の種類をＨＥＰＧ２（ヒト肝癌由来細胞）やＢＡＥ（ウシ大動脈血管内皮細胞）に変更しても同様の結果が得られた。
Example 3 Cell Culture Using Cell Culture Carrier Cells were cultured using the cell culture carrier as follows.
(1) Cells used • CHL (Chinese hamster lung-derived cells)
(2) Medium used: Eagle's minimum medium, 10% fetal bovine serum (3) Cell culture carrier The cell culture carrier prepared in Example 1 was placed on the bottom of a polystyrene cell culture dish with the collagen gel layer facing upward , And a polystyrene cell culture petri dish comparative example only after UV sterilization or electron beam sterilization, after adding the medium and immersing it for 5 minutes, changing the medium three times, and then leaving it for two nights. Infiltrated into cell culture carrier.
(4) Cell seeding Cells previously cultured were collected by trypsin treatment, and the cell concentration was adjusted to 40,000 cells / ml. After discarding the cell and the medium in the petri dish, this cell solution was seeded in the petri dish so that the cell count was 8000 cells / cm 2 and the medium was added.
(5) Culture
The cells were cultured at 37 ° C. for 3 days using a CO 2 incubator.
(6) Results The cell culture carrier prepared in Example 1 of the present invention placed on the bottom of a polystyrene cell culture petri dish has transparency, so that the growth state of cells during cell culture can be observed in detail. There was no problem in cell adhesion and toxicity, and it was almost the same state as the sample of the petri dish for cell culture made of polystyrene alone.
In the cell culture carrier sample of the present invention, there was no problem because the cell sheet was not detached from the polymer hydrogel during cell culture. On the other hand, in the sample B of the comparative example, since the nylon microfilter was opaque, the growing state of the living cells during the cell culture could not be observed.
Similar results were obtained even when the cell type was changed to HEPG2 (human hepatoma-derived cells) or BAE (bovine aortic vascular endothelial cells).
〔実施例４〕細胞シートの剥離
実施例３で培養したサンプルを下記３種類の剥離液および非剥離液に37℃で10分間浸漬したのちの細胞培養担体からの細胞シートの剥離状況を観察した。次いで、剥離した細胞シートをポリスチレン製細胞培養用シャーレ上に置き培地を添加したのち、CO2インキュベーターを用いて37℃で３日間培養し、光学顕微鏡で観察した。結果を表１に示す。本発明の領域で良好な剥離性を示した。また、細胞の生存率も高かった。
（１）剥離液
(a)10×Dulbecco's Phosphate buffered Salineを蒸留水で10倍希釈したもの
(b)Eagle最小培地にカルシウムイオン、マグネシウムイオンの総モル数に対して100モル%の１-ヒドロキシエチリデン-1,1-ジホスホン酸を添加したもの
（２）非剥離液
(c)Eagle最小培地
[Example 4] Peeling of cell sheet The sample cultured in Example 3 was immersed in the following three types of stripping solution and non-peeling solution at 37 ° C for 10 minutes, and the state of peeling of the cell sheet from the cell culture carrier was observed. . Next, the peeled cell sheet was placed on a polystyrene cell culture petri dish, a medium was added, and the cells were cultured at 37 ° C. for 3 days using a CO 2 incubator and observed with an optical microscope. The results are shown in Table 1. Good peelability was exhibited in the region of the present invention. The cell viability was also high.
(1) Stripping solution
(a) 10 × Dulbecco's Phosphate buffered Saline diluted 10 times with distilled water
(b) Eagle's minimum medium with 100 mol% of 1-hydroxyethylidene-1,1-diphosphonic acid added to the total number of moles of calcium and magnesium ions (2) Non-stripping solution
(c) Eagle's minimum medium
〔実施例５〕細胞層の転写
実施例３で細胞培養担体上に培養したサンプルを担体ごと取り出し、ポリスチレン製細胞培養用シャーレに細胞面が接するように置き、CO2インキュベーターを用いて37℃で1日間培養した。このように培養したものを、実施例４の剥離液に10分間浸漬したのち、液を培地に入れ替え、高分子含水ゲル層をピンセットで引っ張り剥離した。その後、培地を入れ替えCO2インキュベーターを用いてさらに37℃で1日間培養した。この細胞をトリパンブルーで染色したのち、光学顕微鏡で観察した。
結果を表２に示す。
本発明の細胞培養担体で良好な結果を得た。なお、サンプルＣは高分子含水ゲル層が剥離せず転写できなかった。また、剥離液(c)については、高分子含水ゲル層が剥離せず、転写できなかった。
[Example 5] Transfer of cell layer The sample cultured on the cell culture carrier in Example 3 was taken out together with the carrier, placed so that the cell surface was in contact with a petri dish for cell culture made of polystyrene, and at 37 ° C using a CO 2 incubator. Cultured for 1 day. What was cultured in this way was immersed in the stripping solution of Example 4 for 10 minutes, then the solution was replaced with a culture medium, and the polymer hydrogel layer was pulled and peeled off with tweezers. Thereafter, the medium was changed, and the cells were further cultured at 37 ° C. for 1 day using a CO 2 incubator. The cells were stained with trypan blue and then observed with an optical microscope.
The results are shown in Table 2.
Good results were obtained with the cell culture carrier of the present invention. In Sample C, the polymer hydrous gel layer did not peel off and could not be transferred. Further, with respect to the stripping solution (c), the polymer hydrous gel layer was not stripped and could not be transferred.
〔実施例６〕細胞層の重層化
UV滅菌を3時間施したポリスチレン製細胞培養用シャーレに実施例３と同様に以下の細胞を培養した。
・CHL（Chinese Hamster Lung Cell）
・BRL（Buffalo Rat Liver 3A, ATCC No. : CRL 1442）
・BAE（Bovine Aortic Endothelial Cell）
シャーレ上に培養した上記各３種の細胞上に、実施例４の剥離液(b)を用いて実施例５と同様に細胞の転写を行い、重層化細胞を得た。この重層化細胞を倍地中４日間培養したのち、トリパンブルーで染色して光学顕微鏡で状況を確認したところ、いずれの重層化細胞も良好に培養されていることがわかった。
[Example 6] Layering of cell layers
The following cells were cultured in the same manner as in Example 3 in a petri dish for cell culture made of polystyrene that had been subjected to UV sterilization for 3 hours.
・ CHL (Chinese Hamster Lung Cell)
・ BRL (Buffalo Rat Liver 3A, ATCC No.: CRL 1442)
・ BAE (Bovine Aortic Endothelial Cell)
On each of the three types of cells cultured on the petri dish, the cells were transferred in the same manner as in Example 5 using the stripping solution (b) of Example 4 to obtain stratified cells. After culturing these stratified cells in a medium for 4 days, staining with trypan blue and confirming the situation with an optical microscope revealed that all the stratified cells were well cultured.
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