JP2006223106A - Cell culture carrier - Google Patents
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- JP2006223106A JP2006223106A JP2005037217A JP2005037217A JP2006223106A JP 2006223106 A JP2006223106 A JP 2006223106A JP 2005037217 A JP2005037217 A JP 2005037217A JP 2005037217 A JP2005037217 A JP 2005037217A JP 2006223106 A JP2006223106 A JP 2006223106A
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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、該細胞培養物を利用した細胞層の重層化方法、該重層化方法により得られる重層化した細胞層に関する。 The present invention relates to a cell culture technique, specifically, a cell culture carrier, a cell culture method using the cell culture carrier, a cell culture obtained by the culture method, and a cell layer using the cell culture And a stratified cell layer 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ポリN-イソプロピルアクリルアミド（以下「PNIPAM」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なうに当たり、37℃の温度条件下、ゲル化したPNIPAM上で培養した細胞をPNIPAM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PNIPAM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（特許文献１：特公平6−104061号公報；非特許文献１：清水達也、岡野光夫、バイオサイエンスとバイオインダストリー、58(12)、851頁(2000)；非特許文献２：大和雅之、岡野光夫、臨外、56(1)、53頁（2001）；非特許文献３：大和雅之、岡野光夫、Materials Integration、13(2)、58頁(2000)；非特許文献４：大和雅之、串田愛、岡野光夫、蛋白質・核酸・酵素、45(10)、72頁(2000)；非特許文献５：大和雅之、廣瀬志弘、播元正美、岡野光夫、蛋白質・核酸・酵素、45(13)、162頁(2000)）。 Poly-N-isopropylacrylamide (hereinafter referred to as “PNIPAM”), which is a temperature-responsive polymer among polymer hydrogels, swells and is liquid at low temperatures, but rapidly undergoes phase transition and shrinks rapidly at around 34 ° C. Turn into. Conventionally, in stratification of cultured cells, cells cultured on gelled PNIPAM under a temperature condition of 37 ° C are stacked on another cell layer together with PNIPAM, and then lowered to 34 ° C or lower to liquefy PNIPAM. And the method of directly superimposing cells was taken (Patent Document 1: Japanese Patent Publication No. 6-1004061; Non-Patent Document 1: Tatsuya Shimizu, Mitsuo Okano, Bioscience and Bioindustry, 58 (12), 851 (2000); Non-Patent Document 2: Masayuki Yamato, Mitsuo Okano, Rin, 56 (1), 53 (2001); Non-Patent Document 3: Masayuki Yamato, Mitsuo Okano, Materials Integration, 13 (2), 58 (2000); Non-Patent Literature 4: Masayuki Yamato, Ai Kushida, Mitsuo Okano, Protein, Nucleic Acid, Enzyme, 45 (10), 72 (2000); Non-Patent Literature 5: Masayuki Yamato, Shihiro Hirose, Hari Masami Moto, Mitsuo Okano, Protein / Nucleic Acid / Enzyme, 45 (13), 162 (2000)).
PNIPAM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PNIPAM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、PNIPAM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PNIPAM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PNIPAM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。
上記の手法の問題点の解決のため、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、及び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の確立につき検討が続けられていた。
When cell culture is performed on PNIPAM, cells usually grow in a single layer. 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 the cell and the PNIPAM that is the basal layer are not adhered to other cells, and an ECM necessary for cell adhesion is not formed.
Therefore, the cell layers of monolayers cultured on PNIPAM were overlaid, and the cell layer was solubilized and removed under conditions of 34 ° C or lower, and the cells were stacked so that the cells were 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 PNIPAM acts as a cytotoxin and inhibits the normal growth of cells, it was an extremely unsuitable and unstable technique for cell stratification.
In order to solve the problems of the above methods, cell culture in a medium in which extracellular matrix components are layered on various gels and gelled, and a medium that can easily remove unnecessary gels after layering are used. The study on the establishment of the culture system was continued.
一方、PNIPAMの温度応答性を応用した例としては、特許文献２（特開平11-349643号公報）にはゼラチンにPNIPAMをグラフト重合させた高分子化合物を用いて製造される細胞培養基材、及び特許文献３（特開平6-157689号公報）にはPNIPAMを主鎖とし水に不溶性の側鎖を有するグラフトコポリマーを主成分とするゲル膜が開示されているが、PNIPAM 自体を含みかつPNIPAMの液状化が制御された膜については、知られていなかった。
本発明は、安定的かつ容易に細胞を重層する技術、及び細胞層を重層した場合に上層部の細胞と下層部の細胞とを充分に接着することができる技術を提供し、これにより、皮膚などの一部組織を除きin vitro条件下では困難とされてきた細胞の階層化技術に道を開くことを目的とする。
具体的には、本発明は、培養した細胞層を短時間で剥離することができかつ細胞の正常な成長を阻害しない細胞培養担体を提供することを目的とする。
The present invention provides a technique for stably and easily overlaying cells, and a technique capable of sufficiently adhering cells in the upper layer portion and cells in the lower layer portion when the cell layers are overlaid. The aim is to open the way to cell stratification technology that has been considered difficult under in vitro conditions except for some tissues.
Specifically, an object of the present invention is to provide a cell culture carrier that can peel a cultured cell layer in a short time and does not inhibit normal cell growth.
本発明者らは上記の課題を解決すべく鋭意研究を行い、温度応答性高分子及び水膨潤性高分子を含むゲル層を有する細胞培養担体が、細胞の成長を阻害することなく、安定的かつ容易な細胞層の剥離及び細胞層の重層化を可能にすることを見出した。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 the cell culture carrier having a gel layer containing a temperature-responsive polymer and a water-swellable polymer is stable without inhibiting cell growth. It was also found that cell layer peeling and cell layer stratification can be easily performed. The present invention has been completed based on these findings.
すなわち本発明は、温度応答性高分子及び水膨潤性高分子を含むゲルを含む細胞培養担体を提供する。本発明の好ましい態様によれば、２個以上の層からなる細胞培養担体であって、温度応答性高分子及び水膨潤性高分子を含むゲル層並びに該ゲル層に隣接する細胞接着性ゲル層を含む細胞培養担体及び細胞培養面最表面が細胞接着性ゲル層である該細胞培養担体が提供される。 That is, the present invention provides a cell culture carrier comprising a gel containing a temperature-responsive polymer and a water-swellable polymer. According to a preferred embodiment of the present invention, a cell culture carrier comprising two or more layers, a gel layer containing a temperature-responsive polymer and a water-swellable polymer, and a cell adhesive gel layer adjacent to the gel layer And a cell culture carrier in which the outermost surface of the cell culture surface is a cell adhesive gel layer.
本発明のさらに好ましい態様によれば、上記いずれかの細胞培養担体において、水膨潤性高分子がポリウレタンである細胞培養担体；上記いずれかの細胞培養担体において、温度応答性高分子の曇点が２５℃以上でありかつ３５℃以下である細胞培養担体；上記いずれかの細胞培養担体において、温度応答性高分子がポリ‐N‐イソプロピルアクリルアミドである細胞培養担体；上記いずれかの細胞培養担体において、細胞接着性ゲルがコラーゲンを含むゲルである細胞培養担体；上記いずれかの細胞培養担体において、温度応答性高分子及び水膨潤性高分子を含むゲル層の一方の面に物理的な補強治具が設けられた細胞培養担体；及び、上記いずれかの細胞培養担体において、該細胞培養担体が電子線、γ線、紫外線のいずれか又は複数を照射することで滅菌された細胞培養担体が提供される。 According to a further preferred aspect of the present invention, in any one of the above cell culture carriers, the cell culture carrier in which the water-swellable polymer is polyurethane; in any of the above cell culture carriers, the cloud point of the temperature-responsive polymer is A cell culture carrier of 25 ° C. or higher and 35 ° C. or lower; in any of the above cell culture carriers; a cell culture carrier in which the temperature-responsive polymer is poly-N-isopropylacrylamide; in any of the above cell culture carriers A cell culture carrier in which the cell adhesion gel is a gel containing collagen; in any one of the above cell culture carriers, physical reinforcement treatment is performed on one surface of the gel layer containing a temperature-responsive polymer and a water-swellable polymer. A cell culture carrier provided with a tool; and any one of the above cell culture carriers, wherein the cell culture carrier is irradiated with one or more of an electron beam, a γ-ray, and an ultraviolet ray. Sterile cell culture support by is provided.
本発明の別の観点からは、上記いずれかの細胞培養担体を用いて細胞を培養する工程を含む細胞の培養方法；上記いずれかの細胞培養担体を用いて培養された細胞培養物が提供される。
本発明のさらに別の観点からは、上記いずれかの細胞培養担体を用いて培養された細胞培養物における温度応答性高分子及び水膨潤性高分子を含むゲル層の膨潤化処理により細胞シートを得る工程を含む細胞培養方法；膨潤化処理を、細胞培養物を温度応答性高分子の曇点以下の温度条件下にすることにより行う該細胞培養方法；上記いずれかの細胞培養方法で得られる細胞シート；上記いずれかの細胞培養担体を用いて培養された細胞培養物を他の細胞培養担体上でさらに培養する工程を含む細胞転写法；該細胞転写法により得られる細胞培養物における温度応答性高分子及び水膨潤性高分子を含むゲル層の膨潤化処理により得られる細胞培養物又は細胞シート；膨潤化処理を、細胞培養物を温度応答性高分子の曇点以下の温度条件下にすることにより行う該細胞培養物又は細胞シート；上記いずれかの細胞培養担体を用いて培養された細胞培養物を他の培養細胞上に培養する工程を含む細胞重層化法；及び、該細胞重層化法により得られる細胞培養物における温度応答性高分子及び水膨潤性高分子を含むゲル層の膨潤化処理により得られる重層化細胞培養物又は重層化細胞シートが提供される。
From another aspect of the present invention, there is provided a cell culture method comprising a step of culturing cells using any of the above cell culture carriers; a cell culture cultured using any of the above cell culture carriers. The
From still another aspect of the present invention, a cell sheet is obtained by swelling treatment of a gel layer containing a temperature-responsive polymer and a water-swellable polymer in a cell culture cultured using any one of the above cell culture carriers. A cell culture method comprising a step of obtaining the cell culture method, wherein the swelling treatment is carried out by subjecting the cell culture to a temperature condition below the cloud point of the temperature-responsive polymer; obtained by any of the cell culture methods described above A cell sheet; a cell transfer method comprising a step of further culturing a cell culture cultured on any one of the above cell culture carriers on another cell culture carrier; a temperature response in the cell culture obtained by the cell transfer method Cell culture or cell sheet obtained by swelling treatment of a gel layer containing a water-soluble polymer and a water-swellable polymer; the swelling treatment is performed under temperature conditions below the cloud point of the temperature-responsive polymer. You A cell stratification method comprising a step of culturing a cell culture cultivated using any of the above cell culture carriers on other cultured cells; and the cell stratification. Provided is a stratified cell culture or a stratified cell sheet obtained by swelling treatment of a gel layer containing a temperature-responsive polymer and a water-swellable polymer in a cell culture obtained by the method.
本発明により、細胞の成長を阻害せず安定的かつ容易に細胞層の剥離が可能であり、かつ細胞層の重層化ができる細胞培養担体が提供される。 According to the present invention, there is provided a cell culture carrier capable of detaching a cell layer stably and easily without inhibiting cell growth and capable of layering cell layers.
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。本発明の細胞培養担体の形状は特に限定されないが、シート状であるのが好ましい。
本明細書において、温度応答性高分子とは温度に従って液状からゲル状に状態が変化するものをいう。本発明において、温度応答性高分子として、昇温すると水に難溶化するポリマーを用いるのが好ましく、温度応答性高分子が水に難溶化する転換点（以下、曇点という）は２５℃〜３５℃であることが好ましい。本発明で用いられる温度応答性高分子の例としては、ポリ−N-イソプロピルアクリルアミド（PNIPAM）及びポリ−N,N-ジエチルアクリルアミドなどの各種ポリアクリルアミド誘導体が挙げられるが、これらに限定されない。温度応答性高分子として好ましくはポリ−N-イソプロピルアクリルアミド及びポリ−N,N-ジエチルアクリルアミド、より好ましくはポリ−N-イソプロピルアクリルアミドが用いられる。
The cell culture carrier means a carrier or support that can be used for culturing cells. The shape of the cell culture carrier of the present invention is not particularly limited, but is preferably a sheet shape.
In this specification, a temperature-responsive polymer refers to a polymer whose state changes from liquid to gel according to temperature. In the present invention, it is preferable to use, as the temperature-responsive polymer, a polymer that is hardly soluble in water when the temperature is raised, and the transition point (hereinafter referred to as cloud point) at which the temperature-responsive polymer is hardly soluble in water is 25 ° C. to It is preferable that it is 35 degreeC. Examples of the temperature-responsive polymer used in the present invention include, but are not limited to, various polyacrylamide derivatives such as poly-N-isopropylacrylamide (PNIPAM) and poly-N, N-diethylacrylamide. As the temperature-responsive polymer, poly-N-isopropylacrylamide and poly-N, N-diethylacrylamide, and more preferably poly-N-isopropylacrylamide are used.
水膨潤性高分子としては、ゲル状になって網目構造を形成し、温度応答性高分子を保持することができ、かつ、水膨潤性が高いものが用いられる。さらに、本発明において、水膨潤性高分子は剥離を容易にするために細胞接着性ゲルとの親和性が低いものが好ましい。
水膨潤性高分子の例としては、ポリウレタン系のポリマーを挙げることができる。ポリウレタン系のポリマーとしては、ポリオール又はオキシド及びジイソシアネートの重付加反応で得られるポリオールポリウレタンウレアが特に好ましい。
ポリオールとしては、例えば、エチレングリコール、ジエチレングリコール、トリエチレングリコール、プロピレングリコール、１，４−ブタンジオール、１，２−シクロヘキサンジオール、１，２，４−ブタントリオール、ネオペンチルグリコール、グリセリン、トリメチロールプロパン、ペンタエリスリトール、トリエタノールアミン、ビスフェノールＡ、シリコンジオール、又はフッ素ジオールなどが挙げられ、オキシドとしては、例えばエチレンオキシド、プロピレンオキシド、ブチレンオキシド、若しくはスチレンオキシド、又はこれら何れかのポリオキシドが挙げられる。ポリオール又はポリオキシドとしては、１種又は２種以上を用いればよく、特にポリエチレンオキシド（分子量10万〜100万）を用いるのが好ましい。
As the water-swellable polymer, a polymer that is gel-like and forms a network structure, can hold the temperature-responsive polymer, and has high water-swellability is used. Furthermore, in the present invention, the water-swellable polymer is preferably one having a low affinity with the cell adhesive gel in order to facilitate peeling.
Examples of water-swellable polymers include polyurethane polymers. As the polyurethane polymer, a polyol polyurethane urea obtained by a polyaddition reaction of polyol or oxide and diisocyanate is particularly preferable.
Examples of the polyol include ethylene glycol, diethylene glycol, triethylene glycol, propylene glycol, 1,4-butanediol, 1,2-cyclohexanediol, 1,2,4-butanetriol, neopentyl glycol, glycerin, trimethylolpropane. , Pentaerythritol, triethanolamine, bisphenol A, silicon diol, or fluorine diol, and the oxide includes, for example, ethylene oxide, propylene oxide, butylene oxide, styrene oxide, or any of these polyoxides. As the polyol or polyoxide, one kind or two or more kinds may be used, and it is particularly preferable to use polyethylene oxide (molecular weight: 100,000 to 1,000,000).
ジイソシアネートとしては、例えば、トリレンジイソシアネート、ジフェニルメタン−４,４'−ジイソシアネート、キシリレンジイソシアネート、ナフタレン−１，５−ジイソシアネート、ｐ−フェニレンジイソシアネート、ｍ−もしくはｐ−テトラメチルキシリレンジイソシアネート、又はトリフェニルメタントリイソシアネートなどの芳香族ジイソシアネート若しくは芳香族トリイソシアネート、水添トリレンジイソシアネート、水添ジフェニルメタン−４,４'−ジイソシアネート、１,４−テトラメチレンジイソシアネート、１,６−ヘキサメチレンジイソシアネート、水添キシリレンジイソシアネート、シクロヘキシル−１，４−ジイソシアネート、又はイソホロンジイソシアネートなどの脂肪族もしくは脂環式ジイソシアネートが挙げられ、また、これらの各種ジイソシアネートモノマーから誘導される２官能のポリイソシヌレネート型ジイソシアネート、アダクト型ジイソシアネート又はビューレット型ジイソシアネートなどのイソシアネートプレポリマーが挙げられる。ジイソシアネートとしては、１種又は２種以上を用いればよく、特にトリレンジイソシアネート、ジフェニルメタン−４,４'−ジイソシアネート、又はナフタレン−１，５−ジイソシアネートが好ましい。 Examples of the diisocyanate include tolylene diisocyanate, diphenylmethane-4,4′-diisocyanate, xylylene diisocyanate, naphthalene-1,5-diisocyanate, p-phenylene diisocyanate, m- or p-tetramethylxylylene diisocyanate, or triphenyl. Aromatic diisocyanates such as methane triisocyanate or aromatic triisocyanate, hydrogenated tolylene diisocyanate, hydrogenated diphenylmethane-4,4′-diisocyanate, 1,4-tetramethylene diisocyanate, 1,6-hexamethylene diisocyanate, hydrogenated xylylene Examples include aliphatic or alicyclic diisocyanates such as range isocyanate, cyclohexyl-1,4-diisocyanate, or isophorone diisocyanate. Is also, polyisobutylene sheet wetting sulfonate type diisocyanate bifunctional derived from these various diisocyanate monomers include isocyanate prepolymers such as adduct-type diisocyanate or biuret type diisocyanate. As the diisocyanate, one or more kinds may be used, and tolylene diisocyanate, diphenylmethane-4,4′-diisocyanate, or naphthalene-1,5-diisocyanate is particularly preferable.
本発明において水膨潤性高分子として用いられるポリオールポリウレタンウレアは特に限定されないが、ポリエチレンオキシド及び芳香族ジイソシアネートの反応で得られるポリオールポリウレタンウレアが特に好ましい。
いかなる特定の理論に拘泥するわけではないが、例えばポリオールポリウレタンウレアはゲル状になって、ジイソシアネート由来部位は分子間水素結合で結ばれたナノ結晶構造のドメインを形成して分子相互間のマクロな流動を防ぐ物理的架橋の役割を果たし温度応答性高分子を保持し、さらに、ポリアルコール由来部位（ポリエーテル鎖）はそれ自体が屈曲性及び伸縮性に富むため保持した温度応答性高分子の状態の変化と相まってゲル全体の伸縮性や親水性を温度によって変化させることができるため培養細胞層の剥離を制御できるものと考えられる。
ポリオールポリウレタンウレアの分子量は好ましくは１万〜５００万であり、より好ましくは５万〜１００万である。市販品として入手可能なポリオールポリウレタンウレアとしては、「ダイアロマー」（商品名、大日精化工業製）などが挙げられる。
The polyol polyurethane urea used as the water-swellable polymer in the present invention is not particularly limited, but a polyol polyurethane urea obtained by a reaction of polyethylene oxide and aromatic diisocyanate is particularly preferable.
Without being bound by any particular theory, for example, polyol polyurethane urea is in the form of a gel and the diisocyanate-derived sites form nanocrystalline domain domains linked by intermolecular hydrogen bonds to form macroscopic molecules between molecules. It plays the role of physical cross-linking to prevent flow and retains the temperature-responsive polymer. Furthermore, the polyalcohol-derived part (polyether chain) itself is highly flexible and stretchable, so it retains the temperature-responsive polymer. It is considered that exfoliation of the cultured cell layer can be controlled because the stretchability and hydrophilicity of the entire gel can be changed by temperature in combination with the change in state.
The molecular weight of the polyol polyurethane urea is preferably 10,000 to 5,000,000, more preferably 50,000 to 1,000,000. Examples of the polyol polyurethane urea available as a commercial product include “Diaromar” (trade name, manufactured by Dainichi Seika Kogyo).
温度応答性高分子及び水膨潤性高分子を含むゲルは、温度応答性高分子及び水膨潤性高分子をそれぞれメタノール又はトルエン等の有機溶媒に溶解した後、２つの溶液を混合して、さらに混合物を乾燥させて得ることができる。温度応答性高分子の含有量は、温度応答性高分子及び水膨潤性高分子の合計質量に対し、好ましくは１０質量％から９０質量％、より好ましくは３０質量％から７０質量％である。温度応答性高分子及び水膨潤性高分子の合計質量に対する温度応答性高分子の含有量が１０％より少ないと、細胞培養中に温度応答性高分子及び水膨潤性高分子を含むゲル層から細胞シートが剥離してしまいやすく、９０％より多いとゲル層のウェット強度が下がり培地中で層が壊れてしまう可能性がある。 The gel containing the temperature-responsive polymer and the water-swellable polymer is prepared by dissolving the temperature-responsive polymer and the water-swellable polymer in an organic solvent such as methanol or toluene, and then mixing the two solutions. The mixture can be obtained by drying. The content of the temperature-responsive polymer is preferably 10% by mass to 90% by mass, and more preferably 30% by mass to 70% by mass with respect to the total mass of the temperature-responsive polymer and the water-swellable polymer. When the content of the temperature-responsive polymer with respect to the total mass of the temperature-responsive polymer and the water-swellable polymer is less than 10%, the gel layer containing the temperature-responsive polymer and the water-swellable polymer is used during cell culture. The cell sheet tends to peel off, and if it exceeds 90%, the wet strength of the gel layer is lowered and the layer may be broken in the medium.
本発明の細胞培養担体において温度応答性高分子及び水膨潤性高分子を含むゲル層の厚さは好ましくは０．０１μm以上１００μm以下、より好ましくは０．５μm以上１０μm以下である。
本明細書において層の厚さは、特に言及のない限り充分に乾燥した状態で計測したものを示す。本明細書においては、この層の厚さを「乾燥膜厚」ということもある。層の厚さの計測は、電子顕微鏡断面像、マイクロメータ膜厚計、エリプソメーター、角度可変XPS、光干渉式膜厚計などを用いて行うことができ、好ましくはマイクロメータ膜厚計、電子顕微鏡断面像、光干渉式膜厚計を用いて行うことができる。
The thickness of the gel layer containing the temperature-responsive polymer and the water-swellable polymer in the cell culture carrier of the present invention is preferably 0.01 μm to 100 μm, more preferably 0.5 μm to 10 μm.
In the present specification, the layer thickness indicates a value measured in a sufficiently dry state unless otherwise specified. In this specification, the thickness of this layer is sometimes referred to as “dry film thickness”. The thickness of the layer can be measured using an electron microscope cross-sectional image, a micrometer film thickness meter, an ellipsometer, an angle variable XPS, a light interference film thickness meter, etc., preferably a micrometer film thickness meter, an electron It can be performed using a microscope cross-sectional image and a light interference film thickness meter.
本発明の細胞培養担体における温度応答性高分子及び水膨潤性高分子を含むゲル層は、一般的に知られている種々の高分子ゲル膜の作製方法を用いて作製することができる。例えば、温度応答性高分子及び水膨潤性高分子を含む溶液をキャストする方法（キャスト法）やバーコーターで塗布する方法（バーコート法）、ギャップコーターで塗布する方法（ギャップコート法）などが挙げられる。これらのうちバーコート塗布法、ギャップコート塗布法が好ましい。 The gel layer containing the temperature-responsive polymer and the water-swellable polymer in the cell culture carrier of the present invention can be prepared using various generally known methods for preparing polymer gel films. For example, a method of casting a solution containing a temperature-responsive polymer and a water-swellable polymer (cast method), a method of applying with a bar coater (bar coat method), a method of applying with a gap coater (gap coat method), etc. Can be mentioned. Of these, the bar coat coating method and the gap coat coating method are preferred.
本発明の細胞培養担体において、好ましくは、温度応答性高分子及び水膨潤性高分子を含むゲル層に隣接して細胞接着性ゲル層が存在する。細胞接着性ゲル層はまた細胞培養面最表面であることが好ましい。
「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よいが、好ましくは層状の細胞外マトリックス成分ゲルである。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、ゼラチン等を例示でき、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、及びゼラチンを例示できる。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。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。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。
In the cell culture carrier of the present invention, there is preferably a cell adhesive gel layer adjacent to a gel layer containing a temperature-responsive polymer and a water-swellable polymer. The cell adhesive gel layer is also preferably the outermost surface of the cell culture surface.
“Cell-adhesive gel layer” means a layered cell-adhesive hydrogel, which may be either a natural or synthetic compound as long as it is non-cytotoxic and adheres to cells under normal culture conditions. Preferably, it is a layered extracellular matrix component gel. The extracellular matrix is generally defined as “a complex body of biological macromolecules that are synthesized outside the cell and are secreted and accumulated outside the cell.” (Biochemical Dictionary (3rd edition) p.570, Tokyo Kagaku Dojin 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, gelatin, etc., among which collagen, atelocollagen, matrigel (gel made of type IV collagen, laminin, heparan sulfate), hyaluronic acid, and gelatin are particularly preferable. It can be illustrated. 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 adhesive component can be performed according to a conventional method. For example, when the cell adhesive component is collagen, a collagen gel can be obtained by incubating a 0.3-0.5% collagen aqueous solution at 37 ° C. for 10-20 minutes. When the extracellular matrix component is gelled, a gelling agent may be used as necessary.
本発明における細胞接着性ゲル層の厚さは0.005μm以上100μm以下が好ましく、0. 005μm以上10μm以下がより好ましく、0. 005μm以上1μm以下がさらに好ましい。本発明の細胞接着性ゲル層が厚いと乾燥時に層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。 The thickness of the cell adhesive gel layer in the present invention is preferably 0.005 μm or more and 100 μm or less, more preferably 0.005 μm or more and 10 μm or less, and further preferably 0.005 μm or more and 1 μm or less. When the cell-adhesive gel layer of the present invention is thick, cracks are generated in the layer when it is dried, and cell transfer becomes extremely difficult.
温度応答性高分子及び水膨潤性高分子を含むゲル層上に細胞接着性ゲル層を形成させる際には、温度応答性高分子及び水膨潤性高分子を含むゲル層と細胞接着性成分ゲル層とを別々に作製した後、両者を重ねてもよいが、温度応答性高分子及び水膨潤性高分子を含むゲル層上に細胞接着性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、温度応答性高分子及び水膨潤性高分子を含むゲル層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で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、市販の細胞接着性ゲル成分水溶液にゲル化調整液を加え、上温度応答性高分子及び水膨潤性高分子を含むゲル層上にキャストし、温度応答性高分子及び水膨潤性高分子を含むゲルに含まれる温度応答性高分子の曇点以上の温度で乾燥させることで、本発明の細胞培養単体が得られる。 When a cell-adhesive gel layer is formed on a gel layer containing a temperature-responsive polymer and a water-swellable polymer, the gel layer containing the temperature-responsive polymer and the water-swellable polymer and the cell-adhesive component gel After the layers are prepared separately, both layers may be overlapped, but after adding the cell-adhesive component-containing aqueous solution on the gel layer containing the temperature-responsive polymer and the water-swellable polymer, the aqueous solution is gelled. It is preferable to do so. The cell adhesive gel layer is obtained by immersing a gel layer containing a temperature-responsive polymer and a water-swellable polymer in a re-adhesive component solution (immersion method), applying (applying method), or casting. (Casting method) can be easily obtained, but any of these methods may be used for preparing the cell culture carrier of the present invention. Of these, the casting method is preferably used. For example, a gelation adjusting solution is added to a commercially available cell adhesive gel component aqueous solution and cast on a gel layer containing an upper temperature responsive polymer and a water swellable polymer, and the temperature responsive polymer and the water swellable polymer are cast. The cell culture simple substance of this invention is obtained by making it dry at the temperature beyond the cloud point of the temperature-responsive polymer contained in the gel containing.
本発明の細胞培養担体上に形成された培養細胞層は、温度応答性高分子及び水膨潤性高分子を含むゲル層の膨潤化処理により細胞シートとして剥離させることができる。膨潤化処理は、本発明の細胞培養担体を用いて培養された細胞培養物を、用いられた温度応答性高分子の曇点以下の温度条件下にすることにより行うのが好ましく、例えば、温度応答性高分子としてPNIPAMを用いているゲル層の膨潤化処理は、該ゲル層を含む細胞培養物を５℃以上３４℃以下、好ましくは１０℃以上３２℃以下の温度条件下にすることにより行うことができる。 The cultured cell layer formed on the cell culture carrier of the present invention can be peeled off as a cell sheet by swelling treatment of a gel layer containing a temperature-responsive polymer and a water-swellable polymer. The swelling treatment is preferably carried out by subjecting the cell culture cultured using the cell culture carrier of the present invention to a temperature condition not higher than the cloud point of the temperature-responsive polymer used. The swelling treatment of the gel layer using PNIPAM as the responsive polymer is performed by subjecting the cell culture containing the gel layer to a temperature condition of 5 ° C. or higher and 34 ° C. or lower, preferably 10 ° C. or higher and 32 ° C. or lower. It can be carried out.
本発明の細胞培養担体としては、膨潤化処理時の操作性を向上させるために、細胞接着性ゲル層と反対側の温度応答性高分子及び水膨潤性高分子を含むゲル層の面に、物理的な補強治具を設けてもよい。物理的な補強治具の材質は、細胞に影響を与えない材質であれば特に限定はないが、金属類（たとえば鉄、ステンレス、チタン、金など）、プラスチック類（たとえばポリスチレン、ポリカーボネート、ポリエチレン、ポリプロピレン、アクリルなど）、陶器などの無機材料類などであり、ステンレス、チタン、プラスチック類が好ましい。 As the cell culture carrier of the present invention, in order to improve the operability during the swelling treatment, on the surface of the gel layer containing the temperature-responsive polymer and the water-swellable polymer on the side opposite to the cell adhesive gel layer, A physical reinforcing jig may be provided. The material of the physical reinforcing jig is not particularly limited as long as it does not affect the cells, but metals (for example, iron, stainless steel, titanium, gold, etc.), plastics (for example, polystyrene, polycarbonate, polyethylene, Polypropylene, acrylic, etc.) and inorganic materials such as ceramics, and stainless steel, titanium and plastics are preferred.
物理的な補強治具は、本発明の細胞培養担体の取り扱い性を向上させることができればいかなる形状をしていてもよいが、板状であることが好ましく、厚さは好ましくは0.1μm以上10 mm以下であり、より好ましくは1μm以上1 mm以下であり、さらに好ましくは10μm以上200μm以下である。
物理的な補強治具は、細胞観察用に本発明の細胞培養担体が見える部分があればその形状は特に限定はないが、円、多角形（三角形、四角形、六角形など）、またはその組み合わせ（扇型など）等が例として挙げられる。そのなかで、円に近い形状であることが好ましい。また、細胞観察用に本発明の細胞培養担体が見える部分は1個でも複数でもよい。また温度応答性高分子及び水膨潤性高分子を含むゲル層の補強治具を接着した面の判別を容易にするために、非対称な形状であることが好ましい。
The physical reinforcing jig may have any shape as long as it can improve the handleability of the cell culture carrier of the present invention, but is preferably plate-shaped and preferably has a thickness of 0.1 μm to 10 μm. mm or less, more preferably 1 μm or more and 1 mm or less, and further preferably 10 μm or more and 200 μm or less.
The shape of the physical reinforcing jig is not particularly limited as long as there is a portion where the cell culture carrier of the present invention can be seen for cell observation, but a circle, a polygon (triangle, square, hexagon, etc.), or a combination thereof (Sector type etc.) etc. are mentioned as an example. Among them, a shape close to a circle is preferable. In addition, the cell culture carrier of the present invention can be seen for cell observation with one or more portions. Further, in order to easily distinguish the surface to which the reinforcing jig of the gel layer containing the temperature-responsive polymer and the water-swellable polymer is bonded, the shape is preferably asymmetric.
物理的な補強治具は、細胞培養に影響を及ぼさない限りいかなる方法で温度応答性高分子及び水膨潤性高分子を含むゲル層に接着させてもよい。たとえば、温度応答性高分子及び水膨潤性高分子を含むゲル層を作製した後市販の接着剤（たとえばアロンアルファ、ボンドなど）を使用して接着させる方法や温度応答性高分子及び水膨潤性高分子を含むゲル層を作成する際に補強治具を未乾燥状態の温度応答性高分子及び水膨潤性高分子を含むゲル層におくことで接着させてもよい。 The physical reinforcing jig may be adhered to the gel layer containing the temperature-responsive polymer and the water-swellable polymer by any method as long as it does not affect the cell culture. For example, a gel layer containing a temperature-responsive polymer and a water-swellable polymer is prepared and then bonded using a commercially available adhesive (for example, Aron Alpha, Bond, etc.). When creating a gel layer containing molecules, a reinforcing jig may be adhered to the gel layer containing a temperature-responsive polymer and a water-swellable polymer in an undried state.
物理的な補強治具は、その材質にもよるが、補助治具の縁が鋭利である場合がある。この場、補助治具の鋭い縁によって温度応答性高分子及び水膨潤性高分子を含むゲルが破けるばかりでなく、取り扱う作業者にも危険を及ぼす懸念がある。したがって、鋭い縁をなくすことが好ましい。鋭い縁をなくす方法として、細胞培養に支障がない限りいかなる方法を用いてもよいが、物理的な研磨（たとえば、やすりなどで磨くなど）や化学処理（たとえば、ケミカルエッチングなど）する方法が挙げられる。本発明において、物理的な補強治具はステンレス製の場合、ケミカルエッチングなどの化学処理を行うことが好ましい。 Depending on the material of the physical reinforcing jig, the edge of the auxiliary jig may be sharp. In this case, the gel containing the temperature-responsive polymer and the water-swellable polymer is not only broken by the sharp edge of the auxiliary jig, but there is also a concern that it may be dangerous for the handling operator. Therefore, it is preferable to eliminate sharp edges. As a method for eliminating sharp edges, any method may be used as long as cell culture is not hindered, but examples include physical polishing (for example, polishing with a file) and chemical processing (for example, chemical etching). It is done. In the present invention, when the physical reinforcing jig is made of stainless steel, it is preferable to perform chemical treatment such as chemical etching.
本発明の細胞培養担体を用いて、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。細胞接着性ゲル層上で細胞を培養する場合には、通常、細胞接着性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 using the cell culture carrier of the present invention include fibroblasts, vascular endothelial cells, chondrocytes, hepatocytes, small intestinal epithelial cells, epidermal keratinocytes, osteoblasts, bone marrow mesenchyme A cell etc. can be illustrated and a fibroblast can be illustrated as a preferable thing. 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 cell adhesive gel layer, it is usually performed until a confluent monolayer cell layer is formed on the cell adhesive gel layer.
本発明の細胞培養担体を用いた細胞の培養は、用いられる温度応答性高分子の曇点より高い温度条件下で行うのが好ましく、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。 The cell culture using the cell culture carrier of the present invention is preferably performed under a temperature condition higher than the cloud point of the temperature-responsive polymer used, and can be specifically 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て培養された細胞培養物は、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。本発明の細胞培養担体上に形成された細胞層は好ましくは細胞接着性ゲル層上に形成された細胞層である。 The cell culture cultured 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 formed on the cell culture carrier of the present invention is preferably a cell layer formed on a cell adhesive gel layer.
温度応答性高分子及び水膨潤性高分子を含むゲル層を膨潤化処理して得られる細胞シートは、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、本発明の細胞培養担体を用いて培養された細胞培養物を細胞培養面が向き合うように、予め培養した細胞上に荷重をかけた状態又はかけない状態で重ね、さらに培養した後温度応答性高分子及び水膨潤性高分子を含むゲル層を膨潤化処理してもよいし、温度応答性高分子及び水膨潤性高分子を含むゲル層を膨潤化して得られる細胞シート同士を重層化してもよいし、温度応答性高分子及び水膨潤性高分子を含むゲル層を膨潤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。また、上記の方法などにより重層化した細胞層を含む細胞シート又は細胞培養物を、さらに別に作製した細胞層に重層化してもよい。別に作製した細胞層としては、本発明の細胞培養担体を用いて培養された細胞培養物の細胞層でもよく、他の細胞培養担体を用いて培養された細胞培養物の細胞層でもよく、また細胞シートでもよい。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。 Since the cell sheet obtained by swelling the gel layer containing the temperature-responsive polymer and the water-swellable polymer contains the cell layer, it can be used for layering and transferring the cell layer. In the layering of the cell layer, the cell culture cultured using the cell culture carrier of the present invention is applied with or without applying a load on the cells cultured in advance so that the cell culture surface faces each other. The gel layer containing the temperature-responsive polymer and the water-swellable polymer may be swelled after further culturing, or the gel layer containing the temperature-responsive polymer and the water-swellable polymer may be swelled. The obtained cell sheets may be layered, or a cell sheet obtained by swelling a gel layer containing a temperature-responsive polymer and a water-swellable polymer may be layered on a separately prepared cell layer. In addition, a cell sheet or cell culture containing a cell layer that has been layered by the above-described method or the like may be layered on a cell layer that has been separately prepared. The cell layer prepared separately may be a cell layer of a cell culture cultured using the cell culture carrier of the present invention, a cell layer of a cell culture cultured using another cell culture carrier, It may be a cell sheet. 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.
細胞層の転写の際には、別の細胞培養用基材上に本発明の細胞培養担体を用いて培養された細胞培養物を細胞培養面が別の細胞培養用基材側になるように荷重をかけた状態またはかけない状態で載せて、さらに培養した後、温度応答性高分子及び水膨潤性高分子を含むゲル層を膨潤化してもよいし、温度応答性高分子及び水膨潤性高分子を含むゲル層を膨潤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。また、転写される細胞培養物は重層化細胞培養物であってもよい。
好ましい重層化ならびに転写方法としては、本発明の細胞培養担体を用いて培養された細胞培養物を予め培養した細胞上もしくは別の細胞培養基材上で培養したのち温度応答性高分子及び水膨潤性高分子を含むゲル層を膨潤化する方法が挙げられる。
When transferring the cell layer, the cell culture cultured on another cell culture substrate using the cell culture carrier of the present invention is placed so that the cell culture surface is on the other cell culture substrate side. The gel layer containing the temperature-responsive polymer and the water-swellable polymer may be swelled after being placed with or without a load and further cultured, or the temperature-responsive polymer and the water-swellable A cell sheet obtained by swelling a gel layer containing a polymer may be transferred to another medium. The cell culture to be transferred may be a stratified cell culture.
As a preferable layering and transfer method, a cell culture cultured using the cell culture carrier of the present invention is cultured on a pre-cultured cell or on another cell culture substrate, and then a temperature-responsive polymer and water swelling are used. And a method of swelling the gel layer containing the conductive polymer.
荷重をかけた状態の細胞の培養法とは、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水膨潤性高分子を含むゲル又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。 As a method for culturing a cell in a state where a load is applied, any method may be used as long as the load is sufficiently applied so that unevenness does not occur in the cell or the substrate to which the cell is transferred. Here, when cells are sealed when applying a load, they suffocate, so that at least one cell culture substrate on the transfer side or the receiving side is a water-permeable gel or a gel containing a water-swellable polymer or It is preferable to be made of these combinations. 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.
荷重をかけた細胞の培養法の荷重は0.1 g/cm2以上50 g/cm2以下であることが好ましく、0.5 g/cm2以上10 g/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制限はないが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。また、荷重をかけた状態の細胞の培養において、培養細胞へのダメージが予想される場合には、本発明の細胞培養担体をプラスチック又は金属製の円筒状の枠やディスク状のシートに固定して培養及び重層化又は転写を行うことにより、ダメージを軽減させることができる。本発明においては、荷重をかけない状態で培養することが好ましい。 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 time for culturing the cell under load is not limited as long as sufficient cell transfer can be realized, but it is preferably 4 hours to 72 hours, more preferably 6 hours to 48 hours. In the case of culturing cells under a load, if damage to the cultured cells is expected, the cell culture carrier of the present invention is fixed to a plastic or metal cylindrical frame or disk-shaped sheet. Damage can be reduced by culturing and layering or transferring. In the present invention, it is preferable to culture without applying a load.
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0.1 kGy以上65 kGy以下が好ましく、1 kGy以上40 kGy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層を変性させ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。
本発明の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よく、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 of 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 denatures the cell adhesive layer. The cell culture carrier thus sterilized can be stored at room temperature for a long time under aseptic conditions.
The sterilization method of the present invention may be carried out singly 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、実施例により本発明を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。
〔例１〕細胞培養担体の作製
（１）細胞培養担体Ａの調製
PNIPAM（曇点３２℃、ALDRICH）の１０％メタノール溶液1.5 g とポリオール・ポリウレタン(ダイアロマー:大日精化工業製)１５％溶液（トルエン−メタノール溶液）1 g を混合し、シリコーン樹脂コートの離型シート上に固形分塗布量が20 g/m2 になるように塗布し、６０℃のオーブン中で2時間乾燥後、塗布膜を剥離して支持体層を作製した。この支持体を水浸・膨潤させた状態で２つの円筒状のプラスチック枠間に挟んで固定した。
約３℃に調整された０．３％コラーゲン水溶液（Cellmatrix I-P：新田ゼラチン（株）製）8 gにEagle' MEM (×10 濃度、Invitrogen Corporation製)1 g を加え、よく攪拌した後、コラーゲンゲル再構成用緩衝液（新田ゼラチン（株）製）1 gを添加した。得られたコラーゲン／培地／ゲル再構成用緩衝液の混合溶液を速やかに上記の支持体層上に、固形分塗布量が5 g/m2 になるように塗布した。室温で約1時間静置後、水洗し、３７℃で約5時間乾燥させて細胞培養担体Ａを得た。
EXAMPLES Hereinafter, although an Example demonstrates this invention further more concretely, the scope of the present invention is not limited to the following Example.
[Example 1] Preparation of cell culture carrier (1) Preparation of cell culture carrier A
Mix PNIPAM (cloud point 32 ° C, ALDRICH) 1.5 g with 10% methanol solution and polyol polyurethane (Diaroma: manufactured by Dainichi Seika Kogyo Co., Ltd.) 15% solution (toluene-methanol solution). The coating was applied on the sheet so that the solid content was 20 g / m 2 , dried in an oven at 60 ° C. for 2 hours, and then the coating film was peeled off to prepare a support layer. This support was immersed and swollen in a state where it was sandwiched between two cylindrical plastic frames.
After adding 1 g of Eagle 'MEM (x10 concentration, manufactured by Invitrogen Corporation) to 8 g of 0.3% collagen aqueous solution (Cellmatrix IP: manufactured by Nitta Gelatin Co., Ltd.) adjusted to about 3 ° C and stirring well, 1 g of a collagen gel reconstitution buffer (Nitta Gelatin Co., Ltd.) was added. The obtained mixed solution of collagen / medium / gel reconstitution buffer was quickly applied onto the support layer so that the solid content was 5 g / m 2 . After standing at room temperature for about 1 hour, washed with water and dried at 37 ° C. for about 5 hours to obtain a cell culture carrier A.
（２）細胞培養担体Ｂの調製
PNIPAM（曇点３２℃、ALDRICH）の１０％メタノール溶液3 g とポリオール・ポリウレタン１５％溶液（トルエン−メタノール溶液）1 g を混合し、ポリエステルベース（厚さ180μm）上に固形分塗布量が5 g/m2 になるように片面に塗布し、６０℃のオーブン中で2時間乾燥させた。乾燥後得られたシートをディスク状（外径21 mm、内径15 mm）にくり抜いた。
上記（１）と同様のコラーゲン／培地／ゲル再構成用緩衝液を離型シート上に、固形分で5 g/m2 になるように塗布し、生成ゲル膜を水洗後、２５℃で一晩乾燥させた。乾燥させたゲル膜をゆっくり剥離し、直径21 mmの円盤状膜に裁断した。得られた円盤状膜を４０℃の水に水浸後、上記で得られたディスク状シートの塗布面上に重ね合わせ、３７℃で約5時間乾燥させて両膜を接着させ細胞培養担体Ｂを得た。
(2) Preparation of cell culture carrier B
Mix 3g of PNIPAM (cloud point 32 ° C, ALDRICH) 10% methanol solution and 1 g polyol / polyurethane 15% solution (toluene-methanol solution). The solid coating amount is 5 on the polyester base (thickness 180μm). It was applied to one side so as to be g / m 2 and dried in an oven at 60 ° C. for 2 hours. The sheet obtained after drying was cut into a disc shape (outer diameter 21 mm, inner diameter 15 mm).
The same collagen / medium / gel reconstitution buffer as in (1) above was applied onto the release sheet so that the solid content was 5 g / m 2 , and the resulting gel membrane was washed with water and then washed at 25 ° C. Dried overnight. The dried gel film was slowly peeled off and cut into a disc-shaped film having a diameter of 21 mm. The obtained disc-shaped membrane is immersed in water at 40 ° C., and then superimposed on the coated surface of the disk-shaped sheet obtained above, and dried at 37 ° C. for about 5 hours to adhere both membranes to the cell culture carrier B. Got.
（３）細胞培養担体Ｃの調製（比較例）
ポリオール・ポリウレタン１５％溶液（トルエン−メタノール溶液）を離型紙上に固形分塗布量が20 g/m2 になるように塗布し、６０℃のオーブン中で2時間乾燥させた。得られた塗布膜を剥離し、水浸・膨潤させた状態で２つの円筒状のプラスチック枠間に挟んで固定した。その上に、PNIPAMの１０％水溶液を固形分塗布量が5 g/m2 になるように塗布し、４０℃のオーブン中で約２時間乾燥させ、細胞培養担体Ｃを得た。
(3) Preparation of cell culture carrier C (comparative example)
A 15% polyol / polyurethane solution (toluene-methanol solution) was applied onto a release paper so that the solid content was 20 g / m 2 and dried in an oven at 60 ° C. for 2 hours. The obtained coating film was peeled off, fixed in a state of being immersed in water and swollen between two cylindrical plastic frames. On top of that, a 10% aqueous solution of PNIPAM was applied so that the solid content was 5 g / m 2 and dried in an oven at 40 ° C. for about 2 hours to obtain cell culture carrier C.
（４）細胞培養担体Ｄの調製（比較例）
上記（３）で得られた細胞培養担体Ｃ上に、３７℃で保温した上記（１）のコラーゲン／培地／ゲル再構成用緩衝液を固形分塗布量が5 g/m2 になるように塗布し、３７℃のオーブン中で約５時間乾燥させ、細胞培養担体Ｄを得た。
(4) Preparation of cell culture carrier D (comparative example)
On the cell culture carrier C obtained in (3) above, the collagen / medium / gel reconstitution buffer (1) kept at 37 ° C. is applied so that the solid content is 5 g / m 2. It was applied and dried in an oven at 37 ° C. for about 5 hours to obtain a cell culture carrier D.
〔例２〕細胞培養担体を用いた細胞の培養
（１）細胞培養
細胞培養担体Ａ，Ｂ，Ｃ，及びＤを細胞接着性層が上向になるように底面に置いたポリスチレン製細胞培養用シャーレ（市販）及び該ポリスチレン製細胞培養用シャーレのみをUV滅菌し、３７℃に保温した培地を添加した。これらのシャーレ（細胞培養担体）に培地を１時間浸潤させた。なお、例２及び例３において培地はEagle'MEM（10%牛胎児血清)を使用した。
予め培養しておいたＢＡＥ細胞（ウシ大動脈血管内皮細胞）をトリプシン処理で回収し、細胞濃度を40000 cell/mlに調製した。上記のシャーレ内の培地を捨てた後、この細胞液を細胞数7500 cell/cm2となるようにシャーレ内に播種した。シャーレに３７℃に保温した培地を添加し、これをCO2インキュベーターを用いて37℃で３日間培養した。
[Example 2] Cell culture using cell culture carrier (1) Cell culture For cell culture made of polystyrene with cell culture carriers A, B, C, and D placed on the bottom surface with the cell adhesive layer facing upward Only a petri dish (commercially available) and the polystyrene cell culture petri dish were UV sterilized, and a medium kept at 37 ° C. was added. A medium was infiltrated into these petri dishes (cell culture carriers) for 1 hour. In Example 2 and Example 3, Eagle'MEM (10% fetal bovine serum) was used as the medium.
Pre-cultured BAE cells (bovine aortic vascular endothelial cells) were collected by trypsin treatment, and the cell concentration was adjusted to 40,000 cells / ml. After discarding the medium in the petri dish, this cell solution was seeded in the petri dish so that the cell number was 7500 cell / cm 2 . A medium kept at 37 ° C. was added to the petri dish, and this was cultured at 37 ° C. for 3 days using a CO 2 incubator.
（２）結果
細胞培養担体Ａ及びＢにおいては、培養中、細胞接着性層（コラーゲン層）は剥離することなく、ＢＡＥ細胞の生育もコントロール（シャーレ単独上）と変わらず正常であった。他方、比較例として作製した細胞培養担体Ｃ及びＤでは、いずれも培地浸漬の数時間後にPNIPAM層が培地中に脱離し、さらに胞培養担体Ｄでは細胞接着性層も剥離していた。また、ＢＡＥ細胞はポリウレタン支持体面上にほとんど観測されず、培地中に浮遊（死滅）していた。
(2) Results In the cell culture carriers A and B, the cell adhesive layer (collagen layer) did not peel during the culture, and the growth of BAE cells was normal as in the control (on the petri dish alone). On the other hand, in the cell culture carriers C and D prepared as comparative examples, the PNIPAM layer was detached into the medium after several hours of immersion in the medium, and the cell adhesion layer was also peeled off in the cell culture carrier D. Further, BAE cells were hardly observed on the surface of the polyurethane support, and were suspended (dead) in the medium.
〔例３〕細胞の重層と細胞シートの剥離
例２で得られたＢＡＥ細胞を培養した細胞培養物（細胞培養担体Ａを用いたもの）を用いて細胞の重層化を行った。この細胞培養物を固定している円筒状プラスチック枠の片方をはずし、予めシャーレ上に用意したＨＥＰＧ２細胞（ヒト肝癌由来細胞）層の上に細胞層が互いに重なるように重層し、さらに３７℃で1日CO2インキュベーター中に静置した。細胞培養担体Ｂ上でＢＡＥ細胞を培養した細胞培養物についても同様に細胞の重層化を行った。
重層化細胞培養物をインキュベーターから取り出し、室温に30分間放冷した後、固定枠及び支持体を取り除いて、倒立型顕微鏡で観察した。
その結果、いずれの重層化細胞培養物においても細胞接着性層に付着したＢＡＥ細胞がＨＥＰＧ２細胞層に重なって正常に生育しているのが確認された。また、支持体層は固定枠とともに除去されており、細胞接着性層と細胞接着性層上に培養されたＢＡＥ細胞のみが、重層化されていることも確認された。
[Example 3] Separation of cell overlay and cell sheet Cells were layered using the cell culture obtained by culturing the BAE cells obtained in Example 2 (using cell culture carrier A). Remove one side of the cylindrical plastic frame that fixes the cell culture, and layer it on the HEPG2 cell (human hepatoma-derived cell) layer prepared in advance on a petri dish so that the cell layers overlap each other. Left in a CO 2 incubator for 1 day. Cell stratification was similarly performed for cell cultures in which BAE cells were cultured on cell culture carrier B.
The stratified cell culture was removed from the incubator and allowed to cool to room temperature for 30 minutes, after which the fixed frame and support were removed and observed with an inverted microscope.
As a result, it was confirmed that in any stratified cell culture, the BAE cells adhering to the cell adhesion layer overlap with the HEPG2 cell layer and grow normally. Further, the support layer was removed together with the fixing frame, and it was confirmed that only the BAE cells cultured on the cell adhesive layer and the cell adhesive layer were layered.
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| WO2020182645A1 (en) | 2019-03-08 | 2020-09-17 | Universität Siegen | Method for separating cells using a stimulus-responsive polymer |
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