JP2006340679A - Method for producing fine particle for cell culture, fine particle for cell culture obtained from the method and method of cell culture by using the fine particle - Google Patents
Method for producing fine particle for cell culture, fine particle for cell culture obtained from the method and method of cell culture by using the fine particle Download PDFInfo
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- 239000010419 fine particle Substances 0.000 title claims abstract description 58
- 238000000034 method Methods 0.000 title claims abstract description 31
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- 239000011259 mixed solution Substances 0.000 claims abstract description 17
- 239000003960 organic solvent Substances 0.000 claims abstract description 15
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- 239000002344 surface layer Substances 0.000 claims abstract description 12
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- IMNFDUFMRHMDMM-UHFFFAOYSA-N N-Heptane Chemical compound CCCCCCC IMNFDUFMRHMDMM-UHFFFAOYSA-N 0.000 claims description 6
- IIEWJVIFRVWJOD-UHFFFAOYSA-N ethylcyclohexane Chemical compound CCC1CCCCC1 IIEWJVIFRVWJOD-UHFFFAOYSA-N 0.000 claims description 6
- UAEPNZWRGJTJPN-UHFFFAOYSA-N methylcyclohexane Chemical compound CC1CCCCC1 UAEPNZWRGJTJPN-UHFFFAOYSA-N 0.000 claims description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerol Natural products OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 5
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- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 claims description 3
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 claims description 3
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 claims description 3
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 claims description 3
- 239000005642 Oleic acid Substances 0.000 claims description 3
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 claims description 3
- 229940057995 liquid paraffin Drugs 0.000 claims description 3
- GYNNXHKOJHMOHS-UHFFFAOYSA-N methyl-cycloheptane Natural products CC1CCCCCC1 GYNNXHKOJHMOHS-UHFFFAOYSA-N 0.000 claims description 3
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 claims description 3
- OYHQOLUKZRVURQ-NTGFUMLPSA-N (9Z,12Z)-9,10,12,13-tetratritiooctadeca-9,12-dienoic acid Chemical compound C(CCCCCCC\C(=C(/C\C(=C(/CCCCC)\[3H])\[3H])\[3H])\[3H])(=O)O OYHQOLUKZRVURQ-NTGFUMLPSA-N 0.000 claims 1
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- OYHQOLUKZRVURQ-IXWMQOLASA-N linoleic acid Natural products CCCCC\C=C/C\C=C\CCCCCCCC(O)=O OYHQOLUKZRVURQ-IXWMQOLASA-N 0.000 description 2
- 229930195734 saturated hydrocarbon Natural products 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
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- 238000005119 centrifugation Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
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Abstract
Description
本発明は、細胞を培養する際の細胞培養担体として好適な細胞培養用微粒子の製造方法及び該方法により得られた細胞培養用微粒子並びにこの微粒子を用いた細胞培養方法に関するものである。 The present invention relates to a method for producing microparticles for cell culture suitable as a cell culture carrier for culturing cells, microparticles for cell culture obtained by the method, and a cell culture method using the microparticles.
細胞は適当な硬い足場がなければ増殖しないことが知られている。そのため細胞の足場材料が適度な硬さを有する必要がある。また、細胞増殖速度を大きくするためには、その足場材料の表面積を大きく設ける必要がある。そのための方法として懸濁培養法が知られている。懸濁培養法は、足場材料、即ち細胞培養担体として適度な硬さと大きな比表面積を有する微粒子を用い、この微粒子に培養対象となる細胞を吸着又は固定化させ、この微粒子表面で細胞を培養するものである。細胞の培養を終えた後は、培養細胞に対してトリプシン含有溶液等の剥離剤を添加して細胞を微粒子から剥離する処理を施す。細胞培養用途として市販されている微粒子としては、Ｃｙｔｏｄｅｘ１（アマシャムバイオサイエンス株式会社製）が知られている（例えば、非特許文献１参照。）。
しかしながら、上記非特許文献１に示される市販の微粒子を細胞培養担体として用いた場合、培養を終えた細胞を微粒子から剥離するためのトリプシン処理を施すと、細胞は微粒子から剥離しても微粒子は破壊されずにそのまま残留してしまうため、細胞と微粒子とを遠心分離等の手段によって分離する必要があった。この分離操作によって培養した細胞の回収率が低くなる問題点があった。
本発明の目的は、懸濁培養法において細胞培養過程で安定であり、かつトリプシン処理により容易に破壊され、トリプシン処理後に培養細胞と微粒子との分離操作を必要としない細胞培養用微粒子の製造方法及び該方法により得られた細胞培養用微粒子並びにこの微粒子を用いた細胞培養方法を提供することにある。
However, when the commercially available fine particles shown in Non-Patent Document 1 are used as a cell culture carrier, if the cells that have been cultured are subjected to trypsin treatment for peeling from the fine particles, Since it remains as it is without being destroyed, it has been necessary to separate cells and fine particles by means such as centrifugation. There was a problem that the recovery rate of the cells cultured by this separation operation was lowered.
An object of the present invention is a method for producing microparticles for cell culture that is stable in a cell culture process in a suspension culture method, is easily destroyed by trypsin treatment, and does not require a separation operation between cultured cells and microparticles after trypsin treatment. It is another object of the present invention to provide a cell culture microparticle obtained by the method and a cell culture method using the microparticle.
請求項１に係る発明は、図１に示すように、室温で液体である有機溶媒に界面活性剤を添加して界面活性剤溶液１１を調製する工程と、界面活性剤溶液１１にコラーゲン又は変性コラーゲン水溶液１２を所定の割合で添加混合することにより乳化させ、混合液中に平均粒径１００〜８００μｍのコラーゲン又は変性コラーゲン液滴１３を形成する工程と、コラーゲン又は変性コラーゲン液滴１３を含む混合液に紫外線を照射して、コラーゲン又は変性コラーゲン液滴１３の表層を硬化させることにより内部にコラーゲン又は変性コラーゲンを封入した平均粒径１００〜８００μｍのコラーゲン又は変性コラーゲン微粒子１４を作製する工程とを含むことを特徴とする細胞培養用微粒子の製造方法である。
請求項１に係る発明では、上記工程を経ることで、微粒子表層のコラーゲン又は変性コラーゲン成分が紫外線照射により変質硬化され、その内部にコラーゲン又は変性コラーゲンが封入された直径１００〜８００μｍの細胞培養用微粒子が得られる。
As shown in FIG. 1, the invention according to claim 1 includes a step of preparing a surfactant solution 11 by adding a surfactant to an organic solvent that is liquid at room temperature, and collagen or denatured in the surfactant solution 11. A step of emulsifying the aqueous collagen solution 12 by adding and mixing at a predetermined ratio to form collagen or denatured collagen droplets 13 having an average particle size of 100 to 800 μm in the mixture, and mixing including the collagen or denatured collagen droplets 13 Irradiating the liquid with ultraviolet rays to harden the surface layer of collagen or denatured collagen droplets 13 to produce collagen or denatured collagen fine particles 14 having an average particle diameter of 100 to 800 μm in which collagen or denatured collagen is encapsulated. It is a manufacturing method of the fine particle for cell cultures characterized by including.
In the invention according to claim 1, through the above steps, the collagen or denatured collagen component in the fine particle surface layer is denatured and hardened by ultraviolet irradiation, and collagen or denatured collagen is encapsulated in the cell culture having a diameter of 100 to 800 μm. Fine particles are obtained.
請求項２に係る発明は、請求項１に係る発明であって、コラーゲン又は変性コラーゲン液滴１３を含む混合液への紫外線照射前に、コラーゲン又は変性コラーゲン液滴１３を含む混合液を有機溶媒により洗浄する工程を更に含む製造方法である。
請求項２に係る発明では、この工程を施すことでコラーゲン又は変性コラーゲン液滴１３の凝集を抑制することができる。
The invention according to claim 2 is the invention according to claim 1, wherein the mixed solution containing collagen or denatured collagen droplets 13 is treated with an organic solvent before the mixture solution containing collagen or denatured collagen droplets 13 is irradiated with ultraviolet rays. It is a manufacturing method further including the process of wash | cleaning by.
In the invention which concerns on Claim 2, aggregation of a collagen or the modified | denatured collagen droplet 13 can be suppressed by performing this process.
請求項３に係る発明は、請求項１に係る発明であって、室温で液体である有機溶媒がイソオクタン、ヘキサン、ヘプタン、シクロヘキサン、メチルシクロヘキサン、エチルシクロヘキサン、流動パラフィン、オレイン酸又はリノール酸であって、界面活性剤がグリセリン脂肪酸エステルである製造方法である。
請求項４に係る発明は、請求項１に係る発明であって、コラーゲン又は変性コラーゲン液滴１３を含む混合液への紫外線照射が波長２４０〜３６５ｎｍの紫外線を１０００μＷ／ｃｍ2以上の強度、１〜１０ｃｍの照射距離で１分〜１０時間照射することにより行われる製造方法である。
請求項４に係る発明では、上記条件で紫外線照射することで、細胞培養に最適な条件の微粒子が得られる。
The invention according to claim 3 is the invention according to claim 1, wherein the organic solvent which is liquid at room temperature is isooctane, hexane, heptane, cyclohexane, methylcyclohexane, ethylcyclohexane, liquid paraffin, oleic acid or linoleic acid. And the surfactant is a glycerin fatty acid ester.
The invention according to claim 4 is the invention according to claim 1, wherein the ultraviolet ray irradiation to the mixed solution containing collagen or denatured collagen droplets 13 has an intensity of 1000 μW / cm 2 or more for ultraviolet rays having a wavelength of 240 to 365 nm, It is a manufacturing method performed by irradiating with an irradiation distance of -10 cm for 1 minute to 10 hours.
In the invention which concerns on Claim 4, the microparticles | fine-particles of the optimal conditions for cell culture are obtained by irradiating with an ultraviolet-ray on the said conditions.
請求項５に係る発明は、請求項２に係る発明であって、コラーゲン又は変性コラーゲン液滴１３を含む混合液を洗浄する有機溶媒がヘキサンである製造方法である。
請求項６に係る発明は、請求項１ないし５いずれか１項に記載の方法により製造され、直径１００〜８００μｍの微粒子であり、微粒子表層のコラーゲン又は変性コラーゲン成分が紫外線照射により変質硬化され、その内部にコラーゲン又は変性コラーゲンが封入されたことを特徴とする細胞培養用微粒子である。
請求項６に係る発明では、細胞培養に最適な大きさであり、細胞の適度な吸着性を有し、変質硬化した表層は細胞培養後に施すトリプシン処理により容易に破壊されるため、トリプシン処理後に培養細胞の分離操作を必要としない。また、内部にコラーゲン又は変性コラーゲンを封入しているため、細胞培養の際にこの微粒子が浮遊状態を保てる程度の比重を有する。
The invention according to claim 5 is the method according to claim 2, wherein the organic solvent for washing the mixed solution containing collagen or denatured collagen droplets 13 is hexane.
The invention according to claim 6 is manufactured by the method according to any one of claims 1 to 5 and is a fine particle having a diameter of 100 to 800 μm, and collagen or denatured collagen component on the fine particle surface is altered and hardened by ultraviolet irradiation, It is a fine particle for cell culture characterized in that collagen or denatured collagen is enclosed inside.
In the invention according to claim 6, since it has an optimum size for cell culture, has an appropriate adsorptivity for cells, and the altered and hardened surface layer is easily destroyed by trypsin treatment performed after cell culture, There is no need to separate cultured cells. Further, since collagen or denatured collagen is encapsulated inside, the microparticles have a specific gravity enough to maintain a floating state during cell culture.
請求項７に係る発明は、請求項６記載の細胞培養用微粒子を細胞の細胞培養担体として用い、細胞を培養液中で培養することを特徴とする細胞培養方法である。
請求項７に係る発明では、この方法により懸濁培養法において細胞を安定して培養することができる。また、担体として用いる本発明の細胞培養用微粒子は細胞培養後に施すトリプシン処理により容易に破壊されるため、トリプシン処理後に培養細胞と微粒子との分離操作を必要としない。従って、培養した細胞の回収率が大幅に向上する。
The invention according to claim 7 is a cell culture method characterized by using the fine particles for cell culture according to claim 6 as a cell culture carrier for cells and culturing the cells in a culture solution.
In the invention which concerns on Claim 7, a cell can be culture | cultivated stably in a suspension culture method by this method. In addition, since the cell culture microparticles of the present invention used as a carrier are easily destroyed by trypsin treatment performed after cell culture, it is not necessary to separate the cultured cells from the microparticles after trypsin treatment. Therefore, the recovery rate of the cultured cells is greatly improved.
本発明の細胞培養用微粒子の製造方法及び該方法により得られた細胞培養用微粒子並びにこの微粒子を用いた細胞培養方法は、懸濁培養法において細胞培養過程で安定であり、かつトリプシン処理により容易に破壊されるため、トリプシン処理後に培養細胞と微粒子との分離操作を必要としない。従って、細胞と微粒子とを分離する操作を施す必要がなくなるため、培養した細胞の回収率が向上する。 The method for producing microparticles for cell culture of the present invention, the microparticles for cell culture obtained by the method, and the cell culture method using the microparticles are stable in the cell culture process in the suspension culture method, and easy by trypsin treatment. Therefore, it is not necessary to separate the cultured cells from the microparticles after trypsin treatment. Therefore, it is not necessary to perform an operation for separating the cells and the fine particles, and the collected cell recovery rate is improved.
次に本発明を実施するための最良の形態を図面に基づいて説明する。
先ず、室温で液体である有機溶媒に界面活性剤を添加して界面活性剤溶液を調製する。室温で液体である有機溶媒としては、飽和又は不飽和炭化水素、高級アルコール、高級脂肪酸が適当である。本発明で室温で液体である有機溶媒とは、１５〜４０℃の範囲内において液体状態を示す溶媒を指す。また、界面活性剤は親水性親油性バランス（ＨＬＢ）が親油性側のものが適当である。このうち、有機溶媒がイソオクタン、ヘキサン、ヘプタン、シクロヘキサン、メチルシクロヘキサン、エチルシクロヘキサン、流動パラフィン、オレイン酸又はリノール酸であって、界面活性剤がグリセリン脂肪酸エステルの組み合わせが好適である。グリセリン脂肪酸エステルのなかでもテトラグリセリン縮合リシノール酸エステルが特に好ましい。
Next, the best mode for carrying out the present invention will be described with reference to the drawings.
First, a surfactant solution is prepared by adding a surfactant to an organic solvent that is liquid at room temperature. As the organic solvent that is liquid at room temperature, saturated or unsaturated hydrocarbons, higher alcohols, and higher fatty acids are suitable. In the present invention, the organic solvent that is liquid at room temperature refers to a solvent that exhibits a liquid state within a range of 15 to 40 ° C. A surfactant having a hydrophilic / lipophilic balance (HLB) of lipophilic side is suitable. Among these, a combination of isooctane, hexane, heptane, cyclohexane, methylcyclohexane, ethylcyclohexane, liquid paraffin, oleic acid, or linoleic acid and a surfactant that is a glycerin fatty acid ester is preferable. Of the glycerin fatty acid esters, tetraglycerin condensed ricinoleic acid esters are particularly preferred.
次いで、図１（ａ）に示すように、界面活性剤溶液１１にコラーゲン又は変性コラーゲン水溶液１２を所定の割合で添加する。使用するコラーゲン又は変性コラーゲン水溶液はブタ由来ゼラチンやウシ由来ゼラチン、魚由来ゼラチン等を５〜２０重量％の水溶液としたものが好適である。界面活性剤溶液へのコラーゲン又は変性コラーゲン水溶液の添加割合は、界面活性剤溶液１００重量％に対して、コラーゲン又は変性コラーゲン水溶液０．３〜３重量％の範囲が好ましい。続いて図１（ｂ）に示すように、所定の割合で混合することにより乳化させ、混合液中に平均粒径１００〜８００μｍのコラーゲン又は変性コラーゲン液滴１３を形成する。この図１（ａ）及び図１（ｂ）に示すように、乳化しやすい試験管等により添加混合することが好ましい。 Next, as shown in FIG. 1A, a collagen or denatured collagen aqueous solution 12 is added to the surfactant solution 11 at a predetermined ratio. The collagen or denatured collagen aqueous solution used is preferably an aqueous solution containing 5 to 20% by weight of porcine derived gelatin, bovine derived gelatin, fish derived gelatin or the like. The ratio of the collagen or the modified collagen aqueous solution added to the surfactant solution is preferably in the range of 0.3 to 3% by weight of the collagen or modified collagen aqueous solution with respect to 100% by weight of the surfactant solution. Subsequently, as shown in FIG. 1B, the mixture is emulsified by mixing at a predetermined ratio, and collagen or denatured collagen droplets 13 having an average particle diameter of 100 to 800 μm are formed in the mixed solution. As shown in FIG. 1 (a) and FIG. 1 (b), it is preferable to add and mix using a test tube or the like that is easily emulsified.
また、形成したコラーゲン又は変性コラーゲン液滴１３を含む混合液への紫外線照射前に、コラーゲン又は変性コラーゲン液滴を含む混合液を有機溶媒により洗浄する工程を施しても良い。この工程を施すことで形成したコラーゲン又は変性コラーゲン液滴の凝集を抑制することができる。コラーゲン又は変性コラーゲン液滴を含む混合液を洗浄する有機溶媒としてはヘキサンが好適である。 Moreover, you may give the process which wash | cleans the liquid mixture containing a collagen or a modified | denatured collagen droplet with an organic solvent before the ultraviolet-ray irradiation to the liquid mixture containing the formed collagen or the modified | denatured collagen droplet 13 is carried out. Aggregation of collagen or denatured collagen droplets formed by applying this step can be suppressed. Hexane is suitable as the organic solvent for washing the mixed solution containing collagen or denatured collagen droplets.
次に、図３に示すように、コラーゲン又は変性コラーゲン液滴を含む混合液に紫外線を照射して、コラーゲン又は変性コラーゲン液滴の表層を硬化させることにより内部にコラーゲン又は変性コラーゲンを封入した平均粒径１００〜８００μｍのコラーゲン又は変性コラーゲン微粒子を作製する。この図１（ｃ）に示すように、コラーゲン又は変性コラーゲン液滴１３を含む混合液をシャーレのような平皿に移し、コラーゲン又は変性コラーゲン液滴に対して均一に、また均等な照射距離ｄで紫外線照射することが好ましい。細胞培養に最適な条件の微粒子を得るためには、コラーゲン又は変性コラーゲン液滴を含む混合液への紫外線照射の条件として、波長２４０〜３６５ｎｍの紫外線を使用し、紫外線の強度を１０００μＷ／ｃｍ2以上とし、照射距離ｄを１〜１０ｃｍとし、１分〜１０時間照射することが好適である。紫外線照射条件は、波長２５４ｎｍの紫外線を使用し、紫外線の強度を３０００〜５０００μＷ／ｃｍ2とし、照射距離ｄを３〜７ｃｍとし、１〜５時間照射することが特に好ましい。紫外線の強度が１０００μＷ／ｃｍ2未満では微粒子表層の硬化が培養環境に耐え得る程度にまで達しないため安定性が保てない。なお、紫外線の強度が１００００μＷ／ｃｍ2を越えると微粒子表層の架橋度を制御することが難しく、トリプシンで分解しない場合があるので、１００００μＷ／ｃｍ2以下の強度で紫外線照射することが好ましい。また、照射距離ｄが１ｃｍ未満では液滴によって架橋度にむらが生じてしまい、１０ｃｍを越えると微粒子表層が十分に硬化しない場合がある。紫外線照射時間が１時間未満では微粒子表層の硬化が培養環境に耐え得る程度にまで達しないため安定性が保てない。具体的には、コラーゲン又は変性コラーゲン微粒子が３７℃の水中で１週間保持することができない。また、紫外線照射時間が５時間を越えると、一旦表層が硬化して形成された微粒子が溶解してしまい、微粒子の原形を保つことができない。但し、紫外線照射距離を短くすれば照射時間は短くてすむので注意が必要である。 Next, as shown in FIG. 3, an average of the collagen or denatured collagen encapsulated by irradiating the mixed solution containing collagen or denatured collagen droplets with ultraviolet rays and curing the surface layer of the collagen or denatured collagen droplets. Collagen or modified collagen fine particles having a particle size of 100 to 800 μm are prepared. As shown in FIG. 1 (c), the mixed solution containing collagen or denatured collagen droplets 13 is transferred to a flat dish such as a petri dish, and uniformly or evenly irradiated to the collagen or denatured collagen droplets. It is preferable to irradiate with ultraviolet rays. In order to obtain fine particles having the optimum conditions for cell culture, ultraviolet rays having a wavelength of 240 to 365 nm are used as conditions for irradiating the mixed solution containing collagen or modified collagen droplets with an ultraviolet intensity of 1000 μW / cm 2. It is preferable that the irradiation distance d is 1 to 10 cm and the irradiation is performed for 1 minute to 10 hours. As the ultraviolet irradiation conditions, it is particularly preferable to use an ultraviolet ray having a wavelength of 254 nm, set the intensity of the ultraviolet ray to 3000 to 5000 μW / cm 2 , set the irradiation distance d to 3 to 7 cm, and irradiate for 1 to 5 hours. If the intensity of the ultraviolet light is less than 1000 μW / cm 2 , the stability of the fine particle surface layer cannot be maintained because it does not reach a level that can withstand the culture environment. If the intensity of the ultraviolet ray exceeds 10,000 μW / cm 2 , it is difficult to control the degree of crosslinking of the fine particle surface layer, and it may not be decomposed by trypsin. Therefore, it is preferable to irradiate the ultraviolet ray with an intensity of 10,000 μW / cm 2 or less. If the irradiation distance d is less than 1 cm, the degree of cross-linking is uneven due to the droplets, and if it exceeds 10 cm, the fine particle surface layer may not be sufficiently cured. If the ultraviolet irradiation time is less than 1 hour, the stability of the fine particle surface layer cannot be maintained because it does not reach the extent that it can withstand the culture environment. Specifically, collagen or denatured collagen particles cannot be kept in water at 37 ° C. for 1 week. If the ultraviolet irradiation time exceeds 5 hours, the fine particles formed once the surface layer is cured are dissolved, and the original shape of the fine particles cannot be maintained. However, it should be noted that the irradiation time can be shortened if the ultraviolet irradiation distance is shortened.
上記工程を経ることにより、微粒子表層のコラーゲン又は変性コラーゲン成分が紫外線照射により変質硬化され、その内部にコラーゲン又は変性コラーゲンが封入された直径１００〜８００μｍの細胞培養用微粒子が得られる。本発明の細胞培養用微粒子は、細胞培養に最適な大きさであり、細胞の適度な吸着性を有し、変質硬化した表層は細胞培養後に施すトリプシン処理により容易に破壊されるため、トリプシン処理後に培養細胞と微粒子との分離操作を必要としない。また、内部にコラーゲン又は変性コラーゲンを封入しているため、細胞培養の際にこの微粒子が浮遊状態を保てる程度の比重を有する。製造する際に使用するコラーゲン又は変性コラーゲンの種類によって変化するが、本発明の微粒子の比重は培養液と同程度、即ち、０．９〜１．１ｇ／ｍｌが好ましい。培養をシーソー型の攪拌装置で行う場合にはこの制限はより緩和される。 By passing through the above steps, collagen or denatured collagen components on the surface of the fine particles are denatured and cured by ultraviolet irradiation, and cell culture fine particles having a diameter of 100 to 800 μm in which collagen or denatured collagen is enclosed are obtained. The microparticles for cell culture of the present invention are optimally sized for cell culture, have an appropriate cell adsorptivity, and the altered and hardened surface layer is easily destroyed by trypsin treatment performed after cell culture. There is no need to separate the cultured cells from the microparticles later. Further, since collagen or denatured collagen is encapsulated inside, the microparticles have a specific gravity enough to maintain a floating state during cell culture. The specific gravity of the fine particles of the present invention is preferably about the same as that of the culture solution, that is, 0.9 to 1.1 g / ml, although it varies depending on the type of collagen or denatured collagen used in production. This restriction is more relaxed when the culture is performed with a seesaw type stirring device.
本発明の細胞培養方法は、本発明の細胞培養用微粒子を細胞の細胞培養担体として用い、細胞を培養液中で培養することを特徴とする。この方法により懸濁培養法において細胞を安定して培養することができる。また、本発明の微粒子は細胞培養後に施すトリプシン処理により容易に破壊されるため、トリプシン処理後に培養細胞と微粒子との分離操作を必要としない。従って、培養した細胞の回収率が大幅に向上する。 The cell culture method of the present invention is characterized in that the cell culture microparticle of the present invention is used as a cell culture carrier for cells and the cells are cultured in a culture solution. By this method, the cells can be stably cultured in the suspension culture method. Moreover, since the microparticles of the present invention are easily destroyed by trypsin treatment performed after cell culture, it is not necessary to separate the cultured cells from the microparticles after trypsin treatment. Therefore, the recovery rate of the cultured cells is greatly improved.
次に本発明の実施例を比較例とともに詳しく説明する。
＜実施例１＞
室温で液体である有機溶媒として鎖状飽和炭化水素であるイソオクタンを、界面活性剤としてテトラグリセリン脂肪酸エステル（坂本薬品工業株式会社製；商品名ＳＹグリスターＣＲ−３１０）をそれぞれ用意した。次いで、イソオクタン１００重量％に対しグリセリン脂肪酸エステルを５重量％の割合で加えて界面活性剤溶液を調製した。また変性コラーゲン水溶液としてブタ由来の１０重量％ゼラチン水溶液（新田ゼラチン株式会社製；商品名タイプＡＰＨ−２５０）を用意した。次に、界面活性剤溶液にこのブタ由来の１０重量％ゼラチン水溶液０．５ｍｌを添加し、この添加液を４０℃に加温しながら攪拌混合して乳化させ、混合液中に変性コラーゲン液滴を形成した。形成した変性コラーゲン液滴を含む混合液を１５℃に保ちながらしばらく攪拌し、混合液にヘキサンを添加して変性コラーゲン液滴を洗浄した。続いてヘキサンを含む混合液に対して、紫外線ランプ（コスモバイオ社製；ＣＳＬ−１００Ｃ）を用い、波長２５４ｎｍ、出力１００Ｗ、強度３４００μＷ／ｃｍ2、照射距離５．５ｃｍの条件で３時間紫外線照射して変性コラーゲン微粒子を作製した。この変性コラーゲン微粒子は光学顕微鏡により確認したところ、内部に変性コラーゲンが内包した平均粒径３００μｍのマイクロカプセルとなっていることが判った。また、得られた変性コラーゲン微粒子は３７℃の水中で１週間の安定性を持つことが確認された。
Next, examples of the present invention will be described in detail together with comparative examples.
<Example 1>
Isooctane, which is a chain saturated hydrocarbon, was prepared as an organic solvent that was liquid at room temperature, and tetraglycerin fatty acid ester (manufactured by Sakamoto Yakuhin Kogyo Co., Ltd .; trade name SY Glyster CR-310) was prepared as a surfactant. Next, a surfactant solution was prepared by adding 5% by weight of glycerin fatty acid ester to 100% by weight of isooctane. Also, a 10% by weight aqueous solution of gelatin derived from swine (manufactured by Nitta Gelatin Co., Ltd .; trade name type APH-250) was prepared as a modified collagen aqueous solution. Next, 0.5 ml of a 10% by weight gelatin aqueous solution derived from swine is added to the surfactant solution, and this added solution is stirred and mixed while being heated to 40 ° C., and denatured collagen droplets are added to the mixed solution. Formed. The mixed solution containing the formed modified collagen droplets was stirred for a while while being maintained at 15 ° C., and hexane was added to the mixed solution to wash the modified collagen droplets. Subsequently, an ultraviolet lamp (manufactured by Cosmo Bio; CSL-100C) is used to irradiate the mixed liquid containing hexane for 3 hours under the conditions of a wavelength of 254 nm, an output of 100 W, an intensity of 3400 μW / cm 2 , and an irradiation distance of 5.5 cm. Thus, modified collagen fine particles were prepared. When the denatured collagen fine particles were confirmed by an optical microscope, it was found that the denatured collagen microparticles were microcapsules having an average particle diameter of 300 μm encapsulated in the denatured collagen. Moreover, it was confirmed that the obtained modified collagen fine particles have stability for 1 week in water at 37 ° C.
＜実施例２＞
紫外線照射時間を５時間とした以外は実施例１と同様にして細胞培養用微粒子を製造した。得られた変性コラーゲン微粒子は３７℃の水中で１週間の安定性を持つことが確認された。
<Example 2>
Fine particles for cell culture were produced in the same manner as in Example 1 except that the ultraviolet irradiation time was changed to 5 hours. It was confirmed that the obtained modified collagen fine particles had a stability of 1 week in water at 37 ° C.
＜比較例１＞
市販されている細胞培養媒体（Ｃｙｔｏｄｅｘ１；アマシャムバイオサイエンス社製）を細胞培養用微粒子として用意した。
<Comparative Example 1>
A commercially available cell culture medium (Cytodex 1; manufactured by Amersham Biosciences) was prepared as fine particles for cell culture.
＜比較試験１＞
実施例１及び２で得られた微粒子並びに比較例１で用意した微粒子を用いて、以下の細胞培養試験を行った。
培養させる細胞として正常のヒト繊維芽細胞（ＷＩ−３８）と、癌化した細胞（ＷＩ−３８−ＶＡ−１３）をそれぞれ用意した。また培養液として１０ｖ／ｖ％のウシ胎児血清を含むＭｉｎｉｍｕｍ Ｅｓｓｅｎｔｉａｌ Ｍｅｄｉｕｍ Ｅａｇｌｅを用意し、シャーレには接着系細胞培養用シャーレと浮遊系細胞培養用シャーレの双方を用いた。接着系細胞培養用シャーレは細胞が付着しやすいようにシャーレの内面に表面コーティング処理されているものであり、浮遊系細胞培養用シャーレには、そのような細胞接着処理が施されていない。
<Comparison test 1>
Using the microparticles obtained in Examples 1 and 2 and the microparticles prepared in Comparative Example 1, the following cell culture test was performed.
Normal human fibroblasts (WI-38) and cancerous cells (WI-38-VA-13) were prepared as cells to be cultured. Also, as a culture solution, a Minimum Essential Medium Eagle containing 10 v / v% fetal bovine serum was prepared, and both an adhesive cell culture petri dish and a floating cell culture petri dish were used as the petri dishes. In the petri dish for adhesion type cell culture, the inner surface of the petri dish is surface-coated so that the cells can easily adhere to the petri dish for suspension type cell culture, and such cell adhesion treatment is not performed.
先ず、シャーレ内に培養液を満たし、細胞と微粒子とを培養液内に入れて、３７℃で２〜３日間保持して細胞を培養した。シーソー型の攪拌機を用い、培養開始後最初の６時間は３０分おきに２分攪拌し、その後は連続攪拌を行った。図２に実施例１の微粒子に細胞を接着させた様子を示す。この図２より明らかなように、実施例１の微粒子表面に細胞が接着しており、実施例１の微粒子は適度な吸着性を有していることが判る。また、実施例２の微粒子についても同様の傾向が得られた。ＷＩ−３８並びにＷＩ−３８−ＶＡ−１３の双方の細胞において培養過程で各微粒子は安定であり、実施例１及び２の微粒子を用いた場合では、細胞接着処理を施していない浮遊系細胞培養用シャーレにおいてＷＩ−３８の細胞が高い増殖傾向を示すことが判った。この結果から、本発明の微粒子は浮遊系細胞培養用シャーレを使用して懸濁培養をするのが適していると考えられる。 First, the petri dish was filled with a culture solution, cells and fine particles were placed in the culture solution, and maintained at 37 ° C. for 2 to 3 days to culture the cells. Using a seesaw type stirrer, stirring was continued for 2 minutes every 30 minutes for the first 6 hours after the start of culture, and then continuous stirring was performed. FIG. 2 shows a state in which cells are adhered to the fine particles of Example 1. As is apparent from FIG. 2, cells are adhered to the surface of the fine particles of Example 1, and it can be seen that the fine particles of Example 1 have an appropriate adsorptivity. The same tendency was obtained for the fine particles of Example 2. In both cells of WI-38 and WI-38-VA-13, each microparticle is stable during the culturing process. When the microparticles of Examples 1 and 2 are used, the suspension cell culture without cell adhesion treatment is performed. It was found that WI-38 cells showed a high growth tendency in the petri dish. From this result, it is considered that the microparticles of the present invention are suitable for suspension culture using a floating cell culture dish.
続いて、細胞培養後にトリプシン水溶液により細胞と微粒子との剥離処理を施した。実施例１の微粒子を用いたシャーレのトリプシン処理を施した後の様子を図３に示す。図３より明らかなように、トリプシン処理後のシャーレ内には微粒子が存在しておらず、トリプシン水溶液により実施例１の微粒子は破壊されたものと考えられる。一方、比較例１のＣｙｔｏｄｅｘ１はトリプシン処理後でも存在しており、この比較例１の微粒子を用いた場合、トリプシン処理後に細胞と微粒子とを分離する手段が必要となる。 Subsequently, the cells were separated from the microparticles with an aqueous trypsin solution after cell culture. FIG. 3 shows a state after the trypsin treatment of the petri dish using the fine particles of Example 1. As apparent from FIG. 3, no fine particles are present in the petri dish after the trypsin treatment, and it is considered that the fine particles of Example 1 were destroyed by the trypsin aqueous solution. On the other hand, Cytodex 1 of Comparative Example 1 exists even after trypsin treatment. When the fine particles of Comparative Example 1 are used, a means for separating cells and fine particles after trypsin treatment is required.
実施例１の微粒子を用いて培養した細胞について、トリプシン処理後に再び培養したところ、図４に示すように、接着系細胞培養用シャーレによって再び培養が可能であった。 When cells cultured using the microparticles of Example 1 were cultured again after trypsin treatment, as shown in FIG. 4, they could be cultured again using an adhesive cell culture petri dish.
１１ 界面活性剤溶液
１２ コラーゲン又は変性コラーゲン含有水溶液
１３ コラーゲン又は変性コラーゲン液滴
１４ コラーゲン又は変性コラーゲン微粒子
DESCRIPTION OF SYMBOLS 11 Surfactant solution 12 Collagen or modified | denatured collagen containing aqueous solution 13 Collagen or modified | denatured collagen droplet 14 Collagen or modified | denatured collagen fine particle
Claims (7)
前記界面活性剤溶液(11)にコラーゲン又は変性コラーゲン含有水溶液(12)を所定の割合で添加混合することにより乳化させ、前記混合液中に平均粒径１００〜８００μｍのコラーゲン又は変性コラーゲン液滴(13)を形成する工程と、
前記コラーゲン又は変性コラーゲン液滴(13)を含む混合液に紫外線を照射して、前記コラーゲン又は変性コラーゲン液滴(13)の表層を硬化させることにより内部にコラーゲン又は変性コラーゲンを封入した平均粒径１００〜８００μｍのコラーゲン又は変性コラーゲン微粒子(14)を作製する工程と
を含むことを特徴とする細胞培養用微粒子の製造方法。 Adding a surfactant to an organic solvent that is liquid at room temperature to prepare a surfactant solution (11);
Collagen or denatured collagen droplets having an average particle diameter of 100 to 800 μm are mixed in the surfactant solution (11) by adding and mixing collagen or a denatured collagen-containing aqueous solution (12) at a predetermined ratio. 13) forming, and
An average particle diameter in which collagen or denatured collagen is encapsulated by irradiating a mixed solution containing the collagen or denatured collagen droplet (13) with ultraviolet rays and hardening the surface layer of the collagen or denatured collagen droplet (13). A step of producing 100-800 μm collagen or denatured collagen fine particles (14).
A cell culture method comprising using the microparticles for cell culture according to claim 6 as a cell culture carrier for cells, and culturing the cells in a culture solution.
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| JP2008256377A (en) * | 2007-03-30 | 2008-10-23 | Fujifilm Corp | Method and instrument for detecting specimen |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5547130A (en) * | 1978-09-28 | 1980-04-03 | Nippi:Kk | Manufacture of collagen bead |
| JPH1147258A (en) * | 1997-07-30 | 1999-02-23 | Menicon Co Ltd | Medical base material containing gelatin and collagen |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5547130A (en) * | 1978-09-28 | 1980-04-03 | Nippi:Kk | Manufacture of collagen bead |
| JPH1147258A (en) * | 1997-07-30 | 1999-02-23 | Menicon Co Ltd | Medical base material containing gelatin and collagen |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2008256377A (en) * | 2007-03-30 | 2008-10-23 | Fujifilm Corp | Method and instrument for detecting specimen |
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