JP2011072233A - Method for producing cell-culturing base material - Google Patents
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Abstract
Description
本発明は細胞を培養するための細胞培養用基材の製造方法に関する。 The present invention relates to a method for producing a cell culture substrate for culturing cells.
従来、細胞を所定の基体上で培養する細胞培養において、細胞の接着性や増殖性向上のため、基体上にコラーゲン等の接着因子を培養面にコーティングした細胞培養用基材が知られている（特許文献１参照）。 Conventionally, in cell culture in which cells are cultured on a predetermined substrate, a cell culture substrate in which an adhesion factor such as collagen is coated on the culture surface is known on the substrate in order to improve cell adhesion and proliferation. (See Patent Document 1).
上述したような細胞培養用基材を用いて細胞培養を行う場合、取り扱いが良く効率のよい細胞増殖が行えることが求められる。本件発明は、保管管理が容易であり、好適な細胞増殖を行うことのできる細胞培養用基材の製造方法を提供することを技術課題とする。 When cell culture is performed using the cell culture substrate as described above, it is required that the cell culture is easy to handle and efficient. This invention makes it a technical subject to provide the manufacturing method of the base material for cell culture which can perform storage management easily and can perform suitable cell growth.
上記課題を解決するために、本発明は以下のような構成を備えることを特徴とする。
（１） 細胞培養用基材の製造方法において、ガラス又は樹脂からなる基体に対して所定の表面処理を行う第１ステップと、該第１ステップにて得られた表面処理済みの前記基体に対して可溶化羊膜組成物をコーティングする第２ステップと、を有することを特徴とする。
（２） （１）の細胞培養用基材の製造方法において、前記可溶化羊膜組成物は、加熱下での酸処理により可溶化した羊膜の可溶化物を媒体中に含む可溶化羊膜組成物であることを特徴とする。
（３） （２）の細胞培養用基材の製造方法において、前記可溶化羊膜組成物は塩基で中和されることにより中性のｐＨを有することを特徴とする。
（４） （３）の細胞培養用基材の製造方法において、前記第１ステップの表面処理は前記基体に対してプラズマを照射することにより行われることを特徴とする。
（５） （１）〜（４）の何れかに記載の細胞培養用基材の製造方法は、前記第２ステップにて得られる前記可溶化羊膜組成物がコーティングされた前記基体に対してγ線による滅菌処理を行う第３ステップと、を有することを特徴とする。
In order to solve the above problems, the present invention is characterized by having the following configuration.
(1) In the method for producing a cell culture substrate, a first step of performing a predetermined surface treatment on a substrate made of glass or resin, and the surface-treated substrate obtained in the first step And a second step of coating the solubilized amniotic membrane composition.
(2) In the method for producing a cell culture substrate according to (1), the solubilized amniotic membrane composition includes a solubilized amniotic membrane solubilized by acid treatment under heating in a medium. It is characterized by being.
(3) In the method for producing a cell culture substrate according to (2), the solubilized amniotic membrane composition has a neutral pH by being neutralized with a base.
(4) In the method for manufacturing a cell culture substrate according to (3), the surface treatment in the first step is performed by irradiating the substrate with plasma.
(5) The method for producing a cell culture substrate according to any one of (1) to (4), wherein the substrate coated with the solubilized amniotic membrane composition obtained in the second step is γ. And a third step of performing a sterilization process using a wire.
本発明によれば、保管管理が容易であり、好適な細胞増殖を行うことのできる細胞培養用基材を得ることができる。 According to the present invention, it is possible to obtain a cell culture substrate that can be easily stored and managed and can perform suitable cell growth.
以下に、本発明の実施形態を説明する。本実施形態の細胞培養用基材は、従来細胞培養用基材に用いられるガラスや樹脂からなる基体を予め表面処理し、この表面処理された基体に対して可溶化羊膜組成物がコーティングされることにより得られるものである。本実施形態で用いる可溶化羊膜組成物は、羊膜を加熱下において酸処理することにより得られたものであり、液体の溶媒中に羊膜の溶解物が含まれたものである。羊膜が由来する動物種は、哺乳動物であれば特に限定されないが、ヒト細胞又はヒトに移植若しくは投与する細胞を培養する場合には、ヒト羊膜を原料とすることが好ましい。 Hereinafter, embodiments of the present invention will be described. In the cell culture substrate of this embodiment, a substrate made of glass or resin, which has been conventionally used as a cell culture substrate, is surface-treated in advance, and the surface-treated substrate is coated with a solubilized amniotic membrane composition. It is obtained by this. The solubilized amniotic membrane composition used in the present embodiment is obtained by acid treatment of amniotic membrane under heating, and a solution of amniotic membrane is contained in a liquid solvent. The animal species from which the amniotic membrane is derived is not particularly limited as long as it is a mammal. However, when culturing human cells or cells to be transplanted or administered to humans, it is preferable to use human amniotic membrane as a raw material.
酸処理に先立ち、羊膜に付着している細胞を除去し、除去しきれない細胞については死滅させることが好ましい。細胞の除去は、羊膜を緩衝溶液中で強く振盪することを繰り返すことにより行うことができる。また、除去しきれなかった細胞を死滅させる処理としては、強アルカリ又はEDTA溶液による処理を挙げることができる。強アルカリとしては、水酸化ナトリウムのようなアルカリ金属水酸化物が好ましい。水酸化ナトリウムの場合、好ましい濃度は特に限定されないが、通常、25ｍＭ〜400ｍＭ、より好ましくは50ｍＭ〜200ｍＭ程度である。また、EDTA溶液中のEDTAの濃度は、通常、5ｍＭ〜80ｍＭ、より好ましくは10ｍＭ〜40ｍＭ程度である。強アルカリ又はEDTAによる処理は、強アルカリ又はEDTA溶液で羊膜を繰り返し洗浄したり、羊膜を強アルカリ又はEDTA溶液中で強く振盪することを繰り返すことにより行うことができる。このような処理を行った場合には、酸処理を行う前に羊膜を水でよく洗浄して強アルカリやEDTAを洗浄除去することが好ましい。 Prior to the acid treatment, it is preferable to remove cells adhering to the amniotic membrane and kill cells that cannot be removed. Cells can be removed by repeatedly shaking the amniotic membrane strongly in a buffer solution. Examples of the treatment for killing cells that could not be removed include treatment with a strong alkali or EDTA solution. As the strong alkali, an alkali metal hydroxide such as sodium hydroxide is preferable. In the case of sodium hydroxide, the preferred concentration is not particularly limited, but is usually about 25 mM to 400 mM, more preferably about 50 mM to 200 mM. The concentration of EDTA in the EDTA solution is usually about 5 mM to 80 mM, more preferably about 10 mM to 40 mM. The treatment with strong alkali or EDTA can be carried out by repeatedly washing the amniotic membrane with a strong alkali or EDTA solution or by repeatedly shaking the amniotic membrane in a strong alkali or EDTA solution. When such treatment is performed, it is preferable to wash and remove the strong alkali and EDTA by thoroughly washing the amniotic membrane with water before the acid treatment.
酸処理に用いる酸は、乾燥時に蒸発することにより、可溶化羊膜組成物を基体上にコーティング、乾燥して得られるコーティング材中に残留しない酸が好ましく、酢酸や塩化水素の水溶液が好ましい。酢酸水溶液の場合、酢酸濃度は特に限定されないが、好ましくは50ｍＭ〜１Ｍ程度、より好ましくは100ｍＭ〜500ｍＭ程度である。酸処理に用いる酸の量（酸溶液の量）は、羊膜を可溶化できる量であれば特に限定されないが、羊膜の湿重量１ｇ当たり5ｍＬ〜100ｍＬ、好ましくは10ｍＬ〜50ｍＬ程度である。
酸処理は、加熱下に行われる。酸処理時の温度は特に限定されないが、通常８０℃〜１００℃であり、好ましくは８５℃〜９５℃である。また、酸処理の時間は、羊膜が完全に溶けるまででよく、通常、６０分間〜９０分間程度である。
The acid used for the acid treatment is preferably an acid that does not remain in the coating material obtained by coating and drying the solubilized amniotic membrane composition on the substrate by evaporating at the time of drying, and an aqueous solution of acetic acid or hydrogen chloride is preferable. In the case of an acetic acid aqueous solution, the concentration of acetic acid is not particularly limited, but is preferably about 50 mM to 1 M, more preferably about 100 mM to 500 mM. The amount of acid used in the acid treatment (amount of acid solution) is not particularly limited as long as it can solubilize the amniotic membrane, but is about 5 to 100 mL, preferably about 10 to 50 mL per 1 g of wet weight of the amniotic membrane.
The acid treatment is performed under heating. Although the temperature at the time of an acid treatment is not specifically limited, Usually, it is 80 to 100 degreeC, Preferably it is 85 to 95 degreeC. The acid treatment time may be until the amniotic membrane is completely dissolved, and is usually about 60 minutes to 90 minutes.
酸処理後は、塩基で処理することにより、組成物を中和することが好ましい。塩基は可溶化羊膜組成物を基体上にコーティング、乾燥して得られるコーティング材中に残留しない塩基が好ましく、アンモニア水が好ましい。アンモニア水の濃度は特に限定されないが、通常25ｍＭ〜400ｍＭ、より好ましくは50ｍＭ〜200ｍＭ程度である。中和後のpHは、中性、すなわちｐH6.5〜7.5程度が好ましい。
上記のようにして、可溶化された羊膜が媒体中に含まれる可溶化羊膜組成物を得ることができる。媒体は水が好ましく、上記の酸処理において酸の水溶液を用いることにより、媒体が水である組成物を容易に得ることができる。また、塩基の中和処理にアンモニア水のような塩基の水溶液を用いることにより、媒体が水である組成物を容易に得ることができる。
After the acid treatment, the composition is preferably neutralized by treatment with a base. The base is preferably a base that does not remain in the coating material obtained by coating and drying the solubilized amniotic membrane composition on a substrate, and ammonia water is preferred. The concentration of the aqueous ammonia is not particularly limited, but is usually about 25 mM to 400 mM, more preferably about 50 mM to 200 mM. The neutralized pH is preferably neutral, that is, about pH 6.5 to 7.5.
As described above, a solubilized amniotic membrane composition containing solubilized amniotic membrane in a medium can be obtained. The medium is preferably water. By using an acid aqueous solution in the above acid treatment, a composition in which the medium is water can be easily obtained. Moreover, the composition whose water is a medium can be easily obtained by using the aqueous solution of the base like ammonia water for the neutralization treatment of the base.
次に、このようにして得られた可溶化羊膜組成物を予め表面処理された基体上にコーティングし、乾燥させることにより本実施形態の細胞培養用基材を得ることができる。基体としては、通常の細胞培養に用いられる基体でよく、例えば培養ディッシュ、シャーレ、マイクロタイタープレートを好適に用いることができる。基体の材質は限定されるものではなく、ガラスや樹脂等（ポリスチレン、ポリカーボネート、ＰＭＭＡ、シクロオレフィンポリマー、シクロオレフィンコポリマー）の通常の細胞培養に用いることのできる材質であればよい。また樹脂では複屈折が少なく、蛍光発色が少ない材料が望まれ、微分干渉顕微鏡や蛍光顕微鏡で観察することができる。
基体に表面処理を行う方法としては、プラズマ表面処理が好適に用いられる。プラズマ表面処理は少なくとも基体のコーティング予定面に対して行われていればよい。また、プラズマ表面処理は略真空中、または大気圧中にて行うことが可能である。プラズマ表面処理を略真空中で行う場合には、排気ポンプ等により装置内を真空にすることのできる真空装置内に基体を置き、真空状態にした上で、不活性ガスまたは活性ガス等を若干量導入し、装置内に設置されたプラズマ発生装置によりプラズマを発生させ、基体の表面処理を行う。装置内に導入される不活性ガスとしては、例えばアルゴン、窒素等が好適に用いられる。また、活性ガスとしては、例えば酸素が好適に用いられる。なお、略真空状態とされるプラズマ表面処理における真空度は、好ましくは１．０×１０-2Ｐａ〜３．０×１０2Ｐａ、より好ましくは１．０×１０-1Ｐａ〜１．３×１０2Ｐａ程度である。また、プラズマ出力条件としては、基体への表面処理が好適に行われる程度であればよく、好ましくは出力１０Ｗ〜１ｋＷ程度、より好ましくは、１００Ｗ〜５００Ｗ程度であり、照射時間（発生時間）は、好ましくは１分〜６０分、より好ましくは３分〜１５分程度である。また、表面処理としては、プラズマ表面処理以外にコロナ放電処理等の既存の表面処理技術を用いることも可能である。
Next, the cell culture substrate of the present embodiment can be obtained by coating the solubilized amniotic membrane composition thus obtained on a previously surface-treated substrate and drying it. The substrate may be a substrate used for normal cell culture. For example, a culture dish, a petri dish, or a microtiter plate can be suitably used. The material of the substrate is not limited, and any material that can be used for normal cell culture of glass, resin, etc. (polystyrene, polycarbonate, PMMA, cycloolefin polymer, cycloolefin copolymer) may be used. In addition, a resin is desired to have a material having little birefringence and little color development of fluorescence, and can be observed with a differential interference microscope or a fluorescence microscope.
As a method for performing surface treatment on the substrate, plasma surface treatment is preferably used. The plasma surface treatment should just be performed with respect to the coating surface of a base | substrate at least. The plasma surface treatment can be performed in a substantially vacuum or atmospheric pressure. When the plasma surface treatment is performed in a substantially vacuum, the substrate is placed in a vacuum device that can be evacuated by an exhaust pump or the like, and after a vacuum state, an inert gas or an active gas is slightly added. The amount is introduced, plasma is generated by a plasma generator installed in the apparatus, and surface treatment of the substrate is performed. As the inert gas introduced into the apparatus, for example, argon, nitrogen or the like is preferably used. As the active gas, for example, oxygen is preferably used. Note that the degree of vacuum in the plasma surface treatment to be in a substantially vacuum state is preferably 1.0 × 10 −2 Pa to 3.0 × 10 2 Pa, more preferably 1.0 × 10 −1 Pa to 1.3. It is about × 10 2 Pa. The plasma output condition may be such that the surface treatment to the substrate is suitably performed, preferably the output is about 10 W to 1 kW, more preferably about 100 W to 500 W, and the irradiation time (generation time) is It is preferably 1 minute to 60 minutes, more preferably about 3 minutes to 15 minutes. In addition to the plasma surface treatment, an existing surface treatment technique such as corona discharge treatment can be used as the surface treatment.
このように表面処理された基体上に可溶化羊膜組成物をコーティングし乾燥させることにより、細胞培養用基材を得る。コーティングは塗布、浸漬、スプレー等の周知の方法により行うことができる。また、乾燥は自然乾燥、減圧乾燥等、周知の方法により行うことができる。また、得られた本実施形態の細胞培養用基材に対して放射線による滅菌を施すこともできる。このような滅菌処理に用いられる放射線としては、γ線、電子線、ｘ線等が用いられ、好ましくはγ線である。放射線を上述した細胞培養用基材に対して滅菌処理可能な所定時間だけ照射し、滅菌を完了させる。γ線による滅菌処理の場合は、１ｋGray〜５０ｋGrayの範囲にて、１時間〜１０時間程度照射を行えばよい。
なお、上述した可溶化羊膜組成物をコーティングして得られる細胞培養用基材は、常温による保管が可能であり、保管管理が行いやすい。
A substrate for cell culture is obtained by coating the solubilized amniotic membrane composition on the surface-treated substrate and drying it. The coating can be performed by a known method such as coating, dipping, spraying or the like. Drying can be performed by a known method such as natural drying or drying under reduced pressure. The obtained cell culture substrate of the present embodiment can be sterilized by radiation. As the radiation used for such sterilization treatment, γ-rays, electron beams, x-rays, and the like are used, and preferably γ-rays. Radiation is irradiated to the above-mentioned cell culture substrate for a predetermined time that can be sterilized to complete sterilization. In the case of sterilization treatment using γ rays, irradiation may be performed in the range of 1 kGray to 50 kGray for about 1 to 10 hours.
Note that the cell culture substrate obtained by coating the above-described solubilized amniotic membrane composition can be stored at room temperature, and is easily stored and managed.
以下、本発明を実験例に基づき具体的に説明する。
＜可溶化羊膜組成物の調製＞
初めに以下の方法により、ヒト羊膜の可溶化物を水中に含む組成物を調製した。
分離羊膜5ｇ（湿重量）を５０mlチューブに入れ、ここに４５mlPBS（−）を加え、室温で５分間強く震盪させる。0.02%のEDTA、45mLにて5回洗浄した後、蒸留脱イオン水４５mlで3回洗浄する。次に、４５mlの２５０mM酢酸を加え、９０℃湯煎で羊膜が溶解するまで、時々震盪させる。羊膜の溶解を確認後、０．１Ｍアンモニア水で中和させ、溶液の吸光度を計測し、羊膜由来タンパク質の濃度を測定した。羊膜由来タンパク質1mg を含む可溶化羊膜溶液を別容器に移し、凍結乾燥にさせ、1mgの粉末状の可溶化羊膜生成物を得た。この凍結乾燥可溶化羊膜生成物を1mLの滅菌蒸留水に溶解し、目的とする可溶化羊膜組成物（１mg／１ml）を得た。
Hereinafter, the present invention will be specifically described based on experimental examples.
<Preparation of solubilized amniotic membrane composition>
First, a composition containing solubilized human amniotic membrane in water was prepared by the following method.
Place 5 g (wet weight) of the separated amniotic membrane into a 50 ml tube, add 45 ml PBS (−) to this, and shake vigorously for 5 minutes at room temperature. Wash 5 times with 45 mL of 0.02% EDTA, then 3 times with 45 ml of distilled deionized water. Next, 45 ml of 250 mM acetic acid is added and occasionally shaken in a 90 ° C. water bath until the amniotic membrane is dissolved. After confirming dissolution of the amniotic membrane, the solution was neutralized with 0.1 M aqueous ammonia, the absorbance of the solution was measured, and the concentration of the amniotic membrane-derived protein was measured. A solubilized amniotic solution containing 1 mg of amnion-derived protein was transferred to another container and freeze-dried to obtain 1 mg of a powdered solubilized amniotic membrane product. This freeze-dried solubilized amniotic membrane product was dissolved in 1 mL of sterile distilled water to obtain the desired solubilized amniotic membrane composition (1 mg / 1 ml).
＜実験例１＞
φ６０ｍｍの培養用ディッシュ（FALCON製 353002 培養面積21cm2）に対してプラズマ照射による表面処理を行った。プラズマ表面処理は、培養用ディッシュを真空プラズマ照射装置（サムコ製 ＰＸ−１０００）内に置き、排気ポンプを用いて一旦、真空状態にした後、酸素を導入し、真空度５．７×１０Ｐａの略真空状態でプラズマを培養用ディッシュに照射することにより行った。プラズマの出力条件としては、出力300W、照射時間350秒にて行った。表面処理された培養用ディッシュの底面に、上述した調製により得られた可溶化羊膜組成物（濃度１mg／１ml）をコーティングした。コーティングに用いる可溶化羊膜組成物の添加量は、3種類（10.5μＬ／dish，21μＬ／dish，105μＬ／dish）とした。可溶化羊膜組成物が添加された３種類の培養用ディッシュ（10.5μＬ／dish，21μＬ／dish，105μＬ／dish）を、減圧乾燥（０．１ＭPa、３時間真空）により完全に乾燥させ、可溶化羊膜相生物がコーティングされた培養用ディッシュを各々得た。３種類のコーティング済培養用ディッシュは、それぞれ培養用ディッシュＡ１（10.5μＬ／dish 可溶化羊膜量0.5μg／cm2），培養用ディッシュＡ２（21μＬ／dish 可溶化羊膜量1.0μg／cm2），培養用ディッシュＡ３（105μＬ／dish 可溶化羊膜量5.0μg／cm2）とした。次に、これらの培養用ディッシュＡ１〜Ａ３上にヒト羊膜間葉細胞（約１×１０5個）を播種し、培養液として１０％ＦＢＳ添加したDMEM／F12を2.0〜2.5mL入れ、３７℃で3日間培養を行った。培養開始から３日後に、トリプシン酵素溶液を用いて、各ディッシュから細胞を分離・回収し、血球算定盤を用いて全細胞数を計測した。この細胞数をもとに、細胞増殖倍率を求めた。結果を表１に示す。
<Experimental example 1>
A surface treatment by plasma irradiation was performed on a φ60 mm dish (353002, culture area 21 cm 2 manufactured by FALCON). In the plasma surface treatment, the culture dish is placed in a vacuum plasma irradiation apparatus (PX-1000 manufactured by Samco), and once evacuated using an exhaust pump, oxygen is introduced, and the degree of vacuum is 5.7 × 10 Pa. This was performed by irradiating the culture dish with plasma in a substantially vacuum state. As the plasma output conditions, the output was 300 W and the irradiation time was 350 seconds. The bottom surface of the surface-treated culture dish was coated with the solubilized amniotic membrane composition (concentration 1 mg / 1 ml) obtained by the above preparation. The amount of solubilized amniotic membrane composition used for coating was three types (10.5 μL / dish, 21 μL / dish, 105 μL / dish). Three types of culture dishes (10.5μL / dish, 21μL / dish, 105μL / dish) to which a solubilized amniotic membrane composition was added were completely dried by solubilization under reduced pressure (0.1 MPa, 3 hours in vacuum) and solubilized. Each culture dish coated with amniotic phase organisms was obtained. The three types of coated culture dishes are culture dish A1 (10.5 μL / dish solubilized amniotic membrane amount 0.5 μg / cm 2 ), culture dish A2 (21 μL / dish solubilized amniotic membrane amount 1.0 μg / cm 2 ), A dish A3 for culture (105 μL / dish solubilized amniotic membrane amount 5.0 μg / cm 2 ) was used. Next, human amniotic mesenchymal cells (about 1 × 10 5 cells) were seeded on these culture dishes A1 to A3, and 2.0 to 2.5 mL of DMEM / F12 supplemented with 10% FBS was added as a culture solution at 37 ° C. For 3 days. Three days after the start of culture, cells were separated and collected from each dish using a trypsin enzyme solution, and the total number of cells was counted using a hemocytometer. Based on this number of cells, the cell growth rate was determined. The results are shown in Table 1.
＜実験例２＞
実験例２では、実験例１に対してプラズマ表面処理を行わず作成した３種類のコーティング済培養用ディッシュを細胞培養に用いた。用いた３種類のコーティング済ディッシュ（プラズマ表面処理なし）をそれぞれ培養用ディッシュＢ１（可溶化羊膜量0.5μg／cm2），培養用ディッシュＢ２（可溶化羊膜量1.0μg／cm2），培養用ディッシュＢ３（可溶化羊膜量5.0μg／cm2）とし、実験例１と同様の細胞培養条件にてヒト羊膜間葉細胞の細胞培養を行い、細胞増殖倍率を求めた。結果を表１に示す。
<Experimental example 2>
In Experimental Example 2, three types of coated culture dishes prepared for Experimental Example 1 without performing plasma surface treatment were used for cell culture. Three kinds of coated dishes (without plasma surface treatment) used were cultured dish B1 (amount of solubilized amniotic membrane 0.5 μg / cm 2 ), culture dish B2 (amount of solubilized amniotic membrane 1.0 μg / cm 2 ), and for culture Cell culture of human amnion mesenchymal cells was carried out under the same cell culture conditions as in Experimental Example 1 with dish B3 (solubilized amniotic membrane amount 5.0 μg / cm 2 ), and the cell growth rate was determined. The results are shown in Table 1.
＜実験例３＞
実験例１で用いた培養用ディッシュ（FALCON製 353002 培養面積21cm2）に対して、可溶化羊膜組成物によるコーティング、及び表面処理の両処理とも行わず、これを培養用ディッシュＣとして、実験例１と同様の細胞培養条件にてヒト羊膜間葉細胞の細胞培養を行い、細胞増殖倍率を求めた。結果を表１に示す。
<Experimental example 3>
The culture dish used in Experimental Example 1 (353002 culture area 21 cm 2 manufactured by FALCON) was not subjected to both the coating with the solubilized amniotic membrane composition and the surface treatment. Cell culture of human amnion mesenchymal cells was performed under the same cell culture conditions as in Example 1, and the cell growth rate was determined. The results are shown in Table 1.
＜実験例４＞
実験例１で用いた培養用ディッシュ（FALCON製 353002 培養面積21cm2）に対して、プラズマ表面処理のみを行ったデッシュを培養用ディッシュＤとし、実験例１と同様の細胞培養条件にてヒト羊膜間葉細胞の細胞培養を行い、細胞増殖倍率を求めた。結果を表１に示す。
<Experimental example 4>
For the culture dish used in Experimental Example 1 (353002 made by FALCON, culture area 21 cm 2 ), the dish on which only the plasma surface treatment was performed was designated as Culture Dish D, and human amniotic membrane under the same cell culture conditions as in Experimental Example 1 The mesenchymal cells were cultured and the cell growth rate was determined. The results are shown in Table 1.
＜実験例５＞
実験例１に示す方法により得られた３種類（可溶化羊膜量0.5μg／cm2，可溶化羊膜量1.0μg／cm2，可溶化溶化羊膜量5.0μg／cm2）の培養用ディッシュＡ１〜Ａ３（表面処理あり、コーティング済）に対してγ線照射による滅菌処理を行い、滅菌処理済みの培養用ディッシュＡ´１〜Ａ´３を得た。γ線照射はγ線照射装置を用いてディッシュに対して10ｋGrayにて5時間行った。照射得られた滅菌処理済みの培養用ディッシュＡ´１〜Ａ´３、及び実験例１にて用いた培養用ディッシュＡ１〜Ａ３を用いてヒト羊膜間葉細胞の細胞培養を行い、細胞増殖倍率、及びγ線非照射ディッシュにおける細胞増殖倍率に対してγ線照射ディッシュの細胞増殖倍率の変化を求めた。細胞培養条件は、培養期間４日とした以外は、実験例１と同条件とした。結果を表２に示す。
<Experimental example 5>
Three types of culture dishes A1 to A3 obtained by the method shown in Experimental Example 1 (solubilized amniotic membrane amount 0.5 μg / cm 2 , solubilized amniotic membrane amount 1.0 μg / cm 2 , solubilized solubilized amniotic membrane amount 5.0 μg / cm 2 ) A3 (with surface treatment and coated) was sterilized by γ-ray irradiation to obtain cultivated dishes A′1 to A′3. The γ-ray irradiation was performed for 5 hours at 10 kGray on the dish using a γ-ray irradiation apparatus. Cell culture of human amnion mesenchymal cells is carried out using the sterilized culture dishes A′1 to A′3 obtained by irradiation and the culture dishes A1 to A3 used in Experimental Example 1. The change in the cell growth rate of the γ-irradiated dish was determined relative to the cell growth rate in the non-γ-irradiated dish. The cell culture conditions were the same as those in Experimental Example 1 except that the culture period was 4 days. The results are shown in Table 2.
＜実験例６＞
実験例２に示す方法により得られた３種類（可溶化羊膜量0.5μg／cm2，可溶化羊膜量1.0μg／cm2，可溶化溶化羊膜量5.0μg／cm2）の培養用ディッシュＢ１〜Ｂ３（表面処理なし、コーティング済）に対して実験例５と同様にγ線照射による滅菌処理を行い、滅菌処理済みの培養用ディッシュＢ´１〜Ｂ´３を得た。得られた滅菌処理済みの培養用ディッシュＢ´１〜Ｂ´３、及び実験例２にて用いた培養用ディッシュＢ１〜Ｂ３を用いて実験例５と同様の細胞培養条件にてヒト羊膜間葉細胞の細胞培養を行い、細胞増殖倍率、及びγ線非照射ディッシュにおける細胞増殖倍率に対してγ線照射ディッシュの細胞増殖倍率の変化を求めた。細胞培養条件は、培養期間４日とした以外は、実験例１と同条件とした。結果を表２に示す。
<Experimental example 6>
Three types of culture dishes B1 to B1 obtained by the method shown in Experimental Example 2 (solubilized amniotic membrane amount 0.5 μg / cm 2 , solubilized amniotic membrane amount 1.0 μg / cm 2 , solubilized solubilized amniotic membrane amount 5.0 μg / cm 2 ) B3 (no surface treatment, coated) was sterilized by γ-ray irradiation in the same manner as in Experimental Example 5 to obtain sterilized culture dishes B′1 to B′3. Using the obtained sterilized culture dishes B′1 to B′3 and the culture dishes B1 to B3 used in Experimental Example 2, human amniotic mesenchyme under the same cell culture conditions as in Experimental Example 5 The cells were cultured, and the change in the cell growth rate of the γ-irradiated dish was determined relative to the cell growth rate and the cell growth rate in the non-γ-irradiated dish. The cell culture conditions were the same as those in Experimental Example 1 except that the culture period was 4 days. The results are shown in Table 2.
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