JP7335971B2 - Nk細胞培養培地用添加組成物、前記添加組成物を用いたnk細胞培養方法、及び前記培養方法で得られた皮膚トラブル改善用化粧料組成物 - Google Patents
Nk細胞培養培地用添加組成物、前記添加組成物を用いたnk細胞培養方法、及び前記培養方法で得られた皮膚トラブル改善用化粧料組成物 Download PDFInfo
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Description
IL-2 2.2mgと500mM L-グルタミン溶液100mLを浮遊細胞培養用基本培地(基本培地にIL-2又はL-グルタミンが既に存在する場合、最終濃度を合わせるために添加量を調整する)に入れて溶かし、最終的に10Lにして基本溶液(B溶液)を製造した。
IL-12 20μgとIL-18 125μgを蒸留水に溶かして10mLにしてサイトカイン溶液(C溶液)を製造した。C溶液1mLを基本溶液(B1溶液)1000mLに溶かしてサイトカイン1-1溶液(C1-1溶液)を製造した。
サイトカイン1-1溶液にリン酸デキサメタゾンナトリウム(Dexamethasone sodium phosphate)が1μg/mLの濃度で含まれるように溶解させ、NK細胞培養培地用添加組成物1を準備した。
(1)A液:抗CD16抗体及び抗CD56抗体がそれぞれ0.01~1.5μg/mLの濃度で含まれるように抗CD16抗体及び抗CD56抗体をB1溶液に溶かして作った溶液
(2)A1溶液:抗CD16抗体が0.1~15μg/mLの濃度で含まれるように抗CD16をC1-1溶液に溶かして作った溶液
(3)A2溶液:抗CD56抗体が0.1~15μg/mLの濃度で含まれるように抗CD56抗体をC1-1溶液に溶かして作った溶液
(4)B1溶液:浮遊細胞培養用基本培地溶液にIL-2を1000~4000IU/mLの濃度となるように作った溶液
(5)B2溶液:B1よりIL-2の濃度が2倍である溶液
(6)C1-1溶液:IL-12が0.5~5ng/mLの濃度、IL-18が2~50ng/mLの濃度で含まれるようにIL-12及びIL-18をB1溶液に溶かして作った溶液
(7)C5溶液:C1-1溶液よりIL-2、IL-18の濃度が5倍である溶液
(8)D溶液:抗CD3抗体が1~12μg/mLの濃度で含まれるように抗CD3抗体をC1-1溶液に溶かして作った溶液
(9)R溶液:サイトカイン(Cytokine)や抗体が含まれておらず、L-グルタミンが3~12mMの濃度で含まれている浮遊細胞培養用基本培地溶液
リンパ球抽出及び自己血漿準備段階を先行特許と同様に行った。
Day1:細胞培養中の細胞にA2溶液1mLを追加した。
Day2:細胞培養中の細胞にD溶液1mLを追加した。
Day3:細胞培養中の細胞にNK細胞培養培地用添加組成物1を5mL追加した。
Day4:必要な場合は、より大きな培養容器に細胞を移して培養する。細胞培養中の細胞にR溶液3mLを追加するか、或いはC1-1溶液10mLで培地を交換する。
Day5:細胞培養中の細胞にA1溶液20mLを追加した。
Day6:細胞培養中の細胞にA溶液20mLを追加した。
Day7:細胞培養中の細胞にB1溶液40mLを追加した。
Day8:細胞培養中の細胞にR溶液300mLを追加した。
Day10:細胞培養中の細胞にR溶液300mLを追加した。
Day11:細胞培養中の細胞にB2溶液300mLとC5溶液50mLを追加した。
Day14~Day19:細胞を収穫した。
Day3にNK細胞培養培地用添加組成物1を添加していない以外は、実施例5と同様の方法でNK細胞を培養した。
制御性T細胞(Treg)抑制用薬物の一つ以上を有効成分として含むNK細胞培養培地用添加組成物がNK細胞培養の際に及ぼす効果を確認するために、実施例5と同様の方法でNK細胞培養の際に得られる細胞増殖結果と、比較例1と同様の方法で得られる細胞増殖結果を観察し、その結果を表1(単位:×107個)、表2、図1~図2bに示した。
制御性T細胞(Treg)抑制用薬物として様々なステロイド剤を含むNK細胞培養培地用添加組成物の効果を確認するために、実施例6~8と同様の方法でNK細胞培養の際に得られる細胞増殖結果を観察し、その結果を表3(単位:×107個)に示した。
NK細胞の割合が約50%程度となる細胞を使用し、NK細胞培養培地用添加組成物に含まれるステロイド剤の濃度を約2倍に高めてday3、day4、day8に添加して培養した以外は、実施例5と同様の方法を行って得られた結果と、NK細胞の割合が約50%程度となる細胞を使用したことを除けば比較例1と同様の方法を行って得られた結果とを比較し、それぞれ図3a及び図3bに示した。
本発明のNK細胞培養培地用添加組成物の効果を確認するために、実施例のように別途のNK細胞培養用添加キットを使用せず、一般的に使用されるNK細胞培養方法を用いて、NK細胞を次のとおりに培養した。リンパ球(1×107)を分離してNK免疫細胞の刺激のためのIL-2、IL-12、IL-18などのサイトカイン(Cytokine)を用いて、細胞刺激後培養2日目に抗CD3抗体を刺激して培養し、CD16、CD56抗体などは全く使用せず、培養3日目にNK細胞培養培地用組成物1を処理した。その後、培地は、IL-2が添加された一般培地を使用した。細胞数の増加に応じて培地を添加しながら細胞を培養した。14日間培養を行い続けた後、その結果を表4、図4a及び図4bに示した。
本発明のNK細胞培養培地用添加組成物の効果を確認するために、実施例のように別途のNK細胞培養用添加キットを使用せず、一般的に使用されるNK細胞培養方法、特に固定化された抗CD3抗体を用いて、次のとおりにNK細胞を培養した。抗CD3抗体をT25フラスコに固定化した後、分離されたリンパ球(0.8×107)を約1時間反応させ、その後、IL-2及びCD16、CD56抗体を用いて培養した。CD16、CD56抗体は2日目、4日目、6日目に刺激をし、培養3日目にNK細胞培養培地用組成物1を処理した。その後、培地は、IL-2が添加された一般培地を使用した。細胞数の増加に応じて培地を添加し、細胞を培養した。14日間培養を行い続けた後、その結果を表5、図5a及び図5bに示した。
本発明のNK細胞培養培地用添加組成物が、活力が低下した細胞の再活性に及ぼす影響力を実験するために、実施例5の方法によってTreg細胞を抑制しつつ培養して25日目になり、活性が低下した老化細胞を用いて再活性化を次のように試みた。既存の培地を除去するために遠心分離して上澄み液を除去し、一般培地(RPMI)に入れて約2時間培養した後、培地をC1溶液で交換して3日培養した。
実施例9で再活性化されたNK細胞の活性度を測定し、その結果を表6に示した。
下記表6に示すように、Tregによって活性が抑制されずに増加することが観察され、細胞の数も急激に減少することは観察されなかった。ただし、測定誤差とも考えられるが、細胞が老化して自然減少する程度の若干の細胞数減少は観察された。この方法は、非常に短時間内(1日~3日)にNK細胞を活性化することができる方法であって、老化細胞だけでなく、末梢血から直ちに分離したPBMC中のNK細胞の活性化はもとより、凍結後に解凍されて活力が低下したNK細胞の再活性化にも非常に効果的であると予測される。
血液内にIL-10の濃度が高いためTreg細胞が多く活性化されているものと思われるヒトからPBMCを得た以外は、実施例5と同様の方法でNK細胞を培養した後、day12に細胞を取り出して既存の培養液を除去し、C1溶液を用いて2日間培養した後、NK細胞を分離して残った培養培地組成物を得た。
実施例10で得られた培養培地組成物の成分を確認し、その結果を表7に示した。
Claims (3)
- 制御性T細胞(Treg)活性抑制用薬物の一つ以上を有効成分として含む、NK細胞を増殖するためのNK細胞培養培地用添加組成物であって、
前記有効成分は、ステロイド剤であって、NK細胞が増殖されるように作用し、
前記ステロイド剤はグルココルチコイド(glucocorticoids)であり、
前記グルココルチコイドは、コルチゾール(Cortisol)、コルチゾン(Cortisone)、プレドニゾロン(Prednisolone)、プレドニゾン(prednisone)、メチルプレドニゾロン(Methylpredni solone)、デキサメタゾン(Dexamethasone)、ベタメタゾン(Betamethasone)、トリアムシノロン(Triamcinolone)、酢酸フルドロコルチゾン(Fludrocortisone acetate)、及び酢酸デオキシコルチコステロン(deoxycorticosterone acetate)よりなる群から選ばれる1つ以上であり、
前記NK細胞培養培地用添加組成物は、さらに、IL-12およびIL-18を含有することを特徴とする、NK細胞培養培地用添加組成物。 - 前記添加組成物は、NK細胞培養段階で前記NK細胞培養培地のT細胞が刺激された後に処理されることを特徴とする、請求項1に記載のNK細胞培養培地用添加組成物。
- 前記ステロイド剤は0.2μg/ml以上の濃度で含まれることを特徴とする、請求項1又は2に記載のNK細胞培養培地用添加組成物。
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US20220177844A1 (en) | 2022-06-09 |
KR102216710B1 (ko) | 2021-02-17 |
WO2020197216A1 (ko) | 2020-10-01 |
JP2022528079A (ja) | 2022-06-08 |
KR20200115838A (ko) | 2020-10-08 |
CN113874491A (zh) | 2021-12-31 |
EP3950936A4 (en) | 2022-12-21 |
EP3950936A1 (en) | 2022-02-09 |
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