WO2016151741A1 - Nk細胞培養容器及びnk細胞培養方法 - Google Patents
Nk細胞培養容器及びnk細胞培養方法 Download PDFInfo
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- 210000000822 natural killer cell Anatomy 0.000 title claims abstract description 89
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0646—Natural killers cells [NK], NKT cells
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- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
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- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
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- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/30—Synthetic polymers
Definitions
- the present invention relates to a NK cell culture vessel and a NK cell culture method.
- ⁇ Immuno-cell therapy for cancer using autologous peripheral blood lymphocytes is classified into ⁇ T lymphocyte therapy, ⁇ T lymphocyte therapy, NK cell therapy, NKT lymphocyte therapy, etc., mainly depending on the proliferating cells.
- lymphocytes are first stimulated strongly using antibodies, cytokines, and certain stimulating substances, so that lymphocytes are activated to determine the direction of differentiation and acquire proliferative ability.
- Activated lymphocytes begin to proliferate in the presence of lymphocyte growth factors (such as interleukin 2) and continue to predominately grow certain lymphocyte subgroups.
- lymphocyte growth factors such as interleukin 2
- anti-CD3 antibody is used for the initial stimulation, after which ⁇ T lymphocytes are continuously expanded in the presence of IL-2 and are usually used after 2 weeks of culture.
- stimulation of lymphocytes with anti-CD3 antibody was carried out by immobilizing the antibody in a plastic culture flask, but it was complicated and could be contaminated with microorganisms.
- a sealed polyethylene bag with an anti-CD3 antibody immobilized on the market is commercially available, can induce stimulation and proliferation of ⁇ T lymphocytes equivalent to or higher than that of a flask, and is provided ready to use. Widely used as a bag with excellent quality and safety.
- a method has been developed in which peripheral blood lymphocytes are stimulated with two antibodies, an anti-CD3 antibody and an anti-CD52 antibody, to suppress unilateral proliferation of T lymphocytes and induce NK cells predominantly.
- This method can also induce NK cells by immobilizing an antibody in a polyethylene resin bag like ⁇ T lymphocytes.
- the method to induce NK cells predominantly by stimulating peripheral blood lymphocytes with two antibodies, anti-CD3 antibody and anti-CD52 antibody, is to immobilize the antibody in a polyethylene resin bag like ⁇ T lymphocytes.
- NK cells can be induced.
- polyethylene resin bags have excellent sealing properties against microbial contamination, but NK cell induction may be low, and there is a need to develop a culture vessel that enhances this low NK cell induction.
- plastic culture flasks have been conventionally used for stimulation of peripheral blood lymphocytes by anti-CD3 antibody and anti-CD52 antibody, but there are cases in which NK cell induction ability is low in this case as well.
- the bag culture requires a step of washing the antibody in the bag before adding peripheral blood lymphocytes. Therefore, development of a simpler culture method and higher NK cell induction efficiency is required.
- a biological sample containing mononuclear cells is partly or entirely formed of a cycloolefin polymer in contact with the biological sample, and anti-CD3 High NK cell proliferation efficiency (high NK cell induction efficiency) is obtained by culturing in a cell culture vessel characterized in that the antibody and anti-CD52 antibody are coated on part or all of the surface in contact with the biological sample.
- the present invention has been completed by confirming that this is possible.
- this invention consists of the following. 1. An NK cell culture vessel for culturing NK cells, Part or all of the surface in contact with the biological sample is formed of a cycloolefin polymer, and An NK cell culture container, wherein an anti-CD3 antibody and an anti-CD52 antibody are coated on a part or all of a surface in contact with the biological sample. 2. A biological sample containing mononuclear cells is coated on a part or all of the surface in contact with the biological sample, wherein part or all of the surface in contact with the biological sample is formed of a cycloolefin polymer and the anti-CD3 antibody and anti-CD52 antibody are in contact with the biological sample.
- a method for culturing NK cells comprising culturing by adding to a cultured vessel. 3.
- a method for culturing NK cells comprising adding a biological sample containing mononuclear cells to a culture vessel containing beads to which an anti-CD3 antibody and an anti-CD52 antibody are bound, and culturing. 4). Adding and culturing a biological sample containing mononuclear cells to a culture container in which part or all of the surface in contact with the biological sample containing beads bound with anti-CD3 antibody and anti-CD52 antibody is formed of cycloolefin polymer NK cell culture method comprising
- the NK cell culture method using the NK cell culture container and antibody-bound beads of the present invention has high NK cell culture efficiency.
- a part or all of the surface in contact with the biological sample is formed of a cycloolefin polymer, and a part or all of the surface in contact with the biological sample is coated with the anti-CD3 antibody and the anti-CD52 antibody.
- a characteristic NK cell culture vessel, and a NK cell culture method using the NK cell culture vessel will be described in detail below.
- NK cells As the NK cell used in the present invention, any NK cell obtained by a method known per se can be used.
- the NK cell donor (donor) and the NK cell recipient (receiver) are preferably of the same type. For example, if the donor is a human, the recipient is a human. More preferably, the NK cell donor and the NK cell recipient are the same individual. For example, if the donor is provider X, the recipient is provider X.
- NK cell culture means NK cell differentiation, stimulation, mutation, induction, maintenance, proliferation, activation and the like, but is not particularly limited.
- NK cells used in the present invention can be obtained by a method known per se (see: Patent No. 5016732). NK cells are obtained by induction from peripheral blood, lymph nodes, thymus, bone marrow, tumor, pleural effusion, ascites or umbilical cord blood, more preferably from peripheral blood mononuclear cells. For example, mononuclear cells containing NK cells can be collected from peripheral blood by specific gravity centrifugation.
- NK cell activation method T lymphocytes and monocytes containing NK cells are stimulated with CD3 agonist (especially anti-CD3 antibody) and CD52 agonist (especially anti-CD52 antibody), so that NK cells are treated with T lymphocytes. It can be activated more than a sphere, and NK cells can be proliferated safely and easily without mixing with K562 or the like.
- NK cells are more than T lymphocytes compared with IL-2 single stimulation. Can also be grown.
- NK cells can be proliferated 1000 times or more (refer to JP 2005-124568).
- the biological sample used in the present invention is not particularly limited as long as NK cells can be cultured by stimulation with anti-CD3 antibody and anti-CD52 antibody, but peripheral blood, lymph node, thymus, bone marrow, tumor, pleural effusion, ascites or umbilical cord Mononuclear cells collected from blood, more preferably peripheral blood mononuclear cells can be listed.
- cycloolefin polymer The cycloolefin polymer used in the present invention (sometimes referred to as “cycloolefin” or “COP”) is preferably composed mainly of COP (50 mass% or more, 60 mass% or more, 70 mass% or more, 80 mass%). % Or more and 90% by mass or more).
- COP means a polymer having a cyclic olefin structure.
- the cycloolefin polymer is marketed, for example, ZEONEX (TM) (ZEONEX: Nippon Zeon Co., Ltd.) etc. can be used.
- the anti-CD3 antibody and anti-CD52 antibody used in the present invention are not particularly limited as long as NK cells can be cultured by contacting with a biological sample.
- the anti-CD3 antibody and anti-CD52 antibody coating are bonded to the contact surface of the cycloolefin polymer or the biological sample of the culture vessel with some force (covalent bond, hydrogen bond, electrostatic interaction, hydrophobic interaction, etc.). Means the state.
- the beads used in the present invention are not particularly limited as long as they can bind an anti-CD3 antibody and an anti-CD52 antibody.
- a preferred bead is a magnetic bead considering that it can be easily recovered.
- NK cell culture vessel In the NK cell culture container of the present invention, at least a part of or the entire surface in contact with the biological sample is formed of a cycloolefin polymer, and the surface on which the anti-CD3 antibody and the anti-CD52 antibody are in contact with the biological sample. Part or all is coated.
- the container body that stores the biological sample, the culture solution, and the like may have any shape, but a back shape is preferable in consideration of transportation, storage, and the like.
- a container main body means the part which accommodates a biological sample, a culture solution, etc., the container main body itself can become a culture container.
- NK cell culture method examples include the following methods. (1) A biological sample containing mononuclear cells is partially or wholly formed of a cycloolefin polymer and a part of the surface in contact with the biological sample, or a part of the surface in contact with the biological sample or the anti-CD3 antibody or anti-CD52 antibody A method of culturing by adding to a culture vessel that is coated all over. (2) A method in which a biological sample containing mononuclear cells is added to a culture vessel containing beads bound with anti-CD3 antibody and anti-CD52 antibody and cultured.
- NK cell culture using cycloolefin polymer bags NK cell culture using a cycloolefin polymer bag was compared with NK cell culture using a polyethylene resin bag. Details are as follows.
- the anti-CD3 antibody and the anti-CD52 antibody were immobilized on a polyethylene resin bag ⁇ tetta (untreated bag) ⁇ , manufactured by Kojin Bio Inc.) and a cycloolefin polymer bag (manufactured by Fukoku), respectively.
- a polyethylene resin bag ⁇ tetta (untreated bag) ⁇ manufactured by Kojin Bio Inc.
- a cycloolefin polymer bag manufactured by Fukoku
- the bag was allowed to stand at 4-8 ° C. for 24 hours, and then both bags were washed twice with 20 ml of PBS. After the washing, 40 ml of a culture solution (Cellex NKGM-1; manufactured by Kojin Bio Inc.) containing 4 ⁇ 10 7 healthy human peripheral blood mononuclear cells (PBMC), 4 ml of autologous plasma, and 500 U / ml of IL-2 are added to both bags. The cells were cultured at 37 ° C., 5% CO 2 .
- a culture solution Cellex NKGM-1; manufactured by Kojin Bio Inc.
- NK cell culture using a cycloolefin resin bag can obtain about 5 times the NK cell culture effect compared to NK cell culture using a polyethylene polymer bag. It was.
- NK cell culture using antibody-bound magnetic beads NK cell culture using antibody-bound magnetic beads was compared with NK cell culture using antibody-bound plastic culture plates. Details are as follows.
- Dynabeads Tosylactivated M-450 (Dynal) was used. Specifically, 100 ⁇ l of beads (4 ⁇ 10 7 beads) were washed twice with PBS, suspended in 1 ml of PBS, anti-CD3 antibody 100 ng / ml (OKT3; manufactured by Janssen Pharma) and anti-CD52 antibody 20 ⁇ g / ml (MabCampath) ; Manufactured by Sanofi) and stirred at room temperature for 24 hours.
- the anti-CD3 antibody and anti-CD52 antibody-bound magnetic beads are washed twice with PBS and suspended in 200 ⁇ l of 0.1% bovine serum albumin-added PBS to prepare an antibody solution containing anti-CD3 antibody and anti-CD52 antibody-bound magnetic beads. did.
- 0.4 ml of the above antibody solution without magnetic beads was added to each well of a 24-well plastic culture plate (Becton Dickinson, catalog number 3047), and allowed to stand at 4-8 ° C for 24 hours. The plate was washed twice with PBS immediately before use to prepare an antibody-binding plate.
- a culture solution containing 10% autologous plasma containing 1 x 10 6 PBMCs from two healthy individuals with relatively low NK cell proliferative capacity and 500 U / ml IL-2 (Cellex NKGM-1; manufactured by Kojin Bio Inc.) 1 ml was added to each well of an antibody-untreated 24-well plate (invention) and an antibody-bound 24-well plate (control). 2.5 ⁇ l (1 ⁇ 10 6 beads) of antibody-bound magnetic beads were added to PBMC added to the antibody-untreated plate. After the addition, the plate was incubated at 37 ° C. with 5% CO 2 .
- the 1/10 solution was transferred to a new plate, and 1 ml of the culture solution and IL-2 200 U / ml were added thereto. Thereafter, the culture was divided appropriately for 10 days, and the culture solution and IL-2 were added.
- 3 ⁇ 10 5 proliferating lymphocytes stimulated with antibody-bound magnetic beads (invention) and antibody-bound plate (control) were collected, and these were collected with fluorescently labeled antibodies (anti-CD3 antibody and anti-CD56 antibody). After staining, the ratio of NK cells (CD3-CD56 +) was detected and compared with a flow cytometer.
- FIG. 2 shows the detection results of the ratio of NK cells (CD3-CD56 +) with a flow cytometer.
- NK cell culture using antibody-bound magnetic beads has about twice the effect of NK cell culture compared to NK cell culture stimulated with an antibody-bound plastic plate. It was.
- a highly efficient NK cell culture container and NK cell culture method can be provided.
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Abstract
Description
最近は、密封ポリエチレン製バッグに抗CD3抗体を固相化させたものが市販され、フラスコと同等かそれ以上のαβTリンパ球の刺激と増殖を誘導でき、またready to useで提供されるため、品質・安全性に優れたバッグとして汎用されるようになっている。
一方、従来、抗CD3抗体と抗CD52抗体による末梢血リンパ球の刺激はプラスチック培養フラスコが使用されてきたが、この場合もNK細胞誘導能が低い症例が存在する。上述したフラスコ培養の問題点のほか、バッグ培養では末梢血リンパ球を入れる前にバッグ内の抗体洗浄の工程が必要なため、培養法がより簡便で、かつNK細胞誘導効率が高い方法の開発が必要である。
1.NK細胞を培養するためのNK細胞培養容器であって、
生体試料と接する面の一部又は全部がシクロオレフィンポリマーで形成されており、並びに、
抗CD3抗体及び抗CD52抗体が該生体試料と接する面の一部又は全部にコーティングされていることを特徴とするNK細胞培養容器。
2.単核球を含む生体試料を、生体試料と接する面の一部又は全部がシクロオレフィンポリマーで形成されており並びに抗CD3抗体及び抗CD52抗体が該生体試料と接する面の一部又は全部にコーティングされている培養容器に添加して培養することを含むNK細胞培養方法。
3.単核球を含む生体試料を、抗CD3抗体及び抗CD52抗体が結合したビーズを収納する培養容器に添加して培養することを含むNK細胞培養方法。
4.単核球を含む生体試料を、抗CD3抗体及び抗CD52抗体が結合したビーズを収納する生体試料と接する面の一部又は全部がシクロオレフィンポリマーで形成された培養容器に添加して培養することを含むNK細胞培養方法。
本発明で使用するNK細胞は、自体公知の方法で取得したいかなるNK細胞も利用することができる。
なお、NK細胞の提供者(ドナー)とNK細胞の被提供者(レシーピエント)は、同種であることが好ましい。例えば、ドナーがヒトの場合には、レシーピエントはヒトである。
また、NK細胞の提供者とNK細胞の被提供者は、同一個体であることがより好ましい。例えば、ドナーが提供者Xの場合には、レシーピエントは提供者Xである。
本発明のNK細胞培養とは、NK細胞の分化、刺激、変異、誘導、維持、増殖、活性化等を意味するが、特に限定されない。
本発明で使用するNK細胞は、自体公知の方法で取得することができる(参照:特許第5016732号)。NK細胞は、末梢血、リンパ節、胸腺、骨髄、腫瘍、胸水、腹水又は臍帯血から採取された単核球、より好ましくは末梢血単核球から誘導して取得する。
例えば、末梢血から比重遠心法により、NK細胞を含む単核球を回収することができる。
NK細胞活性化方法では、Tリンパ球及びNK細胞を含む単核球にCD3アゴニスト(特に、抗CD3抗体)及びCD52アゴニスト(特に、抗CD52抗体)による刺激を与えることにより、NK細胞をTリンパ球よりも活性化させることができ、NK細胞をK562等と混合させることなく安全に且つ簡単に増殖することができる。
特に、Tリンパ球及びNK細胞を含む単核球に抗CD3抗体及び抗CD52抗体による刺激を、IL-2存在下で行うことにより、IL-2単独刺激に比べ、NK細胞をTリンパ球よりも増殖させることができる。また、NK細胞活性化方法を用いればNK細胞を1000倍以上にも増殖させることができる(参照:特開2005-124568)。
本発明で使用する生体試料は、抗CD3抗体及び抗CD52抗体による刺激により、NK細胞を培養することができれば特に限定されないが、末梢血、リンパ節、胸腺、骨髄、腫瘍、胸水、腹水又は臍帯血から採取された単核球、より好ましくは末梢血単核球を列挙することができる。
本発明で使用するシクロオレフィンポリマー(「シクロオレフィン」、「COP」と称する場合がある)は、好ましくは、COPを主成分(50質量%以上、60質量%以上、70質量%以上、80質量%以上、90質量%以上)とする組成物からなる。なお、COPとは、環状オレフィン構造を有するポリマーを意味する。
さらに、シクロオレフィンポリマーは、市販されており、例えば、ZEONEX(TM)(ゼオネックス:日本ゼオン株式会社)等を使用することができる。
本発明で使用する抗CD3抗体及び抗CD52抗体は、生体試料と接触することにより、NK細胞を培養することができれば特に限定されない。また、抗CD3抗体及び抗CD52抗体のコーティングとは、シクロオレフィンポリマー又は培養容器の生体試料の接触面になんらかの力(共有結合、水素結合、静電気的相互作用、疎水的相互作用等)で結合されている状態を意味する。
本発明で使用するビーズは、抗CD3抗体及び抗CD52抗体を結合できれば特に限定されない。特に、好ましいビーズは、回収が容易できることを考慮すれば、磁気ビーズである。
本発明のNK細胞培養容器は、少なくも、生体試料と接する面の一部又は全部がシクロオレフィンポリマーで形成されており、並びに、抗CD3抗体及び抗CD52抗体が該生体試料と接する面の一部又は全部にコーティングされている。
なお、生体試料、培養液等を収納する容器本体は、いかなる形状でも良いが、輸送、収納等を考慮すると、バック形状が好ましい。なお、容器本体とは、生体試料、培養液等を収納する部分を意味するが、容器本体自体が培養容器となることができる。
本発明のNK細胞培養方法は、以下の方法を例示することができる。
(1)単核球を含む生体試料を、生体試料と接する面の一部又は全部がシクロオレフィンポリマーで形成されており並びに抗CD3抗体及び抗CD52抗体が該生体試料と接する面の一部又は全部にコーティングされている培養容器に添加して培養する方法。
(2)単核球を含む生体試料を、抗CD3抗体及び抗CD52抗体が結合したビーズを収納する培養容器に添加して培養する方法。
(3)単核球を含む生体試料を、抗CD3抗体及び抗CD52抗体が結合したビーズを収納する生体試料と接する面の一部又は全部がシクロオレフィンポリマーで形成された培養容器に添加して培養する方法。
シクロオレフィンポリマー性バッグを用いたNK細胞培養を、ポリエチレン樹脂性バッグを用いたNK細胞培養と比較した。詳細は、以下の通りである。
抗CD3抗体及び抗CD52抗体を、それぞれ、ポリエチレン樹脂性バッグ{タゼッタ(未処理バッグ)}、コージンバイオ社製)及びシクロオレフィンポリマー性バッグ(フコク社製)に、固相化した。
具体的には、抗CD3抗体100ng/ml(OKT3;ヤンセンファーマ社製)及び抗CD52抗体20μg/ml(MabCampath;サノフィ社製)をPBS(コージンバイオ社製)に添加して、抗体溶液を調整した。
次に、該抗体溶液9mlを90cm2の面積に調整した両バッグにそれぞれ添加した。
該添加後24時間4~8℃に静置後、両バッグを20mlのPBSで2回洗浄した。
該洗浄後、健常人末梢血単核球(PBMC)4x107を含む培養液(Cellex NKGM-1;コージンバイオ社製)40ml、さらに自己血漿4ml、IL-2 500U/mlを両バックに加えて、5%CO2、37℃で培養した。
さらに、両バッグに適時に適量の培養液を追加し、多数のコロニーが視認できた時点で、1LのCellex NKGM-1(コージンバイオ社製)を含むバッグに移し、適量の培養液とIL-2を追加しながら、さらに10日間培養した。
該培養後、増殖リンパ球を3x105個採取し、これを蛍光標識抗体(抗CD3抗体と抗CD56抗体)で染色し、フローサイトメータでNK細胞(CD3-CD56+)の比率を検出し、比較した。
フローサイトメータでNK細胞(CD3-CD56+)の比率の検出結果を図1に示す。図1に記載の結果から明らかなように、シクロオレフィン樹脂性バッグを用いたNK細胞培養は、ポリエチレンポリマーバッグを用いたNK細胞培養と比較して、約5倍のNK細胞培養効果を得られた。
抗体結合磁気ビーズを用いたNK細胞培養を、抗体結合プラスチック培養プレートを用いたNK細胞培養と比較した。詳細は、以下の通りである。
磁気ビーズは、Dynabeads Tosylactivated M-450 (Dynal社)を使用した。具体的には、ビーズ100μl (4x107 beads)をPBSで2回洗浄後、PBS 1mlに浮遊させ、さらに抗CD3抗体100ng/ml(OKT3;ヤンセンファーマ社製)及び抗CD52抗体20μg/ml(MabCampath;サノフィ社製)を添加し、室温で24時間回転させ撹拌した。撹拌後、抗CD3抗体及び抗CD52抗体結合磁気ビーズをPBSで2回洗浄し、0.1%ウシ血清アルブミン添加PBS 200μlに浮遊させて、抗CD3抗体及び抗CD52抗体結合磁気ビーズを含む抗体液を作成した。
次に、コントロールとして、24穴プラスチック培養プレート(ベクトン・ディキンソン社製、カタログ番号3047)の各穴に、磁気ビーズを含まない上記濃度の抗体液0.4mlを加え、24時間4~8℃で静置し、使用直前にPBSで2回洗浄し、抗体結合プレートを用意した。
次に、比較的NK細胞増殖能の低い2名の健常人由来のPBMC 1x106個を含む自己血漿10%、IL-2 500U/mlを含む培養液(Cellex NKGM-1;コージンバイオ社製)1 mlを、抗体未処理24穴プレート(本発明)及び抗体結合24穴プレート(コントロール)の各穴に添加した。抗体未処理プレートに添加したPBMCに対し、抗体結合磁気ビーズを2.5μl(1x106 beads)を添加した。添加後、プレートを5%CO2、37℃で培養した。
3~4日目にコロニーが多数できたところで、その1/10液量を新しいプレートに移し、これに1 mlの培養液とIL-2 200U/ml添加した。
以後10日間、適宜分割し、培養液、IL-2を追加した。
培養14日目に、抗体結合磁気ビーズ(本発明)及び抗体結合プレート(コントロール)で刺激した増殖リンパ球をそれぞれ3x105個採取し、これを蛍光標識抗体(抗CD3抗体と抗CD56抗体)で染色し、フローサイトメータでNK細胞(CD3-CD56+)の比率を検出、比較した。
フローサイトメータでNK細胞(CD3-CD56+)の比率の検出結果を図2に示す。図2に記載の結果から明らかなように、抗体結合磁気ビーズを用いたNK細胞培養は、抗体結合プラスチックプレートで刺激させたNK細胞培養と比較して、約2倍のNK細胞培養効果を得られた。
Claims (4)
- NK細胞を培養するためのNK細胞培養容器であって、
生体試料と接する面の一部又は全部がシクロオレフィンポリマーで形成されており、並びに、
抗CD3抗体及び抗CD52抗体が該生体試料と接する面の一部又は全部にコーティングされていることを特徴とするNK細胞培養容器。
- 単核球を含む生体試料を、生体試料と接する面の一部又は全部がシクロオレフィンポリマーで形成されており並びに抗CD3抗体及び抗CD52抗体が該生体試料と接する面の一部又は全部にコーティングされている培養容器に添加して培養することを含むNK細胞培養方法。
- 単核球を含む生体試料を、抗CD3抗体及び抗CD52抗体が結合したビーズを収納する培養容器に添加して培養することを含むNK細胞培養方法。
- 単核球を含む生体試料を、抗CD3抗体及び抗CD52抗体が結合したビーズを収納する生体試料と接する面の一部又は全部がシクロオレフィンポリマーで形成された培養容器に添加して培養することを含むNK細胞培養方法。
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