JP5763894B2 - Cell separation method - Google Patents
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- JP5763894B2 JP5763894B2 JP2010148954A JP2010148954A JP5763894B2 JP 5763894 B2 JP5763894 B2 JP 5763894B2 JP 2010148954 A JP2010148954 A JP 2010148954A JP 2010148954 A JP2010148954 A JP 2010148954A JP 5763894 B2 JP5763894 B2 JP 5763894B2
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Description
本発明は、血液等から細胞を分離する方法に関する。 The present invention relates to a method for separating cells from blood or the like.
近年、医学の進歩に伴い、血液中から必要な細胞又は成分のみを分離し、当該細胞を用いた基礎研究や治療への応用が行われるようになってきた。血液から必要とする細胞又は成分を分離・回収する方法には、細胞をその比重に基づいて分離する密度勾配遠心法（例えば、特許文献１）、細胞のサイズや膜表面の抗原を利用し、フィルターや粒子状の担体で細胞を分離する方法（例えば、特許文献２）が用いられている。特に、密度勾配遠心法は、液体状もしくは流動状の密度勾配形成用媒体の上に血液、希釈血液又は血液から事前に回収した細胞を含む溶液等を重層して遠心した後、分離された層のうちの所望の細胞を含有する層のみを回収する事で、必要としない細胞又は成分を実質的に含まないようにすることができるため、より多く用いられてきている。 In recent years, with the advance of medicine, only necessary cells or components are separated from blood and applied to basic research and treatment using the cells. For the method of separating and recovering cells or components required from blood, density gradient centrifugation (for example, Patent Document 1) that separates cells based on their specific gravity, cell size and membrane surface antigen are used, A method of separating cells with a filter or a particulate carrier (for example, Patent Document 2) is used. In particular, the density gradient centrifugation method is a method in which blood, diluted blood, or a solution containing cells collected from blood in advance is layered on a liquid or fluid density gradient forming medium, centrifuged, and then separated. Of these, by collecting only the layer containing the desired cells, it can be made substantially free of unnecessary cells or components.
しかしながら密度勾配遠心法において、より多くの細胞や成分を分離・回収するためには、使用する血液の量も多くする必要があるため、更なる効率の良い分離・回収方法が望まれていた。 However, in order to separate and collect more cells and components in density gradient centrifugation, it is necessary to increase the amount of blood to be used. Therefore, a more efficient separation and collection method has been desired.
本発明の目的は、細胞含有液から必要とする細胞や成分を効率よく分離する方法を提供することにある。 An object of the present invention is to provide a method for efficiently separating necessary cells and components from a cell-containing solution.
本発明者らは、上記の課題を解決すべく鋭意努力した結果、密度勾配遠心法を用いた細胞含有液から細胞を分離する方法において、密度勾配形成用媒体の上に重層する細胞液にあらかじめ血液由来タンパク質又は乳タンパク質を添加しておくことにより、従来の方法に比べて、必要とする細胞の回収率が飛躍的に向上することを見出し、本発明を完成させた。 As a result of diligent efforts to solve the above-mentioned problems, the present inventors have previously applied a cell solution layered on a density gradient forming medium in a method for separating cells from a cell-containing solution using density gradient centrifugation. The inventors have found that by adding blood-derived protein or milk protein in advance, the required cell recovery rate is dramatically improved as compared with conventional methods, and the present invention has been completed.
すなわち本発明を概説すれば、本発明の第１の発明は密度勾配遠心法による細胞からの細胞の分離方法に関し、
１）細胞含有液と血液由来タンパク質、乳タンパク質、又は血液由来タンパク質もしくは乳タンパク質のいずれかを含有する溶液とで細胞液を調製する工程、
２）工程１）で得られた細胞液を密度勾配形成用媒体の上に重層する工程、及び
３）密度勾配遠心分離を行い、分離された細胞を回収する工程、
を包含することを特徴とする。前記方法は、更に、
４）工程３）で回収した細胞を洗浄する工程、及び
５）工程４）で洗浄した細胞を保存する工程
を包含してもよい。
第１の発明の態様としては、血液由来タンパク質が血漿由来タンパク質又は血清由来タンパク質、例えばヒト血清アルブミン、ウシ血清アルブミンである方法が提供される。また、乳タンパク質がカゼインである方法が提供される。更に、血液由来タンパク質含有する溶液が、血漿、血清又はそれらの希釈物である方法が提供される。更に、上記工程４）において、血液由来タンパク質又は乳タンパク質を含有する溶液を用いて回収した細胞を洗浄、及び工程５）において、血液由来タンパク質又は乳タンパク質を含有する溶液を用いて細胞を保存する方法が提供される。
第１の発明の細胞含有液としては、血液、希釈血液、骨髄液、臍帯血及び成分採血液からなる群より選択される試料が挙げられ、また、血液、希釈血液、骨髄液、臍帯血及び成分採血液からなる群より選択される試料から事前に分離された細胞を含む画分が挙げられる。更に、第１の発明の密度勾配形成用媒体としては、ショ糖、グリセロール、デキストラン、メトリザミド、イオディキサノール、ショ糖とエピクロロヒドリンの共重合体、ポリビニルピロリドンの被膜をもつコロイド状シリカ粒子、スクロースポリマー、ジアトリゾ酸、イオヘキソール及びニコデンツからなる群より選択される媒体が挙げられる。
That is, when the present invention is outlined, the first invention of the present invention relates to a method for separating cells from cells by density gradient centrifugation,
1) a step of preparing a cell solution with a cell-containing solution and blood-derived protein, milk protein, or a solution containing either blood-derived protein or milk protein;
2) a step of overlaying the cell fluid obtained in step 1) on a density gradient forming medium, and 3) a step of performing density gradient centrifugation and recovering the separated cells,
It is characterized by including. The method further comprises:
4) A step of washing the cells collected in step 3), and 5) a step of storing the cells washed in step 4) may be included.
As an aspect of the first invention, there is provided a method in which the blood-derived protein is a plasma-derived protein or a serum-derived protein such as human serum albumin or bovine serum albumin. Also provided is a method wherein the milk protein is casein. Furthermore, a method is provided wherein the solution containing blood-derived protein is plasma, serum or a dilution thereof. Furthermore, in step 4), the cells recovered using a solution containing blood-derived protein or milk protein are washed, and in step 5), the cells are stored using a solution containing blood-derived protein or milk protein. A method is provided.
The cell-containing liquid of the first invention includes a sample selected from the group consisting of blood, diluted blood, bone marrow fluid, umbilical cord blood and component blood collection, and also includes blood, diluted blood, bone marrow fluid, umbilical cord blood and Examples thereof include a fraction containing cells previously separated from a sample selected from the group consisting of component blood collection. Further, the density gradient forming medium according to the first invention includes sucrose, glycerol, dextran, metrizamide, iodixanol, a copolymer of sucrose and epichlorohydrin, and colloidal silica having a polyvinylpyrrolidone coating. And a medium selected from the group consisting of particles, sucrose polymer, diatrizoic acid, iohexol and nicodents.
本発明の第２の発明は、密度勾配遠心法による細胞の分離方法における、密度勾配形成用媒体の上に重層する細胞液の調製時の血液由来タンパク質、乳タンパク質、又はそれらタンパク質を含有する溶液の使用に関する。第２の発明の態様としては、血液由来タンパク質が血漿由来タンパク質又は血清由来タンパク質、例えばヒト血清アルブミン、ウシ血清アルブミンである使用が提供される。また、乳タンパク質がカゼインである使用が提供される。また、血液由来タンパク質を含有する溶液が血漿、血清又はそれらの希釈物である使用が提供される。 The second invention of the present invention is a method of separating cells by density gradient centrifugation, wherein blood-derived protein, milk protein, or a solution containing these proteins at the time of preparation of a cell solution layered on a density gradient forming medium About the use of. As an aspect of the second invention, there is provided use in which the blood-derived protein is a plasma-derived protein or a serum-derived protein such as human serum albumin or bovine serum albumin. Also provided is a use wherein the milk protein is casein. Also provided is a use wherein the solution containing blood-derived protein is plasma, serum or a dilution thereof.
本発明の第３の発明は、第１の発明の方法に用いるためのキットに関し、
１）密度勾配形成用媒体、及び
２）血液由来タンパク質及び／又は乳タンパク質、
を含有することを特徴とする。
The third invention of the present invention relates to a kit for use in the method of the first invention,
1) a medium for density gradient formation, and 2) blood-derived protein and / or milk protein,
It is characterized by containing.
本発明により、細胞含有液、特に血液から必要とする細胞を高効率で分離できる事から、採血時の血液量を少なくすることが可能であり、自己の細胞を用いる治療において、患者の負担を軽減することが可能となる。また、血液から効率よく細胞を回収できることから、本発明の方法は基礎研究、医療への応用研究において極めて有用である。 According to the present invention, it is possible to efficiently separate necessary cells from a cell-containing liquid, particularly blood, so that it is possible to reduce the amount of blood at the time of blood collection. It becomes possible to reduce. In addition, since the cells can be efficiently recovered from blood, the method of the present invention is extremely useful in basic research and medical application research.
以下に本発明について詳細に説明する。 The present invention is described in detail below.
本願明細書において「密度勾配形成用媒体」とは、それ自身で又は遠心分離によって密度勾配を形成する液体状又は粒子状の物質を示す。例えば、ショ糖、グリセロール、デキストラン、メトリザミド、イオディキサノール、ショ糖とエピクロロヒドリンの共重合体（例えば後述のＦｉｃｏｌｌ）、ポリビニルピロリドンの被膜をもつコロイド状シリカ粒子（例えば後述のＰｅｒｃｏｌｌ）、スクロースポリマー、ジアトリゾ酸、イオヘキソール、ニコデンツ（ｎｙｃｏｄｅｎｚ）が挙げられ、イオン性又は非イオン性のいずれのものも使用できる。また、Ｆｉｃｏｌｌ、Ｆｉｃｏｌｌ−ＰａｑｕｅやＰｅｒｃｏｌｌ（いずれもＧＥヘルスケア バイオサイエンス社製）、Ｌｙｍｐｈｏｐｒｅｐ、Ｐｏｌｙｍｏｒｐｈｐｒｅｐ、ＯｐｔｉＰｒｅｐ（Ａｘｉｓ−Ｓｈｉｅｌｄ ＰｏＣ ＡＳ社製）、Ｌｙｍｐｈｏｓｅｐａｒｌ（免疫生物研究所製）等の密度勾配形成用として市販されている媒体も好適に使用できる。 In the present specification, the “density gradient forming medium” refers to a liquid or particulate substance that forms a density gradient by itself or by centrifugation. For example, sucrose, glycerol, dextran, metrizamide, iodixanol, a copolymer of sucrose and epichlorohydrin (for example, Ficoll described below), colloidal silica particles having a polyvinylpyrrolidone coating (for example, Percoll described below) Sucrose polymer, diatrizoic acid, iohexol, nycodenz, and either ionic or nonionic can be used. In addition, Ficoll, Ficoll-Paque, Percoll (all manufactured by GE Healthcare Bioscience), Lymphoprep, Polymorphprep, OptiPrep (manufactured by Axis-Shield PoC AS), Lymphosepar (manufactured by Density Biotechnology Institute), etc. A commercially available medium can also be suitably used.
本願明細書において「血液由来タンパク質」とは、血液に含有されるタンパク質を示す。例えば、血液に含有されるタンパク質として血漿由来タンパク質及び／又は血清由来タンパク質が挙げられ、具体的には、血清アルブミン、血清グロブリン等が挙げられる。また血清アルブミンとしては、ヒト血清アルブミン又はウシ血清アルブミンが挙げられる。 In the present specification, “blood-derived protein” refers to a protein contained in blood. For example, proteins derived from blood include plasma-derived proteins and / or serum-derived proteins, and specific examples include serum albumin and serum globulin. Examples of serum albumin include human serum albumin and bovine serum albumin.
本願明細書において「乳タンパク質」とは、乳に含有されるタンパク質を示す。例えば、牛乳の約８０％を占めるカゼインが挙げられる。 As used herein, “milk protein” refers to a protein contained in milk. An example is casein, which accounts for about 80% of milk.
前記のタンパク質は天然由来の材料より単離、精製されたものでもよく、組換え体（微生物や培養細胞）を利用して製造されたものでもよい。 The protein described above may be isolated and purified from naturally-derived materials, or may be produced using recombinants (microorganisms or cultured cells).
本発明の方法においては、血液由来タンパク質又は乳タンパク質は１種のみを使用してもよく、複数のものを混合して使用してもよい。好ましくは単離された血液由来タンパク質又は乳タンパク質、すなわち血液由来又は乳由来の他の成分を実質的に含有しない血液由来タンパク質又は乳タンパク質が本発明に使用される。また、入手可能な組換え血液由来タンパク質又は乳タンパク質を使用してもよい。本発明において、血液由来タンパク質又は乳タンパク質は固体状態のものを直接使用してもよく、これらタンパク質を緩衝液に溶解又は懸濁させ、「血液由来タンパク質又は乳タンパク質を含有する溶液」として使用してもよい。また、血漿、血清又はそれらの希釈物を「血液由来タンパク質含有する溶液」として本発明に使用することもできる。 In the method of the present invention, only one blood-derived protein or milk protein may be used, or a plurality of them may be used in combination. Preferably, isolated blood-derived protein or milk protein, ie blood-derived protein or milk protein substantially free of other components derived from blood or milk is used in the present invention. Also available recombinant blood derived protein or milk protein may be used. In the present invention, blood-derived protein or milk protein may be used directly in a solid state, and these proteins are dissolved or suspended in a buffer solution and used as a “solution containing blood-derived protein or milk protein”. May be. In addition, plasma, serum, or a dilution thereof can be used in the present invention as a “solution containing blood-derived protein”.
本願明細書において「細胞含有液」とは、細胞を含有する液状の組成物を意味する。その種類に限定はなく、任意の細胞含有液に本発明の方法を適用することができる。血液系の細胞の分離を目的とする場合、本発明に使用される細胞含有液としては血液、希釈血液、骨髄液、臍帯血又は成分血液等、生体由来の細胞を含有する試料が例示される。また、組織を材料として調製された細胞懸濁液や培養細胞の懸濁液にも本発明を適用することができる。 In the present specification, the “cell-containing liquid” means a liquid composition containing cells. The type is not limited, and the method of the present invention can be applied to any cell-containing liquid. For the purpose of separating blood cells, examples of the cell-containing liquid used in the present invention include samples containing cells derived from living organisms such as blood, diluted blood, bone marrow fluid, umbilical cord blood, or component blood. . In addition, the present invention can be applied to a cell suspension or a cultured cell suspension prepared using tissue as a material.
前記の細胞含有液は生体試料を公知の操作で分画することにより調製された「細胞含有画分」であってもよい。例えば、血液、希釈血液、骨髄液、臍帯血又は成分採血液（単核球、顆粒球、末梢血幹細胞など、細胞成分を採取することを目的とする成分採血で得られる細胞含有液）等の試料から事前に分離した細胞を含む画分を本発明に使用することができる。 The cell-containing liquid may be a “cell-containing fraction” prepared by fractionating a biological sample by a known operation. For example, blood, diluted blood, bone marrow fluid, umbilical cord blood or component blood collection (cell-containing fluid obtained by component blood collection for the purpose of collecting cell components such as mononuclear cells, granulocytes, peripheral blood stem cells), etc. A fraction containing cells previously separated from a sample can be used in the present invention.
本発明は、当該細胞含有液を材料とした、所望の細胞の分離に利用することができる。本発明により分離される細胞には特に限定はない。特に血液中に存在する細胞が本発明には好適であり、末梢血単核球（ＰＢＭＣ）、多核球、顆粒球、赤血球、造血幹細胞等が挙げられる。 The present invention can be used to separate desired cells using the cell-containing solution as a material. There are no particular limitations on the cells isolated according to the present invention. Particularly, cells present in blood are suitable for the present invention, and examples thereof include peripheral blood mononuclear cells (PBMC), polynuclear cells, granulocytes, erythrocytes, hematopoietic stem cells and the like.
以下、血液からＰＢＭＣを分離回収する方法について説明するが、本発明はこの説明の内容に限定されるものではない。 Hereinafter, a method for separating and recovering PBMC from blood will be described, but the present invention is not limited to the contents of this description.
まず、ドナー（ヒト又は動物）より採血を実施する。得られた血液（例えばヘパリン血）又はその希釈物を常法に従い、例えば２００〜１０００×ｇで５〜４０分間、０〜４０℃にて遠心分離を行い、上清である血漿と分離された細胞含有画分、例えば赤血球層及びＢｕｆｆｙ ｃｏａｔ層を回収する。ＰＢＭＣの製造のためにはＢｕｆｆｙ ｃｏａｔ層を細胞含有液として使用する。
工程１）では、得られた画分、すなわちＢｕｆｆｙ ｃｏａｔ層と、血液由来タンパク質を含有する溶液、例えばヒト血清アルブミンを含む溶液とを混合することで細胞液を調製する。あるいは乳タンパク質を含有する溶液、例えばカゼインを含む溶液と混合し細胞液を調製してもよい。当該溶液は、血液由来タンパク質又は乳タンパク質を含む以外は常法で用いられるもの、例えば生理食塩水、リン酸緩衝生理食塩水等の緩衝液、細胞培養用培地、無血清培地等が使用できるのは当然のことである。血液由来タンパク質を含有する溶液として、血漿、血清又はそれらの希釈液も使用することができる。例えば、前記の遠心分離操作で得ることができる血漿もしくはその希釈液、血液又はその希釈液に抗凝固成分を加えずに凝固させた後に回収した上澄み液（血清）もしくはその希釈液を血液由来タンパク質を含有する溶液として使用してもよい。好ましくは、血漿、血清は非働化処理を行った後に使用される。これら血漿又は血清は、市販されている血漿（製品名：正常ヒト血漿、コージンバイオ社製）又は血清（製品名：Ｈｕｍａｎ ｓｅｒｕｍ，ＡＢ、Ｌｏｎｚａ社製）等を使用いてもよい。また、特に細胞と同じドナー由来の血漿又は血清の使用は、安全性の観点から特に好適である。
First, blood is collected from a donor (human or animal). The obtained blood (for example, heparin blood) or a dilution thereof was centrifuged at 200 to 1000 × g for 5 to 40 minutes at 0 to 40 ° C. according to a conventional method, and separated from the supernatant plasma. Cell-containing fractions such as the red blood cell layer and the Buffy coat layer are collected. For the production of PBMC, the Buffy coat layer is used as the cell-containing solution.
In step 1), a cell solution is prepared by mixing the obtained fraction, that is, the Buffy coat layer, and a solution containing a blood-derived protein, for example, a solution containing human serum albumin. Alternatively, a cell solution may be prepared by mixing with a solution containing milk protein, for example, a solution containing casein. As the solution, those which are used in a conventional manner except for containing blood-derived protein or milk protein, for example, buffer solutions such as physiological saline and phosphate buffered saline, cell culture medium, serum-free medium and the like can be used. Is natural. As a solution containing blood-derived protein, plasma, serum, or a diluted solution thereof can also be used. For example, plasma or a diluted solution thereof obtained by the above centrifugation operation, blood or a supernatant solution (serum) collected after coagulation without adding an anticoagulant component to the diluted solution or a diluted solution thereof is obtained from a blood-derived protein. It may be used as a solution containing Preferably, plasma and serum are used after inactivation treatment. As the plasma or serum, commercially available plasma (product name: normal human plasma, manufactured by Kojin Bio Inc.) or serum (product name: manufactured by Human serum, AB, Lonza) or the like may be used. In particular, the use of plasma or serum derived from the same donor as the cells is particularly suitable from the viewpoint of safety.
上記調製した細胞液中の血液由来タンパク質又は乳タンパク質の最終濃度は、添加もしくは希釈に用いた血液由来タンパク質、乳タンパク質又は血液由来タンパク質もしくは乳タンパク質のいずれかを含有する溶液により細胞液にもたらされる血液由来タンパク質又は乳タンパク質の最終濃度として０．０１〜２５％（Ｗ／Ｖ）が好ましく、好適には０．０５〜１０％（Ｗ／Ｖ）である。また、血液由来タンパク質を含有する溶液として、血漿、血清又はそれらいずれかの希釈物を用いる場合、細胞含有液の液量に対して、０．１〜１００％（Ｖ／Ｖ）に相当する液量を添加すればよく、好適には０．５〜１００％（Ｖ／Ｖ）に相当する液量である。また、血漿、血清の希釈物は、常法で用いられるもの、例えば生理食塩水、リン酸緩衝生理食塩水等の緩衝液、細胞培養用培地、無血清培地等を用いた希釈により調製し、上記液量で細胞含有液に添加すればよい。 The final concentration of blood-derived protein or milk protein in the prepared cell fluid is brought to the cell fluid by a solution containing either blood-derived protein, milk protein, or blood-derived protein or milk protein used for addition or dilution. The final concentration of blood-derived protein or milk protein is preferably 0.01 to 25% (W / V), preferably 0.05 to 10% (W / V). In addition, when plasma, serum, or any one of these dilutions is used as a solution containing a blood-derived protein, a solution corresponding to 0.1 to 100% (V / V) with respect to the amount of the cell-containing solution The amount may be added, and is preferably a liquid amount corresponding to 0.5 to 100% (V / V). In addition, dilutions of plasma and serum are prepared by dilution using those conventionally used, for example, buffer solutions such as physiological saline and phosphate buffered saline, cell culture medium, serum-free medium, and the like. What is necessary is just to add to a cell containing liquid by the said liquid quantity.
次に、工程２）として、調製した細胞液を適切な遠心分離用容器内の密度勾配形成用媒体上に重層し、例えば２００〜１０００×ｇで５〜４０分間、０〜４０℃にて遠心分離を行う。さらに工程３）として、分離された層のうちＰＢＭＣを含む画分である中間層をピペットで回収する。こうして得られたＰＢＭＣ含有画分は、適当な溶液、例えば生理食塩水、リン酸緩衝生理食塩水、細胞培養用培地等に当該回収した中間層を懸濁し、次いで遠心分離を行って上清を除去することにより洗浄を行う。 Next, as step 2), the prepared cell solution is layered on a medium for forming a density gradient in an appropriate centrifugation container, and centrifuged, for example, at 200 to 1000 × g for 5 to 40 minutes at 0 to 40 ° C. Perform separation. Further, as step 3), the intermediate layer which is a fraction containing PBMC among the separated layers is collected with a pipette. The thus obtained PBMC-containing fraction is obtained by suspending the recovered intermediate layer in an appropriate solution such as physiological saline, phosphate buffered saline, cell culture medium, etc., and then centrifuging to obtain a supernatant. Wash by removing.
また、本発明の好適な態様としては、上記記載の工程３）において血液由来タンパク質又は乳タンパク質を含有する溶液が使用できる。例えば、前記の中間層をピペットで回収後、まず初めに血液由来タンパク質又は乳タンパク質を含有する溶液に懸濁し、次いで遠心分離を行って上清を除去する。この操作を数回繰り返すことにより洗浄を行う。この場合、全ての洗浄工程において血液由来タンパク質又は乳タンパク質を含有する溶液を用いてもよく、２回目以降の洗浄時に適当な溶液に変えて行ってもよい。 As a preferred embodiment of the present invention, a solution containing blood-derived protein or milk protein can be used in step 3) described above. For example, after the intermediate layer is collected with a pipette, it is first suspended in a solution containing blood-derived protein or milk protein, and then centrifuged to remove the supernatant. Washing is performed by repeating this operation several times. In this case, a solution containing blood-derived protein or milk protein may be used in all washing steps, or the solution may be changed to an appropriate solution at the second and subsequent washings.
こうして得られたＰＢＭＣの生細胞数及び死細胞数を、例えば自動血球計測装置を用いて算出することにより、ＰＢＭＣの生存率が測定できる。 The viability of PBMC can be measured by calculating the number of live and dead cells of PBMC thus obtained using, for example, an automatic blood cell counter.
上記の操作に準じて、ＰＢＭＣ以外の血液由来細胞も分離することができる。更に、こうして取得された細胞もしくは細胞集団は、他の細胞分離操作（例えば表面抗原を指標とした分離）に供するための材料としてもよい。 According to the above operation, blood-derived cells other than PBMC can also be separated. Furthermore, the cells or cell population thus obtained may be used as a material for other cell separation operations (for example, separation using a surface antigen as an index).
ＰＢＭＣ以外の細胞の調製においては、所望の細胞を含有する試料（体液、組織等）に応じて細胞液の調製を行い、適切な密度勾配形成用媒体、血液由来タンパク質、乳タンパク質又は血液由来タンパク質もしくは乳タンパク質のいずれかを含有する溶液とそれらの濃度、使用条件を適宜設定することにより、本発明の細胞の分離方法を実施することができる。 In the preparation of cells other than PBMC, the cell fluid is prepared according to the sample (body fluid, tissue, etc.) containing the desired cells, and an appropriate density gradient forming medium, blood-derived protein, milk protein or blood-derived protein is prepared. Alternatively, the method for separating cells of the present invention can be carried out by appropriately setting a solution containing any of milk proteins, their concentration, and use conditions.
以上、本発明から、細胞含有液に血液由来タンパク質、乳タンパク質又は血液由来タンパク質もしくは乳タンパク質のいずれかを含有する溶液を別途添加することにより、従来法に比べて必要とする細胞を効率よく分離・回収することが可能となる。 As described above, by separately adding a blood-derived protein, milk protein, or a solution containing either blood-derived protein or milk protein to the cell-containing liquid, the required cells can be efficiently separated as compared with the conventional method.・ It can be collected.
また、本発明により分離・回収した細胞を、血液由来タンパク質又は乳タンパク質を含む溶液で保存することにより、不要な他の成分を含有することなく、細胞濃度を高く保持し、かつ細胞生存率を維持したまま、未凍結状態で長時間保存することが可能である。当該保存方法も本願に包含される。細胞の保存に用いる血液由来タンパク質を含有する溶液として、血漿、血清の希釈物を用いる場合、０．０１〜２５％（Ｖ／Ｖ）の溶液が好ましく、好適には０．０５〜１０％（Ｖ／Ｖ）の溶液である。また、血漿、血清の希釈には、常法で用いられるもの、例えば生理食塩水、リン酸緩衝生理食塩水等の緩衝液、細胞培養用培地、無血清培地等を用いた希釈により調製すればよいが、不要な他の成分を含有しない点から、生理食塩水が好ましい。未凍結状態とは、１〜１０℃、好ましくは４℃である。本発明の保存方法を用いることにより、例えば、４℃で１〜５日間、好適には１〜２日間、細胞濃度を高く保持し、細胞生存率を維持したまま細胞を保存することが可能である。こうして保存された細胞は、使用前に再度工程４）と同様の方法で細胞を洗浄し、使用してもよい。また、血漿及び血清は不要な他の成分を含有していないことから、そのまま使用してもよい。通常、細胞の保存は凍結状態で行い、融解後、洗浄操作を行う必要があるが、本発明の保存方法を用いることにより、これら融解及び洗浄操作を行う必要がないため、これら操作による細胞生存率の低下を防ぐことができ、細胞生存率を維持したまま培養等に使用可能となる。 Further, by storing the cells separated and recovered according to the present invention in a solution containing blood-derived protein or milk protein, the cell concentration can be kept high without containing other unnecessary components, and the cell viability can be increased. It can be stored for a long time in an unfrozen state while being maintained. This storage method is also included in the present application. When using a plasma or serum dilution as a solution containing blood-derived protein used for cell storage, a 0.01 to 25% (V / V) solution is preferable, and preferably 0.05 to 10% ( V / V) solution. In addition, for dilution of plasma and serum, it may be prepared by dilution using a conventional method such as a buffer solution such as physiological saline or phosphate buffered physiological saline, a cell culture medium, a serum-free medium, or the like. Although it is good, physiological saline is preferable because it does not contain other unnecessary components. The unfrozen state is 1 to 10 ° C, preferably 4 ° C. By using the storage method of the present invention, for example, cells can be stored while maintaining a high cell concentration and maintaining cell viability for 1 to 5 days, preferably 1 to 2 days at 4 ° C. is there. The cells thus stored may be used after washing the cells again in the same manner as in step 4) before use. Further, since plasma and serum do not contain other unnecessary components, they may be used as they are. Usually, cells must be stored in a frozen state and must be washed after thawing. However, by using the preservation method of the present invention, it is not necessary to perform these thawing and washing operations. The decrease in the rate can be prevented, and the cell viability can be maintained while maintaining the cell viability.
本発明の第２の発明は、密度勾配遠心法による細胞の分離方法における、密度勾配形成用媒体の上に重層する細胞液の調製時の血液由来タンパク質、乳タンパク質又は血液由来タンパク質もしくは乳タンパク質のいずれかを含有する溶液の使用である。すなわち、本発明により開示された、血液由来タンパク質、乳タンパク質又は血液由来タンパク質もしくは乳タンパク質のいずれかを含有する溶液を、密度勾配遠心法による細胞の分離に供される細胞液の調製に使用する行為は本願発明に包含される。 A second invention of the present invention is a method for separating cells by density gradient centrifugation, wherein blood-derived protein, milk protein, blood-derived protein or milk protein at the time of preparation of a cell solution layered on a density gradient forming medium The use of a solution containing either. That is, the blood-derived protein, milk protein, or a solution containing either blood-derived protein or milk protein disclosed by the present invention is used for preparing a cell solution to be subjected to cell separation by density gradient centrifugation. The act is included in the present invention.
本発明の第３の発明のキットは、本発明の第１の発明の方法に使用される密度勾配形成用媒体、血液由来タンパク質又は乳タンパク質を含有する。好適な態様として、前記の各構成成分に加えてこれらの本発明の第１の発明の細胞分離方法における使用方法を指示する指示書を含むキットが例示される。本発明のキットにおいて、密度勾配形成用媒体、血液由来タンパク質又は乳タンパク質を含有する溶液は、いずれも本発明の細胞分離方法に適した濃度とされていてもよく、用時に希釈して使用される高濃度溶液や用時に溶解又は懸濁して使用される乾燥物もしくは凍結乾燥物とされていてもよい。さらに、本発明のキットは、構成成分の希釈や溶解に使用される溶媒、回収された細胞を洗浄するための洗浄液等を含有してもよい。 The kit of the third invention of the present invention contains the density gradient forming medium, blood-derived protein or milk protein used in the method of the first invention of the present invention. As a preferred embodiment, a kit containing instructions for instructing a method of use in the cell separation method of the first invention of the present invention in addition to each of the above-described components is exemplified. In the kit of the present invention, the density gradient forming medium, blood-derived protein or milk protein-containing solution may all have a concentration suitable for the cell separation method of the present invention. It may be a high-concentration solution or a dried product or a lyophilized product that is dissolved or suspended at the time of use. Furthermore, the kit of the present invention may contain a solvent used for diluting or dissolving the constituent components, a washing solution for washing the collected cells, and the like.
以下に実施例をもって本発明を更に具体的に示すが、本発明は以下の実施例の範囲のみに何ら限定されるものではない。 The present invention will be described more specifically with reference to the following examples. However, the present invention is not limited to the scope of the following examples.
実施例１ ヒト血清アルブミン（ＨＳＡ）含有生理食塩水を用いた新鮮血からのＰＢＭＣ分離
（１）ＰＢＭＣの分離
インフォームド・コンセントの得られたヒト健常人ドナーより、６０ｍＬ分のへパリン加採血を実施した。得られた血液を約１５ｍＬずつコニカルチューブ（ベクトン・ディッキンソン社製又はグライナ―社製）に分注後、７００×ｇ、室温で２０分間遠心し、遠心後の上清である血漿画分とＰＢＭＣを含む細胞画分とに分離した。ＰＢＭＣを含む細胞画分は、ダルベッコＰＢＳ（以下、ＤＰＢＳと記載する。インビトロジェン社製又はニッスイ社製）、０．９６％（Ｗ／Ｖ）ＨＳＡを含む生理食塩水（以下、０．９６％ＨＳＡ／生理食塩水と記載する）、ＲＰＭＩ１６４０（インビトロジェン社製又は和光純薬社製）、又は生理食塩水（テルモ社製）を用いて、それぞれ２０ｍＬにフィルアップし希釈した。得られた希釈細胞液を、Ｆｉｃｏｌｌ−Ｐａｑｕｅ ＰＲＥＭＩＵＭ（ＧＥヘルスケア バイオサイエンス社製）２０ｍＬの上にそれぞれ重層して、７００×ｇ、室温で２０分間遠心した。遠心後、分離した層のうち、ＰＢＭＣ層をピペットで回収し、ＲＰＭＩ１６４０、０．９６％ＨＳＡ／生理食塩水又はリンパ球培養用培地ＧＴ−Ｔ５５１（タカラバイオ社製）をそれぞれ用いて３０ｍＬにフィルアップした後、６５０×ｇ、４℃にて１０分間遠心し、上清を除去した。同様に、６００×ｇ、５００×ｇと段階的に遠心力を落としながら順次遠心操作を行い、計３回洗浄を繰り返した。得られたＰＢＭＣの生細胞数及び生存率（％）を自動血球計測装置（ヌクレオカウンター Ｃｈｅｍｏｍｅｔｅｃ社製）を用いて算出し、以下の式に準じて細胞回収率（％）を算出した。Ｆｉｃｏｌｌ重層時の希釈溶媒と洗浄時の溶媒との関係と得られた結果を表１に示す。表中、ドナー１及び２は異なるドナーの血液を用いて比較を行った結果である。
式：細胞回収率（％）＝各条件で分離した際の回収ＰＢＭＣ細胞数／基本法※で分離した際の回収ＰＢＭＣ細胞数×１００（※基本法とは、Ｆｉｃｏｌｌ重層時の希釈溶媒としてＤＰＢＳを、洗浄時の溶媒としてＲＰＭＩ１６４０を使用する方法を示す。）
Example 1 Separation of PBMC from fresh blood using physiological saline containing human serum albumin (HSA) (1) Separation of PBMC 60 mL of heparinized blood collected from a healthy human donor with informed consent Carried out. About 15 mL of the obtained blood was dispensed into conical tubes (Becton Dickinson or Griener), centrifuged at 700 × g for 20 minutes at room temperature, and the plasma fraction and PBMC as the supernatant after centrifugation. The cell fraction containing was separated. The cell fraction containing PBMC was Dulbecco's PBS (hereinafter referred to as DPBS. Invitrogen or Nissui), 0.96% (W / V) HSA-containing physiological saline (hereinafter 0.96% HSA). / Described as physiological saline), RPMI1640 (manufactured by Invitrogen or Wako Pure Chemical Industries), or physiological saline (manufactured by Terumo), each was filled up to 20 mL and diluted. The obtained diluted cell solution was layered on 20 mL of Ficoll-Paque PREMIUM (manufactured by GE Healthcare Bioscience), and centrifuged at 700 × g and room temperature for 20 minutes. After centrifugation, among the separated layers, the PBMC layer was collected with a pipette and filled to 30 mL using RPMI 1640, 0.96% HSA / saline or lymphocyte culture medium GT-T551 (manufactured by Takara Bio Inc.). Then, the mixture was centrifuged at 650 × g and 4 ° C. for 10 minutes, and the supernatant was removed. Similarly, centrifugal operation was sequentially performed while decreasing the centrifugal force in steps of 600 × g and 500 × g, and washing was repeated a total of three times. The number of viable cells and the survival rate (%) of the obtained PBMC were calculated using an automatic blood cell counter (manufactured by Nucleo Counter Chemometec), and the cell recovery rate (%) was calculated according to the following formula. Table 1 shows the relationship between the diluted solvent during Ficoll overlay and the solvent during washing and the results obtained. In the table, donors 1 and 2 are the results of comparison using blood from different donors.
Formula: Cell recovery rate (%) = number of recovered PBMC cells when separated under each condition / number of recovered PBMC cells when separated by the basic method * 100 (* Basic method is DPBS as a dilution solvent during Ficoll layering, (The method of using RPMI 1640 as a solvent during washing is shown.)
表１に示すように、Ｆｉｃｏｌｌ−Ｐａｑｕｅ ＰＲＥＭＩＵＭ重層時及びその後の細胞洗浄時に０．９６％ＨＳＡ／生理食塩水、すなわち約１％のＨＳＡを含有する生理食塩水を使用することで、基本法、ＲＰＭＩ１６４０の組合せ、生理食塩水とＧＴ−Ｔ５５１の組合せと比較して、細胞の生存率への影響なく、細胞回収率を上げることができた。また、この効果は複数のドナー血液を用いたＰＢＭＣ分離について確認された。従って、本発明の方法は血液からのＰＢＭＣ分離時に好適に使用できることが明らかとなった。 As shown in Table 1, by using 0.96% HSA / saline, that is, physiological saline containing about 1% HSA during Ficoll-Paque PREMIUM overlay and subsequent cell washing, the basic method, RPMI 1640 The cell recovery rate could be increased without affecting the cell viability as compared with the combination of the physiological saline and GT-T551. This effect was also confirmed for PBMC separation using multiple donor blood. Therefore, it has been clarified that the method of the present invention can be suitably used when separating PBMC from blood.
（２）回収ＰＢＭＣの細胞集団の解析
実施例１−（１）で基本法により分離して得られたＰＢＭＣ又は０．９６％ＨＳＡ／生理食塩水を用いて分離して得られたＰＢＭＣを、１％（Ｗ／Ｖ）ウシ血清アルブミン（ＢＳＡ、シグマ社製）を含むＤＰＢＳ（以下１％ＢＳＡ／ＤＰＢＳと記載する）で洗浄した。その後、それぞれのＰＢＭＣを１％ＢＳＡ／ＤＰＢＳに細胞を懸濁し、ＦＩＴＣ標識マウス抗ヒトＣＤ８抗体／ＲＤ１標識マウス抗ヒトＣＤ４抗体／ＰＣ５標識マウス抗ヒトＣＤ３抗体（ベックマンコールター社製）、あるいはＦＩＴＣ標識マウス抗ヒトＣＤ３抗体（ベックマンコールター社製）及びＲＤ１標識マウス抗ヒトＣＤ５６抗体（ベックマンコールター社製）を添加した。ネガティブコントロールとしてＦＩＴＣ標識マウスＩｇＧ１抗体／ＲＤ１標識マウスＩｇＧ１抗体／ＰＣ５標識マウスＩｇＧ１抗体（ベックマンコールター社製）を添加した。氷上で３０分間インキュベートした後、ＡＣＫ Ｌｙｓｉｎｇ Ｂｕｆｆｅｒを添加し、残存赤血球を溶血した。次に、０．１％（Ｗ／Ｖ）ＢＳＡを含むＤＰＢＳで細胞を洗浄し、再度ＤＰＢＳに懸濁した。この細胞をフローサイトメトリー（Ｃｙｔｏｍｉｃｓ ＦＣ５００：ベックマンコールター社製）に供し、各々の細胞集団について、ＣＤ３陽性細胞、ＣＤ４陽性細胞、ＣＤ８陽性細胞、ＣＤ３陰性ＣＤ５６陽性細胞の含有率を算出した。ここで含有率（％）は、全細胞数に占める陽性細胞数の占める割合を示す。結果を表２に示す。表中、ドナー１及び２は異なるドナーのＰＢＭＣを用いて比較を行った結果である。
(2) Analysis of cell population of recovered PBMC PBMC obtained by separation according to the basic method in Example 1- (1) or PBMC obtained by separation using 0.96% HSA / saline It was washed with DPBS (hereinafter referred to as 1% BSA / DPBS) containing% (W / V) bovine serum albumin (BSA, Sigma). Thereafter, the cells were suspended in 1% BSA / DPBS of each PBMC, and FITC-labeled mouse anti-human CD8 antibody / RD1-labeled mouse anti-human CD4 antibody / PC5-labeled mouse anti-human CD3 antibody (manufactured by Beckman Coulter) or FITC-labeled Mouse anti-human CD3 antibody (Beckman Coulter) and RD1-labeled mouse anti-human CD56 antibody (Beckman Coulter) were added. As a negative control, FITC-labeled mouse IgG 1 antibody / RD1-labeled mouse IgG 1 antibody / PC5-labeled mouse IgG 1 antibody (manufactured by Beckman Coulter) was added. After incubating on ice for 30 minutes, ACK Lysing Buffer was added to lyse the remaining red blood cells. Next, the cells were washed with DPBS containing 0.1% (W / V) BSA and resuspended in DPBS. The cells were subjected to flow cytometry (Cytomics FC500: manufactured by Beckman Coulter), and the content ratio of CD3 positive cells, CD4 positive cells, CD8 positive cells, and CD3 negative CD56 positive cells was calculated for each cell population. Here, the content (%) indicates the ratio of the number of positive cells to the total number of cells. The results are shown in Table 2. In the table, donors 1 and 2 are the results of comparison using PBMCs from different donors.
表２に示すように、Ｆｉｃｏｌｌ−Ｐａｑｕｅ ＰＲＥＭＩＵＭ重層時及びその後の細胞洗浄時に０．９６％ＨＳＡ／生理食塩水、すなわち約１％のＨＳＡを含有する生理食塩水を使用して回収されたＰＢＭＣと、基本法で回収されたＰＢＭＣとの間では、そこに含有される各抗原陽性の細胞の割合にほとんど差はなかった。また、複数のドナー血液を用いた場合でも同様な結果であった。すなわち、基本法で回収されたＰＢＭＣと本発明の方法により分離されたＰＢＭＣは、細胞として同じ性質を有するものであることが明らかとなった。このことから、本発明の方法は血液からのＰＢＭＣ分離回収時に好適に使用できることが明らかとなった。 As shown in Table 2, PBMCs recovered using 0.96% HSA / saline, ie, about 1% HSA, during Ficoll-Paque PREMIUM overlay and subsequent cell wash There was almost no difference in the proportion of each antigen-positive cell contained in PBMC collected by the basic method. The same result was obtained when a plurality of donor bloods were used. That is, it became clear that PBMC recovered by the basic method and PBMC separated by the method of the present invention have the same properties as cells. From this, it was revealed that the method of the present invention can be suitably used when separating and recovering PBMC from blood.
実施例２ ＨＳＡ含有生理食塩水を用いた新鮮血からのＰＢＭＣ分離
（１）ＰＢＭＣの分離
実施例１−（１）と同様の方法でインフォームド・コンセントの得られたヒト健常人ドナー血液からＰＢＭＣを分離した。ただし、血漿分離後のＰＢＭＣを含む細胞画分は、ＤＰＢＳ、０．９６％ＨＳＡ／生理食塩水、生理食塩水又はソルデム３Ａ（ブドウ糖、電解質液を含有する維持液、テルモ社製）を用いてそれぞれ別々に希釈を行った。中間層のＰＢＭＣの回収後の洗浄は、ＲＰＭＩ１６４０、０．９６％ＨＳＡ／生理食塩水、生理食塩水又はソルデム３Ａをそれぞれ用いて３回行った。Ｆｉｃｏｌｌ重層時の希釈溶媒と洗浄時の溶媒との関係と、得られた結果を表３に示す。表中、Ｅｘｐ．Ａ及びＢは同一ドナーの血液を用いた実施時期の異なる実験の結果を示す。
Example 2 Separation of PBMC from fresh blood using HSA-containing physiological saline (1) Separation of PBMC From human healthy human donor blood obtained informed consent in the same manner as in Example 1- (1) PBMCs were separated. However, the cell fraction containing PBMCs after plasma separation is obtained using DPBS, 0.96% HSA / saline, saline or Soldem 3A (a maintenance solution containing glucose and electrolyte solution, manufactured by Terumo). Each was diluted separately. Washing of the intermediate layer after recovery of PBMC was performed 3 times using RPMI 1640, 0.96% HSA / saline, saline or Soldem 3A. Table 3 shows the relationship between the diluted solvent during Ficoll overlaying and the solvent during washing, and the results obtained. In the table, Exp. A and B show the results of experiments with different time periods using blood from the same donor.
表３に示すように、Ｆｉｃｏｌｌ−Ｐａｑｕｅ ＰＲＥＭＩＵＭ重層時及びその後の細胞洗浄時に０．９６％ＨＳＡ／生理食塩水、すなわち約１％のＨＳＡを含有する生理食塩水を使用することで、基本法や生理食塩水又はソルデム３Ａの組合わせと比較して、細胞の生存率への影響なく、細胞回収率を上げることができた。一方、生理食塩水及びソルデム３Ａの組合せでは、細胞の生存率が低下した。このことから、本発明の方法は血液からのＰＢＭＣ分離時に好適に使用できることが明らかとなった。 As shown in Table 3, by using 0.96% HSA / saline, that is, a physiological saline containing about 1% HSA at the time of Ficoll-Paque PREMIUM overlay and subsequent cell washing, the basic method and physiological Compared with the combination of saline or Soldem 3A, the cell recovery rate could be increased without affecting the cell viability. On the other hand, the cell viability decreased with the combination of physiological saline and Soldem 3A. From this, it became clear that the method of the present invention can be suitably used when separating PBMC from blood.
実施例３ 得られたＰＢＭＣからのＴ細胞拡大培養
（１）ＰＢＭＣの分離回収
実施例１−（１）と同様の方法で、インフォームド・コンセントの得られたヒト健常人ドナー血液からＰＢＭＣを分離した。その結果を表４に示す。
Example 3 T cell expansion culture from the obtained PBMC (1) Separation and recovery of PBMC In the same manner as in Example 1- (1), PBMC was obtained from human healthy human donor blood from which informed consent was obtained. separated. The results are shown in Table 4.
（２）回収したＰＢＭＣの細胞集団の解析
実施例３−（１）で得られたＰＢＭＣを実施例１−（２）と同様の方法で細胞集団の解析を行った。ただし、実施例１−（２）で使用した抗体に加え、ＦＩＴＣ標識マウス抗ヒトＣＤ１９抗体（ベクトン・ディッキンソン社製）、ＦＩＴＣ標識マウス抗ヒトＣＤ１４抗体（ベクトン・ディッキンソン社製）、ＦＩＴＣ標識マウス抗ヒトＣＤ１５抗体（ベクトン・ディッキンソン社製）も使用した。その結果を表５に示す。すなわち表５は、各々の細胞集団について、ＣＤ３陽性細胞、ＣＤ４陽性細胞、ＣＤ８陽性細胞、ＣＤ３陰性ＣＤ５６陽性細胞、ＣＤ１９陽性細胞、ＣＤ１４陽性細胞及びＣＤ１５陽性細胞の含有率を算出した結果である。
(2) Analysis of recovered PBMC cell population The cell population of the PBMC obtained in Example 3- (1) was analyzed in the same manner as in Example 1- (2). However, in addition to the antibody used in Example 1- (2), FITC-labeled mouse anti-human CD19 antibody (Becton Dickinson), FITC-labeled mouse anti-human CD14 antibody (Becton Dickinson), FITC-labeled mouse anti-antibody Human CD15 antibody (Becton Dickinson) was also used. The results are shown in Table 5. That is, Table 5 shows the results of calculating the content of CD3 positive cells, CD4 positive cells, CD8 positive cells, CD3 negative CD56 positive cells, CD19 positive cells, CD14 positive cells and CD15 positive cells for each cell population.
表５に示すように、Ｆｉｃｏｌｌ−Ｐａｑｕｅ ＰＲＥＭＩＵＭ重層時及びその後の細胞洗浄時に０．９６％ＨＳＡ／生理食塩水、すなわち約１％のＨＳＡを含有する生理食塩水を使用して回収されたＰＢＭＣと、基本法で回収されたＰＢＭＣとの間では、そこに含有される各抗原陽性の細胞の割合に大きな差はなかった。このことから、本発明の方法は血液からのＰＢＭＣ分離時に好適に使用できることが明らかとなった。 As shown in Table 5, PBMC recovered using 0.96% HSA / saline, ie, about 1% HSA, during Ficoll-Paque PREMIUM overlay and subsequent cell wash The proportion of each antigen-positive cell contained therein was not significantly different from the PBMC recovered by the basic method. From this, it became clear that the method of the present invention can be suitably used when separating PBMC from blood.
（３）Ｔ細胞の拡大培養
実施例３−（１）で得られたＰＢＭＣを用いて、Ｔ細胞の拡大培養をおこなった。
抗ＣＤ３抗体ＯＫＴ３（終濃度５μｇ／ｍＬ、ヤンセンファーマ社製）及びレトロネクチン（終濃度２５μｇ／ｍＬ、登録商標、タカラバイオ社製）を含むＡＣＤ−Ａ液（テルモ社製）を培養面積８６ｃｍ２にしたガス透過性培養バッグＣｕｌｔｉＬｉｆｅ（登録商標）２１５（タカラバイオ社製）に１０．４ｍＬ／バッグずつ添加し、ＣＯ２濃度が５％（以下、５％ＣＯ２と記載する）中、３７℃で５時間インキュベートした。更に、上記のバッグは使用前にＲＰＭＩ１６４０で３回洗浄した後、ＯＫＴ３及びレトロネクチン固定化ＣｕｌｔｉＬｉｆｅ２１５として実験に供した。
(3) T cell expansion culture Using the PBMC obtained in Example 3- (1), T cell expansion culture was performed.
An ACD-A solution (manufactured by Terumo) containing anti-CD3 antibody OKT3 (final concentration 5 μg / mL, manufactured by Janssen Pharma) and RetroNectin (final concentration 25 μg / mL, registered trademark, manufactured by Takara Bio Inc.) was applied to a culture area of 86 cm 2 . 10.4 mL / bag was added to the gas-permeable culture bag, MultiLife (registered trademark) 215 (Takara Bio Inc.), and the CO 2 concentration was 5% (hereinafter referred to as 5% CO 2 ) at 37 ° C. Incubated for 5 hours. Further, the bag was washed three times with RPMI 1640 before use, and then subjected to experiments as OKT3 and retronectin-immobilized MultiLife 215.
実施例３−（１）で分離したＰＢＭＣ １．２×１０７ｃｅｌｌｓを、１．０％（Ｖ／Ｖ）非働化済み血漿を含むＧＴ−Ｔ５５１（以下血漿含有ＧＴ−Ｔ５５１と記載）１２０ｍＬに懸濁し、上記で作製したＯＫＴ３及びレトロネクチン固定化ＣｕｌｔｉＬｉｆｅ２１５に添加した。終濃度２００Ｕ／ｍＬとなるようにＩＬ−２（製剤名；Ｐｒｏｌｅｕｋｉｎ、カイロン社製）を添加し、５％ＣＯ２中３７℃で培養を開始した（培養０日目）。培養開始４日目に、各ＣｕｌｔｉＬｉｆｅ２１５内の細胞液を均一な懸濁液とし、一部を希釈して、培養面積を４３０ｃｍ２にした何も固定化していないガス透過性培養バッグＣｕｌｔｉＬｉｆｅ（登録商標）Ｅｖａ（タカラバイオ社製）に移した。この際、細胞液を４０ｍＬ添加し、血漿含有ＧＴ−Ｔ５５１を２９６ｍＬ添加した。終濃度２００Ｕ／ｍＬとなるようにＩＬ−２を添加し、５％ＣＯ２中３７℃で培養した。培養を継続し、培養開始７日目には各ＣｕｌｔｉＬｉｆｅＥｖａに細胞液と等量の血漿を含まないＧＴ−Ｔ５５１を添加し２倍希釈した後、いずれも終濃度２００Ｕ／ｍＬとなるようにＩＬ−２を添加した。培養開始１０日目に自動血球計測装置（ヌクレオカウンター、Ｃｈｅｍｏｍｅｔｅｃ社製）を用いて生細胞数を計測し、培養開始時の細胞数と比較し、拡大培養率を算出した。また、得られた培養後の細胞の細胞集団を実施例１−（２）と同様の方法で解析し、ＣＤ３陽性細胞含有率、ＣＤ４陽性細胞含有率、ＣＤ８陽性細胞含有率、及びＣＤ３陰性ＣＤ５６陽性細胞含有率を算出した。その結果を表６に示す。 PBMC 1.2 × 10 7 cells separated in Example 3- (1) were added to 120 mL of GT-T551 (hereinafter referred to as plasma-containing GT-T551) containing 1.0% (V / V) inactivated plasma. The suspension was added to OKT3 and retronectin-immobilized MultiLife 215 prepared above. IL-2 (formulation name: Proleukin, manufactured by Chiron) was added to a final concentration of 200 U / mL, and culture was started at 37 ° C. in 5% CO 2 (culture day 0). On the 4th day from the start of the culture, the cell solution in each CultureLife 215 was made into a uniform suspension, a part of the culture solution was diluted to a culture area of 430 cm 2 , and nothing was immobilized on the gas-permeable culture bag CultureLife (registered trademark) ) Eva (manufactured by Takara Bio Inc.). At this time, 40 mL of cell fluid was added, and 296 mL of plasma-containing GT-T551 was added. IL-2 was added to a final concentration of 200 U / mL, and the cells were cultured at 37 ° C. in 5% CO 2 . On the 7th day from the start of the culture, GT-T551 containing no plasma equivalent to the cell solution was added to each CultureLifeEva and diluted 2-fold, and then IL- so that the final concentration was 200 U / mL. 2 was added. On the 10th day from the start of culture, the number of viable cells was measured using an automatic blood cell counter (Nucleo Counter, manufactured by Chemometec), and compared with the number of cells at the start of culture, the expansion culture rate was calculated. Further, the obtained cell population of the cultured cells was analyzed by the same method as in Example 1- (2), and the CD3 positive cell content rate, the CD4 positive cell content rate, the CD8 positive cell content rate, and the CD3 negative CD56 The positive cell content was calculated. The results are shown in Table 6.
表６に示すように、どちらの血液分離方法で得られたＰＢＭＣもスタート細胞数を同じとした拡大培養において、拡大培養率、培養後の細胞の構成に差はなかった。すなわち、基本法で回収されたＰＢＭＣと本発明の方法により分離されたＰＢＭＣは、細胞として同じ性質を有するものであることが明らかとなった。従って、本発明の方法は血液からのＰＢＭＣ分離回収時に好適に使用できることが明らかとなった。 As shown in Table 6, there was no difference in the expansion culture rate and the cell configuration after the culture in PBMCs obtained by either blood separation method in the same number of start cells. That is, it became clear that PBMC recovered by the basic method and PBMC separated by the method of the present invention have the same properties as cells. Therefore, it has been clarified that the method of the present invention can be suitably used when separating and recovering PBMC from blood.
実施例４ ＨＳＡ含有生理食塩水を用いた新鮮血からのＰＢＭＣ分離回収
（１）ＰＢＭＣの分離回収
実施例１−（１）と同様の方法で、インフォームド・コンセントの得られたヒト健常人ドナー血液からＰＢＭＣを分離した。ただし、血漿分離後のＰＢＭＣを含む細胞画分は、ＤＰＢＳ、０．１％（Ｖ／Ｗ）ＨＳＡを含む生理食塩水又は０．３％（Ｖ／Ｗ）ＨＳＡを含む生理食塩水（以下、０．１％ＨＳＡ／生理食塩水、０．３％ＨＳＡ／生理食塩水とそれぞれ記載する）、０．９６％ＨＳＡ／生理食塩水を用いてそれぞれ別々に希釈した。中間層のＰＢＭＣの回収後の洗浄は、それぞれＲＰＭＩ１６４０、０．１％ＨＳＡ／生理食塩水、０．３％ＨＳＡ／生理食塩水、０．９６％ＨＳＡ／生理食塩水を用いて３回行った。その結果を表７に示す。表中、ドナー３及び４は異なるドナーの血液を用いて比較を行った結果である。
Example 4 Separation and collection of PBMC from fresh blood using HSA-containing physiological saline (1) Separation and collection of PBMC In the same manner as in Example 1- (1), a healthy human subject with informed consent PBMC were separated from donor blood. However, the cell fraction containing PBMC after plasma separation is DPBS, physiological saline containing 0.1% (V / W) HSA or physiological saline containing 0.3% (V / W) HSA (hereinafter, 0.1% HSA / saline solution and 0.3% HSA / saline solution) and 0.96% HSA / saline solution, respectively. Washing of the intermediate layer after recovery of PBMC was performed three times using RPMI 1640, 0.1% HSA / saline, 0.3% HSA / saline, and 0.96% HSA / saline, respectively. . The results are shown in Table 7. In the table, donors 3 and 4 are the results of comparison using blood from different donors.
表７に示すように、Ｆｉｃｏｌｌ−Ｐａｑｕｅ ＰＲＥＭＩＵＭ重層時及びその後の細胞洗浄時に０．１％〜約１％の範囲でＨＳＡを含む生理食塩水を使用することで、基本法と比較して、細胞の生存率への影響なく、細胞回収率を上げることができた。また、複数のドナー血液を用いた場合でも同様な結果であった。このことから、本発明の方法は血液からのＰＢＭＣ分離回収時に好適に使用できることが明らかとなった。 As shown in Table 7, when using physiological saline containing HSA in the range of 0.1% to about 1% at the time of Ficoll-Paque PREMIUM overlay and subsequent cell washing, The cell recovery rate could be increased without affecting the survival rate. The same result was obtained when a plurality of donor bloods were used. From this, it was revealed that the method of the present invention can be suitably used when separating and recovering PBMC from blood.
実施例５ ＨＳＡ含有生理食塩水を用いた成分採血液からのＰＢＭＣ分離回収
インフォームド・コンセントの得られたヒト健常人ドナーより、成分採血を実施した。ここでいう成分採血とは、単核球採取を目的とした採血である。得られた成分採血液をＤＰＢＳ又は０．９６％ＨＳＡ／生理食塩水で希釈後、Ｆｉｃｏｌｌ−ｐａｑｕｅ ＰＲＥＭＩＵＭ上に重層して７００×ｇで２０分間遠心した。中間層のＰＢＭＣをピペットで回収後、それぞれＲＰＭＩ１６４０又は０．９６％ＨＳＡ／生理食塩水を用いて３回洗浄した。その結果を表８に示す。表中、ドナー５及び６は異なるドナーの成分採血液を用いて比較を行った結果である。
Example 5 Separation and recovery of PBMC from component blood using HSA-containing physiological saline Component blood was collected from a healthy human donor with informed consent. The component blood collection here refers to blood collection for the purpose of collecting mononuclear cells. The obtained component blood sample was diluted with DPBS or 0.96% HSA / saline, layered on Ficoll-paque PREMIUM, and centrifuged at 700 × g for 20 minutes. The intermediate layer PBMCs were collected with a pipette and then washed three times with RPMI 1640 or 0.96% HSA / saline, respectively. The results are shown in Table 8. In the table, donors 5 and 6 are the results of comparison using component blood from different donors.
表８に示すように、Ｆｉｃｏｌｌ−Ｐａｑｕｅ ＰＲＥＭＩＵＭ重層時及びその後の細胞洗浄時に０．９６％ＨＳＡ／生理食塩水、すなわち約１％のＨＳＡを含有する生理食塩水を使用することで、基本法と比較して、細胞の生存率への影響なく、細胞回収率を上げることができた。また、複数のドナーの成分採血液を用いた場合でも同様な結果であった。このことから、本発明の方法は細胞含有液の違いに関係なく、成分採血液からのＰＢＭＣ分離回収時においても好適に使用できることが明らかとなった。 As shown in Table 8, compared with the basic method by using 0.96% HSA / saline, that is, a physiological saline containing about 1% HSA during Ficoll-Paque PREMIUM overlay and subsequent cell washing Thus, the cell recovery rate could be increased without affecting the cell viability. In addition, similar results were obtained when component blood samples from a plurality of donors were used. From this, it has been clarified that the method of the present invention can be suitably used at the time of PBMC separation and recovery from component blood regardless of the difference in the cell-containing liquid.
実施例６ ＨＳＡ含有生理食塩水を用いた新鮮血からのＰＢＭＣ分離回収
（１）ＰＢＭＣの分離回収
実施例１−（１）と同様の方法で、インフォームド・コンセントの得られたヒト健常人ドナー血液からＰＢＭＣを分離した。ただし、血漿分離後のＰＢＭＣを含む細胞画分は、ＤＰＢＳ、０．９６％ＨＳＡ／生理食塩水又は４．２％（Ｗ／Ｖ）ＨＳＡを含む生理食塩水（以下、４．２％ＨＳＡ／生理食塩水と記載する）を用いてそれぞれ別々に希釈した。中間層のＰＢＭＣの回収後の洗浄は、それぞれＲＰＭＩ１６４０、０．９６％ＨＳＡ／生理食塩水、４．２％ＨＳＡ/生理食塩水を用いて３回行った。その結果を表９に示す。
Example 6 Separation and collection of PBMC from fresh blood using HSA-containing physiological saline (1) Separation and collection of PBMC In the same manner as in Example 1- (1), a healthy human subject with informed consent PBMC were separated from donor blood. However, the cell fraction containing PBMCs after plasma separation is DPBS, 0.96% HSA / saline or 4.2% (W / V) saline (hereinafter referred to as 4.2% HSA / (Referred to as “saline solution”). Washing of the intermediate layer after recovery of PBMC was performed three times using RPMI 1640, 0.96% HSA / saline, and 4.2% HSA / saline, respectively. The results are shown in Table 9.
表９に示すように、Ｆｉｃｏｌｌ−Ｐａｑｕｅ ＰＲＥＭＩＵＭ重層時及びその後の細胞洗浄時に０．９６％〜４．２％の範囲でＨＳＡを含む生理食塩水を使用することで、基本法と比較して、細胞の生存率への影響なく、細胞回収率を上げることができた。このことから、本発明の方法は血液からのＰＢＭＣ分離回収時に好適に使用されることが明らかとなった。 As shown in Table 9, by using physiological saline containing HSA in the range of 0.96% to 4.2% at the time of Ficoll-Paque PREMIUM overlay and subsequent cell washing, cells were compared with the basic method. The cell recovery rate could be increased without affecting the survival rate. From this, it has been clarified that the method of the present invention is suitably used at the time of separating and recovering PBMC from blood.
実施例７ ＢＳＡ含有生理食塩水を用いた新鮮血からのＰＢＭＣ分離回収
（１）ＰＢＭＣの分離回収
実施例１−（１）と同様の方法で、インフォームド・コンセントの得られたヒト健常人ドナー血液からＰＢＭＣを分離した。ただし、血漿分離後のＰＢＭＣを含む細胞画分は、ＤＰＢＳ、又は１％（Ｗ／Ｖ）ＢＳＡを含む生理食塩水（以下、１％ＢＳＡ／生理食塩水と記載する）を用いてそれぞれ別々に希釈した。中間層のＰＢＭＣの回収後の洗浄は、それぞれＲＰＭＩ１６４０、１％ＢＳＡ／生理食塩水を用いて３回行った。その結果を表１０に示す。
Example 7 Separation and collection of PBMC from fresh blood using BSA-containing physiological saline (1) Separation and collection of PBMC In the same manner as in Example 1- (1), a healthy human subject with informed consent PBMC were separated from donor blood. However, the cell fraction containing PBMC after plasma separation was separately prepared using DPBS or physiological saline containing 1% (W / V) BSA (hereinafter referred to as 1% BSA / physiological saline). Diluted. Washing of the intermediate layer after recovery of PBMC was performed three times using RPMI 1640 and 1% BSA / saline, respectively. The results are shown in Table 10.
表１０に示すように、Ｆｉｃｏｌｌ−Ｐａｑｕｅ ＰＲＥＭＩＵＭ重層時及びその後の細胞洗浄時に１％ＢＳＡ／生理食塩水を使用することで、基本法と比較して、細胞の生存率への影響なく、細胞回収率を上げることができた。このことから、本発明の方法は血液からのＰＢＭＣ分離回収時に好適に使用されることが明らかとなった。 As shown in Table 10, by using 1% BSA / saline at the time of Ficoll-Paque PREMIUM overlaying and subsequent cell washing, the cell recovery rate was not affected as compared to the basic method without affecting the cell viability. I was able to raise. From this, it has been clarified that the method of the present invention is suitably used at the time of separating and recovering PBMC from blood.
実施例８ ＨＳＡ含有生理食塩水を用いた新鮮血からのＰＢＭＣ分離回収
（１）ＰＢＭＣの分離回収
実施例１−（１）と同様の方法で、インフォームド・コンセントの得られたヒト健常人ドナー血液からＰＢＭＣを分離した。ただし、血漿分離後のＰＢＭＣを含む細胞画分は、ＤＰＢＳ、５％（Ｗ／Ｖ）ＨＳＡを含む生理食塩水（以下５％ＨＳＡ／生理食塩水と記載する）及びＦｉｃｏｌｌ−Ｐａｑｕｅ ＰＲＥＭＩＵＭの添付資料に血液分離時の推奨バッファーとして記載されているＢｌａｎｃｅｄ ｓｏｌｔ ｓｏｌｕｔｉｏｎ（以下、推奨バッファーと記載する）を用いてそれぞれ別々に希釈した。推奨バッファーは、１ｇのＤ−グルコース（ナカライテスク社製）、０．００７４ｇの塩化カルシウム２水和物（ナカライテスク社製）、０．１９９２ｇの塩化マグネシウム６水和物（ナカライテスク社製）、０．４０２６ｇの塩化カリウム（ナカライテスク社製）、１７．５６５ｇのＴｒｉｓ（ナカライテスク社製）を蒸留水（大塚製薬社製）で溶解し、５Ｎ塩酸（和光純薬社製）でｐＨ７．６に調製後、蒸留水で１Ｌにメスアップしてからフィルターユニット（旭テクノグラス社製）で滅菌したバッファーＡと、８．１９ｇの塩化ナトリウム（ナカライテスク社製）を蒸留水で１Ｌにメスアップしてからオートクレーブ滅菌したバッファーＢとを１：９の割合で混合して調製した。中間層のＰＢＭＣの回収後の洗浄は、それぞれＲＰＭＩ１６４０、５％ＨＳＡ／生理食塩水及び推奨バッファーを用いて３回行った。その結果を表１１に示す。
Example 8 Separation and collection of PBMC from fresh blood using physiological saline containing HSA (1) Separation and collection of PBMC In the same manner as in Example 1- (1), a healthy human subject with informed consent PBMC were separated from donor blood. However, the cell fraction containing PBMC after plasma separation is DPBS, physiological saline containing 5% (W / V) HSA (hereinafter referred to as 5% HSA / physiological saline), and attached documents of Ficoll-Paque PREMIUM. Each was diluted separately using a branched salt solution (hereinafter referred to as a recommended buffer) described as a recommended buffer during blood separation. The recommended buffer is 1 g of D-glucose (Nacalai Tesque), 0.0074 g of calcium chloride dihydrate (Nacalai Tesque), 0.1992 g of magnesium chloride hexahydrate (Nacalai Tesque), 0.4026 g of potassium chloride (manufactured by Nacalai Tesque), 17.565 g of Tris (manufactured by Nacalai Tesque) are dissolved in distilled water (manufactured by Otsuka Pharmaceutical Co., Ltd.), and the pH is 7.6 with 5N hydrochloric acid (manufactured by Wako Pure Chemical Industries, Ltd.). After making up to 1 L with distilled water, buffer A sterilized with a filter unit (Asahi Techno Glass) and 8.19 g of sodium chloride (Nacalai Tesque) were made up to 1 L with distilled water. Then, it was prepared by mixing with autoclaved buffer B at a ratio of 1: 9. Washing of the intermediate layer after recovery of PBMC was performed 3 times using RPMI 1640, 5% HSA / saline, and recommended buffer, respectively. The results are shown in Table 11.
表１１に示すように、Ｆｉｃｏｌｌ−Ｐａｑｕｅ ＰＲＥＭＩＵＭ重層時及びその後の細胞洗浄時に５％のＨＳＡを含む生理食塩水を使用することで、基本法と比較して、細胞の生存率への影響なく、細胞回収率を上げることができた。また、実施例４、実施例６及び実施例８の結果から、０．１％〜５％の範囲でＨＳＡを含む生理食塩水を使用する事で、基本法と比較して、細胞の生存率への影響なく、細胞回収率を上げられることが明らかとなった。さらにはＨＳＡを含む生理食塩水を使用することは、分離媒体（Ｆｉｃｏｌｌ−Ｐａｑｕｅ ＰＲＥＭＩＵＭ）販売元が血液分離時に推奨するバッファーと比較して、細胞の生存率への影響なく、細胞回収率を上げることができた。このことから、本発明の方法は血液からのＰＢＭＣ分離回収時に好適に使用できることが明らかとなった。 As shown in Table 11, by using physiological saline containing 5% HSA at the time of Ficoll-Paque PREMIUM overlay and subsequent cell washing, the cell viability was not affected as compared with the basic method. The recovery rate could be increased. In addition, from the results of Example 4, Example 6 and Example 8, by using a physiological saline containing HSA in the range of 0.1% to 5%, the cell viability is improved as compared with the basic method. It was clarified that the cell recovery rate can be increased without the influence of. Furthermore, the use of physiological saline containing HSA increases the cell recovery rate without affecting the cell viability compared to the buffer recommended by the separation medium (Ficoll-Paque PREMIUM) vendor during blood separation. I was able to. From this, it was revealed that the method of the present invention can be suitably used when separating and recovering PBMC from blood.
（２）回収したＰＢＭＣの細胞集団の解析
実施例８−（１）で得られたＰＢＭＣを実施例２−（２）と同様の方法で細胞集団の解析を行った。ただし、ＡＣＫ Ｌｙｓｉｎｇ Ｂｕｆｆｅｒで残存赤血球の溶血処理は行わなかった。その結果を表１２に示す。
(2) Analysis of cell population of recovered PBMC The cell population of PBMC obtained in Example 8- (1) was analyzed in the same manner as in Example 2- (2). However, the erythrocyte was not subjected to hemolysis treatment with ACK Lysing Buffer. The results are shown in Table 12.
表１２に示すように、Ｆｉｃｏｌｌ−Ｐａｑｕｅ ＰＲＥＭＩＵＭ重層時及びその後の細胞洗浄時に５％ＨＳＡ／生理食塩水、推奨バッファーを使用して回収されたＰＢＭＣと、基本法で回収されたＰＢＭＣとの間では、含有される各抗原陽性の細胞の割合にほとんど差はなかった。また同様に５％のＨＳＡを含む生理食塩水を使用することは、推奨バッファーと比較しても、含有される各抗原陽性細胞の割合にほとんど差はなかった。すなわち、基本法で回収されたＰＢＭＣと本発明の方法により分離されたＰＢＭＣは、得られる各細胞の含有率がほぼ同じものであることが明らかとなった。このことから、本発明の方法は血液からのＰＢＭＣ分離回収時に好適に使用されることが明らかとなった。 As shown in Table 12, between the PBMC recovered using the 5% HSA / saline solution and the recommended buffer at the time of Ficoll-Paque PREMIUM overlay and subsequent cell washing, and the PBMC recovered by the basic method, There was little difference in the proportion of each antigen-positive cell contained. Similarly, the use of physiological saline containing 5% HSA showed almost no difference in the proportion of each antigen-positive cell contained even when compared with the recommended buffer. That is, it was revealed that the content of each cell obtained was almost the same between the PBMC recovered by the basic method and the PBMC separated by the method of the present invention. From this, it has been clarified that the method of the present invention is suitably used at the time of separating and recovering PBMC from blood.
実施例９ カゼインナトリウム含有生理食塩水を用いた新鮮血からのＰＢＭＣ分離回収
（１）ＰＢＭＣの分離回収
実施例１−（１）と同様の方法で、インフォームド・コンセントの得られたヒト健常人ドナー血液からＰＢＭＣを分離した。ただし、血漿分離後のＰＢＭＣを含む細胞画分は、ＤＰＢＳ、１％のカゼインナトリウム（和光純薬社製）を含む生理食塩水（以下、１％カゼインＮａ／生理食塩水と記載する）及び０．２％のカゼインナトリウムを含む生理食塩水（以下、０．２％カゼインＮａ／生理食塩水と記載する）を用いてそれぞれ別々に希釈した。中間層のＰＢＭＣの回収後の洗浄は、それぞれＲＰＭＩ１６４０、１％カゼインＮａ／生理食塩水及び０．２％カゼインＮａ／生理食塩水を用いて３回行った。その結果を表１３に示す。
Example 9 Separation and collection of PBMC from fresh blood using sodium casein-containing physiological saline (1) Separation and collection of PBMC In the same manner as in Example 1- (1), human healthy subject informed consent was obtained. PBMC were isolated from human donor blood. However, the cell fraction containing PBMCs after plasma separation is composed of physiological saline (hereinafter referred to as 1% casein Na / saline) containing DPBS, 1% sodium casein (manufactured by Wako Pure Chemical Industries, Ltd.) and 0 Each sample was diluted separately with physiological saline containing 2% sodium casein (hereinafter referred to as 0.2% casein Na / saline). Washing of the intermediate layer after recovery of PBMC was performed 3 times using RPMI 1640, 1% casein Na / saline, and 0.2% casein Na / saline, respectively. The results are shown in Table 13.
表１３に示すように、Ｆｉｃｏｌｌ−Ｐａｑｕｅ ＰＲＥＭＩＵＭ重層時及びその後の細胞洗浄時に１％又は０．２％のカゼインナトリウムを含む生理食塩水を使用することで、基本法と比較して、細胞の生存率への影響なく、細胞回収率を上げることができた。また、複数のドナー血液を用いた場合でも同様な結果であった。このことから、本発明の方法は血液からのＰＢＭＣ分離回収時に好適に使用できることが明らかとなった。 As shown in Table 13, by using physiological saline containing 1% or 0.2% sodium caseinate at the time of Ficoll-Paque PREMIUM overlay and subsequent cell washing, cell viability was compared with the basic method. The cell recovery rate could be increased without any effect on the cell. The same result was obtained when a plurality of donor bloods were used. From this, it was revealed that the method of the present invention can be suitably used when separating and recovering PBMC from blood.
（２）回収したＰＢＭＣの細胞集団の解析
実施例９−（１）で得られたＰＢＭＣを実施例２−（２）と同様の方法で細胞集団の解析を行った。ただし、ＡＣＫ Ｌｙｓｉｎｇ Ｂｕｆｆｅｒで残存赤血球の溶血処理を行わなかった。その結果を表１４に示す。
(2) Analysis of cell population of recovered PBMC The cell population of PBMC obtained in Example 9- (1) was analyzed in the same manner as in Example 2- (2). However, the erythrocyte was not subjected to hemolysis treatment with ACK Lysing Buffer. The results are shown in Table 14.
表１４に示すように、Ｆｉｃｏｌｌ−Ｐａｑｕｅ ＰＲＥＭＩＵＭ重層時及びその後の細胞洗浄時に５％ＨＳＡ／生理食塩水、１％又は０．２％のカゼインナトリウムを含有する生理食塩水を使用して回収されたＰＢＭＣと、基本法で回収されたＰＢＭＣとの間では、そこに含有される各抗原陽性の細胞の割合にほとんど差はなかった。また、複数のドナー血液を用いた場合でも同様な結果であった。すなわち、基本法で回収されたＰＢＭＣと本発明の方法により分離されたＰＢＭＣは、得られる各細胞の含有率がほぼ同じものであることが明らかとなった。このことから、本発明の方法は血液からのＰＢＭＣ分離回収時に好適に使用できることが明らかとなった。 As shown in Table 14, recovered using Ficoll-Paque PREMIUM overlay and subsequent cell wash using saline containing 5% HSA / saline, 1% or 0.2% sodium caseinate. There was almost no difference in the proportion of each antigen-positive cell contained in PBMC and PBMC recovered by the basic method. The same result was obtained when a plurality of donor bloods were used. That is, it was revealed that the content of each cell obtained was almost the same between the PBMC recovered by the basic method and the PBMC separated by the method of the present invention. From this, it was revealed that the method of the present invention can be suitably used when separating and recovering PBMC from blood.
（３）ＰＢＭＣの保存
実施例９−（１）で得られたドナー８のＰＢＭＣをＲＰＭＩ１６４０に懸濁後、ＣＰ−１（極東製薬工業社製）と２５％ＨＳＡ（製剤名ブミネート、バクスター社製）を１７：８の割合で混合した保存液を等量加えて液体窒素中にて保存した。使用時には、これら保存ＰＢＭＣを３７℃水浴中にて急速融解し、１０μｇ／ｍＬのＤＮａｓｅ（カルビオケム社製）を含むＧＴ−Ｔ５５１（タカラバイオ社製）で洗浄後、各実験に供した。
(3) Preservation of PBMC After suspending PBMC of donor 8 obtained in Example 9- (1) in RPMI 1640, CP-1 (manufactured by Kyokuto Pharmaceutical Co., Ltd.) and 25% HSA (formulation name Buminate, manufactured by Baxter) ) Was added at an equal volume of 17: 8 and stored in liquid nitrogen. At the time of use, these stored PBMCs were rapidly melted in a 37 ° C. water bath, washed with GT-T551 (Takara Bio) containing 10 μg / mL DNase (Calbiochem), and then used for each experiment.
（４）Ｔ細胞の拡大培養
実施例９−（３）で得られたドナー８のＰＢＭＣを用いて、Ｔ細胞の拡大培養を行った。
ＯＫＴ３（終濃度０．３μｇ／ｍＬ）を含むＤＰＢＳを培養面積３．８ｃｍ２の１２ウエルプレート（コーニング社製）に０．４５ｍＬ／ウエルずつ添加し、５％ＣＯ２中３７℃で５時間インキュベートした。また、上記の１２ウエルプレートは使用前に培養器材から当該ＤＰＢＳを吸引除去後、各ウエルをＤＰＢＳで２回、ＲＰＭＩ１６４０で１回洗浄し各実験に供した。
(4) Expanded culture of T cells Using the PBMC of donor 8 obtained in Example 9- (3), expanded culture of T cells was performed.
DPBS containing OKT3 (final concentration 0.3 μg / mL) was added to a 12-well plate (manufactured by Corning) at a culture area of 3.8 cm 2 at 0.45 mL / well and incubated at 37 ° C. in 5% CO 2 for 5 hours. did. In addition, the 12-well plate was aspirated and removed from the culture equipment before use, and then each well was washed twice with DPBS and once with RPMI 1640 and used for each experiment.
実施例９−（３）で得られたドナー８のＰＢＭＣ ０．５３×１０６ｃｅｌｌｓを、血漿含有ＧＴ−Ｔ５５１ ５．３ｍＬに懸濁し、上記で作製したＯＫＴ３固定化１２ウエルプレートに添加した。終濃度２００Ｕ／ｍＬとなるようにＩＬ−２を添加し、５％ＣＯ２中３７℃で培養を開始した（培養０日目）。培養開始４日目に、各１２ウエルプレート内の細胞液を均一な懸濁液とし、一部を血漿含有ＧＴ−Ｔ５５１で約１２倍希釈して、この希釈液約７．８ｍＬを培養面積が１０ｃｍ２の何も固定化していないＴ２５細胞培養フラスコ（コーニング社製）を立てたものに移した。終濃度２００Ｕ／ｍＬとなるようにＩＬ−２を添加し、５％ＣＯ２中３７℃で培養した。培養を継続し、培養開始７日目には各フラスコに細胞液と等量の血漿を含まないＧＴ−Ｔ５５１を添加し２倍希釈した後、いずれも終濃度２００Ｕ／ｍＬとなるようにＩＬ−２を添加した。培養開始１１日目にトリパンブルー染色法にて生細胞数を計測し、培養開始時の細胞数と比較し、拡大培養率を算出した。また、得られた培養後の細胞の細胞集団を実施例１−（２）と同様の方法で解析し、ＣＤ３陽性細胞含有率、ＣＤ４陽性細胞含有率、ＣＤ８陽性細胞含有率、及びＣＤ３陰性ＣＤ５６陽性細胞含有率を算出した。表中の基本法の値はＮ＝２の平均値を示し、１％カゼインＮａ／生理食塩水はＮ＝１の値を示す。その結果を表１５に示す。 PBMC 0.53 × 10 6 cells of donor 8 obtained in Example 9- (3) were suspended in 5.3 mL of plasma-containing GT-T551 and added to the OKT3-immobilized 12-well plate prepared above. IL-2 was added to a final concentration of 200 U / mL, and culture was started at 37 ° C. in 5% CO 2 (culture day 0). On the 4th day from the start of the culture, the cell solution in each 12-well plate was made into a uniform suspension, and a part thereof was diluted about 12-fold with plasma-containing GT-T551. A 10 cm 2 nothing-immobilized T25 cell culture flask (Corning) was transferred to a standing one. IL-2 was added to a final concentration of 200 U / mL, and the cells were cultured at 37 ° C. in 5% CO 2 . The culture was continued, and on day 7 after the start of culture, GT-T551 containing no plasma equivalent to the cell solution was added to each flask and diluted 2-fold, and then IL- was added so that the final concentration was 200 U / mL. 2 was added. On the 11th day from the start of culture, the number of viable cells was counted by trypan blue staining, and compared with the number of cells at the start of culture, the expansion culture rate was calculated. Further, the obtained cell population of the cultured cells was analyzed by the same method as in Example 1- (2), and the CD3 positive cell content rate, the CD4 positive cell content rate, the CD8 positive cell content rate, and the CD3 negative CD56 The positive cell content was calculated. The value of the basic method in the table shows the average value of N = 2, and 1% casein Na / saline shows the value of N = 1. The results are shown in Table 15.
表１５に示すように、１％カゼインＮａ／生理食塩水を用いた血液分離方法で得られたＰＢＭＣもスタート細胞数を同じとした拡大培養において、拡大培養率、培養後の細胞の構成に差はなかった。すなわち、基本法で回収されたＰＢＭＣと本発明の方法により分離されたＰＢＭＣは、細胞として同じ性質を有するものであることが明らかとなった。従って、本発明の方法は血液からのＰＢＭＣ分離回収時に好適に使用されることが明らかとなった。 As shown in Table 15, PBMCs obtained by the blood separation method using 1% casein Na / saline also differed in the expansion culture rate and the cell configuration after the culture in the expansion culture with the same start cell number. There was no. That is, it became clear that PBMC recovered by the basic method and PBMC separated by the method of the present invention have the same properties as cells. Therefore, it has been clarified that the method of the present invention is suitably used when separating and recovering PBMC from blood.
実施例１０ ＮｙｃｏＰｒｅｐ １．０７７での新鮮血からのＰＢＭＣ分離回収
実施例１−（１）と同様の方法で、インフォームド・コンセントの得られたヒト健常人ドナー血液からＰＢＭＣを分離した。ただし、密度勾配形成用媒体としてＦｉｃｏｌｌ−Ｐａｑｕｅ ＰＲＥＭＩＵＭ、及びＮｙｃｏＰｒｅｐ １．０７７（ＡＸＩＳ−ＳＨＩＥＬＤ社製）を用いた。また、血漿分離後のＰＢＭＣを含む細胞画分は、ＤＰＢＳ、５％ＨＳＡ／生理食塩水を用いてそれぞれ別々に希釈した。中間層のＰＢＭＣの回収後の洗浄は、分離剤ごとにそれぞれＲＰＭＩ１６４０、５％ＨＳＡ／生理食塩水を用いて３回行った。その結果を表１６に示す。
Example 10 PBMC Separation and Recovery from Fresh Blood with NycoPrep 1.077 PBMC was separated from human healthy human donor blood with informed consent in the same manner as in Example 1- (1). However, Ficoll-Paque PREMIUM and NycoPrep 1.077 (manufactured by AXIS-SHIELD) were used as the density gradient forming medium. The cell fraction containing PBMC after plasma separation was diluted separately with DPBS, 5% HSA / saline. Washing of the intermediate layer after recovery of PBMC was performed three times for each separating agent using RPMI 1640 and 5% HSA / saline. The results are shown in Table 16.
表１６に示すように、Ｆｉｃｏｌｌ−Ｐａｑｕｅ ＰＲＥＭＩＵＭを密度勾配形成用媒体として使用した場合と同等に、ＮｙｃｏＰｒｅｐ １．０７７重層時及びその後の細胞洗浄時に５％ＨＳＡ／生理食塩水を使用することで、細胞の生存率への影響なく、細胞回収率を上げることができた。このことから、密度勾配形成用媒体によらず本発明の方法は血液からのＰＢＭＣ分離回収時に好適に使用できることが明らかとなった。 As shown in Table 16, using 5% HSA / saline at the time of NycoPrep 1.077 layering and subsequent cell washing, equivalent to the case where Ficoll-Paque PREMIUM is used as the density gradient forming medium, The cell recovery rate could be increased without affecting the cell viability. From this, it became clear that the method of the present invention can be suitably used at the time of PBMC separation and recovery from blood regardless of the density gradient forming medium.
実施例１１ ＨＳＡ含有生理食塩水を用いた成分採血液からのＰＢＭＣ分離回収
（１）ＰＢＭＣの分離回収
インフォームド・コンセントの得られたヒト健常人ドナーより、成分採血を実施した。得られた成分採血液を０．９６％又は５％ＨＳＡ／生理食塩水で希釈後、Ｆｉｃｏｌｌ−ｐａｑｕｅ ＰＲＥＭＩＵＭ上に重層して７００×ｇで２０分間遠心した。中間層のＰＢＭＣをピペットで回収後、それぞれ０．９６％又は５％ＨＳＡ／生理食塩水を用いて３回洗浄した。結果を表１７に示す。
Example 11 Separation and recovery of PBMC from component blood using HSA-containing physiological saline (1) Separation and recovery of PBMC Component blood was collected from a healthy human donor with informed consent. The obtained component blood sample was diluted with 0.96% or 5% HSA / saline, layered on Ficoll-paque PREMIUM, and centrifuged at 700 × g for 20 minutes. The intermediate layer PBMCs were collected with a pipette and then washed three times with 0.96% or 5% HSA / saline, respectively. The results are shown in Table 17.
表１７に示すように、Ｆｉｃｏｌｌ−Ｐａｑｕｅ ＰＲＥＭＩＵＭ重層時及びその後の細胞洗浄時に５％ＨＳＡ／生理食塩水を使用することで、０．９６％ＨＳＡ／生理食塩水、すなわち約１％のＨＳＡを含有する生理食塩水を使用した場合と比較して、細胞の生存率への影響なく、細胞回収率を上げることができた。なお両条件とも基本法に比べ高い細胞回収率を示した。これらのことから、本発明の方法は成分採血液からのＰＢＭＣ分離回収時においても好適に使用できることが明らかとなった。 As shown in Table 17, 0.96% HSA / saline, that is, about 1% HSA is contained by using 5% HSA / saline at the time of Ficoll-Paque PREMIUM overlay and subsequent cell washing. Compared with using normal saline, the cell recovery rate could be increased without affecting the cell viability. Both conditions showed higher cell recovery than the basic method. From these facts, it has been clarified that the method of the present invention can be suitably used even when PBMC is separated and collected from component blood.
実施例１２ ＨＳＡ含有生理食塩水を用いた新鮮血からのＰＢＭＣ分離回収
（１）ＰＢＭＣの分離回収
実施例１−（１）と同様の方法で、インフォームド・コンセントの得られたヒト健常人ドナー血液からＰＢＭＣを分離した。ただし、血漿分離後のＰＢＭＣを含む細胞画分は、ＤＰＢＳ、０．９６％ＨＳＡ（製剤名ブミネート、バクスター社製）／生理食塩水又は０．９６％ＨＳＡ（製剤名アルブミナー、ＣＳＬベーリング社製）／生理食塩水を用いてそれぞれ別々に希釈した。中間層のＰＢＭＣの回収後の洗浄は、表１８記載の洗浄時溶媒を用いて３回行った。その結果を表１８に示す。
Example 12 Separation and collection of PBMC from fresh blood using HSA-containing physiological saline (1) Separation and collection of PBMC In the same manner as in Example 1- (1), a healthy human subject with informed consent PBMC were separated from donor blood. However, the cell fraction containing PBMC after plasma separation is DPBS, 0.96% HSA (formulation name Buminate, manufactured by Baxter) / saline or 0.96% HSA (formulation name Albuminer, manufactured by CSL Bering) / Diluted separately with physiological saline. Washing of the intermediate layer after the recovery of PBMC was performed three times using the washing solvent shown in Table 18. The results are shown in Table 18.
表１８に示すように、Ｆｉｃｏｌｌ−Ｐａｑｕｅ ＰＲＥＭＩＵＭ重層時及びその後の細胞洗浄時に０．９６％ＨＳＡ／生理食塩水、すなわち約１％のＨＳＡを含有する生理食塩水を使用することで、ＨＳＡの製造元によらず、基本法と比較して、細胞の生存率への影響なく、細胞回収率を上げることができた。このことから、本発明の方法は血液からのＰＢＭＣ分離回収時に好適に使用できることが明らかとなった。 As shown in Table 18, by using 0.96% HSA / saline, that is, a saline containing about 1% HSA, during Ficoll-Paque PREMIUM overlay and subsequent cell washing, the manufacturer of HSA Regardless of the basic method, the cell recovery rate could be increased without affecting the cell viability. From this, it was revealed that the method of the present invention can be suitably used when separating and recovering PBMC from blood.
実施例１３ Ｈｉｓｔｏｐａｑｕｅ １．０７７での新鮮血からのＰＢＭＣ分離回収
（１）ＰＢＭＣの分離回収
実施例１−（１）と同様の方法で、インフォームド・コンセントの得られたヒト健常人ドナー血液からＰＢＭＣを分離した。ただし、密度勾配形成用媒体としてＦｉｃｏｌｌ−Ｐａｑｕｅ ＰＲＥＭＩＵＭとＨｉｓｔｏｐａｑｕｅ １．０７７（シグマ社製）を用いた。また、血漿分離後のＰＢＭＣを含む細胞画分は、ＤＰＢＳ、５％ＨＳＡ／生理食塩水を用いてそれぞれ別々に希釈した。中間層のＰＢＭＣの回収後の洗浄は、分離剤ごとにそれぞれＲＰＭＩ１６４０、５％ＨＳＡ／生理食塩水を用いて３回行った。その結果を表１９に示す。
Example 13 Separation and collection of PBMC from fresh blood with Histopaque 1.077 (1) Separation and collection of PBMC In the same manner as in Example 1- (1), human healthy human donor blood obtained informed consent PBMCs were separated from However, Ficoll-Paque PREMIUM and Histopaque 1.077 (manufactured by Sigma) were used as the density gradient forming medium. The cell fraction containing PBMC after plasma separation was diluted separately with DPBS, 5% HSA / saline. Washing of the intermediate layer after recovery of PBMC was performed three times for each separating agent using RPMI 1640 and 5% HSA / saline. The results are shown in Table 19.
表１９に示すように、Ｆｉｃｏｌｌ−Ｐａｑｕｅ ＰＲＥＭＩＵＭを密度勾配形成用媒体として使用した場合と同等に、Ｈｉｓｔｏｐａｑｕｅ １．０７７重層時及びその後の細胞洗浄時に５％ＨＳＡ／生理食塩水を使用することで、細胞の生存率への影響なく、細胞回収率を上げることができた。このことから、密度勾配形成用媒体によらず本発明の方法は血液からのＰＢＭＣ分離回収時に好適に使用できることが明らかとなった。 As shown in Table 19, using Ficoll-Paque PREMIUM as a density gradient forming medium, using 5% HSA / saline at the time of Histopaque 1.077 layering and subsequent cell washing, The cell recovery rate could be increased without affecting the cell viability. From this, it became clear that the method of the present invention can be suitably used at the time of PBMC separation and recovery from blood regardless of the density gradient forming medium.
実施例１４ ＨＳＡ含有生理食塩水を用いたＰＢＭＣの保存
（１）ＰＢＭＣの分離回収と保存
実施例１−（１）と同様の方法で、インフォームド・コンセントの得られたヒト健常人ドナー血液からＰＢＭＣを分離した。ただし、血漿分離後のＰＢＭＣを含む細胞画分は、０．９６％ＨＳＡ／生理食塩水を用いて２５ｍＬにフィルアップし希釈し、中間層のＰＢＭＣの回収後の洗浄は、０．９６％ＨＳＡ／生理食塩水を用いて３回行った。この際、遠心はすべて室温で行った。得られたＰＢＭＣを３本のコニカルチューブに分注し、５００×ｇ、室温で５分間遠心し、上清を除去した。各コニカルチューブについて、細胞濃度が１×１０７ｃｅｌｌｓ／ｍＬとなるように２．５％ＨＳＡ／生理食塩水、ＣＰＳ−１（細胞科学研究所社製）、２．５％ＨＳＡ／ＣＰＳ−１に懸濁し、４℃暗所で保存した。各溶媒で保存当日、１日後、２日後の細胞濃度及び生存率を表２０に示す。
Example 14 Preservation of PBMC using HSA-containing physiological saline (1) Separation and collection and storage of PBMC In the same manner as in Example 1- (1), human healthy donor blood from which informed consent was obtained PBMCs were separated from However, the cell fraction containing PBMC after plasma separation was filled up and diluted to 25 mL with 0.96% HSA / saline, and washing after recovery of PBMC in the intermediate layer was 0.96% HSA. / Performed 3 times with physiological saline. At this time, all centrifugation was performed at room temperature. The obtained PBMC was dispensed into three conical tubes, centrifuged at 500 × g for 5 minutes at room temperature, and the supernatant was removed. For each conical tube, 2.5% HSA / saline, CPS-1 (manufactured by Cell Science Laboratories), 2.5% HSA / CPS-1 so that the cell concentration is 1 × 10 7 cells / mL. And stored in the dark at 4 ° C. Table 20 shows the cell concentration and viability after 1 day, 2 days after storage on each solvent.
表２０に示すように、ＰＢＭＣを４℃保存する際に、２．５％ＨＳＡ／生理食塩水、２．５％ＨＳＡを添加したＣＰＳ−１溶液を使用することで、添加していないＣＰＳ−１と比較して、細胞濃度を高く保持した状態で保存可能であることが示された。このことから、２．５％ＨＳＡは細胞の４℃保存時に好適に使用できることが明らかとなった。ＰＢＭＣの保存は、凍結状態で行われることが常法であるが、4℃という未凍結状態での保存時に本方法は好適に使用される。また、ＨＳＡ／生理食塩水で保存可能なことから、保存後に細胞を使用する際、ＣＰＳ−１の成分を除く洗浄工程が不要であり、保存していた細胞をそのまま使用可能であることが明らかとなった。 As shown in Table 20, when PBMC was stored at 4 ° C., a CPS-1 solution added with 2.5% HSA / saline and 2.5% HSA was used. Compared with 1, it was shown that the cell concentration could be preserved in a high state. From this, it became clear that 2.5% HSA can be suitably used when the cells are stored at 4 ° C. Although PBMC is usually stored in a frozen state, this method is preferably used when stored in an unfrozen state at 4 ° C. In addition, since it can be stored in HSA / saline, it is clear that when the cells are used after storage, a washing step for removing the components of CPS-1 is unnecessary, and the stored cells can be used as they are. It became.
（２）４℃保存ＰＢＭＣの細胞集団の解析
実施例１４−（１）で保存したＰＢＭＣを実施例１−（２）と同様の方法で細胞集団の解析を行った。ただし、ＦＩＴＣ標識マウス抗ヒトＣＤ３抗体、あるいはＥＣＤ標識マウス抗ヒトＣＤ４抗体及びＰＣ５標識マウス抗ヒトＣＤ８抗体を使用し、ネガティブコントロールとしてＦＩＴＣ標識マウスＩｇＧ１抗体／ＲＤ１標識マウスＩｇＧ１抗体／ＰＣ５標識マウスＩｇＧ１抗体及びＥＣＤ標識マウスＩｇＧ１抗体を使用した。結果を表２１に示す。
(2) Analysis of cell population of PBMC stored at 4 ° C. The cell population of PBMC stored in Example 14- (1) was analyzed in the same manner as in Example 1- (2). However, FITC-labeled mouse anti-human CD3 antibody, or using ECD-labeled mouse anti-human CD4 antibody and PC5-labeled mouse anti-human CD8 antibody, FITC-labeled mouse IgG 1 antibody / RD1-labeled mouse IgG 1 antibody / PC5-labeled mouse as a negative control, IgG 1 antibody and ECD labeled mouse IgG 1 antibody were used. The results are shown in Table 21.
表２１に示すように、２．５％ＨＳＡを添加した保存液中のＰＢＭＣと添加していない保存液中のＰＢＭＣとの間では、含有される各抗原陽性の細胞の割合に大きな差はなかった。このことから、ＨＳＡはＰＢＭＣ４℃保存時に好適に使用されることが明らかとなった。 As shown in Table 21, there is no significant difference in the proportion of each antigen-positive cell contained between the PBMC in the preservation solution added with 2.5% HSA and the PBMC in the preservation solution not added. It was. From this, it became clear that HSA is suitably used during PBMC storage at 4 ° C.
実施例１５ カゼインナトリウム含有生理食塩水を用いた新鮮血からのＰＢＭＣ分離回収（血液分離工程別）
（１）ＰＢＭＣの分離回収
実施例１−（１）と同様の方法で、インフォームド・コンセントの得られたヒト健常人ドナー血液からＰＢＭＣを分離した。ただし、血漿分離後のＰＢＭＣを含む細胞画分は、ＤＰＢＳ、１％カゼインＮａ／生理食塩水及び１％ＨＳＡ／生理食塩水を用いてそれぞれ別々に希釈した。中間層のＰＢＭＣの回収後の洗浄は、それぞれＲＰＭＩ１６４０、１％カゼインＮａ／生理食塩水又は１％ＨＳＡ／生理食塩水を用いて行った。このとき、各洗浄溶媒を用いて４５ｍＬにフィルアップし、遠心は全て室温で行った。結果を表２２に示す。
Example 15 PBMC separation and recovery from fresh blood using sodium casein-containing physiological saline (by blood separation step)
(1) Separation and recovery of PBMC PBMC was separated from human healthy donor blood from which informed consent was obtained in the same manner as in Example 1- (1). However, the cell fraction containing PBMC after plasma separation was diluted separately with DPBS, 1% casein Na / saline and 1% HSA / saline. Washing after recovery of PBMC in the intermediate layer was performed using RPMI 1640, 1% casein Na / saline or 1% HSA / saline, respectively. At this time, it filled up to 45 mL using each washing | cleaning solvent, and all centrifugation was performed at room temperature. The results are shown in Table 22.
表２２に示すように、Ｆｉｃｏｌｌ−Ｐａｑｕｅ ＰＲＥＭＩＵＭ重層時及びその後の細胞洗浄時に１％カゼインナトリウムおよび１％ＨＳＡを含む生理食塩水を使用することで、基本法と比較して、細胞の生存率への影響なく、細胞回収率を上げることができた。また、１％カゼインＮａ／生理食塩水は、Ｆｉｃｏｌｌ−Ｐａｑｕｅ ＰＲＥＭＩＵＭ重層時または細胞洗浄時のいずれかに使用した場合でも、基本法と比較して細胞回収率を上げることが示された。このことから、本発明の方法は血液からのＰＢＭＣ分離回収の分離工程にかかわらず好適に使用されることが明らかとなった。 As shown in Table 22, by using physiological saline containing 1% sodium caseinate and 1% HSA during Ficoll-Paque PREMIUM overlay and subsequent cell washing, cell viability was improved compared to the basic method. The cell recovery rate could be increased without any effect. Moreover, it was shown that 1% casein Na / saline increases the cell recovery rate compared to the basic method even when used in either Ficoll-Paque PREMIUM overlay or cell washing. From this, it became clear that the method of the present invention is suitably used regardless of the separation step of PBMC separation / recovery from blood.
本発明により、従来の分離方法と比べ、高い効率で必要とする細胞を分離する方法が提供される。本発明により、細胞含有液と血液由来タンパク質、乳タンパク質、又は血液由来タンパク質もしくは乳タンパク質のいずれかを含有する溶液で細胞液を調製する工程を付加するだけという簡便な方法で、細胞液から必要とする細胞を効率よく分離できることから、本発明は基礎研究、医療への応用研究において極めて有用である。 The present invention provides a method for separating required cells with higher efficiency than conventional separation methods. In accordance with the present invention, a simple method of adding a step of preparing a cell solution with a cell-containing solution and blood-derived protein, milk protein, or a solution containing either blood-derived protein or milk protein is necessary from the cell solution. Therefore, the present invention is extremely useful in basic research and medical application research.
Claims (8)
２）工程１）で得られた細胞液を密度勾配形成用媒体の上に重層する工程、及び
３）密度勾配遠心分離を行い、分離された末梢血単核球を回収する工程、
を包含することを特徴とする末梢血単核球の分離方法。 1) A step of preparing a cell solution with blood or a Buffy coat layer collected from blood and serum albumin , casein , or a solution containing either serum albumin or casein ,
2) layering the cell fluid obtained in step 1) on the density gradient forming medium, and 3) performing density gradient centrifugation and recovering the separated peripheral blood mononuclear cells ,
A method for separating peripheral blood mononuclear cells , comprising:
４）工程３）で回収した末梢血単核球を洗浄する工程、を包含する請求項１記載の方法。 further,
4) The method according to claim 1, further comprising the step of washing the peripheral blood mononuclear cells collected in step 3).
５）工程４）で洗浄した末梢血単核球を保存する工程、
を包含する請求項２記載の方法。 further,
5) storing the peripheral blood mononuclear cells washed in step 4);
The method of claim 2 comprising:
１）密度勾配形成用媒体、
２）血清アルブミン及び／又はカゼイン。 A kit for use in the method according to any one of claims 1 to 5, comprising the following 1) and 2);
1 ) density gradient forming medium,
2 ) Serum albumin and / or casein .
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