JP2013034436A - Method for concentrating cell suspension - Google Patents
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本発明は、細胞懸濁液処理器具、特に、簡便かつ迅速に細胞含有組成物を無菌的に製造することを可能にする細胞分離材を充填した細胞懸濁液処理器具を用いた細胞懸濁液の濃縮方法に関するものである。 The present invention relates to a cell suspension treatment device, particularly a cell suspension using a cell suspension treatment device filled with a cell separation material that enables aseptic production of a cell-containing composition in a simple and rapid manner. The present invention relates to a liquid concentration method.
近年、細胞含有組成物は、様々な疾患の治療に用いられている。具体的には、例えば、細胞含有組成物は、骨壊死の治療に用いられている。 In recent years, cell-containing compositions have been used for the treatment of various diseases. Specifically, for example, the cell-containing composition is used for the treatment of osteonecrosis.
骨壊死は、外傷や血管病変あるいは骨細胞への直接の傷害によって、骨細胞、骨基質、骨髄細胞の死滅または破壊が起こった状態である。 Osteonecrosis is a condition in which bone cells, bone matrix, or bone marrow cells are killed or destroyed by trauma, vascular lesions, or direct injury to bone cells.
骨壊死の治療法としては、例えば、骨に分化する間葉系幹細胞をβ−リン酸三カルシウム等の細胞支持体に付与して、細胞含有組成物を調製した後に、該細胞含有組成物を生体に投与する方法が開発されている。該方法で、骨欠損部の修復速度が高まり、骨壊死を治療することができる。 As a treatment method for osteonecrosis, for example, a mesenchymal stem cell that differentiates into bone is applied to a cell support such as β-tricalcium phosphate, and a cell-containing composition is prepared. Methods for administration to living bodies have been developed. By this method, the repair speed of the bone defect is increased and osteonecrosis can be treated.
上記細胞含有組成物の投与により治療効果を高めるには、投与する細胞濃度や細胞数を高める必要があり、そのため多量の生体試料を用いることや、細胞を培養することがしばしば行われている。これら大量の細胞を含んだ試料を生体に投与することは現実的に困難である場合があり、しばしば、培養、採取した試料を濃縮することが行われている。このような濃縮として、特許文献１に記載したような遠心分離による濃縮が行われている。
しかしながら、遠心分離による方法は、細胞に遠心によるストレスがかかることや、処理している細胞含有組成物が大気と接触する可能性や、また、閉鎖的に処理を行おうとすると装置が大型になることが懸念される。
In order to enhance the therapeutic effect by administration of the cell-containing composition, it is necessary to increase the cell concentration and the number of cells to be administered. For this reason, a large amount of biological sample is used and cells are often cultured. In some cases, it is practically difficult to administer a sample containing such a large amount of cells to a living body, and it is often performed to concentrate a sample collected and cultured. As such concentration, concentration by centrifugation as described in
However, in the method using centrifugation, the cell is subjected to stress due to centrifugation, the cell-containing composition being treated may come into contact with the atmosphere, and the apparatus becomes large when the treatment is performed in a closed manner. There is concern.
そこで、閉鎖系で簡便に目的細胞を回収可能な分離方法として特許文献２にあるようなフィルターを用いた方法が開示されている。これら分離材を用いた細胞懸濁液処理器具により、閉鎖系で簡便に目的の細胞を分離することが行われている。
しかしながら、上記のようなフィルターを用いた細胞懸濁液処理器具では、大量の細胞を処理する場合、フィルターへの細胞の目詰まり等が生じることで細胞回収率の低下が懸念される。
Therefore, a method using a filter as disclosed in
However, in the cell suspension processing apparatus using the filter as described above, when a large amount of cells are processed, there is a concern that the cell recovery rate is lowered due to clogging of the cells to the filter.
本発明は、上記問題点に鑑みなされたものであって、その目的は、簡便かつ迅速に、大量の細胞懸濁液を処理することである。 The present invention has been made in view of the above problems, and an object of the present invention is to process a large amount of cell suspension simply and quickly.
本発明者らは、上記課題に鑑み鋭意検討した結果、繊維径の異なる複数の不織布フィルターを特定の順序で配することにとより、高い細胞回収率を確保しつつ、ろ過時の流速を高められることを見出し、本発明を完成させるに至った。 As a result of intensive studies in view of the above problems, the present inventors have arranged a plurality of non-woven fabric filters having different fiber diameters in a specific order, and increased the flow rate during filtration while ensuring a high cell recovery rate. As a result, the present invention has been completed.
すなわち、本発明は、細胞懸濁液を、入口と出口を有し、不織布が積層されたフィルターが充填された容器に導入し、細胞を実質的に含まないろ過液を排出させた後、該容器に回収液を導入して、該フィルターに補足されている細胞を回収する細胞濃縮方法であって、
（ａ）各層の平均繊維径は１．５μｍ以上であり、
（ｂ）少なくとも２層は、平均繊維径５．０μｍ以下であり、
（ｃ）平均繊維径５．０μｍ以下の層のみに着目した場合に、近接する２層は、入口側の層の平均繊維径をＵ、出口側の平均繊維径をＤとして、いずれも
（ｃ１）Ｕ＞Ｄ かつ Ｕ−Ｄ≧０．５、または
（ｃ２）Ｕ＜Ｄ
の関係を満たす（ただし、少なくとも１箇所は上記（ｃ１）の関係を満たす）ことを特徴とする、細胞濃縮方法である。
That is, the present invention introduces the cell suspension into a container filled with a filter having an inlet and an outlet and laminated with a nonwoven fabric, and after discharging the filtrate substantially free of cells, A cell concentration method for introducing a collection liquid into a container and collecting cells captured by the filter,
(A) The average fiber diameter of each layer is 1.5 μm or more,
(B) At least two layers have an average fiber diameter of 5.0 μm or less,
(C) When paying attention only to a layer having an average fiber diameter of 5.0 μm or less, the two adjacent layers have U as the average fiber diameter of the layer on the inlet side and D as the average fiber diameter on the outlet side. ) U> D and U−D ≧ 0.5, or (c2) U <D
(However, at least one location satisfies the relationship (c1) above).
本発明の方法によれば、フィルターへの細胞の目詰まりの発生を抑え、大量の細胞懸濁液を迅速かつ高回収率で処理することができる。 According to the method of the present invention, the occurrence of clogging of cells in the filter can be suppressed, and a large amount of cell suspension can be processed quickly and at a high recovery rate.
以下に、本発明の細胞懸濁液の濃縮方法について説明する。
本発明の細胞懸濁液の濃縮方法は、細胞懸濁液を、細胞を捕捉する不織布からなるフィルターが充填された処理器に導入し、細胞を実質的に含まないろ過液を排出させた後、該処理器に回収液を導入して該フィルターに捕捉されている細胞を回収し濃縮する方法である。
Below, the concentration method of the cell suspension of this invention is demonstrated.
In the method for concentrating a cell suspension according to the present invention, the cell suspension is introduced into a processor filled with a filter made of a nonwoven fabric for capturing cells, and the filtrate substantially free of cells is discharged. In this method, the collection liquid is introduced into the processor to collect and concentrate the cells captured by the filter.
本方法において、細胞懸濁液を構成する細胞とは、特に限定されないが、例えば人工多能性幹細胞（ｉＰＳ細胞）、胚性幹細胞（ＥＳ細胞）、間葉系幹細胞、脂肪由来間葉系細胞、脂肪由来間質幹細胞、多能性成体幹細胞、骨髄ストローマ細胞、造血幹細胞等の多分化能を有する生体幹細胞、Ｔ細胞、Ｂ細胞、キラーＴ細胞（細胞障害性Ｔ細胞）、ＮＫ細胞、ＮＫＴ細胞、制御性Ｔ細胞などのリンパ球系の細胞、マクロファージ、単球、樹状細胞、顆粒球、赤血球、血小板など、神経細胞、筋細胞、線維芽細胞、肝細胞、心筋細胞などの体細胞または、遺伝子の導入や分化などの処理を行った細胞が例示される。 In the present method, the cells constituting the cell suspension are not particularly limited. For example, artificial pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), mesenchymal stem cells, adipose-derived mesenchymal cells Adipose-derived stromal stem cells, pluripotent adult stem cells, bone marrow stromal cells, hematopoietic stem cells, and other multipotent biological stem cells, T cells, B cells, killer T cells (cytotoxic T cells), NK cells, NKT Cells, lymphoid cells such as regulatory T cells, macrophages, monocytes, dendritic cells, granulocytes, erythrocytes, platelets, etc., somatic cells such as nerve cells, muscle cells, fibroblasts, hepatocytes, cardiomyocytes, etc. Alternatively, cells subjected to treatment such as gene introduction and differentiation are exemplified.
また、細胞懸濁液としては、細胞を含む懸濁液であれば特に限定されず用いることができるが、例えば、脂肪、皮膚、血管、角膜、口腔、腎臓、肝臓、膵臓、心臓、神経、筋肉、前立腺、腸、羊膜、胎盤、臍帯などの生体組織を酵素処理や破砕処理や抽出処理や分解処理や超音波処理などをした後の懸濁液、血液や骨髄液、臍帯血などの体液、血液や骨髄液を密度勾配遠心処理やろ過処理や酵素処理や分解処理や超音波処理などの前処理をして調製された細胞懸濁液等が例示される。 In addition, the cell suspension can be used without any particular limitation as long as it is a suspension containing cells. For example, fat, skin, blood vessel, cornea, oral cavity, kidney, liver, pancreas, heart, nerve, Suspensions after body tissue such as muscle, prostate, intestine, amniotic membrane, placenta, and umbilical cord are subjected to enzyme treatment, crushing treatment, extraction treatment, degradation treatment, ultrasonic treatment, and body fluids such as blood, bone marrow fluid, and cord blood Examples thereof include cell suspensions prepared by subjecting blood and bone marrow fluid to pretreatment such as density gradient centrifugation, filtration, enzyme treatment, degradation treatment, and ultrasonic treatment.
また、上記に例示した細胞を生体外で培養液や刺激因子などを用いて培養や増殖などをした後の細胞懸濁液であってもよい。
細胞を捕捉する不織布の材料としては、特に制限なく用いることができる。例えば、ポリプロピレン、ポリエチレン、高密度ポリエチレン、低密度ポリエチレン等のポリオレフィン、ポリエステル、塩化ビニル、ポリビニルアルコール、塩化ビニリデン、レーヨン、ビニロン、ポリスチレン、アクリル（ポリメチルメタクリレート、ポリヒドロキシエチルメタクリレート、ポリアクロニトリル、ポリアクリル酸、ポリアクリレート等）、ナイロン、ポリウレタン、ポリイミド、アラミド、ポリアミド、キュプラ、ケブラー、カーボン、フェノール、テトロン、パルプ、麻、セルロース、ケナフ、キチン、キトサン、ガラス、綿等を挙げることができる。中でも、ポリエステル、ポリプロピレン、アクリル、レーヨン、ナイロン等の高分子を好適に用いることができる。上記細胞分離材は、これらの材質のうち、単一の材質からなってもよいし、複数の材質を組み合わせた複合材からなってもよい。
Further, it may be a cell suspension after the cells exemplified above are cultured or proliferated in vitro using a culture solution or a stimulating factor.
The nonwoven fabric material that traps cells can be used without particular limitation. For example, polyolefin such as polypropylene, polyethylene, high density polyethylene, low density polyethylene, polyester, vinyl chloride, polyvinyl alcohol, vinylidene chloride, rayon, vinylon, polystyrene, acrylic (polymethyl methacrylate, polyhydroxyethyl methacrylate, polyacrylonitrile, poly Acrylic acid, polyacrylate, etc.), nylon, polyurethane, polyimide, aramid, polyamide, cupra, kevlar, carbon, phenol, tetron, pulp, hemp, cellulose, kenaf, chitin, chitosan, glass, cotton and the like. Among these, polymers such as polyester, polypropylene, acrylic, rayon, and nylon can be suitably used. The cell separation material may be composed of a single material among these materials, or may be composed of a composite material obtained by combining a plurality of materials.
フィルターを充填する処理器としては、アクリロニトリルブタジエンスチレンターポリマー等のアクリロニトリルポリマー；ポリテトラフルオロエチレン、ポリクロロトリフルオロエチレン、テトラフルオロエチレンとヘキサフルオロプロピレンのコポリマー、ポリ塩化ビニル等のハロゲン化ポリマー；ポリアミド、ポリイミド、ポリスルホン、ポリカーボネート、ポリエチレン、ポリプロピレン、ポリビニルクロリドアクリルコポリマー、ポリカーボネートアクリロニトリルブタジエンスチレン、ポリスチレン、ポリメチルペンテン等を使用できるが、特に耐滅菌性を有する素材、具体的にはポリプロピレン、ポリ塩化ビニル、ポリエチレン、ポリイミド、ポリカーボネート、ポリスルホン、ポリメチルペンテン、ポリスチレン等のブロック共重合体を使用することが好ましい。 The processing unit for filling the filter includes acrylonitrile polymer such as acrylonitrile butadiene styrene terpolymer; halogenated polymer such as polytetrafluoroethylene, polychlorotrifluoroethylene, copolymer of tetrafluoroethylene and hexafluoropropylene, polyvinyl chloride; polyamide , Polyimide, polysulfone, polycarbonate, polyethylene, polypropylene, polyvinyl chloride acrylic copolymer, polycarbonate acrylonitrile butadiene styrene, polystyrene, polymethylpentene, etc. can be used, but particularly sterilization resistant materials, specifically polypropylene, polyvinyl chloride, Blocks such as polyethylene, polyimide, polycarbonate, polysulfone, polymethylpentene, and polystyrene It is preferable to use a copolymer.
本発明における細胞を実質的に含まないろ過液とは、ろ過液の細胞濃度が、細胞懸濁液処理器具入り口より投入される細胞懸濁液中の細胞濃度の１％以下であるろ過液を指す。これ以上の細胞がろ過液側に含まれる場合、効率的な濃縮が行えないことが懸念される。
細胞をフィルターで捕捉した後に導入する回収液としては、特に限定されるものではなく、細胞に対して負の影響を与えない溶液であればよい。例えば、生理的食塩液やリンゲル液など注射用剤として一般的に用いられる溶液、リン酸緩衝液等の緩衝液、αＭＥＭ培地やＤＭＥＭ培地等の細胞培養用の培地等を挙げることができる。これら例示した溶液の中でも、細胞への負の影響が小さいことから、細胞培養用培地や生理食塩水を好ましく用いることができる。
The filtrate substantially free of cells in the present invention refers to a filtrate in which the cell concentration of the filtrate is 1% or less of the cell concentration in the cell suspension introduced from the cell suspension treatment apparatus inlet. Point to. When more cells than this are contained on the filtrate side, there is a concern that efficient concentration cannot be performed.
The collection liquid to be introduced after capturing the cells with a filter is not particularly limited, and any solution that does not negatively affect the cells may be used. Examples thereof include a solution generally used as an injectable agent such as physiological saline and Ringer's solution, a buffer solution such as a phosphate buffer, a cell culture medium such as an αMEM medium and a DMEM medium, and the like. Among these exemplified solutions, a medium for cell culture and physiological saline can be preferably used since the negative influence on cells is small.
また、上記回収液には、細胞保護の観点から、タンパクを添加してもよい。上記物質は特に限定されるものではないが、具体的には、例えば、血漿、血清、アルブミン等を挙げることができる。 Moreover, you may add protein to the said collection | recovery liquid from a viewpoint of cell protection. Although the said substance is not specifically limited, For example, plasma, serum, albumin etc. can be mentioned.
さらに、上記回収液には、粘張度を上げるための物質を添加してもよい。上記物質は特に限定されるものではないが、具体的には、例えば、アルブミン、フィブリノーゲン、グロブリン、デキストラン、ヒドロキシエチルスターチ、ヒドロキシエチルセルロース等を挙げることができる。このような物質を添加することにより、細胞分離材に捕捉された細胞の回収率を向上させることができる。 Furthermore, you may add the substance for raising the degree of viscosity to the said collection | recovery liquid. Although the said substance is not specifically limited, For example, albumin, fibrinogen, globulin, dextran, hydroxyethyl starch, hydroxyethyl cellulose etc. can be mentioned. By adding such a substance, it is possible to improve the recovery rate of the cells captured by the cell separation material.
また、回収液を導入する方法としては、特に限定しないが、回収液の流速は剪断力を高め、細胞を高率で回収するためにできるだけ高速が好ましいが、内圧上昇によるフィルターとチューブ等の接続部のはずれや、細胞へのダメージを起こさない流速に制御することが好ましい。また、回収液をフィルターに導入する手段は、シリンジポンプ、ブラッドポンプ、ペリスタポンプ等の装置を用いるものや、簡便法としてシリンジを手で押す方法、液体を貯留したバッグを押しつぶして液流を惹起する方法、落差処理等が挙げられる。更に、細胞の回収率をより高めるために、フィルターに振動を加えるとか、ストップドフロー等を行ってもよい。 The method for introducing the recovery liquid is not particularly limited, but the flow rate of the recovery liquid is preferably as high as possible in order to increase the shear force and recover the cells at a high rate. It is preferable to control the flow rate so as not to cause separation of parts and damage to cells. In addition, the means for introducing the recovered liquid into the filter uses a device such as a syringe pump, a blood pump, or a peristaltic pump, a method of pushing the syringe by hand as a simple method, or crushing a bag storing the liquid to induce a liquid flow Method, drop processing, and the like. Furthermore, in order to further increase the cell recovery rate, vibration may be applied to the filter, or stopped flow may be performed.
本発明の方法においては、不織布からなるフィルターが積層された積層フィルターが用いられる。本発明で用いられる積層フィルターは、平均繊維径が異なる少なくとも２層を有し、細胞懸濁液入り口側から１層目、２層目というように定義される。繊維径の異なる層を２層以上有するフィルターとすることにより、細胞を捕捉する箇所が分散され、目詰まりの発生が抑制されるとともに、フィルターからの細胞の分離・回収も効率的に行うことができる。なお、繊維径が同じ不織布が連続して積層された部分は、積層された不織布の枚数によらず１層として扱う。 In the method of the present invention, a laminated filter in which filters made of nonwoven fabric are laminated is used. The multilayer filter used in the present invention has at least two layers having different average fiber diameters and is defined as the first layer and the second layer from the cell suspension inlet side. By using a filter having two or more layers having different fiber diameters, the locations where cells are captured are dispersed, clogging is suppressed, and the cells can be efficiently separated and collected from the filter. it can. In addition, the part by which the nonwoven fabric with the same fiber diameter was laminated | stacked continuously is handled as one layer irrespective of the number of laminated nonwoven fabrics.
本発明で用いられる不織布は、いずれの層においても平均繊維径が１．５μｍ以上であることを特徴とする。平均繊維径の上限は特に制限されないが、積層フィルターのうち少なくとも２層は、平均繊維径が５．０μｍ以下（即ち、平均繊維径の範囲が１．５〜５．０μｍ）の不織布で構成されている必要がある。 The nonwoven fabric used in the present invention is characterized in that the average fiber diameter is 1.5 μm or more in any layer. The upper limit of the average fiber diameter is not particularly limited, but at least two layers of the laminated filter are composed of a nonwoven fabric having an average fiber diameter of 5.0 μm or less (that is, the average fiber diameter range is 1.5 to 5.0 μm). Need to be.
さらに、本発明で用いる積層フィルターにおいては、平均繊維径５．０μｍ以下の層のみに着目した場合に、近接する２層は、入口側の層の平均繊維径をＵ、出口側の平均繊維径をＤとして、いずれも、
（ｃ１）Ｕ＞Ｄ かつ Ｕ−Ｄ≧０．５、または
（ｃ２）Ｕ＜Ｄの関係を満たす
（ただし、少なくとも１箇所は上記（ｃ１）の関係を満たす）ことを特徴とする。
Furthermore, in the multilayer filter used in the present invention, when attention is paid only to a layer having an average fiber diameter of 5.0 μm or less, the two adjacent layers are U, the average fiber diameter of the inlet side layer, and the average fiber diameter of the outlet side. As D,
(C1) U> D and UD ≧ 0.5, or (c2) U <D is satisfied (however, at least one location satisfies the relationship (c1)).
例えば、平均繊維径が各々、１層目 ４．５μｍ、２層目 ３．０μｍ、３層目 ２．０μｍの場合、１層目と２層目、および、２層目と３層目はそれぞれ、前記（ｃ１）の関係を満たし、本発明で用いる積層フィルターに該当する。また、平均繊維径が各々、１層目 ４．５μｍ、２層目 ４．７μｍ、３層目 ３．０μｍの場合、１層目と２層目は前記（ｃ２）の関係を満たし、２層目と３層目は前記（ｃ１）の関係を満たすため、本発明で用いる積層フィルターに該当する。 For example, when the average fiber diameter is 4.5 μm for the first layer, 3.0 μm for the second layer, and 2.0 μm for the third layer, the first layer and the second layer, and the second layer and the third layer are respectively This satisfies the relationship (c1) and corresponds to the multilayer filter used in the present invention. When the average fiber diameter is 4.5 μm for the first layer, 4.7 μm for the second layer, and 3.0 μm for the third layer, the first layer and the second layer satisfy the relationship (c2) described above. Since the eye and the third layer satisfy the relationship (c1), they correspond to the multilayer filter used in the present invention.
なお、本発明においては、少なくとも（ｃ１）１箇所は前記（ｃ１）の関係を満たす必要がある。即ち、平均繊維径が１層目 ４．５μｍ、２層目 ４．３μｍの２層のみからなるフィルター等は、本発明で用いるフィルターには該当しない。細胞は成長の程度により細胞径の違いがあるが、平均繊維径が１．５〜５．０μｍからなる層を、上記（ｃ１）を満たすように配置することにより、細胞種でも細胞の大きさによる影響を抑えて、効率よく細胞を捕捉、回収することができる。 In the present invention, at least one (c1) needs to satisfy the relationship (c1). That is, a filter composed of only two layers having an average fiber diameter of 4.5 μm for the first layer and 4.3 μm for the second layer does not correspond to the filter used in the present invention. Cells have different cell diameters depending on the degree of growth, but by arranging a layer having an average fiber diameter of 1.5 to 5.0 μm so as to satisfy the above (c1), the cell size is the same as the cell type. It is possible to efficiently capture and collect cells while suppressing the influence of the above.
また、本発明に用いる積層フィルターにおいて、前記（ｃ１）または（ｃ２）の関係を満たすか否かは、平均繊維径が５．０μｍ以下（即ち、平均繊維径の範囲が１．５μｍ〜５．０μｍ）の層だけに注目して判断する。例えば、平均繊維径が１層目 １０μｍ、２層目 ４．５μｍ、３層目 ３．５μｍ、４層目 ２．５μｍの場合には、１層目は判断対象外とし、２層目と３層目、および３層目と４層目の関係で判断すればよい。同様に、１層目 ４．５μｍ、２層目 １０μｍ、３層目 ３．５μｍ、４層目 ２．５μｍの場合には、２層目を判断対象外とし、１層目と３層目、および、３層目と４層目の関係で判断すればよい。即ち、本発明で用いる積層フィルターは、平均繊維径の範囲が１．５〜５．０μｍの層のみならず、平均繊維径が５．０μｍ超の不織布からなる層を有していてもよいことを意味する。 Further, in the multilayer filter used in the present invention, whether or not the relationship (c1) or (c2) is satisfied is determined based on whether the average fiber diameter is 5.0 μm or less (that is, the average fiber diameter ranges from 1.5 μm to 5. Judgment is made by paying attention only to the 0 μm layer. For example, when the average fiber diameter is 10 μm for the first layer, 4.5 μm for the second layer, 3.5 μm for the third layer, and 2.5 μm for the fourth layer, the first layer is excluded from the judgment and the second and third layers The determination may be made based on the layer and the relationship between the third and fourth layers. Similarly, when the first layer is 4.5 μm, the second layer is 10 μm, the third layer is 3.5 μm, the fourth layer is 2.5 μm, the second layer is excluded from the judgment, and the first layer, the third layer, The determination may be made based on the relationship between the third and fourth layers. That is, the multilayer filter used in the present invention may have not only a layer having an average fiber diameter of 1.5 to 5.0 μm but also a layer made of a nonwoven fabric having an average fiber diameter of more than 5.0 μm. Means.
本発明に用いられる積層フィルターは、３層以上からなることが好ましく、平均繊維径が１．５〜５．０μｍの層だけに着目しても３層以上を有することが、より好ましい。 The multilayer filter used in the present invention is preferably composed of three or more layers, and more preferably has three or more layers even when attention is paid only to a layer having an average fiber diameter of 1.5 to 5.0 μm.
また、本発明に用いられる積層フィルターは、途中に平均繊維径５．０μｍ超の層を挟むことなく、平均繊維径１．５〜５．０μｍの層が連続して存在することが好ましい。更に、連続して存在する平均繊維径１．５〜５．０μｍの層同士は、全て前記（ｃ１）の関係であることが、より好ましい。即ち、本発明に用いられる積層フィルターとしては、それぞれ平均繊維径１．５〜５．０μｍである層が、入口側から出口側に向かって、平均繊維径が差分０．５μｍ以上で順に小さくなる構成であるのが特に好ましく、繊維径が最も細い層は、平均繊維径が２．０μｍ以下（即ち１．５〜２．０μｍの範囲）であるのが最も好ましい。フィルターの目詰まりをより効果的に防止するため、上記好ましい態様において、最も入口側に平均繊維径５．０μ超の層を追加してもよい。 In the multilayer filter used in the present invention, it is preferable that layers having an average fiber diameter of 1.5 to 5.0 μm are continuously present without interposing a layer having an average fiber diameter of more than 5.0 μm in the middle. Furthermore, it is more preferable that all the layers having an average fiber diameter of 1.5 to 5.0 μm that are continuously present have the relationship (c1). That is, as the multilayer filter used in the present invention, each layer having an average fiber diameter of 1.5 to 5.0 μm decreases in order with an average fiber diameter of 0.5 μm or more from the inlet side toward the outlet side. It is particularly preferable that the layer has the smallest fiber diameter, and the average fiber diameter is most preferably 2.0 μm or less (that is, in the range of 1.5 to 2.0 μm). In order to prevent clogging of the filter more effectively, in the above preferred embodiment, a layer having an average fiber diameter of more than 5.0 μm may be added on the most inlet side.
本発明でいう繊維径とは、繊維軸に対して直角方向の繊維の幅であり、繊維径の測定は、不織布からなるフィルターを走査型電子顕微鏡にて写真撮影し、写真に記載されたスケールから求めた繊維径の計算値を平均することにより求めることが出来る。平均繊維径とは、上記のように測定した繊維径の平均値を意味しており、５０個以上、望ましくは１００個以上の平均値である。但し、繊維が多数に重なりあった場合、他繊維が邪魔をしてその幅が測定できない場合、著しく直径の異なる繊維が混在している場合などは、そのデータは除いて繊維径を算出する。また、太さの大きく異なる、例えば７μｍ以上繊維径の異なる複数の繊維から構成される不織布の場合には、繊維径が細い方が分離効率への影響が大きいため、別々に繊維径を計算し、細い繊維径をその不織布の繊維径とする。 The fiber diameter referred to in the present invention is the width of the fiber in a direction perpendicular to the fiber axis, and the fiber diameter is measured by taking a photograph of a filter made of a nonwoven fabric with a scanning electron microscope and describing the scale. It can be obtained by averaging the calculated fiber diameter values obtained from An average fiber diameter means the average value of the fiber diameter measured as mentioned above, and is an average value of 50 or more, desirably 100 or more. However, when a large number of fibers are overlapped, when other fibers are obstructed and the width cannot be measured, or when fibers having remarkably different diameters are mixed, the fiber diameter is calculated excluding the data. In addition, in the case of a nonwoven fabric composed of a plurality of fibers having greatly different thicknesses, for example, 7 μm or more, the fiber diameter is calculated separately because the smaller the fiber diameter, the greater the influence on the separation efficiency. The thin fiber diameter is defined as the fiber diameter of the nonwoven fabric.
本発明の濃縮方法においては、予めプライミング処理を行ってもよい。プライミングとは、細胞懸濁液をフィルターに導入する前に、フィルターに溶液を導入することで、フィルター内の気泡除去や不織布を溶液で浸すことを行うことである。 In the concentration method of the present invention, a priming process may be performed in advance. Priming refers to removing bubbles in the filter and immersing the nonwoven fabric with the solution by introducing the solution into the filter before introducing the cell suspension into the filter.
特に、蛋白質を含む溶液でプライミングすることで、効率的な細胞の捕捉と回収を行うことができる。蛋白質を含む溶液とは、例えば、血漿、血清などの溶液、血漿、血清、アルブミン等を含む細胞培養用培地や生理食塩水を例示することができる。特に、細胞を培養した培養液と同じ組成であることが、細胞への影響等を考慮すると好ましい。 In particular, efficient cell capture and recovery can be performed by priming with a solution containing a protein. Examples of the protein-containing solution include a solution such as plasma and serum, a cell culture medium and physiological saline containing plasma, serum, albumin, and the like. In particular, it is preferable that the composition is the same as the culture solution in which the cells are cultured in consideration of the influence on the cells.
本発明でいう細胞懸濁液の濃縮方法において、不織布からなるフィルター通過時の細胞懸濁液の線速度は、３ｃｍ／ｍｉｎ以下であることが好ましい。２．５ｃｍ以下であることがより好ましく、２ｃｍ以下であることが更に好ましい。３ｃｍ／ｍｉｎ以上の線速度でフィルターに導入した場合、細胞がフィルターに効率的に捕捉されず、通過してしまう懸念がある。 In the cell suspension concentration method referred to in the present invention, the linear velocity of the cell suspension when passing through a filter made of a nonwoven fabric is preferably 3 cm / min or less. It is more preferably 2.5 cm or less, and further preferably 2 cm or less. When introduced into a filter at a linear velocity of 3 cm / min or more, there is a concern that cells are not efficiently captured by the filter and pass through.
本発明の濃縮方法によれば、通常７０％以上の回収率で細胞の分離・回収が可能である。回収率は、より好ましくは７５％以上、更に好ましくは８０％以上、特に好ましくは８５％以上である。 According to the concentration method of the present invention, it is possible to separate and collect cells with a recovery rate of usually 70% or more. The recovery rate is more preferably 75% or more, still more preferably 80% or more, and particularly preferably 85% or more.
本発明により分離される細胞は、白血病治療、心筋再生や血管再生、幹細胞疲弊疾患、骨疾患、軟骨疾患、虚血性疾患、血管系疾患、神経病、やけど、慢性炎症、心疾患、免疫不全、クーロン病等の疾患、豊胸、しわとり、美容成形、組織陥没症等の組織増大などの再生医療、Ｔ細胞療法、ＮＫＴ細胞療法、樹状細胞移入療法などの免疫療法、遺伝子導入した細胞を用いる遺伝子療法などに用いることも可能であるが、これらに限定されるものではない。また、分離された細胞をスキャフォールドなどの構造材料に播種して治療に用いることも可能である。 Cells isolated according to the present invention include leukemia treatment, myocardial regeneration and vascular regeneration, stem cell exhaustion disease, bone disease, cartilage disease, ischemic disease, vascular disease, neurological disease, burn, chronic inflammation, heart disease, immunodeficiency, Regenerative medicine such as coulomb disease, breast augmentation, wrinkle removal, cosmetic molding, tissue enlargement such as tissue depression, immunotherapy such as T cell therapy, NKT cell therapy, dendritic cell transfer therapy, and gene-transferred cells Although it can be used for the gene therapy used, it is not limited to these. In addition, the separated cells can be seeded on a structural material such as a scaffold and used for treatment.
以下、実験結果を用いて本発明を説明する。なお、ここで細胞回収率とは、回収後の細胞懸濁液中の細胞数を、カラムに通過させた細胞数で除した値で、値が高いほど回収効率に優れていることを示す。細胞通過率とは、カラムを通過した後の細胞懸濁液中の細胞数を、カラムに通過させた細胞数で除した値である。細胞数は、細胞懸濁液を血球カウンター（シスメックス、Ｋ−４５００）により測定し、白血球画分の細胞濃度を本実施例での細胞濃度として算出し、細胞懸濁液量と細胞濃度より算出した。 Hereinafter, the present invention will be described using experimental results. Here, the cell recovery rate is a value obtained by dividing the number of cells in the recovered cell suspension by the number of cells passed through the column, and indicates that the higher the value, the better the recovery efficiency. The cell passage rate is a value obtained by dividing the number of cells in the cell suspension after passing through the column by the number of cells passed through the column. The number of cells is determined by measuring the cell suspension with a blood cell counter (Sysmex, K-4500), calculating the cell concentration of the leukocyte fraction as the cell concentration in this example, and calculating from the cell suspension amount and the cell concentration. did.
各実験例における細胞濃縮の方法は、所定の枚数の不織布を、半径３６ｍｍ高さ１２ｍｍの円柱状のカラムに充填した細胞処理器具を用いて行った。入り口にチューブおよびローラークレンメを取り付け、所定の溶液でプライミング操作を行った。所定の速度になるよう調整し、重量による自然落下により、所定量の細胞懸濁液を通液した。通液後、出口側より、シリンジを用いて回収液５０ｍｌを手押しによりカラムに押し込み、捕捉された細胞を回収した。回収された細胞懸濁液中および通過したろ液中の細胞数を測定し、回収率および通過率として算出した。 The cell concentration method in each experimental example was performed using a cell processing instrument in which a predetermined number of nonwoven fabrics were packed into a columnar column having a radius of 36 mm and a height of 12 mm. A tube and a roller clamp were attached to the inlet, and a priming operation was performed with a predetermined solution. It adjusted so that it might become a predetermined | prescribed speed | rate, and passed the predetermined amount cell suspension by the natural fall by weight. After passing through, 50 ml of the collected liquid was manually pushed into the column from the outlet side using a syringe, and the captured cells were collected. The number of cells in the collected cell suspension and in the filtrate that passed through was measured and calculated as the recovery rate and the passing rate.
〔実施例１〕
半径３６ｍｍ高さ１２ｍｍのカラムに、細胞懸濁液の入り口側よりポリブチレンテレフタレート製不織布（繊維径４．６μｍ、目付け７０ｇ／ｍ２）１６枚、ポリブチレンテレフタレート製不織布（繊維径２．９μｍ、目付け７０ｇ／ｍ２）２０枚を積層状態で充填し、まず１０％ＦＢＳ添加ＲＰＭＩ１６４０培地４５ｍｌを入口側よりシリンジを用いて手押しで通液した。次に、Ｊｕｋａｔ細胞がＲＰＭＩ１６４０培地に懸濁された細胞懸濁液（ｊｕｋａｔ細胞濃度２．１ｘ１０６ｃｅｌｌｓ/ｍｌ）１０００ｍｌを線速度１．６ｃｍ/ｍｉｎ．でカラム入り口より通液した。その後、通液とは逆方向より０．２％Ａｌｂ添加ＲＰＭＩ１６４０培地５０ｍｌをシリンジを用いて手押しで押し込み、細胞を回収した。結果、細胞回収率および通過率は８２％、０％であった。
[Example 1]
In a column with a radius of 36 mm and a height of 12 mm, 16 non-woven fabrics made of polybutylene terephthalate (fiber diameter 4.6 μm, basis weight 70 g / m 2 ), polybutylene terephthalate nonwoven fabric (fiber diameter 2.9 μm, from the entrance side of the cell suspension) 20 sheets (weight per unit area: 70 g / m 2 ) were filled in a laminated state, and 45 ml of 10% FBS-added RPMI1640 medium was first manually passed from the inlet side using a syringe. Next, 1000 ml of a cell suspension (jukat cell concentration 2.1 × 10 6 cells / ml) in which Jukat cells are suspended in RPMI 1640 medium was applied at a linear velocity of 1.6 cm / min. Was passed through the column inlet. Thereafter, 50 ml of 0.2% Alb-added RPMI 1640 medium was pushed by hand with a syringe in the direction opposite to the flow direction, and the cells were collected. As a result, the cell recovery rate and passage rate were 82% and 0%.
〔実施例２〕
半径３６ｍｍ高さ１２ｍｍのカラムに、細胞懸濁液の入り口側よりポリブチレンテレフタレート製不織布（繊維径４．６μｍ、目付け７０ｇ／ｍ２）１６枚、ポリブチレンテレフタレート製不織布（繊維径２．９μｍ、目付け７０ｇ／ｍ２）１６枚、ポリブチレンテレフタレート製不織布（繊維径１．７μｍ、目付け２０ｇ／ｍ２）１２枚、を積層状態で充填し、まず１０％ＦＢＳ添加ＲＰＭＩ１６４０培地４５ｍｌを入口側よりシリンジを用いて手押しで通液した。次に、ヒトＴ細胞性白血病由来株細胞（ｊｕｋａｔ細胞）がＲＰＭＩ１６４０培地に懸濁された細胞懸濁液（ｊｕｋａｔ細胞濃度２．１ｘ１０６ｃｅｌｌｓ/ｍｌ）１０００ｍｌを線速度１．６ｃｍ/ｍｉｎ．でカラム入り口より通液した。その後、通液とは逆方向より０．２％Ａｌｂ添加ＲＰＭＩ１６４０培地５０ｍｌをシリンジを用いて手押しで押し込み、細胞を回収した。結果、細胞回収率および通過率は８７％、０％であった。
[Example 2]
In a column with a radius of 36 mm and a height of 12 mm, 16 non-woven fabrics made of polybutylene terephthalate (fiber diameter 4.6 μm, basis weight 70 g / m 2 ), polybutylene terephthalate nonwoven fabric (fiber diameter 2.9 μm, from the entrance side of the cell suspension) 16 sheets of weight per unit area 70 g / m 2 ) and 12 sheets of polybutylene terephthalate non-woven fabric (fiber diameter 1.7 μm, weight per unit area 20 g / m 2 ) were filled in a laminated state. First, 45 ml of 10% FBS-added RPMI 1640 medium was injected from the inlet side The solution was passed by hand using Next, 1000 ml of a cell suspension (jukat cell concentration: 2.1 × 10 6 cells / ml) in which human T-cell leukemia-derived strain cells (jukat cells) are suspended in RPMI 1640 medium was applied at a linear velocity of 1.6 cm / min. Was passed through the column inlet. Thereafter, 50 ml of 0.2% Alb-added RPMI 1640 medium was pushed by hand with a syringe in the direction opposite to the flow direction, and the cells were collected. As a result, the cell recovery rate and passage rate were 87% and 0%.
〔実施例３〕
半径３６ｍｍ高さ１２ｍｍのカラムに、細胞懸濁液の入り口側よりポリブチレンテレフタレート製不織布（繊維径４．６μｍ、目付け７０ｇ／ｍ２）１６枚、ポリブチレンテレフタレート製不織布（繊維径２．９μｍ、目付け７０ｇ／ｍ２）１６枚、ポリブチレンテレフタレート製不織布（繊維径１．７μｍ、目付け２０ｇ／ｍ２）１２枚、を積層状態で充填し、まず１０％ＦＢＳ添加ＲＰＭＩ１６４０培地４５ｍｌを入口側よりシリンジを用いて手押しで通液した。次に、Ｊｕｋａｔ細胞がＲＰＭＩ１６４０培地に懸濁された細胞懸濁液（ｊｕｋａｔ細胞濃度２．１ｘ１０６ｃｅｌｌｓ/ｍｌ）１０００ｍｌを線速度０．８ｃｍ/ｍｉｎ．でカラム入り口より通液した。その後、通液とは逆方向より０．２％Ａｌｂ添加ＲＰＭＩ１６４０培地５０ｍｌをシリンジを用いて手押しで押し込み、細胞を回収した。結果、細胞回収率および通過率は９２％、０％であった。
Example 3
In a column with a radius of 36 mm and a height of 12 mm, 16 non-woven fabrics made of polybutylene terephthalate (fiber diameter 4.6 μm, basis weight 70 g / m 2 ), polybutylene terephthalate nonwoven fabric (fiber diameter 2.9 μm, from the entrance side of the cell suspension) 16 sheets of weight per unit area 70 g / m 2 ) and 12 sheets of polybutylene terephthalate non-woven fabric (fiber diameter 1.7 μm, weight per unit area 20 g / m 2 ) were filled in a laminated state. First, 45 ml of 10% FBS-added RPMI 1640 medium was injected from the inlet side The solution was passed by hand using Next, 1000 ml of a cell suspension (jukat cell concentration 2.1 × 10 6 cells / ml) in which Jukat cells are suspended in RPMI 1640 medium was added at a linear velocity of 0.8 cm / min. Was passed through the column inlet. Thereafter, 50 ml of 0.2% Alb-added RPMI 1640 medium was pushed by hand with a syringe in the direction opposite to the flow direction, and the cells were collected. As a result, the cell recovery rate and passage rate were 92% and 0%.
〔実施例４〕
半径３６ｍｍ高さ１２ｍｍのカラムに、細胞懸濁液の入り口側よりポリブチレンテレフタレート製不織布（繊維径４．６μｍ、目付け７０ｇ／ｍ２）１６枚、ポリブチレンテレフタレート製不織布（繊維径２．９μｍ、目付け７０ｇ／ｍ２）１６枚、ポリブチレンテレフタレート製不織布（繊維径１．７μｍ、目付け２０ｇ／ｍ２）１２枚、を積層状態で充填し、まず生理食塩水４５ｍｌを入口側よりシリンジを用いて手押しで通液した。次に、Ｊｕｋａｔ細胞がＲＰＭＩ１６４０培地に懸濁された細胞懸濁液（ｊｕｋａｔ細胞濃度２．１ｘ１０６ｃｅｌｌｓ/ｍｌ）１０００ｍｌを線速度１．６ｃｍ/ｍｉｎ．でカラム入り口より通液した。その後、通液とは逆方向より０．２％Ａｌｂ添加ＲＰＭＩ１６４０培地５０ｍｌをシリンジを用いて手押しで押し込み、細胞を回収した。結果、細胞回収率および通過率は７８％、０％であった。
Example 4
In a column with a radius of 36 mm and a height of 12 mm, 16 non-woven fabrics made of polybutylene terephthalate (fiber diameter 4.6 μm, basis weight 70 g / m 2 ), polybutylene terephthalate nonwoven fabric (fiber diameter 2.9 μm, from the entrance side of the cell suspension) basis weight 70g / m 2) 16 sheets, polybutylene terephthalate nonwoven fabric (fiber diameter 1.7 [mu] m, basis weight 20g / m 2) 12 sheets, the filled in a stacked state, first, using a syringe from the inlet side saline 45ml The liquid was passed by hand. Next, 1000 ml of a cell suspension (jukat cell concentration 2.1 × 10 6 cells / ml) in which Jukat cells are suspended in RPMI 1640 medium was applied at a linear velocity of 1.6 cm / min. Was passed through the column inlet. Thereafter, 50 ml of 0.2% Alb-added RPMI 1640 medium was pushed by hand with a syringe in the direction opposite to the flow direction, and the cells were collected. As a result, the cell recovery rate and passage rate were 78% and 0%.
〔実施例５〕
半径３６ｍｍ高さ１２ｍｍのカラムに、細胞懸濁液の入り口側よりポリブチレンテレフタレート製不織布（繊維径４．６μｍ、目付け７０ｇ／ｍ２）１６枚、ポリブチレンテレフタレート製不織布（繊維径２．９μｍ、目付け７０ｇ／ｍ２）１６枚、ポリブチレンテレフタレート製不織布（繊維径１．７μｍ、目付け２０ｇ／ｍ２）１２枚、を積層状態で充填し、まず１０％ＦＢＳ添加ＲＰＭＩ１６４０培地４５ｍｌを入口側よりシリンジを用いて手押しで通液した。次に、Ｊｕｋａｔ細胞がＲＰＭＩ１６４０培地に懸濁された細胞懸濁液（ｊｕｋａｔ細胞濃度２．１ｘ１０６ｃｅｌｌｓ/ｍｌ）１０００ｍｌを線速度４．０ｃｍ/ｍｉｎ．でカラム入り口より通液した。その後、通液とは逆方向より０．２％Ａｌｂ添加ＲＰＭＩ１６４０培地５０ｍｌをシリンジを用いて手押しで押し込み、細胞を回収した。結果、細胞回収率および通過率は７９％、０％であった。
Example 5
In a column with a radius of 36 mm and a height of 12 mm, 16 non-woven fabrics made of polybutylene terephthalate (fiber diameter 4.6 μm, basis weight 70 g / m 2 ), polybutylene terephthalate nonwoven fabric (fiber diameter 2.9 μm, from the entrance side of the cell suspension) 16 sheets of weight per unit area 70 g / m 2 ) and 12 sheets of polybutylene terephthalate non-woven fabric (fiber diameter 1.7 μm, weight per unit area 20 g / m 2 ) were filled in a laminated state. First, 45 ml of 10% FBS-added RPMI 1640 medium was injected from the inlet side The solution was passed by hand using Next, 1000 ml of a cell suspension (jukat cell concentration 2.1 × 10 6 cells / ml) in which Jukat cells are suspended in RPMI 1640 medium was applied at a linear velocity of 4.0 cm / min. Was passed through the column inlet. Thereafter, 50 ml of 0.2% Alb-added RPMI 1640 medium was pushed by hand with a syringe in the direction opposite to the flow direction, and the cells were collected. As a result, the cell recovery rate and passage rate were 79% and 0%.
〔比較例１〕
半径３６ｍｍ高さ１２ｍｍのカラムに、細胞懸濁液の入り口側よりポリブチレンテレフタレート製不織布（繊維径４．６μｍ、目付け７０ｇ／ｍ２）１６枚、ポリブチレンテレフタレート製不織布（繊維径４．３μｍ、目付け７０ｇ／ｍ２）２０枚を積層状態で充填し、まず１０％ＦＢＳ添加ＲＰＭＩ１６４０培地４５ｍｌを入口側よりシリンジを用いて手押しで通液した。次に、Ｊｕｋａｔ細胞がＲＰＭＩ１６４０培地に懸濁された細胞懸濁液（ｊｕｋａｔ細胞濃度２．１ｘ１０６ｃｅｌｌｓ/ｍｌ）１０００ｍｌを線速度１．６ｃｍ/ｍｉｎ．でカラム入り口より通液した。その後、通液とは逆方向より０．２％Ａｌｂ添加ＲＰＭＩ１６４０培地５０ｍｌをシリンジを用いて手押しで押し込み、細胞を回収した。結果、細胞回収率および通過率は６９％、１６％であった。
[Comparative Example 1]
In a column with a radius of 36 mm and a height of 12 mm, 16 non-woven fabrics made of polybutylene terephthalate (fiber diameter 4.6 μm, basis weight 70 g / m 2 ), polybutylene terephthalate nonwoven fabric (fiber diameter 4.3 μm, from the inlet side of the cell suspension) 20 sheets (weight per unit area: 70 g / m 2 ) were filled in a laminated state, and 45 ml of 10% FBS-added RPMI1640 medium was first manually passed from the inlet side using a syringe. Next, 1000 ml of a cell suspension (jukat cell concentration 2.1 × 10 6 cells / ml) in which Jukat cells are suspended in RPMI 1640 medium was applied at a linear velocity of 1.6 cm / min. Was passed through the column inlet. Thereafter, 50 ml of 0.2% Alb-added RPMI 1640 medium was pushed by hand with a syringe in the direction opposite to the flow direction, and the cells were collected. As a result, the cell recovery rate and passage rate were 69% and 16%.
〔比較例２〕
半径３６ｍｍ高さ１２ｍｍのカラムに、細胞懸濁液の入り口側よりポリブチレンテレフタレート製不織布（繊維径２．９μｍ、目付け７０ｇ／ｍ２）３６枚を積層状態で充填し、まず１０％ＦＢＳ添加ＲＰＭＩ１６４０培地４５ｍｌを入口側よりシリンジを用いて手押しで通液した。次に、Ｊｕｋａｔ細胞がＲＰＭＩ１６４０培地に懸濁された細胞懸濁液（ｊｕｋａｔ細胞濃度２．１ｘ１０６ｃｅｌｌｓ/ｍｌ）１０００ｍｌを線速度１．６ｃｍ/ｍｉｎ．でカラム入り口より通液した。その後、通液とは逆方向より０．２％Ａｌｂ添加ＲＰＭＩ１６４０培地５０ｍｌ押し込み、細胞を回収しようとしたが、閉塞し、回収できなかった。
[Comparative Example 2]
A column of 36 mm in radius and 12 mm in height was filled with 36 non-woven fabrics made of polybutylene terephthalate (fiber diameter 2.9 μm, basis weight 70 g / m 2 ) from the entrance side of the cell suspension in a laminated state. First, RPMI 1640 with 10% FBS added 45 ml of the medium was passed by hand from the inlet side using a syringe. Next, 1000 ml of a cell suspension (jukat cell concentration 2.1 × 10 6 cells / ml) in which Jukat cells are suspended in RPMI 1640 medium was applied at a linear velocity of 1.6 cm / min. Was passed through the column inlet. Thereafter, 50 ml of RPMI 1640 medium supplemented with 0.2% Alb was pushed in from the direction opposite to the flow direction to try to recover the cells, but the cells were blocked but could not be recovered.
Claims (9)
（ａ）各層の平均繊維径は１．５μｍ以上であり、
（ｂ）少なくとも２層は、平均繊維径５．０μｍ以下であり、
（ｃ）平均繊維径５．０μｍ以下の層のみに着目した場合に、近接する２層は、入口側の層の平均繊維径をＵ、出口側の平均繊維径をＤとして、いずれも
（ｃ１）Ｕ＞Ｄ かつ Ｕ−Ｄ≧０．５、または
（ｃ２）Ｕ＜Ｄ
の関係を満たす（ただし、少なくとも１箇所は上記（ｃ１）の関係を満たす）ことを特徴とする、細胞濃縮方法。 The cell suspension is introduced into a container having an inlet and an outlet, and filled with a filter laminated with a nonwoven fabric. After draining the filtrate substantially free of cells, the recovered liquid is introduced into the container. A cell concentration method for recovering cells supplemented by the filter,
(A) The average fiber diameter of each layer is 1.5 μm or more,
(B) At least two layers have an average fiber diameter of 5.0 μm or less,
(C) When paying attention only to a layer having an average fiber diameter of 5.0 μm or less, the two adjacent layers have U as the average fiber diameter of the layer on the inlet side and D as the average fiber diameter on the outlet side. ) U> D and U−D ≧ 0.5, or (c2) U <D
(However, at least one location satisfies the relationship (c1) above).
Ｕ＞Ｄ かつ Ｕ−Ｄ≧０．５
の関係を満たす、請求項１〜８のいずれかに記載の細胞濃縮方法。 When attention is paid to two adjacent layers having an average fiber diameter of 5.0 μm or less, the average fiber diameter of the layer on the inlet side is U, and the average fiber diameter on the outlet side is D, both of which U> D and U−D ≧ 0 .5
The cell concentration method according to any one of claims 1 to 8, which satisfies the above relationship.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2016116465A (en) * | 2014-12-19 | 2016-06-30 | 株式会社カネカ | Production method of cell concentrated solution |
| JP2017079660A (en) * | 2015-10-29 | 2017-05-18 | 株式会社カネカ | Method for producing cell concentrated solution using cell separation filter |
| WO2020196649A1 (en) * | 2019-03-27 | 2020-10-01 | 株式会社カネカ | Cell production device, cell production method, and server, system and device to be used therefor |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0892001A (en) * | 1994-09-26 | 1996-04-09 | Asahi Medical Co Ltd | Method for storing cell group |
| JP2000083649A (en) * | 1998-09-14 | 2000-03-28 | Terumo Corp | Device for separating and recovering cell and separation and recovery of cell |
| JP2000217887A (en) * | 1999-02-01 | 2000-08-08 | Nissho Corp | Method for recovering useful cell from frozen cell and instrument therefor |
| JP2002238996A (en) * | 2001-02-15 | 2002-08-27 | Asahi Medical Co Ltd | Leukocyte-removing filter device and leukocyte-removing method |
| JP2005082567A (en) * | 2003-09-11 | 2005-03-31 | Asahi Kasei Medical Co Ltd | Method for producing cellular composition for treatment by vascularization and system for producing the same |
| JP2008086235A (en) * | 2006-09-29 | 2008-04-17 | Kaneka Corp | Method for recovering cell and cell-recovering filter |
| JP2009284860A (en) * | 2008-05-30 | 2009-12-10 | Asahi Kasei Kuraray Medical Co Ltd | Method for concentrating mononuclear cell and platelet |
-
2011
- 2011-08-09 JP JP2011174107A patent/JP2013034436A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0892001A (en) * | 1994-09-26 | 1996-04-09 | Asahi Medical Co Ltd | Method for storing cell group |
| JP2000083649A (en) * | 1998-09-14 | 2000-03-28 | Terumo Corp | Device for separating and recovering cell and separation and recovery of cell |
| JP2000217887A (en) * | 1999-02-01 | 2000-08-08 | Nissho Corp | Method for recovering useful cell from frozen cell and instrument therefor |
| JP2002238996A (en) * | 2001-02-15 | 2002-08-27 | Asahi Medical Co Ltd | Leukocyte-removing filter device and leukocyte-removing method |
| JP2005082567A (en) * | 2003-09-11 | 2005-03-31 | Asahi Kasei Medical Co Ltd | Method for producing cellular composition for treatment by vascularization and system for producing the same |
| JP2008086235A (en) * | 2006-09-29 | 2008-04-17 | Kaneka Corp | Method for recovering cell and cell-recovering filter |
| JP2009284860A (en) * | 2008-05-30 | 2009-12-10 | Asahi Kasei Kuraray Medical Co Ltd | Method for concentrating mononuclear cell and platelet |
Non-Patent Citations (2)
| Title |
|---|
| JPN6015035595; 医工学治療 Ｖｏｌ．１２， Ｎｏ．３, 2000, ｐ．８５０-８５４ * |
| JPN6015035598; 繊維と工業 Ｖｏｌ．６１， Ｎｏ．８, 2005, ｐ．２１５-２１６ * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2016116465A (en) * | 2014-12-19 | 2016-06-30 | 株式会社カネカ | Production method of cell concentrated solution |
| JP2017079660A (en) * | 2015-10-29 | 2017-05-18 | 株式会社カネカ | Method for producing cell concentrated solution using cell separation filter |
| WO2020196649A1 (en) * | 2019-03-27 | 2020-10-01 | 株式会社カネカ | Cell production device, cell production method, and server, system and device to be used therefor |
| JP7483687B2 (en) | 2019-03-27 | 2024-05-15 | 株式会社カネカ | Cell manufacturing device, cell manufacturing method, and server, system, and device used therein |
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