JP2011010582A - Filter for separating adult stem cell, and method of separating the same - Google Patents

Filter for separating adult stem cell, and method of separating the same Download PDF

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JP2011010582A
JP2011010582A JP2009156381A JP2009156381A JP2011010582A JP 2011010582 A JP2011010582 A JP 2011010582A JP 2009156381 A JP2009156381 A JP 2009156381A JP 2009156381 A JP2009156381 A JP 2009156381A JP 2011010582 A JP2011010582 A JP 2011010582A
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Akira Kobayashi
明 小林
Shinya Yoshida
進也 吉田
Kiichi Watanabe
基一 渡邊
Masaru Nakatani
勝 中谷
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Kaneka Corp
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Abstract

PROBLEM TO BE SOLVED: To provide a cell separation filter for selectively separating/recovering adhesive adult stem cells from a body fluid containing adult stem cells, such as bone marrow, peripheral blood, cord blood, and menstruation blood.SOLUTION: The cell separation filter including a cell separator capable of capturing/recovering adhesive adult stem cells from a body fluid such as bone marrow, peripheral blood, cord blood, and menstruation blood, is characterized in that a numerical value (cm) derived by dividing the thickness (cm) of the cell separator by the filtration area (cm) thereof is not lower than 0.05 and not higher than 1.0 when the cell separator is charged into a container composed of a body-fluid inflow section and a body-fluid outflow section. Cells extremely effective as a source of cells for use in regenerative medicine and causing few side effects, with a low degree of contamination by erythrocytes and leukocytes, can be made available by using the filter. By integrating a culture bag with the cell separation filter, the preparation of intended cells including extracting and amplifying the same can be accomplished in a closed system, whereby highly safe cells for use in medicine can be prepared.

Description

本発明は、骨髄、末梢血、臍帯血、及び月経血などの体液から付着性の成体幹細胞を選択的に捕捉・回収するための細胞分離フィルター、及び該フィルターを使用した成体幹細胞分離方法を提供する技術分野に関するものである。   The present invention provides a cell separation filter for selectively capturing and collecting adherent adult stem cells from body fluids such as bone marrow, peripheral blood, umbilical cord blood, and menstrual blood, and an adult stem cell separation method using the filter It is related to the technical field.

骨髄液、臍帯血中には近年になって骨、軟骨、筋肉、脂肪など多様な細胞に分化し得る性質を持った付着性の成体幹細胞が存在することが明らかになってきており（特許文献１、非特許文献１、２、３）該細胞を効率良く分離・増幅させる方法は再生医療発展の見地から極めて重要である。また、付着性の成体幹細胞は骨髄液中に成人で１０4から１０6個に1つ程度の数という非常に存在頻度が少ないことが報告されており（非特許文献４）、該細胞画分を分離・濃縮後に回収する方法が種々検討されている。 In recent years, it has become clear that bone marrow fluid and umbilical cord blood have adherent adult stem cells that have the property of differentiating into various cells such as bone, cartilage, muscle, and fat. 1. Non-patent documents 1, 2, 3) A method for efficiently separating and amplifying the cells is extremely important from the viewpoint of the development of regenerative medicine. In addition, it has been reported that adherent adult stem cells are present in bone marrow fluid at an extremely low frequency of about 1 in 10 4 to 10 6 in adults (Non-patent Document 4). Various methods for recovering after separation and concentration have been studied.

例えばPittengerらは、密度勾配分離方法であるフィコールパック分画法を用いた脂肪、軟骨、骨細胞への分化を（非特許文献４）、関谷らは、フィコールバック分画法およびデキストランを用いた軟骨への分化を試みている（非特許文献５、６）。   For example, Pittenger et al. Differentiated into fat, cartilage, and bone cells using Ficoll-pack fractionation, which is a density gradient separation method (Non-patent Document 4), and Sekiya et al. Used Ficoll-back fractionation and dextran. Attempts to differentiate into cartilage (Non-Patent Documents 5 and 6).

しかしながら、上記フィコールは医薬品ＧＭＰに準拠して製造されておらず、デキストランを用いた自然沈降方法では赤血球層以外の画分に付着性の成体幹細胞が共存して分離されるため、必ずしも最善の方法ではない。   However, the ficoll is not manufactured according to the pharmaceutical GMP, and the natural precipitation method using dextran separates adherent adult stem cells in fractions other than the erythrocyte layer. is not.

また、これらの方法は、細胞分離液と細胞を分けるために遠心分離器を使用して細胞を数回洗浄する操作が必要であり、操作性が煩雑、遠心操作による細胞のダメージや、開放系での操作によるコンタミネーションの危険がともなう。   In addition, these methods require the operation of washing the cells several times using a centrifuge to separate the cell separation solution from the cells, and the operability is complicated. There is a risk of contamination due to the operation at.

このような理由から、実際に付着性の成体幹細胞を分離、回収する場合には、骨髄液や臍帯血をそのまま培養して、非付着性の細胞を洗浄し、付着性の細胞を得るという例が多数報告されている（例えば非特許文献７）。   For these reasons, when actually separating and collecting adherent adult stem cells, bone marrow fluid or umbilical cord blood is cultured as it is, non-adherent cells are washed, and adherent cells are obtained. Have been reported (for example, Non-Patent Document 7).

一方で最近、末梢血や臍帯血をフィルターに通液し、単核球や造血幹細胞（ＣＤ３４陽性細胞）をフィルターに捕捉させ、高粘度の溶液を通液方向とは逆方向から流入し、捕捉細胞を回収するフィルター分離法が開示されている（特許文献２、３）。   On the other hand, recently, peripheral blood and umbilical cord blood are passed through a filter, and mononuclear cells and hematopoietic stem cells (CD34 positive cells) are captured by the filter. A filter separation method for recovering cells is disclosed (Patent Documents 2 and 3).

しかし、これらのフィルターは、末梢血、あるいは臍帯血中の白血球が全てフィルターに捕捉されることが特徴的であり、そのためフィルターの孔が細かく、血餅や細胞凝集塊、捕捉細胞などによりフィルターの目詰まりが生じやすい。また最近では、月経血の中に幹細胞が豊富に含まれているとの報告があり、月経血中から幹細胞を分離する際にもフィルターの目詰まりが生じやすいという問題点がある。   However, these filters are characterized in that all white blood cells in peripheral blood or umbilical cord blood are captured by the filter, and therefore, the pores of the filter are fine, and the filter may be blocked by clots, cell clumps, captured cells, etc. Clogging is likely to occur. Recently, there are reports that stem cells are abundant in menstrual blood, and there is a problem that clogging of the filter is likely to occur when separating stem cells from menstrual blood.

目詰まりを回避する方法として、フィルターの孔を大きくすると必要な細胞がフィルターを通過して捕捉率が大幅に低下してしまうため、フィルターの厚みを厚くすると、捕捉細胞の回収液の剪断応力がフィルターの抵抗で低下してしまい、細胞回収率が大幅に低下するという問題がある。   As a method of avoiding clogging, if the pores of the filter are enlarged, the necessary cells pass through the filter and the capture rate is greatly reduced. There exists a problem that it falls with the resistance of a filter and a cell recovery rate falls significantly.

一方で、赤血球、白血球を実質的に通過させる細胞分離材を充填した幹細胞分離フィルターが開示されているが（特許文献４）、フィルター自体の濾過面積を抑えつつ、目詰りをなくし、夾雑細胞の除去、目的細胞の回収率を向上させるという課題を同時に満たすフィルター技術は知られていない。   On the other hand, a stem cell separation filter filled with a cell separation material that substantially allows red blood cells and white blood cells to pass therethrough is disclosed (Patent Document 4). However, while suppressing the filtration area of the filter itself, clogging is eliminated, There is no known filter technology that simultaneously satisfies the problem of removing and improving the recovery rate of target cells.

国際公開第０１／８３７０９号International Publication No. 01/83709 特開平１０−３１３８５５号公報Japanese Patent Laid-Open No. 10-313855 特表２００７−５３０６９１号公報Special Table 2007-530691 国際公開第０７／０４６５０１号International Publication No. 07/046501

Pliard A. et al.:Conversion of an Immortilized Mesodermal Progenitor Cell Towards Osteogenic, Chondrogenic,or Adipogenic Pathways. J. Cell Biol. 130(6):1461-72(1995)Pliard A. et al .: Conversion of an Immortilized Mesodermal Progenitor Cell Towards Osteogenic, Chondrogenic, or Adipogenic Pathways. J. Cell Biol. 130 (6): 1461-72 (1995) Mackay A. M.et al.:Chondrogenic differentiation of cultured human mesenchymal Stem Cells from Marrow,Tissue Engineering 4(4):415-428(1998)Mackay A. M. et al .: Chondrogenic differentiation of cultured human mesenchymal Stem Cells from Marrow, Tissue Engineering 4 (4): 415-428 (1998) Angele P.et al.:Engineering of Osteochondoral Tissue with Bone Marrow Mesenchymal Progenitor Cells in a Derivatived Hyaluronan Geration Composite Sponge, Tissue Engineering 5(6):545-553(1999)Angele P. et al .: Engineering of Osteochondoral Tissue with Bone Marrow Mesenchymal Progenitor Cells in a Derivatived Hyaluronan Geration Composite Sponge, Tissue Engineering 5 (6): 545-553 (1999) Pittenger.et al. Multilineage Potential of Adult Human Mesenchymal Stem Cells,Science 284:143-147(1999)Pittenger. Et al. Multilineage Potential of Adult Human Mesenchymal Stem Cells, Science 284: 143-147 (1999) Sekiya. et al. In vitro Cartilage Formation by human adult Stem Cells from Bone Marrow Stroma defines the sequence cellular and molecular events during chondrogenesis,Developmental Biology 7(99):4397-4402(2002)Sekiya. Et al. In vitro Cartilage Formation by human adult Stem Cells from Bone Marrow Stroma defines the sequence cellular and molecular events during chondrogenesis, Developmental Biology 7 (99): 4397-4402 (2002) Wakitani.et al. Human autologus culture expanded Bone Marrow Mesenchymal Cell Transplantation for repair of Cartilage defects in Osteoarthritic Knees,OsteoArthritis Reserch Society International (2002)10,199-206.Wakitani.et al.Human autologus culture expanded Bone Marrow Mesenchymal Cell Transplantation for repair of Cartilage defects in Osteoarthritic Knees, OsteoArthritis Reserch Society International (2002) 10,199-206. Tsutsumi.et al.Retention of Multilineage Differentiation Potential of Mesenchymal Cells During Proliferation in Response to FGF,Biochemical and Biophysical Reserch Communications 288,413-419(2001).Tsutsumi. Et al. Retention of Multilineage Differentiation Potential of Mesenchymal Cells During Proliferation in Response to FGF, Biochemical and Biophysical Reserch Communications 288, 413-419 (2001).

本発明の課題は、成体幹細胞を含む体液（特に骨髄液）をフィルターで濾過する際に、目詰まりすることなく、また細胞回収液を注入して成体幹細胞を回収する際に、高収率で回収することが可能な成体幹細胞分離フィルター、及び該フィルターを使用した成体幹細胞分離方法を提供することにある。   The subject of the present invention is that when filtering body fluid (especially bone marrow fluid) containing adult stem cells without clogging, and when recovering adult stem cells by injecting the cell recovery solution, the yield is high. An adult stem cell separation filter that can be collected and an adult stem cell separation method using the filter.

発明者らは、上記課題を解決すべく鋭意検討した結果、驚くべきことに骨髄液、末梢血、臍帯血、及び月経血などの成体幹細胞を含む体液中から成体幹細胞を回収する方法において、付着性の成体幹細胞を捕捉・回収が可能な細胞分離材の濾過面積と充填時の厚みが特定の比率にあると、従来フィルターより小さい面積でも目詰まりがなく、高収率で成体幹細胞の分離、回収が可能であることを見出した。したがって、本発明が提供するのは以下の通りである：
（１）骨髄、末梢血、臍帯血、及び月経血などの体液から付着性の成体幹細胞を捕捉・回収が可能な細胞分離材を体液流入部と体液流出部を有する容器に充填した時に、細胞分離材の厚み（ｃｍ）を濾過面積（ｃｍ2）で割った値（ｃｍ-1）が０．０５以上１．０以下であることを特徴とする細胞分離フィルター、
（２）体液流入部あるいは、体液流入部以外の体液流入側に、細胞分離材内に留まっている体液を洗浄するための洗浄液流入部、及び細胞分離材に捕捉された細胞を回収するための容器を備え、また体液流出部あるいは、体液流出部以外の体液流出部側に、細胞分離材に捕捉された細胞を回収するための細胞回収液流入部を備えた（１）に記載の細胞分離フィルター、
（３）細胞分離材が不織布であることを特徴とする（１）または（２）に記載の細胞分離フィルター、
（４）細胞分離材が赤血球、白血球を実質的に通過させることを特徴とする（１）〜（３）のいずれかに記載の細胞分離フィルター、
（５）細胞分離材に捕捉された細胞を回収するための容器が、細胞培養可能な容器であることを特徴とする（１）〜（４）のいずれかに記載の細胞分離フィルター、
（６）骨髄、末梢血、臍帯血、及び月経血などの体液を、体液流入部と体液流出部を有する容器に充填した時に、細胞分離材の厚み（ｃｍ）を濾過面積（ｃｍ2）で割った値（ｃｍ-1）が、０．０５以上１．０以下であることを特徴とする細胞分離フィルターに通液し、除去対象細胞含有液を排出させた後に、該フィルター内に留まった除去対象細胞をさらに排出するための洗浄液を通液し、さらに該フィルターに捕捉された細胞を回収するため体液流出部あるいは、体液流出部以外の体液流出部側に備えた細胞回収液流入部より細胞回収液を流入し、体液流入部あるいは体液流入部以外の体液流入側に設置した細胞回収容器に該フィルターに捕捉された細胞を回収する成体幹細胞分離方法、
（７）細胞分離材が赤血球、白血球を実質的に通過させることを特徴とする（６）に記載の成体幹細胞分離方法。 As a result of intensive studies to solve the above problems, the inventors have surprisingly found that in a method for recovering adult stem cells from body fluids containing adult stem cells such as bone marrow fluid, peripheral blood, umbilical cord blood, and menstrual blood, When the filtration area of the cell separation material that can capture and recover adult adult stem cells and the thickness at the time of filling are in a certain ratio, there is no clogging even with an area smaller than the conventional filter, separation of adult stem cells in high yield, It was found that recovery was possible. Accordingly, the present invention provides the following:
(1) When a cell separation material capable of capturing and collecting adherent adult stem cells from body fluids such as bone marrow, peripheral blood, umbilical cord blood, and menstrual blood is filled into a container having a body fluid inflow portion and a body fluid outflow portion, A cell separation filter, wherein a value (cm -1 ) obtained by dividing the thickness (cm) of the separation material by the filtration area (cm 2 ) is 0.05 or more and 1.0 or less,
(2) A body fluid inflow portion or a body fluid inflow side other than the body fluid inflow portion, a body fluid inflow portion for washing body fluid remaining in the cell separation material, and a cell for collecting cells captured by the cell separation material The cell separation according to (1), comprising a container, and further comprising a cell recovery fluid inflow portion for recovering cells captured by the cell separation material on the body fluid outflow portion or on the body fluid outflow portion other than the body fluid outflow portion. filter,
(3) The cell separation filter according to (1) or (2), wherein the cell separation material is a nonwoven fabric,
(4) The cell separation filter according to any one of (1) to (3), wherein the cell separation material substantially allows red blood cells and white blood cells to pass through.
(5) The cell separation filter according to any one of (1) to (4), wherein the container for collecting the cells captured by the cell separation material is a container capable of cell culture.
(6) When a body fluid such as bone marrow, peripheral blood, umbilical cord blood, and menstrual blood is filled in a container having a body fluid inflow portion and a body fluid outflow portion, the thickness (cm) of the cell separation material is expressed by the filtration area (cm 2 ). The divided value (cm −1 ) was passed through a cell separation filter characterized by being 0.05 or more and 1.0 or less, and after the draining target cell-containing solution was discharged, it remained in the filter. From the body fluid outflow part or the cell recovery liquid inflow part provided on the side of the body fluid outflow part other than the body fluid outflow part for passing the washing liquid for further discharging the cells to be removed and further collecting the cells captured by the filter An adult stem cell separation method for collecting cells captured by the filter into a cell collection container installed on a body fluid inflow side other than a body fluid inflow part or a body fluid inflow part,
(7) The adult stem cell separation method according to (6), wherein the cell separation material substantially allows red blood cells and white blood cells to pass therethrough.

本発明により得られる細胞分離フィルターを使用することにより、骨髄、末梢血、臍帯血、及び月経血などの成体幹細胞を含む体液中から多種多様な細胞、臓器に分化し得る能力を有している成体幹細胞を選択的に分離・回収することが可能になる。   By using the cell separation filter obtained by the present invention, it has the ability to differentiate into a wide variety of cells and organs from body fluids including adult stem cells such as bone marrow, peripheral blood, umbilical cord blood, and menstrual blood. Adult stem cells can be selectively separated and collected.

また該細胞分離フィルターを使用した分離方法で回収した細胞は、増幅させずにそのまま、あるいは閉鎖系で増幅させることが可能となり、心筋再生や血管再生などの再生医療や細胞医療に用いる治療用細胞を調製するためのフィルターとして提供することが可能となる。   In addition, the cells recovered by the separation method using the cell separation filter can be amplified as they are without being amplified or in a closed system, and are used for regenerative medicine such as myocardial regeneration and vascular regeneration and therapeutic cells used for cell medicine. It becomes possible to provide as a filter for preparing.

本発明は、骨髄、末梢血、臍帯血、及び月経血などの体液中から多種多様な細胞、臓器に分化し得る能力を有している成体幹細胞を選択的に分離・回収し、必要に応じ、回収直後に閉鎖系で細胞増幅可能なバックを備えた細胞分離フィルターを提供できることにある。   The present invention selectively isolates and collects adult stem cells having the ability to differentiate into a wide variety of cells and organs from body fluids such as bone marrow, peripheral blood, umbilical cord blood, and menstrual blood. Thus, it is possible to provide a cell separation filter having a bag capable of amplifying cells in a closed system immediately after collection.

該細胞分離フィルターの使用方法の概略を次に記す。   An outline of how to use the cell separation filter is described below.

まず、該細胞分離フィルターに、体液入口側から体液を通液することにより、赤血球、白血球や血小板は実質的に捕捉されずに体液出口側より流出し、細胞分離フィルター内に目的細胞を捕捉することが可能である。次に、洗浄液を同方向から通液することにより、細胞分離フィルター内に溜まっている赤血球、白血球、血小板の大多数を洗浄除去することが可能である。さらに、体液出口側からすなわち、洗浄液を流した方向とは逆方向から、細胞回収液を流すことにより、上記目的細胞を高い効率で分離回収することが可能である。   First, by passing a body fluid from the body fluid inlet side to the cell separation filter, red blood cells, white blood cells and platelets flow out from the body fluid outlet side without being substantially captured, and the target cells are captured in the cell separation filter. It is possible. Next, it is possible to wash and remove the majority of red blood cells, white blood cells, and platelets accumulated in the cell separation filter by passing the washing solution from the same direction. Furthermore, the target cells can be separated and recovered with high efficiency by flowing the cell recovery solution from the body fluid outlet side, that is, from the direction opposite to the direction in which the cleaning solution is supplied.

以下、本発明を詳細に説明する。   Hereinafter, the present invention will be described in detail.

本発明における体液とは血液、骨髄液、臍帯血、及び月経血など成体幹細胞を含むものを指す。またこれ以外に、該体液の稀釈液、あるいはフィコール、パーコール、ヒドロキシエチルスターチ（ＨＥＳ）、バクティナーチューブ、リンフォプレップ等を使用し、比重密度遠心分離法により前処理を施して調製された細胞懸濁液なども使用できる。   The body fluid in the present invention refers to those containing adult stem cells such as blood, bone marrow fluid, umbilical cord blood, and menstrual blood. In addition to this, cells prepared by subjecting the body fluid to dilution, or using Ficoll, Percoll, Hydroxyethyl Starch (HES), Bactiner tube, Lymphprep, etc., and pretreatment by specific gravity centrifugation Suspensions can also be used.

本発明における付着性の成体幹細胞とは、培養皿に付着して増殖し、分化能を有することを特徴とする細胞をいう。具体的には、間葉系幹細胞、多能性体性幹細胞（Multipotent Adult Progenitor Cells:MAPCs）など多分化能を有する細胞等を指す。   The adherent adult stem cell in the present invention refers to a cell characterized in that it grows by attaching to a culture dish and has differentiation ability. Specifically, it refers to cells having pluripotency such as mesenchymal stem cells and multipotent adult progenitor cells (MAPCs).

間葉系幹細胞とは、体液中から分離され、自己増殖を繰り返す能力を有し、下流の細胞系譜への分化が可能な細胞を指す。   A mesenchymal stem cell refers to a cell that is isolated from a body fluid, has the ability to repeat self-proliferation, and can differentiate into a downstream cell lineage.

この間葉系幹細胞は、分化誘導因子により中胚葉系の細胞、例えば、骨芽細胞や軟骨細胞などへ分化する細胞である。多能性体性幹細胞とは、分化誘導因子により中胚葉系以外の細胞、例えば、神経細胞、肝細胞にも分化する可能性のある細胞をいうが、本発明はこれらに限定されるものではない。   This mesenchymal stem cell is a cell that differentiates into a mesodermal cell such as an osteoblast or a chondrocyte by a differentiation-inducing factor. A pluripotent somatic stem cell refers to a cell that can differentiate into a cell other than the mesodermal system, for example, a nerve cell or a hepatocyte, by a differentiation-inducing factor, but the present invention is not limited thereto. Absent.

ここでいう培養皿とは、一般的に細胞培養に使用されているポリスチレン製の細胞培養シャーレやフラスコ等が挙げられ、また、該シャーレやフラスコに、コラーゲンやフィブロネクチン、ラミニンなどの細胞外マトリックス成分のタンパク質や、ヒアルロン酸、コンドロイチン硫酸やデルマタン硫酸などの多糖類をコーティングしたものでもよい。   Examples of the culture dish used here include polystyrene cell culture dishes and flasks generally used for cell culture. In addition, extracellular matrix components such as collagen, fibronectin, and laminin are added to the dishes and flasks. Or a protein coated with a polysaccharide such as hyaluronic acid, chondroitin sulfate or dermatan sulfate.

次に本発明における細胞分離材について説明する。   Next, the cell separation material in the present invention will be described.

本発明における成体幹細胞を捕捉・回収が可能な細胞分離材とは、連通孔構造の多孔質体形状でも、繊維の集合体、織物などでも良いが、不織布であることがより好ましい。   The cell separation material capable of capturing and collecting adult stem cells in the present invention may be in the form of a porous body having a communicating hole structure, an aggregate of fibers, a woven fabric, or the like, but is preferably a nonwoven fabric.

細胞分離材が不織布である場合には、不織布の目開きは、目的細胞の捕捉性から短径が３μｍ以上で、長径が１２０μｍ以下であることが好ましい。３μｍより小さいと、赤血球や白血球、血小板の詰りが生じやすくなる。また１２０μｍより大きいと目的細胞の捕捉が困難となる。赤血球や白血球の除去率からより好ましくは、５μｍ〜８０μｍ、赤血球や白血球の除去効率、及び目的細胞の回収率から、さらに好ましくは、５μｍ〜７０μｍである。   When the cell separation material is a non-woven fabric, it is preferable that the mesh size of the non-woven fabric is 3 μm or more in the short diameter and 120 μm or less in the long diameter in order to capture target cells. If it is smaller than 3 μm, clogging of red blood cells, white blood cells, and platelets tends to occur. On the other hand, if it is larger than 120 μm, it becomes difficult to capture target cells. More preferably, it is 5 μm to 80 μm from the removal rate of red blood cells and white blood cells, and more preferably 5 μm to 70 μm from the removal efficiency of red blood cells and white blood cells and the recovery rate of target cells.

不織布の目開きとは、例えば、下記方法により求めることができる。   The opening of the nonwoven fabric can be determined, for example, by the following method.

細胞分離材を走査型電子顕微鏡にて写真撮影し、異なる２本以上の繊維が交差することにより形成される実質的な孔の長径、および短径を画像解析装置にて５０ポイント以上測定し、それぞれの平均値を求める。すなわち請求項記載の目開きの範囲とは、下限値が上記のようにして求めた短径の平均値を、上限値が長径の平均値を示す。   The cell separation material is photographed with a scanning electron microscope, and the major and minor diameters of the substantial holes formed by the crossing of two or more different fibers are measured with an image analyzer at least 50 points. Find the average of each. In other words, the range of the apertures described in the claims refers to the average value of the minor axis obtained by the lower limit as described above, and the average value of the major axis of the upper limit.

また不織布の繊維径は、目的細胞の回収率から、３〜４０μｍが好ましい。３μｍより細いと白血球との相互作用が高まり、赤血球や白血球の除去（通過）効率が低くなる。また４０μｍより太いと有効接触面積の低下やショートパスが起こりやすくなり、目的細胞の回収率の低下につながる。目的細胞と細胞分離材との相互作用を上げ、収率を上げるためには、５μｍ〜３５μｍがより好ましい。さらに好ましくは５μｍから３０μｍである。   Further, the fiber diameter of the nonwoven fabric is preferably 3 to 40 μm from the recovery rate of the target cells. If it is thinner than 3 μm, the interaction with leukocytes increases, and the removal (passage) efficiency of red blood cells and leukocytes decreases. On the other hand, if the thickness is larger than 40 μm, the effective contact area is likely to be reduced and a short pass is likely to occur, leading to a reduction in the recovery rate of target cells. In order to increase the interaction between the target cell and the cell separation material and increase the yield, 5 μm to 35 μm is more preferable. More preferably, it is 5 μm to 30 μm.

また、赤血球や白血球の除去効率、及び目的細胞の回収率から、細胞分離フィルターの目付け（ｇ／ｍ2）／厚み（ｍ）＝Ｋ（ｇ／ｍ3）は、１．０×１０4≦Ｋ≦１．０×１０6であることが好ましく、赤血球や白血球の除去効率から２．５×１０4≦Ｋ≦７．５×１０5がより好ましく、さらに好ましくは５．０×１０4≦Ｋ≦５．０×１０5である。 Further, based on the removal efficiency of red blood cells and white blood cells and the recovery rate of target cells, the basis weight (g / m 2 ) / thickness (m) = K (g / m 3 ) of the cell separation filter is 1.0 × 10 4 ≦ It is preferable that K ≦ 1.0 × 10 6 , and 2.5 × 10 4 ≦ K ≦ 7.5 × 10 5 is more preferable, and 5.0 × 10 4 ≦ is more preferable from the removal efficiency of red blood cells and white blood cells. K ≦ 5.0 × 10 5 .

細胞分離材の材質は、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などのポリオレフィン、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、アクリロニトリル、アクリル酸、アクリル酸エステルなど）、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、ポリアクリレート、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、綿などの少なくとも１種より選択される材質が好ましい。   Cell separation material is made of 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, Acrylonitrile, acrylic acid, acrylic ester, etc.), nylon, polyurethane, polyimide, aramid, polyamide, cupra, kevlar, carbon, polyacrylate, phenol, tetron, pulp, hemp, cellulose, kenaf, chitin, chitosan, glass, cotton, etc. A material selected from at least one of the above is preferred.

より好ましくは、ポリエステル、ポリスチレン、アクリル、レーヨン、ポリオレフィン、ビニロン、ナイロン、ポリウレタンなどの少なくとも１種より選択される合成高分子である。   More preferably, it is a synthetic polymer selected from at least one of polyester, polystyrene, acrylic, rayon, polyolefin, vinylon, nylon, polyurethane and the like.

２種以上の合成高分子を組み合わせる場合は、その組み合わせに特に限定はないが、ポリエステルおよびポリプロピレン、またはレーヨンおよびポリオレフィン、またはポリエステル、レーヨンおよびビニロンなどからなる組み合わせが挙げられる。   When two or more kinds of synthetic polymers are combined, the combination is not particularly limited, and examples thereof include polyester and polypropylene, or rayon and polyolefin, or a combination of polyester, rayon, and vinylon.

２種類以上の合成高分子を組み合わせる場合の繊維の形態としては、１本の繊維が異成分同士の合成高分子よりなる繊維、あるいは異成分同士が剥離分割した分割繊維でもよい。   When the two or more kinds of synthetic polymers are combined, the form of the fiber may be a fiber in which one fiber is made of a synthetic polymer of different components, or a split fiber in which different components are separated from each other.

また、成分の異なる合成高分子単独よりなる繊維をそれぞれ複合化した形態でもよい。ここでいう複合化とは、特に限定はないが２種類以上の繊維が混在した状態より構成される形態、あるいは合成高分子単独よりなる形態をそれぞれ張り合わせたものなど挙げられるが、本発明はこれに限定されるものではない。   Moreover, the form which each compounded the fiber which consists of synthetic polymers with different components individually may be sufficient. The term “composite” as used herein is not particularly limited, and examples thereof include a form composed of a state in which two or more kinds of fibers are mixed or a form composed of synthetic polymers alone. It is not limited to.

本発明で言う濾過面積とは、細胞分離材を容器に充填したときに体液と接触可能な最表面の細胞分離材面積を言う。すなわち、容器に充填したときの細胞分離材のシール部は除く。また細胞分離材内部も含めた表面積とは異なる。   The filtration area referred to in the present invention refers to the area of the outermost cell separation material that can come into contact with body fluid when the container is filled with the cell separation material. That is, the sealing part of the cell separating material when the container is filled is excluded. Moreover, it differs from the surface area including the inside of the cell separation material.

細胞分離材の厚みとは、細胞分離材を容器に充填したときの厚みを言う。本発明においては、先に記した成体幹細胞を捕捉・回収が可能な細胞分離材を使用するが、同時に細胞分離材を容器に充填したときに、細胞分離材の厚み（ｃｍ）を濾過面積（ｃｍ2）で割った値（ｃｍ-1）が、０．０５以上１．０以下になるように容器に充填することが好ましい。０．０５より小さいと細胞分離材の厚みが薄すぎ、回収目的細胞である成体幹細胞が、フィルター出口側より漏れ出し捕捉率の低下に繋がる。逆に１．０より大きいと、細胞分離材が厚くなり過ぎ、フィルター内に捕捉された成体幹細胞を細胞回収液にて回収する際に、細胞分離材の抵抗により細胞を回収する際の剪断応力が低下し、細胞回収率の低下をきたす。 The thickness of the cell separation material means the thickness when the cell separation material is filled in a container. In the present invention, the cell separation material capable of capturing and recovering the adult stem cells described above is used. At the same time, when the cell separation material is filled in a container, the thickness (cm) of the cell separation material is adjusted to the filtration area ( The container is preferably filled so that the value (cm −1 ) divided by cm 2 ) is 0.05 or more and 1.0 or less. If it is less than 0.05, the thickness of the cell separation material is too thin, and adult stem cells, which are target cells for collection, leak out from the filter outlet side, leading to a decrease in capture rate. On the other hand, if the ratio is larger than 1.0, the cell separation material becomes too thick, and when the adult stem cells captured in the filter are collected with the cell collection solution, the shear stress when collecting the cells due to the resistance of the cell separation material Decreases, and the cell recovery rate decreases.

フィルター内への捕捉率を上げ、かつ捕捉された成体幹細胞の回収率を上げるためには、より好ましくは０．０７以上０．７５以下、さらに好ましくは、０．０９以上０．５０以下である。   In order to increase the capture rate into the filter and increase the recovery rate of the captured adult stem cells, it is more preferably 0.07 or more and 0.75 or less, and further preferably 0.09 or more and 0.50 or less. .

細胞分離材の濾過面積は、前記比率の範囲内ならば特に限定はなく、体液処理量との関係から任意に設定できる。すなわち、体液処理量が少ない場合は、細胞のロス等の問題から、濾過面積を必要以上に大きくする必要はなく、また処理液量が多い場合には、濾過面積が小さすぎるとフィルターの詰まり等が生じるため、処理量に応じて適切な濾過面積のフィルターを選定することが出来る。例えば、体液処理量が０．１ｍｌから２００ｍｌ程度の場合、フィルターの濾過面積は、０．１ｃｍ2〜２０ｃｍ2程度が好ましいが、これに限定されない。 The filtration area of the cell separation material is not particularly limited as long as it is within the above range, and can be arbitrarily set from the relationship with the body fluid treatment amount. That is, when the amount of body fluid treatment is small, it is not necessary to increase the filtration area more than necessary due to problems such as cell loss. When the amount of treatment liquid is large, if the filtration area is too small, the filter is clogged. Therefore, it is possible to select a filter having an appropriate filtration area according to the processing amount. For example, if the body fluid treatment amount of about 200ml from 0.1 ml, filtration area of the filter is preferably 0.1 cm 2 to 20 cm 2 approximately, but not limited to.

本発明で言う赤血球、白血球を実質的に通過させるとは、該細胞分離材により前記細胞がほとんど通過するが、若干捕捉されていてもよいことを意味する。細胞分離材による赤血球、白血球の除去率（通過率）は、赤血球が８０％以上、白血球が５０％以上であることが好ましい。細胞分離フィルターの性能面から、より好ましくは赤血球が８５％以上、白血球が６０％以上さらに好ましくは、赤血球が９０％以上、白血球が７０％以上である。   In the present invention, substantially passing red blood cells and white blood cells means that the cells are almost passed by the cell separating material, but may be slightly captured. The removal rate (passage rate) of red blood cells and white blood cells by the cell separation material is preferably 80% or more for red blood cells and 50% or more for white blood cells. From the viewpoint of the performance of the cell separation filter, more preferably, red blood cells are 85% or more, white blood cells are 60% or more, more preferably, red blood cells are 90% or more, and white blood cells are 70% or more.

細胞分離フィルターは、体液の入口および出口を有するもの、さらには細胞洗浄液や細胞回収液の入口および出口を有するもの、さらには回収した細胞をそのまま培養するための培養バックなどを備えた容器内に、細胞分離材が充填されてなるものが好ましい。   The cell separation filter has a body fluid inlet and outlet, a cell washing liquid and a cell recovery liquid inlet and outlet, and a culture bag for culturing the recovered cells as they are. Those filled with a cell separation material are preferred.

具体的には、細胞分離フィルターに、体液を送液するための体液流入部、および細胞分離材を通過した体液を排出するための体液流出部を有しており、さらに体液流入部あるいは、体液流入側に独立して細胞分離材内に留まっている体液を洗浄するための洗浄液流入部を備え、また体液流出部あるいは、体液流出部側に独立して細胞分離材に捕捉された細胞を、体液の流れとは逆方向から細胞回収液を流して細胞を回収するための細胞回収液流入部を備えていてもよい。また回収された細胞を培養するための、培養バックを備えていてもよい。   Specifically, the cell separation filter has a body fluid inflow portion for feeding body fluid and a body fluid outflow portion for discharging body fluid that has passed through the cell separation material, and further includes a body fluid inflow portion or body fluid. Provided with a washing fluid inflow part for washing the body fluid remaining in the cell separation material independently on the inflow side, and the cells captured by the cell separation material independently on the body fluid outflow part or the body fluid outflow part side, You may provide the cell collection liquid inflow part for flowing a cell collection liquid from the direction opposite to the flow of a bodily fluid, and collect | recovering cells. Further, a culture bag for culturing the collected cells may be provided.

培養用バックは、細胞分離材に捕捉された細胞を含む細胞回収液を閉鎖系で回収できるように、体液流入部あるいは細胞洗浄液流入部、あるいは体液流入側に独立して備えることができる。細胞懸濁液を回収した後は、バックを細胞分離フィルターから切り離して培養することができる。   The culture bag can be provided independently at the body fluid inflow part, the cell washing liquid inflow part, or the body fluid inflow side so that the cell recovery liquid containing the cells captured by the cell separation material can be recovered in a closed system. After collecting the cell suspension, the bag can be separated from the cell separation filter and cultured.

つぎに成体幹細胞の分離回収方法について説明する。   Next, a method for separating and collecting adult stem cells will be described.

１）液送液工程；該細胞分離フィルターに体液を通液する際には、体液を入れた容器から送液回路を通じて自然落下で送液しても、ポンプにより通液しても良い。また体液を入れたシリンジを直接、該フィルターに接続し、手でシリンジを押して注入してもよい。ポンプにより通液する場合の流速は、０．１ｍｌ／ｍｉｎ〜１００ｍｌ／ｍｉｎ程度が挙げられるが、本発明はこれに限定されるものではない。   1) Liquid feeding step: When the body fluid is passed through the cell separation filter, the body fluid may be sent from the container in which the body fluid is placed through a liquid feeding circuit by natural fall or through a pump. Alternatively, a syringe containing body fluid may be directly connected to the filter and injected by pushing the syringe by hand. The flow rate when the liquid is passed by the pump includes about 0.1 ml / min to 100 ml / min, but the present invention is not limited to this.

２）細胞洗浄工程；細胞洗浄液は、生理的食塩液、リンゲル液、細胞培養に使用する培地、リン酸緩衝液などの一般的な緩衝液が挙げられるが、安全面から生理的食塩液が好ましい。洗浄は、回路を通じて自然落下で送液しても、ポンプにより通液しても良い。ポンプにより通液する場合の流速は、０．１ｍｌ／ｍｉｎ〜１００ｍｌ／ｍｉｎ程度が挙げられる。洗浄量は、細胞分離フィルター容量により異なるが、該フィルター容積の約１〜５倍程度の体積で洗浄することが好ましい。   2) Cell washing step; Examples of the cell washing solution include physiological saline, Ringer's solution, medium used for cell culture, and general buffers such as phosphate buffer, but physiological saline is preferable from the viewpoint of safety. Cleaning may be carried out by natural fall through the circuit or may be passed through a pump. The flow rate when the liquid is passed by the pump is about 0.1 ml / min to 100 ml / min. The washing amount varies depending on the cell separation filter volume, but it is preferable to wash with a volume of about 1 to 5 times the filter volume.

３）細胞回収工程；細胞回収液は体液を流した方向とは逆方向から入れ、細胞を回収する。細胞回収液は、等張液であれば特に限定はないが、生理的食塩液やリンゲル液など注射用剤として使用実績のあるものや、緩衝液、細胞培養用の培地などが挙げられる。また細胞分離フィルターに捕捉された細胞の回収率を上げるため、細胞回収液の粘張度を上げてもよい。   3) Cell recovery step: The cell recovery solution is added in the direction opposite to the direction in which the body fluid is flowed to recover the cells. The cell recovery solution is not particularly limited as long as it is an isotonic solution, and examples thereof include those that have been used as injectable agents such as physiological saline and Ringer's solution, buffer solutions, and cell culture media. Moreover, in order to increase the recovery rate of the cells captured by the cell separation filter, the viscosity of the cell recovery solution may be increased.

そのために上記細胞回収液にアルブミン、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などを添加することができる。本発明はこれに限定されるものではない。   For that purpose, albumin, fibrinogen, globulin, dextran, hydroxyethyl starch, hydroxyethylcellulose, etc. can be added to the cell recovery solution. The present invention is not limited to this.

バック内に回収した細胞を増幅させる場合は、細胞回収液に培養液（例えば、Ｄｕｌｂｅｃｃｏ ＭＥＭ（日水），α−ＭＥＭ（GIBCO BRL社製），ＭＥＭ（日水），ＩＭＥＭ（日水），ＲＰＭＩ−１６４０（日水）培地など）を使用し、フィルター付属の培養用バックに直接回収する方法などが挙げられる。この培養液には、必要に応じて血清を５〜２０％程度添加しても良い。   In the case of amplifying the cells collected in the bag, a culture solution (for example, Dulbecco MEM (Nissui), α-MEM (GIBCO BRL), MEM (Nissui), IMEM (Nissui)), RPMI-1640 (Nissui) medium etc.) and the like and the like are directly collected in the culture bag attached to the filter. About 5 to 20% of serum may be added to this culture solution as necessary.

つぎにバックに細胞を回収後、必要量の培養液をバックに満たし、フィルターから取り外し、そのまま培養する。バック素材としては、酸素透過性が高く、細胞の付着性が高い素材が好ましい。   Next, after collecting the cells in the bag, the bag is filled with a necessary amount of culture solution, removed from the filter, and cultured as it is. As the back material, a material having high oxygen permeability and high cell adhesion is preferable.

酸素透過性が高い素材としては、ポリメチルペンテン、環状ポリオレフィン、オレフィン系熱可塑性エラストマー、スチレン系熱可塑性エラストマー、ポリアミド系熱可塑性エラストマーなどが挙げられる。   Examples of the material having high oxygen permeability include polymethylpentene, cyclic polyolefin, olefin-based thermoplastic elastomer, styrene-based thermoplastic elastomer, polyamide-based thermoplastic elastomer, and the like.

また細胞の付着性が高い素材としては、ポリスチレン、ポリプロピレン、塩化ビニルなどが挙げられる。上記酸素透過性が高いバックに細胞付着性が高い素材を固定化するなどの方法で、高い酸素透過性を維持したまま、細胞接着性を付与するなどの方法が挙げられるが、本発明はこれに限定されるものではない。   Examples of materials with high cell adhesion include polystyrene, polypropylene, and vinyl chloride. Examples of the method of immobilizing a material having high cell adhesion on the bag having high oxygen permeability include a method of imparting cell adhesion while maintaining high oxygen permeability. It is not limited to.

また該バックは、一般的に使用されている血液バックのような形状をしていてもよいが、平板状のカートリッジ方式でもよい。回収細胞を培養工程を経ずそのまま、患部などに注入する場合は、生理食塩液等点滴などに使用実績のある等張液など、安全性が保障されている細胞回収液を使用することが好ましい。   The bag may be shaped like a commonly used blood bag, but may be a flat cartridge type. When the recovered cells are injected directly into the affected area without passing through the culturing step, it is preferable to use a cell recovery solution that guarantees safety, such as an isotonic solution that has been used for infusions such as physiological saline. .

細胞回収液を細胞分離フィルターに注入し、目的細胞を回収する時は、細胞回収液をシリンジなどに予め入れておき、シリンジのプランジャーを手などで勢いよく押し出すことにより実現できる。回収液量、および流速はフィルター容量により異なるが、フィルター容積の１〜３倍量程度の細胞回収液を、流速０．５ｍｌ／ｓｅｃ〜５ｍｌ／ｓｅｃ程度で注入することが好ましい。本発明はこれに限定されるものではない。   When the cell collection liquid is injected into the cell separation filter and the target cells are collected, the cell collection liquid is put in a syringe or the like in advance, and the plunger of the syringe is pushed out by hand. Although the amount of collected liquid and the flow rate vary depending on the filter volume, it is preferable to inject a cell collected solution of about 1 to 3 times the filter volume at a flow rate of about 0.5 ml / sec to 5 ml / sec. The present invention is not limited to this.

バック内に回収された細胞をバックのまま培養する際の条件としては特に限定されないが、例えば、培地としてGIBCO BRL社製のαMEM培地に15〜20％の牛胎児血清を添加したものを用い、３７℃にてＣＯ2インキュベータ内で培養することが望ましい。 Although it is not particularly limited as a condition when culturing the cells collected in the bag as it is, for example, using a medium supplemented with 15-20% fetal calf serum to the GMEM BRL αMEM medium, It is desirable to culture in a CO 2 incubator at 37 ° C.

培地交換は、培地を吸い取り新しい培地を等量加えてもよいが、培地を抜き取らずに、新しい培地を適宜加えていってもよい。特にバック培養の場合は、新しい培地を加えていくことにより、細胞の分離から増幅までの一連の工程を閉鎖系で実施することが出来、コンタミネーションの防止や作業効率の大幅な向上につながる。   For medium exchange, the medium may be sucked up and an equal amount of new medium may be added, but a new medium may be added as appropriate without removing the medium. In particular, in the case of back culture, by adding a new medium, a series of steps from cell separation to amplification can be carried out in a closed system, leading to prevention of contamination and significant improvement in work efficiency.

細胞を剥離する場合は、キレート剤やディスパーゼ、コラゲナーゼなどの細胞剥離剤、好ましくはトリプシンを用いて剥離、回収することができる。また上記培養にあたっては、分化誘導剤を添加し、各種細胞に分化させることができる。   When cells are detached, they can be detached and collected using a cell remover such as a chelating agent, dispase or collagenase, preferably trypsin. In the above culture, a differentiation inducer can be added to differentiate into various cells.

分化誘導剤としては特に限定されないが、軟骨への分化誘導剤としてはデキサメサゾン、TGFβ、インスリン、トランスフェリン、エタノールアミン、プロリン、アスコルビン酸等が挙げられ、骨への分化誘導剤としてはデキサメサゾン、βグリセロリン酸、ビタミンC等が挙げられ、心筋への分化誘導剤としてはEGF、PDGF、5−アザシチジン等が挙げられ、神経への分化誘導剤としてはbHLH、EGF、FGF−2等が挙げられ、血管への分化誘導剤としてはbFGF、VEGF等が挙げられる。   The differentiation inducing agent is not particularly limited, but examples of the differentiation inducing agent for cartilage include dexamethasone, TGFβ, insulin, transferrin, ethanolamine, proline, ascorbic acid, etc. Acid, vitamin C, and the like, EGF, PDGF, 5-azacytidine and the like as the myocardial differentiation inducer, bHLH, EGF, FGF-2 and the like as the neuronal differentiation inducer, blood vessels Examples of differentiation-inducing agents include bFGF and VEGF.

本細胞分離フィルターは、骨髄、末梢血、臍帯血、及び月経血などの体液から付着性の成体幹細胞を選択的に捕捉・回収することが可能であり、回収細胞をバック内に収納し、そのまま培養することにより、細胞の分離、回収から増幅まで一貫して閉鎖系で処理することが可能な細胞分離フィルターを提供するものである。   This cell separation filter can selectively capture and collect adherent adult stem cells from bodily fluids such as bone marrow, peripheral blood, umbilical cord blood, and menstrual blood. By culturing, a cell separation filter is provided that can be processed in a closed system consistently from separation, recovery and amplification of cells.

以下に実施例により本発明を詳細に説明するが、本発明はこれらの実施例に限定されるものではない。   EXAMPLES The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

（実施例１）
細胞分離フィルターを使用した骨髄液中からの成体幹細胞分離：
細胞ソース：
体重約３０Ｋｇの家畜ブタに筋肉注射にてケタラール、セラクタールを注入し、その後ネンブタールを静脈注射にて追加することにより麻酔を行った。１０ｍｌのシリンジに約２０ＩＵ／ｍｌになるように予めヘパリンを入れておき、腸骨より１５Ｇの穿刺針を用いて骨髄液を採取した。次に採取した骨髄プールにヘパリンを最終濃度で５０ＩＵ／ｍｌになるように添加して、十分に転倒混和を行った。 Example 1
Adult stem cell separation from bone marrow fluid using cell separation filter:
Cell source:
Anesthesia was performed by injecting ketalal or cerectal by intramuscular injection into domestic pigs weighing approximately 30 kg, and then adding nembutal by intravenous injection. Heparin was put in advance in a 10 ml syringe so as to be about 20 IU / ml, and bone marrow fluid was collected from the iliac using a 15 G puncture needle. Next, heparin was added to the collected bone marrow pool to a final concentration of 50 IU / ml, and the mixture was thoroughly inverted.

＜細胞分離性能評価＞
出入口を供えた内径２．２ｃｍの円筒状のハウジングに、レーヨンとポリエチレンからなる不織布Ｋ（目付け（ｇ／ｍ2）／厚み（ｍ））＝１．８×１０5（９５／（５．２×１０-4））、繊維径＝１５±１０μｍ、目開き＝５〜５０μｍを充填し、不織布の上下を外径２．２ｃｍ、内径１．８ｃｍのリングにて挟み込む（充填時不織布厚み０．３ｃｍ）ことにより、不織布を充填した細胞分離フィルターを作製した。なお充填時不織布厚み（ｃｍ）／濾過面積（ｃｍ2）＝０．１２（ｃｍ-1）であった。 <Evaluation of cell separation performance>
Nonwoven fabric K made of rayon and polyethylene (weight per unit (g / m 2 ) / thickness (m)) = 1.8 × 10 5 (95 / (5.2) × 10 −4 )), fiber diameter = 15 ± 10 μm, mesh opening = 5 to 50 μm, and sandwich the upper and lower sides of the nonwoven fabric with a ring having an outer diameter of 2.2 cm and an inner diameter of 1.8 cm (nonwoven fabric thickness 0. 3 cm), a cell separation filter filled with a nonwoven fabric was produced. In addition, it was non-woven fabric thickness (cm) at the time of filling / filtration area (cm 2 ) = 0.12 (cm −1 ).

次に、該細胞分離フィルター体積の約１０倍量の生理食塩液にて不織布の洗浄を行い、シリンジポンプにて骨髄液３（ｍｌ）を空筒線速度１．６（ｃｍ／ｍｉｎ）で通液し、フィルター出口側血球数の測定を自動血球計測装置（シスメックスＫ−４５００）にて実施した。血球の通過率は、フィルター通過前の血球数で、フィルター通過後の対応する細胞数を割ることにより求めた。その結果、赤血球の通過率は９８％、白血球の通過率は７２％、血小板の通過率は５９％であった。   Next, the nonwoven fabric was washed with a physiological saline solution of about 10 times the volume of the cell separation filter, and the bone marrow fluid 3 (ml) was passed through the syringe pump at an empty tube linear velocity of 1.6 (cm / min). Then, the number of blood cells on the outlet side of the filter was measured with an automatic blood cell counter (Sysmex K-4500). The passing rate of blood cells was determined by dividing the number of cells after passing through the filter by the number of blood cells before passing through the filter. As a result, the red blood cell passage rate was 98%, the white blood cell passage rate was 72%, and the platelet passage rate was 59%.

次に同方向から生理食塩液２０ｍｌを同流速にて流すことにより、赤血球や白血球、血小板の洗浄除去を行い、牛胎児血清１０％を含む細胞培養液（α−ＭＥＭ培地）３０ｍｌを骨髄液を流した方向と逆方向から勢いよく流すことにより、目的とする細胞画分を回収した。回収した細胞懸濁液１０ｍｌをポリスチレン製シャーレ（直径１０ｃｍ、ＩＷＡＫＩ社）に移し、３７℃、ＣＯ2インキュベーター内で培養を行った。２〜３日ごとに培地交換し、培養開始９日後にクリスタルバイオレットでコロニーを染色して出現したコロニー数を測定した。また、フィルター通過後の骨髄液に関しても、通過液体積の１／３量を塩化アンモニウムにて溶血させ、その後生理食塩液にて１回洗浄後、回収液と同条件にて培養を行い、出現したコロニー数を測定した。その結果、回収液側の出現コロニー数は、２２０個／シャーレ（骨髄液１ｍｌ相当）であった。また通過液側のコロニー数は、２３個／シャーレ（骨髄液１ｍｌ相当）であった。 Next, 20 ml of physiological saline is flowed from the same direction at the same flow rate to wash and remove red blood cells, white blood cells, and platelets, and 30 ml of cell culture solution (α-MEM medium) containing fetal bovine serum 10% is added to bone marrow fluid. The desired cell fraction was collected by vigorously flowing from the direction opposite to the flow direction. 10 ml of the recovered cell suspension was transferred to a petri dish made of polystyrene (diameter 10 cm, IWAKI), and cultured in a CO 2 incubator at 37 ° C. The medium was changed every 2 to 3 days, and the number of colonies that appeared after staining colonies with crystal violet was measured 9 days after the start of the culture. In addition, the bone marrow fluid that has passed through the filter is also hemolyzed with ammonium chloride for 1/3 of the volume of the fluid passing through, then washed once with physiological saline, and cultured under the same conditions as the recovered solution. Colonies were counted. As a result, the number of appearance colonies on the collected liquid side was 220 / petri dish (equivalent to 1 ml of bone marrow fluid). The number of colonies on the passing liquid side was 23 / petri dish (equivalent to 1 ml of bone marrow fluid).

（実施例２）
ハウジングの内径を１．８ｃｍ、不織布の上下を外径１．８ｃｍ、内径１．４ｃｍのリングにて挟み込んだ（充填時不織布厚み０．６ｃｍ）フィルターを使用した以外は、実施例１と同様の方法で、細胞分離フィルターを作製し、細胞通過率、コロニー数を求めた。なお充填時不織布厚み（ｃｍ）／濾過面積（ｃｍ2）＝０．３９（ｃｍ-1）であった。その結果、血球の通過率は、それぞれ赤血球が９７％、白血球が６７％、血小板が５５％であった。また細胞回収液から求めたコロニー数は、２３９個／シャーレ（骨髄液１ｍｌ相当）であった。また通過液側のコロニー数は、８個／シャーレ（骨髄液１ｍｌ相当）であった。 (Example 2)
Except for using a filter in which the inner diameter of the housing is 1.8 cm and the upper and lower sides of the nonwoven fabric are sandwiched between rings with an outer diameter of 1.8 cm and an inner diameter of 1.4 cm (nonwoven fabric thickness when filled: 0.6 cm), the same as in Example 1 By the method, a cell separation filter was prepared, and the cell passage rate and the number of colonies were determined. In addition, it was non-woven fabric thickness at the time of filling (cm) / filtration area (cm 2 ) = 0.39 (cm −1 ). As a result, the blood cell passage rates were 97% for red blood cells, 67% for white blood cells, and 55% for platelets. The number of colonies obtained from the cell recovery solution was 239 / petri dish (equivalent to 1 ml of bone marrow fluid). The number of colonies on the passing liquid side was 8 / petri dish (equivalent to 1 ml of bone marrow fluid).

＜比較例１＞
ハウジングの内径を２．６ｃｍ、不織布の上下を外径２．６ｃｍ、内径２．２ｃｍのリングにて挟み込んだ（充填時不織布厚み０．１５ｃｍ）フィルターを使用した以外以外は、実施例１と同様の方法で、細胞分離フィルターを作製し、細胞通過率、コロニー数を求めた。なお充填時不織布厚み（ｃｍ）／濾過面積（ｃｍ2）＝０．０４（ｃｍ-1）であった。その結果、血球の通過率は、それぞれ赤血球が９９％、白血球が８５％、血小板が６８％であった。また細胞回収液から求めたコロニー数は、１５４ 個／シャーレ（骨髄液１ｍｌ相当）であった。また通過液側のコロニー数は、４８個／シャーレ（骨髄液１ｍｌ相当）であった。 <Comparative Example 1>
Except for using a filter in which the inner diameter of the housing is 2.6 cm, and the upper and lower sides of the nonwoven fabric are sandwiched by rings having an outer diameter of 2.6 cm and an inner diameter of 2.2 cm (nonwoven fabric thickness when filled: 0.15 cm). In this manner, a cell separation filter was prepared, and the cell passage rate and the number of colonies were determined. In addition, it was non-woven fabric thickness at the time of filling (cm) / filtration area (cm 2 ) = 0.04 (cm −1 ). As a result, the passing rate of blood cells was 99% for red blood cells, 85% for white blood cells, and 68% for platelets. The number of colonies determined from the cell recovery solution was 154 / petri dish (equivalent to 1 ml of bone marrow fluid). The number of colonies on the flow-through side was 48 / petri dish (equivalent to 1 ml of bone marrow fluid).

＜比較例２＞
ハウジングの内径を１．４ｃｍ、不織布の上下を外径１．４ｃｍ、内径１．０ｃｍのリングにて挟み込んだ（充填時不織布厚み０．９ｃｍ）フィルターを使用した以外は、実施例１と同様の方法で、細胞分離フィルターを作製し、細胞通過率、コロニー数を求めた。なお充填時不織布厚み（ｃｍ）／濾過面積（ｃｍ2）＝１．１５（ｃｍ-1）であった。その結果、血球の通過率は、それぞれ赤血球が９６％、白血球が６１％、血小板が５１％であった。また細胞回収液から求めたコロニー数は、１７５個／シャーレ（骨髄液１ｍｌ相当）であった。また通過液側のコロニー数は、２個／シャーレ（骨髄液１ｍｌ相当）であった。 <Comparative Example 2>
Except for using a filter in which the inner diameter of the housing is 1.4 cm and the upper and lower sides of the nonwoven fabric are sandwiched by rings having an outer diameter of 1.4 cm and an inner diameter of 1.0 cm (nonwoven fabric thickness when filled: 0.9 cm), the same as in Example 1 By the method, a cell separation filter was prepared, and the cell passage rate and the number of colonies were determined. In addition, it was non-woven fabric thickness at the time of filling (cm) / filtration area (cm 2 ) = 1.15 (cm −1 ). As a result, the passing rate of blood cells was 96% for red blood cells, 61% for white blood cells, and 51% for platelets. The number of colonies determined from the cell recovery solution was 175 / petri dish (equivalent to 1 ml of bone marrow fluid). The number of colonies on the flow-through side was 2 / petri dish (equivalent to 1 ml of bone marrow fluid).

（実施例３）
ハウジングの内径を２．６ｃｍ、不織布の上下を外径２．６ｃｍ、内径２．２ｃｍのリングにて挟み込んだ（充填時不織布厚み０．６ｃｍ）フィルターを使用し、骨髄液を２５ｍｌ処理した以外は、実施例１と同様の方法で細胞分離フィルターを作製し、細胞通過率、コロニー数を求めた。なお充填時不織布厚み（ｃｍ）／濾過面積（ｃｍ2）＝０．１６（ｃｍ-1）であった。その結果、血球の通過率は、それぞれ赤血球が９８％、白血球が７８％、血小板が６６％であった。また細胞回収液から求めたコロニー数は、２２８個／シャーレ（骨髄液１ｍｌ相当）であった。また通過液側のコロニー数は、１２個／シャーレ（骨髄液１ｍｌ相当）であった。 (Example 3)
Except that the inner diameter of the housing is 2.6 cm, the upper and lower sides of the nonwoven fabric are sandwiched between rings with an outer diameter of 2.6 cm and an inner diameter of 2.2 cm (nonwoven fabric thickness 0.6 cm when filled), and 25 ml of bone marrow fluid is treated. A cell separation filter was prepared in the same manner as in Example 1, and the cell passage rate and the number of colonies were determined. In addition, it was non-woven fabric thickness at the time of filling (cm) / filtration area (cm 2 ) = 0.16 (cm −1 ). As a result, the blood cell passage rates were 98% for red blood cells, 78% for white blood cells, and 66% for platelets. The number of colonies determined from the cell recovery solution was 228 / petri dish (equivalent to 1 ml of bone marrow fluid). The number of colonies on the passing liquid side was 12 / petri dish (equivalent to 1 ml of bone marrow fluid).

（実施例４）
ハウジングの内径を２．２ｃｍ、不織布の上下を外径２．２ｃｍ、内径１．８ｃｍのリングにて挟み込んだ（充填時不織布厚み０．９ｃｍ）フィルターを使用し、骨髄液を２５ｍｌ処理した以外は実施例１と同様の方法で、細胞分離フィルターを作製し、細胞通過率、コロニー数を求めた。なお充填時不織布厚み（ｃｍ）／濾過面積（ｃｍ2）＝０．３５（ｃｍ-1）であった。その結果、血球の通過率は、それぞれ赤血球が９７％、白血球が７３％、血小板が６１％であった。また細胞回収液から求めたコロニー数は、２２６個／シャーレ（骨髄液１ｍｌ相当）であった。また通過液側のコロニー数は、４個／シャーレ（骨髄液１ｍｌ相当）であった。 Example 4
Except that the inner diameter of the housing is 2.2 cm, the upper and lower sides of the nonwoven fabric are sandwiched between rings with an outer diameter of 2.2 cm and an inner diameter of 1.8 cm (nonwoven fabric thickness 0.9 cm when filled), and 25 ml of bone marrow fluid is treated. A cell separation filter was prepared in the same manner as in Example 1, and the cell passage rate and the number of colonies were determined. In addition, it was non-woven fabric thickness (cm) at the time of filling / filtration area (cm 2 ) = 0.35 (cm −1 ). As a result, the blood cell passage rates were 97% for red blood cells, 73% for white blood cells, and 61% for platelets. The number of colonies determined from the cell recovery solution was 226 / petri dish (equivalent to 1 ml of bone marrow fluid). The number of colonies on the flow-through side was 4 / petri dish (equivalent to 1 ml of bone marrow fluid).

＜比較例３＞
ハウジングの内径を１．４ｃｍ、不織布の上下を外径１．４ｃｍ、内径１．０ｃｍのリングにて挟み込んだ（充填時不織布厚み０．９ｃｍ）フィルターを使用し、骨髄液を２５ｍｌ処理した以外は、実施例１と同様の方法で、細胞分離フィルターを作製し、細胞通過率、コロニー数を求めた。なお充填時不織布厚み（ｃｍ）／濾過面積（ｃｍ2）＝１．１５（ｃｍ-1）であった。その結果、血球の通過率は、それぞれ赤血球が９７％、白血球が６８％、血小板が５７％であった。また細胞回収液から求めたコロニー数は、１６１個／シャーレ（骨髄液１ｍｌ相当）であった。また通過液側のコロニー数は、７個／シャーレ（骨髄液１ｍｌ相当）であった。 <Comparative Example 3>
Except that the inner diameter of the housing was 1.4 cm, the upper and lower sides of the nonwoven fabric were sandwiched by rings with an outer diameter of 1.4 cm and an inner diameter of 1.0 cm (nonwoven fabric thickness 0.9 cm when filled), and 25 ml of bone marrow fluid was treated. A cell separation filter was prepared in the same manner as in Example 1, and the cell passage rate and the number of colonies were determined. In addition, it was non-woven fabric thickness at the time of filling (cm) / filtration area (cm 2 ) = 1.15 (cm −1 ). As a result, the blood cell passage rates were 97% for red blood cells, 68% for white blood cells, and 57% for platelets. The number of colonies determined from the cell recovery solution was 161 / petri dish (equivalent to 1 ml of bone marrow fluid). The number of colonies on the passing liquid side was 7 / petri dish (equivalent to 1 ml of bone marrow fluid).

なお、実施例１〜４、及び比較例１〜３の白血球、赤血球、及び血小板の通過率を表１に示した。   In addition, Table 1 shows the white blood cell, red blood cell, and platelet passage rates of Examples 1 to 4 and Comparative Examples 1 to 3.

Figure 2011010582
Figure 2011010582

また、実施例１〜４、及び比較例１〜３の細胞回収液、及びフィルター通過液の幹細胞コロニー出現数を表２に示した。   In addition, Table 2 shows the number of stem cell colonies appearing in the cell collection liquids of Examples 1 to 4 and Comparative Examples 1 to 3 and the filter passage liquid.

Figure 2011010582
Figure 2011010582

以上の結果から、本発明の細胞分離フィルター及び、該方法を使用することにより、夾雑細胞を除去しつつ、成体幹細胞をロスなく、安定的に回収できることがわかる。しかも本細胞分離フィルターは、体液中から成体幹細胞を分離、回収、増幅まで閉鎖系で処理することができ、コンタミネーションの防止という観点からも有用なフィルターであることがわかる。   From the above results, it can be seen that by using the cell separation filter of the present invention and the method, adult stem cells can be stably recovered without loss while removing contaminating cells. In addition, the present cell separation filter can be treated in a closed system from the separation, recovery and amplification of adult stem cells from body fluids, and it can be seen that it is a useful filter from the viewpoint of preventing contamination.

Claims (7)

骨髄、末梢血、臍帯血、及び月経血などの体液から付着性の成体幹細胞を捕捉・回収が可能な細胞分離材を体液流入部と体液流出部を有する容器に充填した時に、細胞分離材の厚み（ｃｍ）を濾過面積（ｃｍ2）で割った値（ｃｍ-1）が０．０５以上１．０以下であることを特徴とする細胞分離フィルター。 When a cell separation material capable of capturing and collecting adherent adult stem cells from body fluids such as bone marrow, peripheral blood, umbilical cord blood, and menstrual blood is filled into a container having a body fluid inflow portion and a body fluid outflow portion, A cell separation filter having a thickness (cm) divided by a filtration area (cm 2 ) (cm −1 ) of 0.05 or more and 1.0 or less. 体液流入部あるいは、体液流入部以外の体液流入側に、細胞分離材内に留まっている体液を洗浄するための洗浄液流入部、及び細胞分離材に捕捉された細胞を回収するための容器を備え、また体液流出部あるいは、体液流出部以外の体液流出部側に、細胞分離材に捕捉された細胞を回収するための細胞回収液流入部を備えた請求項１に記載の細胞分離フィルター。 Provided with a body fluid inflow portion or a body fluid inflow side other than the body fluid inflow portion, a washing fluid inflow portion for washing the body fluid remaining in the cell separation material, and a container for collecting the cells captured by the cell separation material The cell separation filter according to claim 1, further comprising a cell recovery fluid inflow portion for recovering cells trapped by the cell separation material on the body fluid outflow portion or a body fluid outflow portion other than the body fluid outflow portion. 細胞分離材が不織布であることを特徴とする請求項１または２に記載の細胞分離フィルター。 The cell separation filter according to claim 1 or 2, wherein the cell separation material is a nonwoven fabric. 細胞分離材が赤血球、白血球を実質的に通過させることを特徴とする請求項１〜３のいずれかに記載の細胞分離フィルター。 The cell separation filter according to any one of claims 1 to 3, wherein the cell separation material substantially allows red blood cells and white blood cells to pass therethrough. 細胞分離材に捕捉された細胞を回収するための容器が、細胞培養可能な容器であることを特徴とする請求項１〜４のいずれかに記載の細胞分離フィルター。 The cell separation filter according to any one of claims 1 to 4, wherein the container for collecting the cells trapped by the cell separation material is a container capable of cell culture. 骨髄、末梢血、臍帯血、及び月経血などの体液を、体液流入部と体液流出部を有する容器に充填した時に、細胞分離材の厚み（ｃｍ）を濾過面積（ｃｍ2）で割った値（ｃｍ-1）が、０．０５以上１．０以下であることを特徴とする細胞分離フィルターに通液し、除去対象細胞含有液を排出させた後に、該フィルター内に留まった除去対象細胞をさらに排出するための洗浄液を通液し、さらに該フィルターに捕捉された細胞を回収するため体液流出部あるいは、体液流出部以外の体液流出部側に備えた細胞回収液流入部より細胞回収液を流入し、体液流入部あるいは体液流入部以外の体液流入側に設置した細胞回収容器に該フィルターに捕捉された細胞を回収する成体幹細胞分離方法。 A value obtained by dividing the thickness (cm) of the cell separation material by the filtration area (cm 2 ) when a body fluid such as bone marrow, peripheral blood, umbilical cord blood, and menstrual blood is filled in a container having a body fluid inflow portion and a body fluid outflow portion. (Cm −1 ) is passed through a cell separation filter characterized by being not less than 0.05 and not more than 1.0, and after removing the removal target cell-containing solution, the removal target cells remaining in the filter The cell recovery solution from the body fluid outflow part or the cell recovery liquid inflow part provided on the side of the body fluid outflow part other than the body fluid outflow part to collect the cells captured by the filter. An adult stem cell separation method in which cells trapped in the filter are collected in a body fluid inflow portion or a cell collection container installed on a body fluid inflow side other than the body fluid inflow portion. 細胞分離材が赤血球、白血球を実質的に通過させることを特徴とする請求項６に記載の成体幹細胞分離方法。 The adult stem cell separation method according to claim 6, wherein the cell separation material substantially allows red blood cells and white blood cells to pass therethrough.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016047444A1 (en) * 2014-09-24 2016-03-31 株式会社カネカ Cell separation material and cell separation method
EP3070164A1 (en) 2015-03-20 2016-09-21 Medical Biobank Swiss Institute SA/AG (MBSI) System and method for providing isolated concentrated multipotent stromal cells for administration

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016047444A1 (en) * 2014-09-24 2016-03-31 株式会社カネカ Cell separation material and cell separation method
JPWO2016047444A1 (en) * 2014-09-24 2017-07-06 株式会社カネカ Cell separation material and cell separation method
EP3070164A1 (en) 2015-03-20 2016-09-21 Medical Biobank Swiss Institute SA/AG (MBSI) System and method for providing isolated concentrated multipotent stromal cells for administration

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