JP2003009851A - Method for hematopoietic stem cell collection - Google Patents
Method for hematopoietic stem cell collectionInfo
- Publication number
- JP2003009851A JP2003009851A JP2001194428A JP2001194428A JP2003009851A JP 2003009851 A JP2003009851 A JP 2003009851A JP 2001194428 A JP2001194428 A JP 2001194428A JP 2001194428 A JP2001194428 A JP 2001194428A JP 2003009851 A JP2003009851 A JP 2003009851A
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- JP
- Japan
- Prior art keywords
- hematopoietic stem
- blood
- stem cells
- peripheral
- collecting
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は、末梢循環血液中の
造血幹細胞および／または造血前駆細胞（以下、末梢循
環血液中の造血幹細胞および／または造血前駆細胞を、
造血幹細胞、と略す）を増加させる方法、それに用いる
器具および増加させた造血幹細胞を採取する方法に関す
る。得られた細胞は、造血幹細胞を用いて行う各種疾患
の治療及び免疫学や細胞生物学などの基礎科学分野で用
いることができる。TECHNICAL FIELD The present invention relates to hematopoietic stem cells and / or hematopoietic progenitor cells in peripheral circulating blood (hereinafter, hematopoietic stem cells and / or hematopoietic progenitor cells in peripheral circulating blood,
The present invention relates to a method for increasing hematopoietic stem cells, a device used for the method, and a method for collecting the increased hematopoietic stem cells. The obtained cells can be used in the fields of basic science such as treatment of various diseases using hematopoietic stem cells and immunology and cell biology.
【０００２】[0002]
【従来の技術】造血幹細胞移植は、白血病や再生不良性
貧血の根治療法として確立している。造血幹細胞移植の
代表的なものは、骨髄にそのソースをもとめる骨髄移植
であるが、近年、いわゆる末梢血幹細胞移植も注目を集
めている。これは、通常は、ほとんど造血幹細胞が存在
していない末梢循環血液中にも、ある種の薬剤を投与す
ることによりその存在比率が増加するという現象に着目
して、ドナー（自家移植の場合は移植患者自身）にある
種の薬剤を投与し、末梢循環血液中の造血幹細胞を増加
させてから遠心型血球採取装置により造血幹細胞を含む
細胞集団を採取し、移植に用いるものである。Background Art Hematopoietic stem cell transplantation has been established as a root treatment method for leukemia and aplastic anemia. A typical example of hematopoietic stem cell transplantation is bone marrow transplantation in which the source is found in bone marrow, but in recent years, so-called peripheral blood stem cell transplantation has also attracted attention. This is because the donor (in the case of autologous transplantation, in the case of autologous transplantation) focuses on the phenomenon that the presence ratio of certain drugs increases even in the peripheral circulating blood in which almost no hematopoietic stem cells normally exist. This is to administer a certain drug to the transplant patient itself) to increase the amount of hematopoietic stem cells in the peripheral circulating blood, and then collect a cell population containing hematopoietic stem cells by a centrifugal blood cell sampling device and use it for transplantation.
【０００３】この方法は、骨髄移植の際に必要とされる
ドナーの全身麻酔を必要としないため、安全性が高いこ
とが利点とされている。しかしながら、ここで用いられ
る薬剤は抗癌剤や顆粒球コロニー刺激因子（Ｇ−ＣＳ
Ｆ）であるために、新たな問題点が存在する。すなわ
ち、抗癌剤は臓器障害など、各種の重篤な副作用がある
だけでなく、それ自身発癌性を有することが危惧されて
おり、一方、Ｇ−ＣＳＦも骨痛などの副作用があるだけ
でなく、白血病細胞の増殖を刺激することが知られてお
り、また長期の安全性については未確認である。さら
に、これらの薬剤はいずれも高価なことも問題であり、
昨今の医療費削減に逆行するものである。すなわち、従
来の、薬剤を用いて末梢循環血液中の造血幹細胞を増加
させる方法は安全上およびコストの面で問題を有してお
り、より安全で安価な造血幹細胞の増加方法が待望され
ている。This method is advantageous in that it is highly safe because it does not require general anesthesia of the donor, which is required for bone marrow transplantation. However, the drugs used here are anticancer drugs and granulocyte colony stimulating factor (G-CS).
Due to F), there are new problems. That is, it is feared that the anti-cancer agent not only has various serious side effects such as organ damage but also has carcinogenicity by itself, while G-CSF not only has side effects such as bone pain, It is known to stimulate the proliferation of leukemic cells, and its long-term safety has not been confirmed. Furthermore, the problem is that all of these drugs are expensive,
It goes against the recent reduction in medical costs. That is, the conventional method of increasing hematopoietic stem cells in peripheral blood using a drug has a problem in safety and cost, and a safer and cheaper method of increasing hematopoietic stem cells is desired. .
【０００４】特公平８−２５８８７号公報には、ドナー
の血液が生物学的に活性化され、その生体が一定の機能
的な状態になるように、ドナーに１０分から２４時間に
わたり作用を施した後、一定量の生物学的に活性な血液
をこのドナーから採取し、これをインキュベーション
し、血清を分離して保存する方法が開示されている。た
とえば、睡眠作用血清を作るために、ドナーを２０〜２
４時間眠らせずにおいてから血清を採取し、この血清を
不眠症治療に用いることが記載されている。しかしなが
ら、同技術は血清中の生理活性物質の増加を目的とした
もので、循環末梢血液中の特定細胞の増加に関する記述
はない。In Japanese Examined Patent Publication No. 8-25887, the donor is acted on for 10 minutes to 24 hours so that the donor's blood is biologically activated and the living body is in a certain functional state. Later, a method is disclosed in which an amount of biologically active blood is taken from this donor, which is incubated and the serum is separated and stored. For example, to produce sleep-acting serum, 20 to 2 donors are used.
It is described that serum is collected after 4 hours of sleep without sleep and this serum is used for treating insomnia. However, this technique aims to increase the amount of physiologically active substances in serum, and there is no description regarding the increase of specific cells in circulating peripheral blood.
【０００５】[0005]
【発明が解決しようとする課題】本発明の目的は、安全
性が高く、かつ、安価な循環末梢血液中の造血幹細胞を
増加させる方法、それに用いられる器具および造血幹細
胞を含む細胞集団を採取する方法を提供することにあ
る。DISCLOSURE OF THE INVENTION An object of the present invention is to provide a highly safe and inexpensive method for increasing hematopoietic stem cells in circulating peripheral blood, a device used therefor, and a cell population containing hematopoietic stem cells. To provide a method.
【０００６】[0006]
【課題を解決するための手段】本発明者は、かかる課題
を解決すべく鋭意検討を進めた結果、造血幹細胞分野と
は全く異分野で用いられている人工腎臓用血液透析器の
利用に本発明の糸口を見出した。すなわち、血液透析治
療においては、透析開始後に一過性の白血球減少が見ら
れ、その後回復することはしばしば発生する事象である
ことが知られているが、本発明者はその回復期に、単に
成熟した白血球が回復してくるだけなく、造血幹細胞が
骨髄から動員され増加しており、それが成熟白血球の増
加につながっており、したがって、血液透析を施行中の
患者の循環末梢血中には造血幹細胞がきわめて高頻度に
存在しているに違いないという大胆な仮説を立てた。そ
して、従来、血液透析分野では全く無縁であったＣＤ３
４抗原陽性細胞の存在を、これも血液透析分野の研究で
は稀にしか活用されていないフローサイトメトリー法で
解析することにより、そして透析分野では全く実施され
ていない造血コロニーアッセイを実施することにより上
記の仮説を実証し、本発明を完成するに至った。Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventor has found that the present invention is applied to the use of a hemodialyzer for an artificial kidney, which is used in a completely different field from the field of hematopoietic stem cells. The clue of the invention was found. That is, in hemodialysis treatment, it is known that a transient leukopenia is observed after the initiation of dialysis, and that recovery thereafter is an event that often occurs. In addition to the recovery of mature leukocytes, hematopoietic stem cells are mobilized and increased from the bone marrow, which leads to an increase in mature leukocytes, and therefore in the circulating peripheral blood of patients undergoing hemodialysis. He made a bold hypothesis that hematopoietic stem cells must be present at a very high frequency. And, in the past, CD3 was completely unrelated in the field of hemodialysis.
By analyzing the presence of 4-antigen-positive cells by flow cytometry, which is also rarely used in studies in the field of hemodialysis, and by performing a hematopoietic colony assay, which has never been performed in the field of dialysis. The above hypothesis was verified, and the present invention was completed.
【０００７】すなわち、本発明は、末梢循環血液を、末
梢循環血液中の白血球濃度を減少させる材料に接触させ
る工程を含むことを特徴とする末梢循環血液中の造血幹
細胞を増加させる方法、末梢循環血液中の白血球濃度を
減少させる材料が、液体導入口と液体導出口を具備する
容器に充填されていることを特徴とする末梢血中の造血
幹細胞増加器具および末梢循環血液を末梢循環血液中の
白血球濃度を減少させる材料に接触させて末梢循環血液
中の造血幹細胞の頻度を上昇させ、次いで、造血幹細胞
を含む細胞集団を採取することを特徴とする造血幹細胞
の採取方法である。[0007] That is, the present invention comprises a method of increasing the number of hematopoietic stem cells in the peripheral circulating blood, which comprises the step of contacting the peripheral circulating blood with a material that reduces the leukocyte concentration in the peripheral circulating blood. A material for reducing leukocyte concentration in blood is filled with a container having a liquid inlet and a liquid outlet, and a device for increasing hematopoietic stem cells in peripheral blood and peripheral circulating blood A method for collecting hematopoietic stem cells, which comprises contacting with a material that reduces leukocyte concentration to increase the frequency of hematopoietic stem cells in peripheral circulating blood, and then collecting a cell population containing hematopoietic stem cells.
【０００８】[0008]
【発明実施の形態】以下、本発明を詳細に説明する。本
発明でいう末梢循環血液とは、ヒトを含む動物の末梢血
管を流れている血液のことである。本発明でいう末梢循
環血液中の白血球濃度を減少させる材料とは、白血球と
接触した際に白血球が吸着する材料のことで、直接的に
白血球濃度を減少させる材料や、白血球が材料と接触
し、白血球の機能および／または構造が変化して生体内
の何らかの組織に捕捉されるにより、いわば間接的に白
血球濃度を減少させる材料があるが、そのどちらでもよ
い。具体的には、通常用いられている医用材料であれば
いかなる材料も使用できるが、成形性、滅菌性や細胞毒
性が低いという点で好ましいものを例示すると、ポリス
ルホン、ポリエチレン、ポリプロピレン、ポリスチレ
ン、アクリル樹脂、ナイロン、ポリエステル、ポリカー
ボネート、ポリアクリルアミド、ポリウレタンなどの合
成高分子、アガロース、セルロース、酢酸セルロース、
キチン、キトサン、アルギン酸塩などの天然高分子、ヒ
ドロキシアパタイト、ガラス、アルミナ、チタニアなど
の無機材料、ステンレス、チタン、アルミニムなどの金
属などが挙げられる。BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below. The peripheral blood circulation in the present invention refers to blood flowing in peripheral blood vessels of animals including humans. The material that reduces the leukocyte concentration in the peripheral circulation blood in the present invention is a material that leukocytes are adsorbed when it comes into contact with leukocytes, and a material that directly reduces the leukocyte concentration or leukocytes contact with the material. There are materials that indirectly reduce the leukocyte concentration by changing the function and / or structure of leukocytes and capturing them in some tissue in the living body, but either of them may be used. Specifically, any material can be used as long as it is a commonly used medical material, but examples of preferable materials in terms of moldability, sterilization and low cytotoxicity include polysulfone, polyethylene, polypropylene, polystyrene, and acrylic. Resin, nylon, polyester, polycarbonate, polyacrylamide, synthetic polymers such as polyurethane, agarose, cellulose, cellulose acetate,
Examples thereof include natural polymers such as chitin, chitosan and alginate, inorganic materials such as hydroxyapatite, glass, alumina and titania, and metals such as stainless steel, titanium and aluminum.
【０００９】一般的に、合成高分子は直接的に白血球濃
度を減少させる材料、セルロースや酢酸セルロースは間
接的に白血球濃度を減少させる材料といわれている。後
者は血液中の補体がこれらの材料に存在する水酸基（Ｏ
Ｈ基）と接触することにより活性化され、それが白血球
の活性化など表面構造変化につながるといわれている。
これら材料の形状としては、中空糸、繊維塊、不織布、
織布、スポンジ状多孔体（膜を含む）、ビーズなどが挙
げられる。Generally, it is said that synthetic polymers directly reduce leukocyte concentration, and cellulose and cellulose acetate indirectly reduce leukocyte concentration. The latter is a hydroxyl group (O) whose complement in blood is present in these materials.
It is said to be activated by contact with H group), which leads to surface structure changes such as activation of leukocytes.
The shapes of these materials include hollow fibers, fiber lumps, non-woven fabrics,
Examples thereof include woven cloth, sponge-like porous material (including membrane), beads and the like.
【００１０】本発明における造血幹細胞増加器具は、前
記白血球濃度を減少させる材料を液体導入口と液体導出
口を具備する容器に充填したものである。液体導入口と
液体導出口を有する容器の材質としては、成形性や滅菌
性に優れ、細胞毒性が低いという点で好ましいものを例
示すると、ポリエチレン、ポリプロピレン、ポリスチレ
ン、アクリル樹脂、ナイロン、ポリエステル、ポリカー
ボネート、ポリアクリルアミド、ポリウレタン、塩化ビ
ニルなどの合成高分子、ヒドロキシアパタイト、ガラ
ス、アルミナ、チタニアなどの無機材料、ステンレス、
チタン、アルミニウムなどの金属が挙げられるが、これ
らに限定されるものではない。容器の構造としては、形
状は直方体、立方体、円柱形、楕円柱形などが挙げられ
るが、いずれの形状でもよい。また滅菌済みの医療用具
として市販されている血液透析器や白血球吸着器を用い
ることができるが、これらに限定されるものではなく、
前述の材料を用いて適宜製造してもよい。The hematopoietic stem cell increasing device according to the present invention is one in which the material for reducing the white blood cell concentration is filled in a container having a liquid inlet and a liquid outlet. As the material of the container having the liquid inlet and the liquid outlet, preferred examples in terms of excellent moldability and sterilization property and low cytotoxicity are polyethylene, polypropylene, polystyrene, acrylic resin, nylon, polyester, polycarbonate. , Synthetic polymers such as polyacrylamide, polyurethane, vinyl chloride, inorganic materials such as hydroxyapatite, glass, alumina, titania, stainless steel,
Examples include metals such as titanium and aluminum, but are not limited to these. Examples of the structure of the container include a rectangular parallelepiped, a cube, a cylinder, and an elliptic cylinder, but any shape may be used. Further, hemodialyzers and leukocyte adsorbers that are commercially available as sterilized medical devices can be used, but are not limited to these.
You may produce suitably using the above-mentioned material.
【００１１】これらの方法により末梢循環血液中の造血
幹細胞の頻度を上昇させた後、そのまま採血（全血）す
ることによって造血幹細胞の頻度の高い血液を得ること
ができるが、ここには末梢血幹細胞移植においては不要
の赤血球が多量に混入している。もちろん、この血液を
比重遠心法や赤血球沈降法といったｉｎ ｖｉｔｒｏ法
により赤血球除去してもよいが、遠心型血球分離装置を
用いる方法や白血球吸着器を用いる方法を用いると新た
に赤血球を除去する工程を付加する必要が無いのでより
好ましい。By increasing the frequency of hematopoietic stem cells in peripheral blood by these methods and then collecting blood (whole blood) as it is, blood having a high frequency of hematopoietic stem cells can be obtained. In stem cell transplantation, a large amount of unnecessary red blood cells are mixed. Of course, this blood may be removed by an in vitro method such as a gravity centrifuge method or an erythrocyte sedimentation method, but a step of newly removing the erythrocyte by a method using a centrifugal blood cell separator or a method using a leukocyte adsorber Is more preferable because it is not necessary to add.
【００１２】遠心型血球分離装置を用いる方法は、末梢
循環血液中の造血幹細胞の頻度を上昇させた後に、末梢
循環血液を遠心型血球分離装置に通液して造血幹細胞含
有白血球画分を採取する。白血球吸着器を用いる方法
は、末梢循環血液を白血球吸着器に通液して造血幹細胞
含有白血球画分を吸着させた後、吸着器を体外循環装置
から取り外し、血液入口側または血液出口側から液体を
導入して捕捉されている吸着した造血幹細胞含有白血球
画分を回収する。導入する液体としては、細胞に悪影響
を及ぼさない液体であればいかなる液体も使用可能であ
るが、いくつかを例示すると、生理食塩水、ダルベッコ
リン酸塩緩衝液（Ｄ−ＰＢＳ）やハンクス液（ＨＢＳ
Ｓ）などの緩衝液、ＲＰＭＩ１６４０などの培地が挙げ
られる。これらの液体に、細胞保護、栄養補給、抗凝固
性付与、凍結保存時の凍害防止、粘度向上（回収率向上
に有効な場合がある）などの目的で、必要に応じてアル
ブミン、グロブリン、グルコース、サッカロース、トレ
ハロース、クエン酸化合物、ＥＤＴＡ、ジメチルスルホ
キシド、デキストラン、ポリビニルピロリドン、グリセ
リン、キチン誘導体、ヒドロキシエチルデンプン、ゼラ
チンなどを添加してもよい。この液体には、液体単体の
みならず、空気、アルゴン、窒素など細胞に悪影響を及
ぼさない気体を混合したものも含まれる。In the method using the centrifugal blood cell separator, the frequency of hematopoietic stem cells in the peripheral blood circulation is increased, and then the peripheral blood circulation is passed through the centrifugal blood cell separator to collect the white blood cell fraction containing hematopoietic stem cells. To do. The method using the leukocyte adsorber is to pass the peripheral circulating blood through the leukocyte adsorber to adsorb the hematopoietic stem cell-containing leukocyte fraction, and then remove the adsorber from the extracorporeal circulation device and liquid from the blood inlet side or blood outlet side. And the adsorbed hematopoietic stem cell-containing leukocyte fraction that has been captured is collected. As the liquid to be introduced, any liquid can be used as long as it does not adversely affect cells, but some examples include physiological saline, Dulbecco's phosphate buffer (D-PBS) and Hanks' solution ( HBS
Buffer solutions such as S) and culture media such as RPMI1640. If necessary, albumin, globulin, glucose may be added to these liquids for the purposes of cell protection, nutritional supplementation, anticoagulant protection, frost damage prevention during cryopreservation, and viscosity improvement (may be effective in improving recovery rate). , Saccharose, trehalose, citric acid compounds, EDTA, dimethyl sulfoxide, dextran, polyvinylpyrrolidone, glycerin, chitin derivatives, hydroxyethyl starch, gelatin and the like may be added. This liquid includes not only a liquid alone but also a mixture of air, argon, nitrogen, and other gases that do not adversely affect cells.
【００１３】本発明で得られた造血幹細胞は、そのま
ま、あるいは必要に応じてさらなる分離精製、培養、活
性化、分化誘導、増幅、遺伝子導入、凍結保存などの各
種処理が施された後、各種疾患の治療及び免疫学や細胞
生物学などの基礎科学分野の研究に用いられる。以下
に、実施例により本発明を具体的に説明するが、本発明
はこれにより限定されるものではない。The hematopoietic stem cells obtained in the present invention may be subjected to various treatments as they are or, if necessary, after further separation and purification, culture, activation, differentiation induction, amplification, gene transfer, cryopreservation, etc. It is used for treatment of diseases and research in basic science fields such as immunology and cell biology. Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited thereto.
【００１４】[0014]
【実施例１】市販のセルロース製透析器（膜厚約１５μ
ｍ、内径約１８０μｍのセルロース中空糸を膜面積１．
５ｍ2になるように、ポリカーボネート製円筒容器に充
填したもの。この円筒容器は長手方向の両端のキャップ
に血液導入口と血液導出口を有し中空糸内部に血液が導
入、導出されるようになっている。また容器側面には中
空糸外側に透析液を通液する透析液導入口と透析液導出
口を具備している）による透析（抗凝固剤としてヘパリ
ンを使用、血液流量２００ｍｌ／分、透析液流量５００
ｍｌ／分）を行っている慢性腎不全患者から、透析開始
直後と開始１時間後に透析回路のサンプリングポートを
通じて１０ｍｌの血液を採血した。この血液をフローサ
イトメトリー法によるＣＤ３４陽性細胞解析とメチルセ
ルロース培地を用いたコロニーアッセイ法に供した。Example 1 A commercially available cellulose dialyzer (film thickness: about 15 μm)
m, an inner diameter of about 180 μm, and a cellulose hollow fiber having a membrane area of 1.
What was filled in a cylindrical cylinder made of polycarbonate so as to be 5 m 2 . This cylindrical container has a blood inlet and a blood outlet in caps at both ends in the longitudinal direction so that blood can be introduced and discharged into the hollow fiber. In addition, the side of the container is equipped with a dialysate inlet and a dialysate outlet for passing dialysate outside the hollow fiber) (using heparin as an anticoagulant, blood flow rate 200 ml / min, dialysate flow rate) 500
Immediately after the start of dialysis and one hour after the start of dialysis, 10 ml of blood was collected from a chronic renal failure patient who is performing (ml / min). The blood was subjected to a CD34-positive cell analysis by a flow cytometry method and a colony assay method using a methylcellulose medium.
【００１５】フローサイトメトリー法によるＣＤ３４陽
性細胞測定はＦＩＴＣ標識ＣＤ３４抗体を用い、ＳＳＣ
−ＦＩＴＣに展開する方法（宮崎、他：日常診療と血
液、５巻２号、２１〜２３ページ、１９９５年）により
測定した。コロニーアッセイ法はあらかじめサイトカイ
ン類が添加されているメチルセルロース培地であるＭｅ
ｔｈｏＣｕｌｔ ＧＦ４４３４Ｖ（商標）（ベリタス社
製）を用い、同社の操作マニュアルにしたがって培養を
実施（５×１０4有核細胞を播種）し、２週間後に顕微
鏡下でＣＦＵ−ＧＭ（顆粒球・マクロファージ系前駆細
胞）、ＢＦＵ−Ｅ（赤血球系前駆細胞）およびＣＦＵ−
Ｍｉｘ（前記２種よりも未分化な前駆細胞）を計数し
た。その結果を表１に示す。The FITC-labeled CD34 antibody was used to measure CD34-positive cells by flow cytometry using SSC.
-It was measured by the method developed in FITC (Miyazaki et al .: Daily medical care and blood, Vol. 5, No. 2, 21 to 23, 1995). The colony assay method is Me which is a methylcellulose medium to which cytokines have been added in advance.
Using thoCult GF4434V (trademark) (manufactured by Veritas), culture was carried out (seeding 5 × 10 4 nucleated cells) according to the operation manual of the company, and 2 weeks later, CFU-GM (granulocyte / macrophage system was observed under a microscope. Progenitor cells), BFU-E (erythroid progenitor cells) and CFU-
Mix (progenitor cells that are less differentiated than the above two types) was counted. The results are shown in Table 1.
【００１６】この結果から、本発明の方法により得られ
た細胞集団には、造血幹細胞の表面マーカーであるＣＤ
３４陽性細胞が通常状態の末梢血よりも高率に存在し、
またその細胞はｉｎ ｖｉｔｒｏ造血活性を有している
ことが明らかになった。From these results, the cell population obtained by the method of the present invention was found to include CD, which is a surface marker of hematopoietic stem cells.
34 positive cells were present at a higher rate than in normal peripheral blood,
In addition, it was revealed that the cells have in vitro hematopoietic activity.
【００１７】[0017]
【実施例２】末梢循環血液よりも造血幹細胞の頻度が上
昇した血液を白血球吸着器で採取する例のモデル実験を
行った。
１．モデル血液
通常の末梢循環血液よりはるかに多量の造血幹細胞を含
有するといわれている臍帯血を造血幹細胞の頻度が上昇
した血液のモデルとして用いた。
２．白血球吸着器
容器外寸（縦×横×厚み）４１×４１×１８ｍｍで液体
流出口と液体流入口を対角線上に持つポリカーボネート
製容器に、平均繊維径２．３μｍのポリエステル不織布
１８枚と平均繊維径１２μｍのポリエステル不織布１６
枚を充填し白血球吸着器を製作した。
３．細胞分離回収操作
２．の白血球吸着器の入口側と出口側に、先端にルアー
ロックアダプター（メス）を具備した塩化ビニル製チュ
ーブを接続した。入口側のルアーロックアダプター（メ
ス）に臍帯血の入った１００ｍｌのディスポーザブルシ
リンジを接続し、シリンジのプランジャーを押すことで
血液を白血球吸着器に通液した。なお、出口側には１０
０ｍｌのプラスチックビーカーを置き、出口側から導出
される血液を受けとめた。全ての血液を通液し終わった
後、出口側のルアーロックアダプター（メス）に生理食
塩水５０ｍｌが入った５０ｍｌのディスポーザブルシリ
ンジを接続し、プランジャーを強く押すことで、白血球
吸着器の中に生理食塩水を導入し、吸着されている細胞
を入口側に接続されているディスポーザブルシリンジに
回収した。
４．分析及び結果
実施例１と同様のフローサイトメトリー法で分析を行っ
た。その結果を表２に示す。Example 2 A model experiment of an example in which blood having a higher frequency of hematopoietic stem cells than in peripheral blood was collected by a leukocyte adsorber was conducted. 1. Model blood Umbilical cord blood, which is said to contain much more hematopoietic stem cells than normal peripheral blood, was used as a model of blood with an increased frequency of hematopoietic stem cells. 2. Leukocyte adsorber container Outer dimensions (length x width x thickness) 41 x 41 x 18 mm polycarbonate container with liquid outlet and liquid inlet on the diagonal, 18 polyester nonwoven fabrics with average fiber diameter 2.3 μm and average fiber Polyester nonwoven fabric 16 with a diameter of 12 μm
A white blood cell adsorber was manufactured by filling the sheets. 3. Cell separation and collection operation 2. A tube made of vinyl chloride having a luer lock adapter (female) at its tip was connected to the inlet side and the outlet side of the leukocyte adsorber. A 100 ml disposable syringe containing cord blood was connected to the luer lock adapter (female) on the inlet side, and the blood was passed through the leukocyte adsorber by pushing the plunger of the syringe. In addition, 10 at the exit side
A 0 ml plastic beaker was placed to receive the blood drawn from the outlet side. After passing all the blood, connect a 50 ml disposable syringe containing 50 ml of physiological saline to the luer lock adapter (female) on the outlet side, and press the plunger strongly to put it into the leukocyte adsorber. Physiological saline was introduced, and the adsorbed cells were collected in a disposable syringe connected to the inlet side. 4. Analysis and Results Analysis was performed by the same flow cytometry method as in Example 1. The results are shown in Table 2.
【００１８】この結果から、モデル血液として用いた臍
帯血は通常の末梢循環血よりもはるかに高頻度で造血幹
細胞（ＣＤ３４陽性細胞）を含むため、本実施例のモデ
ルとして適切であること、また本モデル血液を通液した
白血球吸着器から造血幹細胞を高率に回収できることが
明らかになった。From these results, the umbilical cord blood used as a model blood contains hematopoietic stem cells (CD34-positive cells) at a much higher frequency than normal peripheral circulating blood, and is therefore suitable as a model for this example. It was clarified that hematopoietic stem cells can be collected at a high rate from the leukocyte adsorber in which this model blood was passed.
【００１９】[0019]
【表１】 [Table 1]
【００２０】[0020]
【表２】 [Table 2]
【００２１】[0021]
【発明の効果】本発明によれば、すでに医療用具として
使用されている市販の血液透析器や白血球吸着器を用い
ることにより、末梢循環血液中の造血幹細胞の比率を増
加させることができるので、安全性が高く、簡便、か
つ、安価に循環末梢血液中の造血幹細胞を増加させ、造
血幹細胞を採取することができる。したがって、本発明
は、末梢血幹細胞移植の発展に寄与するところがきわめ
て大きい。According to the present invention, the ratio of hematopoietic stem cells in the peripheral circulating blood can be increased by using a commercially available hemodialyzer or leukocyte adsorber which has already been used as a medical device. It is highly safe, convenient, and inexpensive to increase hematopoietic stem cells in circulating peripheral blood and collect hematopoietic stem cells. Therefore, the present invention greatly contributes to the development of peripheral blood stem cell transplantation.
Claims (8)
球濃度を減少させる材料に接触させる工程を含むことを
特徴とする末梢循環血液中の造血幹細胞を増加させる方
法。1. A method for increasing the number of hematopoietic stem cells in peripheral circulating blood, which comprises the step of contacting the peripheral circulating blood with a material that reduces the leukocyte concentration in the peripheral circulating blood.
る材料に接触させる工程が、末梢循環血液を血液透析器
または白血球吸着器に通液することからなる請求項１記
載の造血幹細胞を増加させる方法。2. The number of hematopoietic stem cells according to claim 1, wherein the step of contacting with the material for reducing the leukocyte concentration in the peripheral circulating blood comprises passing the peripheral circulating blood through a hemodialyzer or a leukocyte adsorber. Method.
る材料が、液体導入口と液体導出口を具備する容器に充
填されていることを特徴とする末梢血中の造血幹細胞増
加器具。3. A device for increasing hematopoietic stem cells in peripheral blood, characterized in that a container having a liquid inlet and a liquid outlet is filled with a material that reduces the white blood cell concentration in peripheral blood circulation.
る材料が、中空糸、繊維塊、不織布、織布、多孔体およ
びビーズから選ばれた少なくとも一種である請求項３記
載の末梢血中の造血幹細胞増加器具。4. The peripheral blood in the peripheral blood according to claim 3, wherein the material for reducing the leukocyte concentration in peripheral blood is at least one selected from hollow fibers, fiber lumps, non-woven fabrics, woven fabrics, porous bodies and beads. Device for increasing hematopoietic stem cells.
る材料が補体活性化能を有するものである請求項３記載
の末梢血中の造血幹細胞増加器具。5. The device for increasing hematopoietic stem cells in peripheral blood according to claim 3, wherein the material for reducing the leukocyte concentration in peripheral blood has complement activating ability.
濃度を減少させる材料に接触させて末梢循環血液中の造
血幹細胞の頻度を上昇させ、次いで、造血幹細胞を含む
細胞集団を採取することを特徴とする造血幹細胞の採取
方法。6. A method of contacting peripheral circulating blood with a material that reduces leukocyte concentration in peripheral circulating blood to increase the frequency of hematopoietic stem cells in peripheral circulating blood, and then collecting a cell population containing hematopoietic stem cells. A characteristic method for collecting hematopoietic stem cells.
法が、造血幹細胞の頻度が上昇した末梢循環血液を遠心
型血球分離装置により採取することからなる請求項６記
載の造血幹細胞の採取方法。7. The method for collecting hematopoietic stem cells according to claim 6, wherein the method for collecting a cell population containing hematopoietic stem cells comprises collecting peripheral circulating blood having an increased frequency of hematopoietic stem cells by a centrifugal blood cell separator.
法が、造血幹細胞の頻度が上昇した末梢循環血液を白血
球吸着器に通液し、吸着された白血球を吸着器から回収
することからなる請求項６記載の造血幹細胞の採取方
法。8. A method for collecting a cell population containing hematopoietic stem cells, which comprises passing peripheral circulating blood having an increased frequency of hematopoietic stem cells through a leukocyte adsorber and collecting the adsorbed leukocytes from the adsorber. Item 7. A method for collecting hematopoietic stem cells according to Item 6.
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|---|---|---|---|
| JP2001194428A JP4833443B2 (en) | 2001-06-27 | 2001-06-27 | How to collect hematopoietic stem cells |
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| JP4833443B2 JP4833443B2 (en) | 2011-12-07 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2010515503A (en) * | 2007-01-13 | 2010-05-13 | メムブラーナ ゲゼルシャフト ミット ベシュレンクテル ハフツング | Device for removing white blood cells from blood |
| JP2011515084A (en) * | 2008-03-18 | 2011-05-19 | セールス・エンジニアリング・アクチェンゲゼルシャフト | Kit for collecting blood for producing stem cells, preferably peripheral blood |
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|---|---|---|---|---|
| WO1994028919A1 (en) * | 1993-06-08 | 1994-12-22 | Ajinomoto Co., Inc. | Hematopoietic cell proliferation accelerator |
| JP2000505284A (en) * | 1996-02-05 | 2000-05-09 | セル ジェネシス，インコーポレイテッド | Transduced human hematopoietic stem cells |
| JP2000507203A (en) * | 1995-12-20 | 2000-06-13 | ロシュ ダイアグノスティクス ゲゼルシャフト ミット ベシュレンクテル ハフツング | Combination of G-CSF with chemotherapeutic agent for stem cell mobilization |
-
2001
- 2001-06-27 JP JP2001194428A patent/JP4833443B2/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994028919A1 (en) * | 1993-06-08 | 1994-12-22 | Ajinomoto Co., Inc. | Hematopoietic cell proliferation accelerator |
| JP2000507203A (en) * | 1995-12-20 | 2000-06-13 | ロシュ ダイアグノスティクス ゲゼルシャフト ミット ベシュレンクテル ハフツング | Combination of G-CSF with chemotherapeutic agent for stem cell mobilization |
| JP2000505284A (en) * | 1996-02-05 | 2000-05-09 | セル ジェネシス，インコーポレイテッド | Transduced human hematopoietic stem cells |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2010515503A (en) * | 2007-01-13 | 2010-05-13 | メムブラーナ ゲゼルシャフト ミット ベシュレンクテル ハフツング | Device for removing white blood cells from blood |
| JP2011515084A (en) * | 2008-03-18 | 2011-05-19 | セールス・エンジニアリング・アクチェンゲゼルシャフト | Kit for collecting blood for producing stem cells, preferably peripheral blood |
| US8500712B2 (en) | 2008-03-18 | 2013-08-06 | Thankstem Srl | Kit for collecting blood, preferably peripheral blood, for the production of stem cells |
Also Published As
| Publication number | Publication date |
|---|---|
| JP4833443B2 (en) | 2011-12-07 |
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