JP2013252126A - Dextran-containing mammalian cell suspension for prevention of pulmonary embolism formation - Google Patents
Dextran-containing mammalian cell suspension for prevention of pulmonary embolism formation Download PDFInfo
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- JP2013252126A JP2013252126A JP2012230496A JP2012230496A JP2013252126A JP 2013252126 A JP2013252126 A JP 2013252126A JP 2012230496 A JP2012230496 A JP 2012230496A JP 2012230496 A JP2012230496 A JP 2012230496A JP 2013252126 A JP2013252126 A JP 2013252126A
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- 229940095064 tartrate Drugs 0.000 description 1
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- 239000002562 thickening agent Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000000472 traumatic effect Effects 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 1
- 229940070710 valerate Drugs 0.000 description 1
- NQPDZGIKBAWPEJ-UHFFFAOYSA-N valeric acid Chemical compound CCCCC(O)=O NQPDZGIKBAWPEJ-UHFFFAOYSA-N 0.000 description 1
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Abstract
Description
本発明は、哺乳動物細胞及びデキストラン若しくはその誘導体又はそれらの塩を含む、哺乳動物細胞を血管経由で投与する際の肺塞栓形成を予防するための哺乳動物細胞懸濁液や、デキストラン若しくはその誘導体又はそれらの塩を有効成分として含む、哺乳動物細胞を血管経由で投与する際の肺塞栓形成の予防剤等に関する。 The present invention relates to a mammalian cell suspension for preventing pulmonary embolism when a mammalian cell is administered via a blood vessel, a dextran or a derivative thereof, comprising the mammalian cell and dextran or a derivative thereof or a salt thereof. Alternatively, the present invention relates to a prophylactic agent for pulmonary embolism when a mammalian cell is administered via a blood vessel, containing a salt thereof as an active ingredient.
近年、幹細胞研究の急速な発展によって再生医療への気運は高まっており、その知識や理解は、研究者のみならず、一般にも広く普及してきている。幹細胞を用いた再生医療は、幹細胞が有する自己複製能と多分化能や、幹細胞が分泌する因子を利用して、様々な疾患で損傷を受けた細胞や組織の機能を回復させることを目的とした医療である。白血病や再生不良性貧血などの血液難病の患者に骨髄移植すると、造血系幹細胞が患者体内に生着し、ほぼ一生にわたって造血能の維持が可能となる。また、最近では多くの研究者が造血幹細胞以外の幹細胞を用いた臨床応用を目指し、中枢神経、末梢神経、骨髄、小腸などにおける幹細胞を同定し、外傷性疾患や組織変性疾患に対する組織幹細胞の移植治療が実践され始めている（非特許文献１〜３）。 In recent years, the rapid development of stem cell research has increased the momentum toward regenerative medicine, and its knowledge and understanding have become widespread not only by researchers but also generally. Regenerative medicine using stem cells aims to restore the functions of cells and tissues damaged by various diseases by utilizing the self-replicating ability and pluripotency of stem cells and factors secreted by stem cells. Medical care. When bone marrow transplantation is performed in patients with intractable blood diseases such as leukemia and aplastic anemia, hematopoietic stem cells are engrafted in the patient's body, and hematopoietic ability can be maintained almost throughout the life. Recently, many researchers have identified stem cells in central nerves, peripheral nerves, bone marrow, small intestine, etc., aiming for clinical application using stem cells other than hematopoietic stem cells, and transplanting tissue stem cells for traumatic diseases and tissue degenerative diseases Treatment has begun to be practiced (Non-Patent Documents 1 to 3).
哺乳動物幹細胞が目的の損傷部位（患部）に効率良く移植されるためには、幹細胞の移植方法（移植ルート）の最適化を図ることが、重要であると考えられている。現在までのところ、幹細胞の移植方法として、主に４種類の方法、すなわち、定位的[直接]移植法、髄腔内[脳脊髄内]移植法、経静脈的移植法、及び経動脈的移植法が知られている。このうち、定位的移植法は、幹細胞を直接患部へ移入する方法である。定位的移植法を用いると、侵襲がやや大きいが、直接患部に投与するため、生着するドナー幹細胞が多く、このため投与する幹細胞数を少なくすることができる。また、髄腔内移植法は、脳室穿刺により幹細胞を髄腔内に移入する方法である。髄腔内移植法は、主に脳梗塞、脳挫傷、脊髄損傷などの脳疾患を治療するために検討されている方法であるが、脳室穿刺が新たな脳損傷を招くリスクが潜在するなど、臨床応用する上でまだ多くの課題が残っている。 In order for mammalian stem cells to be transplanted efficiently to a target injury site (affected site), it is considered important to optimize the stem cell transplantation method (transplant route). To date, there are mainly four types of stem cell transplantation methods: stereotactic [direct] transplantation, intrathecal [intracerebral spinal cord] transplantation, transvenous transplantation, and transarterial transplantation. The law is known. Among these, stereotactic transplantation is a method in which stem cells are directly transferred to the affected area. When stereotactic transplantation is used, the invasion is somewhat large, but since it is administered directly to the affected area, there are many engrafted donor stem cells, and therefore the number of stem cells to be administered can be reduced. The intrathecal transplantation method is a method of transferring stem cells into the medullary canal by ventricular puncture. Intrathecal transplantation is a method that has been studied mainly to treat brain diseases such as cerebral infarction, cerebral contusion, and spinal cord injury, but there is a potential risk that ventricular puncture may cause new brain damage. Many problems still remain in clinical application.
経静脈的移植法及び経動脈的移植法は、幹細胞等の細胞をそれぞれ静脈及び動脈内へ移入する方法（血管経由投与法）である。血管経由投与法を用いると、生着するドナー肝細胞の数が定位的移植法を用いた場合と比べ少ないものの、低侵襲であるとともに、全身に幹細胞等の細胞や幹細胞等の細胞が分泌する因子を循環させることができる。現在のところ、臨床で行われている血管経由投与法として、脳梗塞疾患に対して間葉系幹細胞（Mesenchymal Stem Cells；ＭＳＣ）を経静脈的に移植する方法や脳梗塞疾患に対して単核球を経静脈的に移植する方法や、Ｉ型糖尿病患者に膵島細胞を経静脈的に移植する方法などが知られている。 The transvenous transplantation method and the transarterial transplantation method are methods in which cells such as stem cells are transferred into veins and arteries, respectively (administration via blood vessels). When using the vascular administration method, the number of donor hepatocytes to engraft is less than when using stereotaxic transplantation, but it is minimally invasive and secretes cells such as stem cells and stem cells throughout the body. Factors can be circulated. At present, as a method for administration via blood vessels, which is practiced in clinical practice, a method of transplanting mesenchymal stem cells (MSC) intravenously for cerebral infarction disease or mononuclear for cerebral infarction disease A method of transplanting spheres intravenously, a method of transplanting islet cells intravenously in type I diabetic patients, and the like are known.
血管経由投与法を用いて幹細胞等の細胞を移植した場合のリスクとして、経動脈又は経静脈投与した細胞が、肺を通過する際に肺動脈の毛細血管で詰まった状態（肺塞栓）になり、その結果、肺機能や心機能の低下（肺塞栓症）を招き、場合によっては死に至る危険性があることが指摘されている。肺塞栓形成や肺塞栓症を未然に防ぐために、臨床の現場では幹細胞投与中の末梢血酸素分圧をパルスオキシメータでモニターしながら、幹細胞の移植が行われている。 As a risk when transplanting cells such as stem cells using the vascular administration method, cells that have been transarterally or intravenously administered are clogged with capillaries of the pulmonary artery (pulmonary embolism) when passing through the lung, As a result, it has been pointed out that there is a risk of pulmonary function or cardiac function (pulmonary embolism) and possibly death. In order to prevent pulmonary embolism and pulmonary embolism, stem cells are transplanted while monitoring peripheral oxygen partial pressure during stem cell administration with a pulse oximeter in clinical practice.
他方、デキストラン（dextran）は、グルコースからなる多糖類の１種であり、増粘剤
、保湿剤等として、医薬品、化粧品の分野で汎用されている。しかしながら、デキストランを含有する溶液に幹細胞等の細胞を懸濁し、細胞懸濁液を血管経由で投与した場合、細胞による肺塞栓形成のリスクが低減することや肺塞栓症が予防されるかどうかは不明であった。
On the other hand, dextran is a kind of polysaccharide consisting of glucose, and is widely used as a thickener, moisturizer, etc. in the fields of pharmaceuticals and cosmetics. However, if cells such as stem cells are suspended in a solution containing dextran and the cell suspension is administered via a blood vessel, the risk of pulmonary embolism due to cells is reduced and pulmonary embolism is prevented. It was unknown.
本発明の課題は、哺乳動物幹細胞等の哺乳動物細胞を血管経由で投与する際の肺塞栓形成を予防することができる哺乳動物細胞懸濁液や、哺乳動物細胞を血管経由で投与する際の肺塞栓形成の予防剤を提供することにある。 An object of the present invention is to provide a mammalian cell suspension that can prevent pulmonary embolism when a mammalian cell such as a mammalian stem cell is administered via a blood vessel, or a mammalian cell suspension that is administered via a blood vessel. The object is to provide a preventive agent for pulmonary embolism.
本発明者らは、デキストランを含有する溶液に哺乳動物幹細胞を懸濁すると、哺乳動物幹細胞の凝集が抑制されることや生存率の低下が抑制されることを見いだしている（特願２０１０−２５１２７３）。幹細胞移植による肺塞栓形成の原因として、幹細胞が凝集した細胞塊が肺動脈の毛細血管を塞いでいることが考えられるが、幹細胞、例えば、間葉系幹細胞（Mesenchymal Stem Cells；ＭＳＣ）の大きさは１０〜５０μｍであるのに対して、肺動脈の毛細血管は内径が１０μｍであり、ＭＳＣの方が肺動脈の毛細血管よりも大きいので、単純に細胞の凝集を阻害しただけでは、幹細胞による肺塞栓形成を阻害ないことも十分考えられた。本発明者らは、デキストランを含有する乳酸リンゲル溶液に脂肪組織由来間葉系幹細胞（adipose tissue-derived mesenchymal stem cell；ＡＴ−ＭＳＣ）を懸濁し、ＡＴ−ＭＳＣ懸濁デキストラン含有乳酸リンゲル溶液をブタに静脈投与して検証したところ、デキストランを含有する細胞懸濁液を用いてＡＴ−ＭＳＣを投与すると、投与中又は投与後における肺動脈圧の上昇を抑制できることや、血中酸素分圧の低下を抑制できることを見いだし、本発明を完成するに至った。 The present inventors have found that suspension of mammalian stem cells in a solution containing dextran suppresses aggregation of mammalian stem cells and suppresses a decrease in survival rate (Japanese Patent Application No. 2010-251273). ). As a cause of the formation of pulmonary embolism by stem cell transplantation, it is conceivable that the aggregate of stem cells is blocking capillaries of pulmonary arteries. The pulmonary artery capillaries have an inner diameter of 10 μm, whereas the MSCs are larger than the pulmonary artery capillaries, whereas pulmonary embolization by stem cells can be achieved simply by inhibiting cell aggregation. It was thought enough not to interfere with. The present inventors suspend adipose tissue-derived mesenchymal stem cells (AT-MSCs) in a lactate Ringer solution containing dextran, and suspend the AT-MSC suspension dextran-containing lactate Ringer solution in pigs. When AT-MSC was administered using a cell suspension containing dextran, it was possible to suppress an increase in pulmonary arterial pressure during administration or after administration, and to reduce blood oxygen partial pressure. It has been found that it can be suppressed, and the present invention has been completed.
すなわち本発明は、（１）哺乳動物細胞及びデキストラン若しくはその誘導体又はそれらの塩を含む、哺乳動物細胞を血管経由で投与する際の肺塞栓形成を予防するための哺乳動物細胞懸濁液や、（２）哺乳動物細胞が哺乳動物幹細胞である、上記（１）記載の哺乳動物細胞懸濁液や、（３）哺乳動物幹細胞が哺乳動物間葉系幹細胞又は哺乳動物多能性幹細胞である、上記（２）記載の哺乳動物細胞懸濁液や、（４）哺乳動物細胞が単一細胞の状態にある哺乳動物細胞を含む、上記（１）〜（３）のいずれか記載の哺乳動物細胞懸濁液や、（５）デキストラン若しくはその誘導体又はそれらの塩の濃度が０．１〜２０％の範囲内である、上記（１）〜（４）のいずれか記載の哺乳動物細胞懸濁液に関する。 That is, the present invention includes (1) a mammalian cell suspension for preventing pulmonary embolism when a mammalian cell is administered via a blood vessel, including the mammalian cell and dextran or a derivative thereof or a salt thereof, (2) the mammalian cell is a mammalian stem cell, the mammalian cell suspension according to (1) above, or (3) the mammalian stem cell is a mammalian mesenchymal stem cell or a mammalian pluripotent stem cell. The mammalian cell suspension according to any one of (1) to (3) above, comprising the mammalian cell suspension according to (2), or (4) a mammalian cell in which the mammalian cell is in a single cell state. The mammalian cell suspension according to any one of (1) to (4) above, wherein the concentration of the suspension or (5) dextran or a derivative thereof or a salt thereof is in the range of 0.1 to 20%. About.
また本発明は、（６）デキストラン若しくはその誘導体又はそれらの塩を有効成分として含む、哺乳動物細胞を血管経由で投与する際の肺塞栓形成の予防剤や、（７）哺乳動物細胞が哺乳動物幹細胞である、上記（６）記載の予防剤や、（８）哺乳動物幹細胞が哺乳動物間葉系幹細胞又は哺乳動物多能性幹細胞である、上記（７）記載の予防剤に関する。 The present invention also relates to (6) an agent for preventing pulmonary embolism when a mammalian cell is administered via a blood vessel, comprising dextran or a derivative thereof or a salt thereof as an active ingredient, and (7) the mammalian cell is a mammal. The prophylactic agent according to (6), which is a stem cell, and (8) the prophylactic agent according to (7), wherein the mammalian stem cell is a mammalian mesenchymal stem cell or a mammalian pluripotent stem cell.
さらに本発明は、（９）哺乳動物細胞を血管経由で投与する際の肺塞栓形成の予防剤を調製するためのデキストラン若しくはその誘導体又はそれらの塩の使用や、（１０）哺乳動物細胞が哺乳動物幹細胞である、上記（９）記載の使用に関する。 Furthermore, the present invention relates to (9) use of dextran or a derivative thereof or a salt thereof for preparing a preventive agent for pulmonary embolism when a mammalian cell is administered via a blood vessel, or (10) a mammalian cell is sucked. The use according to (9) above, which is an animal stem cell.
本発明によると、哺乳動物幹細胞等の哺乳動物細胞を血管経由で投与する際の肺塞栓形成のリスクを低減することができ、肺塞栓症を発症するリスクを低下することができる。 According to the present invention, the risk of pulmonary embolism formation when mammalian cells such as mammalian stem cells are administered via blood vessels can be reduced, and the risk of developing pulmonary embolism can be reduced.
本発明の哺乳動物幹細胞等の哺乳動物細胞を血管経由で投与する際の肺塞栓形成を予防するための哺乳動物細胞懸濁液としては、哺乳動物幹細胞等の哺乳動物細胞及びデキストラン若しくはその誘導体又はそれらの塩（以下、デキストラン類という）を含む懸濁液であれば特に制限されず、また、本発明の哺乳動物幹細胞等の哺乳動物細胞を血管経由で投与する際の肺塞栓形成の予防剤としては、デキストラン類を有効成分として含む組成物であれば特に制限されず、上記哺乳動物としては、マウス、ラット、ハムスター、モルモット等のげっ歯類、ウサギ等のウサギ目、ブタ、ウシ、ヤギ、ウマ、ヒツジ等の有蹄目、イヌ、ネコ等のネコ目、ヒト、サル、アカゲザル、カニクイザル、マーモセット、オランウータン、チンパンジーなどの霊長類等を例示することが、中でも、マウス、ブタ、ヒトを好適に例示することができ、また上記哺乳動物細胞としては、再生医療等に血管経由で投与される哺乳動物幹細胞の他に、Ｉ型糖尿病患者に経静脈投与される哺乳動物の膵島細胞、がん患者に経静脈投与される哺乳動物の樹状細胞やナチュラルキラー細胞、アルファ・ベータ（αβ）Ｔ細胞、ガンマ・デルタ（γδ）Ｔ細胞、細胞障害性Ｔ細胞（cytotoxic T lymphocyte；ＣＴＬ）等を例示することができる。 Mammalian cell suspensions for preventing pulmonary embolism when administering mammalian cells such as mammalian stem cells of the present invention via blood vessels include mammalian cells such as mammalian stem cells and dextran or derivatives thereof, or The suspension is not particularly limited as long as it is a suspension containing a salt thereof (hereinafter referred to as dextran), and a preventive agent for pulmonary embolism when a mammalian cell such as the mammalian stem cell of the present invention is administered via a blood vessel. Is not particularly limited as long as it is a composition containing dextran as an active ingredient. Examples of mammals include rodents such as mice, rats, hamsters, and guinea pigs, rabbits such as rabbits, pigs, cows, and goats. Ghosts such as horses, sheep, cats such as dogs and cats, spirits such as humans, monkeys, rhesus monkeys, cynomolgus monkeys, marmosets, orangutans, chimpanzees Among them, mice, pigs, and humans can be preferably exemplified, and examples of the mammalian cells include I stem cells other than mammalian stem cells administered via revascularization to regenerative medicine and the like. Mammalian islet cells intravenously administered to patients with type 2 diabetes, mammalian dendritic cells and natural killer cells intravenously administered to cancer patients, alpha-beta (αβ) T cells, gamma-delta (γδ) Examples thereof include T cells and cytotoxic T cells (CTL).
また上記「幹細胞」とは、自己複製能及び分化・増殖能を有する未熟な細胞を意味する。幹細胞には、分化能力に応じて、多能性幹細胞（pluripotent stem ce11）、複能性幹細胞（multipotent stem ce11）、単能性幹細胞（unipotent stem ce11）等の亜集団が含まれる。多能性幹細胞とは、それ自体では個体になることができないが、生体を構成する全ての組織や細胞へ分化し得る能力を有する細胞を意味する。複能性幹細胞とは、全ての種類ではないが、複数種の組織や細胞へ分化し得る能力を有する細胞を意味する。単能性幹細胞とは、特定の組織や細胞へ分化し得る能力を有する細胞を意味する。 The “stem cell” means an immature cell having self-renewal ability and differentiation / proliferation ability. The stem cells include subpopulations such as pluripotent stem cells (pluripotent stem ce11), multipotent stem cells (multipotent stem ce11), and unipotent stem cells (unipotent stem ce11) depending on the differentiation ability. A pluripotent stem cell means a cell that cannot be an individual by itself, but has the ability to differentiate into all tissues and cells constituting a living body. A multipotent stem cell means a cell having the ability to differentiate into multiple types of tissues and cells, although not all types. A unipotent stem cell means a cell having the ability to differentiate into a specific tissue or cell.
多能性幹細胞としては、胚性幹細胞（ＥＳ細胞）、ＥＧ細胞、ｉＰＳ細胞等を挙げることができる。ＥＳ細胞は、内部細胞塊をフィーダー細胞上又はＬＩＦを含む培地中で培養することにより製造することができる。ＥＳ細胞の製造方法は、例えば、ＷＯ９６／２２３６２、ＷＯ０２／１０１０５７、ＵＳ５，８４３，７８０、ＵＳ６，２００，８０６、ＵＳ６，２８０，７１８等に記載されている。ＥＧ細胞は、始原生殖細胞をｍＳＣＦ、ＬＩＦ及びｂＦＧＦを含む培地中で培養することにより製造することができる（Ce11,70: 841-847, 1992）。ｉＰＳ細胞は、体細胞（例えば線維芽細胞、皮膚細胞等）にＯｃｔ３／４、Ｓｏｘ２及びＫｌｆ４（必要に応じて更にｃ−Ｍｙｃ又はｎ−Ｍｙｃ）等のリプログラミング因子を導入することにより製造することができる（Ce11,126:p.663-676,2006;Nature,448:p.313-317,2007;Nat Biotechno1,26;p.101-106,2008;Cel1 131:p.861‐872,2007;Science,318:p.1917-1920,2007;Ce11 Stem Cells 1:p.55-70,2007;Nat Biotechnol,25:p.1177-1181,2007;Nature,448:p.318-324,2007;Cell Stem Cells 2:p.10-12,2008;Nature,451:p.141-146,2008;Science,318:p.1917-1920,2007）。体細胞の核を核移植することによって作製された初期胚を培養することによって樹立した幹細胞も、多能性幹細胞としてまた好ましい（Nature,385,810（1997）；Science,280,1256（1998）；Nature Biotechnology,17,456（1999）；Nature,394,369（1998）；Nature Genetics,22,127（1999）；Proc.Nat1.Acad.Sci.USA,96,14984（1999））、Rideout IIIら（Nature Genetics,24,109（2000））。 Examples of pluripotent stem cells include embryonic stem cells (ES cells), EG cells, iPS cells, and the like. ES cells can be produced by culturing the inner cell mass on feeder cells or in a medium containing LIF. Methods for producing ES cells are described in, for example, WO96 / 22362, WO02 / 101057, US5,843,780, US6,200,806, US6,280,718 and the like. EG cells can be produced by culturing primordial germ cells in a medium containing mSCF, LIF and bFGF (Ce11, 70: 841-847, 1992). iPS cells are produced by introducing reprogramming factors such as Oct3 / 4, Sox2 and Klf4 (c-Myc or n-Myc as necessary) into somatic cells (eg, fibroblasts, skin cells, etc.). (Ce11,126: p.663-676,2006; Nature, 448: p.313-317,2007; Nat Biotechno1,26; p.101-106,2008; Cel1 131: p.861-872 2007; Science, 318: p.1917-1920,2007; Ce11 Stem Cells 1: p.55-70,2007; Nat Biotechnol, 25: p.1177-1181,2007; Nature, 448: p.318-324, 2007; Cell Stem Cells 2: p.10-12,2008; Nature, 451: p.141-146,2008; Science, 318: p.1917-1920,2007). Stem cells established by culturing early embryos produced by nuclear transfer of somatic cell nuclei are also preferred as pluripotent stem cells (Nature, 385, 810 (1997); Science, 280, 1256 (1998); Nature Biotechnology, 17, 456 (1999); Nature, 394, 369 (1998); Nature Genetics, 22, 127 (1999); Proc. Nat1. Acad. Sci. USA, 96, 14984 (1999)), Rideout III et al. (Nature Genetics, 24, 109 (2000) )).
複能性幹細胞としては、脂肪細胞、骨細胞、軟骨細胞、脂肪細胞等の細胞に分化可能な間葉系幹細胞、白血球、赤血球、血小板等の血球系細胞に分化可能な造血系幹細胞、ニューロン、アストロサイト、オリゴデンドロサイト等の細胞に分化可能な神経系幹細胞、骨髄幹細胞、生殖幹細胞等の体性幹細胞等を挙げることができる。複能性幹細胞は、好ましくは間葉系幹細胞である。間葉系幹細胞とは、骨芽細胞、軟骨芽細胞及び脂肪芽細胞の全て又はいくつかへの分化が可能な幹細胞を意味する。機能性幹細胞は、自体公知の方法により、生体から単離することができる。例えば、間葉系幹細胞は、哺乳動物の骨髄、脂肪組織、末梢血、臍帯血等から公知の一般的な方法で採取することができる。例えば、骨髄穿刺後の造血幹細胞等の培養、継代によりヒト間葉系幹細胞を単離することができる（Journal of Autoimmunity，30（2008）163-171）。複能性幹細胞は、上記多能性幹細胞を適切な誘導条件下で培養することによっても得ることができる。 Multipotent stem cells include mesenchymal stem cells that can differentiate into cells such as adipocytes, bone cells, chondrocytes, adipocytes, hematopoietic stem cells that can differentiate into blood cells such as leukocytes, erythrocytes, and platelets, neurons, Examples include neural stem cells that can differentiate into cells such as astrocytes and oligodendrocytes, somatic stem cells such as bone marrow stem cells and germ stem cells. The multipotent stem cell is preferably a mesenchymal stem cell. By mesenchymal stem cell is meant a stem cell that can differentiate into all or some of osteoblasts, chondroblasts and lipoblasts. Functional stem cells can be isolated from a living body by a method known per se. For example, mesenchymal stem cells can be collected by known general methods from mammalian bone marrow, adipose tissue, peripheral blood, umbilical cord blood and the like. For example, human mesenchymal stem cells can be isolated by culture and passage of hematopoietic stem cells after bone marrow puncture (Journal of Autoimmunity, 30 (2008) 163-171). Multipotent stem cells can also be obtained by culturing the pluripotent stem cells under appropriate induction conditions.
本発明の哺乳動物細胞懸濁液中に含まれる幹細胞等の細胞として、付着性の細胞を例示することができる。付着性の細胞は懸濁液中で凝集しやすいが、本発明の懸濁液にはデキストラン類が含まれるため、この凝集を効果的に抑制することができる。本明細書中、「付着性」細胞とは、足場に接着することで生存、増殖、物質の生産を行なうことができる足場依存性の細胞を意味する。付着性幹細胞としては、多能性幹細胞、間葉系幹細胞、神経系幹細胞、骨髄幹細胞、生殖幹細胞等を挙げることができる。付着性幹細胞は、好ましくは、間葉系幹細胞又は多能性幹細胞である。 Examples of cells such as stem cells contained in the mammalian cell suspension of the present invention include adherent cells. Adherent cells are likely to aggregate in the suspension, but since the suspension of the present invention contains dextrans, this aggregation can be effectively suppressed. In the present specification, the “adhesive” cell means an anchorage-dependent cell that can survive, proliferate, and produce a substance by adhering to the scaffold. Examples of adherent stem cells include pluripotent stem cells, mesenchymal stem cells, nervous system stem cells, bone marrow stem cells, and reproductive stem cells. The adherent stem cells are preferably mesenchymal stem cells or pluripotent stem cells.
哺乳動物細胞は生体内から分離されたものであっても、インビトロで継代培養されたものであってもよい。また、本発明の哺乳動物細胞懸濁液に含まれる哺乳動物細胞（集団）は、単離又は精製されていることが好ましい。本明細書中、「単離または精製」とは、目的とする成分以外の成分を除去する操作が施されていることを意味する。単離または精製された哺乳動物細胞の純度（全細胞数に対する、哺乳動物幹細胞数等の目的とする細胞の割合）は、通常３０％以上、好ましくは５０％以上、より好ましくは７０％以上、更に好ましくは９０％以上（例えば１００％）である。 Mammalian cells may be isolated from the living body or subcultured in vitro. Moreover, it is preferable that the mammalian cell (population) contained in the mammalian cell suspension of the present invention is isolated or purified. In the present specification, “isolation or purification” means that an operation for removing components other than the target component has been performed. The purity of the isolated or purified mammalian cells (the ratio of the target cells such as the number of mammalian stem cells to the total number of cells) is usually 30% or more, preferably 50% or more, more preferably 70% or more, More preferably, it is 90% or more (for example, 100%).
本発明の懸濁液に含まれる哺乳動物細胞（集団）は、単一細胞（シングルセル）の状態の哺乳動物細胞を含むことが好ましい。本明細書において、「単一細胞の状態」とは、他の細胞と寄り集まって塊を形成していないこと（即ち、凝集していない状態）を意味する。単一細胞の状態の哺乳動物細胞は、インビトロで培養した哺乳動物細胞をトリプシン／ＥＤＴＡ等で酵素処理することにより調製することができる。哺乳動物細胞中に含まれる単一細胞の状態の哺乳動物細胞の割合は、通常７０％以上、好ましくは９０％以上、より好ましくは９５％以上、更に好ましくは９９％以上（例えば１００％）である。単一細胞の状態の細胞の割合は、哺乳動物細胞をＰＢＳに分散し、これを顕微鏡下で観察し、無作為に選択された複数個（例、１０００個）の細胞について凝集の有無を調べることにより決定することができる。 The mammalian cells (population) contained in the suspension of the present invention preferably include mammalian cells in a single cell (single cell) state. In the present specification, the “single cell state” means that the cells do not gather together to form a lump (that is, they are not aggregated). Mammalian cells in a single cell state can be prepared by enzymatic treatment of mammalian cells cultured in vitro with trypsin / EDTA or the like. The ratio of mammalian cells in a single cell state contained in mammalian cells is usually 70% or more, preferably 90% or more, more preferably 95% or more, and further preferably 99% or more (for example, 100%). is there. The percentage of cells in a single cell state is determined by dispersing mammalian cells in PBS and observing them under a microscope, and examining the presence or absence of aggregation of a plurality of randomly selected cells (eg, 1000). Can be determined.
本発明の懸濁液において、哺乳動物細胞は好ましくは浮遊している。本明細書において、「浮遊」とは、細胞が、懸濁液を収容した容器の内壁に接触することなく、懸濁液中に保持されていることをいう。 In the suspension of the present invention, the mammalian cells are preferably floating. In this specification, “floating” means that cells are held in the suspension without contacting the inner wall of the container containing the suspension.
本発明において用いられるデキストラン類におけるデキストランとしては、Ｄ−グルコースからなる多糖（Ｃ６Ｈ１０Ｏ５）ｎであって、α１→６結合を主鎖とするものであれば特に制限されず、デキストランの重量平均分子量（Ｍｗ）としては、例えば、デキストラン４０（Ｍｗ＝４００００）、デキストラン７０（Ｍｗ＝７００００）などを挙げることができる。これらデキストランは、化学合成、微生物による生産、酵素による生産等のいずれの公知の方法で製造することができるが、市販品を用いることもできる。例えば、低分子デキストランＬ注(大塚製薬工場社製)、デキストラン７０（東京化成工業社製）などの市販品を挙げることができる。 The dextran used in the present invention is not particularly limited as long as it is a polysaccharide (C 6 H 10 O 5 ) n consisting of D-glucose and having an α1 → 6 bond as the main chain. Examples of the weight average molecular weight (Mw) include dextran 40 (Mw = 40000) and dextran 70 (Mw = 70000). These dextrans can be produced by any known method such as chemical synthesis, production by microorganisms, production by enzymes, etc., but commercially available products can also be used. For example, commercially available products such as low molecular weight dextran L injection (manufactured by Otsuka Pharmaceutical Factory) and dextran 70 (manufactured by Tokyo Chemical Industry Co., Ltd.) can be mentioned.
本発明において用いられるデキストラン類におけるデキストラン誘導体としては、デキストラン硫酸、カルボキシル化デキストラン、ジエチルアミノエチル（ＤＥＡＥ）−デキストランなどが含まれる。 Dextran derivatives in the dextrans used in the present invention include dextran sulfate, carboxylated dextran, diethylaminoethyl (DEAE) -dextran and the like.
本発明において用いられるデキストラン類におけるデキストラン又はその誘導体の塩としては、例えば塩酸塩、臭化水素酸塩、ヨウ化水素酸塩、リン酸塩、硝酸塩、硫酸塩、酢酸塩、プロピオン酸塩、トルエンスルホン酸塩、コハク酸塩、シュウ酸塩、乳酸塩、酒石酸塩、グリコール酸塩、メタンスルホン酸塩、酪酸塩、吉草酸塩、クエン酸塩、フマル酸塩、マレイン酸塩、リンゴ酸塩等の酸付加塩や、ナトリウム塩、カリウム塩、カルシウム塩等の金属塩や、アンモニウム塩、アルキルアンモニウム塩などを挙げることができる。なお、これらの塩は使用時において溶液として用いられ、その作用は、デキストランの場合と同効なものが好ましい。これらの塩類は、水和物又は溶媒和物を形成していてもよく、またいずれかを単独で又は２種以上を適宜組み合わせて用いることができる。 Examples of the salt of dextran or a derivative thereof used in the present invention include hydrochloride, hydrobromide, hydroiodide, phosphate, nitrate, sulfate, acetate, propionate, toluene Sulfonate, succinate, oxalate, lactate, tartrate, glycolate, methanesulfonate, butyrate, valerate, citrate, fumarate, maleate, malate, etc. Acid addition salts, metal salts such as sodium salts, potassium salts and calcium salts, ammonium salts and alkylammonium salts. These salts are used as a solution at the time of use, and the action is preferably the same as that of dextran. These salts may form a hydrate or a solvate, and can be used alone or in combination of two or more.
本発明の哺乳動物細胞懸濁液や肺塞栓形成予防剤に適用されるデキストラン類濃度としては、幹細胞等の細胞による肺塞栓形成を予防できる濃度であればよく、懸濁する細胞数や細胞濃度に応じて適宜選択することができるが、哺乳動物細胞の凝集や生存率低下を抑制するのに十分な濃度が好ましい。デキストラン類の濃度が高いほど肺塞栓形成を予防できる効果の他、凝集や生存率低下を抑制する効果は高くなるが、デキストラン類濃度が高すぎると、細胞の生存率に悪影響を及ぼす可能性がある。例えば、本発明の哺乳動物細胞懸濁液や肺塞栓形成予防剤に適用されるデキストラン類濃度は、通常、０．１％以上、好ましくは、０．５％以上、より好ましくは１．０％以上である。また、斡細胞の生存率への悪影響を回避する観点から、懸濁液中のデキストラン類の濃度は、例えば２０％以下、好ましくは１０％以下、より好ましくは７％以下、更に好ましくは５％以下である。従って、懸濁液中のデキストラン類の濃度は、例えば、０．１〜２０％、好ましくは０．５〜１０％、より好ましくは１．０〜７．０％、更に好ましくは１．０〜５．０％である。 The concentration of dextran applied to the mammalian cell suspension or the pulmonary embolization prevention agent of the present invention may be any concentration that can prevent pulmonary embolization by cells such as stem cells. The concentration may be appropriately selected depending on the concentration, but a concentration sufficient to suppress aggregation of mammalian cells and a decrease in survival rate is preferable. The higher the concentration of dextran, the more effective it is to prevent pulmonary embolism, and the more effective it is to suppress aggregation and decrease in the survival rate.However, if the concentration of dextran is too high, the cell viability may be adversely affected. is there. For example, the concentration of dextran applied to the mammalian cell suspension or the pulmonary embolism prevention agent of the present invention is usually 0.1% or more, preferably 0.5% or more, more preferably 1.0%. That's it. From the viewpoint of avoiding adverse effects on the viability of sputum cells, the concentration of dextran in the suspension is, for example, 20% or less, preferably 10% or less, more preferably 7% or less, and even more preferably 5%. It is as follows. Therefore, the concentration of dextran in the suspension is, for example, 0.1 to 20%, preferably 0.5 to 10%, more preferably 1.0 to 7.0%, and still more preferably 1.0 to 5.0%.
本発明において、血管経由で投与する際の「肺塞栓形成」とは、経動脈又は経静脈移植（投与）された哺乳動物細胞が肺の末梢の動脈系、例えば１又は２以上の肺動脈の毛細血管を塞いだ状態を形成することを意味する。前記移植された哺乳動物細胞が肺の末梢の動脈系を「塞いだ状態」とは、必ずしも細胞は肺の末梢の動脈系中で停止している必要はなく、当該箇所での血流を妨害していれば十分である。また、この場合の血流は完全に停止している必要はなく、広い意味で肺機能の低下を生じさせる程度の血流の低下が生じていれば十分である。前記「肺機能」とは、第一義的には大気からの酸素の血中への取り込みを意味するが、二酸化炭素やその他のガスの排出も含む。細胞による肺塞栓形成が起こると、肺動脈の毛細血管における血流が低下し、頻呼吸（呼吸数の増加）や頻脈（心拍数の増加）が生じたり、肺動脈圧などの血圧が上昇したり、あるいは血中酸素分圧が低下する。このため、細胞移植による肺塞栓の形成を予防することにより、肺塞栓による頻呼吸や頻脈や血圧上昇や血中酸素分圧低下等のリスクを低減することができる。また細胞移植により肺塞栓が形成されると、場合によっては、肺機能の低下や、心機能（血液を動脈の内部に移動させる機能や心臓が血液を静脈の外に移動させる機能等）の低下などの肺塞栓症を発症する。さらに、本発明における肺塞栓形成には、哺乳動物細胞が肺の末梢の動脈系に加えてそれ以外の血管を塞いだ状態を形成する場合も含まれる。 In the present invention, “pulmonary embolization” when administered via a blood vessel means transmural or intravenous implantation (administration) of mammalian cells in the peripheral arterial system of the lung, for example, one or more capillaries of the pulmonary artery. It means forming a state in which blood vessels are blocked. The state in which the transplanted mammalian cells “clog” the peripheral arterial system of the lung does not necessarily mean that the cell has stopped in the peripheral arterial system of the lung, and obstructs the blood flow at that location. It is enough if you do. In this case, the blood flow does not need to be completely stopped, and it is sufficient that the blood flow is reduced to such a degree that the lung function is reduced in a broad sense. The “pulmonary function” primarily means the uptake of oxygen from the atmosphere into the blood, but also includes the discharge of carbon dioxide and other gases. When pulmonary embolization occurs due to cells, blood flow in capillaries of the pulmonary artery decreases, causing tachypnea (increased respiratory rate) and tachycardia (increased heart rate), and blood pressure such as pulmonary artery pressure increases Or, the partial pressure of oxygen in the blood decreases. For this reason, by preventing the formation of pulmonary embolism by cell transplantation, it is possible to reduce risks such as tachypnea, tachycardia, blood pressure increase and blood oxygen partial pressure decrease due to pulmonary embolism. In addition, when pulmonary embolism is formed by cell transplantation, depending on the case, the lung function may decrease or the cardiac function (the function of moving blood into the artery or the function of moving the blood out of the vein) may decrease. Develops pulmonary embolism. Furthermore, pulmonary embolization in the present invention includes cases where mammalian cells form a state in which other blood vessels are blocked in addition to the peripheral arterial system of the lung.
本発明の哺乳動物細胞懸濁液においては、哺乳動物細胞がふつうデキストラン類を有効成分として含む生理的水溶液中に懸濁されている。他方、本発明の肺塞栓形成の予防剤は、液体タイプと非液体タイプとに大別される。液体タイプ予防剤においては、ふつうデキストラン類を有効成分として含む生理的水溶液として構成され、かかる液体タイプ予防剤に哺乳動物細胞を懸濁すると、本発明の哺乳動物細胞懸濁液を調製することができる。また、非液体タイプ予防剤においては、ふつう哺乳動物細胞が懸濁された生理的水溶液に添加される粉体等のデキストラン類含有物として構成され、かかる非液体タイプ予防剤を哺乳動物細胞が懸濁された生理的水溶液に添加すると、本発明の哺乳動物細胞懸濁液を調製することができる。 In the mammalian cell suspension of the present invention, the mammalian cells are usually suspended in a physiological aqueous solution containing dextran as an active ingredient. On the other hand, the preventive agent for pulmonary embolization according to the present invention is roughly classified into a liquid type and a non-liquid type. The liquid type preventive agent is usually constituted as a physiological aqueous solution containing dextran as an active ingredient, and the mammalian cell suspension of the present invention can be prepared by suspending mammalian cells in such a liquid type preventive agent. it can. In addition, the non-liquid type preventive agent is usually configured as a dextran-containing material such as powder added to a physiological aqueous solution in which mammalian cells are suspended, and the mammalian cell suspends the non-liquid type preventive agent. When added to a turbid physiological aqueous solution, the mammalian cell suspension of the present invention can be prepared.
さらに、本発明の哺乳動物細胞懸濁液の異なる態様としては、哺乳動物細胞及びデキストラン類を含む哺乳動物細胞懸濁液を血管経由で投与する幹細胞等の細胞移植における、肺塞栓形成を予防する方法を挙げることができ、また、本発明の肺塞栓形成の予防剤の異なる態様としては、哺乳動物細胞を血管経由で投与する際の肺塞栓形成の予防剤を調製するためのデキストラン類の使用を挙げることができる。 Furthermore, as a different aspect of the mammalian cell suspension of the present invention, pulmonary embolism is prevented in cell transplantation of stem cells or the like in which a mammalian cell suspension containing mammalian cells and dextrans is administered via blood vessels. As another embodiment of the preventive agent for pulmonary embolism according to the present invention, use of dextran for preparing a prophylactic agent for pulmonary embolism when a mammalian cell is administered via a blood vessel. Can be mentioned.
上記生理的水溶液としては、例えば、生理食塩水、リン酸緩衝化生理食塩水、トリス緩衝化生理食塩水、ＨＥＰＥＳ緩衝化生理食塩水、リンゲル液（乳酸リンゲル液、酢酸リンゲル液、重炭酸リンゲル液等）、５％グルコース水溶液、哺乳動物培養用の液体培地、等張剤（ブドウ糖、Ｄ−ソルビトール、Ｄ−マンニトール、ラクトース、塩化ナトリウム等）の水溶液などの等張水溶液を挙げることができ、これらの中でもリンゲル液が好ましく、乳酸リンゲル液、酢酸リンゲル液又は重炭酸リンゲル液がより好ましく、乳酸リンゲル液がさらに好ましい。本明細書において「等張」とは、浸透圧が２５０〜３８０ｍＯｓｍ／ｌの範囲内であることを意味する。生理的水溶液は、更に安定剤（例えば、ヒト血清アルブミン、ポリエチレングリコールなど）、緩衝剤（例えば、リン酸塩緩衝液、酢酸ナトリウム緩衝液）、キレート剤（例えば、ＥＤＴＡ、ＥＧＴＡ、クエン酸、サリチレート）、溶解補助剤、保存剤、酸化防止剤等を含むことができる。 Examples of the physiological aqueous solution include physiological saline, phosphate buffered physiological saline, Tris buffered physiological saline, HEPES buffered physiological saline, Ringer's solution (lactate Ringer's solution, acetated Ringer's solution, bicarbonated Ringer's solution, etc.), 5 % Isotonic aqueous solution such as aqueous solution of glucose solution, mammalian culture medium, isotonic agent (glucose, D-sorbitol, D-mannitol, lactose, sodium chloride, etc.), among them Ringer's solution Preferably, lactate Ringer solution, acetate Ringer solution or bicarbonate Ringer solution is more preferable, and lactate Ringer solution is more preferable. As used herein, “isotonic” means that the osmotic pressure is in the range of 250 to 380 mOsm / l. The physiological aqueous solution further contains a stabilizer (eg, human serum albumin, polyethylene glycol, etc.), a buffer (eg, phosphate buffer, sodium acetate buffer), a chelating agent (eg, EDTA, EGTA, citric acid, salicylate). ), Solubilizers, preservatives, antioxidants, and the like.
本発明者らにより、デキストラン類同様に、肺塞栓形成の予防作用を有することが明らかにされているトレハロース類をデキストラン類と併用することができる。 Like the dextrans, the present inventors can use trehalose, which has been shown to have a prophylactic action against pulmonary embolism, in combination with the dextran.
本発明の細胞懸濁液に含まれる幹細胞等の細胞は、浮遊した状態で存在し、通常浮遊した状態の細胞は凝集しやすいが、本発明のデキストラン類による効果により、細胞凝集が抑制され、単一細胞の状態を長時間維持することができる。 Cells such as stem cells contained in the cell suspension of the present invention are present in a suspended state, and normally suspended cells are likely to aggregate, but due to the effects of the dextrans of the present invention, cell aggregation is suppressed, The state of a single cell can be maintained for a long time.
哺乳動物細胞のデキストラン類を含む生理的水溶液中への懸濁は、ピペッティングやタッピング等の当該技術分野における周知の方法により実施することができる。
本発明の哺乳動物細胞懸濁液の温度は、通常０〜３７℃、好ましくは０〜２５℃の範囲内である。本発明の懸濁液中の哺乳動物細胞の密度は、幹細胞等の細胞による肺塞栓形成を予防できる密度であればよいが、デキストラン類による哺乳動物細胞の凝集や生存率の低下を抑制する効果が達成される密度がより好ましく、通常１０３〜１０１０個／ｍｌの範囲内である。
Suspension of mammalian cells in a physiological aqueous solution containing dextran can be carried out by methods well known in the art such as pipetting and tapping.
The temperature of the mammalian cell suspension of the present invention is usually in the range of 0 to 37 ° C, preferably 0 to 25 ° C. The density of the mammalian cells in the suspension of the present invention may be any density that can prevent the formation of pulmonary embolism by cells such as stem cells, but the effect of suppressing the aggregation of mammalian cells and the decrease in the survival rate due to dextrans Is more preferable, and is usually in the range of 10 3 to 10 10 pieces / ml.
本発明の哺乳動物細胞懸濁液では、デキストラン類により哺乳動物細胞の凝集が抑制されているので、これを用いて細胞移植を実施することにより、細胞の凝集物が、カニューレ中に詰まってしまうリスクを低減することができる。また、本発明の哺乳動物細胞懸濁液では、デキストラン類により、懸濁液中の哺乳動物細胞の生存率の低下が抑制されるので、本発明の懸濁液を用いれば、より状態のよい細胞により細胞移植を実施することができ、治療効果の向上が期待できる。 In the mammalian cell suspension of the present invention, the aggregation of mammalian cells is suppressed by dextrans, so that cell aggregates are clogged in the cannula by performing cell transplantation using this. Risk can be reduced. In addition, in the mammalian cell suspension of the present invention, dextrans suppress a decrease in the survival rate of mammalian cells in the suspension. Cell transplantation can be performed with cells, and an improvement in therapeutic effect can be expected.
本発明の哺乳動物細胞懸濁液や肺塞栓形成の予防剤は、適切な滅菌容器内に収容することにより、哺乳動物細胞懸濁液製剤や肺塞栓形成の予防製剤として製造することができる。かかる容器としては、ボトル、バイアル、シリンジ、輸液バッグ等の可塑性のバッグ、試験管などを挙げることができる。これらの容器は、ガラス又はプラスチックのような各種の材料から形成し得る。また、哺乳動物細胞懸濁液製剤用の容器には、容器内の本発明の哺乳動物細胞懸濁液を患者に点滴注入可能とするように、カニューレ及び／又は注射針を接続することができる。 The preventive agent for mammalian cell suspension or pulmonary embolism formation of the present invention can be produced as a mammalian cell suspension preparation or a preventive agent for pulmonary embolism formation by storing in a suitable sterile container. Examples of such containers include plastic bags such as bottles, vials, syringes, infusion bags, and test tubes. These containers may be formed from a variety of materials such as glass or plastic. In addition, a cannula and / or injection needle can be connected to the container for mammalian cell suspension preparation so that the mammalian cell suspension of the present invention in the container can be instilled into a patient. .
以下、実施例により本発明をより具体的に説明するが、本発明の技術的範囲はこれらの例示に限定されるものではない。 EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.
１．本発明の幹細胞懸濁用溶液が幹細胞集団による肺塞栓形成を阻害するため用いることができることの確認（１）
１−１ 方法
１−１−１ ミニブタ静脈投与に用いるＡＴ−ＭＳＣ懸濁液の調製
〔１〕ヒトＡＴ−ＭＳＣ（Cambrex、日本代理店:TaKaRa、京都、日本）を解凍し、培養皿（直径９ｃｍのnon-coatプラスチック製）(nunc、東京、日本)にて培養した。
４回の継代後に２８８枚の培養皿まで増殖させた。２８８枚の培養皿のうち、１４４枚をＰＢＳ（−）にて３回洗浄し、×１０トリプシンＥＤＴＡ（GIBCO、東京、日本）をＰＢＳ（−）にて１０倍希釈した溶液を１ｍＬ／培養皿の条件で加えた。
〔２〕３７℃、ＣＯ２インキュベーターにて３０〜６０秒間静置し、１０％ウシ胎児血清（ＦＢＳ）（GIBCO、東京、日本）を含むαＭＥＭ（GIBCO、東京、日本）を３ｍＬ／培養皿の条件で添加して、酵素処理を停止させ、細胞を５０ｍＬ遠心チューブ（Falcon、東京、日本）にて回収し、１２００ｒｐｍ、５分の条件で遠心処理を行った。
〔３〕上清をアスピレーターにて除去し、４０ｍＬのＰＢＳ（−）を加えてピペッティングして細胞を再懸濁し、１２００ｒｐｍ、５分の条件で遠心し、培地及びトリプシンＥＤＴＡを除去する洗浄操作を計３回繰り返した。
〔４〕重量平均分子量が４００００のデキストラン（デキストラン４０）を３０ｍｇ／ｍｌの濃度で含有する、乳酸リンゲル液（デキストラン４０加）（サヴィオゾール輸液）(大塚製薬工場、徳島、日本)と、重量平均分子量が４００００のデキストラン（デキストラン４０）を１００ｍｇ／ｍｌの濃度で含有する、低分子デキストランＬ注(大塚製薬工場、徳島、日本)とを等量添加して６．５％（６５ｍｇ／ｍｌ）デキストラン含有乳酸リンゲル液を作製し、輸液バッグに加えた後、適量の液を注射器にて吸い取り、洗浄ＰＢＳ（−）を除去した細胞に加えて、再度懸濁（３０Ｇ注射針［ニプロ、東京、日本］と５ｍＬシリンジ［ニプロ、東京、日本］を用いて、３回の吸排出）し、ＡＴ−ＭＳＣ懸濁デキストラン含有乳酸リンゲル液（７．５×１０５細胞／ｍＬ）を作製した。一方、コントロールとして、生理食塩液(大塚製薬工場、徳島、日本)を細胞懸濁液として用い、ＡＴ−ＭＳＣ懸濁生理食塩液（８．４×１０５細胞／ｍＬ）を作製した。
〔５〕２種類のＡＴ−ＭＳＣ懸濁液（ＡＴ−ＭＳＣ懸濁生理食塩液及びＡＴ−ＭＳＣ懸濁デキストラン含有乳酸リンゲル液）をそれぞれ別の輸液バッグに戻し、複数回、輸液バッグを揺らして細胞を撹拌し、細胞点滴投与（移植）用として準備した。
1. Confirmation that the solution for suspending stem cells of the present invention can be used to inhibit pulmonary embolization by a stem cell population (1)
1-1 Method 1-1-1 Preparation of AT-MSC Suspension Used for Mini Pig Intravenous Administration [1] Human AT-MSC (Cambrex, Japan Agency: TaKaRa, Kyoto, Japan) is thawed and cultured dish (diameter The cells were cultured in 9 cm non-coat plastic (nunc, Tokyo, Japan).
After 4 passages, 288 culture dishes were grown. Of 288 culture dishes, 144 were washed 3 times with PBS (−), and 10 mL of a solution obtained by diluting × 10 trypsin EDTA (GIBCO, Tokyo, Japan) with PBS (−) was 1 mL / culture dish. It was added under the conditions.
[2] Allow to stand at 37 ° C. in a CO 2 incubator for 30 to 60 seconds, and add 3 ml of αMEM (GIBCO, Tokyo, Japan) containing 10% fetal bovine serum (FBS) (GIBCO, Tokyo, Japan) to a culture dish. The enzyme treatment was stopped under the conditions, and the cells were collected in a 50 mL centrifuge tube (Falcon, Tokyo, Japan), and centrifuged at 1200 rpm for 5 minutes.
[3] Remove the supernatant with an aspirator, add 40 mL of PBS (-), pipette to resuspend the cells, and centrifuge at 1200 rpm for 5 minutes to remove the medium and trypsin EDTA. Was repeated three times.
[4] Lactated Ringer's solution (dextran 40 added) (Savizol infusion) (Otsuka Pharmaceutical Factory, Tokushima, Japan) containing dextran (dextran 40) having a weight average molecular weight of 40,000 at a concentration of 30 mg / ml, and weight average molecular weight Contains 6.5% (65 mg / ml) dextran by adding an equal amount of low molecular weight dextran L injection (Otsuka Pharmaceutical Factory, Tokushima, Japan) containing 40000 dextran (dextran 40) at a concentration of 100 mg / ml After preparing the lactated Ringer's solution and adding it to the infusion bag, suck the appropriate amount of solution with a syringe, add the washed PBS (-) to the removed cells, and resuspend (30G syringe needle [Nipro, Tokyo, Japan] Using a 5 mL syringe [Nipro, Tokyo, Japan], sucking and discharging 3 times), AT-MSC suspended dextran-containing lactated Ringer's solution (7. 5 × 10 5 cells / mL). On the other hand, as a control, an physiological saline solution (Otsuka Pharmaceutical Factory, Tokushima, Japan) was used as a cell suspension to prepare an AT-MSC suspension physiological saline solution (8.4 × 10 5 cells / mL).
[5] Return two types of AT-MSC suspensions (AT-MSC suspension physiological saline and AT-MSC suspension dextran-containing lactated Ringer's solution) to separate infusion bags, and shake the infusion bag multiple times to obtain cells. Was stirred and prepared for cell infusion (transplantation).
１−１−２ ＡＴ−ＭＳＣ懸濁液のミニブタへの静脈投与及び肺動脈圧測定
〔１〕麻酔下にて体重約１５ｋｇのクラウンミニブタ(ジャパンファーム、鹿児島、日本)の右外頚静脈よりスワンガンツカテーテル(エドワーズ)を挿入した。
〔２〕トランスデューサーを用いて圧測定を行いながら、カテーテルを肺動脈へと進めた。カテーテルの位置をＸ線透視下(Ｃ−アーム)で再確認し、固定した。
〔３〕ＡＴ−ＭＳＣ懸濁液投与前の肺動脈圧を測定した。
〔４〕カットダウンしたルートを用いて、上記２種類のＡＴ−ＭＳＣ懸濁液を５ｍＬ/分の流速にて４０分間２００ｍＬでそれぞれのブタへ移植した（ＡＴ−ＭＳＣ懸濁生理食塩液；ｎ＝１、１．５×１０８細胞／個体、ＡＴ−ＭＳＣ懸濁デキストラン含有乳酸リンゲル液；ｎ＝１、１．７×１０８細胞／個体）。肺動脈圧の計測は経時的に６０分間行なった。
1-1-2 Intravenous administration of AT-MSC suspension to minipig and measurement of pulmonary artery pressure [1] Swanganz catheter from right external jugular vein of crown minipig (Japan Farm, Kagoshima, Japan) weighing approximately 15 kg under anesthesia (Edwards) was inserted.
[2] The catheter was advanced to the pulmonary artery while measuring pressure using a transducer. The position of the catheter was reconfirmed under fluoroscopy (C-arm) and fixed.
[3] The pulmonary artery pressure before administration of the AT-MSC suspension was measured.
[4] Using the cut-down route, the above two types of AT-MSC suspensions were transplanted into each pig at 200 mL for 40 minutes at a flow rate of 5 mL / min (AT-MSC suspension physiological saline; n = 1, 1.5 × 10 8 cells / individual, AT-MSC suspension dextran-containing lactated Ringer's solution; n = 1, 1.7 × 10 8 cells / individual). The pulmonary artery pressure was measured for 60 minutes over time.
１−２ 結果
投与（移植）開始から５分ごとに肺動脈圧を測定した値を、投与開始時の肺動脈圧で標準化した相対値として図1に示す。生理食塩水に懸濁したＡＴ−ＭＳＣを投与したブタでは、投与開始後から肺動脈圧が上昇し始め、投与後１５分において投与前と比べ肺動脈圧が１６ｍｍＨｇ上昇した。肺動脈圧の上昇は、投与終了とともに徐々に下降を始めたが、投与終了後２０分経過した時点（投与後６０分）でも投与前と比較して９ｍｍＨｇ程度の上昇が認められた。一方、デキストラン含有乳酸リンゲル液に懸濁したＡＴ−ＭＳＣを投与したブタでは、投与開始５分後に最大３ｍｍＨｇの肺動脈圧の上昇が認められたものの、３ｍｍＨｇ以下のごく僅かの肺動脈圧の上昇しか計測されず、投与終了後２０分経過した時点（投与後６０分）では投与前と同じ肺動脈圧になっていた。以上の結果は、デキストランを含有する溶液に懸濁したＡＴ−ＭＳＣを移植すると、肺動脈圧上昇を抑制できることを示している。すなわち、デキストラン不含溶液を用いてＡＴ−ＭＳＣを移植すると、ＡＴ−ＭＳＣが肺、特に肺の毛細血管に集積することにより肺塞栓が形成され、肺における血流が低下し、肺動脈圧が上昇するが、デキストランが含有する溶液を用いてＭＳＣを移植すると、ＡＴ−ＭＳＣの肺における集積（肺塞栓の形成）が抑制され、肺における血流が低下せず、肺動脈圧は上昇しないと考えられる。
1-2 Results The values obtained by measuring the pulmonary artery pressure every 5 minutes from the start of administration (transplantation) are shown in FIG. 1 as relative values normalized by the pulmonary artery pressure at the start of administration. In pigs administered with AT-MSC suspended in physiological saline, pulmonary artery pressure began to increase after the start of administration, and pulmonary artery pressure increased by 16 mmHg 15 minutes after administration compared to before administration. The increase in pulmonary arterial pressure started to decrease gradually with the end of administration, but an increase of about 9 mmHg was observed even when 20 minutes had passed after the end of administration (60 minutes after administration) compared to before administration. On the other hand, in pigs administered with AT-MSC suspended in dextran-containing lactated Ringer's solution, a pulmonary artery pressure increase of 3 mmHg at the maximum was observed 5 minutes after the start of administration, but only a slight increase in pulmonary artery pressure of 3 mmHg or less was measured. However, at the time when 20 minutes passed after the end of administration (60 minutes after administration), the pulmonary artery pressure was the same as that before administration. The above results indicate that an increase in pulmonary artery pressure can be suppressed by transplanting AT-MSC suspended in a solution containing dextran. That is, when AT-MSC is transplanted using a dextran-free solution, AT-MSC accumulates in the lungs, particularly in the pulmonary capillaries, forming pulmonary emboli, reducing blood flow in the lungs and increasing pulmonary artery pressure. However, when MSC is transplanted using a solution containing dextran, AT-MSC accumulation in the lung (formation of pulmonary embolism) is suppressed, blood flow in the lung does not decrease, and pulmonary artery pressure does not increase. .
２．本発明の幹細胞懸濁用溶液が幹細胞集団による肺塞栓形成を阻害するため用いることができることの確認（２）
２−１ 方法
２−１−１ マイクロミニブタ静脈投与に用いるＡＴ−ＭＳＣ懸濁液の調製
〔１〕ブタの皮下脂肪組織から樹立したブタＡＴ−ＭＳＣを事前に凍結保存した細胞チューブを解凍し、培養皿（直径９ｃｍのnon-coatプラスチック製）(nunc、東京、日本)にて培養した。なお、解凍時の継代数は４代目である。
解凍培養後、４回の継代後に２４０枚の培養皿まで増殖させた。２４０枚の培養皿のうち、１２０枚をＰＢＳ（−）にて３回洗浄し、０．５％トリプシンＥＤＴＡ（GIBCO、東京、日本）をＰＢＳ（−）にて１０倍希釈した溶液を１ｍＬ／培養皿の条件で加えた。
〔２〕３７℃、ＣＯ２インキュベーターにて３０〜６０秒間静置し、１０％ウシ胎児血清（ＦＢＳ）（GIBCO、東京、日本）を含むαＭＥＭ（GIBCO、東京、日本）を３ｍＬ／培養皿の条件で添加して、酵素処理を停止させ、細胞を５０ｍＬ遠心チューブ（Falcon、東京、日本）にて回収し、１２００ｒｐｍ、５分の条件で遠心処理を行った。
〔３〕上清をアスピレーターにて除去し、４０ｍＬのＰＢＳ（−）を加えてピペッティングして細胞を再懸濁し、１２００ｒｐｍ、５分の条件で遠心し、培地及びトリプシンＥＤＴＡを除去する洗浄操作を計３回繰り返した。
〔４〕重量平均分子量が４００００のデキストラン（デキストラン４０）を３０ｍｇ／ｍｌの濃度で含有する、乳酸リンゲル液（デキストラン４０加）（サヴィオゾール輸液）(大塚製薬工場、徳島、日本)と、重量平均分子量が４００００のデキストラン（デキストラン４０）を１００ｍｇ／ｍｌの濃度で含有する、低分子デキストランＬ注(大塚製薬工場、徳島、日本)とを等量添加して６．５％（６５ｍｇ／ｍｌ）デキストラン含有乳酸リンゲル液１００ｍＬを作製し、輸液バッグに加えた後、適量の液を注射器にて吸い取り、洗浄ＰＢＳ（−）を除去した細胞に加えて、再度懸濁（３０Ｇ注射針［ニプロ、東京、日本］と５ｍＬシリンジ［ニプロ、東京、日本］を用いて、３回の吸排出）し、ＡＴ−ＭＳＣ懸濁デキストラン含有乳酸リンゲル液（１．５×１０６細胞／ｍＬ）を作製した。一方、コントロールとして、２４０枚準備した培養皿の内、１２０枚から細胞を同様の操作を経て回収し、生理食塩液(大塚製薬工場、徳島、日本)を細胞懸濁液として用い、ＡＴ−ＭＳＣ懸濁生理食塩液（１．７×１０６細胞／ｍＬ）を作製した。なお、各輸液バッグには１００ｍＬの細胞を懸濁した液を充填している。
〔５〕２種類のＡＴ−ＭＳＣ懸濁液（ＡＴ−ＭＳＣ懸濁生理食塩液及びＡＴ−ＭＳＣ懸濁デキストラン含有乳酸リンゲル液）をそれぞれ別の輸液バッグに戻し、複数回、輸液バッグを揺らして細胞を撹拌し、細胞点滴投与（移植）用として準備した。
2. Confirmation that the solution for suspending stem cells of the present invention can be used to inhibit pulmonary embolization by a stem cell population (2)
2-1 Method 2-1-1 Preparation of AT-MSC Suspension Used for Microminiature Pig Intravenous Administration [1] Thaw a cell tube in which porcine AT-MSC established from porcine subcutaneous adipose tissue was cryopreserved in advance, The cells were cultured in a culture dish (made of non-coat plastic having a diameter of 9 cm) (nunc, Tokyo, Japan). The passage number at the time of thawing is the fourth generation.
After thawing culture, it was grown to 240 culture dishes after 4 passages. Of the 240 culture dishes, 120 were washed 3 times with PBS (−), and a solution of 0.5% trypsin EDTA (GIBCO, Tokyo, Japan) diluted 10-fold with PBS (−) was 1 mL / Added under conditions of culture dishes.
[2] Allow to stand at 37 ° C. in a CO 2 incubator for 30 to 60 seconds, and add 3 ml of αMEM (GIBCO, Tokyo, Japan) containing 10% fetal bovine serum (FBS) (GIBCO, Tokyo, Japan) to a culture dish. The enzyme treatment was stopped under the conditions, and the cells were collected in a 50 mL centrifuge tube (Falcon, Tokyo, Japan), and centrifuged at 1200 rpm for 5 minutes.
[3] Remove the supernatant with an aspirator, add 40 mL of PBS (-), pipette to resuspend the cells, and centrifuge at 1200 rpm for 5 minutes to remove the medium and trypsin EDTA. Was repeated three times.
[4] Lactated Ringer's solution (dextran 40 added) (Savizol infusion) (Otsuka Pharmaceutical Factory, Tokushima, Japan) containing dextran (dextran 40) having a weight average molecular weight of 40,000 at a concentration of 30 mg / ml, and weight average molecular weight Contains 6.5% (65 mg / ml) dextran by adding an equal amount of low molecular weight dextran L injection (Otsuka Pharmaceutical Factory, Tokushima, Japan) containing 40000 dextran (dextran 40) at a concentration of 100 mg / ml After preparing 100 mL of lactated Ringer's solution and adding it to the infusion bag, suck the appropriate amount of solution with a syringe, add it to the cells from which the washed PBS (-) has been removed, and resuspend (30G needle [Nipro, Tokyo, Japan] And 5 mL syringe [Nipro, Tokyo, Japan] 3 times suction and discharge), AT-MSC suspension dextran-containing lactate ringe A liquid (1.5 × 10 6 cells / mL) was prepared. On the other hand, as a control, cells were collected from 120 of the 240 culture dishes prepared through the same operation, and physiological saline (Otsuka Pharmaceutical Factory, Tokushima, Japan) was used as the cell suspension. Suspension physiological saline (1.7 × 10 6 cells / mL) was prepared. Each infusion bag is filled with a solution in which 100 mL of cells are suspended.
[5] Return two types of AT-MSC suspensions (AT-MSC suspension physiological saline and AT-MSC suspension dextran-containing lactated Ringer's solution) to separate infusion bags, and shake the infusion bag multiple times to obtain cells. Was stirred and prepared for cell infusion (transplantation).
２−１−２ ＡＴ−ＭＳＣ懸濁液のマイクロミニブタへの静脈投与及び血液ガス測定
〔１〕麻酔下（投与麻酔及び純酸素無し）にて体重約８〜９ｋｇのマイクロミニブタ(富士マイクラ、静岡、日本)の右外頸から静脈へカテーテルを留置した。さらに耳静脈に点滴ラインを確保した。
〔２〕ｉＳＴＡＴと専用カートリッジを用いてＡラインから約２．５ｍＬの採血を行い、血中酸素分圧測定及びサチュレーションモニターにて心拍数や血圧が安定化することを確認した。なお、血中酸素分圧測定に関しては５分毎に実施し、２回連続して安定（同一値）したことを確認した。
〔３〕耳静脈より２０分間、各細胞を添加した懸濁液を点滴の要領で移植した（ＡＴ−ＭＳＣ懸濁生理食塩液；ｎ＝１、１．５×１０８細胞／個体、ＡＴ−ＭＳＣ懸濁デキストラン含有乳酸リンゲル液；ｎ＝１、１．７×１０８細胞／個体）。移植開始時から５分毎に血中酸素分圧の測定を行い、移植開始から６０分までを測定した。
2-1-2 Intravenous administration of AT-MSC suspension to micro-mini pigs and blood gas measurement [1] Micro-mini pigs (Fuji Mikura, Shizuoka) weighing about 8-9 kg under anesthesia (no administration anesthesia and no pure oxygen) , Japan) from the right external neck to the vein. In addition, an infusion line was secured in the ear vein.
[2] About 2.5 mL of blood was collected from the A line using iSTAT and a dedicated cartridge, and it was confirmed that the heart rate and blood pressure were stabilized by measuring blood oxygen partial pressure and a saturation monitor. The blood oxygen partial pressure measurement was performed every 5 minutes, and it was confirmed that the blood oxygen partial pressure was stabilized twice (same value).
[3] A suspension to which each cell was added from the ear vein for 20 minutes was transplanted in the manner of infusion (AT-MSC suspension physiological saline; n = 1, 1.5 × 10 8 cells / individual, AT- MSC suspended dextran-containing lactated Ringer's solution; n = 1, 1.7 × 10 8 cells / individual). The blood oxygen partial pressure was measured every 5 minutes from the start of transplantation, and measured until 60 minutes from the start of transplantation.
２−２ 結果
投与（移植）開始から５分ごとに血中酸素分圧を測定した値を、投与開始時の血中酸素分圧で標準化した相対値として図２や表１に示す。生理食塩液に懸濁したＡＴ−ＭＳＣを投与したブタでは、投与開始後から血中酸素分圧が低下し始め、投与後１５分において投与前と比べ血中酸素分圧が約１９％低下した（図２、表１）。細胞投与開始後２５分経過した時点で投与前と比較して血中酸素分圧が約４２％低下した。一方、デキストラン含有乳酸リンゲル液に懸濁したＡＴ−ＭＳＣを投与したブタでは、投与開始からほぼ一定の血中酸素分圧値を示し、血中酸素分圧の低下は認められなかった。以上の結果は、デキストランを含有する溶液に懸濁したＡＴ−ＭＳＣを静脈投与すると、血中酸素分圧低下を抑制できることを示している。すなわち、デキストラン不含溶液を用いてＡＴ−ＭＳＣを移植すると、ＡＴ−ＭＳＣが肺、特に肺の毛細血管に集積することにより肺塞栓が形成され、肺における血流が低下し、血中酸素分圧の低下が起こるが、デキストランを含有する溶液を用いてＭＳＣを移植すると、ＡＴ−ＭＳＣの肺における集積（肺塞栓の形成）が抑制され、肺における血流が低下せず、血中酸素分圧は低下しないと考えられる。
2-2 Results FIG. 2 and Table 1 show the values obtained by measuring the blood oxygen partial pressure every 5 minutes from the start of administration (transplantation) as the relative values normalized by the blood oxygen partial pressure at the start of administration. In pigs administered AT-MSC suspended in physiological saline, the blood oxygen partial pressure began to decrease after the start of administration, and the blood oxygen partial pressure decreased by about 19% at 15 minutes after administration compared to before administration. (FIG. 2, Table 1). At 25 minutes after the start of cell administration, the blood oxygen partial pressure decreased by about 42% compared to before administration. On the other hand, pigs administered with AT-MSC suspended in dextran-containing lactated Ringer's solution showed an almost constant blood oxygen partial pressure value from the start of administration, and no decrease in blood oxygen partial pressure was observed. The above results indicate that a decrease in blood oxygen partial pressure can be suppressed by intravenous administration of AT-MSC suspended in a solution containing dextran. That is, when AT-MSC is transplanted using a dextran-free solution, AT-MSC accumulates in the lungs, particularly in the pulmonary capillaries, thereby forming a pulmonary embolism, lowering blood flow in the lung, Although pressure drop occurs, when MSC is transplanted using a solution containing dextran, accumulation of AT-MSC in the lung (formation of pulmonary embolism) is suppressed, blood flow in the lung does not decrease, and blood oxygen content is reduced. The pressure is not expected to drop.
本発明によると、ＭＳＣなどの幹細胞等の細胞の移植時に、肺塞栓形成を予防したり、肺塞栓症の発症リスクを低減することができるので、再生医療等における移植医療分野やがん治療分野で有用である。 According to the present invention, at the time of transplantation of cells such as stem cells such as MSC, pulmonary embolism can be prevented and the risk of developing pulmonary embolism can be reduced. It is useful in.
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| JPN6013028416; '肺塞栓症とその予防について-党員における予防違法ガイドラインの試作-' 昭和医会誌 Ｖｏｌ． ６３， Ｎｏ． ４, 2003, ｐ． ３９２-４０４ * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPWO2018123628A1 (en) * | 2016-12-28 | 2019-10-31 | ロート製薬株式会社 | Cell pharmaceutical composition, disease treatment kit and cell suspension solution |
| JP2019193627A (en) * | 2018-04-25 | 2019-11-07 | セルトラスト・アニマル・セラピューティクス株式会社 | Method for preserving cells and cell suspension |
Also Published As
| Publication number | Publication date |
|---|---|
| TW201406402A (en) | 2014-02-16 |
| WO2013168402A1 (en) | 2013-11-14 |
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