JP6958350B2 - Method for producing stem cell culture supernatant - Google Patents

Method for producing stem cell culture supernatant Download PDF

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JP6958350B2
JP6958350B2 JP2017506604A JP2017506604A JP6958350B2 JP 6958350 B2 JP6958350 B2 JP 6958350B2 JP 2017506604 A JP2017506604 A JP 2017506604A JP 2017506604 A JP2017506604 A JP 2017506604A JP 6958350 B2 JP6958350 B2 JP 6958350B2
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JPWO2016148230A1 (en
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達哉 山口
真希子 平岡
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Toyobo Co Ltd
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06Animal cells or tissues; Human cells or tissues
    • C12N5/0602Vertebrate cells
    • C12N5/0652Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662Stem cells
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12MAPPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/10Hollow fibers or tubes
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells

Description

本発明は、幹細胞の培養上清を製造する方法に関する。 The present invention relates to a method for producing a culture supernatant of stem cells.

幹細胞である間葉系幹細胞は、体性幹細胞の一種であり、間葉系の細胞、即ち、骨細胞、心筋細胞、軟骨細胞、脂肪細胞などへの分化能を有することから、骨や血管、心筋の再構築などの再生医療への応用が期待されている。また、間葉系幹細胞は抗炎症作用や免疫調節等の作用も有することから、肝疾患や移植片対宿主病、自己免疫疾患など、既に様々な疾患に対して幅広く利用されていることが知られている。 Mesenchymal stem cells, which are stem cells, are a type of somatic stem cells and have the ability to differentiate into mesenchymal cells, that is, bone cells, myocardial cells, chondrocytes, adipocytes, etc. It is expected to be applied to regenerative medicine such as myocardial reconstruction. In addition, since mesenchymal stem cells also have anti-inflammatory and immunomodulatory effects, it is known that they are already widely used for various diseases such as liver disease, graft-versus-host disease, and autoimmune disease. Has been done.

こうした中、細胞移植治療において生体内に移植されたこれらの間葉系幹細胞の働きには、間葉系細胞から分泌される種々のサイトカイン等の生理活性物質が大きく寄与していることが明らかにされてきた。 Under these circumstances, it is clear that physiologically active substances such as various cytokines secreted from mesenchymal cells greatly contribute to the function of these mesenchymal stem cells transplanted in vivo in cell transplantation therapy. It has been.

間葉系幹細胞をインビトロで培養した際には、培養液中にこうした生理活性物質が放出されることになる。そこで、間葉系幹細胞の培養に使用した培養液を回収し、細胞から放出される物質を多く含むこの培養液を利用して、組織を再生すること等に成功した例が報告されている。上田らは、ラットを用いた実験で、骨髄間葉系幹細胞の培養上清が骨の再生能力を持つことを示した(非特許文献1)。この中で、骨髄間葉系幹細胞の培養上清中には、インスリン様成長因子(IGF)や血管内皮細胞増殖因子(VEGF)などが多く含まれており、これらの因子が組織の再生などに関わっていることが示唆されている(非特許文献1)。上田らは、この他にも、間葉系幹細胞の培養上清が創傷治癒効果、歯周組織再生促進効果を持つことを報告している(非特許文献2、非特許文献3)。 When mesenchymal stem cells are cultured in vitro, these bioactive substances are released into the culture medium. Therefore, it has been reported that the culture medium used for culturing mesenchymal stem cells was recovered, and the tissue was successfully regenerated by using this culture medium containing a large amount of substances released from the cells. In an experiment using rats, Ueda et al. Showed that the culture supernatant of bone marrow mesenchymal stem cells has the ability to regenerate bone (Non-Patent Document 1). Among these, the culture supernatant of bone marrow mesenchymal stem cells contains a large amount of insulin-like growth factor (IGF) and vascular endothelial growth factor (VEGF), and these factors are used for tissue regeneration and the like. It has been suggested that it is involved (Non-Patent Document 1). Ueda et al. Also reported that the culture supernatant of mesenchymal stem cells has a wound healing effect and a periodontal tissue regeneration promoting effect (Non-Patent Documents 2 and 3).

また、有村らは、骨髄間葉系幹細胞の培養上清が抗炎症作用を有し、腸炎の予防・治療効果を示すことを報告している(特許文献1)。さらに、Suらは、間葉系幹細胞の培養上清の抗アレルギー作用について報告している(非特許文献4)。 In addition, Arimura et al. Reported that the culture supernatant of bone marrow mesenchymal stem cells has an anti-inflammatory effect and exhibits a preventive / therapeutic effect on enteritis (Patent Document 1). Furthermore, Su et al. Reported the antiallergic effect of the culture supernatant of mesenchymal stem cells (Non-Patent Document 4).

更に最近では、細胞から分泌される、エキソソームと呼ばれる小胞が、様々なタンパク質やRNAを含み、これが細胞間の伝達を担う物質である可能性が指摘されている。
以上のことから、細胞培養においては、それによって得られる細胞だけでなく、培養上清もまた非常に貴重なものであり、それを効率よく作製することは極めて重要なことである。
More recently, it has been pointed out that vesicles called exosomes, which are secreted from cells, contain various proteins and RNA, which may be substances responsible for cell-cell transmission.
From the above, in cell culture, not only the cells obtained thereby but also the culture supernatant are extremely valuable, and it is extremely important to efficiently produce them.

特開2013−018756号公報Japanese Unexamined Patent Publication No. 2013-018756

Tissue Engineering PartA.2012;18:1479−1489Tissue Engineering Part A. 2012; 18: 1479-1489 Cytotherapy,2012;14(10):1171−1181Cytotherapy, 2012; 14 (10): 1171-1181 Cytotherapy,2015;17(4):369−381Cytotherapy, 2015; 17 (4): 369-381 The Journal of allergy and clinical immunology,2015;136(2):423−432The Journal of allergy and clinical immunology, 2015; 136 (2): 423-432

本発明の課題は、優れた組織再生能、あるいは優れた疾患治癒作用、あるいは高い生理活性を持つ、幹細胞の培養上清を作製する方法に関する。 An object of the present invention relates to a method for preparing a culture supernatant of stem cells having excellent tissue regeneration ability, excellent disease healing effect, or high physiological activity.

上述のように、間葉系幹細胞などの幹細胞を培養した培養液中には、種々の作用を有する生理活性物質等が含まれており、これらが組織再生や疾患治癒の効果に寄与している。こうした細胞の培養上清は、通常、フラスコやシャーレに細胞を植え込み、これを一定期間培養した後に、このとき培養に使用した培養液を回収することにより得られる。 As described above, the culture medium in which stem cells such as mesenchymal stem cells are cultured contains physiologically active substances having various actions, which contribute to the effects of tissue regeneration and disease healing. .. The culture supernatant of such cells is usually obtained by implanting the cells in a flask or a petri dish, culturing the cells for a certain period of time, and then collecting the culture solution used for the culture at this time.

この場合、培養液中に放出される生理活性物質の量は、細胞の数に比例して大きくなる。このため、細胞数が多ければ良いが、例えば間葉系幹細胞のような接着系細胞はシャーレやフラスコの底面に一層に広がって増殖するため、細胞から放出される生理活性物質を出来るだけ多く、高濃度に含む培養上清を回収するためには、この培養方法は決して効率が良いとは言えない。 In this case, the amount of the physiologically active substance released into the culture medium increases in proportion to the number of cells. For this reason, it is sufficient if the number of cells is large, but since adherent cells such as mesenchymal stem cells spread further to the bottom of the petri dish or flask and proliferate, as much bioactive substance as possible is released from the cells. In order to recover the culture supernatant contained in a high concentration, this culture method cannot be said to be efficient.

即ち、従来の細胞培養方法においては、細胞数に対し、接触する培養液の容量が大きいため、培養上清中に分泌される有効な成分は希釈され、その含有濃度は薄くなってしまう。このため、得られた培養上清を組織再生等に利用する際には、培養上清を濃縮するとか、凍結乾燥を行うなどの必要性が生ずる。 That is, in the conventional cell culture method, since the volume of the culture solution to be contacted is large with respect to the number of cells, the effective component secreted in the culture supernatant is diluted and its content concentration becomes thin. Therefore, when the obtained culture supernatant is used for tissue regeneration or the like, it is necessary to concentrate the culture supernatant or freeze-dry it.

こうした背景により、培養した細胞から分泌される生理活性物質をなるべく高濃度で含む培養上清を効率良く作製する培養形態および方法が求められている。 Against this background, there is a demand for a culture form and method for efficiently producing a culture supernatant containing a physiologically active substance secreted from cultured cells at a concentration as high as possible.

本発明者らは、上記課題に対し鋭意検討を行った結果、上記課題を解決できることを見出し、本発明を完成するに至った。即ち、本願発明の概要は以下の通りである。
1.幹細胞培養上清液の製造方法であって、以下の[a]から[c]の工程を含む、方法。
[a]細胞培養容器内の透過性膜中空糸の内表面に播種した幹細胞に培養液を0.01〜1mm/minの線速度で供給し、細胞が播種されていない側に培養液を0.02〜5mm/minの線速度で供給して前記幹細胞を培養する工程
[b]前記幹細胞を培養する際に、前記培養液に血清を添加した培養液を用いて前記幹細胞を増殖する工程
[c]増殖した幹細胞に無血清培養液または低血清培養液を接触させて得られた培養上清を回収する工程
2.前記細胞培養容器は、透過性膜中空糸を格納したモジュールであって、前記中空糸の内腔と連通する2つの開口部と、前記モジュールの内側かつ前記中空糸の外腔と連通する2つの開口部を有するものである、1に記載の方法。
3.以下の[d]から[g]の構成を含む細胞培養装置を用いて前記幹細胞の培養を行う、1または2に記載の方法。
[d]前記細胞培養容器
[e]少なくとも1つの培養液貯留容器
[f]前記細胞培養容器と前記培養液貯留容器とを接続する流路網
[g]前記流路網に設けられ、前記細胞培養容器への培養液の供給および/または前記細胞培養容器からの培養液の排出を制御する手段
4.前記透過性膜中空糸の孔半径が、1〜500Åであることを特徴とする、1から3のいずれかに記載の方法。
5.前記幹細胞が間葉系幹細胞であることを特徴とする、1からのいずれかに記載の方法。
As a result of diligent studies on the above problems, the present inventors have found that the above problems can be solved, and have completed the present invention. That is, the outline of the present invention is as follows.
1. 1. A method for producing a stem cell culture supernatant, which comprises the following steps [a] to [c].
[A] The culture solution is supplied to the stem cells seeded on the inner surface of the permeable membrane hollow thread in the cell culture vessel at a linear velocity of 0.01 to 1 mm / min, and the culture solution is 0 on the side where the cells are not seeded. A step of culturing the stem cells by supplying the cells at a linear velocity of .02 to 5 mm / min [b] A step of proliferating the stem cells using a culture solution in which serum is added to the culture solution when culturing the stem cells [b] c] Step of contacting the proliferated stem cells with a serum-free culture medium or a low-serum culture medium and collecting the obtained culture supernatant. The cell culture vessel is a module that stores a permeable membrane hollow fiber, and has two openings that communicate with the inner cavity of the hollow fiber and two openings that communicate with the inside of the module and the outer cavity of the hollow fiber. The method according to 1, wherein the method has an opening.
3. 3. The method according to 1 or 2, wherein the stem cells are cultured using a cell culture apparatus containing the following configurations [d] to [g].
[D] The cell culture container [e] At least one culture solution storage container [f] A flow path network connecting the cell culture container and the culture solution storage container [g] The cell provided in the flow path network. 4. Means for controlling the supply of the culture solution to the culture container and / or the discharge of the culture solution from the cell culture container. The method according to any one of 1 to 3, wherein the pore radius of the permeable membrane hollow fiber is 1 to 500 Å.
5. The method according to any one of 1 to 4 , wherein the stem cell is a mesenchymal stem cell.

例えば、従来法であるシャーレを用いた細胞培養においては、シャーレ底面に生える細胞は、細胞数が3〜4万個/cm程度に達する。直径約10cmのシャーレであれば、約2,000,000〜3,000,000個まで細胞が増殖し、これに対し10ml程度の培養液を用いる。直径10cm×高さ1.5cm、即ち容器容積120cmから、培養上清が10ml回収される。For example, in cell culture using dish a conventional method, cells that grows in a Petri dish bottom, the cell number reached approximately 30,000 to 40,000 pieces / cm 2. In a petri dish with a diameter of about 10 cm, about 2,000,000 to 3,000,000 cells grow, and about 10 ml of a culture solution is used. 10 ml of the culture supernatant is collected from a diameter of 10 cm × a height of 1.5 cm, that is, a container volume of 120 cm 3.

これに対し、本発明では、透過性膜中空糸のモジュールを用いた培養を行い、かつ、この中空糸モジュール内を極めて低速度で培養液を通過させるため、細胞数に比して回収する培養上清が従来法に比べ、大幅に少ない。例えば、数百本の透過性膜中空糸が収納された10cm長の太さ約1cmの中空糸モジュールにおいては、細胞数は10,000,000個以上に達するが、細胞に直接接して流れる培養液の総量は、10mlに満たず、対細胞数比は従来法の約1/5で、培養上清中に分泌される物質濃度は逆に約5倍となる。また、容器容積は約8cmであるため、容積あたりの回収効率も向上する。
即ち、本発明により、培養する間葉系幹細胞から分泌される生理活性物質を高濃度に含む培養上清液を、簡便に効率よく作製することが可能となった。
On the other hand, in the present invention, the culture using the permeable membrane hollow fiber module is performed, and the culture solution is passed through the hollow fiber module at an extremely low speed, so that the culture is collected in comparison with the number of cells. The amount of supernatant is significantly smaller than that of the conventional method. For example, in a 10 cm long hollow fiber module having a thickness of about 1 cm and containing hundreds of permeable membrane hollow fibers, the number of cells reaches 10,000,000 or more, but the culture flows in direct contact with the cells. The total volume of the liquid is less than 10 ml, the cell number ratio is about 1/5 of the conventional method, and the concentration of the substance secreted in the culture supernatant is conversely about 5 times. Moreover, since the container volume is about 8 cm 3 , the recovery efficiency per volume is also improved.
That is, according to the present invention, it has become possible to easily and efficiently prepare a culture supernatant containing a high concentration of a physiologically active substance secreted from mesenchymal stem cells to be cultured.

本発明の幹細胞培養上清製造実施に用いる中空糸モジュールの構成例を示すものである。The configuration example of the hollow fiber module used for carrying out the production of the stem cell culture supernatant of the present invention is shown. 本発明の幹細胞培養上清製造実施に用いる細胞培養形態の例を、模式図で示すものである。An example of the cell culture form used for carrying out the production of the stem cell culture supernatant of the present invention is shown in a schematic diagram. 本発明の実施例1における培養上清作製のスケジュールを示すものである。It shows the schedule of the culture supernatant preparation in Example 1 of this invention. 本発明の実施例1における培養上清中のVEGF濃度を示すものである。It shows the VEGF concentration in the culture supernatant in Example 1 of the present invention. 本発明の実施例1における培養上清中のIGF濃度を示すものである。It shows the IGF concentration in the culture supernatant in Example 1 of the present invention. 本発明の実施例1における培養上清によるTNF−α産生抑制効果を示すものである。It shows the effect of suppressing TNF-α production by the culture supernatant in Example 1 of the present invention.

本発明の実施態様の一つは、以下の[a]から[c]の工程を含む、幹細胞培養上清液(馴化培地、コンディションドメディウム)の製造方法である。
[a]細胞培養容器内の透過性膜中空糸表面に播種した幹細胞に培養液を供給する工程
[b]前記幹細胞に前記培養液を接触させて前記幹細胞を培養する工程
[c]前記幹細胞が分泌した成分を含む培養液を回収する工程
One of the embodiments of the present invention is a method for producing a stem cell culture supernatant (conditioned medium, conditioned medium), which comprises the following steps [a] to [c].
[A] A step of supplying a culture solution to stem cells seeded on the surface of a permeable membrane hollow thread in a cell culture vessel [b] A step of bringing the culture solution into contact with the stem cells to culture the stem cells [c] The stem cells Step of collecting the culture solution containing the secreted components

(細胞培養上清液)
本発明において、細胞の培養上清とは、細胞を一定期間(数時間から数日)培養した際に、細胞に直接または間接に接触していた培養液を細胞と分離して得られるものを言う。培養液馴化培地、コンディションドメディウム(Conditioned medium)などと同意である。
(Cell culture supernatant)
In the present invention, the cell culture supernatant is obtained by separating a culture solution that has been in direct or indirect contact with a cell from the cell when the cell is cultured for a certain period of time (several hours to several days). To tell. Consent with culture medium, Conditioned medium, etc.

(培養の対象となる幹細胞)
本発明に係る対象の一つとなる幹細胞としては、特に限定されるものではないが、骨髄間葉系幹細胞、あるいは脂肪組織由来間葉系幹細胞が好適である。プライマリー細胞に限らず、遺伝子改変等によって株化された間葉系幹細胞も用いることが出来る。動物種も特に限定されず、ヒト、マウス、ラット等のいずれの動物由来のものも使用できる。また、間葉系幹細胞以外の接着性を有する種々の体性幹細胞にも好適に使用できる。
(Stem cells to be cultured)
The stem cell that is one of the subjects of the present invention is not particularly limited, but bone marrow mesenchymal stem cells or adipose tissue-derived mesenchymal stem cells are preferable. Not limited to primary cells, mesenchymal stem cells established by gene modification or the like can also be used. The animal species is not particularly limited, and any animal-derived animal such as human, mouse, or rat can be used. Further, it can be suitably used for various somatic stem cells having adhesiveness other than mesenchymal stem cells.

(培養液)
本発明の幹細胞培養上清液の製造方法に用いる培養液の組成等は、特に限定されない。例えば、DMEM、αMEM、RPMI1640などを基礎培地とし、これに適宜、動物血、細胞増殖因子、ホルモンなどを添加することにより調製されたものが使用できる。
(Culture solution)
The composition of the culture solution used in the method for producing the stem cell culture supernatant of the present invention is not particularly limited. For example, DMEM, αMEM, RPMI1640 or the like is used as a basal medium, and those prepared by adding animal blood, cell growth factors, hormones and the like as appropriate can be used.

本発明に用いる培養液は、場合によっては動物血清を含まないことが好ましいことがある。これは、動物血清には細胞増殖因子等の生理活性物質が豊富に含まれるため、時にはこれらの生理活性物質の存在が、培養上清を使用する際に目的の妨げとなるとか、マイナスに作用するなどの可能性があるためである。 In some cases, the culture medium used in the present invention preferably does not contain animal serum. This is because animal serum is rich in bioactive substances such as cell growth factors, and sometimes the presence of these bioactive substances interferes with the purpose of using the culture supernatant or has a negative effect. This is because there is a possibility of doing so.

(透過性膜中空糸)
本発明で使用する透過性膜中空糸は、細胞を中空糸内腔に保持でき、溶液や低分子の物質を透過させるような構造をとることができるものであれば、特に限定されるものではない。
(Transparent membrane hollow fiber)
The permeable membrane hollow fiber used in the present invention is not particularly limited as long as it can hold cells in the hollow fiber lumen and can have a structure that allows a solution or a small molecule substance to permeate. No.

本発明において透過性膜中空糸を使用する理由の一つは、培養面積あたりの細胞成育数がシャーレ等のプラスチック素材よりも多いためである。何故なら、膜中空糸上に接着し増殖する細胞は、プラスチック上のように大きく伸展しないため、単位面積あたりに接着する細胞数は、膜中空糸上の方がプラスチック上よりも数倍多くなるためである。更に、膜中空糸を緊密に束ねることにより、単位体積あたりの培養面積を格段に増やすことが出来る。 One of the reasons for using the permeable membrane hollow fiber in the present invention is that the number of cells growing per culture area is larger than that of a plastic material such as a petri dish. This is because the cells that adhere to and proliferate on the hollow fiber membrane do not stretch as much as on the plastic, so the number of cells that adhere to the hollow fiber membrane per unit area is several times higher on the hollow fiber membrane than on the plastic membrane. Because. Further, by tightly bundling the membrane hollow fibers, the culture area per unit volume can be remarkably increased.

本発明で使用する透過性膜中空糸の素材は、特に限定されないが、例えば、セルロースアセテート、セルローストリアセテートおよび再生セルロースなどのセルロース系材料、ポリスルホンおよびポリエーテルスルホンなどのポリスルホン系材料、ポリエチレン、ポリプロピレン、ポリスチレン、ポリビニルアルコール、ポリメチルメタクリレート、ポリアクリロニトリル、フッ素系樹脂、ポリアミド、ポリフッ化ビニリデン(PVDF)等の熱可塑性高分子による骨格構造を有する不溶性担体が好適に利用できる。また、これらの誘導体が主成分であっても良い。 The material of the permeable membrane hollow yarn used in the present invention is not particularly limited, and for example, cellulose-based materials such as cellulose acetate, cellulose triacetate and regenerated cellulose, polysulfone-based materials such as polysulfone and polyethersulfone, polyethylene, polypropylene, and the like. An insoluble carrier having a skeletal structure made of a thermoplastic polymer such as polystyrene, polyvinyl alcohol, polymethylmethacrylate, polyacrylonitrile, fluororesin, polyamide, and polyvinylidene fluoride (PVDF) can be preferably used. Further, these derivatives may be the main component.

本発明で使用する透過性膜中空糸は、前記の素材に化学的に修飾を加えたものであっても良い。例えば、本発明で使用する透過性膜中空糸は、親水化処理されていることが好ましい。透過性膜中空糸を親水化処理することにより、培養細胞への培養液等の液体成分の供給が容易になる。透過性膜中空糸を親水化処理する方法としては、例えば、透過性膜中空糸をエチレン−ビニルアルコール共重合体等の親水性高分子や、グリセリン、エタノールで処理する方法が挙げられる。あるいは、使用する細胞に応じて、中空糸への接着向上のため、コラーゲンやフィブロネクチン等のコーティング剤を使用しても良い。 The permeable membrane hollow fiber used in the present invention may be a material obtained by chemically modifying the above-mentioned material. For example, the permeable membrane hollow fiber used in the present invention is preferably hydrophilized. By hydrophilizing the permeable membrane hollow fiber, it becomes easy to supply a liquid component such as a culture solution to cultured cells. Examples of the method for hydrophilizing the permeable membrane hollow fiber include a method for treating the permeable membrane hollow fiber with a hydrophilic polymer such as an ethylene-vinyl alcohol copolymer, glycerin, or ethanol. Alternatively, depending on the cells used, a coating agent such as collagen or fibronectin may be used to improve the adhesion to the hollow fiber.

本発明で使用する透過性膜中空糸の大きさについても特に制限はないが、中空糸の内径の好ましい下限は10μmであり、さらに好ましくは20μm、さらに好ましくは50μmである。一方、中空糸の内径の好ましい上限は1000μm、より好ましくは500μmである。膜厚についても、特に制限はないが、中空糸が適度の強度を保ち、かつ物質の透過性も良好な範囲で設定すればよく、例えば10〜200μm程度が好ましい。 The size of the permeable membrane hollow fiber used in the present invention is also not particularly limited, but the lower limit of the inner diameter of the hollow fiber is preferably 10 μm, more preferably 20 μm, and further preferably 50 μm. On the other hand, the preferable upper limit of the inner diameter of the hollow fiber is 1000 μm, more preferably 500 μm. The film thickness is also not particularly limited, but the hollow fiber may be set within a range in which the hollow fiber maintains an appropriate strength and the permeability of the substance is also good, and is preferably about 10 to 200 μm, for example.

本発明で使用する透過性膜中空糸の孔径は、細胞は通過させないが水、塩類、比較的分子量の小さい栄養分などの培養液成分は通過させる通孔であれば、特に限定されるものではないが、細胞の培養、ならびに培養上清を回収することを考慮すると、物質交換がある程度効率的な孔径を有し、かつ細胞が産生した生理活性物質等の有用成分を透過しないことが必要である。そのため、透過性膜中空糸の孔径(孔半径)は、1〜500Åが好ましく、5〜300Åがより好ましく、10〜150Åがさらに好ましい。 The pore size of the permeable membrane hollow thread used in the present invention is not particularly limited as long as it allows cells to pass through but allows passage of culture solution components such as water, salts, and nutrients having a relatively small molecular weight. However, considering cell culture and recovery of culture supernatant, it is necessary that substance exchange has a pore size that is efficient to some extent and that useful components such as physiologically active substances produced by cells do not permeate. .. Therefore, the pore diameter (hole radius) of the permeable membrane hollow fiber is preferably 1 to 500 Å, more preferably 5 to 300 Å, and even more preferably 10 to 150 Å.

透過性膜中空糸の孔径は、透過性膜中空糸の透過性と密接に関連する。透過性膜中空糸の透過性は、例えば、単位時間・単位面積あたりの液通過量で示される。本発明で使用する透過性膜中空糸において、透過性は特に限定されるものではないが、例えば、1〜1000ml/m/hr/mmHg、更に好ましくは10〜500ml/m/hr/mmHgが好適である。The pore size of the permeable membrane hollow fiber is closely related to the permeability of the permeable membrane hollow fiber. Permeability The permeability of the hollow fiber membrane is indicated by, for example, the amount of liquid passing per unit time and unit area. In the permeable membrane hollow fiber used in the present invention, the permeability is not particularly limited, but for example, 1 to 1000 ml / m 2 / hr / mmHg, more preferably 10 to 500 ml / m 2 / hr / mmHg. Is preferable.

(幹細胞培養上清液の製造工程)
本発明の幹細胞培養上清液の製造方法は、以下の工程を含む。
[a]透過性膜中空糸内腔に存在する幹細胞に培養液を供給する工程
[b]前記培養液を連続的または間欠的に前記中空糸内腔を移動(通過)させることにより前記幹細胞に前記培養液を接触させて前記幹細胞を培養する工程
[c]前記工程により得られた培養液を回収する工程
その順序は、例えば[a][b][c]の順に行えばよいが、操作の途中で2つ以上の工程が重複してもよい。ただし、[b]の工程開始が[a]の工程開始に先んじることはなく、[c]の工程開始が[b]の工程開始に先んじることはない。
(Manufacturing process of stem cell culture supernatant)
The method for producing a stem cell culture supernatant of the present invention includes the following steps.
[A] Step of supplying the culture solution to the stem cells existing in the permeable membrane hollow fiber cavity [b] The culture solution is continuously or intermittently moved (passed) through the hollow fiber cavity to the stem cells. Step of culturing the stem cells by contacting the culture broth [c] Step of collecting the culture broth obtained by the step The order may be, for example, [a] [b] [c]. Two or more steps may be overlapped in the middle of. However, the start of the process of [b] does not precede the start of the process of [a], and the start of the process of [c] does not precede the start of the process of [b].

本発明の幹細胞培養上清液の製造方法は、前記以外の制限は特になく、例えば、前記中空糸を用い、幹細胞の培養を中空糸内腔側で行えば良い。たとえば、幹細胞を培養する際は、幹細胞縣濁液を中空糸内腔に注入すること等により幹細胞を中空糸表面に播種し、播種終了後、培養液を幹細胞が存在する中空糸内腔に灌流させる等して供給する。次いで、内腔側にて培養を行う。たとえば、供給された培養液を、連続的または間欠的に前記中空糸内腔を移動(通過)させることにより、前記幹細胞に前記培養液を接触させて前記幹細胞を培養する。なお、間欠的な移動(通過)とは、一時的に培養液の中空糸内での流れを止めたり進めたりする工程を繰り返すことを指す。ここで流れを止めたり進めたりする間隔設定は特に制限されず、等間隔でも不規則でもよい。培養液は、細胞に必要な養分や酸素などを供給し、逆に老廃物を排出する役割を有する。培養期間中、ガス交換および、細胞への栄養供給、老廃物除去のため、内腔側、外腔側ともに、培地を供給し続けることが好ましい。その際、ペリスタポンプ等を用いることにより、適切な速度で供給・排出し、あるいは循環させることができる。このようにして一定期間培養を行った後の培養液を回収すれば、本発明の幹細胞培養上清液を得ることができる。 The method for producing the stem cell culture supernatant of the present invention is not particularly limited, and for example, the stem cells may be cultured on the hollow fiber lumen side using the hollow fiber. For example, when culturing stem cells, the stem cells are seeded on the surface of the hollow fiber by injecting a stem cell turbid solution into the hollow fiber lumen, and after the seeding is completed, the culture solution is perfused into the hollow fiber lumen where the stem cells are present. Supply by letting. Next, culture is performed on the lumen side. For example, the supplied culture solution is continuously or intermittently moved (passed) through the hollow fiber lumen to bring the culture solution into contact with the stem cells to culture the stem cells. The intermittent movement (passage) refers to repeating the process of temporarily stopping or advancing the flow of the culture solution in the hollow fiber. Here, the interval setting for stopping or advancing the flow is not particularly limited, and may be evenly spaced or irregular. The culture solution has a role of supplying necessary nutrients and oxygen to cells, and conversely discharging waste products. During the culture period, it is preferable to continue to supply the medium on both the luminal side and the outer luminal side for gas exchange, nutrient supply to cells, and removal of waste products. At that time, by using a perista pump or the like, it is possible to supply / discharge or circulate at an appropriate speed. By collecting the culture broth after culturing for a certain period of time in this way, the stem cell culture supernatant of the present invention can be obtained.

(中空糸モジュール)
本発明の幹細胞培養上清液の製造方法で使用する透過性膜中空糸は、筒状容器に数十本〜数万本の中空糸が束ねられた中空糸束を格納してモジュール化し、細胞培養容器としてもよい(このようなモジュールを、以下、中空糸モジュールとも呼ぶ)。中空糸モジュールには、培養基材に培地を供給することができる開口部と、培地を排出することができる開口部とを、それぞれ1つ以上有している必要がある。
(Hollow fiber module)
The permeable membrane hollow fiber used in the method for producing the stem cell culture supernatant of the present invention is modularized by storing a hollow fiber bundle in which dozens to tens of thousands of hollow fibers are bundled in a tubular container to form cells. It may be used as a culture container (hereinafter, such a module is also referred to as a hollow fiber module). The hollow fiber module needs to have at least one opening capable of supplying the medium to the culture substrate and one opening capable of discharging the medium.

このような透過性膜中空糸を用いたモジュールの構成は特に限定されないが、例えば図1に示すように、4つの開口部(端部導管および側部導管)を有するモジュールケース1本体に透過性膜中空糸2が適宜必要な本数束ねられて充填されている形態が挙げられる。この形態においては、前記4つの開口部のうち、2つの端部導管3はそれぞれ前記中空糸束の両端において各中空糸の中空部を閉塞しないように適当なシール材(例えば、ポリウレタン系ポッティング剤)により接続されており、前記端部導管3の一方から導入された液体などが中空糸内腔を通ってもう一方の端部導管3から導出される(すなわち、一方向に流れる)ように構成されている。一方、前記開口部のうち、残りの2つの側部導管4の内側の空間はそれぞれ前記モジュールケース1の内側であってかつ前記中空糸の外腔である空間(以下、単に「外腔側」とも呼ぶ。)と接続しており、前記側部導管4の一方から導入された液体などが膜中空糸の外腔側を通ってもう一方の側部導管4から導出される(すなわち、一方向に流れる)ように構成されている。 The configuration of the module using such a permeable membrane hollow fiber is not particularly limited, but as shown in FIG. 1, for example, it is permeable to the main body of the module case 1 having four openings (end conduit and side conduit). Examples thereof include a form in which a required number of hollow fiber membranes 2 are bundled and filled. In this embodiment, of the four openings, the two end conduits 3 are suitable sealing materials (for example, polyurethane-based potting agents) so as not to block the hollow portions of the hollow fibers at both ends of the hollow fiber bundle. ), And the liquid or the like introduced from one of the end conduits 3 is led out from the other end conduit 3 (that is, flows in one direction) even though it passes through the hollow fiber lumen. Has been done. On the other hand, in the opening, the space inside the remaining two side ducts 4 is the space inside the module case 1 and the outer cavity of the hollow fiber (hereinafter, simply "outer cavity side"). It is connected to (also referred to as), and liquid or the like introduced from one of the side ducts 4 is led out from the other side duct 4 even though it passes through the outer cavity side of the membrane hollow fiber (that is, one direction). It is configured to flow to).

本発明の幹細胞培養上清液の製造方法に、培養容器として前記中空糸モジュールを用いる場合、例えば、幹細胞懸濁液を前記端部導管より注入することにより播種し、播種終了後、培養液を幹細胞が存在する中空糸内腔に灌流させる等して供給すればよい。また、培養時には、中空糸内腔側への培養液の灌流と同時に、中空糸外腔側へも培地を側部導管より注入し灌流させることが好ましい。 When the hollow thread module is used as the culture vessel in the method for producing the stem cell culture supernatant of the present invention, for example, the stem cell suspension is seeded by injecting it from the end conduit, and after the seeding is completed, the culture solution is used. It may be supplied by perfusing the hollow thread lumen in which the stem cells are present. Further, at the time of culturing, it is preferable that the culture medium is perfused to the hollow fiber lumen side at the same time as the medium is injected from the side duct to the hollow fiber outer lumen side.

前記中空糸または中空糸モジュールは、適切な方法で滅菌し供給されることが好ましい。滅菌方法については、特に制限はないが、例えば、加圧加熱滅菌、ガンマ線滅菌、エチレンオキサイドガス滅菌等が挙げられる。 The hollow fiber or hollow fiber module is preferably sterilized and supplied by an appropriate method. The sterilization method is not particularly limited, and examples thereof include pressure heat sterilization, gamma ray sterilization, and ethylene oxide gas sterilization.

培養容器に中空糸モジュールを用いることにより、本発明の実施がより効率的に実施可能となる。中空糸モジュール内腔の細胞へ新鮮な培地を低速度で均一に供給することができ、培養上清の回収が効率良く実施できる。 By using the hollow fiber module in the culture vessel, the present invention can be carried out more efficiently. The fresh medium can be uniformly supplied to the cells in the lumen of the hollow fiber module at a low speed, and the culture supernatant can be efficiently collected.

(本発明に用いる細胞培養システム)
本発明の幹細胞培養上清液の製造方法においては、以下の[d]から[g]の構成を含む細胞培養システムを用いて培養を行えばよい。
[d]前記細胞培養容器
[e]少なくとも1つの培養液貯留容器
[f]前記細胞培養容器と前記培養液貯留容器とを接続する流路網
[g]前記流路網に設けられ、前記細胞培養容器への培養液の供給、および/または前記細胞培養容器からの培養液の排出を制御する手段
(Cell culture system used in the present invention)
In the method for producing a stem cell culture supernatant of the present invention, culturing may be performed using a cell culture system containing the following configurations [d] to [g].
[D] The cell culture container [e] At least one culture solution storage container [f] A flow path network connecting the cell culture container and the culture solution storage container [g] The cell provided in the flow path network. Means for controlling the supply of the culture solution to the culture container and / or the discharge of the culture solution from the cell culture container.

前記システムで用いる細胞培養容器については、多孔性膜中空糸を用いたモジュールであって、前記中空糸の両端からそれぞれ内腔と接続する2つの開口部と、外腔と接続する2つの開口部を有する細胞培養容器であれば、特に限定されない。 The cell culture vessel used in the system is a module using a porous membrane hollow fiber, and has two openings connected to the inner cavity and two openings connected to the outer cavity from both ends of the hollow fiber. It is not particularly limited as long as it is a cell culture vessel having.

(培養液貯留容器)
前記システムで用いる培養液貯留容器の形態や素材については特に限定されないが、ガス透過性を有することが望ましい。前記システムの一つの態様においては、前記中空糸モジュールを含む培養装置を構成し、それをCOインキュベーター内に設置して培養上清の作製を実施してもよいが、その際、ガス透過性を有する培養液貯留容器を用いれば、気泡発生などの問題点を有するガス交換用チューブを設置することなく、簡便に培養を行うことができる。このような培養液貯留容器としては、例えば、市販の細胞培養バッグ(ニプロ社製、東洋製罐社製のものなどが入手できる。)、プラスチック製ボトルなどが挙げられる。
(Culture storage container)
The form and material of the culture solution storage container used in the system are not particularly limited, but it is desirable that the culture solution storage container has gas permeability. In one aspect of the system, a culture apparatus including the hollow thread module may be configured and placed in a CO 2 incubator to prepare a culture supernatant, in which case gas permeability is achieved. By using the culture solution storage container having the above, it is possible to easily carry out the culture without installing a gas exchange tube having a problem such as bubble generation. Examples of such a culture solution storage container include commercially available cell culture bags (manufactured by Nipro, Toyo Seikan, etc. are available), plastic bottles, and the like.

(流路網)
前記システムにおいて、前記細胞培養容器にはいくつかの流路(流路網)が接続している。その構成(配管)は特に限定されないが、少なくとも前記細胞培養容器と前記液体培地貯留容器との接続部分を含み、前記液体培地貯留容器から前記液体培地貯留容器への培地の供給が可能なように配管されている必要がある。このような流路網として、少なくとも(1)前記細胞培養容器と前記培養液貯留容器の中空糸内腔側の開口部との接続部分、および(2)前記細胞培養容器と前記培養液貯留容器の中空糸外腔側の開口部との接続部分を含む流路網、が挙げられる。なお、本明細書において「接続」とは、直接繋がっている場合、および、配管などの流路を介して繋がっている場合のいずれであってもよい。
(Channel network)
In the system, several channels (channel networks) are connected to the cell culture vessel. The configuration (pipe) is not particularly limited, but includes at least a connection portion between the cell culture container and the liquid medium storage container so that the medium can be supplied from the liquid medium storage container to the liquid medium storage container. Must be piped. As such a flow path network, at least (1) the connection portion between the cell culture container and the opening on the hollow thread lumen side of the culture solution storage container, and (2) the cell culture container and the culture solution storage container. A flow path network including a connection portion with an opening on the outer cavity side of the hollow thread. In addition, in this specification, "connection" may be either the case where it is directly connected or the case where it is connected through a flow path such as a pipe.

前記システムに用いる配管の素材は特に限定されない。例えば、シリコンチューブや、マスターフレックス(登録商標)、C−フレックス(登録商標)、タイゴン(登録商標)、ファーメド(登録商標)BPT、ファーマピュア(登録商標)、ノルプレン(登録商標)、サニテック(登録商標)などを用いることができる。前記液体培地貯留容器と前記細胞培養容器とを接続する部分の流路管については、ガス透過性を有する流路管を用いることが好ましい。例えば、シリコンチューブ等が好適に利用できる。 The material of the piping used in the system is not particularly limited. For example, Silicon Tube, Masterflex (registered trademark), C-Flex (registered trademark), Tygon (registered trademark), Farmed (registered trademark) BPT, Pharmapure (registered trademark), Norprene (registered trademark), Sanitech (registered trademark). Trademark) and the like can be used. As the flow path tube of the portion connecting the liquid medium storage container and the cell culture container, it is preferable to use a flow path tube having gas permeability. For example, a silicon tube or the like can be preferably used.

(培養液の供給、排出などを制御する手段)
前記システムには、前記流路網に設けられ、前記細胞培養容器への培養液の供給、および/または前記細胞培養容器からの培養液の排出を制御する手段が設けられている。細胞懸濁液や培養液の供給方法の形態は、特に限定されるものではないが、ペリスタポンプを用いた灌流や、培養液貯留容器が培養バッグなど軟質のものである場合は前記容器にローラーを当てて培地を絞り出す方法等が好適である。ポンプの設置場所は特に限定されないが、液体がポンプ部分を通過する際にゴミが混入する等のリスクを避けるため、排出側に設置することが好ましい。
(Means to control the supply and discharge of the culture solution)
The system is provided with means provided in the channel network to control the supply of the culture solution to the cell culture vessel and / or the discharge of the culture solution from the cell culture vessel. The form of the method for supplying the cell suspension or the culture medium is not particularly limited, but perfusion using a perista pump or when the culture medium storage container is a soft one such as a culture bag, a roller is placed in the container. A method of squeezing out the medium by hitting it is preferable. The location of the pump is not particularly limited, but it is preferable to install the pump on the discharge side in order to avoid the risk of dust being mixed in when the liquid passes through the pump portion.

(細胞培養システムの一態様)
上記の中空糸モジュールや培養液貯留容器、ペリスタポンプなどにより構成される培養システムをCOインキュベーター内に設置し、本発明の実施の一つの態様例としたものを図2に示す。図2において、5および6はそれぞれ、ガス透過性を有する培養液貯留容器(細胞培養バッグなど)である。培養液貯留容器5からは、中空糸モジュール7の端部導管3aに、流路が接続され、培養液貯留容器5に入っている培地が中空糸内腔側に移送できるようになっている。他方、培養液貯留容器6からは、中空糸モジュール7の側部導管4aに、流路が接続され、培養液貯留容器6に入っている培地が中空糸外腔側に移送できるようになっている。中空糸モジュール7の内腔側を通過した培養液は、端部導管3bから排出され、ペリスタポンプ8を経由して、培養上清回収容器11まで流路が接続され、中空糸モジュール内腔を通過した培養上清が回収できるようになっている。一方、中空糸モジュール7の外腔側を通過した培養液は、側部導管4bから排出され、ペリスタポンプ9を経由して、廃液回収容器10に回収される。
(One aspect of cell culture system)
FIG. 2 shows an example of an embodiment of the present invention in which a culture system composed of the above-mentioned hollow fiber module, culture solution storage container, perista pump, etc. is installed in a CO 2 incubator. In FIG. 2, 5 and 6 are gas-permeable culture medium storage containers (cell culture bag, etc.), respectively. From the culture solution storage container 5, a flow path is connected to the end conduit 3a of the hollow thread module 7, so that the medium contained in the culture solution storage container 5 can be transferred to the hollow thread lumen side. On the other hand, from the culture solution storage container 6, a flow path is connected to the side conduit 4a of the hollow fiber module 7, so that the medium contained in the culture solution storage container 6 can be transferred to the hollow fiber outer cavity side. There is. The culture solution that has passed through the lumen side of the hollow fiber module 7 is discharged from the end conduit 3b, and the flow path is connected to the culture supernatant collection container 11 via the perista pump 8 and passes through the hollow fiber module lumen. The culture supernatant can be collected. On the other hand, the culture solution that has passed through the outer cavity side of the hollow fiber module 7 is discharged from the side conduit 4b and collected in the waste liquid recovery container 10 via the perista pump 9.

(中空糸内腔における幹細胞の培養)
前記システムを用いて本発明の幹細胞培養上清液の製造方法を実施する場合、例えば、前記の透過性膜中空糸モジュールを用いる場合、中空糸への培地の供給は、内腔側および外腔側の2ルートが必要である。その際、培地の供給にあたり、培養液貯留容器は内腔側および外腔側で別々のものを用いても良いし、1つの容器から内腔側および外腔側の両方に培地を供給しても良い。また、中空糸内腔側と外腔側の水圧差や浸透圧差により、内腔から外腔、あるいは外腔から内腔へと培養液が流れることは極力抑えることが望ましい。何故なら、中空糸内腔を流れてきた培養液には、細胞が放出した物質が多く含まれるのに対し、中空糸外腔側には細胞が存在しないため、外腔側を流れる培養液中には細胞が放出する物質は、極めて少量であるためである。即ち、中空糸の一端の開口部から内腔へと流入した培養液は、なるべく外腔への流出や外腔からの流入の干渉などを受けず、そのまま、もう一方の開口部から流出していくことが望ましい。
(Culturing stem cells in the hollow fiber lumen)
When the method for producing the stem cell culture supernatant of the present invention is carried out using the system, for example, when the permeable membrane hollow fiber module is used, the medium is supplied to the hollow fiber on the luminal side and the outer cavity. Two routes on the side are required. At that time, when supplying the medium, separate culture medium storage containers may be used on the lumen side and the outer lumen side, or the medium is supplied from one container to both the lumen side and the outer lumen side. Is also good. Further, it is desirable to suppress the flow of the culture solution from the lumen to the lumen or from the lumen to the lumen as much as possible due to the water pressure difference or the osmotic pressure difference between the hollow fiber lumen side and the outer lumen side. This is because the culture medium flowing through the hollow thread lumen contains a large amount of substances released by cells, whereas the culture solution flowing through the hollow thread outer cavity side contains no cells. This is because the amount of substances released by cells is extremely small. That is, the culture solution that has flowed into the lumen from the opening at one end of the hollow fiber is not affected by the outflow to the outer cavity or the interference of the inflow from the outer cavity as much as possible, and flows out from the other opening as it is. It is desirable to go.

(培養上清を作製するための培養液)
培養に用いられる培養液は特に限定されず、間葉系幹細胞の培養液として通常用いられるものであれば良いが、幹細胞の培養上清液を治療等の目的で生体に適用する場合には、培養液中の動物血清がなるべく少ない、あるいは動物血清を含まないことが望ましい。このため、血清を含む培養液を用いて幹細胞を培養する場合、充分に細胞が増えた状態になったところで低血清培養液や無血清培養液に交換し、最終的に血清含有量が少ない、または血清を含まない培養上清液を得ることが出来る。また、無血清でも幹細胞の培養を可能とするよう成分を調整された培養液を用いて最初から細胞培養を行い、血清を含まない培養上清液を得ることも可能である。
(Culture solution for preparing culture supernatant)
The culture medium used for culturing is not particularly limited as long as it is usually used as a culture medium for mesenchymal stem cells, but when the culture supernatant of stem cells is applied to a living body for the purpose of treatment or the like, it may be used. It is desirable that the culture medium contains as little animal serum as possible or does not contain animal serum. Therefore, when culturing stem cells using a culture medium containing serum, the cells are replaced with a low serum culture medium or a serum-free culture medium when the cells are sufficiently increased, and finally the serum content is low. Alternatively, a culture supernatant containing no serum can be obtained. It is also possible to obtain a serum-free culture supernatant by culturing cells from the beginning using a culture medium whose components have been adjusted so that stem cells can be cultured even without serum.

(培養上清の回収)
前記のような理由により、本発明により培養上清を得るには、中空糸内腔を流れてきた培養液は、中空糸外腔を流れてきた培養液とは区別して、容器に回収することが好ましい。
(Recovery of culture supernatant)
For the above reasons, in order to obtain the culture supernatant according to the present invention, the culture solution flowing through the hollow fiber lumen should be collected in a container separately from the culture solution flowing through the hollow fiber outer cavity. Is preferable.

(培養液が流れる速度)
細胞培養における培養液、特に中空糸内腔を細胞に接して流れる培養液の流速については、流速を厳密に制御することが好ましい。流速が遅すぎると、細胞への栄養供給が十分になされず、細胞が増殖しにくくなる。逆に、流速が速すぎても、細胞周囲の環境変化が激しく、細胞が周りの環境に馴染めず、細胞が増殖しにくくなる。このように、培養液、特に中空糸内腔を流れる培養液の流速は、細胞増殖度合いや環境に応じて、調整することが好ましい。細胞増殖度合いを調べる方法は、特に限定されないが、培養液中のグルコースや乳酸塩の濃度等の測定結果をもとに行うことが出来る。細胞が播種された側に流す培養液の好ましい流速は、0.01〜1mm/minである。一方、細胞が播種されていない側に流す培養液の好ましい流速は、0.02〜5mm/minである。
(Speed at which the culture solution flows)
It is preferable to strictly control the flow velocity of the culture medium in cell culture, particularly the culture medium flowing in contact with the cells through the hollow thread lumen. If the flow velocity is too slow, the nutrient supply to the cells is insufficient and the cells are difficult to proliferate. On the contrary, even if the flow velocity is too fast, the environment around the cell changes drastically, the cell does not adapt to the surrounding environment, and the cell becomes difficult to proliferate. As described above, the flow velocity of the culture solution, particularly the culture solution flowing through the hollow fiber lumen, is preferably adjusted according to the degree of cell proliferation and the environment. The method for examining the cell proliferation degree is not particularly limited, but it can be performed based on the measurement results such as the concentration of glucose and lactate in the culture solution. The preferred flow rate of the culture medium flowing to the side where the cells are seeded is 0.01 to 1 mm / min. On the other hand, the preferable flow rate of the culture solution to be flowed to the side where the cells are not seeded is 0.02 to 5 mm / min.

本発明において、中空糸内腔を流れる培養液、即ち、前記培養液貯留容器から中空糸モジュールの中空糸内腔を通過し、培養上清回収容器へと流れる培養液の流れは、一方向であることが好ましい。ここで、一方向とは、中空糸モジュールへの培地の導入から導出のルートが常に同じ方向であることを言う。具体的には、例えば、中空糸モジュールにおける培地の導入口と導出口がそれぞれ1つずつ存在し、培地が常に導入口から導出口に向けて流れる形態が挙げられる。一方、中空糸の外腔側を流れる培養液については、一方向に流れても良いし、一旦、細胞培養容器から導出された培地が再度導入口から導入される、いわゆる循環式であってもよい。 In the present invention, the flow of the culture solution flowing through the hollow thread lumen, that is, the culture solution flowing from the culture solution storage container through the hollow thread lumen of the hollow thread module to the culture supernatant recovery container is unidirectional. It is preferable to have. Here, unidirectional means that the route from the introduction of the medium into the hollow fiber module to the derivation is always the same direction. Specifically, for example, there is a form in which there is one inlet and one outlet for the medium in the hollow fiber module, and the medium always flows from the inlet to the outlet. On the other hand, the culture solution flowing on the outer cavity side of the hollow fiber may flow in one direction, or may be a so-called circulation type in which the medium once drawn out from the cell culture vessel is introduced again from the introduction port. good.

(1)中空糸モジュールによる間葉系幹細胞の培養及び培養上清の作製
細胞播種前に予めコラーゲン(新田ゼラチン)をコートした中空糸モジュール(中空糸はPES/PVP製、内径200μm、外径260μm、膜厚30μm、孔半径75Å、東洋紡)を用い、図2に示すものと同様の構成装置をCOインキュベーター内に設置し、本実施例を行った。中空糸内腔にヒト骨髄間葉系幹細胞(CELL APPLICATIONS Inc.)を播種(播種細胞数は、5.0×10cells/モジュール)した。このとき、中空糸モジュール(中空糸内径基準)の総培養面積は98cmであるため、細胞播種密度は、約5100cells/cmであった。培養液は、培養開始(細胞播種)から96時間後までは、10%ウシ胎児血清(ライフテクノロジーズ)を添加したDMEM GlutaMAX(ライフテクノロジーズ)を用い、培養上清を採取する96時間以降は、MF−medium,間葉系幹細胞増殖培地(東洋紡)を用いた。MF−mediumは、血清を1%しか含まない低血清培養液である。
(1) Culturing mesenchymal stem cells using a hollow fiber module and preparing a culture supernatant A hollow fiber module (hollow fiber is made of PES / PVP, inner diameter 200 μm, outer diameter) coated with collagen (Nitta Gelatin) in advance before cell seeding. Using 260 μm, a film thickness of 30 μm, a hole radius of 75 Å, and Toyo Boseki), the same components as those shown in FIG. 2 were installed in a CO 2 incubator, and this example was carried out. Seeded hollow fiber lumen human bone marrow mesenchymal stem cells (CELL APPLICATIONS Inc.) (seeding cell number, 5.0 × 10 5 cells / modules) was. At this time, since the total culture area of the hollow fiber module (based on the inner diameter of the hollow fiber) was 98 cm 2 , the cell seeding density was about 5100 cells / cm 2 . As the culture medium, DMEM GlutaMAX (Life Technologies) supplemented with 10% fetal bovine serum (Life Technologies) was used from the start of culture (cell seeding) to 96 hours after the culture supernatant was collected, and after 96 hours, MF was used. -Medium, mesenchymal stem cell growth medium (Toyo Boseki) was used. MF-medium is a low serum culture medium containing only 1% serum.

図3に、本発明の方法による培養上清作製のスケジュールを示す。細胞播種(培養開始)から7日間(168時間後)の培養を実施した。この間、中空糸内腔を流れる培養液の流速(線速度)は、細胞播種を行ってから96時間後までは、平均0.066mm/min、96時間後から144時間後までは、平均0.20mm/min、144時間後から168時間後までは、平均0.33mm/minとした。一方、中空糸外腔を流れる培養液の速度は、培養開始から終了まで、3.4mm/minとした。細胞培養上清は、培養開始96時間後から168時間後までの72時間分を回収し、後の性能評価に用いるまで、4℃に保存した。培養上清の量は、計7.9mlであった。尚、流速については、中空糸内腔、外腔それぞれから流出する流量を測定し、中空糸断面積等をもとに算出した。培養から168時間後に細胞をトリプシンで消化、剥離回収し、細胞数をカウントした結果、1.2×10個の細胞が回収された。FIG. 3 shows a schedule for preparing a culture supernatant by the method of the present invention. Culturing was carried out for 7 days (168 hours later) from cell seeding (start of culturing). During this period, the flow velocity (linear velocity) of the culture solution flowing through the hollow thread lumen was 0.066 mm / min on average from 96 hours after cell seeding, and 0. From 20 mm / min, 144 hours to 168 hours, the average was 0.33 mm / min. On the other hand, the velocity of the culture solution flowing through the outer cavity of the hollow fiber was 3.4 mm / min from the start to the end of the culture. The cell culture supernatant was collected for 72 hours from 96 hours to 168 hours after the start of culture, and stored at 4 ° C. until it was used for later performance evaluation. The total amount of culture supernatant was 7.9 ml. The flow velocity was calculated based on the cross-sectional area of the hollow fiber by measuring the flow rate flowing out from each of the hollow fiber lumen and the outer cavity. Digested cells 168 hours after incubation with trypsin, peeling recovered, the results obtained by counting the number of cells, 1.2 × 10 7 cells were recovered.

(2)シャーレを用いた間葉系幹細胞の培養及び培養上清の作製(従来法)
2枚のコラーゲンコートシャーレ(培養面積55cm、旭テクノガラス)にヒト骨髄間葉系幹細胞(CELL APPLICATIONS Inc.)を細胞播種密度が約5100cells/cmとなるよう播種した。培養液は、実施例1と同様に、細胞播種から96時間までは、10%ウシ胎児血清(ライフテクノロジーズ)を添加したDMEM GlutaMAX(ライフテクノロジーズ)を用い、96時間以降はMF−medium,間葉系幹細胞増殖培地(東洋紡)に培地を交換した。
図3に、従来法による培養上清作製のスケジュールを示す。培養開始48時間後、および96時間後に、培養液交換を実施した。その後、培養液交換をせず、培養開始から168時間で100%コンフルエントに達したところで培養を終了した。この最後の培地交換から培養終了までの72時間の培養を行った培養液を培養上清として回収した。培養上清の量は、計10.0mlであった。培養から168時間後に細胞をトリプシンで消化、剥離回収し、細胞数をカウントした結果、2.6×10個の細胞が回収された。
(2) Culturing mesenchymal stem cells using a petri dish and preparing a culture supernatant (conventional method)
Human bone marrow mesenchymal stem cells (CELL APPLICATIONS Inc.) were seeded on two collagen-coated petri dishes (culture area 55 cm 2, Asahi Techno Glass) so that the cell seeding density was about 5100 cells / cm 2 . As the culture medium, DMEM GlutaMAX (Life Technologies) supplemented with 10% fetal bovine serum (Life Technologies) was used from cell seeding to 96 hours after cell seeding, and after 96 hours, MF-media and mesenchymal were used. The medium was replaced with a line stem cell growth medium (Toyobo).
FIG. 3 shows the schedule for preparing the culture supernatant by the conventional method. Culture solution exchange was performed 48 hours and 96 hours after the start of culturing. Then, without exchanging the culture medium, the culture was terminated when 100% confluence was reached within 168 hours from the start of the culture. The culture broth obtained after culturing for 72 hours from the last medium exchange to the end of culturing was collected as a culture supernatant. The total amount of the culture supernatant was 10.0 ml. After 168 hours from culturing, the cells were digested with trypsin, exfoliated and collected, and the number of cells was counted. As a result, 2.6 × 10 6 cells were collected.

(3)回収した培養上清の評価(VEGFおよびIGFの濃度測定)
上記、(1)および(2)の方法にて回収したそれぞれの培養上清について、組織再生に関わる性能を評価するため、培養上清に含まれるVEGF(血管内皮細胞増殖因子)およびIGF(インスリン様成長因子)の濃度を測定した。
VEGFの定量には、human VEGF ELISA Kit(R&D Systems)を用いた。
(3) Evaluation of collected culture supernatant (measurement of VEGF and IGF concentrations)
VEGF (Vascular Endothelial Growth Factor) and IGF (Insulin) contained in the culture supernatants are included in the culture supernatants in order to evaluate the performance related to tissue regeneration for each of the culture supernatants collected by the above methods (1) and (2). The concentration of (like growth factor) was measured.
Human VEGF ELISA Kit (R & D Systems) was used for the quantification of VEGF.

培養上清中のVEGF濃度を測定した結果を図4に示す。この結果、従来法にて回収した培養上清中のVEGF濃度が、561.6pg/mlであったのに対し、本発明により回収された培養上清中では、3597.1pg/mlであり、明らかな高濃度を示した。また、この濃度から、72時間の培養時間中に回収された総VEGF量は、従来法では、5616pgであるのに対し、本発明では、28417.1pgであった。
IGFの定量には、human IGF−I ELISA Kit(R&D Systems)を用いた。
The result of measuring the VEGF concentration in the culture supernatant is shown in FIG. As a result, the VEGF concentration in the culture supernatant recovered by the conventional method was 561.6 pg / ml, whereas it was 3597.1 pg / ml in the culture supernatant recovered by the present invention. It showed a clear high concentration. Further, from this concentration, the total amount of VEGF recovered during the culture time of 72 hours was 5616 pg in the conventional method, whereas it was 28417.1 pg in the present invention.
Human IGF-I ELISA Kit (R & D Systems) was used for the quantification of IGF.

培養上清中のIGF濃度を測定した結果を図5に示す。この結果、従来法にて回収した培養上清中のIGF濃度が、1864.6pg/mlであったのに対し、本発明により回収された培養上清中では、13798.0pg/mlであり、明らかな高濃度を示した。また、この濃度から、72時間の培養時間中に回収された総VEGF量は、従来法では、18.6ngであるのに対し、本発明では、109.0ngであった。 The result of measuring the IGF concentration in the culture supernatant is shown in FIG. As a result, the IGF concentration in the culture supernatant recovered by the conventional method was 1864.6 pg / ml, whereas in the culture supernatant recovered by the present invention, it was 3798.0 pg / ml. It showed a clear high concentration. Further, from this concentration, the total amount of VEGF recovered during the culturing time of 72 hours was 18.6 ng in the conventional method, whereas it was 109.0 ng in the present invention.

(4)回収した培養上清の評価(活性化マクロファージに対する効果測定)
上記、(1)および(2)の方法にて回収したそれぞれの培養上清について、抗炎症作用を評価した。これは、マクロファージ様細胞株であるRAW264.7細胞(大日本製薬)を、リポ多糖類(LPS)で刺激し、刺激により産生される炎症性サイトカイン(TNF−α)の産生を測定することにより評価した。
測定は以下のように実施した。即ち、RAW264.7細胞を、10%ウシ胎児血清(ライフテクノロジーズ)を含むRPMI1640培地(ライフテクノロジーズ)に懸濁し、24ウェルプレートに5×10個/ウェルで播種した。24時間培養後に、培養液の半分を前記(1)または(2)の培養上清に交換した。この1時間後、LPSを最終100ng/mlとなるよう添加し、更に3時間培養した後、培養液を回収し、Mouse TNF−αELISA Kit(R&D Systems)を用いて、培養液中のTNF−αを測定した。
培養上清によるTNF−α産生抑制効果を図6に示す。この結果、本発明の方法により作製した間葉系幹細胞の培養上清は、従来法で作製した培養上清に比べ、顕著にTNF−αの産生を抑制していることがわかった。
(4) Evaluation of collected culture supernatant (measurement of effect on activated macrophages)
The anti-inflammatory effect was evaluated for each of the culture supernatants collected by the above methods (1) and (2). This is done by stimulating RAW264.7 cells (Dainippon Pharmaceutical Co., Ltd.), which is a macrophage-like cell line, with lipopolysaccharide (LPS) and measuring the production of inflammatory cytokines (TNF-α) produced by the stimulation. evaluated.
The measurement was carried out as follows. That is, RAW264.7 cells were suspended in RPMI1640 medium (Life Technologies) containing 10% fetal bovine serum (Life Technologies) and seeded on a 24-well plate at 5 × 10 5 cells / well. After culturing for 24 hours, half of the culture broth was replaced with the culture supernatant of (1) or (2). After 1 hour, LPS was added to a final 100 ng / ml, and after further culturing for 3 hours, the culture broth was collected, and TNF-α in the culture broth was collected using Mouse TNF-αELISA Kit (R & D Systems). Was measured.
The effect of suppressing TNF-α production by the culture supernatant is shown in FIG. As a result, it was found that the culture supernatant of the mesenchymal stem cells prepared by the method of the present invention significantly suppressed the production of TNF-α as compared with the culture supernatant prepared by the conventional method.

前記(3)及び(4)に示された結果より、本発明により作製された幹細胞の培養上清は、従来のものに比べ、生理活性物質に富み、組織再生や抗炎症作用に優れたものであると言える。 From the results shown in (3) and (4) above, the culture supernatant of the stem cells produced by the present invention is richer in physiologically active substances and excellent in tissue regeneration and anti-inflammatory action as compared with the conventional ones. It can be said that.

本発明により、組織再生や抗炎症など、様々な作用を有する幹細胞の培養上清を簡便かつ高効率に製造することを可能とする。 INDUSTRIAL APPLICABILITY According to the present invention, it is possible to easily and efficiently produce a culture supernatant of stem cells having various actions such as tissue regeneration and anti-inflammatory.

1 モジュールケース
2 透過性膜中空糸
3 端部導管
4 側部導管
5、6 培養液貯留容器
7 細胞培養容器(中空糸モジュール)
8、9 ペリスタポンプ
10 廃液回収容器
11 培養上清回収容器
1 Module case 2 Permeable membrane hollow fiber 3 End conduit 4 Side conduit 5, 6 Culture solution storage container 7 Cell culture container (hollow fiber module)
8, 9 Perista pump 10 Waste liquid recovery container 11 Culture supernatant recovery container

Claims (5)

幹細胞培養上清液の製造方法であって、以下の[a]から[c]の工程を含む、方法。
[a]細胞培養容器内の透過性膜中空糸の内表面に播種した幹細胞に培養液を0.01〜1mm/minの線速度で供給し、細胞が播種されていない側に培養液を0.02〜5mm/minの線速度で供給して前記幹細胞を培養する工程
[b]前記幹細胞を培養する際に、前記培養液に血清を添加した培養液を用いて前記幹細胞を増殖する工程
[c]増殖した幹細胞に無血清培養液または低血清培養液を接触させて得られた培養上清を回収する工程
A method for producing a stem cell culture supernatant, which comprises the following steps [a] to [c].
[A] The culture solution is supplied to the stem cells seeded on the inner surface of the permeable membrane hollow thread in the cell culture vessel at a linear velocity of 0.01 to 1 mm / min, and the culture solution is 0 on the side where the cells are not seeded. A step of culturing the stem cells by supplying the cells at a linear velocity of .02 to 5 mm / min [b] A step of proliferating the stem cells using a culture solution in which serum is added to the culture solution when culturing the stem cells [b] c] A step of contacting the proliferated stem cells with a serum-free culture medium or a low-serum culture medium and collecting the obtained culture supernatant.
前記細胞培養容器は、透過性膜中空糸を格納したモジュールであって、前記中空糸の内腔と連通する2つの開口部と、前記モジュールの内側かつ前記中空糸の外腔と連通する2つの開口部を有するものである、請求項1に記載の方法。 The cell culture vessel is a module that stores a permeable membrane hollow fiber, and has two openings that communicate with the inner cavity of the hollow fiber and two openings that communicate with the inside of the module and the outer cavity of the hollow fiber. The method according to claim 1, wherein the method has an opening. 以下の[d]から[g]の構成を含む細胞培養装置を用いて前記幹細胞の培養を行う、請求項1または2に記載の方法。
[d]前記細胞培養容器
[e]少なくとも1つの培養液貯留容器
[f]前記細胞培養容器と前記培養液貯留容器とを接続する流路網
[g]前記流路網に設けられ、前記細胞培養容器への培養液の供給および/または前記細胞培養容器からの培養液の排出を制御する手段
The method according to claim 1 or 2, wherein the stem cells are cultured using a cell culture apparatus containing the following configurations [d] to [g].
[D] The cell culture container [e] At least one culture solution storage container [f] A flow path network connecting the cell culture container and the culture solution storage container [g] The cell provided in the flow path network. Means for controlling the supply of the culture solution to the culture container and / or the discharge of the culture solution from the cell culture container.
前記透過性膜中空糸の孔半径が、1〜500Åであることを特徴とする、請求項1から3のいずれかに記載の方法。 The method according to any one of claims 1 to 3, wherein the pore radius of the permeable membrane hollow fiber is 1 to 500 Å. 前記幹細胞が間葉系幹細胞であることを特徴とする、請求項1からのいずれかに記載の方法。
The method according to any one of claims 1 to 4 , wherein the stem cell is a mesenchymal stem cell.
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