JP6674727B2 - How to remove endotoxin from cells - Google Patents
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- JP6674727B2 JP6674727B2 JP2013196850A JP2013196850A JP6674727B2 JP 6674727 B2 JP6674727 B2 JP 6674727B2 JP 2013196850 A JP2013196850 A JP 2013196850A JP 2013196850 A JP2013196850 A JP 2013196850A JP 6674727 B2 JP6674727 B2 JP 6674727B2
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Description
本発明は、細胞からエンドトキシンを除去する方法、該方法を用いた細胞培養物製造方法、該製造方法により製造された細胞培養物などに関する。 The present invention relates to a method for removing endotoxin from cells, a method for producing a cell culture using the method, a cell culture produced by the method, and the like.
エンドトキシンは、グラム陰性菌の細胞壁表面に存在するリポ多糖複合体であり、体内に入ると、極めて微量であっても発熱、血圧低下、白血球数減少、血液凝固異常、血管内皮細胞障害、毛細血管透過性亢進などの症状を引き起こし、ＤＩＣ（播種性血管内凝固症候群）、ＭＯＦ（多臓器不全）、エンドトキシンショックなどの致死率の高い疾患の原因ともなる。したがって、生体に対して使用する医薬品や医療器具などにおいては、こうした有害事象の発生を回避するため、エンドトキシンの厳重な管理が必要となる。しかしながら、エンドトキシンは環境中に広く存在しており、比較的容易に汚染が起こるため、エンドトキシンの除去または不活化のための手法が研究されている。エンドトキシンを除去する手法としては、例えば、限外ろ過膜や逆浸透膜などによるろ過、イオン交換クロマトグラフィー、蒸留、活性炭、ポリミキシンＢ、ヒスチジン、ヒスタミン、酸化チタン（特許文献１）、ケイ酸アルミニウム（特許文献２）などの種々の吸着剤による吸着などが、エンドトキシンを不活化する手法としては、例えば、熱、酸、アルカリによる処理などが、それぞれ知られている。しかしながら、いずれの方法も多くの問題点を含んでおり、現実的に応用可能な絶対的な方法は未だ見出されていない。 Endotoxin is a lipopolysaccharide complex present on the cell wall surface of Gram-negative bacteria.When it enters the body, even in very small amounts, it produces fever, a decrease in blood pressure, a decrease in white blood cells, abnormal blood coagulation, vascular endothelial cell disorders, and capillary blood vessels. It causes symptoms such as hyperpermeability and causes high lethality such as DIC (disseminated intravascular coagulation), MOF (multi-organ failure), and endotoxin shock. Therefore, strict control of endotoxin is required in medicines and medical devices used for living bodies in order to avoid such adverse events. However, since endotoxin is widely present in the environment and relatively easily contaminated, methods for removing or inactivating endotoxin have been studied. As a method for removing endotoxin, for example, filtration using an ultrafiltration membrane or a reverse osmosis membrane, ion exchange chromatography, distillation, activated carbon, polymyxin B, histidine, histamine, titanium oxide (Patent Document 1), aluminum silicate ( As a method for inactivating endotoxin by adsorption with various adsorbents such as Patent Document 2), for example, treatment with heat, acid, or alkali is known, respectively. However, each method has many problems, and an absolute method that can be applied practically has not been found yet.
一方、近年、損傷した組織等の修復のために、種々の細胞を移植する試みが行われている。例えば、狭心症、心筋梗塞などの虚血性心疾患により損傷した心筋組織の修復のために、胎児心筋細胞、骨格筋芽細胞、間葉系幹細胞、心臓幹細胞、ＥＳ細胞等の利用が試みられている（非特許文献１）。このような試みの一環として、スキャフォールドを利用して形成した細胞構造物や、細胞をシート状に形成したシート状細胞培養物が開発されてきた（特許文献３）。シート状細胞培養物の治療への応用については、火傷などによる皮膚損傷に対する培養表皮シートの利用、角膜損傷に対する角膜上皮シート状細胞培養物の利用、食道ガン内視鏡的切除に対する口腔粘膜シート状細胞培養物の利用などの検討が進められている。
このような細胞培養物を臨床応用する場合には、移植を受けるレシピエントに対する安全性を確保するうえで、エンドトキシンの管理が重要となる。しかしながら、上述のような従来のエンドトキシンの除去や不活化のための手法は、医療用化合物や医療器具などの非生物を対象としたものであり、生体である細胞に適用可能な手法は未だ知られていない。
On the other hand, in recent years, attempts have been made to transplant various cells to repair damaged tissues and the like. For example, in order to repair myocardial tissue damaged by ischemic heart disease such as angina pectoris and myocardial infarction, utilization of fetal cardiomyocytes, skeletal myoblasts, mesenchymal stem cells, cardiac stem cells, ES cells, etc. has been attempted. (Non-Patent Document 1). As one of such attempts, a cell structure formed using a scaffold and a sheet-shaped cell culture in which cells are formed in a sheet shape have been developed (Patent Document 3). For the application of sheet-shaped cell culture to treatment, use of cultured epidermis sheet for skin damage due to burns, use of corneal epithelial sheet-shaped cell culture for corneal injury, oral mucosal sheet for endoscopic resection of esophageal cancer Studies on the use of cell cultures and the like are underway.
When clinically applying such a cell culture, it is important to control endotoxin in order to ensure safety for recipients receiving transplantation. However, the conventional methods for removing or inactivating endotoxin as described above are intended for non-living organisms such as medical compounds and medical instruments, and methods applicable to living cells are still unknown. Not been.
本発明の目的は、細胞に適用可能なエンドトキシン除去方法を提供することにある。 An object of the present invention is to provide an endotoxin removal method applicable to cells.
本発明者は、上記課題を解決するために鋭意研究を進める中、細胞懸濁液を、細胞凝集阻害剤を含む洗浄液で繰り返し２回以上洗浄することにより、細胞からエンドトキシンを効果的に除去できることを見出し、本発明を完成させた。 In order to solve the above-mentioned problems, the inventor of the present invention has been able to effectively remove endotoxin from cells by repeatedly washing the cell suspension twice or more with a washing solution containing a cell aggregation inhibitor, while proceeding intensive studies. And completed the present invention.
すなわち、本発明は以下に関する。
[１]細胞懸濁液を、細胞凝集阻害剤を含む洗浄液で繰り返し２回以上洗浄するステップを含む、細胞からエンドトキシンを除去する方法。
[２]洗浄するステップにおいて、細胞懸濁液を、細胞凝集阻害剤を含む洗浄液で繰り返し３〜１０回洗浄する、[１]に記載の方法。
[３]洗浄するステップにおいて、細胞懸濁液を、細胞凝集阻害剤を含む洗浄液で繰り返し６回洗浄する、[２]に記載の方法。
[４]細胞凝集阻害剤が、アルブミン、トレハロース、ヒドロキシエチルデンプン、デキストラン、局所麻酔薬からなる群から選択される、[１]〜[３]のいずれかに記載の方法。
[５][１]〜[４]のいずれかに記載の方法を含む、医療用細胞培養物の製造方法。
[６][５]に記載の方法で製造された、医療用細胞培養物。
[７][６]に記載の医療用細胞培養物を含む、医薬組成物。
[８]対象において疾患を処置する方法であって、[６]に記載の細胞培養物または[７]に記載の医薬組成物の有効量をそれ必要とする対象に移植するステップを含む、前記方法。
That is, the present invention relates to the following.
[1] A method for removing endotoxin from cells, comprising a step of repeatedly washing a cell suspension twice or more with a washing solution containing a cell aggregation inhibitor.
[2] The method according to [1], wherein in the washing step, the cell suspension is repeatedly washed 3 to 10 times with a washing solution containing a cell aggregation inhibitor.
[3] The method according to [2], wherein in the washing step, the cell suspension is repeatedly washed six times with a washing solution containing a cell aggregation inhibitor.
[4] The method according to any one of [1] to [3], wherein the cell aggregation inhibitor is selected from the group consisting of albumin, trehalose, hydroxyethyl starch, dextran, and a local anesthetic.
[5] A method for producing a medical cell culture, comprising the method according to any one of [1] to [4].
[6] A medical cell culture produced by the method according to [5].
[7] A pharmaceutical composition comprising the medical cell culture according to [6].
[8] A method for treating a disease in a subject, comprising the step of transplanting an effective amount of the cell culture according to [6] or the pharmaceutical composition according to [7] into a subject in need thereof. Method.
本発明のエンドトキシン除去方法により、生体に有害な量のエンドトキシンを含まない、安全性の高い医療用細胞培養物を製造することができるため、エンドトキシンによるリスクを回避することが可能となり、医療用細胞培養物を用いた処置の普及拡大が期待できる。また、本発明のエンドトキシン除去方法は細胞への負担が少なく、細胞培養物の治療活性を維持することができる。 The endotoxin removal method of the present invention does not contain an endotoxin in a harmful amount to a living body, and thus a highly safe medical cell culture can be produced. The spread of treatment using cultures can be expected. In addition, the method for removing endotoxin of the present invention exerts less burden on cells, and can maintain the therapeutic activity of the cell culture.
本明細書において別様に定義されない限り、本明細書で用いる全ての技術用語および科学用語は、当業者が通常理解しているものと同じ意味を有する。本明細書中で参照する全ての特許、出願、公開された出願および他の出版物は、その全体を参照により本明細書に援用する。 Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All patents, applications, published applications and other publications referred to herein are hereby incorporated by reference in their entirety.
本発明の一側面は、細胞懸濁液を、細胞凝集阻害剤を含む洗浄液で繰り返し２回以上洗浄するステップを含む、細胞からエンドトキシンを除去する方法（以下、「除去方法」と略す場合もある）に関する。
本発明の除去方法における細胞は特に限定されず、任意の種類のものを包含するが、医療目的で使用されるものが好ましい。医療目的としては、限定されずに例えば疾患の処置、移植医療、再生医療などが挙げられる。かかる目的で使用する細胞としては、限定されずに、例えば、血液疾患などの治療に用いる造血幹細胞、免疫療法などに用いるリンパ球、樹状細胞などの免疫細胞、シート状細胞培養物を形成し得る細胞、例えば、筋芽細胞（例えば、骨格筋芽細胞など）、間葉系幹細胞（例えば、骨髄、脂肪組織、末梢血、皮膚、毛根、筋組織、子宮内膜、胎盤、臍帯血由来のものなど）、心筋細胞、線維芽細胞、心臓幹細胞、胚性幹細胞、ｉＰＳ細胞、滑膜細胞、軟骨細胞、上皮細胞（例えば、口腔粘膜上皮細胞、網膜色素上皮細胞、鼻粘膜上皮細胞など）、内皮細胞（例えば、血管内皮細胞など）、肝細胞（例えば、肝実質細胞など）、膵細胞（例えば、膵島細胞など）、腎細胞、副腎細胞、歯根膜細胞、歯肉細胞、骨膜細胞、皮膚細胞等が挙げられる。
One aspect of the present invention is a method for removing endotoxin from cells, which comprises a step of repeatedly washing a cell suspension with a washing solution containing a cell aggregation inhibitor two or more times (hereinafter, sometimes referred to as a “removal method” in some cases). ).
Cells in the removal method of the present invention are not particularly limited, and include any type of cells, but those used for medical purposes are preferred. Medical purposes include, but are not limited to, for example, treatment of disease, transplantation medicine, regenerative medicine, and the like. The cells used for this purpose are not limited, and include, for example, hematopoietic stem cells used for treatment of blood diseases and the like, lymphocytes used for immunotherapy and the like, immune cells such as dendritic cells, and sheet-shaped cell cultures. Obtained cells, such as those derived from myoblasts (eg, skeletal myoblasts), mesenchymal stem cells (eg, bone marrow, adipose tissue, peripheral blood, skin, hair root, muscle tissue, endometrium, placenta, cord blood) , Cardiomyocytes, fibroblasts, cardiac stem cells, embryonic stem cells, iPS cells, synovial cells, chondrocytes, epithelial cells (eg, oral mucosal epithelial cells, retinal pigment epithelial cells, nasal mucosal epithelial cells, etc.), Endothelial cells (eg, vascular endothelial cells), hepatocytes (eg, hepatic parenchymal cells), pancreatic cells (eg, pancreatic islet cells), kidney cells, adrenal cells, periodontal ligament cells, gingival cells, periosteal cells, skin cells Etc.
細胞懸濁液は、細胞が、液体媒体中に浮遊した状態の液体を指す。液体媒体としては、細胞の操作に通常使用する任意の媒体、限定されずに、例えば、生理食塩水、リンゲル液、ハンクス平衡塩液などの平衡塩液、ＰＢＳ（リン酸緩衝生理食塩水）、液体培地などを用いることができる。浮遊細胞であれば、培養中の細胞を含む液体培地をそのまま細胞懸濁液としてもよいが、培地を上記の液体媒体と置換して細胞懸濁液を調製することも可能である。付着細胞であれば、培養中の細胞を基材から剥離し、これを上記の液体媒体に懸濁して細胞懸濁液を調製することができる。 Cell suspension refers to a liquid in which cells are suspended in a liquid medium. The liquid medium may be any medium commonly used for cell manipulation, including, but not limited to, physiological saline, Ringer's solution, balanced salt solutions such as Hanks' balanced salt solution, PBS (phosphate buffered saline), liquid A medium or the like can be used. If the cells are suspension cells, a liquid medium containing the cells being cultured may be used as it is as a cell suspension, but it is also possible to prepare a cell suspension by replacing the medium with the above liquid medium. If it is an adherent cell, the cell in culture can be detached from the substrate, and this can be suspended in the above-mentioned liquid medium to prepare a cell suspension.
本発明の除去方法における細胞凝集阻害剤は、細胞を洗浄する際に細胞同士の凝集を阻害できる物質であれば特に限定されず、下記実施例で用いたアルブミンのほか、細胞凝集阻害作用が知られている既知の任意の化合物、例えば、トレハロース、ヒドロキシエチルデンプン、デキストラン（WO 2012/063870）、局所麻酔薬（特開2006-42693）などを含む。アルブミンとしては、血清アルブミン、特に同種の血清アルブミンを用いることが好ましいが、卵アルブミン、乳アルブミン、筋アルブミンなどを用いることもできる。ここで「同種」とは、細胞を投与する対象（レシピエント）と同一の種の生物に由来することを意味する。例えば、投与する対象がヒトである場合、ヒトアルブミンが同種のアルブミンに該当する。局所麻酔薬としては、限定されずに、例えば、リドカイン、メピバカイン、ジブカイン、ブピバカイン、プロピトカイン等のアミド型局所麻酔薬、コカイン、プロカイン、クロロカイン、テトラカイン等のエステル型局所麻酔薬などが挙げられる。 The cell aggregation inhibitor in the removal method of the present invention is not particularly limited as long as it is a substance that can inhibit cell-cell aggregation when washing the cells. In addition to albumin used in the following examples, the cell aggregation inhibitor is known to have a cell aggregation inhibitory effect. And any known compounds such as trehalose, hydroxyethyl starch, dextran (WO 2012/063870), local anesthetics (JP-A 2006-42693), and the like. As albumin, it is preferable to use serum albumin, particularly serum albumin of the same species, but egg albumin, milk albumin, muscle albumin and the like can also be used. Here, “same species” means that they are derived from an organism of the same species as the subject (recipient) to which the cells are administered. For example, when the subject to be administered is a human, human albumin corresponds to the same kind of albumin. Examples of the local anesthetic include, but are not limited to, amide-type local anesthetics such as lidocaine, mepivacaine, dibucaine, bupivacaine, and propitocaine, and ester-type local anesthetics such as cocaine, procaine, chlorocaine, and tetracaine.
細胞凝集阻害剤の濃度は、細胞を洗浄する際に細胞同士の凝集を阻害できるものであれば特に限定されないが、例えば、アルブミンであれば、好ましくは０．１〜５％（ｗ/ｖ）、より好ましくは０．２５〜２.５％（ｗ/ｖ）であり、トレハロースであれば、好ましくは４．５３〜３６２．４ｍｇ／ｍｌ、より好ましくは１５．１〜１８１．２ｍｇ／ｍｌであり、ヒドロキシエチルデンプンであれば、好ましくは１〜５００ｍｇ／ｍｌ、より好ましくは１０〜１００ｍｇ／ｍｌであり、デキストランであれば、好ましくは１〜５００ｍｇ／ｍｌ、より好ましくは１０〜２００ｍｇ／ｍｌ、より一層好ましくは３０〜１２５ｍｇ／ｍｌ、さらに好ましくは３０〜１００ｍｇ／ｍｌ、さらにより好ましくは６５〜１００ｍｇ／ｍｌであり、局所麻酔薬であれば、好ましくは０．１〜５重量％、より好ましくは０．５〜２.５重量％、最も好ましくは約１重量％である。 The concentration of the cell aggregation inhibitor is not particularly limited as long as the cell aggregation can be inhibited when the cells are washed. For example, in the case of albumin, preferably 0.1 to 5% (w / v) , More preferably 0.25 to 2.5% (w / v), and if it is trehalose, it is preferably 4.53 to 362.4 mg / ml, more preferably 15.1 to 181.2 mg / ml. Yes, if it is hydroxyethyl starch, it is preferably 1 to 500 mg / ml, more preferably 10 to 100 mg / ml, and if it is dextran, it is preferably 1 to 500 mg / ml, more preferably 10 to 200 mg / ml, It is still more preferably 30 to 125 mg / ml, still more preferably 30 to 100 mg / ml, even more preferably 65 to 100 mg / ml. If, preferably 0.1 to 5% by weight, more preferably 0.5 to 2.5 wt%, and most preferably from about 1 wt%.
本発明の除去方法における洗浄液は、細胞凝集阻害剤が液体であれば、細胞凝集阻害剤のみから構成されてもよいし、細胞凝集阻害剤以外の成分、例えば希釈剤、緩衝剤、ｐＨ調整剤、浸透圧調整剤などを含んでいてもよい。細胞凝集阻害剤が固体の場合、洗浄液は、典型的には、上記成分のほか、細胞凝集阻害剤を溶解または懸濁する溶媒を含む。溶媒としては、限定されずに、例えば、生理食塩水、リンゲル液、ハンクス平衡塩液などの平衡塩液、ＰＢＳ（リン酸緩衝生理食塩水）、液体培地などが挙げられる。洗浄液は、細胞へのダメージを低減する観点から、体液の浸透圧と等しい等張液であることが好ましい。 The washing solution in the removal method of the present invention may be composed of only the cell aggregation inhibitor, as long as the cell aggregation inhibitor is a liquid, or a component other than the cell aggregation inhibitor, for example, a diluent, a buffer, and a pH adjuster. And an osmotic pressure adjusting agent. When the cell aggregation inhibitor is a solid, the washing solution typically contains a solvent that dissolves or suspends the cell aggregation inhibitor in addition to the above components. Examples of the solvent include, but are not limited to, physiological saline, Ringer's solution, a balanced salt solution such as Hanks' balanced salt solution, PBS (phosphate buffered saline), and a liquid medium. The washing solution is preferably an isotonic solution equal to the osmotic pressure of body fluid from the viewpoint of reducing damage to cells.
本発明の除去方法における洗浄は、培地の置換目的で行なわれている１〜２回程度のすすぎによる洗浄操作とは異なり、洗浄液に置換懸濁し、この細胞に混和撹拌操作を加えることにより洗浄を行うものをいう。この混和撹拌操作は、ピペッティングなどにより細胞に対して比較的強い力が加えられるものである。撹拌操作は、１回の洗浄あたり５回程度のピペッティングによる撹拌であることがエンドトキシンの効率的な除去と細胞の損傷防止の観点から望ましい。また洗浄回数は、洗浄液を交換して、２回以上行うことがエンドトキシンの除去に有効であるが、一方、１０回を超えて洗浄しても、洗浄効果は上がらず、むしろ細胞へのストレスが大きくなることが懸念されるため、２〜１０回とすることが好ましく、例えば、３〜９回、４〜８回または５〜７回とすることができる。中でも洗浄回数を６回とすることが特に好ましい。洗浄液の交換は、典型的には、洗浄液に懸濁させた細胞を遠心処理により沈殿させ、上清を廃棄し、沈殿した細胞を新たな洗浄液に懸濁することにより行うことができるが、同様の効果が得られる他の方法、例えば、細胞と洗浄液を濾過により分離する手法などを用いてもよい。 The washing in the removal method of the present invention is different from the washing operation by rinsing about 1 to 2 times performed for the purpose of replacing the culture medium, and the washing is performed by replacing and suspending the cells with a washing solution and adding a mixing and stirring operation to the cells. What you do. In the mixing and stirring operation, a relatively strong force is applied to the cells by pipetting or the like. The stirring operation is preferably performed by pipetting about 5 times per washing from the viewpoint of efficient removal of endotoxin and prevention of cell damage. It is effective to remove the endotoxin at least twice by changing the washing solution, but on the other hand, even if washing is performed more than 10 times, the washing effect is not improved, and the stress on the cells is rather reduced. Since there is a concern that the size will increase, the number is preferably 2 to 10 times, for example, 3 to 9 times, 4 to 8 times or 5 to 7 times. Among them, it is particularly preferable to set the number of times of washing to six times. The exchange of the washing solution is typically performed by sedimenting the cells suspended in the washing solution by centrifugation, discarding the supernatant, and suspending the precipitated cells in a new washing solution. Other methods that can obtain the effect described above, for example, a method of separating cells and a washing solution by filtration may be used.
本発明の除去方法において洗浄に用いる容器としては、細胞操作に通常用いるものを用いることができるが、エンドトキシンを吸着しやすい材質のものが、エンドトキシンのより効率的な除去の観点から好ましい。かかる材質としては、例えば、ポリプロピレン、ポリエチレン、ポリスチレンなどの疎水性物質が挙げられる。また、エンドトキシンを吸着することが知られている物質でコーティングされた容器を使用することもできる。エンドトキシンを吸着することが知られている物質としては、限定されずに、例えば、活性炭、ポリミキシンＢ、ヒスチジン、ヒスタミン、酸化チタン、ケイ酸アルミニウムなどが挙げられる。 As the container used for washing in the removal method of the present invention, a container usually used for cell manipulation can be used, but a material that easily adsorbs endotoxin is preferable from the viewpoint of more efficient endotoxin removal. Examples of such a material include hydrophobic substances such as polypropylene, polyethylene, and polystyrene. It is also possible to use containers coated with substances known to adsorb endotoxin. Examples of substances known to adsorb endotoxin include, but are not limited to, activated carbon, polymyxin B, histidine, histamine, titanium oxide, aluminum silicate, and the like.
本発明の別の側面は、本発明の除去方法を含む、医療用細胞培養物の製造方法（以下、「製造方法」と略す場合がある）、および、当該製造方法によって製造された医療用細胞培養物（以下、「細胞培養物」と略す場合がある）に関する。
本発明において「医療用」とは、医療用途、限定されずに例えば、疾患の処置、移植医療、再生医療などの用途に使用されることを意味する。したがって、本発明の「医療用細胞培養物」は、医療製品に求められる種々の基準、例えば、エンドトキシンに関する基準などを満たすものである。かかる基準としては、例えば、行政当局によって定められたものが挙げられる。例えば、現行の第十六改正日本薬局方において、エンドトキシンの規格値はＫ／Ｍとして設定され、ここでＫは発熱を誘起するといわれる体重１ｋｇ当たりのエンドトキシンの量（ＥＵ／ｋｇ）であり、投与経路による区分に基づき、脊髄腔内投与では０．２ＥＵ／ｋｇ、それ以外では５．０ＥＵ／ｋｇと設定されており、Ｍは体重１ｋｇ当たり１回に投与される最大量（頻回または持続的に投与される場合、Ｍは１時間以内に投与される最大総量）と設定されている。したがって、６０ｋｇの成人に最大で１回に細胞培養物１０ｍｌを静脈内注射する場合、上記規格値は３０ＥＵ／ｍｌとなる。ただし、これは単なる例示であり、当業者であれば、種々の状況に適合した基準値や規格値を適宜決定することができる。なお、医療は、ヒト医療および獣医療の両方を含む。
Another aspect of the present invention is a method for producing a medical cell culture, which includes the removal method of the present invention (hereinafter, may be abbreviated as “production method”), and a medical cell produced by the production method. A culture (hereinafter sometimes abbreviated as “cell culture”).
In the present invention, “medical use” means to be used for medical use, such as, but not limited to, treatment of disease, transplantation medicine, regenerative medicine and the like. Therefore, the “medical cell culture” of the present invention satisfies various standards required for medical products, for example, standards relating to endotoxin. Such criteria include, for example, those set by administrative authorities. For example, in the current 16th revision Japanese Pharmacopoeia, the standard value of endotoxin is set as K / M, where K is the amount of endotoxin per kg of body weight (EU / kg) which is said to induce fever, Based on the classification by administration route, it is set to 0.2 EU / kg for intrathecal administration and 5.0 EU / kg for other cases, and M is the maximum dose administered once per kg of body weight (frequent or continuous). M is set to be the maximum total dose administered within one hour). Therefore, when a 60 kg adult is intravenously injected with a maximum of 10 ml of the cell culture at a time, the above-mentioned standard value is 30 EU / ml. However, this is merely an example, and those skilled in the art can appropriately determine a reference value or a standard value suitable for various situations. The medical treatment includes both human medicine and veterinary medicine.
本発明の好ましい態様において、細胞培養物は、３×１０７個の細胞を含む３ｍｌの細胞懸濁液において、１０ＥＵ／ｍｌ以上、好ましくは５ＥＵ／ｍｌ以上、より好ましくは１ＥＵ／ｍｌ以上、さらに好ましくは０．５ＥＵ／ｍｌ以上、より一層好ましくは０．２５ＥＵ／ｍｌ以上、特に好ましくは０．１３ＥＵ／ｍｌ以上のエンドトキシンを含まない。また、本発明の細胞培養物は、好ましくは、エンドトキシンを検出可能なレベルで含まない。エンドトキシンの検出は、既知の任意の方法、例えば、限定されずに、比濁法（例えばカイネティック−比濁法）、ゲル化法、比色法、合成基質法などのリムルス法などを用いることができる。 In a preferred embodiment of the invention, the cell culture is at least 10 EU / ml, preferably at least 5 EU / ml, more preferably at least 1 EU / ml, in a 3 ml cell suspension containing 3 × 10 7 cells. It is preferably free of endotoxin of 0.5 EU / ml or more, more preferably 0.25 EU / ml or more, particularly preferably 0.13 EU / ml or more. Also, the cell cultures of the present invention preferably do not contain detectable levels of endotoxin. The endotoxin can be detected by any known method, for example, and without limitation, a turbidity method (eg, a kinetic-turbidimetric method), a gelling method, a colorimetric method, a Limulus method such as a synthetic substrate method, or the like. Can be.
本発明における細胞培養物は、細胞懸濁液や細胞構造体などの、医療用途に適した種々の形態をとることができる。細胞構造体は、シート状、柱状、塊状、栓状などの種々の形状であってよい。本発明においては、治療効果の高さなどの観点から、シート状の細胞構造体、すなわち、シート状細胞培養物が好ましい。「シート状細胞培養物」は、細胞が互いに連結してシート状になったものをいい、典型的には１の細胞層からなるものであるが、２以上の細胞層から構成されるものも含む。細胞同士は、直接（接着分子などの細胞要素を介するものを含む）および／または介在物質を介して、互いに連結していてもよい。介在物質としては、細胞同士を少なくとも物理的（機械的）に連結し得る物質であれば特に限定されないが、例えば、細胞外マトリックスなどが挙げられる。介在物質は、好ましくは細胞由来のもの、特に、細胞培養物を構成する細胞に由来するものである。細胞は少なくとも物理的（機械的）に連結されるが、さらに機能的、例えば、化学的、電気的に連結されてもよい。 The cell culture in the present invention can take various forms suitable for medical use, such as a cell suspension and a cell structure. The cell structure may have various shapes such as a sheet shape, a column shape, a block shape, and a plug shape. In the present invention, a sheet-shaped cell structure, that is, a sheet-shaped cell culture, is preferable from the viewpoint of high therapeutic effect and the like. “Sheet-shaped cell culture” refers to a sheet-like cell in which cells are connected to each other, and is typically composed of one cell layer, but may be composed of two or more cell layers. Including. The cells may be connected to each other directly (including via a cellular element such as an adhesion molecule) and / or via an intermediary substance. The intervening substance is not particularly limited as long as it is a substance capable of at least physically (mechanically) connecting cells, and examples thereof include an extracellular matrix. The mediator is preferably of cell origin, in particular of the cells that make up the cell culture. Cells are at least physically (mechanically) linked, but may be further functionally linked, eg, chemically or electrically.
本発明の細胞培養物は、生体適合性や治療効果の高さなどの観点から、スキャフォールド（支持体）を含まないことが好ましい。スキャフォールドは、その表面上および／またはその内部に細胞を付着させ、細胞培養物の物理的一体性を維持するために当該技術分野において用いられることがあり、例えば、ポリビニリデンジフルオリド（ＰＶＤＦ）製の膜等が知られているが、本発明の好ましい細胞培養物は、かかるスキャフォールドがなくともその物理的一体性を維持することができるものである。また、本発明の細胞培養物は、好ましくは、細胞培養物を構成する細胞由来の物質のみからなり、それら以外の物質を含まない。 The cell culture of the present invention preferably does not contain a scaffold (support) from the viewpoints of biocompatibility and high therapeutic effect. Scaffolds may be used in the art to attach cells to and / or on their surface and maintain the physical integrity of the cell culture, for example, polyvinylidene difluoride (PVDF) Although membranes and the like are known, preferred cell cultures of the present invention are those that can maintain their physical integrity without such a scaffold. Further, the cell culture of the present invention preferably comprises only substances derived from the cells constituting the cell culture, and does not contain any other substances.
本発明の細胞培養物に用いる細胞は、細胞培養物による治療が可能な任意の多細胞生物（例えば動物など）に由来し得る。かかる生物には、限定されずに、例えば、ヒト、非ヒト霊長類、イヌ、ネコ、ブタ、ウマ、ヤギ、ヒツジ、げっ歯目動物（例えば、マウス、ラット、ハムスター、モルモットなど）、ウサギなどの哺乳動物が含まれる。また、細胞培養物の形成に用いる細胞は１種類のみであってもよいが、２種類以上の細胞を用いることもできる。本発明の好ましい態様において、細胞培養物を形成する細胞が２種類以上ある場合、最も多い細胞の比率（純度）は、細胞培養物製造終了時において、６０％以上、好ましくは７０％以上、より好ましくは７５％以上である。 The cells used in the cell culture of the present invention can be derived from any multicellular organism (eg, an animal) that can be treated with the cell culture. Such organisms include, without limitation, humans, non-human primates, dogs, cats, pigs, horses, goats, sheep, rodents (eg, mice, rats, hamsters, guinea pigs, etc.), rabbits, etc. Mammals. Further, only one type of cell may be used for forming a cell culture, but two or more types of cells may be used. In a preferred embodiment of the present invention, when there are two or more types of cells forming a cell culture, the ratio (purity) of the largest number of cells at the end of cell culture production is 60% or more, preferably 70% or more, It is preferably at least 75%.
細胞は異種由来細胞であっても同種由来細胞であってもよい。ここで「異種由来細胞」は、細胞培養物が移植に用いられる場合、そのレシピエントとは異なる種の生物に由来する細胞を意味する。例えば、レシピエントがヒトである場合、サルやブタに由来する細胞などが異種由来細胞に該当する。また、「同種由来細胞」は、レシピエントと同一の種の生物に由来する細胞を意味する。例えば、レシピエントがヒトである場合、ヒト細胞が同種由来細胞に該当する。同種由来細胞は、自己由来細胞（自己細胞または自家細胞ともいう）、すなわち、レシピエントに由来する細胞と、同種非自己由来細胞（他家細胞ともいう）を含む。自己由来細胞は、移植しても拒絶反応が生じないため、本発明においては好ましい。しかしながら、異種由来細胞や同種非自己由来細胞を利用することも可能である。異種由来細胞や同種非自己由来細胞を利用する場合は、拒絶反応を抑制するため、免疫抑制処置が必要となることがある。なお、本明細書中で、自己由来細胞以外の細胞、すなわち、異種由来細胞と同種非自己由来細胞を非自己由来細胞と総称することもある。本発明の一態様において、細胞は自家細胞または他家細胞である。本発明の一態様において、細胞は自家細胞である。本発明の別の態様において、細胞は他家細胞である。 The cells may be xenogenic cells or allogeneic cells. As used herein, “heterologous cells” means cells derived from an organism of a different species from the recipient when a cell culture is used for transplantation. For example, when the recipient is a human, cells derived from monkeys and pigs correspond to xenogeneic cells. "Allogeneic cell" means a cell derived from an organism of the same species as the recipient. For example, when the recipient is human, human cells correspond to cells derived from the same species. Allogeneic cells include autologous cells (also called autologous cells or autologous cells), that is, cells derived from the recipient and allogeneic non-autologous cells (also called allogeneic cells). Autologous cells are preferred in the present invention because rejection does not occur even after transplantation. However, it is also possible to use xenogeneic cells or allogeneic non-autologous cells. When xenogeneic cells or allogeneic non-autologous cells are used, immunosuppressive treatment may be necessary to suppress rejection. In this specification, cells other than autologous cells, that is, non-autologous cells of the same species as cells of xenogeneic origin may be collectively referred to as non-autologous cells. In one aspect of the invention, the cells are autologous cells or allogeneic cells. In one aspect of the invention, the cells are autologous cells. In another aspect of the invention, the cells are allogeneic cells.
本発明の細胞培養物は、種々の疾患、特に組織の異常に関連する疾患の処置に有用である。したがって、一態様において、本発明の細胞培養物は、組織の異常に関連する疾患の処置に用いるためのものである。処置の対象となる組織としては、限定されずに、例えば、心筋、角膜、網膜、食道、皮膚、関節、軟骨、肝臓、膵臓、歯肉、腎臓、甲状腺、骨格筋、中耳、骨髄などが挙げられる。また、処置の対象となる疾患としては、限定されずに、例えば、心疾患（例えば、心筋傷害（心筋梗塞、心外傷）、心筋症など）、角膜疾患（例えば、角膜上皮幹細胞疲弊症、角膜損傷（熱・化学腐食）、角膜潰瘍、角膜混濁、角膜穿孔、角膜瘢痕、スティーブンス・ジョンソン症候群、眼類天疱瘡など）、網膜疾患（例えば、網膜色素変性症、加齢黄斑変性症など）、食道疾患（例えば、食道手術（食道ガン除去）後の食道の炎症・狭窄の予防など）、皮膚疾患（例えば、皮膚損傷（外傷、熱傷）など）、関節疾患（例えば、変形性関節炎など）、軟骨疾患（例えば、軟骨の損傷など）、肝疾患（例えば、慢性肝疾患など）、膵臓疾患（例えば、糖尿病など）、歯科疾患（例えば、歯周病など）、腎臓疾患（例えば、腎不全、腎性貧血、腎性骨異栄養症など）、甲状腺疾患（例えば、甲状腺機能低下症など）、筋疾患（例えば、筋損傷、筋炎など）、中耳疾患（例えば、中耳炎など）、骨髄疾患（例えば、白血病、再生不良性貧血、免疫不全疾患など）が挙げられる。 The cell culture of the present invention is useful for treating various diseases, particularly diseases related to tissue abnormalities. Thus, in one aspect, the cell culture of the present invention is for use in treating a disease associated with a tissue abnormality. The tissues to be treated include, but are not limited to, for example, myocardium, cornea, retina, esophagus, skin, joints, cartilage, liver, pancreas, gingiva, kidney, thyroid, skeletal muscle, middle ear, bone marrow, and the like. Can be Examples of the disease to be treated include, but are not limited to, heart disease (eg, myocardial injury (myocardial infarction, cardiac trauma), cardiomyopathy, etc.), corneal disease (eg, corneal epithelial stem cell exhaustion, cornea) Injury (thermal / chemical erosion), corneal ulcer, corneal opacity, corneal perforation, corneal scar, Stevens-Johnson syndrome, ocular pemphigus, etc., retinal diseases (eg, retinitis pigmentosa, age-related macular degeneration, etc.) Esophageal disease (eg, prevention of esophageal inflammation / stenosis after esophageal surgery (removal of esophageal cancer)), skin disease (eg, skin damage (trauma, burn), etc.), joint disease (eg, osteoarthritis, etc.) Cartilage disease (eg, cartilage damage), liver disease (eg, chronic liver disease), pancreatic disease (eg, diabetes), dental disease (eg, periodontal disease), kidney disease (eg, renal failure) , Renal anemia, kidney Osteodystrophy, etc.), thyroid disease (eg, hypothyroidism, etc.), muscular disease (eg, muscle damage, myositis, etc.), middle ear disease (eg, otitis media, etc.), bone marrow disease (eg, leukemia, poor regeneration) Anemia, immunodeficiency disease, etc.).
本発明の細胞培養物が上記疾患に有用であることは、例えば、特許文献３、非特許文献１、Arauchi et al., Tissue Eng Part A. 2009 Dec;15(12):3943-9、Ito et al., Tissue Eng. 2005 Mar-Apr;11(3-4):489-96、Yaji et al., Biomaterials. 2009 Feb;30(5):797-803、Yaguchi et al., Acta Otolaryngol. 2007 Oct;127(10):1038-44、Watanabe et al., Transplantation. 2011 Apr 15;91(7):700-6、Shimizu et al., Biomaterials. 2009 Oct;30(30):5943-9、Ebihara et al., Biomaterials. 2012 May;33(15):3846-51、Takagi et al., World J Gastroenterol. 2012 Oct 7;18(37):5145-50などに記載されている。 The usefulness of the cell culture of the present invention for the above-mentioned diseases is, for example, described in Patent Document 3, Non-patent Document 1, Arauchi et al., Tissue Eng Part A. 2009 Dec; 15 (12): 3943-9, Ito et al., Tissue Eng. 2005 Mar-Apr; 11 (3-4): 489-96, Yaji et al., Biomaterials.2009 Feb; 30 (5): 797-803, Yaguchi et al., Acta Otolaryngol. 2007 Oct; 127 (10): 1038-44, Watanabe et al., Transplantation. 2011 Apr 15; 91 (7): 700-6, Shimizu et al., Biomaterials.2009 Oct; 30 (30): 5943-9 Ebihara et al., Biomaterials. 2012 May; 33 (15): 3846-51, Takagi et al., World J Gastroenterol. 2012 Oct 7; 18 (37): 5145-50.
本発明の細胞培養物は、処置の対象となる組織に適用し、これを修復、再生するために使用することもできるが、ホルモンなどの生理活性物質の給源として、処置の対象となる組織以外の部位（例えば、皮下組織など）に移植することもできる（例えば、Arauchi et al., Tissue Eng Part A. 2009 Dec;15(12):3943-9、Shimizu et al., Biomaterials. 2009 Oct;30(30):5943-9など）。また、本発明のシート状細胞培養物を注射可能な大きさに断片化し、これを処置が必要な部位に注射することもできる（Wang et al., Cardiovasc Res. 2008 Feb 1;77(3):515-24）。 The cell culture of the present invention can be applied to a tissue to be treated, and used for repairing and regenerating the same. However, as a source of a physiologically active substance such as a hormone, a tissue other than the tissue to be treated is used. (Eg, Arauchi et al., Tissue Eng Part A. 2009 Dec; 15 (12): 3943-9, Shimizu et al., Biomaterials. 2009 Oct;). 30 (30): 5943-9). In addition, the sheet-shaped cell culture of the present invention can be fragmented into an injectable size and injected into a site requiring treatment (Wang et al., Cardiovasc Res. 2008 Feb 1; 77 (3) : 515-24).
本発明の細胞培養物は、種々の追加成分、例えば、薬学的に許容し得る担体や、細胞培養物の生存性、生着性および／または機能などを高める成分、対象疾患の処置に有用な他の有効成分などをさらに含んでいてもよい。かかる追加成分としては、既知の任意のものを使用することができ、当業者はこれらの追加成分について精通している。また、本発明の細胞培養物は、細胞培養物の生存性、生着性および／または機能などを高める成分や、対象疾患の処置に有用な他の有効成分などと併用することができる。 The cell culture of the present invention is useful for treating a variety of additional components, such as pharmaceutically acceptable carriers, components that enhance the viability, engraftment and / or function of the cell culture, and the diseases of interest. It may further contain other active ingredients and the like. As such additional components, any known components can be used, and those skilled in the art are familiar with these additional components. In addition, the cell culture of the present invention can be used in combination with a component that enhances the survival, engraftment, and / or function of the cell culture, and other active components that are useful for treating a target disease.
本発明の細胞培養物は、本発明の除去方法でエンドトキシンが除去された細胞を用いて、既知の任意の方法により製造することができる。例えば、シート状細胞培養物の製造方法は、特許文献３、特開2010-081829、特開2010-226962、特開2010-226991、特開2011-110368、特開2011-115058、特開2011-172925などに記載されている。本発明の製造方法で用いる器具や、培地、試薬、添加物などの材料は、いずれもエンドトキシンを含まないことが好ましい。 The cell culture of the present invention can be produced by any known method using cells from which endotoxin has been removed by the removal method of the present invention. For example, a method for producing a sheet-shaped cell culture is disclosed in Patent Document 3, JP 2010-081829, JP 2010-226962, JP 2010-226991, JP 2011-110368, JP 2011-115058, JP 2011-15058. 172925. It is preferable that all of the instruments, media, reagents, additives, and other materials used in the production method of the present invention do not contain endotoxin.
本発明の製造方法の特に好ましい態様は、以下のステップを含む：
（１）細胞懸濁液を、細胞凝集阻害剤を含む洗浄液で繰り返し２回以上洗浄するステップ、
（２）ステップ（１）で得られた細胞を凍結するステップ、
（３）凍結した細胞を解凍するステップ、
（４）シート状細胞培養物を形成するステップ、
（５）形成されたシート状細胞培養物を回収するステップ。
（１）の洗浄するステップは、本発明の除去方法についてすでに上記したとおりである。
A particularly preferred embodiment of the production method according to the invention comprises the following steps:
(1) a step of repeatedly washing the cell suspension twice or more with a washing solution containing a cell aggregation inhibitor;
(2) freezing the cells obtained in step (1),
(3) thawing the frozen cells,
(4) forming a sheet-shaped cell culture;
(5) A step of collecting the formed sheet-shaped cell culture.
The washing step (1) is as described above for the removal method of the present invention.
（２）の細胞を凍結するステップは、既知の任意の手法により行うことができる。かかる手法としては、限定されずに、例えば、容器内の細胞を、凍結手段、例えば、フリーザー、ディープフリーザー、低温の媒体（例えば、液体窒素等）に供することなどが挙げられる。凍結手段の温度は、容器内の細胞集団の一部、好ましくは全体を凍結させ得る温度であれば特に限定されないが、典型的には０℃以下、好ましくは−２０℃以下、より好ましくは−４０℃以下、さらに好ましくは−８０℃以下である。また、凍結操作における冷却速度は、凍結解凍後の細胞の生存率や機能を大きく損なうものでなければ特に限定されないが、典型的には４℃から冷却を始めて−８０℃に達するまで１〜５時間、好ましくは２〜４時間、特に約３時間かける程度の冷却速度である。具体的には、例えば、０．４６℃／分の速度で冷却することができる。かかる冷却速度は、所望の温度に設定した凍結手段に、細胞を含む容器を直接、または、凍結処理容器に収容して供することにより達成することができる。凍結処理容器は、容器内の温度の下降速度を所定の速度に制御する機能を有していてもよい。かかる凍結処理容器としては、既知の任意のもの、例えば、BICELL(R)（日本フリーザー）などを用いることができる。 The step (2) of freezing the cells can be performed by any known technique. Such a technique includes, but is not limited to, for example, subjecting the cells in the container to a freezing means, for example, a freezer, a deep freezer, or a low-temperature medium (for example, liquid nitrogen or the like). The temperature of the freezing means is not particularly limited as long as it can freeze a part, preferably the whole, of the cell population in the container, but is typically 0 ° C. or lower, preferably −20 ° C. or lower, more preferably −20 ° C. or lower. It is 40 ° C or lower, more preferably -80 ° C or lower. The cooling rate in the freezing operation is not particularly limited as long as the viability and function of the cells after freezing and thawing are not significantly impaired. Typically, the cooling rate is 1 to 5 from 4 ° C. until the temperature reaches −80 ° C. The cooling rate is in the order of hours, preferably 2 to 4 hours, especially about 3 hours. Specifically, for example, cooling can be performed at a rate of 0.46 ° C./min. Such a cooling rate can be achieved by providing a container containing cells directly to a freezing means set to a desired temperature or by providing the container containing the cells in a freezing treatment container. The freezing treatment container may have a function of controlling the rate of temperature decrease in the container to a predetermined speed. Such freezing vessel of a known arbitrary, for example, can be used as BICELL (R) (Nippon freezer).
凍結操作は、細胞を培養液や生理緩衝液などに浸漬させたまま行ってもよいが、細胞を凍結・解凍操作から保護するための凍結保護剤を培養液に加えたり、培養液を凍結保護剤を含む凍結保存液と置換するなどの処理を施したうえで行ってもよい。したがって、本発明の製造方法は、培養液に凍結保護剤を添加するステップ、または、培養液を凍結保存液に置換するステップをさらに含んでもよい。培養液を凍結保存液に置換する場合、凍結時に細胞が浸漬している液に有効濃度の凍結保護剤が含まれていれば、培養液を実質的に全て除去してから凍結保存液を添加しても、培養液を一部残したまま凍結保存液を添加してもよい。ここで、「有効濃度」とは、凍結保護剤が、毒性を示すことなく、凍結保護効果、例えば、凍結保護剤を用いない場合と比べた、凍結解凍後の細胞の生存率、活力、機能などの低下抑制効果を示す濃度を意味する。かかる濃度は当業者に知られているか、ルーチンの実験などにより適宜決定することができる。 The freezing operation may be performed while the cells are immersed in a culture solution or physiological buffer, but a cryoprotectant to protect the cells from freezing and thawing operations may be added to the culture solution, or the culture solution may be cryoprotected. It may be performed after treatment such as replacement with a cryopreservation solution containing the agent. Therefore, the production method of the present invention may further include a step of adding a cryoprotectant to the culture solution, or a step of replacing the culture solution with a cryopreservation solution. When replacing the culture solution with a cryopreservation solution, if the liquid in which the cells are immersed during freezing contains an effective concentration of a cryoprotectant, virtually all of the culture solution is removed before adding the cryopreservation solution. Alternatively, the cryopreservation solution may be added while leaving a part of the culture solution. Here, the “effective concentration” means that the cryoprotectant does not show toxicity and has a cryoprotective effect, for example, cell viability, vitality, and function after cryothawing as compared with the case where no cryoprotectant is used. Etc. means a concentration which exhibits a reduction suppressing effect. Such a concentration is known to those skilled in the art or can be appropriately determined by routine experiments and the like.
凍結保護剤は、細胞に対して凍結保護作用を示すものであれば特に限定されずに、例えば、ジメチルスルホキシド（ＤＭＳＯ）、グリセロール、エチレングリコール、プロピレングリコール、セリシン、プロパンジオール、デキストラン、ポリビニルピロリドン、ポリビニルアルコール、ヒドロキシエチルデンプン、コンドロイチン硫酸、ポリエチレングリコール、ホルムアミド、アセトアミド、アドニトール、ペルセイトール、ラフィノース、ラクトース、トレハロース、スクロース、マンニトールなどを含む。凍結保護剤は、単独で用いても、２種または３種以上を組み合わせて用いてもよい。 The cryoprotective agent is not particularly limited as long as it exhibits a cryoprotective effect on cells, and examples thereof include dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, propylene glycol, sericin, propanediol, dextran, polyvinylpyrrolidone, Polyvinyl alcohol, hydroxyethyl starch, chondroitin sulfate, polyethylene glycol, formamide, acetamide, adonitol, perseitol, raffinose, lactose, trehalose, sucrose, mannitol and the like. The cryoprotectants may be used alone or in combination of two or more.
培養液への凍結保護剤の添加濃度、または、凍結保存液中の凍結保護剤の濃度は、上記で定義した有効濃度であれば特に限定されず、典型的には、例えば、培養液または凍結保存液全体に対して２〜２０％（ｖ／ｖ）である。しかしながら、この濃度範囲からは外れるが、それぞれの凍結保護剤について知られているか、実験的に決定した代替的な使用濃度を採用することもでき、かかる濃度も本発明の範囲内である。 The concentration of the cryoprotectant added to the culture solution or the concentration of the cryoprotectant in the cryopreservation solution is not particularly limited as long as it is an effective concentration as defined above. It is 2 to 20% (v / v) based on the whole storage solution. However, although outside this concentration range, alternative use concentrations known or experimentally determined for each cryoprotectant may be employed, and such concentrations are also within the scope of the invention.
（３）の凍結した細胞を解凍するステップは、既知の任意の細胞解凍手法により行うことができ、典型的には、例えば、凍結した細胞を、解凍手段、例えば、凍結温度より高い温度の固形、液状もしくはガス状の媒体（例えば、水）、ウォーターバス、インキュベーター、恒温器などに供したり、または、凍結した細胞を、凍結温度より高い温度の媒体（例えば、培養液）で浸漬することにより達成されるが、これに限定されない。解凍手段または浸漬媒体の温度は、細胞を所望の時間内に解凍できる温度であれば特に限定されないが、典型的には４〜５０℃、好ましくは３０℃〜４０℃、より好ましくは３６〜３８℃である。また、解凍時間は、解凍後の細胞の生存率や機能を大きく損なうものでなければ特に限定されないが、典型的には２分以内であり、特に２０秒以内とすることで生存率の低下を大幅に抑制することができる。解凍時間は、例えば、解凍手段または浸漬媒体の温度、凍結時の培養液または凍結保存液の容量もしくは組成などを変化させて調節することができる。 The step (3) of thawing the frozen cells can be performed by any known cell thawing method. Typically, for example, the frozen cells are thawed by a thawing means, for example, a solid at a temperature higher than the freezing temperature. By subjecting it to a liquid or gaseous medium (eg, water), a water bath, an incubator, an incubator, or by immersing the frozen cells in a medium at a temperature higher than the freezing temperature (eg, a culture solution). Achieved, but not limited to. The temperature of the thawing means or the immersion medium is not particularly limited as long as the cells can be thawed within a desired time, but is typically 4 to 50 ° C, preferably 30 to 40 ° C, more preferably 36 to 38 ° C. ° C. The thawing time is not particularly limited as long as the survival rate and function of the cells after thawing are not significantly impaired. However, the thawing time is typically within 2 minutes, and particularly within 20 seconds, the reduction in the survival rate can be prevented. It can be greatly suppressed. The thawing time can be adjusted by, for example, changing the temperature of the thawing means or the immersion medium, and the volume or composition of the culture solution or the cryopreservation solution at the time of freezing.
（４）のシート状細胞培養物を形成するステップは、既知の任意の手法により行うことができる。かかる手法としては、限定されずに、例えば、特許文献３、特開2010-081829、特開2010-226962、特開2010-226991、特開2011-110368、特開2011-115058、特開2011-172925などに記載されたものが挙げられる。シート状細胞培養物を形成するステップは、細胞を培養基材上に播種するステップ、および、播種した細胞をシート化するステップを含んでもよい。 The step (4) of forming a sheet-shaped cell culture can be performed by any known technique. Such a method is not limited, for example, Patent Document 3, JP 2010-081829, JP 2010-226962, JP 2010-226991, JP 2011-110368, JP 2011-115058, JP 2011- 172925 and the like. The step of forming a sheet-shaped cell culture may include a step of seeding the cells on a culture substrate, and a step of sheeting the seeded cells.
培養基材は、細胞がその上でシート状細胞培養物を形成し得るものであれば特に限定されず、例えば、種々の材質の容器、容器中の固形もしくは半固形の表面などを含む。容器は、培養液などの液体を透過させない構造・材料が好ましい。かかる材料としては、限定することなく、例えば、ポリエチレン、ポリプロピレン、テフロン（登録商標）、ポリエチレンテレフタレート、ポリメチルメタクリレート、ナイロン６，６、ポリビニルアルコール、セルロース、シリコン、ポリスチレン、ガラス、ポリアクリルアミド、ポリジメチルアクリルアミド、金属（例えば、鉄、ステンレス、アルミニウム、銅、真鍮）等が挙げられる。また、容器は、少なくとも１つの平坦な面を有することが好ましい。かかる容器の例としては、限定することなく、例えば、細胞培養皿、細胞培養ボトルなどが挙げられる。また、容器は、その内部に固形もしくは半固形の表面を有してもよい。固形の表面としては、上記のごとき種々の材料のプレートや容器などが、半固形の表面としては、ゲル、軟質のポリマーマトリックスなどが挙げられる。培養基材は、上記材料を用いて作製してもよいし、市販のものを利用してもよい。好ましい培養基材としては、限定することなく、例えば、シート状細胞培養物の形成に適した、接着性の表面を有する基材が挙げられる。具体的には、親水性の表面を有する基材、例えば、コロナ放電処理したポリスチレン、コラーゲンゲルや親水性ポリマーなどの親水性化合物を該表面にコーティングした基材、さらには、コラーゲン、フィブロネクチン、ラミニン、ビトロネクチン、プロテオグリカン、グリコサミノグリカンなどの細胞外マトリックスや、カドヘリンファミリー、セレクチンファミリー、インテグリンファミリーなどの細胞接着因子などを表面にコーティングした基材などが挙げられる。また、かかる基材は市販されている（例えば、Corning(R) TC-Treated Culture Dish、Corningなど）。 The culture substrate is not particularly limited as long as the cells can form a sheet-shaped cell culture thereon, and includes, for example, containers of various materials, solid or semi-solid surfaces in the containers, and the like. The container is preferably made of a structure / material that does not allow the passage of a liquid such as a culture solution. Examples of such a material include, but are not limited to, polyethylene, polypropylene, Teflon (registered trademark), polyethylene terephthalate, polymethyl methacrylate, nylon 6,6, polyvinyl alcohol, cellulose, silicon, polystyrene, glass, polyacrylamide, and polydimethyl. Acrylamide, metal (eg, iron, stainless steel, aluminum, copper, brass) and the like can be mentioned. Also, the container preferably has at least one flat surface. Examples of such containers include, without limitation, cell culture dishes, cell culture bottles, and the like. Further, the container may have a solid or semi-solid surface inside. Examples of the solid surface include plates and containers made of various materials as described above, and examples of the semi-solid surface include a gel and a soft polymer matrix. The culture substrate may be prepared using the above materials, or a commercially available substrate may be used. Preferred culture substrates include, but are not limited to, substrates having an adhesive surface, suitable for forming sheet-like cell cultures, for example. Specifically, a substrate having a hydrophilic surface, for example, corona discharge-treated polystyrene, a substrate having a hydrophilic compound such as a collagen gel or a hydrophilic polymer coated on the surface, further, collagen, fibronectin, laminin And extracellular matrices such as vitronectin, proteoglycan, and glycosaminoglycan, and substrates coated on the surface with cell adhesion factors such as cadherin family, selectin family, integrin family, and the like. Further, such substrates are commercially available (e.g., Corning (R) TC-Treated Culture Dish, Corning , etc.).
培養基材は、刺激、例えば、温度や光に応答して物性が変化する材料で表面が被覆されていてもよい。かかる材料としては、限定されずに、例えば、（メタ）アクリルアミド化合物、Ｎ−アルキル置換（メタ）アクリルアミド誘導体（例えば、Ｎ−エチルアクリルアミド、Ｎ−ｎ−プロピルアクリルアミド、Ｎ−ｎ−プロピルメタクリルアミド、Ｎ−イソプロピルアクリルアミド、Ｎ−イソプロピルメタクリルアミド、Ｎ−シクロプロピルアクリルアミド、Ｎ−シクロプロピルメタクリルアミド、Ｎ−エトキシエチルアクリルアミド、Ｎ−エトキシエチルメタクリルアミド、Ｎ−テトラヒドロフルフリルアクリルアミド、Ｎ−テトラヒドロフルフリルメタクリルアミド等）、Ｎ，Ｎ−ジアルキル置換（メタ）アクリルアミド誘導体（例えば、Ｎ，Ｎ−ジメチル（メタ）アクリルアミド、Ｎ，Ｎ−エチルメチルアクリルアミド、Ｎ，Ｎ−ジエチルアクリルアミド等）、環状基を有する（メタ）アクリルアミド誘導体（例えば、１−（１−オキソ−２−プロペニル）−ピロリジン、１−（１−オキソ−２−プロペニル）−ピペリジン、４−（１−オキソ−２−プロペニル）−モルホリン、１−（１−オキソ−２−メチル−２−プロペニル）−ピロリジン、１−（１−オキソ−２−メチル−２−プロペニル）−ピペリジン、４−（１−オキソ−２−メチル−２−プロペニル）−モルホリン等）、またはビニルエーテル誘導体（例えば、メチルビニルエーテル）のホモポリマーまたはコポリマーからなる温度応答性材料、アゾベンゼン基を有する光吸収性高分子、トリフェニルメタンロイコハイドロオキシドのビニル誘導体とアクリルアミド系単量体との共重合体、および、スピロベンゾピランを含むＮ−イソプロピルアクリルアミドゲル等の光応答性材料などの公知のものを用いることができる（例えば、特開平2-211865、特開2003-33177参照）。これらの材料に所定の刺激を与えることによりその物性、例えば、親水性や疎水性を変化させ、同材料上に付着した細胞培養物の剥離を促進することができる。温度応答性材料で被覆された培養皿は市販されており（例えば、CellSeed Inc.のUpCell(R)）、これらを本発明の製造方法に使用することができる。 The culture substrate may be coated on its surface with a material whose properties change in response to a stimulus, for example, temperature or light. Examples of such materials include, but are not limited to, (meth) acrylamide compounds, N-alkyl-substituted (meth) acrylamide derivatives (eg, N-ethylacrylamide, Nn-propylacrylamide, Nn-propylmethacrylamide, N-isopropylacrylamide, N-isopropylmethacrylamide, N-cyclopropylacrylamide, N-cyclopropylmethacrylamide, N-ethoxyethylacrylamide, N-ethoxyethylmethacrylamide, N-tetrahydrofurfurylacrylamide, N-tetrahydrofurfuryl methacryl Amide), N, N-dialkyl-substituted (meth) acrylamide derivatives (eg, N, N-dimethyl (meth) acrylamide, N, N-ethylmethylacrylamide, N, N-diethyl Acrylamide), (meth) acrylamide derivatives having a cyclic group (eg, 1- (1-oxo-2-propenyl) -pyrrolidine, 1- (1-oxo-2-propenyl) -piperidine, 4- (1-oxo) -2-propenyl) -morpholine, 1- (1-oxo-2-methyl-2-propenyl) -pyrrolidine, 1- (1-oxo-2-methyl-2-propenyl) -piperidine, 4- (1-oxo) -2-methyl-2-propenyl) -morpholine or the like, a temperature-responsive material comprising a homopolymer or copolymer of a vinyl ether derivative (eg, methyl vinyl ether), a light-absorbing polymer having an azobenzene group, triphenylmethane leucohydro. A copolymer of a vinyl derivative of an oxide and an acrylamide monomer, and spirobenzopyra Can be used to include N- and isopropyl acrylamide gels known, such as photoresponsive materials (e.g., JP-A-2-211865, see JP-2003-33177). By applying a predetermined stimulus to these materials, their physical properties, for example, hydrophilicity or hydrophobicity, can be changed, and the detachment of the cell culture adhered on the materials can be promoted. Culture dishes coated with a temperature-responsive materials are commercially available (e.g., UpCell of CellSeed Inc. (R)) can be used, these the production method of the present invention.
培養基材への細胞の播種は、既知の任意の手法および条件で行うことができる。培養基材への細胞の播種は、例えば、細胞を培養液に懸濁した細胞懸濁液を培養基材（培養容器）に注入することにより行ってもよい。細胞懸濁液の注入には、スポイトやピペットなど、細胞懸濁液の注入操作に適した器具を用いることができる。 Seeding of the cells into the culture substrate can be performed by any known method and conditions. Seeding of the cells into the culture substrate may be performed, for example, by injecting a cell suspension in which the cells are suspended in a culture solution into the culture substrate (culture container). For injection of the cell suspension, a device suitable for the operation of injecting the cell suspension, such as a dropper or a pipette, can be used.
播種した細胞をシート化するステップも、既知の任意の手法および条件で行うことができる。かかる手法の非限定例は、例えば、特許文献３、特開2010-081829、特開2010-226962、特開2010-226991、特開2011-110368、特開2011-115058、特開2011-172925などに記載されている。細胞のシート化は、細胞同士が接着分子や、細胞外マトリックスなどの細胞間接着機構を介して互いに接着することにより達成されると考えられている。したがって、播種した細胞をシート化するステップは、例えば、細胞を、細胞間接着を形成する条件下で培養することにより達成することができる。かかる条件は、細胞間接着を形成することができればいかなるものであってもよいが、通常は一般的な細胞培養条件と同様の条件であれば細胞間接着を形成することができる。当業者であれば、播種する細胞の種類に応じて最適な条件を選択することができる。本明細書において、播種した細胞をシート化するための培養を、「シート化培養」と呼ぶ場合もある。 The step of sheeting the seeded cells can also be performed by any known method and conditions. Non-limiting examples of such a method, for example, Patent Document 3, JP 2010-081829, JP 2010-226962, JP 2010-226991, JP 2011-110368, JP 2011-115058, JP 2011-172925 and the like It is described in. It is believed that cell sheeting is achieved by cells adhering to each other via intercellular adhesion mechanisms such as adhesion molecules and extracellular matrix. Therefore, the step of forming the seeded cells into a sheet can be achieved, for example, by culturing the cells under conditions that form intercellular adhesion. Such conditions may be any as long as they can form cell-cell adhesion, but usually, cell-cell adhesion can be formed under the same conditions as general cell culture conditions. Those skilled in the art can select optimal conditions according to the type of cells to be seeded. In this specification, the culture for forming the seeded cells into sheets may be referred to as “sheet culture”.
培養に用いる細胞培養液（単に「培養液」もしくは「培地」と呼ぶ場合もある）は、細胞の生存を維持できるものであれば特に限定されないが、典型的には、アミノ酸、ビタミン類、電解質を主成分としたものが利用できる。本発明の一態様において、培養液は、細胞培養用の基礎培地をベースにしたものである。かかる基礎培地には、限定されずに、例えば、ＤＭＥＭ、ＭＥＭ、Ｆ１２、ＤＭＥ、ＲＰＭＩ１６４０、ＭＣＤＢ（ＭＣＤＢ１０２、１０４、１０７、１２０、１３１、１５３、１９９など）、Ｌ１５、ＳｋＢＭ、ＲＩＴＣ８０−７などが含まれる。これらの基礎培地の多くは市販されており、その組成も公知となっている。
基礎培地は、標準的な組成のまま（例えば、市販されたままの状態で）用いてもよいし、細胞種や細胞条件に応じてその組成を適宜変更してもよい。したがって、本発明に用いる基礎培地は、公知の組成のものに限定されず、１または２以上の成分が追加、除去、増量もしくは減量されたものを含む。
The cell culture solution (sometimes simply referred to as “culture solution” or “medium”) used for the culture is not particularly limited as long as it can maintain the survival of the cells, but typically includes amino acids, vitamins, and electrolytes. Can be used. In one aspect of the present invention, the culture solution is based on a basal medium for cell culture. Examples of such a basal medium include, but are not limited to, DMEM, MEM, F12, DME, RPMI1640, MCDB (MCDB102, 104, 107, 120, 131, 153, 199, etc.), L15, SkBM, RITC80-7 and the like. included. Many of these basal media are commercially available and their compositions are also known.
The basal medium may be used with its standard composition (for example, as it is commercially available), or its composition may be appropriately changed depending on the cell type and cell conditions. Therefore, the basal medium used in the present invention is not limited to those having a known composition, and includes those in which one or more components have been added, removed, increased or reduced in weight.
細胞の培養は、当該技術分野で通常なされている条件で行うことができる。例えば、典型的な培養条件としては、３７℃、５％ＣＯ２での培養が挙げられる。培養は任意の大きさおよび形状の容器で行うことができる。細胞培養物の大きさや形状は、培養容器の細胞付着面の大きさ・形状を調整すること、または、培養容器の細胞付着面に、所望の大きさ・形状の型枠を設置し、その内部で細胞を培養することなどにより任意に調節することができる。 The culturing of the cells can be performed under the conditions usually performed in the art. For example, typical culture conditions include culture at 37 ° C., 5% CO 2 . The culture can be performed in a container of any size and shape. The size and shape of the cell culture can be adjusted by adjusting the size and shape of the cell attachment surface of the culture container, or by installing a mold of the desired size and shape on the cell attachment surface of the culture container, and It can be adjusted arbitrarily by, for example, culturing the cells with.
（５）におけるシート状細胞培養物の回収は、シート状細胞培養物が少なくとも部分的に、シート構造を保ったまま、足場となっている培養基材から遊離（剥離）できれば特に限定されず、例えば、タンパク質分解酵素（例えばトリプシンなど）による酵素処理および／またはピペッティングなどの機械的処理によって行うことができる。また、細胞を、刺激、例えば、温度や光に応答して物性が変化する材料で表面を被覆した培養基材上で培養して細胞培養物を形成した場合には、所定の刺激を加えることで、非酵素的に遊離することもできる。 The collection of the sheet-shaped cell culture in (5) is not particularly limited as long as the sheet-shaped cell culture can be at least partially released (peeled off) from the culture substrate serving as a scaffold while maintaining the sheet structure. For example, it can be performed by an enzymatic treatment with a protease (for example, trypsin) and / or a mechanical treatment such as pipetting. In addition, when a cell culture is formed by culturing cells on a culture substrate whose surface is coated with a material whose physical properties change in response to temperature or light, for example, a material, the predetermined stimulus is applied. Thus, it can be released non-enzymatically.
本発明の別の側面は、本発明の細胞培養物を含む、医薬組成物に関する。
本発明の医薬組成物は、本発明の細胞培養物に加えて、種々の追加成分、例えば、薬学的に許容し得る担体や、細胞培養物の生存性、生着性および／または機能などを高める成分、対象疾患の処置に有用な他の有効成分などを含んでいてもよい。かかる追加成分としては、既知の任意のものを使用することができ、当業者はこれらの追加成分について精通している。また、本発明の医薬組成物は、細胞培養物の生存性、生着性および／または機能などを高める成分や、対象疾患の処置に有用な他の有効成分などと併用することができる。一態様において、本発明の医薬組成物は、組織の異常に関連する疾患の処置に用いるためのものである。処置の対象となる組織や疾患は、本発明の細胞培養物について上記したとおりである。
Another aspect of the present invention relates to a pharmaceutical composition comprising the cell culture of the present invention.
The pharmaceutical composition of the present invention may contain, in addition to the cell culture of the present invention, various additional components, such as a pharmaceutically acceptable carrier and the viability, engraftment and / or function of the cell culture. It may contain a component that enhances, other active components useful for treating the target disease, and the like. As such additional components, any known components can be used, and those skilled in the art are familiar with these additional components. Further, the pharmaceutical composition of the present invention can be used in combination with a component that enhances the survival, engraftment, and / or function of a cell culture, and other active components that are useful for treating a target disease. In one embodiment, the pharmaceutical composition of the present invention is for use in treating a disease associated with a tissue abnormality. The tissue or disease to be treated is as described above for the cell culture of the present invention.
本発明の別の側面は、対象において疾患を処置する方法であって、本発明の細胞培養物または医薬組成物の有効量をそれ必要とする対象に投与するステップを含む方法に関する。本発明の処置方法の対象となる組織や疾患は、本発明の細胞培養物について上記したとおりである。また、本発明の処置方法においては、細胞培養物の生存性、生着性および／または機能などを高める成分や、対象疾患の処置に有用な他の有効成分などを、本発明の細胞培養物または医薬組成物と併用することができる。 Another aspect of the present invention relates to a method of treating a disease in a subject, comprising administering to a subject in need thereof an effective amount of a cell culture or pharmaceutical composition of the present invention. The tissues and diseases to be treated by the treatment method of the present invention are as described above for the cell culture of the present invention. In the treatment method of the present invention, the cell culture of the present invention contains components that enhance the viability, engraftment and / or function of the cell culture, and other active components useful for treating the target disease. Alternatively, it can be used in combination with a pharmaceutical composition.
本発明の処置方法は、本発明の製造方法に従って、細胞培養物を製造するステップをさらに含んでもよい。本発明の処置方法は、細胞培養物を製造するステップの前に、対象から細胞培養物を製造するための細胞または細胞の給源となる組織を採取するステップをさらに含んでもよい。一態様において、細胞または細胞の給源となる組織を採取する対象は、細胞培養物の投与を処置を受ける対象と同一の個体である。別の態様において、細胞または細胞の給源となる組織を採取する対象は、細胞培養物の投与を処置を受ける対象とは同種の別個体である。別の態様において、細胞または細胞の給源となる組織を採取する対象は、細胞培養物の投与を処置を受ける対象とは異種の個体である。 The treatment method of the present invention may further include a step of producing a cell culture according to the production method of the present invention. The treatment method of the present invention may further include, before the step of producing the cell culture, collecting a cell or a tissue that is a source of the cell for producing the cell culture from the subject. In one embodiment, the subject from whom the cells or tissue from which the cells are sourced is harvested is the same individual as the subject who is to be treated for administration of the cell culture. In another embodiment, the subject from whom the cells or the tissue from which the cells are sourced is harvested is a homologous separate from the subject who is to be treated for administration of the cell culture. In another embodiment, the subject from whom the cells or tissue from which the cells are sourced is harvested is an individual that is heterogeneous to the subject to be treated for administration of the cell culture.
本発明において、用語「対象」は、任意の生物個体、好ましくは動物、さらに好ましくは哺乳動物、さらに好ましくはヒトの個体を意味する。本発明において、対象は健常であっても、何らかの疾患に罹患していてもよいものとするが、疾患の処置が企図される場合には、典型的には当該疾患に罹患しているか、罹患するリスクを有する対象を意味する。 In the context of the present invention, the term “subject” means any living individual, preferably an animal, more preferably a mammal, more preferably a human individual. In the present invention, the subject may be healthy or suffer from any disease, but if treatment of the disease is contemplated, typically the subject is suffering from or suffering from the disease. Means at risk of doing
また、用語「処置」は、疾患の治癒、一時的寛解または予防などを目的とする医学的に許容される全ての種類の予防的および／または治療的介入を包含するものとする。例えば、「処置」の用語は、疾患の進行の遅延または停止、病変の退縮または消失、当該疾患発症の予防または再発の防止などを含む、種々の目的の医学的に許容される介入を包含する。 Also, the term "treatment" is intended to include all types of medically acceptable prophylactic and / or therapeutic interventions, such as for the cure, temporary remission or prevention of disease. For example, the term "treatment" encompasses medically acceptable interventions for a variety of purposes, including slowing or stopping the progression of a disease, regressing or eliminating lesions, preventing the onset of the disease or preventing its recurrence, and the like. .
本発明において、有効量とは、例えば、疾患の発症や再発を抑制し、症状を軽減し、または進行を遅延もしくは停止し得る量（例えば、細胞培養物に含まれる細胞数、細胞培養物のサイズ、重量など）であり、好ましくは、当該疾患の発症および再発を予防し、または当該疾患を治癒する量である。また、投与による利益を超える悪影響が生じない量が好ましい。かかる量は、例えば、マウス、ラット、イヌまたはブタなどの実験動物や疾患モデル動物における試験などにより適宜決定することができ、このような試験法は当業者によく知られている。また、処置の対象となる組織病変の大きさは、有効量決定のための重要な指標となり得る。 In the present invention, an effective amount is, for example, an amount capable of suppressing the onset and recurrence of a disease, reducing symptoms, or delaying or stopping the progress (for example, the number of cells contained in a cell culture, Size, weight, etc.), and preferably an amount that prevents the onset and recurrence of the disease or cures the disease. Also preferred is an amount that does not cause adverse effects beyond the benefit of administration. Such an amount can be appropriately determined, for example, by a test in a laboratory animal such as a mouse, a rat, a dog or a pig, or a disease model animal, and such a test method is well known to those skilled in the art. In addition, the size of a tissue lesion to be treated can be an important index for determining an effective amount.
本発明の細胞培養物および医薬組成物は、静脈内、筋肉内、皮下、局所、動脈内、門脈内、心室内、腹腔内等の種々の経路から投与することができる。シート状細胞培養物の場合は、投与方法として、典型的には組織への直接的な適用が挙げられるが、シート状細胞培養物の断片を用いる場合には、注射による投与が可能な種々の経路、例えば、静脈内、筋肉内、皮下、局所、動脈内、門脈内、心室内、腹腔内等の経路から投与してもよい。
投与頻度は、典型的には１回の処置につき１回であるが、所望の効果が得られない場合には、複数回投与することも可能である。
The cell culture and the pharmaceutical composition of the present invention can be administered from various routes such as intravenous, intramuscular, subcutaneous, topical, intraarterial, intraportal, intraventricular, and intraperitoneal. In the case of a sheet-shaped cell culture, the administration method typically includes direct application to a tissue.However, when a fragment of a sheet-shaped cell culture is used, various methods that can be administered by injection are used. Administration may be by any route, for example, intravenous, intramuscular, subcutaneous, topical, intraarterial, intraportal, intraventricular, intraperitoneal, and the like.
The frequency of administration is typically once per treatment, but multiple administrations are possible if the desired effect is not obtained.
以下に、本発明を実施例を参照してより詳細に説明するが、これは本発明の特定の具体例を示すものであり、本発明はこれに限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to Examples, which show specific embodiments of the present invention, and the present invention is not limited thereto.
実施例１ 細胞からのエンドトキシンの除去
２．５ｇのヒトアルブミン（一般社団法人日本血液製剤機構製）を５００ｍＬのハンクス平衡塩液に加え、ヒトアルブミンを０．５ｗ/ｖ％の濃度で含む洗浄液を調製した。３×１０７個のヒト骨格筋芽細胞を３０ｍＬの洗浄液に懸濁した細胞懸濁液を５０ｍＬコニカルチューブに入れ、そこにエンドトキシン（E. coli UKT-B株由来、和光純薬工業（株）製）を添加した。細胞懸濁液を１００μＬ採取し、その後エンドトキシン量の測定に供した。細胞懸濁液を２４０×ｇで７分間遠心して上清を廃棄した。チューブに洗浄液を２８．５ｍＬ加え、ピペッティングにより細胞を十分に撹拌した後、２４０×ｇで７分間遠心して上清を廃棄した。この操作を６回繰り返した。６回目の洗浄操作で、上清を廃棄した後に残った細胞を３ｍＬの洗浄液に懸濁し、エンドトキシン量の測定に供した。また、未使用の洗浄液もエンドトキシン量の測定に供した。なお、洗浄液には、一般的な作業条件下で混入する可能性のあるエンドトキシンの影響を評価するために、予め一定量のエンドトキシンを添加しておいた。各検体のエンドトキシン量は、トキシノメーターＥＴ−６０００およびリムルスＥＳ−ＩＩシングルテストワコー（いずれも和光純薬工業（株）製）を用いて比濁時間法（カイネティック−比濁法）にて測定した。結果を下表に示す。
Example 1 Removal of Endotoxin from Cells 2.5 g of human albumin (manufactured by Japan Blood Products Organization) was added to 500 mL of Hanks balanced salt solution, and a washing solution containing human albumin at a concentration of 0.5 w / v% was added. Prepared. A cell suspension obtained by suspending 3 × 10 7 human skeletal myoblasts in a 30 mL washing solution was placed in a 50 mL conical tube, and an endotoxin (from E. coli UKT-B strain, Wako Pure Chemical Industries, Ltd.) was added thereto. Was added. 100 μL of the cell suspension was collected and then used for measurement of the amount of endotoxin. The cell suspension was centrifuged at 240 × g for 7 minutes and the supernatant was discarded. 28.5 mL of the washing solution was added to the tube, the cells were sufficiently stirred by pipetting, and then centrifuged at 240 × g for 7 minutes to discard the supernatant. This operation was repeated six times. In the sixth washing operation, cells remaining after discarding the supernatant were suspended in 3 mL of a washing solution, and used for measurement of the amount of endotoxin. An unused washing solution was also used for measurement of the amount of endotoxin. Note that a certain amount of endotoxin was added to the washing solution in advance in order to evaluate the effect of endotoxin that might be mixed under general working conditions. The endotoxin amount of each sample was determined by a turbidimetric time method (kinetic-turbidimetric method) using a toxinometer ET-6000 and Limulus ES-II Single Test Wako (both manufactured by Wako Pure Chemical Industries, Ltd.). It was measured. The results are shown in the table below.
クリアランス（％）は、「（添加エンドトキシン量−洗浄後のエンドトキシン量）÷添加エンドトキシン量×１００」で計算した。
上記結果が示すとおり、本発明の除去方法により、細胞培養物に添加したエンドトキシンを、検出限界未満となるまで、実に９９．９５％超も除去することができた。
The clearance (%) was calculated by “(the amount of added endotoxin−the amount of endotoxin after washing) ÷ the amount of added endotoxin × 100”.
As shown by the above results, the removal method of the present invention was able to remove as much as 99.95% of endotoxin added to the cell culture until the endotoxin became below the detection limit.
Claims (2)
前記洗浄は、細胞懸濁液から収集した細胞を洗浄液に懸濁し、この細胞に混和撹拌操作を加えることであり、
洗浄前後それぞれの細胞懸濁液の細胞密度が少なくとも１×１０６細胞／ｍｌであり、
洗浄後の細胞懸濁液が１０ＥＵ／ｍｌ以上のエンドトキシンを含まず、
洗浄後のエンドトキシンのクリアランス（％）が９９．９５％超である、前記方法。 A method for removing endotoxin from a cell suspension , comprising a step of repeatedly washing the cell suspension with a washing solution containing albumin six times or more,
The washing is to suspend cells collected from the cell suspension in a washing solution, and to add a mixing and stirring operation to the cells,
The cell density of each cell suspension before and after washing is at least 1 × 10 6 cells / ml,
The cell suspension after washing does not contain endotoxin of 10 EU / ml or more,
Such a method, wherein the endotoxin clearance (%) after washing is greater than 99.95%.
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