JP2021038268A - Regenerative therapeutic composition and method for producing regenerative therapeutic composition - Google Patents
Regenerative therapeutic composition and method for producing regenerative therapeutic composition Download PDFInfo
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- JP2021038268A JP2021038268A JP2020203946A JP2020203946A JP2021038268A JP 2021038268 A JP2021038268 A JP 2021038268A JP 2020203946 A JP2020203946 A JP 2020203946A JP 2020203946 A JP2020203946 A JP 2020203946A JP 2021038268 A JP2021038268 A JP 2021038268A
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Abstract
Description
本発明は、再生治療用組成物及び再生治療用組成物の製造方法に関する。さらに詳しくは、歯髄由来幹細胞を利用した再生治療用組成物及び再生治療用組成物の製造方法に関する。 The present invention relates to a composition for regenerative therapy and a method for producing the composition for regenerative therapy. More specifically, the present invention relates to a composition for regenerative treatment using dental pulp-derived stem cells and a method for producing the composition for regenerative treatment.
従来の医療では治療困難な疾病に対する汎用的な代替技術として、幹細胞を利用した再生医療が注目されている。かかる幹細胞を用いた再生医療は、全ての難病にとっての新しい臨床方法における有望なツールであり、例えば、胚性幹細胞（ＥＳ細胞）、誘導多能性幹細胞（ｉＰＳ細胞）、及び体性幹細胞を初めとする種々の幹細胞が報告されている。また、体性幹細胞のうち、骨髄、脂肪組織、皮膚、臍帯、胎盤等の種々の組織から単離される間葉系幹細胞（Ｍｅｓｅｎｃｈｙｍａｌ Ｓｔｅｍ Ｃｅｌｌ：ＭＳＣ）が、再生医療における治療（再生治療）の臨床的応用として特に用いられてきた。 Regenerative medicine using stem cells is attracting attention as a general-purpose alternative technology for diseases that are difficult to treat with conventional medicine. Regenerative medicine using such stem cells is a promising tool in new clinical methods for all intractable diseases, including, for example, embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), and somatic stem cells. Various stem cells have been reported. In addition, among somatic stem cells, mesenchymal stem cells (MSCs) isolated from various tissues such as bone marrow, adipose tissue, skin, umbilical cord, and placenta are used for clinical treatment (regenerative treatment) in regenerative medicine. It has been used especially as an application.
しかしながら、細胞採取に伴うドナーへの侵襲及び採取した個人により分泌されるサイトカイン量も一定でないことが確認されている。また、採取から投与までは一定期間の培養時間が必要であり、急性期への対応は不可能であった。ここで、サイトカイン（Ｃｙｔｏｋｉｎｅ）とは、細胞から分泌される生理活性蛋白質であり、細胞間の相互作用に関与する生理活性物質の総称を指す。かかるサイトカインは、標的組織の内在性幹細胞に対する誘導シグナルとして機能し、細胞の増殖、分化、細胞死、機能発現等の多様な細胞応答を引き起こすための有効成分となる。 However, it has been confirmed that the invasion of donors associated with cell collection and the amount of cytokines secreted by the collected individuals are not constant. In addition, a certain period of culture time was required from collection to administration, and it was impossible to cope with the acute phase. Here, cytokine (Cytokine) is a physiologically active protein secreted from cells, and refers to a general term for physiologically active substances involved in cell-cell interactions. Such cytokines function as induction signals for endogenous stem cells of target tissues, and serve as active ingredients for inducing various cellular responses such as cell proliferation, differentiation, cell death, and functional expression.
また、本発明者等は採取した線維芽細胞を顔面皮膚へ注入することによって肌の状態を再生させる治療に関わってきたが（例えば、非特許文献１を参照。）、細胞の培養状態と臨床結果において相関を推測させる知見を得ている。 In addition, the present inventors have been involved in the treatment of regenerating the skin condition by injecting the collected fibroblasts into the facial skin (see, for example, Non-Patent Document 1), but the cell culture condition and clinical practice. We have obtained findings that make us infer the correlation in the results.
一方、移植した細胞の動向に関して、移植後に細胞は残らないことがわかってきている（例えば、非特許文献２等。）。幹細胞自体を再生医療に使用するに当たっては、前記のような問題点があるため、近年、種々の幹細胞自体を使用するのではなく、それら幹細胞自体が産生する種々の生体因子、例えば、前記したサイトカインとなる、各種の成長因子を使用する方法が検討されてきた（例えば、特許文献１等を参照。）。 On the other hand, regarding the trend of transplanted cells, it has been found that no cells remain after transplantation (for example, Non-Patent Document 2 etc.). In using the stem cells themselves for regenerative medicine, there are the above-mentioned problems. Therefore, in recent years, instead of using various stem cells themselves, various biological factors produced by the stem cells themselves, such as the above-mentioned cytokines, have been used. A method of using various growth factors has been studied (see, for example, Patent Document 1 and the like).
しかしながら、再生治療用組成物を用いるに際して、歯髄由来幹細胞培養上清に含まれる、細胞再生のために有効な成分となるサイトカインについて、その種類や濃度の有効な選択については検討されていないのが実情であった。 However, when using the composition for regenerative therapy, the effective selection of the type and concentration of cytokines contained in the pulp-derived stem cell culture supernatant, which are effective components for cell regeneration, has not been investigated. It was the actual situation.
本発明は、前記の課題に鑑みてなされたものであり、歯髄由来幹細胞を培養することによって得られた幹細胞培養上清が含むサイトカインのうち、特定の種類のサイトカイン及びその濃度に着目した再生治療用組成物及び再生治療用組成物の製造方法を提供することにある。 The present invention has been made in view of the above problems, and among the cytokines contained in the stem cell culture supernatant obtained by culturing dental pulp-derived stem cells, a specific type of cytokine and a regenerative therapy focusing on the concentration thereof. The present invention is to provide a method for producing a composition for use and a composition for regenerative therapy.
前記の課題を解決するために、本発明に係る再生治療用組成物は、歯髄由来幹細胞自体を含まない歯髄由来幹細胞培養上清を含んでなる再生治療用組成物であって、
前記歯髄由来幹細胞培養上清が含有するサイトカインである、血管内皮細胞増殖因子（ＶＥＧＦ）の濃度が１５０〜５０００ｐｇ／ｍｌであることを特徴とする。
In order to solve the above-mentioned problems, the composition for regenerative treatment according to the present invention is a composition for regenerative treatment containing a pulp-derived stem cell culture supernatant that does not contain dental pulp-derived stem cells themselves.
The concentration of vascular endothelial growth factor (VEGF), which is a cytokine contained in the pulp-derived stem cell culture supernatant, is 150 to 5000 pg / ml.
本発明に係る再生治療用組成物は、前記した本発明において、前記歯髄由来幹細胞培養上清が含有するサイトカインである、インシュリン様成長因子（ＩＧＦ）の濃度が８０〜１０００ｐｇ／ｍｌであることを特徴とする。 The composition for regenerative therapy according to the present invention has the above-mentioned present invention in which the concentration of insulin-like growth factor (IGF), which is a cytokine contained in the pulp-derived stem cell culture supernatant, is 80 to 1000 pg / ml. It is a feature.
本発明に係る再生治療用組成物は、前記した本発明において、前記歯髄由来幹細胞培養上清が含有するカルシウムイオン（Ｃａ２＋）の濃度が６．５〜８．０ｍｇ／ｄｌであることを特徴とする。 The composition for regenerative therapy according to the present invention is characterized in that, in the present invention described above , the concentration of calcium ions (Ca 2+ ) contained in the pulp-derived stem cell culture supernatant is 6.5 to 8.0 mg / dl. And.
本発明に係る再生治療用組成物は、前記した本発明において、癌、認知症、肝硬変、アルツハイマー病、パーキンソン病、関節リュウマチ、脳梗塞、心筋梗塞、アトピー性皮膚炎、花粉症、しわの増加、白髪、脱毛、老眼、近眼、ドライアイ、ドライマウス、歯周病、筋力低下、骨粗鬆症、糖尿病、更年期障害、不妊及び勃起不全から選ばれる少なくとも１つの症状の改善または予防に用いられることを特徴とする。 In the present invention described above, the composition for regenerative treatment according to the present invention has an increase in cancer, dementia, liver cirrhosis, Alzheimer's disease, Parkinson's disease, rheumatoid arthritis, cerebral infarction, myocardial infarction, atopic dermatitis, hay fever, and wrinkles. , White hair, hair loss, old eye, myopic eye, dry eye, dry mouth, periodontal disease, muscle weakness, osteoporosis, diabetes, climacteric disorder, infertility and erectile dysfunction. And.
本発明に係る再生治療用組成物の製造方法は、歯髄由来幹細胞を培養して歯髄由来幹細胞培養上清を得て、
当該歯髄由来幹細胞培養上清を歯髄由来幹細胞自体を含まないように処理して、
前記歯髄由来幹細胞培養上清が含有するサイトカインである、血管内皮細胞増殖因子（ＶＥＧＦ）の濃度が１５０〜５０００ｐｇ／ｍｌとなるようにすることを特徴とする。
In the method for producing a composition for regenerative therapy according to the present invention, pulp-derived stem cells are cultured to obtain a pulp-derived stem cell culture supernatant.
The pulp-derived stem cell culture supernatant was treated so as not to contain the pulp-derived stem cells themselves.
The concentration of vascular endothelial growth factor (VEGF), which is a cytokine contained in the pulp-derived stem cell culture supernatant, is set to 150 to 5000 pg / ml.
本発明に係る再生治療用組成物の製造方法は、前記した本発明において、前記歯髄由来幹細胞培養上清が含有するサイトカインである、インシュリン様成長因子（ＩＧＦ）の濃度が８０〜１０００ｐｇ／ｍｌとなるようにすることを特徴とする。 In the method for producing a composition for regenerative therapy according to the present invention, in the above-mentioned invention, the concentration of insulin-like growth factor (IGF), which is a cytokine contained in the pulp-derived stem cell culture supernatant, is 80 to 1000 pg / ml. It is characterized by being.
本発明に係る再生治療用組成物の製造方法は、前記した本発明において、前記歯髄由来幹細胞培養上清が含有するカルシウムイオン（Ｃａ２＋）の濃度が６．５〜８．０ｍｇ／ｄｌとなるようにすることを特徴とする。 In the method for producing a composition for regenerative therapy according to the present invention, the concentration of calcium ions (Ca 2+ ) contained in the pulp-derived stem cell culture supernatant in the present invention is 6.5 to 8.0 mg / dl. It is characterized by doing so.
本発明に係る再生治療用組成物の製造方法は、前記した本発明において、前記血管内皮細胞増殖因子（ＶＥＧＦ）、インシュリン様成長因子（ＩＧＦ）、カルシウムイオン（Ｃａ２＋）の濃度のうち少なくとも１つを前記の濃度とするために、前記処理の後に濃度調整の操作を行うことを特徴とする。 The method for producing a composition for regenerative therapy according to the present invention is the method for producing a composition for regenerative treatment according to the present invention, wherein at least one of the concentrations of the vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), and calcium ion (Ca 2+). In order to obtain the above-mentioned concentration, the concentration adjustment operation is performed after the above-mentioned treatment.
本発明に係る再生治療用組成物の製造方法は、前記した本発明において、前記濃度調整の操作が、濃縮、希釈、遠心処理、溶媒の置換、透析、凍結、乾燥、凍結乾燥、脱塩及び保存からなる群より選択される少なくとも１つの操作であることを特徴とする。 In the method for producing a composition for regenerative therapy according to the present invention, in the above-mentioned invention, the operation of concentration adjustment includes concentration, dilution, centrifugation, solvent substitution, dialysis, freezing, drying, freeze-drying, desalting and It is characterized in that it is at least one operation selected from the group consisting of preservation.
本発明によれば、従来の医療では治療困難な疾病に対する汎用的な代替技術として、歯髄由来幹細胞を利用した再生治療用組成物を用いた再生医療を実施するにあたり、歯髄由来幹細胞培養上清が含むサイトカインのうち、血管内皮細胞増殖因子（ＶＥＧＦ）の濃度を特定範囲とすることにより、再生治療に有益と考えられるサイトカインが細胞へ有効に作用し、細胞増殖能が良好となり、再生治療に優れた再生治療用組成物を効率よく提供することができる。 According to the present invention, as a general-purpose alternative technique for diseases that are difficult to treat by conventional medical treatment, when performing regenerative medicine using a composition for regenerative treatment using dental pulp-derived stem cells, a dental pulp-derived stem cell culture supernatant is used. By setting the concentration of vascular endothelial cell proliferation factor (VEGF) among the included cytokines in a specific range, cytokines that are considered to be beneficial for regenerative therapy act effectively on cells, improve cell proliferation ability, and are excellent in regenerative therapy. The composition for regenerative medicine can be efficiently provided.
以下、本発明の一態様について説明する。本発明は、歯髄由来幹細胞自体を含まない歯髄由来幹細胞培養上清を含んでなる再生治療用組成物であり、かかる歯髄由来幹細胞培養上清に含まれる、細胞を再生するための有効な成分（サイトカイン）のうち、血管内皮細胞増殖因子（Ｖａｓｃｕｌａｒ Ｅｎｄｏｔｈｅｌｉａｌ Ｇｒｏｗｔｈ Ｆａｃｔｏr：ＶＥＧＦ）の濃度を特定範囲とした再生治療用組成物及び再生治療用組成物の製造方法である。 Hereinafter, one aspect of the present invention will be described. The present invention is a composition for regenerative treatment comprising a dental pulp-derived stem cell culture supernatant that does not contain the dental pulp-derived stem cells themselves, and is an effective component for regenerating cells contained in the dental pulp-derived stem cell culture supernatant (the present invention). Among the cytokines), it is a method for producing a composition for regenerative treatment and a composition for regenerative treatment in which the concentration of vascular endothelial growth factor (VEGF) is within a specific range.
ここで、本発明の対象とされる「再生治療用組成物」における「再生治療」とは、再生対象となる組織へ直接的及び間接的に作用する細胞並びに生理活性物質により再生を促進する治療方法である。再生治療を実施するにあたっては、体内に現存する細胞に対して何らかの影響を及ぼすことができる物質である必要がある。また、再生治療を実現するにあたっては、かかる物質によって細胞が増殖することが不可欠であるため、再生治療用組成物は、細胞の増殖能が良好な、細胞の増殖を促進するような効果のある組成物である必要がある。 Here, the "regenerative therapy" in the "composition for regenerative therapy" which is the subject of the present invention is a treatment which promotes regeneration by cells and physiologically active substances that act directly and indirectly on the tissue to be regenerated. The method. In carrying out regenerative therapy, it is necessary to have a substance that can have some effect on the cells existing in the body. Further, in order to realize regenerative therapy, it is indispensable for cells to proliferate by such a substance. Therefore, the composition for regenerative therapy has a good cell proliferative ability and has an effect of promoting cell proliferation. Must be a composition.
再生治療用組成物は、いわゆる「損傷部治療用組成物」を包含する治療組成物として使用される。損傷部治療用組成物における「損傷部治療」とは、一般に、標的組織の損傷部を修復ないしは回復することを指し、例えば、再生治療用組成物を、標的組織を有する患者に、標的組織の損傷部を修復するために有効な量で投与することにより、損傷部の細胞を再生、増殖させ、損傷部を治療することができる。また、標的組織の再生対象部位を再生するために有効な量で投与することにより、損傷部だけでなく、再生対象部の細胞の再生、増殖を促進するような、再生治療に用いることができる。 The composition for regenerative treatment is used as a therapeutic composition including a so-called "composition for treating an injured part". "Treatment of an injured part" in a composition for treating an injured part generally refers to repairing or recovering an injured part of a target tissue, for example, a composition for regenerative treatment is applied to a patient having the target tissue of the target tissue. When administered in an amount effective for repairing the injured part, the cells in the injured part can be regenerated and proliferated, and the injured part can be treated. In addition, by administering an amount effective for regenerating the regeneration target site of the target tissue, it can be used for regeneration therapy that promotes the regeneration and proliferation of cells not only in the damaged portion but also in the regeneration target portion. ..
ここでいう「損傷部」とは、組織に物理的または生理的に欠陥が生じて、本来の機能を発揮できなくなった組織上の部位を意味し、外傷のみならず、組織の物理的または生理的欠陥に起因した傷害部、障害部または疾患部も包含する概念として用いられる。 The term "damaged part" as used herein means a part of a tissue that is physically or physiologically defective and cannot perform its original function, and is not only a trauma but also a physical or physiological part of the tissue. It is also used as a concept that includes an injured part, a damaged part, or a diseased part caused by a physical defect.
また、「修復」とは、損傷部の細胞を再生、増殖させることにより、標的組織における損傷によって失われた機能の一部または全部が、損傷時における損傷部の機能と比較して維持または回復していることを意味し、組織の機能が回復することのみならず、機能的な組織として再生することも広く包含する。なお、機能が維持または回復していることの評価については、損傷した組織において異なるが、外観、対象となる機能の程度を評価するために通常用いられるアッセイ等に基づいて実施するようにすればよい。 In addition, "repair" means that by regenerating and proliferating cells in the damaged part, some or all of the functions lost due to the damage in the target tissue are maintained or restored as compared with the functions of the damaged part at the time of damage. It means that the tissue is not only restored to its function, but also regenerated as a functional tissue. The evaluation of maintenance or recovery of function differs depending on the damaged tissue, but it should be performed based on an assay usually used to evaluate the appearance and the degree of the target function. Good.
幹細胞の供給源としては、近年、歯髄が注目されており、本発明では、歯髄に由来する体性幹細胞（歯髄幹細胞）を使用することができる。歯髄由来幹細胞は、例えば、ヒトから脱落あるいは抜去された乳歯、永久歯に由来する体性幹細胞を用いることができる。自己増殖能と多分化能を併せ持つ新規な幹細胞集団として、乳歯の歯髄由来幹細胞（Ｓｔｅｍ Ｃｅｌｌｓ ｆｒｏｍ Ｅｘｆｏｌｉａｔｅｄ Ｄｅｃｉｄｕｏｕｓ Ｔｅｅｔｈ：ＳＨＥＤ）や、永久歯の歯髄由来幹細胞（Ｄｅｎｔａｌ Ｐｕｌｐ Ｓｔｅｍ Ｃｅｌｌｓ：ＤＰＳＣｓ）が同定されている。 In recent years, dental pulp has been attracting attention as a source of stem cells, and in the present invention, somatic stem cells (dental pulp stem cells) derived from dental pulp can be used. As the pulp-derived stem cells, for example, somatic stem cells derived from deciduous teeth or permanent teeth that have been shed or removed from humans can be used. Dental pulp-derived stem cells (Stem Cells from Exfoliated Decidous Stem: SHED) of deciduous teeth and dental pulp-derived stem cells (DPSCs) of permanent teeth have been identified as a novel stem cell population having both autoproliferative ability and pluripotency. ..
本発明によれば、歯髄由来幹細胞を培養して得られた歯髄由来幹細胞培養上清を再生治療用組成物の有効成分として用いる。かかる歯髄由来幹細胞培養上清は、サイトカインの混合物が含まれているため、例えば、損傷部に適用されると、損傷部における細胞を再生、増殖させ、その結果、損傷部を有する組織を修復することができる。また、損傷部だけでなく、細胞の増殖を促進するような、再生治療に用いることができる。 According to the present invention, a pulp-derived stem cell culture supernatant obtained by culturing dental pulp-derived stem cells is used as an active ingredient of a composition for regenerative therapy. Since such pulp-derived stem cell culture supernatant contains a mixture of cytokines, for example, when applied to an injured part, it regenerates and proliferates cells in the injured part, and as a result, repairs the tissue having the injured part. be able to. In addition, it can be used for regenerative therapy that promotes cell proliferation as well as the damaged part.
歯髄由来幹細胞培養上清中のサイトカインの混合物は、標的組織の内在性幹細胞に対する誘導シグナルとして作用することにより、かかる内在性幹細胞が分化し、増殖し得ると推論し得る。その結果、標的組織の損傷部での細胞の増殖及び、細胞外マトリクスの生成などが行われ得る。これらのことから、損傷部を有する組織は、標的組織の内在性幹細胞のこのような再生能に基づいて修復することができると考えられる。 It can be inferred that the mixture of cytokines in the pulp-derived stem cell culture supernatant can differentiate and proliferate such endogenous stem cells by acting as an inducing signal to the endogenous stem cells of the target tissue. As a result, cell proliferation in the damaged part of the target tissue, generation of extracellular matrix, and the like can be performed. From these facts, it is considered that the tissue having the damaged part can be repaired based on such regenerative ability of the endogenous stem cells of the target tissue.
再生治療用組成物は、サイトカインの混合物が含まれる歯髄由来幹細胞培養上清からなることによって、体内のサイトカイン分泌を促進するものである。歯髄由来幹細胞等の間葉系由来の体性幹細胞は、例えば、血管内皮細胞増殖因子（ＶＥＧＦ）、インシュリン様成長因子（ＩＧＦ）、肝細胞増殖因子（ＨＧＦ）、血小板由来成長因子（ＰＤＧＦ）、形質転換成長因子−ベータ（ＴＧＦ−β）−１及び−３、ＴＧＦ−α、ＫＧＦ、ＨＢＥＧＦ、ＳＰＡＲＣ等の種々のサイトカインを産生し得ると考えられており、本発明では、歯髄由来幹細胞培養上清が、少なくとも所定濃度の血管内皮細胞増殖因子（ＶＥＧＦ）を含むようにしている。 The composition for regenerative therapy promotes cytokine secretion in the body by comprising a pulp-derived stem cell culture supernatant containing a mixture of cytokines. Somatic stem cells derived from the mesenchymal system such as dental pulp-derived stem cells include, for example, vascular endothelial cell growth factor (VEGF), insulin-like growth factor (IGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), and the like. It is believed that various cytokines such as transformed growth factor-beta (TGF-β) -1 and -3, TGF-α, KGF, HBEGF, SPARC can be produced, and in the present invention, on dental pulp-derived stem cell culture. Qing is made to contain at least a predetermined concentration of vascular endothelial cell growth factor (VEGF).
歯髄由来幹細胞培養上清には、その他、インシュリン様成長因子（ＩＧＦ）や、肝細胞増殖因子（ＨＧＦ）、血小板由来成長因子（ＰＤＧＦ）及び形質転換成長因子−ベータ（ＴＧＦ−β）等のうちの少なくとも１種を含むことが好ましい。また、サイトカイン以外にも、サイトカイン遺伝子の発現誘導に至るために重要と考えられている、カルシウムイオン（Ｃａ２＋）（以下、単に「カルシウムイオン」とする場合がある。）が含まれることが好ましい。歯髄由来幹細胞培養上清は、前記のインシュリン様成長因子（ＩＧＦ）等のサイトカインやカルシウムイオン（Ｃａ２＋）を含むことが特に好ましく、歯髄由来幹細胞等の間葉系由来の体性幹細胞は、これらインシュリン様成長因子（ＩＧＦ）等のサイトカインやカルシウムイオンを産生し得ると考えられている。 In addition, insulin-like growth factor (IGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), transformed growth factor-beta (TGF-β), etc. are included in the dental pulp-derived stem cell culture supernatant. It is preferable to contain at least one of. In addition to cytokines, it is preferable that calcium ions (Ca 2+ ) (hereinafter, may be simply referred to as “calcium ions”), which are considered to be important for inducing the expression of cytokine genes, are contained. .. It is particularly preferable that the pulp-derived stem cell culture supernatant contains cytokines such as the above-mentioned insulin-like growth factor (IGF) and calcium ions (Ca 2+ ), and mesenchymal-derived somatic stem cells such as dental pulp-derived stem cells are these. It is believed that cytokines such as insulin-like growth factor (IGF) and calcium ions can be produced.
前記のサイトカインの濃度が特定範囲であれば、サイトカインの分泌が好適に促進され、細胞の再生効果や損傷部の治療効果を向上させやすくなると考えられ、本発明にあっては、前記のサイトカインのうち、使用される歯髄由来幹細胞培養上清における血管内皮細胞増殖因子（ＶＥＧＦ）の濃度に着目し、再生治療用組成物としている。なお、サイトカイン濃度は、市販品である各サイトカインのＥＬＩＳＡキット等を使用することによって簡便に測定することができる。また、カルシウムイオンの濃度に関しても、例えば、市販品であるメタロアッセイ カルシウム 測定（ＯＣＰＣ）（メタロジェニクス（株）製）等を使用することにより簡便に測定することができる。 When the concentration of the cytokine is within a specific range, it is considered that the secretion of the cytokine is preferably promoted, and it is easy to improve the cell regeneration effect and the therapeutic effect of the damaged part. Of these, we focused on the concentration of vascular endothelial growth factor (VEGF) in the dentin-derived stem cell culture supernatant used, and used it as a composition for regenerative therapy. The cytokine concentration can be easily measured by using a commercially available ELISA kit or the like for each cytokine. Further, the concentration of calcium ions can also be easily measured by using, for example, a commercially available metallogenic calcium measurement (OCPC) (manufactured by Metallogenics Co., Ltd.).
本発明で使用される歯髄由来幹細胞培養上清は、歯髄から得られる幹細胞（歯髄由来幹細胞）を培養して得られた培養上清を意味し、「歯髄由来幹細胞培養上清」は、歯髄由来幹細胞を培養して得られ、かつ細胞成分（歯髄由来幹細胞自体）を含まない培養液と定義される。かかる歯髄由来幹細胞培養上清は、例えば、培養後に細胞成分（歯髄由来幹細胞自体）を分離除去することによって、本発明の再生治療用組成物として使用可能な歯髄由来幹細胞培養上清を得ることができる。分離除去するための処理としては、特に制限はないが、具体的には、例えば、遠心処理、透析及び膜分離等の各種処理等を適宜施すことで、細胞成分（歯髄由来幹細胞自体）を含まない培養上清を得ることができる。 The pulp-derived stem cell culture supernatant used in the present invention means a culture supernatant obtained by culturing stem cells (pulp-derived stem cells) obtained from the pulp, and "pulp-derived stem cell culture supernatant" is derived from the pulp. It is defined as a culture solution obtained by culturing stem cells and containing no cellular components (pulp-derived stem cells themselves). From the pulp-derived stem cell culture supernatant, for example, by separating and removing the cell component (pulp-derived stem cell itself) after culturing, a pulp-derived stem cell culture supernatant that can be used as the composition for regenerative treatment of the present invention can be obtained. it can. The treatment for separation and removal is not particularly limited, but specifically, cell components (dental pulp-derived stem cells themselves) are contained by appropriately performing various treatments such as centrifugation, dialysis and membrane separation. No culture supernatant can be obtained.
本発明において、「未処理」の歯髄由来幹細胞培養上清とは、例えば、遠心処理、透析及び膜分離等の各種処理がされず、歯髄由来幹細胞自体が分離除去されていない（歯髄由来幹細胞を含有する）歯髄由来幹細胞培養上清を指し、「処理済（処理された）」の歯髄由来幹細胞培養上清とは、前記した処理がされ、歯髄由来幹細胞自体を含有しない歯髄由来幹細胞培養上清を指す。 In the present invention, the “untreated” pulp-derived stem cell culture supernatant is not subjected to various treatments such as centrifugation, dialysis, and membrane separation, and the pulp-derived stem cells themselves are not separated and removed (pulp-derived stem cells are not separated and removed). Refers to the pulp-derived stem cell culture supernatant (containing), and the “treated (treated)” pulp-derived stem cell culture supernatant is the pulp-derived stem cell culture supernatant that has been treated as described above and does not contain the pulp-derived stem cells themselves. Point to.
また、「未調整の幹細胞培養上清」と表す場合は、歯髄由来幹細胞を培養して得られ、前記の処理がされて歯髄由来幹細胞自体が分離除去された歯髄由来幹細胞培養上清（処理済みの歯髄由来幹細胞培養上清）そのもの、あるいはかかる培養上清を変質したり体積変化させたりしないで保存したものをいい、歯髄由来幹細胞培養上清の原液と称する場合がある。ここで、「調整」とは、未調整の歯髄由来幹細胞培養上清（歯髄由来幹細胞培養上清の原液）に対して、濃縮、希釈、遠心処理、溶媒の置換、透析、凍結、乾燥、凍結乾燥、脱塩、及び保存等のうち少なくとも１つの操作を行って濃度を増減させることをいう。 In addition, when it is expressed as "unadjusted stem cell culture supernatant", it is obtained by culturing dental pulp-derived stem cells, and the dental pulp-derived stem cells themselves are separated and removed by the above-mentioned treatment (treated). The pulp-derived stem cell culture supernatant) itself, or the culture supernatant preserved without alteration or volume change, may be referred to as a stock solution of the pulp-derived stem cell culture supernatant. Here, "adjustment" means concentration, dilution, centrifugation, solvent replacement, dialysis, freezing, drying, and freezing of an unadjusted pulp-derived stem cell culture supernatant (stock solution of pulp-derived stem cell culture supernatant). It means increasing or decreasing the concentration by performing at least one operation such as drying, desalting, and storage.
なお、後記する歯髄由来幹細胞培養上清の製造においては、これを含む再生治療用組成物を適用する患者自身の幹細胞を用いてもよいが、必ずしもかかる幹細胞を用いなければならないという制限はない。加えて、ヒトの幹細胞を用いてよいことはもちろんであるが、ヒトの幹細胞だけでなく、ヒト以外の哺乳類（ウシ、ウマ、ブタ、サル及び羊等。）から得られた歯髄由来幹細胞の培養上清（非ヒト由来の歯髄由来幹細胞培養上清）についても、ヒト及びヒト以外の前記哺乳類に対しての臨床応用がすすめられている。このことは結果的に動物関連産業の発展に大きく貢献することができる。 In the production of the pulp-derived stem cell culture supernatant described later, the patient's own stem cells to which the composition for regenerative therapy containing the same may be used, but there is no limitation that such stem cells must be used. In addition, it goes without saying that human stem cells may be used, but not only human stem cells, but also cultures of dental pulp-derived stem cells obtained from non-human mammals (cattle, horses, pigs, monkeys, sheep, etc.). The supernatant (non-human-derived dental pulp-derived stem cell culture supernatant) is also being clinically applied to humans and the mammals other than humans. As a result, this can greatly contribute to the development of the animal-related industry.
以下、再生治療用組成物を構成し、必要により濃度調整の対象となる、歯髄由来幹細胞培養上清の製造方法の一例を説明する。ここで、歯髄由来幹細胞培養上清を得るには、歯髄由来幹細胞を培養等する必要があるため、まず、歯髄由来幹細胞を得る方法の一例を説明する。 Hereinafter, an example of a method for producing a pulp-derived stem cell culture supernatant, which constitutes a composition for regenerative treatment and is subject to concentration adjustment as necessary, will be described. Here, since it is necessary to culture the pulp-derived stem cells in order to obtain the pulp-derived stem cell culture supernatant, first, an example of the method for obtaining the pulp-derived stem cells will be described.
（Ａ）歯髄由来幹細胞の製造：
前記したように、「歯髄由来幹細胞」とは、脱落または抜去した歯牙の中に存在する歯髄組織内に含まれる細胞のことを指す。採取した歯髄組織から歯髄由来幹細胞を得るには、歯髄細胞を培養等すればよく、例えば、以下の手順によって行うことができる。
(A) Production of pulp-derived stem cells:
As described above, the “pulp-derived stem cell” refers to a cell contained in the pulp tissue present in the shed or removed tooth. In order to obtain dental pulp-derived stem cells from the collected dental pulp tissue, the dental pulp cells may be cultured, for example, by the following procedure.
（１）歯髄の採取：
脱落または抜去したヒトの乳歯や永久歯から採取した歯髄組織から、歯髄由来幹細胞を付着性細胞として選別することができる。
(1) Collection of pulp:
Pulp-derived stem cells can be selected as adherent cells from pulp tissue collected from shed or removed human deciduous or permanent teeth.
例えば、脱落または抜去した歯牙の歯冠部を分割し、歯科用リーマーにて歯髄組織を回収する。具体的には、自然に脱落した乳歯（または抜歯した乳歯、或いは永久歯等。）等の歯牙を、例えば、クロロヘキシジン液やポビドンヨード液（イソジン（登録商標）液）等で消毒した後、歯冠部を分割し歯科用リーマー等によって歯髄組織を回収するようにすればよい。 For example, the crown of a tooth that has fallen off or has been removed is divided, and pulp tissue is collected with a dental reamer. Specifically, teeth such as naturally shed deciduous teeth (or extracted deciduous teeth, permanent teeth, etc.) are disinfected with, for example, chlorohexidine solution or povidone iodine solution (isodine (registered trademark) solution), and then the crown portion. The pulp tissue may be collected by a dental reamer or the like.
（２）酵素処理：
（１）で分離・回収して採取した歯髄組織をコラゲナーゼ等で処理を行い、処理を行った組織及び細胞を回収する。例えば、採取した歯髄組織を基本培地（例えば、１０％ウシ血清・抗生物質含有ダルベッコ変法イーグル培地（Ｄｕｌｂｅｃｃｏ’ｓ Ｍ ｏｄｉｆｉｅｄ Ｅａｇｌｅ’ｓ Ｍｅｄｉｕｍ、以下、「ＤＭＥＭ」とする場合もある。）等。）に懸濁し、例えば、２ｍｇ／ｍｌのコラゲナーゼ及びディスパーゼで３７℃、１時間処理するようにする。そして、例えば、５分間の遠心操作（例えば、６００〜５０００×ｇ）により、酵素処理後の歯髄組織、歯髄細胞を回収する。
(2) Enzyme treatment:
The dental pulp tissue separated / collected and collected in (1) is treated with collagenase or the like, and the treated tissue and cells are collected. For example, the collected dental pulp tissue is used as a basal medium (for example, Dulbecco's Modified Eagle's Medium containing 10% bovine serum and antibiotics (hereinafter, may be referred to as “DMEM”) and the like. ), For example, treated with 2 mg / ml of collagenase and dispase at 37 ° C. for 1 hour. Then, for example, the dental pulp tissue and dental pulp cells after the enzyme treatment are collected by centrifugation for 5 minutes (for example, 600 to 5000 × g).
（３）細胞培養：
前記した処理を行い、回収した組織及び細胞を４ｃｃの５体積％〜１５体積％のウシ血清を含有した、例えば、５０〜１５０ユニット／ｍｌの抗生物質を含有するダルベッコ変法イーグル培地（ＤＭＥＭ）あるいは間葉系幹細胞用培地に懸濁し、付着性細胞培養用ディッシュ、６ウェルへ播種する。
(3) Cell culture:
Dalveco's Modified Eagle's Medium (DMEM) containing 4 cc of 5% by volume to 15% by volume of bovine sera, eg, 50 to 150 units / ml of antibiotics, in the tissues and cells recovered by the above treatment. Alternatively, it is suspended in a medium for mesenchymal stem cells and seeded in 6 wells of a dish for adhering cell culture.
次に、５体積％の二酸化炭素（以下、ＣＯ２とする場合がある。）雰囲気下、約３７℃に調整したインキュベーターで培養する。サブコンフルエント（培養容器の表面の約７０面積％を細胞が占める状態を示す。）またはコンフルエントに達したときに細胞を０．０５体積％トリプシン・ＥＤＴＡにて、例えば、５分間、３７℃で処理する。ディッシュから剥離した歯髄由来幹細胞を直径１０ｃｍの付着性細胞培養用ディッシュに播種し、拡大培養を行うようにする。 Next, the cells are cultured in an incubator adjusted to about 37 ° C. in an atmosphere of 5% by volume of carbon dioxide (hereinafter, may be referred to as CO 2). When the cells reach subconfluent (indicating a state in which cells occupy about 70 area% of the surface of the culture vessel) or confluent, the cells are treated with 0.05% by volume trypsin / EDTA, for example, at 37 ° C. for 5 minutes. To do. Dental pulp-derived stem cells exfoliated from the dish are seeded in a dish for adhering cell culture having a diameter of 10 cm, and expanded culture is performed.
継代培養は、繰り返し行ってもよく、細胞培養は、例えば、継代培養を１〜１５回行い、必要な細胞数（例えば、約１×１０７個／ｍｌ）まで増殖させることが好ましい。以上の培養の後、細胞を回収して保存することにしてもよい。なお、このように保存し、将来の骨・軟骨・神経などの疾患（特に難治性疾患等。）の治療に適用すれば、安全でかつ低侵襲な自己細胞による再生医療が提供できることになるため好ましい。 Subculture may be repeated, the cell culture, for example, perform subculturing 15 times, the required number of cells (e.g., about 1 × 10 7 cells / ml) are preferably grown up. After the above culture, the cells may be collected and stored. If it is preserved in this way and applied to the treatment of future diseases such as bone, cartilage, and nerves (especially intractable diseases, etc.), it will be possible to provide safe and minimally invasive regenerative medicine using autologous cells. preferable.
（４）細胞の回収：
そして、例えば、トリプシン処理等で培養容器から細胞を剥離した後、所定の条件（例えば、６００〜５０００×ｇが好ましく、より好ましくは７５０〜５０００×ｇ）で遠心処理を施して細胞（付着性細胞）を採取して、歯髄由来幹細胞を回収することができる。
(4) Cell recovery:
Then, for example, after exfoliating the cells from the culture vessel by trypsin treatment or the like, the cells (adhesiveness) are subjected to centrifugation under predetermined conditions (for example, 600 to 5000 × g is preferable, and more preferably 750 to 5000 × g). Cells) can be collected to collect dental pulp-derived stem cells.
（Ｂ）歯髄由来幹細胞培養上清の製造：
次に、歯髄由来幹細胞培養上清の製造の一例を説明する。まず、前記した方法で得られた歯髄由来幹細胞を、基本培地、例えば、血清として１０体積％のＦＢＳ等の動物血清を加えた培地（前記したＤＭＥＭ等。）を用いて、例えば、５体積％ＣＯ２雰囲気下、３７℃の条件下に、例えば、２４〜４８時間培養するようにする。その後、血清を含まないＤＭＥＭへ置換し、さらに、例えば、一定の時間（例えば、２４〜７２時間等。）培養を行って、未処理の歯髄由来幹細胞培養上清を得る。
(B) Production of pulp-derived stem cell culture supernatant:
Next, an example of producing a pulp-derived stem cell culture supernatant will be described. First, the dental pulp-derived stem cells obtained by the above-mentioned method are added to a basal medium, for example, a medium (DMEM or the like described above) to which animal serum such as FBS of 10% by volume is added as serum, for example, 5% by volume. The cells are cultured in a CO 2 atmosphere at 37 ° C. for, for example, 24-48 hours. Then, it is replaced with DMEM containing no serum, and further cultured for a certain period of time (for example, 24-72 hours, etc.) to obtain an untreated dental pulp-derived stem cell culture supernatant.
（Ｃ）歯髄由来幹細胞培養上清の処理（歯髄由来幹細胞の除去）：
得られた未処理の歯髄由来幹細胞培養上清から歯髄由来幹細胞（歯髄由来幹細胞自体）を取り除くため、前記の一定の時間経過後、例えば、６００〜５０００×ｇで３〜７分間遠心処理を行うことにより、歯髄由来幹細胞（歯髄由来幹細胞自体）を含まない（歯髄由来幹細胞を取り除いた）、処理済みの歯髄由来幹細胞培養上清を得ることができる。他の処理としては、歯髄由来幹細胞を通過させない分離膜を通過させる等の処理により、歯髄由来幹細胞を含まない（歯髄由来幹細胞を取り除いた）、処理済みの歯髄由来幹細胞培養上清を得ることができる。
(C) Treatment of pulp-derived stem cell culture supernatant (removal of pulp-derived stem cells):
In order to remove the pulp-derived stem cells (pulp-derived stem cells themselves) from the obtained untreated pulp-derived stem cell culture supernatant, after a certain period of time, for example, centrifugation is performed at 600 to 5000 × g for 3 to 7 minutes. This makes it possible to obtain a treated pulp-derived stem cell culture supernatant that does not contain pulp-derived stem cells (pulp-derived stem cells themselves) (from which pulp-derived stem cells have been removed). As another treatment, it is possible to obtain a treated pulp-derived stem cell culture supernatant that does not contain pulp-derived stem cells (the pulp-derived stem cells have been removed) by a treatment such as passing through a separation membrane that does not allow pulp-derived stem cells to pass through. it can.
なお、歯髄由来幹細胞培養上清に用いる歯髄由来幹細胞の継代数の制限は特にないが、標的組織の改善や予防能力、及び標的となる組織の種類の幅広さという観点から、５〜１５とすることが好ましい。 There is no particular limitation on the number of passages of pulp-derived stem cells used in the pulp-derived stem cell culture supernatant, but the number is 5 to 15 from the viewpoint of improving and preventing target tissues and the wide range of target tissues. Is preferable.
（Ｄ）歯髄由来幹細胞培養上清における血管内皮細胞増殖因子（ＶＥＧＦ）等の濃度の選択：
前記の方法で得られた、歯髄由来幹細胞を含まない（処理済みの）歯髄由来幹細胞培養上清は、種々の成長因子を含むが、回収後の成長因子の量（濃度）は均一でない。本発明にあっては、分泌するサイトカインのうち、少なくとも血管内皮細胞増殖因子（ＶＥＧＦ）を指標にし、濃度を測定し基準化のデータとし、再生治療に必要な血管の再生及び細胞増殖に関わる指標として用いるようにしている。
(D) Selection of concentration of vascular endothelial growth factor (VEGF), etc. in dental pulp-derived stem cell culture supernatant:
The pulp-derived stem cell-free (treated) pulp-derived stem cell culture supernatant obtained by the above method contains various growth factors, but the amount (concentration) of the growth factors after recovery is not uniform. In the present invention, among the secreted cytokines, at least vascular endothelial growth factor (VEGF) is used as an index, the concentration is measured and used as standardized data, and an index related to vascular regeneration and cell proliferation necessary for regenerative therapy is used. I am trying to use it as.
本発明にあっては、歯髄由来幹細胞を含まない歯髄由来幹細胞培養上清が含有するサイトカインである、血管内皮細胞増殖因子（ＶＥＧＦ）の濃度は１５０〜５０００ｐｇ／ｍｌとする。ＶＥＧＦの濃度をかかる範囲とすることにより、再生治療に有益と考えられるサイトカインが細胞へ有効に作用し、細胞増殖能が良好となり、再生治療に役立つものとなる。前記したように、再生治療を実現するにあたっては、適用により細胞が増殖することが不可欠であるため、細胞の増殖能が良好であれば、細胞の増殖を促進するような効果のある組成物となる。一方、ＶＥＧＦの濃度が１５０ｐｇ／ｍｌより低いと、細胞増殖能が期待できない場合があり、５０００ｐｇ／ｍｌより高いと、濃度を高くすることに対して期待したほどの細胞増殖能とならない場合があるとともに、そのような高濃度のＶＥＧＦの製造も困難となる場合がある。ＶＥＧＦの濃度は２００〜４０００ｐｇ／ｍｌとすることが好ましく、２００〜３６００ｐｇ／ｍｌとすることが特に好ましい。 In the present invention, the concentration of vascular endothelial growth factor (VEGF), which is a cytokine contained in the pulp-derived stem cell culture supernatant containing no pulp-derived stem cells, is 150 to 5000 pg / ml. By setting the concentration of VEGF in such a range, cytokines considered to be beneficial for regenerative therapy act effectively on cells, the cell proliferation ability is improved, and the cells are useful for regenerative therapy. As described above, in order to realize regenerative therapy, it is indispensable for cells to proliferate by application. Therefore, if the proliferative ability of cells is good, the composition has an effect of promoting cell proliferation. Become. On the other hand, if the concentration of VEGF is lower than 150 pg / ml, the cell proliferation ability may not be expected, and if it is higher than 5000 pg / ml, the cell proliferation ability may not be as expected for increasing the concentration. At the same time, it may be difficult to produce such a high concentration of VEGF. The concentration of VEGF is preferably 200 to 4000 pg / ml, particularly preferably 200 to 3600 pg / ml.
また、必要により、歯髄由来幹細胞培養上清が含有するサイトカインである、インシュリン様成長因子（ＩＧＦ）の濃度について、一定範囲のものを選択することが好ましい。かかるＩＧＦの濃度は８０〜１０００ｐｇ／ｍｌとすることが好ましく、ＩＧＦの濃度をかかる範囲とすることにより、増殖能がさらに良好となる。ＩＧＦの濃度は１００〜７００ｐｇ／ｍｌとすることがさらに好ましく、１２０〜６００ｐｇ／ｍｌとすることが特に好ましい。 Further, if necessary, it is preferable to select a concentration of insulin-like growth factor (IGF), which is a cytokine contained in the pulp-derived stem cell culture supernatant, in a certain range. The concentration of such IGF is preferably 80 to 1000 pg / ml, and by setting the concentration of IGF in such a range, the proliferative ability is further improved. The concentration of IGF is more preferably 100 to 700 pg / ml, and particularly preferably 120 to 600 pg / ml.
また、サイトカイン遺伝子の発現誘導に至るために重要と考えられる、カルシウムイオン（Ｃａ２＋）の濃度についても、一定範囲のものを選択することが好ましい。カルシウムイオンの濃度は６．５〜８．０ｍｇ／ｄｌとすることが好ましく、カルシウムイオンの濃度をかかる範囲とすることにより、サイトカイン遺伝子の発現を誘導し、その結果増殖能がさらに良好となる。カルシウムイオンの濃度は６．５〜７．５ｍｇ／ｄｌとすることがさらに好ましく、６．５〜７．０ｍｇ／ｄｌとすることが特に好ましい。 In addition, it is preferable to select a calcium ion (Ca 2+ ) concentration in a certain range, which is considered to be important for inducing the expression of the cytokine gene. The concentration of calcium ions is preferably 6.5 to 8.0 mg / dl, and by setting the concentration of calcium ions in such a range, the expression of cytokine genes is induced, and as a result, the proliferative ability is further improved. The concentration of calcium ions is more preferably 6.5 to 7.5 mg / dl, and particularly preferably 6.5 to 7.0 mg / dl.
サイトカインである血管内皮細胞増殖因子（ＶＥＧＦ）の濃度、ないしは血管内皮細胞増殖因子（ＶＥＧＦ）、インシュリン様成長因子（ＩＧＦ）及びカルシウムイオンの濃度の少なくとも１つを前記の範囲とするためには、歯髄由来幹細胞培養上清を得るに際し、前記（Ｃ）で得られた処理済みの歯髄由来幹細胞培養上清（歯髄由来幹細胞培養上清の原液）をそのまま用いるようにしてもよい。 In order to set at least one of the concentrations of vascular endothelial growth factor (VEGF), which is a cytokine, or the concentrations of vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), and calcium ions in the above range, When obtaining the tooth pulp-derived stem cell culture supernatant, the treated tooth pulp-derived stem cell culture supernatant (stock solution of the tooth pulp-derived stem cell culture supernatant) obtained in (C) above may be used as it is.
また、かかる血管内皮細胞増殖因子（ＶＥＧＦ）等の濃度を所望の濃度とするため、歯髄由来幹細胞培養上清を濃縮、希釈することにより濃度調整するようにしてもよい。濃度調整する操作としては、特に制限はなく、従来公知の方法、例えば、濃縮、希釈、遠心処理、溶媒の置換、透析、凍結、乾燥、凍結乾燥、脱塩及び保存等からなる群より選択される少なくとも１つの操作を行うことにより、最適化した濃度に調整するようにすることが好ましい。これらの操作は、１種を単独で、あるいは２種以上を組み合わせて用いるようにしてもよく、また、必要により繰り返して行うようにすればよい。 Further, in order to make the concentration of such vascular endothelial growth factor (VEGF) or the like a desired concentration, the concentration may be adjusted by concentrating and diluting the pulp-derived stem cell culture supernatant. The operation for adjusting the concentration is not particularly limited, and is selected from the group consisting of conventionally known methods such as concentration, dilution, centrifugation, solvent substitution, dialysis, freezing, drying, freeze-drying, desalting and storage. It is preferable to adjust to the optimized concentration by performing at least one operation. These operations may be performed individually by 1 type or in combination of 2 or more types, and may be repeated as necessary.
濃縮、希釈等の操作による濃度調整は、歯髄由来幹細胞培養上清の原液に対して、濃度が概ね１／４（０．２５）〜１０倍となることを目安に行うようにすればよいが、かかる範囲には制限されず、原液における血管内皮細胞増殖因子（ＶＥＧＦ）等の濃度や所望の濃度等に応じて適宜決定すればよい。なお、歯髄由来幹細胞培養上清の原液におけるＶＥＧＦ、インシュリン様成長因子（ＩＧＦ）及びカルシウムイオンの濃度については、原料となる歯髄由来幹細胞の状態に左右され、また、前記した濃縮や希釈等の操作を繰り返しても、ある一定の濃度で横ばいとなったりする等、濃縮や希釈の操作等に応じた濃度とならない場合もあり、前記した濃縮や希釈等の種々の操作を臨機応変に適用することが好ましい。 The concentration may be adjusted by operations such as concentration and dilution so that the concentration is approximately 1/4 (0.25) to 10 times that of the stock solution of the dental pulp-derived stem cell culture supernatant. The range is not limited to this, and it may be appropriately determined according to the concentration of vascular endothelial growth factor (VEGF) or the like in the stock solution, the desired concentration, and the like. The concentrations of VEGF, insulin-like growth factor (IGF) and calcium ions in the stock solution of the pulp-derived stem cell culture supernatant depend on the state of the pulp-derived stem cells as the raw material, and the above-mentioned operations such as concentration and dilution Even if the above steps are repeated, the concentration may not be adjusted according to the concentration or dilution operation, such as the concentration leveling off at a certain level. Therefore, the above-mentioned various operations such as concentration or dilution may be applied flexibly. Is preferable.
前記の調整方法は、従来公知の手段を適用することができる。なお、濃縮及び希釈の方法の具体例としては、例えば、濃縮は下記のスピンカラム法（スピンカラム濃縮法）や、エタノール沈殿濃縮法等が挙げられ、これらの濃縮方法は、必要により繰り返して行うようにすればよい。希釈の方法としては製造に用いた培養液原液（例えば、再生治療用組成物製造に用いたＤＭＥＭ原液等。）を用いて、目的とする濃度まで、必要により繰り返して希釈を行えばよい。 Conventionally known means can be applied to the above-mentioned adjustment method. Specific examples of the concentration and dilution methods include the following spin column method (spin column concentration method) and ethanol precipitation concentration method for concentration, and these concentration methods are repeated as necessary. You can do it like this. As a method of dilution, a culture solution stock solution used for production (for example, DMEM stock solution used for production of a composition for regenerative treatment) may be used, and dilution may be repeated as necessary to a desired concentration.
スピンカラム法（スピンカラム濃縮法）とは、歯髄由来幹細胞培養上清をＡｍｉｃｏｎ Ｕｌｔｒａ Ｃｅｎｔｒｉｆｕｇａｌ Ｆｉ ｌｔｅｒ Ｕｎｉｔｓ−１０Ｋ（ミリポア社製）等を用いて濃縮する方法であり、最大で７５倍の濃縮が可能である。具体的な手順の一例は下記のとおりである。なお、下記の手順において示す容量、時間等の条件は、あくまでも一例であり、歯髄由来幹細胞培養上清の濃度や状態等に応じて適宜変更することができる（以下、「エタノール沈殿濃縮法」についても同じ。）。 The spin column method (spin column concentration method) is a method in which the pulp-derived stem cell culture supernatant is concentrated using Amicon Ultra Centrifugal Filter Units-10K (manufactured by Millipore), etc., and can be concentrated up to 75 times. Is. An example of a specific procedure is as follows. The conditions such as volume and time shown in the following procedure are merely examples, and can be appropriately changed according to the concentration and condition of the pulp-derived stem cell culture supernatant (hereinafter, "ethanol precipitation concentration method"). Is the same.).
（ｉ）まず、歯髄由来幹細胞培養上清（最大１５ｍｌ）をＡｍｉｃｏｎ Ｕｌｔｒａ Ｃｅｎｔｒｉｆｕｇａｌ Ｆｉｌｔｅｒ Ｕｎｉｔｓ−１０Ｋへ投入し、４０００×ｇで約６０分間遠心し、２００μｌまで濃縮するようにする。
（ii）前記したＡｍｉｃｏｎ Ｕｌｔｒａ Ｃｅｎｔｒｉｆｕｇａｌ Ｆｉｌｔｅｒ Ｕｎｉｔｓ−１０Ｋへ培養上清と同量の滅菌したリン酸緩衝生理食塩水（Ｐｈｏｓｐ ｈａｔｅ Ｂｕｆｆｅｒｅｄ Ｓａｌｉｎｅ、以下、「ＰＢＳ」という）を投入し、再度４０００×ｇで約６０分間遠心し、ベース溶液をＰＢＳへ置換する。
（iii）得られた溶液２００μｌをマイクロテストチューブへ回収し、濃縮した歯髄由来幹細胞培養上清とする。
(I) First, the pulp-derived stem cell culture supernatant (up to 15 ml) is put into Amicon Ultra Centrifugal Filter Units-10K and centrifuged at 4000 × g for about 60 minutes to concentrate to 200 μl.
(Ii) Add the same amount of sterilized phosphate buffered saline (Phosphatate Buffered Saline, hereinafter referred to as "PBS") to the above-mentioned Amicon Ultra Centrifugal Filter Units-10K in the same amount as the culture supernatant, and add it again at 4000 xg. Centrifuge for about 60 minutes to replace the base solution with PBS.
(Iii) 200 μl of the obtained solution is collected in a microtest tube and used as a concentrated pulp-derived stem cell culture supernatant.
歯髄由来幹細胞培養上清を、エタノール沈殿法を用いて濃縮する（最大１０倍濃縮）方法（エタノール沈殿濃縮法）の具体的な手順の一例は次のとおりである。 An example of a specific procedure of a method of concentrating a pulp-derived stem cell culture supernatant using an ethanol precipitation method (maximum 10-fold concentration) (ethanol precipitation concentration method) is as follows.
（ｉ）培養上清５ｍｌに対し１００体積％エタノール４５ｍｌを加え、混和し、−２０℃で６０分間放置する。
（ii）４℃、１５０００×ｇで１５分間遠心する。
（iii）上澄みを除去し、９０％エタノール１０ｍｌを加え、よく攪拌する。
（iv）４℃、１５０００×ｇで５分間遠心する。
（ｖ）上澄みを除去し、得られたペレットを滅菌水５００μｌに溶解し、マイクロテストチューブへ回収し、濃縮した歯髄由来幹細胞培養上清とする。
(I) Add 45 ml of 100% by volume ethanol to 5 ml of the culture supernatant, mix, and leave at −20 ° C. for 60 minutes.
(Ii) Centrifuge at 4 ° C. and 15000 × g for 15 minutes.
(Iii) Remove the supernatant, add 10 ml of 90% ethanol, and stir well.
(Iv) Centrifuge at 4 ° C. and 15000 xg for 5 minutes.
(V) The supernatant is removed, and the obtained pellet is dissolved in 500 μl of sterilized water and collected in a microtest tube to prepare a concentrated pulp-derived stem cell culture supernatant.
なお、本発明で用いられる歯髄由来幹細胞培養上清は、凍結乾燥物としてもよく、良好な保存安定性が得られる。歯髄由来幹細胞培養上清の凍結乾燥方法としては、凍結乾燥を実施するために通常行われている方法を適用することができ、例えば、以下（ｉ）〜（iv）の方法を挙げることができる。なお、下記の方法や挙げている条件（温度や凍結期間等。）はあくまでも一例である。 The pulp-derived stem cell culture supernatant used in the present invention may be freeze-dried, and good storage stability can be obtained. As a method for freeze-drying the pulp-derived stem cell culture supernatant, a method usually used for freeze-drying can be applied, and examples thereof include the following methods (i) to (iv). .. The following methods and the listed conditions (temperature, freezing period, etc.) are just examples.
（ｉ）前記の方法で得られた歯髄由来幹細胞培養上清またはそれを濃縮したものを−２００℃〜−２０℃で２時間から半日凍結する。
（ii）凍結後、サンプルチューブの蓋を開放し、凍結乾燥機へセットする。
（iii）１〜２日間凍結乾燥を行う。
（iv）凍結乾燥して得られたものを歯髄由来幹細胞培養上清の凍結乾燥物とする（−２００℃〜−２０℃で保存可能である。）。
(I) The pulp-derived stem cell culture supernatant obtained by the above method or a concentrated product thereof is frozen at −200 ° C. to −20 ° C. for 2 hours to half a day.
(Ii) After freezing, open the lid of the sample tube and set it in the freeze dryer.
(Iii) Freeze-dry for 1 to 2 days.
(Iv) The product obtained by freeze-drying is used as a freeze-dried product of the pulp-derived stem cell culture supernatant (it can be stored at −200 ° C. to −20 ° C.).
以上説明した本発明によれば、従来の医療では治療困難な疾病に対する汎用的な代替技術として、歯髄由来幹細胞を利用した再生治療用組成物を用いた再生医療を実施するにあたり、歯髄由来幹細胞培養上清が含むサイトカインのうち、血管内皮細胞増殖因子（ＶＥＧＦ）を特定範囲とすることにより、再生治療に有益と考えられるサイトカインが細胞へ有効に作用し、細胞増殖能が良好となり、再生治療に優れた再生治療用組成物を効率よく提供することができる。 According to the present invention described above, as a general-purpose alternative technique for diseases that are difficult to treat by conventional medical treatment, in carrying out regenerative medicine using a composition for regenerative treatment using dental pulp-derived stem cells, dental pulp-derived stem cell culture By setting the vascular endothelial cell proliferation factor (VEGF) in a specific range among the cytokines contained in the supernatant, cytokines that are considered to be beneficial for regenerative therapy act effectively on cells, improving cell proliferation ability and for regenerative therapy. An excellent composition for regenerative medicine can be efficiently provided.
本発明で得られた再生治療用組成物は、各種の再生治療、例えば、癌、認知症、肝硬変、アルツハイマー病、パーキンソン病、関節リュウマチ、脳梗塞、心筋梗塞、アトピー性皮膚炎、花粉症、しわの増加、白髪、脱毛、老眼、近眼、ドライアイ、ドライマウス、歯周病、筋力低下、骨粗鬆症、糖尿病、更年期障害、不妊及び勃起不全等の各種症状に対する改善または予防に用いることができる。かかる再生治療用組成物は、例えば、注射剤、経口剤、点鼻剤、経肺投与剤、点眼剤または塗布剤等の剤型、形態等として用いることができる。 The composition for regenerative treatment obtained in the present invention includes various regenerative treatments such as cancer, dementia, liver cirrhosis, Alzheimer's disease, Parkinson's disease, rheumatoid arthritis, cerebral infarction, myocardial infarction, atopic dermatitis, pollinosis, and the like. It can be used for improvement or prevention of various symptoms such as increased wrinkles, gray hair, hair loss, old eyes, myopic eyes, dry eyes, dry mouth, periodontal disease, muscle weakness, osteoporosis, diabetes, menopausal disorders, infertility and erectile dysfunction. Such a composition for regenerative treatment can be used, for example, as a dosage form, form, etc. of an injection, an oral preparation, a nasal drop, a pulmonary administration, an eye drop, a coating, and the like.
なお、以上説明した態様は、本発明の一態様を示したものであって、本発明は、前記した実施形態に限定されるものではなく、本発明の構成を備え、目的及び効果を達成できる範囲内での変形や改良が、本発明の内容に含まれるものであることはいうまでもない。また、本発明を実施する際における具体的な構造及び形状等は、本発明の目的及び効果を達成できる範囲内において、他の構造や形状等としても問題はない。本発明は前記した各実施形態に限定されるものではなく、本発明の目的を達成できる範囲での変形や改良は、本発明に含まれるものである。 It should be noted that the embodiments described above show one aspect of the present invention, and the present invention is not limited to the above-described embodiment, but includes the configuration of the present invention and can achieve the object and the effect. Needless to say, modifications and improvements within the scope are included in the content of the present invention. Further, the specific structure, shape, etc. in carrying out the present invention may be any other structure, shape, etc. within the range in which the object and effect of the present invention can be achieved. The present invention is not limited to the above-described embodiments, and modifications and improvements within the range in which the object of the present invention can be achieved are included in the present invention.
例えば、前記の実施形態では、歯髄由来幹細胞及び歯髄由来幹細胞培養上清を得るための手法の一例を説明したが、歯髄由来幹細胞等を得るための手段は前記した内容には限定されず、所望の歯髄由来幹細胞等を得るための他の手段を使用することができる。同様に、処理済みの歯髄由来幹細胞培養上清の濃度を調整する方法についても、血管内皮細胞増殖因子（ＶＥＧＦ）の濃度を１５０〜５０００ｐｇ／ｍｌとするための他の手段を用いるようにしてもよい。
その他、本発明の実施の際の具体的な構造及び形状等は、本発明の目的を達成できる範囲で他の構造等としてもよい。
For example, in the above-described embodiment, an example of a method for obtaining pulp-derived stem cells and pulp-derived stem cell culture supernatant has been described, but the means for obtaining dental pulp-derived stem cells and the like is not limited to the above-mentioned contents and is desired. Other means for obtaining pulp-derived stem cells and the like can be used. Similarly, for the method of adjusting the concentration of the treated pulp-derived stem cell culture supernatant, other means for adjusting the concentration of vascular endothelial growth factor (VEGF) to 150 to 5000 pg / ml may be used. Good.
In addition, the specific structure, shape, etc. at the time of carrying out the present invention may be other structures, etc. as long as the object of the present invention can be achieved.
以下、実施例等に基づき本発明をさらに詳細に説明するが、本発明は、これらに限定されるものではない。 Hereinafter, the present invention will be described in more detail based on Examples and the like, but the present invention is not limited thereto.
（１）歯髄由来幹細胞培養上清の製造：
歯髄由来幹細胞培養上清は前記した（Ａ）〜（Ｃ）の方法により調製を行った。具体的には下記のとおりである。
(1) Production of pulp-derived stem cell culture supernatant:
The pulp-derived stem cell culture supernatant was prepared by the methods (A) to (C) described above. Specifically, it is as follows.
（１−１）歯髄由来幹細胞の製造：
下記（ｉ）〜（iv）の方法を用いて、歯髄由来幹細胞を製造した。
(1-1) Production of pulp-derived stem cells:
Dental pulp-derived stem cells were produced using the methods (i) to (iv) below.
（ｉ）歯髄の採取：
脱落または抜去したヒトの乳歯から採取した歯髄細胞から、歯髄由来幹細胞を付着性細胞として選別した。採取した乳歯の歯牙をクロロヘキシジンやポビドンヨード液（イソジン（登録商標）液）で消毒した後、歯冠部を分割し歯科用リーマーにて歯髄組織を回収するようにした。
(I) Collection of pulp:
From the pulp cells collected from the shed or removed human deciduous teeth, pulp-derived stem cells were selected as adherent cells. After disinfecting the collected deciduous teeth with chlorohexidine or povidone iodine solution (Isodine (registered trademark) solution), the crown was divided and the pulp tissue was collected with a dental reamer.
（ii）酵素処理：
（ｉ）で分離・回収して採取した歯髄組織を基本培地（１０％ウシ血清・抗生物質含有ダルベッコ変法イーグル培地（ＤＭＥＭ）等。）に懸濁し、２ｍｇ／ｍｌのコラゲナーゼ及びディスパーゼで３７℃、１時間処理するようにした。そして、５分間の７７７×ｇの遠心操作により、酵素処理後の歯髄組織、歯髄細胞を回収した。
(Ii) Enzyme treatment:
The dental pulp tissue separated / recovered and collected in (i) is suspended in a basal medium (10% bovine serum / antibiotic-containing modified Dulbecco's modified Eagle's medium (DMEM), etc.) and mixed with 2 mg / ml collagenase and dispase at 37 ° C. I tried to process for 1 hour. Then, the dental pulp tissue and dental pulp cells after the enzyme treatment were collected by centrifugation at 777 × g for 5 minutes.
（iii）細胞培養：
前記した処理を行い、回収した組織及び細胞を４ｃｃの５体積％〜１５体積％のウシ血清を含有した１００００ユニット／ｍｌのペニシリン、１００００μｇ／ｍｌのストレプトマイシン、及び２５μｇ／ｍｌのアンホテリシンＢを含有するダルベッコ変法イーグル培地（ＤＭＥＭ）に懸濁し、付着性細胞培養用ディッシュ、６ウェルへ播種した。
(Iii) Cell culture:
The tissues and cells recovered by the above treatment contain 10000 units / ml penicillin containing 4 cc of 5% by volume to 15% by volume bovine serum, 10000 μg / ml streptomycin, and 25 μg / ml amphotericin B. It was suspended in Dalveco's modified Eagle's medium (DMEM) and seeded in 6 wells of adherent cell culture dishes.
５体積％のＣＯ２雰囲気下、３７℃に調整したインキュベーターで培養した。サブコンフルエント（培養容器の表面の約７０面積％を細胞が占める状態）またはコンフルエントに達したときに細胞を０．０５体積％トリプシン・ＥＤＴＡにて５分間、３７℃で処理する。そして、ディッシュから剥離した歯髄由来幹細胞を直径１０ｃｍの付着性細胞培養用ディッシュに播種し拡大培養を行った。なお。細胞培養は、継代培養を３回行い、必要な細胞数（約１×１０７個／ｍｌ）まで増殖させた。 The cells were cultured in an incubator adjusted to 37 ° C. under a CO 2 atmosphere of 5% by volume. When the cells reach subconfluent (a state in which cells occupy about 70 area% of the surface of the culture vessel) or confluent, the cells are treated with 0.05% by volume trypsin EDTA for 5 minutes at 37 ° C. Then, the pulp-derived stem cells exfoliated from the dish were seeded in a dish for adhering cell culture having a diameter of 10 cm, and expanded culture was performed. In addition. Cell culture was performed subcultured three times and grown to the required number of cells (about 1 × 10 7 cells / ml).
（iv）細胞の回収：
トリプシン処理等で培養容器から（iii）で培養した細胞を剥離した後、７７７×ｇで遠心処理を施すことによって、細胞（付着性細胞）を採取して、歯髄由来幹細胞を回収した。
(Iv) Cell recovery:
After peeling the cells cultured in (iii) from the culture vessel by trypsin treatment or the like, the cells (adherent cells) were collected by centrifuging at 777 × g to collect dental pulp-derived stem cells.
（１−２）歯髄由来幹細胞培養上清の製造：
（１−１）で得られた歯髄由来幹細胞を、基本培地、血清として１０体積％のＦＢＳ等の動物血清を加えた培地（前記したＤＭＥＭ等。）を用いて、５体積％ＣＯ２雰囲気下、３７℃の条件下に、４８時間培養するようにした。なお、歯髄由来幹細胞培養上清に用いる歯髄由来幹細胞の継代培養は、８回までとした。
(1-2) Production of pulp-derived stem cell culture supernatant:
The dental pulp-derived stem cells obtained in (1-1) are used as a basal medium and a medium (DMEM or the like described above) containing 10% by volume of animal serum such as FBS as serum under a 5% by volume CO 2 atmosphere. , 37 ° C., for 48 hours. The subculture of the pulp-derived stem cells used in the pulp-derived stem cell culture supernatant was limited to 8 times.
前記の培養の後、血清を含まないＤＭＥＭへ置換して、さらに４８時間培養を行うようにした（処理前の歯髄由来幹細胞培養上清とした。）。 After the above culture, the cells were replaced with DMEM containing no serum, and the culture was further carried out for 48 hours (the pulp-derived stem cell culture supernatant before treatment was used).
４８時間経過後、歯髄由来幹細胞（歯髄由来幹細胞自体）を通過させない分離膜を通過させる処理を行い、歯髄由来幹細胞を取り除いて、歯髄由来幹細胞（歯髄由来幹細胞自体）を含まない、処理済みの歯髄由来幹細胞培養上清を得た。 After 48 hours, a treatment is performed to allow the pulp-derived stem cells (pulp-derived stem cells themselves) to pass through a separation membrane, and the pulp-derived stem cells are removed to remove the pulp-derived stem cells (the pulp-derived stem cells themselves). Derived stem cell culture supernatant was obtained.
（２）血管内皮細胞増殖因子（ＶＥＧＦ）等の濃度の調整：
得られた歯髄由来幹細胞培養上清について、サイトカインである血管内皮細胞増殖因子（ＶＥＧＦ）及びインシュリン様成長因子（ＩＧＦ）の濃度を測定したところ、ＶＥＧＦの濃度は９８７．８ｐｇ／ｍｌ、ＩＧＦの濃度は５１４．２ｐｇ／ｍｌであった。また、カルシウムイオンの濃度は６．６ｍｇ／ｄｌであった。
(2) Adjustment of concentration of vascular endothelial growth factor (VEGF), etc .:
When the concentrations of vascular endothelial growth factor (VEGF) and insulin-like growth factor (IGF), which are cytokines, were measured in the obtained dental pulp-derived stem cell culture supernatant, the concentration of VEGF was 987.8 pg / ml, and the concentration of IGF. Was 514.2 pg / ml. The concentration of calcium ions was 6.6 mg / dl.
なお、これらの濃度の測定のうち、サイトカインである血管内皮細胞増殖因子（ＶＥＧＦ）及びインシュリン様成長因子（ＩＧＦ）の濃度の測定は、市販品である各サイトカインのＥＬＩＳＡキットにより行った。また、カルシウムイオンの濃度は、市販品であるメタロアッセイ カルシウム 測定（ＯＣＰＣ）（メタロジェニクス（株）製）を使用して行った。 Among the measurements of these concentrations, the concentrations of the cytokines vascular endothelial growth factor (VEGF) and insulin-like growth factor (IGF) were measured using a commercially available ELISA kit for each cytokine. The concentration of calcium ions was measured using a commercially available metallogenic calcium measurement (OCPC) (manufactured by Metallogenics Co., Ltd.).
これをサンプル４（実施例４）とし、歯髄由来幹細胞培養上清におけるＶＥＧＦ、ＩＧＦ及びカルシウムイオンの濃度が、サンプル４を含め表１に示す６つの濃度となるように、濃度を調整するようにした。なお、サンプル４の濃度より濃度が低いサンプル５、サンプル６については下記の操作によりサンプル４の歯髄由来幹細胞培養上清を希釈して再生治療用組成物（順に、実施例５、比較例１）とした。 This was used as sample 4 (Example 4), and the concentrations were adjusted so that the concentrations of VEGF, IGF, and calcium ions in the pulp-derived stem cell culture supernatant were the six concentrations shown in Table 1 including sample 4. did. For Samples 5 and 6 having a concentration lower than that of Sample 4, the pulp-derived stem cell culture supernatant of Sample 4 was diluted by the following operation to prepare a composition for regenerative treatment (in order, Example 5 and Comparative Example 1). And said.
また、サンプル４の濃度より濃度が高いサンプル１ないしサンプル３についても下記の操作によりサンプル４を濃縮して、再生治療用組成物（順に、実施例１ないし実施例３）とした。なお、希釈や濃縮の操作は、必要により繰り返して行うようにした。 Further, with respect to Samples 1 to 3 having a concentration higher than that of Sample 4, Sample 4 was concentrated by the following operation to obtain a composition for regenerative treatment (in order, Examples 1 to 3). The dilution and concentration operations were repeated as necessary.
（希釈方法）
目的とする濃度まで、再生治療用組成物製造に用いたＤＭＥＭ原液を用いて希釈を行った。
(Dilution method)
Dilution was performed using the DMEM stock solution used in the production of the composition for regenerative treatment to the desired concentration.
（濃縮方法）
下記のスピンカラム法にて、目的とする濃度まで濃縮を行った。
(Concentration method)
Concentration was performed to the desired concentration by the following spin column method.
（ｉ）歯髄由来幹細胞培養上清（最大１５ｍｌ）をＡｍｉｃｏｎ Ｕｌｔｒａ Ｃｅｎｔｒｉｆｕｇａｌ Ｆｉｌｔｅｒ Ｕｎｉｔｓ−１０Ｋへ投入し、４０００×ｇで約６０分間遠心し、２００μｌまで濃縮するようにした。
（ii）前記したＡｍｉｃｏｎ Ｕｌｔｒａ Ｃｅｎｔｒｉｆｕｇａｌ Ｆｉｌｔｅｒ Ｕｎｉｔｓ−１０Ｋへ培養上清と同量の滅菌したＰＢＳを投入し、再度４０００×ｇで約６０分間遠心し、ベース溶液をＰＢＳへ置換した。
（iii）得られた溶液２００μｌをマイクロテストチューブへ回収し、濃縮した歯髄由来幹細胞培養上清とした。
（iv）目的とした濃度より低かった場合、前記（ｉ）〜（iii）の操作を繰り返した。
(I) The pulp-derived stem cell culture supernatant (maximum 15 ml) was added to Amicon Ultra Centrifugal Filter Units-10K and centrifuged at 4000 × g for about 60 minutes to concentrate to 200 μl.
(Ii) The same amount of sterilized PBS as the culture supernatant was added to the above-mentioned Amicon Ultra Centrifugal Filter Units-10K, and the mixture was centrifuged again at 4000 × g for about 60 minutes to replace the base solution with PBS.
(Iii) 200 μl of the obtained solution was collected in a microtest tube and used as a concentrated pulp-derived stem cell culture supernatant.
(Iv) When the concentration was lower than the target concentration, the above operations (i) to (iii) were repeated.
（３）ヒト骨髄由来幹細胞の調製：
ヒト骨髄由来幹細胞（Ｂｏｎｅ Ｍａｒｒｏｗ Ｍｅｓｅｎｃｈｙｍａｌ ｓｔｅｍ ｃｅｌｌｓ、骨髄間葉系幹細胞：ＢＭ）は、ロンザジャパン株式会社から購入し、同社の取扱説明書に従って培養した。
(3) Preparation of human bone marrow-derived stem cells:
Human bone marrow-derived stem cells (Bone Marrow Mesenchymal stem cells, bone marrow mesenchymal stem cells: BM) were purchased from Ronza Japan Co., Ltd. and cultured according to the company's instruction manual.
次に、培養して得られたヒト骨髄由来幹細胞について、前記取扱説明書と同様の方法に従い、継代培養を３回繰り返し行い、細胞数が５．０×１０４個／ｍｌとなるように６ウェル（６ｗｅｌｌ）のディッシュプレートへ播種を行った。 Next, for the human bone marrow-derived stem cells obtained by culturing, subculture was repeated 3 times according to the same method as in the above instruction manual so that the number of cells was 5.0 × 10 4 cells / ml. Seeding was performed on a 6-well dish plate.
［試験例１］
ヒト骨髄由来幹細胞の増殖能の確認：
（３）で得られたヒト骨髄由来幹細胞に対して、（２）で得られたサンプル１ないしサンプル６の再生治療用組成物を培養液として用いて、ヒト骨髄由来幹細胞の増殖を確認した。また、ヒト骨髄由来幹細胞に対して培養能があることで知られる１０％ＦＢＳ含有ＤＭＥＭについても同様にしてヒト骨髄由来幹細胞の増殖を確認した。なお、サンプル１ないしサンプル６の評価は、ｎ＝３で行った。
[Test Example 1]
Confirmation of proliferative capacity of human bone marrow-derived stem cells:
With respect to the human bone marrow-derived stem cells obtained in (3), the proliferation of human bone marrow-derived stem cells was confirmed by using the regenerative therapeutic composition of Samples 1 to 6 obtained in (2) as a culture medium. In addition, the proliferation of human bone marrow-derived stem cells was also confirmed in DMEM containing 10% FBS, which is known to have the ability to culture human bone marrow-derived stem cells. The evaluation of Samples 1 to 6 was performed with n = 3.
そして、一般に動物実験等で細胞移植に用いられる培養条件である１０％ＦＢＳ培養液の細胞による増殖能を基準として、サンプル１ないしサンプル６の再生治療用組成物の増殖能を評価した。評価は、１０％ＦＢＳ細胞による増殖率を１とした場合における、サンプル１ないしサンプル６の再生治療用組成物の増殖率（相対値であり、ｎ＝３の平均値。）を確認して、結果の数値がｔ検定により５％有意差のない値となった０．６以上（１０％ＦＢＳ細胞による増殖能の６割以上を達成できる。）である場合を、再生治療用組成物として十分な増殖能がある（表１では「○」と記載した。）と評価した。一方、結果の数値が０．６未満である場合を、再生治療用組成物としては増殖能が不十分（表１では「×」と記載した。）と評価した。評価結果を表１に示す。 Then, the proliferative ability of the regenerative therapeutic composition of Samples 1 to 6 was evaluated based on the proliferative ability of the 10% FBS culture solution, which is a culture condition generally used for cell transplantation in animal experiments and the like. In the evaluation, the proliferation rate (relative value, average value of n = 3) of the composition for regenerative treatment of Samples 1 to 6 when the proliferation rate by 10% FBS cells was set to 1, was confirmed. When the value of the result is 0.6 or more (60% or more of the proliferative ability by 10% FBS cells can be achieved), which is a value with no significant difference of 5% by the t-test, it is sufficient as a composition for regenerative therapy. It was evaluated as having a proliferative ability (indicated as "○" in Table 1). On the other hand, when the numerical value of the result was less than 0.6, it was evaluated that the proliferative ability was insufficient as the composition for regenerative therapy (indicated as "x" in Table 1). The evaluation results are shown in Table 1.
（評価結果）
表１に示すように、血管内皮細胞増殖因子（ＶＥＧＦ）の濃度が１５０〜５０００ｐｇ／ｍｌであるサンプル１ないしサンプル５（実施例１ないし実施例５）の再生治療用組成物は、指標とした１０％ＦＢＳ細胞の増殖率に対して０．６以上の増殖率を示した（評価結果：○）。このように、サンプル１ないしサンプル５（実施例１ないし実施例５）は、再生治療用組成物として増殖能が良好となり、再生治療に有益と考えられるサイトカインが、細胞の増殖への影響があることが推測される機能を有することが確認された。 As shown in Table 1, the composition for regenerative treatment of Samples 1 to 5 (Examples 1 to 5) having a concentration of vascular endothelial growth factor (VEGF) of 150 to 5000 pg / ml was used as an index. The growth rate was 0.6 or more with respect to the growth rate of 10% FBS cells (evaluation result: ◯). As described above, Samples 1 to 5 (Examples 1 to 5) have good proliferative ability as a composition for regenerative therapy, and cytokines considered to be beneficial for regenerative therapy have an effect on cell proliferation. It was confirmed that it has a function that is presumed to be.
また、サンプル１ないしサンプル５（実施例１ないし実施例５）のインシュリン様成長因子（ＩＧＦ）の濃度は８０〜１０００ｐｇ／ｍｌ、カルシウムイオンの濃度は６．５〜８．０ｍｇ／ｄｌの範囲内であった。 In addition, the concentration of insulin-like growth factor (IGF) in Samples 1 to 5 (Examples 1 to 5) is in the range of 80 to 1000 pg / ml, and the concentration of calcium ions is in the range of 6.5 to 8.0 mg / dl. Met.
一方、ＶＥＧＦの濃度が１５０ｐｇ／ｍｌを下回るサンプル６（比較例１）の再生治療用組成物は、１０％ＦＢＳ細胞の増殖率に対して０．５であり（評価結果：×）、有意に増殖活性が低下することが確認された。なお、サンプル６（比較例１）は、ＩＧＦの濃度も８０ｐｇ／ｍｌ、カルシウムイオンの濃度も６．５ｍｇ／ｄｌをそれぞれ下回っているものである。 On the other hand, the composition for regenerative treatment of Sample 6 (Comparative Example 1) having a VEGF concentration of less than 150 pg / ml was 0.5 with respect to the proliferation rate of 10% FBS cells (evaluation result: ×), which was significantly significant. It was confirmed that the proliferative activity was reduced. In Sample 6 (Comparative Example 1), the IGF concentration was also lower than 80 pg / ml, and the calcium ion concentration was also lower than 6.5 mg / dl.
以上の結果から、歯髄由来幹細胞培養上清について、少なくともＶＥＧＦを所定の濃度とすることによって、増殖能が良好となるように最適化された再生治療用組成物となることが確認できた。 From the above results, it was confirmed that the composition for regenerative therapy optimized for improving the proliferative ability of the dental pulp-derived stem cell culture supernatant by adjusting the concentration of at least VEGF to a predetermined concentration.
本発明は、歯髄由来幹細胞を利用して効果的な再生治療用組成物を提供する手段として有利に利用することができ、産業上の利用可能性は高いものである。 INDUSTRIAL APPLICABILITY The present invention can be advantageously used as a means for providing an effective composition for regenerative therapy by utilizing dental pulp-derived stem cells, and has high industrial applicability.
Claims (9)
前記歯髄由来幹細胞培養上清が含有するサイトカインである、血管内皮細胞増殖因子（ＶＥＧＦ）の濃度が１５０〜５０００ｐｇ／ｍｌであることを特徴とする再生治療用組成物。 A composition for regenerative therapy containing a pulp-derived stem cell culture supernatant that does not contain dental pulp-derived stem cells themselves.
A composition for regenerative therapy, wherein the concentration of vascular endothelial growth factor (VEGF), which is a cytokine contained in the pulp-derived stem cell culture supernatant, is 150 to 5000 pg / ml.
当該歯髄由来幹細胞培養上清を歯髄由来幹細胞自体を含まないように処理して、
前記歯髄由来幹細胞培養上清が含有するサイトカインである、血管内皮細胞増殖因子（ＶＥＧＦ）の濃度が１５０〜５０００ｐｇ／ｍｌとなるようにすることを特徴とする再生治療用組成物の製造方法。 Culture the pulp-derived stem cells to obtain a pulp-derived stem cell culture supernatant,
The pulp-derived stem cell culture supernatant was treated so as not to contain the pulp-derived stem cells themselves.
A method for producing a composition for regenerative therapy, which comprises adjusting the concentration of vascular endothelial growth factor (VEGF), which is a cytokine contained in the pulp-derived stem cell culture supernatant, to 150 to 5000 pg / ml.
The claim is characterized in that the concentration adjusting operation is at least one operation selected from the group consisting of concentration, dilution, centrifugation, solvent replacement, dialysis, freezing, drying, lyophilization, desalting and storage. Item 8. The method for producing a composition for regenerative therapy according to Item 8.
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