JP2012205599A - Three-dimensional cell culturing method, and composition for cell transplantation - Google Patents
Three-dimensional cell culturing method, and composition for cell transplantation Download PDFInfo
- Publication number
- JP2012205599A JP2012205599A JP2012171027A JP2012171027A JP2012205599A JP 2012205599 A JP2012205599 A JP 2012205599A JP 2012171027 A JP2012171027 A JP 2012171027A JP 2012171027 A JP2012171027 A JP 2012171027A JP 2012205599 A JP2012205599 A JP 2012205599A
- Authority
- JP
- Japan
- Prior art keywords
- cells
- cell
- human
- composition
- mps
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
本発明は、マイクロ粒子と血漿添加による各種体性多能性細胞の細胞培養方法及び細胞移植用組成物に係り、詳細には低分子ヘパリンとプロタミンとから構成されるマイクロ粒子と、細胞増殖又は分化誘導を促進させるサイトカインと、血漿添加によるゲル化培地を用いた接着性細胞及び造血系浮遊細胞等の体外３次元細胞培養方法及び再生医療等に用いられる細胞移植用組成物に関するものである。 The present invention relates to a method for culturing various somatic pluripotent cells by addition of microparticles and plasma, and a composition for cell transplantation. Specifically, the present invention relates to microparticles composed of low molecular weight heparin and protamine, The present invention relates to a composition for cell transplantation used for in vitro three-dimensional cell culture methods such as adherent cells and hematopoietic suspension cells using a cytokine that promotes differentiation induction and a gelled medium by adding plasma, and regenerative medicine.
相応する電荷を有する高分子間静電反応により生じる多重電解質複合体は、電荷を帯びた生体高分子の生体内挙動の研究モデルとなる。さらに、ユニークな組成や構造からなる多重電解質複合体は、可溶性ナノ粒子の形成、複合化マイクロ粒子の形成（コアセルベーション）、非結晶性沈殿物等を形成し、バイオテクノロジーや医学の分野での適用が可能である。例えば、タンパク質と核酸よりなる複合体形成は転写過程に影響を及ぼすと考えられる。また、低分子ヘパリン／プロタミン マイクロ粒子（ＬＨ／Ｐ ＭＰｓ）に加えて、ＤＮＡ・キトサン複合体マイクロ（ナノ）粒子がそれぞれ遺伝子キャリア及び薬剤キャリアとして記述された。 Multi-electrolyte complexes generated by electrostatic reactions between polymers with corresponding charges serve as research models for in vivo behavior of charged biopolymers. In addition, multi-electrolyte composites with a unique composition and structure form soluble nanoparticles, complex microparticles (coacervation), non-crystalline precipitates, etc. in the fields of biotechnology and medicine. Can be applied. For example, the formation of a complex consisting of a protein and a nucleic acid is thought to affect the transcription process. In addition to low molecular weight heparin / protamine microparticles (LH / P MPs), DNA / chitosan complex micro (nano) particles were described as a gene carrier and a drug carrier, respectively.
ところで、骨髄や脂肪組織を含んだ多くを生体組織には、組織の維持や修復のため多分化能を保持した前駆体細胞である生体幹細胞が含まれていることが知られている。特に骨髄細胞は造血系幹細胞や間葉系幹細胞を含み、各種血液細胞のみならず、皮膚、骨、軟骨、血管、筋肉の細胞に分化しそれぞれの組織再生を促進する。同様に、脂肪組織由来間葉系細胞も皮膚、骨、軟骨、血管、筋肉の細胞に分化することが知られており、人において豊富な幹細胞資源となる脂肪組織に大きな注目が集まっている。 By the way, it is known that living tissue including many bone marrow and adipose tissue contains living stem cells, which are precursor cells having pluripotency for maintaining and repairing tissues. In particular, bone marrow cells include hematopoietic stem cells and mesenchymal stem cells and differentiate into not only various blood cells but also skin, bone, cartilage, blood vessel, and muscle cells to promote the regeneration of each tissue. Similarly, adipose tissue-derived mesenchymal cells are also known to differentiate into skin, bone, cartilage, blood vessel, and muscle cells, and a great deal of attention is being focused on adipose tissue, which is an abundant stem cell resource in humans.
しかしながら、各種体性幹細胞や前駆体細胞の収率は低く、培養には高濃度（１０％以上）の異種動物の血清、フィーダー細胞、そしてコラーゲンや腫瘍生成基底膜であるマトリゲルをマトリックスとして使用する必要があり、ウィルスや未知病原体による感染及び抗原性付与の問題が避けられない。よって、上記体性幹細胞や前駆体細胞を効率的、且つ安全に異種動物の血清培地、フィーダー細胞、そしてコラーゲンやマトリゲルを使用しないマトリックス内で増殖させる培養法が求められている。さらに再生医療における細胞移植には、生体内でのその生着性及び増殖性を高める有効な細胞キャリアを併用する必要がある。 However, the yield of various somatic stem cells and progenitor cells is low, and high concentrations (over 10%) of sera from different animals, feeder cells, and Matrigel, which is collagen and tumorigenic basement membrane, are used as a matrix for culturing. The problem of infection and antigenicity imparted by viruses and unknown pathogens is inevitable. Therefore, there is a need for a culture method in which the somatic stem cells and precursor cells are efficiently and safely grown in a heterogeneous animal serum medium, feeder cells, and a matrix that does not use collagen or matrigel. Furthermore, for cell transplantation in regenerative medicine, it is necessary to use an effective cell carrier that enhances the engraftment and proliferation in vivo.
ところで、血液を凝固させ血小板や血液凝固因子（フィブリノーゲン等) を除いたものが血清(serum) であり、血漿は抗凝固剤（クエン酸等）入りの採血管を用いて採血した血液を遠心分離して細胞成分（赤血球や白血球）を除いたものが血小板を含んだ血漿（プラズマ) である。この血漿を添加した培地は、混合後１０分以内に室温で容易にゲル化することが見出されている。 By the way, blood is clotted and platelets and blood coagulation factors (fibrinogen, etc.) are removed, and serum is serum. Plasma is centrifuged using a blood collection tube containing an anticoagulant (citric acid, etc.). The plasma (plasma) containing platelets is obtained by removing cellular components (red blood cells and white blood cells). This medium supplemented with plasma has been found to gel easily at room temperature within 10 minutes after mixing.
そこで、近年では、血漿の添加によるゲル化に着目した血漿ゲル（Ｐｌａｓｍａ ｇｅｌ）は、細胞増殖及び分化誘導を促進する様々な増殖因子やサイトカイン等の生理活性物質を含有し運搬する血小板を多く含み、再生医療等多くの分野で様々な応用研究が進められている。 Therefore, in recent years, plasma gels that focus on gelation by the addition of plasma contain a large amount of platelets containing and transporting various growth factors and cytokines and other physiologically active substances that promote cell proliferation and differentiation induction. Various applied researches are being promoted in many fields such as regenerative medicine.
これらの血小板に運搬される生理活性物質は、血小板由来増殖因子（ＰＤＧＦｓ）、線維芽増殖因子（ＦＧＦｓ）、肝細胞増殖因子（ＨＧＦ）、トランスフォーミング増殖因子（ＴＧＦｓ）、血管内皮増殖因子（ＶＥＧＦｓ）等２０を超え、これらのほとんどはヘパリン結合性であることが知られている。さらに、形成外科の分野でも創傷治癒、変形の改善（含雛取り）、育毛等幅広い臨床応用が期待されており、また血管新生療法や再生医療等の臨床の場でも既に適用されているが、その効果はさほど高くないのが現状である。 The physiologically active substances delivered to these platelets are platelet-derived growth factors (PDGFs), fibroblast growth factors (FGFs), hepatocyte growth factors (HGF), transforming growth factors (TGFs), vascular endothelial growth factors (VEGFs). ) Etc., and most of these are known to be heparin-binding. Furthermore, in the field of plastic surgery, a wide range of clinical applications such as wound healing, deformation improvement (including chick removal) and hair growth are expected, and it has already been applied in clinical settings such as angiogenesis therapy and regenerative medicine. The effect is not so high.
このように各分野において、血漿ゲルの有効性をさらに高めるため、貴重な患者本人の血液製剤である血漿に含有されている生理活性物質の活性を維持したまま、より有効に局所に保持、徐放が可能な、安全な薬剤キャリアの出現が求められていた。 As described above, in each field, in order to further increase the effectiveness of plasma gel, the activity of the physiologically active substance contained in plasma, which is a valuable patient's own blood product, is maintained and more effectively maintained locally. The emergence of safe drug carriers that can be released has been demanded.
そこで、本発明者等は、鋭意研究の結果、低分子へパリン（ＬＨ）とプロタミン（Ｐ）との組み合わせを用いることにより、簡便に平均粒径１．５μｍ程度（０．５μｍ以上３μｍ未満）から、さらには平均粒径１００ｎｍの微粒子を作成でき、得られた微粒子をコートしたプレートが徐放性の薬物包接或いは細胞接着マトリックスとして使用できることを見出した（詳細は、特許文献１及び非特許文献１〜５を参照）。 As a result of intensive studies, the present inventors have simply used a combination of low molecular weight heparin (LH) and protamine (P), so that the average particle size is about 1.5 μm (0.5 μm or more and less than 3 μm). Furthermore, it was found that fine particles having an average particle diameter of 100 nm can be prepared, and the plate coated with the obtained fine particles can be used as a sustained-release drug inclusion or cell adhesion matrix (for details, see Patent Document 1 and Non-patent Document). Reference 1-5).
すなわち、このＬＨ／Ｐ ＭＰｓ−コートマトリックスが、塩基性線維芽細胞増殖因子（ＦＧＦ）−２及び血小板に含まれる様々なヘパリン結合性増殖因子やサイトカイン等の生理活性物質を吸着してそれらの活性を保護し、徐放するマトリックスとして有効であること、また上記ＬＨ／Ｐ ＭＰｓに吸着・保護され活性を保持した生理活性物質が、生体内で生理活性物質含有ＬＨ／Ｐ ＭＰｓ複合体の生分解に伴い周辺部位へ徐放され、生体局所での血管新生や肉芽形成の促進に寄与することを実証した。 That is, this LH / P MPs-coat matrix adsorbs physiologically active substances such as basic fibroblast growth factor (FGF) -2 and various heparin-binding growth factors and cytokines contained in platelets and their activity. Is effective as a matrix that protects and slowly releases, and the bioactive substance that is adsorbed and protected by the LH / P MPs and retains its activity is biodegraded in vivo in the LH / P MPs complex containing the physiologically active substance. As a result, it was demonstrated that it was gradually released to the surrounding area and contributed to the promotion of angiogenesis and granulation in the living body.
ところで、近年ヒトや動物の細胞を生体外で培養し増幅及び分化誘導を行い、その機能を利用して人工臓器や人工組織として利用する研究が活発に行なわれている。しかしながら、各種細胞の中でも、特に再生医療での移植用細胞として需要の大きい骨髄及び脂肪組織由来の間葉系幹細胞については、ウィルスや未知病原体による感染、抗原性付与の問題を有する異種動物の血清、フィーダー細胞、そしてコラーゲンやマトリゲル等のマトリックスとしての使用を排除し、多分化能を維持したまま安全に増幅する培養法が未だ確立されていない。 By the way, in recent years, researches have been actively conducted in which human and animal cells are cultured in vitro, amplified and differentiated, and used as artificial organs or artificial tissues by utilizing their functions. However, among various cells, especially for mesenchymal stem cells derived from bone marrow and adipose tissue, which are in great demand as cells for transplantation in regenerative medicine, the sera of heterologous animals that have problems of infection by viruses and unknown pathogens, and the addition of antigenicity No culture method has been established yet that eliminates the use of feeder cells and matrices such as collagen and matrigel, and allows safe amplification while maintaining pluripotency.
特に、造血系前駆体細胞或いは血管内皮前駆体細胞として機能することが知られているＣＤ３４陽性造血系前駆体細胞についての体外増幅についての確立された方法はなく、その需要に比して採取できる量が非常に少ないため、成人に移植することが困難となっていた。従って、安全性や安定した供給等の問題点から異種動物の血清、フィーダー細胞、そしてコラーゲンやマトリゲル等のマトリックスとしての使用を排除した体外大量増幅システムの開発が必要である。 In particular, there is no established method for in vitro amplification of CD34 positive hematopoietic progenitor cells known to function as hematopoietic progenitor cells or vascular endothelial progenitor cells, which can be collected relative to their demand The amount was so small that it was difficult to transplant to adults. Therefore, it is necessary to develop an extracorporeal mass amplification system that eliminates the use of sera from different animals, feeder cells, and matrices such as collagen and matrigel from problems such as safety and stable supply.
さらに細胞移植は、細胞の有する脆弱性や分散性により、必要な場所に有効に移植細胞を生着、増殖させることに困難を極めているのが現状であり、安全で有効な細胞運搬システムの出現が求められている。 Furthermore, cell transplantation is currently difficult to engraft and proliferate transplanted cells effectively where necessary due to the vulnerability and dispersibility of cells, and the emergence of safe and effective cell delivery systems. Is required.
そこで、本発明は上記問題点に鑑みてなされたものであり、骨髄由来間葉系幹細胞や脂肪組織由来間葉系細胞、ＣＤ３４陽性造血系前駆体細胞、植皮用皮膚構築に不可欠な線維芽細胞、末梢血流障害の治療に応用が期待される血管内皮細胞等の再生医療に必要な接着性細胞又は造血系浮遊細胞を高い増殖率で培養することができ、且つ人体に対する安全性が高く、さらには生体内での生着性及び細胞生存性を顕著に高めた３次元細胞培養方法及び細胞移植用組成物を提供することを目的とするものである。 Therefore, the present invention has been made in view of the above problems, and bone marrow-derived mesenchymal stem cells, adipose tissue-derived mesenchymal cells, CD34-positive hematopoietic progenitor cells, and fibroblasts essential for skin construction for skin grafting. Adhesive cells or hematopoietic suspension cells necessary for regenerative medicine such as vascular endothelial cells, which are expected to be applied in the treatment of peripheral blood flow disorders, can be cultured at a high growth rate, and are highly safe for the human body. Furthermore, it is an object of the present invention to provide a three-dimensional cell culture method and a cell transplant composition that have significantly enhanced engraftment and cell viability in vivo.
上記した目的を達成するため、請求項１記載の３次元細胞培養方法は、再生医療に用いる間葉系幹細胞や腫瘍細胞を含む接着性細胞と、
低分子ヘパリンとプロタミンとからなる平均粒径が０．１〜５μｍであるＬＨ／Ｐ ＭＰｓと、
前記接着性細胞の用途に応じて任意に選択されるサイトカインと、
容積比０．５〜２５％のヒト血漿と、
で構成されるゲル状組成物内で前記接着性細胞の培養を行うことを特徴とする。
In order to achieve the above-described object, the three-dimensional cell culture method according to claim 1 comprises an adhesive cell containing mesenchymal stem cells and tumor cells used for regenerative medicine,
LH / P MPs having an average particle size of 0.1 to 5 μm consisting of low molecular weight heparin and protamine;
A cytokine arbitrarily selected according to the use of the adhesive cells;
Human plasma with a volume ratio of 0.5-25%,
The adherent cells are cultured in a gel composition composed of:
請求項２記載の細胞移植用組成物は、再生医療に用いる間葉系幹細胞や腫瘍細胞を含む接着性細胞と、
低分子ヘパリンとプロタミンとからなる平均粒径が０．１〜５μｍであるＬＨ／Ｐ ＭＰｓと、
前記接着性細胞の用途に応じて任意に選択されるサイトカインと、
容積比０．５〜２５％のヒト血漿と、
で構成されるゲル状組成物であることを特徴とする。
The composition for cell transplantation according to claim 2, an adhesive cell containing mesenchymal stem cells and tumor cells used for regenerative medicine,
LH / P MPs having an average particle size of 0.1 to 5 μm consisting of low molecular weight heparin and protamine;
A cytokine arbitrarily selected according to the use of the adhesive cells;
Human plasma with a volume ratio of 0.5-25%,
It is a gel-like composition comprised by this, It is characterized by the above-mentioned.
請求項３記載の３次元細胞培養方法は、再生医療に用いるＣＤ３４陽性造血系前駆体細胞を含む造血系浮遊細胞と、
低分子ヘパリンとプロタミンとからなる平均粒径が０．１〜５μｍであるＬＨ／Ｐ ＭＰｓと、
前記造血系浮遊細胞の用途に応じて任意に選択されるサイトカインと、
容積比０．５〜２５％のヒト血漿と、
で構成されるゲル状組成物内で前記造血系浮遊細胞の培養を行うことを特徴とする。
The three-dimensional cell culture method according to claim 3, wherein hematopoietic suspension cells containing CD34 positive hematopoietic precursor cells used for regenerative medicine;
LH / P MPs having an average particle size of 0.1 to 5 μm consisting of low molecular weight heparin and protamine;
A cytokine arbitrarily selected according to the use of the hematopoietic suspension cells,
Human plasma with a volume ratio of 0.5-25%,
The hematopoietic suspension cells are cultured in a gel composition comprising:
請求項４記載の細胞移植用組成物は、再生医療に用いるＣＤ３４陽性造血系前駆体細胞を含む造血系浮遊細胞と、
低分子ヘパリンとプロタミンとからなる平均粒径が０．１〜５μｍであるＬＨ／Ｐ ＭＰｓと、
前記造血系浮遊細胞の用途に応じて任意に選択されるサイトカインと、
容積比０．５〜２５％のヒト血漿と、
で構成されるゲル状組成物であることを特徴とする。
The composition for cell transplantation according to claim 4, wherein hematopoietic suspension cells containing CD34 positive hematopoietic precursor cells used for regenerative medicine;
LH / P MPs having an average particle size of 0.1 to 5 μm consisting of low molecular weight heparin and protamine;
A cytokine arbitrarily selected according to the use of the hematopoietic suspension cells,
Human plasma with a volume ratio of 0.5-25%,
It is a gel-like composition comprised by this, It is characterized by the above-mentioned.
本発明によれば、再生医療に用いられる接着性細胞や造血系浮遊細胞と、低分子ヘパリンとプロタミンとからなる平均粒径が０．１〜５μｍであるＬＨ／Ｐ ＭＰｓと、使用する細胞の用途に応じて任意に選択されるサイトカインと、容積比０．５〜２５％のヒト血漿とで構成されるゲル状の細胞移植用組成物を用いるため、細針を装着したシリンジでも注入可能となる。 According to the present invention, adhesive cells and hematopoietic suspension cells used for regenerative medicine, LH / P MPs having an average particle size of 0.1 to 5 μm consisting of low molecular weight heparin and protamine, and the cells to be used Because it uses a gel-like composition for cell transplantation composed of cytokines arbitrarily selected according to the application and human plasma with a volume ratio of 0.5 to 25%, it can be injected with a syringe equipped with a fine needle. Become.
また、局所への移植後、適度な粘稠性により注入箇所に留まり、また各種接着性細胞や各種造血系浮遊細胞の分散を限定するとともに、生着性、増殖性及び多分化能に優れた局所細胞移植のための運搬システムとして適用することができる。 In addition, after transplantation to the local area, it stays at the injection site with an appropriate viscosity, and it limits the dispersion of various adhesive cells and various hematopoietic suspension cells, and is excellent in engraftment, proliferation, and pluripotency. It can be applied as a delivery system for local cell transplantation.
以下、本発明を実施するための形態について、添付した図面を参照しながら詳細に説明する。また、この実施の形態によりこの発明が限定されるものではなく、この形態に基づいて当業者などによりなされる実施可能な他の形態、実施例及び運用技術などはすべて本発明の範疇に含まれる。 Hereinafter, embodiments for carrying out the present invention will be described in detail with reference to the accompanying drawings. In addition, the present invention is not limited by this embodiment, and all other forms, examples, operation techniques, and the like that can be implemented by those skilled in the art based on this form are included in the scope of the present invention. .
本発明に係る３次元細胞培養方法は、再生医療に用いられる各種細胞（接着性細胞又は造血系浮遊細胞、より好ましくは移植対象となる患者の自己細胞）と、低分子ヘパリンとプロタミンから構成されるＬＨ／Ｐ ＭＰｓと、同種血漿（好ましくは自己血漿）及び用途に応じて適宜選択される適切なサイトカインを含有した培地からなる血漿ゲルと、で構成されている。 The three-dimensional cell culture method according to the present invention is composed of various cells (adherent cells or hematopoietic suspension cells, more preferably patient autologous cells to be transplanted) used in regenerative medicine, low molecular weight heparin and protamine. LH / P MPs, and plasma gel composed of a homogenous plasma (preferably autologous plasma) and a medium containing an appropriate cytokine appropriately selected according to the application.
再生医療に用いられる細胞としては、「接着性細胞」と「造血系浮遊細胞」とに大別される。
「接着性細胞」としては、例えばヒト脂肪組織由来間葉系細胞やヒト骨髄由来間葉系幹細胞等の種々の間葉系幹細胞及び血管内皮細胞及びその前駆体細胞、線維芽細胞及びその前駆体細胞、血管内皮細胞及びその前駆体細胞、肝実質細胞及びその前駆体細胞、滕β細胞及びその前駆体細胞、平滑筋細胞及びその前駆体細胞、軟骨細胞及びその前駆体細胞、筋芽細胞、骨芽細胞、癌細胞等の種々の腫瘍細胞（例えばヒト肝癌細胞株（Ｈｕｈ７））がある。
また、「造血系浮遊細胞」としては、例えばヒト赤血白血病細胞（ＴＦ−１）やヒト骨髄由来ＣＤ３４造血系前駆体細胞等の種々の造血系前駆体細胞、胚性幹細胞（Embryonic stem cells：ＥＳ細胞) 、人工多能性幹細胞（Induced pluripotent stem cells：ｉＰＳ細胞）等がある。
なお、上記各種細胞は、用途に応じて適宜選択又は任意に組み合わせて使用することができる。
Cells used for regenerative medicine are roughly classified into “adhesive cells” and “hematopoietic suspension cells”.
Examples of “adhesive cells” include various mesenchymal stem cells such as human adipose tissue-derived mesenchymal cells and human bone marrow-derived mesenchymal stem cells, vascular endothelial cells and precursor cells thereof, fibroblasts and precursors thereof. Cells, vascular endothelial cells and their precursor cells, hepatocytes and their precursor cells, 滕 β cells and their precursor cells, smooth muscle cells and their precursor cells, chondrocytes and their precursor cells, myoblasts, There are various tumor cells such as osteoblasts and cancer cells (eg, human hepatoma cell line (Huh7)).
Examples of “hematopoietic floating cells” include various hematopoietic precursor cells such as human erythroleukemia cells (TF-1) and human bone marrow-derived CD34 hematopoietic progenitor cells, embryonic stem cells: ES cells), induced pluripotent stem cells (iPS cells), and the like.
The various cells can be appropriately selected or arbitrarily combined depending on the use.
低分子ヘパリン／プロタミン マイクロ粒子（ＬＨ／Ｐ ＭＰｓ）は、低分子ヘパリンとプロタミンとを含むマイクロ粒子である。本発明で用いられる低分子ヘパリンは、一般に天然ヘパリン（分子量１５０００〜２００００Ｄａ程度）を解重合して得られる低分子量のヘパリンである。その平均分子量の上限は、プロタミンと混合することにより微粒子を形成できる程度のものであればよく、一般的には１００００、好ましくは９０００、より好ましくは８０００、さらに好ましくは６０００であり、数平均分子量の下限は、通常は１０００、好ましくは３０００、より好ましくは４０００である。 Low molecular weight heparin / protamine microparticles (LH / P MPs) are microparticles containing low molecular weight heparin and protamine. The low molecular weight heparin used in the present invention is generally a low molecular weight heparin obtained by depolymerizing natural heparin (molecular weight of about 15,000 to 20000 Da). The upper limit of the average molecular weight is only required to be such that fine particles can be formed by mixing with protamine, and is generally 10,000, preferably 9000, more preferably 8000, more preferably 6000, and the number average molecular weight. Is usually 1000, preferably 3000, and more preferably 4000.
なお、数平均分子量において通常（天然）のヘパリンと区別できれば十分であり、例えば、１００００以上、或いは２００００以上の分子量を持つ分画を含んでいても、全体として平均分子量が約１００００未満であれば本発明における低分子ヘパリンとして使用できる。本発明では、ヘパリンの平均分子量として、分子量が既知の標準物質を用いたゲル浸透クロマトグラフィー法によって測定される数平均分子量を用いる。 In addition, it is sufficient that the number average molecular weight can be distinguished from normal (natural) heparin. For example, even if a fraction having a molecular weight of 10,000 or more or 20,000 or more is included, the average molecular weight is less than about 10,000 as a whole. It can be used as a low molecular weight heparin in the present invention. In the present invention, the number average molecular weight measured by gel permeation chromatography using a standard substance having a known molecular weight is used as the average molecular weight of heparin.
例えば、ブタの小腸粘膜由来のヘパリンを亜硝酸分解して得られる解重合ヘパリン（ダルテパリン）は、フラグミン（商品名）として市販されており、４０００〜６０００の平均分子量を有する。同様に、ブタの小腸粘膜由来のヘパリンを亜硝酸分解して得られる解重合ヘパリン（レビパリン）はローモリン（商品名）として市販されている。また、ウシ又はブタ腸粘膜由来のヘパリンを過酸化水素と酢酸第二銅により分解して得られる解重合ヘパリン（パルナパリン）は、ローヘパ（商品名）として市販されており、これらは何れも本発明における低分子ヘパリンとして使用できる。 For example, depolymerized heparin (dalteparin) obtained by nitrous acid decomposition of heparin derived from porcine small intestinal mucosa is commercially available as Fragmin (trade name) and has an average molecular weight of 4000 to 6000. Similarly, depolymerized heparin (reviparin) obtained by nitrous acid decomposition of heparin derived from porcine small intestinal mucosa is commercially available as lomorin (trade name). In addition, depolymerized heparin (parnaparin) obtained by decomposing heparin derived from bovine or porcine intestinal mucosa with hydrogen peroxide and cupric acetate is commercially available as Lohepa (trade name), both of which are the present invention. Can be used as low molecular weight heparin.
ヘパリンは単独では抗凝固作用を持たず、血漿中のATIII と結合することによってその作用を発揮し、第IIa 因子、第XIIa因子、第XIa 因子、第Xa因子、第IXa 因子などの凝固系酵素を阻害、不活化する。一方、本発明で使用する低分子ヘパリンは抗第XIIa、抗第Xa因子活性を持つものの、第IIa 因子、第XIa 因子、第IXa 因子に対する阻害活性は軽微であることが医薬品として明らかにされているので、創傷部位に注入しても当該部位における出血傾向を助長することなく使用できる。 Heparin alone does not have an anticoagulant effect, but exerts its effect by binding to plasma ATIII, and coagulation enzymes such as factor IIa, factor XIIa, factor XIa, factor Xa, factor IXa Inhibits and inactivates. On the other hand, although low molecular weight heparin used in the present invention has anti-factor XIIa and anti-factor Xa activities, it has been clarified as a pharmaceutical that its inhibitory activity against factor IIa, factor XIa and factor IXa is slight. Therefore, even if it is injected into the wound site, it can be used without promoting the bleeding tendency at the site.
低分子ヘパリンとしては、上記の市販されているものを使用してもよいし、或いは、過ヨウ素酸酸化により低分子化したヘパリンや、特異的脱硫酸化ヘパリンなども好適に用いることができる。このような低分子量のヘパリンを使用することにより、プロタミンと混合した際に好適な微粒子を得ることができる。 As the low molecular weight heparin, the above-mentioned commercially available one may be used, or heparin having a low molecular weight by periodate oxidation, specific desulfated heparin, or the like can be suitably used. By using such low molecular weight heparin, suitable fine particles can be obtained when mixed with protamine.
本発明で用いるプロタミンは、動物の精子の核中でＤＮＡと結合して存在する塩基性の高いタンパク質として知られている。一般的には、２７〜６５残基からなる低分子量タンパク質であり、アミノ酸の４０〜７０％をアルギニンが占めると言われている。プロタミンも医薬品として市販されており、本発明では市販のプロタミンをそのまま使用することができる。 The protamine used in the present invention is known as a highly basic protein that is present in the sperm nucleus of an animal by binding to DNA. Generally, it is a low molecular weight protein consisting of 27 to 65 residues, and it is said that arginine occupies 40 to 70% of amino acids. Protamine is also commercially available as a pharmaceutical. In the present invention, commercially available protamine can be used as it is.
本発明の好適態様では、低分子ヘパリン溶液にプロタミン溶液を滴下して低分子ヘパリンとプロタミンとを含む粒子を生成させる。例えば、ダルテパリン等の低分子ヘパリンの水溶液に、プロタミン水溶液を後述する割合になるよう滴下し、ボルテックスなどでさらに攪拌することによって粒子を得る。低分子ヘパリンおよびプロタミンの濃度は各々設定することができ、好適にはそれぞれ０．０１〜３０ｍｇ／ｍｌである。このとき、低分子ヘパリンおよびプロタミンの濃度を調節することによって、得られる粒子のサイズを制御することができる。 In a preferred embodiment of the present invention, a protamine solution is dropped into a low molecular weight heparin solution to produce particles containing low molecular weight heparin and protamine. For example, a protamine aqueous solution is dropped into an aqueous solution of a low molecular weight heparin such as dalteparin at a ratio described later, and particles are obtained by further stirring with a vortex or the like. The concentrations of low molecular weight heparin and protamine can be set respectively, and are preferably 0.01 to 30 mg / ml, respectively. At this time, the size of the obtained particles can be controlled by adjusting the concentrations of low molecular weight heparin and protamine.
例えば、低分子ヘパリンおよびプロタミンの濃度がそれぞれ１〜２０ｍｇ／ｍｌ程度の溶液を用いると、０．５〜１０μｍ程度の平均粒子径をもつ粒子を得ることができ、低分子ヘパリンおよびプロタミンの濃度がそれぞれ０．０５〜０．５ｍｇ／ｍｌ程度の溶液を用いると、５０〜２００ｎｍ程度の平均粒子径をもつ粒子を得ることができる。 For example, when a solution having low molecular weight heparin and protamine concentrations of about 1 to 20 mg / ml is used, particles having an average particle diameter of about 0.5 to 10 μm can be obtained. When a solution of about 0.05 to 0.5 mg / ml is used, particles having an average particle diameter of about 50 to 200 nm can be obtained.
低分子ヘパリンとプロタミンとの混合重量比については、プロタミンに対する低分子ヘパリンの重量を等量、或いはやや過剰することが、粒子の収率向上の点で好ましく、過剰なプロタミンの存在は不溶性のペースト状沈殿物が生成する懸念がある。従って、低分子ヘパリンの溶液にプロタミンの溶液を滴下する場合には、滴下後の溶液内における低分子ヘパリン／プロタミンの重量比は、好ましくは１／１〜５／１であり、より好ましくは１／１〜２／１である。 As for the mixing weight ratio of low molecular weight heparin and protamine, it is preferable that the weight of low molecular weight heparin with respect to protamine is equal or slightly excessive from the viewpoint of improving the yield of particles, and the presence of excessive protamine is an insoluble paste. There is concern about the formation of a precipitate. Therefore, when the protamine solution is dropped into the low molecular weight heparin solution, the weight ratio of the low molecular weight heparin / protamine in the dropped solution is preferably 1/1 to 5/1, more preferably 1 / 1 to 2/1.
すなわち、上記低分子ヘパリン溶液に含まれる低分子ヘパリン１重量部に対して、好ましくは０．２〜１重量部、より好ましくは０．５〜１重量部のプロタミンが添加されるようにプロタミン溶液が滴下される。 That is, the protamine solution is added such that 0.2 to 1 part by weight, more preferably 0.5 to 1 part by weight of protamine is added to 1 part by weight of the low molecular weight heparin contained in the low molecular weight heparin solution. Is dripped.
このように作成されたＬＨ／Ｐ ＭＰｓは、血漿中に含まれる多くのへパリン結合性増殖因子やサイトカイン等生理活性物質を担持することができ、担持された生理活性物質を熱やトリプシン等タンパク質分解酵素による不活化要因から保護して活性を維持延長することができ、さらに骨髄や脂肪組織由来の間葉系幹細胞等様々な接着性細胞の表面に接合し、細胞凝集体（スフェロイド）を形成する。 The LH / P MPs prepared in this way can carry a large amount of physiologically active substances such as heparin-binding growth factors and cytokines contained in plasma, and the loaded physiologically active substances can be converted into proteins such as heat and trypsin. Protects against inactivation by degrading enzymes and maintains and prolongs the activity, and also joins the surface of various adherent cells such as bone marrow and adipose tissue-derived mesenchymal stem cells to form cell aggregates (spheroids) To do.
ヒト血漿は、健常ボランティアから０．１〜８m1の２％クエン酸溶液の入った採血管に血液を採血し、所定条件（例えば、１５分間、１７００ｒｐｍ（Table Top 冷却遠心機 2800,ローター：RS 240,クボタ））で遠心し、生じた３つの層、底部の赤（白）血球画分、赤（白）血球画分から上部（１ｃｍ）を多血小板血漿（Platelet Rich Plasma ：ＰＲＰ）画分、さらに上部を少血小板血漿（Platelet Poor Plasma ：ＰＰＰ）として採取し、ＰＲＰとＰＰＰを合わせたものである。 Human plasma is collected from a healthy volunteer in a blood collection tube containing a 0.1% to 8 ml 1% 2% citric acid solution, and is subjected to predetermined conditions (for example, 1700 rpm (Table Top cooling centrifuge 2800, rotor: RS 240). , Kubota)), and the resulting three layers, the bottom red (white) blood cell fraction, the red (white) blood cell fraction from the top (1 cm) to the platelet rich plasma (PRP) fraction, The upper part is collected as platelet low plasma (PPP), and PRP and PPP are combined.
なお、本発明では、採血した血液の有効利用とゲル形成効率の観点から培地・血漿ゲル化剤としてヒト血漿を使用したが、血小板とともにそこに包含されたサイトカインが濃縮されているＰＲＰ或いは血漿ゲル化に優れたＰＰＰの用途別使用も可能である。また、安全性の面からヒトへの臨床適用には、自己（autogenous）血漿の使用が望ましい。 In the present invention, human plasma is used as a medium / plasma gelling agent from the viewpoint of effective utilization of collected blood and gel formation efficiency. However, PRP or plasma gel in which cytokines contained therein are concentrated together with platelets is used. It is also possible to use PPPs excellent in conversion for different purposes. From the viewpoint of safety, the use of autogenous plasma is desirable for clinical application to humans.
サイトカインとしては、一例としてインターロイキン（Interleukin ：ＩＬ）、幹細胞因子（Stem cell factor：ＳＣＦ）、上皮増殖因子（Epidermal Growth Factor ：ＥＧＦ）、線維芽細胞増殖因子（Fibroblast Growth Factor：ＦＧＦ）、塩基性線維芽細胞増殖因子（basic fibroblast growth factor：ＦＧＦ−２）、血小板由来増殖因子（Platelet-Derived Growth Factor：ＰＤＧＦ）、肝細胞増殖因子（Hepatocyto Growth Factor：ＨＧＦ）、トランスフォーミング増殖因子（Transforming growth factor：ＴＧＦ）、トロンボポエチン（Thrombopoietin：ＴＰＯ）、コロニー刺激因子（Coloney-Stimulating Factor：ＣＳＦ）、顆粒球コロニー刺激因子（Granulocyte-Coloney-Stimulating Factor：Ｇ−ＣＳＦ）、顆粒球マクロファージコロニー刺激因子（Granulocyte-macrophage-colony stimulating factor：ＧＭ−ＣＳＦ）、ＦＬｔ３−ｌｉｇａｎｄ（Human Fms-related tyrosin kinase 3 ligand ）等があり、使用する接着性細胞又は造血系浮遊細胞に応じて任意に選択して添加する。 As cytokines, for example, interleukin (IL), stem cell factor (SCF), epidermal growth factor (EGF), fibroblast growth factor (FGF), basic Fibroblast growth factor (FGF-2), Platelet-Derived Growth Factor (PDGF), Hepatocyto Growth Factor (HGF), Transforming growth factor : TGF), thrombopoietin (TPO), colony-stimulating factor (CSF), granulocyte-coloney-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (Granulocyte- macrophage-colony stimulating factor (GM-CSF), FLt3-li and (Human Fms-related tyrosin kinase 3 ligand) have such added arbitrarily selected depending on the adherent cell or hematopoietic floating cells used.
培地としては、用途に応じて選択された各種細胞に適切なサイトカインに応じて選択される細胞培養培地として、例えばＤＭＥＭ（Dulbecco's Modified Eagle Medium）、ＨＰＧＭ（Hematopoietic Progenitor Growth Medium）等を使用する。 As the medium, for example, DMEM (Dulbecco's Modified Eagle Medium), HPGM (Hematopoietic Progenitor Growth Medium), or the like is used as a cell culture medium selected according to cytokines appropriate for various cells selected according to the use.
本発明の細胞移植用組成物におけるゲル化の条件としては、血小板及び血液凝固因子（フィブリノーゲン等）を含めた血漿を用いているため、採血時に採血管に入れる抗凝固剤としてのクエン酸（２％液）を採血量に対して１〜２％に制限、或いは添加するＬＨ／Ｐ ＭＰｓを０．２ｍｇ／ｍｌ以下に制限することによって適度な粘稠性を有するとともに増殖した細胞の分散性を限定し、生着性及び増殖性に優れた組成物を得ることができる。 なお、このゲル化メカニズムは、人体が怪我をしたときに出血した血液が凝固（ゲル化）する血液凝固作用と同一である。 As the gelation conditions in the composition for cell transplantation of the present invention, since plasma containing platelets and blood coagulation factors (fibrinogen etc.) is used, citric acid (2 % Solution) is limited to 1 to 2% of the collected blood amount, or LH / P MPs to be added is limited to 0.2 mg / ml or less, so that the dispersibility of the proliferated cells is moderately viscous. It is possible to obtain a composition that is limited and has excellent engraftment and proliferation properties. This gelation mechanism is the same as the blood coagulation action in which blood that bleeds coagulates (gelates) when the human body is injured.
以上説明したように、本発明の細胞移植用組成物は、適切なサイトカイン及びＬＨ／Ｐ ＭＰｓを添加した血漿（より好ましく自己血漿）からなるゲル状を成しているため、接着性或いは造血性浮遊性細胞に対して、安全性の面で問題のある異種動物の血清、フィーダー細胞、そしてコラーゲンやマトリゲル等のマトリックスとしての使用を排除し、対象として、骨髄由来間葉系幹細胞及び脂肪組織由来間葉系細胞及び血管内皮細胞及びその前駆体細胞、線維芽細胞及びその前駆体細胞、肝実質細胞及びその前駆体細胞、膵β細胞及びその前駆体細胞、平滑筋細胞及びその前駆体細胞、軟骨細胞及びその前駆体細胞等の接着性・体性細胞全般に有効である。さらに、ヒト骨髄由来ＣＤ３４陽性造血系前駆体細胞或いはＴＦ−１細胞等の造血系浮遊細胞に加えて、躋帯血や末梢血由来ＣＤ３４陽性造血系前駆体細胞、骨髄や末梢血中の単核球細胞の選択的増幅及び分化誘導に適切なサイトカインの添加を伴う適応が可能である。 As described above, since the composition for cell transplantation of the present invention is in the form of a gel composed of plasma (more preferably autologous plasma) added with appropriate cytokines and LH / P MPs, it has adhesiveness or hematopoietic properties. Eliminate the use of floating cells as sera, feeder cells, and matrices of collagen and matrigel, etc., from bone marrow-derived mesenchymal stem cells and adipose tissue Mesenchymal cells and vascular endothelial cells and precursor cells thereof, fibroblasts and precursor cells thereof, hepatocytes and precursor cells thereof, pancreatic β cells and precursor cells thereof, smooth muscle cells and precursor cells thereof, It is effective for all adherent and somatic cells such as chondrocytes and their precursor cells. Furthermore, in addition to hematopoietic floating cells such as human bone marrow-derived CD34-positive hematopoietic progenitor cells or TF-1 cells, mononuclear cells in cord blood and peripheral blood-derived CD34-positive hematopoietic progenitor cells, bone marrow and peripheral blood Adaptation with the addition of appropriate cytokines for selective amplification and differentiation induction of sphere cells is possible.
また、本発明の細胞増殖用組成物を用いた３次元細胞培養方法は、用途に応じた所望の細胞及びμｍ〜ｎｍオーダーの微粒子を血漿ゲルに包含させた軟ゲル状の複合体という形態的特徴を有するので、特に注射器やカテーテルなどを介して、容易に体内に注入する細胞運搬用組成物としても機能する。さらに、適度な粘稠性と徐放性を有するため、生体局所に注入した際に、組成物内で３次元的に増殖した状態で局所に組成物を留まらせつつ、サイトカインにより分化・増殖した細胞及び細胞が生成した有効成分を周辺部位に徐放させることができる。 In addition, the three-dimensional cell culture method using the composition for cell proliferation of the present invention has a morphological form called a soft gel complex in which desired cells and microparticles in the order of μm to nm are included in a plasma gel depending on the application. Since it has characteristics, it also functions as a cell-transporting composition that can be easily injected into the body, particularly via a syringe or catheter. Furthermore, since it has moderate viscosity and sustained release properties, when injected into a living body, it is differentiated and proliferated by cytokines while retaining the composition locally in a state of three-dimensional growth in the composition. The cell and the active ingredient produced by the cell can be gradually released to the peripheral site.
また、本発明の細胞増殖用組成物によって増幅させた各種細胞は、トリプシン／ＥＤＴＡ溶液による所定時間（１０分以内）溶解することで容易に回収することができる。 In addition, various cells amplified by the cell growth composition of the present invention can be easily recovered by lysis for a predetermined time (within 10 minutes) using a trypsin / EDTA solution.
ところで、上述した形態において、細胞移植用組成物に含有させる細胞は特に制限はないが、本培地・血漿ゲル包接担体に担持されやすく、体内において生着及び増殖が望まれる再生医療に有用な同種移植用細胞（より好ましくは自己細胞）とするのが好ましい。また、異種移植用細胞導入への適用も可能である。例えば、後述する実施例７で示すように、ヒト肝癌細胞（Ｈｕｈ７）を免疫不全マウス（ヌードマウス）に移植する際に、本細胞移植用組成物の使用は、腫癌細胞の生着性・成長性を大きく高めることが確認された。 By the way, in the above-mentioned form, the cell to be contained in the cell transplant composition is not particularly limited, but it is easily carried on the present medium / plasma gel inclusion carrier and is useful for regenerative medicine in which engraftment and proliferation are desired in the body. It is preferable to use allograft cells (more preferably autologous cells). Moreover, application to introduction of cells for xenotransplantation is also possible. For example, as shown in Example 7 to be described later, when transplanting human hepatoma cells (Huh7) into immunodeficient mice (nude mice), the use of the present cell transplantation composition is effective for engraftment of tumor cancer cells. It was confirmed that the growth potential was greatly improved.
また、本発明の細胞移植用組成物に添加されるサイトカインは、ＬＨ／Ｐ ＭＰｓの構成成分である低分子へパリン又はプロタミンとの親和性が高い薬物、例えばＦＧＦ−２等のへパリン結合性増殖因子又はサイトカインから選択される活性物質を含有させるのが好ましい。特に、ヒト血漿は患者自身から採血した血液から調製できるもので、そこに含まれる増殖因子やサイトカインは、殆どがヘパリン結合性であることが知られている。また、本発明の細胞移植用組成物において、使用する媒体としては水性媒体が好ましい。 In addition, the cytokine added to the composition for cell transplantation of the present invention is a drug having a high affinity for low molecular heparin or protamine, which is a constituent component of LH / P MPs, for example, heparin binding property such as FGF-2. It is preferable to contain an active substance selected from growth factors or cytokines. In particular, human plasma can be prepared from blood collected from a patient himself, and it is known that most of the growth factors and cytokines contained therein are heparin-binding. In the cell transplant composition of the present invention, an aqueous medium is preferable as a medium to be used.
さらに、上述した細胞移植用組成物を用いた３次元細胞培養方法は、ヒト骨髄由来間葉系幹細胞やヒト脂肪組織由来間葉系細胞を含む接着性細胞、ＴＦ−１やヒト骨髄由来ＣＤ３４造血系前駆体細胞を含む造血系浮遊細胞に限定されるものではなく、同種血漿、望ましくは自己血漿を用いることで、例えば躋帯血や末梢血由来ＣＤ３４陽性造血系前駆体細胞、マウス、ラット、兎等他種動物の骨髄由来間葉系幹細胞及び脂肪組織由来間葉系細胞及び血管内皮細胞及びその前駆体細胞、線維芽細胞及びその前駆体細胞、血管内皮細胞及びその前駆体細胞、肝実質細胞及びその前駆体細胞、肺β細胞及びその前駆体細胞、平滑筋細胞及びその前駆体細胞、軟骨細胞及びその前駆体細胞、筋芽細胞、骨芽細胞等の様々な接着細胞に適切な増殖因子やサイトカイン等の生理活性物質の添加を伴う適応が可能である。 Furthermore, the three-dimensional cell culture method using the above-described cell transplantation composition is a method of forming hematopoietic cells including human bone marrow-derived mesenchymal stem cells or human adipose tissue-derived mesenchymal cells, TF-1, or human bone marrow-derived CD34 hematopoiesis. It is not limited to hematopoietic suspension cells including systemic progenitor cells, but by using allogeneic plasma, desirably autologous plasma, for example, CD34 positive hematopoietic progenitor cells derived from cord blood or peripheral blood, mouse, rat, Bone marrow-derived mesenchymal stem cells and adipose tissue-derived mesenchymal stem cells and vascular endothelial cells and their precursor cells, fibroblasts and their precursor cells, vascular endothelial cells and their precursor cells, liver parenchyma Proliferation suitable for various adherent cells such as cells and their precursor cells, lung β cells and their precursor cells, smooth muscle cells and their precursor cells, chondrocytes and their precursor cells, myoblasts, osteoblasts, etc. Factors and It is possible adaptation with the addition of a physiologically active substance such as cytokine.
以下、本発明による実施例を示す。なお、本発明はこれらの実施例に記載された態様に限定されるわけではなく、前・後記の趣旨に照らし合わせて設計変更することは何れも本発明の技術的範囲に含まれるものである。 Examples according to the present invention will be described below. It should be noted that the present invention is not limited to the embodiments described in these examples, and any design changes in light of the gist of the preceding and following descriptions are included in the technical scope of the present invention. .
［低分子ヘパリン／プロタミン マイクロ粒子（ＬＰ／Ｐ ＭＰｓ）の作製］
低分子ヘパリンとして、市販のダルテパリンであるフラグミン注射液（商品名）（６．４ｍｇ／ｍｌ、１０００ＩＵ／ｍｌ、ヘパリンの数平均分子量は約５０００、キッセイ薬品工業株式会社）を使用した。この低分子ヘパリン溶液に対して、市販のプロタミン注射液（１０ｍｇ／ｍｌ：持田製薬株式会社) をボルテックスで撹拌しながら7 ：3 の容積比になるまで滴下し混合することで得た。
[Production of low molecular weight heparin / protamine microparticles (LP / P MPs)]
As low molecular weight heparin, a commercially available dalteparin fragmin injection solution (trade name) (6.4 mg / ml, 1000 IU / ml, the number average molecular weight of heparin is about 5000, Kissei Pharmaceutical Co., Ltd.) was used. A commercially available protamine injection solution (10 mg / ml: Mochida Pharmaceutical Co., Ltd.) was added dropwise to this low molecular weight heparin solution while stirring by vortexing until a volume ratio of 7: 3 was obtained.
［ヒト血漿の採取］
健常ボランティアから０．１〜８m1の２％クエン酸溶液の入った採血管に４０ｍｌの血液を採血し、１５分間、１７００ｒｐｍ（Table Top 冷却遠心機 2800,ローター：RS 240,クボタ）で遠心した。生じた３つの層、底部の赤（白）血球画分、赤（白）血球画分から上部（１ｃｍ）を多血小板血漿（Platelet Rich Plasma ：ＰＲＰ）画分、さらに上部を少血小板血漿（Platelet Poor Plasma ：ＰＰＰ）として採取した。ＰＲＰとＰＰＰを合わせたがヒト血漿である。なお、４０ｍｌのヒト血液から４〜５ｍｌのＰＲＰ、１５〜２０ｍｌのＰＰＰ、２０〜２４ｍｌのヒト血漿が得られた。ＰＲＰの血小板濃度は７９．６±１０．２×１０4 ／μｌ、ＰＰＰは３．６±０．６×１０4 ／μｌ、血漿は１８．２±３．６×１０4 ／μｌであった。
[Collecting human plasma]
40 ml of blood was collected from a healthy volunteer into a blood collection tube containing a 0.1% to 8 ml 1% 2% citric acid solution, and centrifuged at 1700 rpm (Table Top cooling centrifuge 2800, rotor: RS 240, Kubota) for 15 minutes. The resulting three layers, the red (white) blood cell fraction at the bottom, the upper (1 cm) fraction from the red (white) blood cell fraction, the platelet rich plasma (PRP) fraction, and the upper part, the platelet low plasma (Platelet Poor) Plasma: collected as PPP). Human plasma is a combination of PRP and PPP. In addition, 4 to 5 ml of PRP, 15 to 20 ml of PPP, and 20 to 24 ml of human plasma were obtained from 40 ml of human blood. The platelet concentration of PRP was 79.6 ± 10.2 × 10 4 / μl, PPP was 3.6 ± 0.6 × 10 4 / μl, and plasma was 18.2 ± 3.6 × 10 4 / μl. .
［実施例１］
作製したＬＨ／Ｐ ＭＰｓ（０．１ｍｇ／ｍｌ）と、サイトカインとしてＦＧＦ−２（５ｎｇ／ｍｌ）を含む無血清ＤＭＥＭ培地と、ヒト血漿（容積比４％）とを加えて作製したゲル状の細胞移植用組成物内を用いて、ヒト骨髄由来間葉系幹細胞及びヒト脂肪組織由来間葉系細胞に対する体外３次元培養増幅を実施した。
[Example 1]
A gel-like product prepared by adding the prepared LH / P MPs (0.1 mg / ml), serum-free DMEM medium containing FGF-2 (5 ng / ml) as a cytokine, and human plasma (volume ratio 4%). Using the inside of the composition for cell transplantation, in vitro three-dimensional culture amplification was performed on human bone marrow-derived mesenchymal stem cells and human adipose tissue-derived mesenchymal cells.
図１（ａ）に示すように、２４ウエル組織培養プレートに、約５０，０００のヒト骨髄由来間葉系幹細胞を作製した細胞移植用組成物内に加えて播種し、５日間体外３次元培養を行った（図中「■」）。比較対象として、ＦＧＦ−２（５ｎｇ／ｍｌ）のみを含むヒト血漿（容積比４％）ＤＭＥＭ（図中「□」）、ＬＨ／Ｐ ＭＰｓのみを含むヒト血漿（容積比４％）ＤＭＥＭ（図中「△」）、ＬＨ／Ｐ ＭＰｓ及びＦＧＦ−２の両者を含まないヒト血漿（容積比４％）ＤＭＥＭ（図中「●」）を３次元培養ネガティブコントロールとし、５日間の細胞増殖を比較した。
また、他の比較対象として、図１（ｂ）に示すように、ＬＨ／Ｐ ＭＰｓ及びＦＧＦ−２を含む無血清ＤＭＥＭ培地においてヒト血漿に代えてヒト血清（容積比４％）を加えたＤＭＥＭ（図中「■」）、ＦＧＦ−２（５ｎｇ／ｍｌ）のみを含むヒト血清（容積比４％）ＤＭＥＭ（図中「□」）、ＬＨ／Ｐ ＭＰｓのみを含むヒト血清（容積比４％）ＤＭＥＭ（図中「△」）、ＬＨ／Ｐ ＭＰｓ及びＦＧＦ−２の両者を含まないヒト血清（容積比１０％）ＤＭＥＭ（図中「○」）、ヒト血清（容積比４％）ＤＭＥＭ（図中「●」）を用いて、上記ヒト骨髄由来間葉系幹細胞を体外２次元培養し、５日間の細胞増殖を比較した。なお、各図中の縦軸は、細胞増幅（Cell growth ）を示す。
As shown in FIG. 1 (a), in a 24-well tissue culture plate, about 50,000 human bone marrow-derived mesenchymal stem cells were seeded in addition to the prepared cell transplantation composition, and cultured in vitro for three days. (“■” in the figure). As comparison targets, human plasma (volume ratio 4%) DMEM (4% in the figure) containing only FGF-2 (5 ng / ml), human plasma (volume ratio 4%) DMEM containing only LH / P MPs (Figure) Middle “△”), human plasma (volume ratio 4%) DMEM (“●” in the figure) that does not contain both LH / P MPs and FGF-2 was used as a three-dimensional culture negative control to compare cell growth for 5 days did.
As another comparison object, as shown in FIG. 1 (b), DMEM in which human serum (volume ratio 4%) was added instead of human plasma in a serum-free DMEM medium containing LH / P MPs and FGF-2. (“■” in the figure), human serum containing only FGF-2 (5 ng / ml) (volume ratio 4%) DMEM (“□” in the figure), human serum containing only LH / P MPs (volume ratio 4%) ) DMEM (“△” in the figure), human serum not containing both LH / P MPs and FGF-2 (volume ratio 10%) DMEM (“◯” in the figure), human serum (volume ratio 4%) DMEM ( The human bone marrow-derived mesenchymal stem cells were cultured in vitro two-dimensionally using “●” in the figure, and the cell proliferation for 5 days was compared. In addition, the vertical axis | shaft in each figure shows cell amplification (Cell growth).
図２（ａ）は、ヒト骨髄由来間葉系幹細胞について上記作製した細胞移植用組成物内での体外３次元培養の培養３日目の顕微鏡写真であり、図２（ｂ）はＬＨ／Ｐ ＭＰｓ、ＦＧＦ−２及びヒト血清（容積比４％）を加えた２次元培養の培養３日目の顕微鏡写真である。 FIG. 2 (a) is a photomicrograph of the third day of culture of in vitro three-dimensional culture in the above-prepared cell transplant composition for human bone marrow-derived mesenchymal stem cells, and FIG. 2 (b) shows LH / P It is the microscope picture of the culture | cultivation 3rd day of the two-dimensional culture which added MPs, FGF-2, and human serum (volume ratio 4%).
また、図３（ａ）に示すように、２４ウエル組織培養プレートに、約５０，０００のヒト脂肪組織由来間葉系細胞を作製した細胞移植用組成物内に加えて播種し、５日間体外３次元培養を行った（図中「■」）。比較対象として、ＦＧＦ−２（５ｎｇ／ｍｌ）のみを含むヒト血漿（容積比４％）ＤＭＥＭ（図中「□」）、ＬＨ／Ｐ ＭＰｓのみを含むヒト血漿（容積比４％）ＤＭＥＭ（図中「△」）、ＬＨ／Ｐ ＭＰｓ及びＦＧＦ−２の両者を含まないヒト血漿（容積比４％）ＤＭＥＭ（図中「●」）を３次元培養ネガティブコントロールとし、５日間の細胞増殖を比較した。
また、他の比較対象として、図３（ｂ）に示すように、ＬＨ／Ｐ ＭＰｓ及びＦＧＦ−２を含む無血清ＤＭＥＭ培地においてヒト血漿に代えてヒト血清（容積比４％）を加えたＤＭＥＭ（図中「■」）、ＦＧＦ−２（５ｎｇ／ｍｌ）のみを含むヒト血清（容積比４％）ＤＭＥＭ（図中「□」）、ＬＨ／Ｐ ＭＰｓのみを含むヒト血清（容積比４％）ＤＭＥＭ（図中「△」）、ＬＨ／Ｐ ＭＰｓ及びＦＧＦ−２の両者を含まないヒト血清（容積比１０％）ＤＭＥＭ（図中「○」）、ヒト血清（容積比４％）ＤＭＥＭ（図中「●」）を用いて、上記ヒト脂肪組織由来間葉系細胞を体外２次元培養し、５日間の細胞増殖を比較した。なお、各図中の縦軸は、細胞増幅（Cell growth ）を示す。
In addition, as shown in FIG. 3 (a), in a 24-well tissue culture plate, about 50,000 human adipose tissue-derived mesenchymal cells were seeded in addition to the prepared cell transplantation composition and in vitro for 5 days. Three-dimensional culture was performed (“■” in the figure). As comparison targets, human plasma (volume ratio 4%) DMEM (4% in the figure) containing only FGF-2 (5 ng / ml), human plasma (volume ratio 4%) DMEM containing only LH / P MPs (Figure) Middle “△”), human plasma (volume ratio 4%) DMEM (“●” in the figure) that does not contain both LH / P MPs and FGF-2 was used as a three-dimensional culture negative control to compare cell growth for 5 days did.
As another comparison object, as shown in FIG. 3 (b), DMEM in which human serum (volume ratio 4%) was added instead of human plasma in a serum-free DMEM medium containing LH / P MPs and FGF-2. (“■” in the figure), human serum containing only FGF-2 (5 ng / ml) (volume ratio 4%) DMEM (“□” in the figure), human serum containing only LH / P MPs (volume ratio 4%) ) DMEM (“△” in the figure), human serum not containing both LH / P MPs and FGF-2 (volume ratio 10%) DMEM (“◯” in the figure), human serum (volume ratio 4%) DMEM ( Using the “●” in the figure, the human adipose tissue-derived mesenchymal cells were cultured in vitro two-dimensionally, and the cell growth for 5 days was compared. In addition, the vertical axis | shaft in each figure shows cell amplification (Cell growth).
図４（ａ）は、ヒト脂肪組織由来間葉系細胞について上記作製した細胞移植用組成物内での体外３次元培養の培養３日目の顕微鏡写真であり、図４（ｂ）はＬＨ／Ｐ ＭＰｓ、ＦＧＦ−２及びヒト血清（容積比４％）を加えた２次元培養の培養３日目の顕微鏡写真である。 FIG. 4 (a) is a photomicrograph of the third day of culture of in vitro three-dimensional culture in the cell transplant composition prepared above for human adipose tissue-derived mesenchymal cells, and FIG. 4 (b) shows LH / It is the microscope picture of the culture | cultivation 3rd day of the two-dimensional culture which added PMPs, FGF-2, and human serum (volume ratio 4%).
なお、本実施例では、細胞培養後の細胞移植用組成物に対し、トリプシン・ＥＤＴＡ溶液で１０分以内に溶解してその中の増殖細胞を分雛し、血球計測器を用いて細胞数を計測した。 In this example, the cell transplanted composition after cell culture was dissolved in a trypsin / EDTA solution within 10 minutes, and the proliferating cells therein were classified, and the number of cells was determined using a hemocytometer. Measured.
図１〜４に示すように、比較結果として、両細胞ともＬＨ／Ｐ ＭＰｓ及びＦＧＦ−２を含む無血清ＤＭＥＭ培地にヒト血漿（容積比４％）を加えた本発明の細胞移植用組成物内において最大の細胞増殖を示した。 As shown in FIGS. 1 to 4, as a comparison result, the cell transplantation composition of the present invention in which human plasma (volume ratio 4%) is added to a serum-free DMEM medium containing LH / P MPs and FGF-2 for both cells. Showed maximum cell proliferation.
［実施例２］
作製したＬＨ／Ｐ ＭＰｓ（０．１ｍｇ／ｍｌ）と、ＦＧＦ−２（５ｎｇ／ｍｌ）を含む無血清ＤＭＥＭと、ヒト血漿（容積比４％）とを加えて作製した本発明に係る細胞移植用組成物内で、体外３次元培養増幅させたヒト骨髄由来間葉系幹細胞及びヒト脂肪組織由来間葉系細胞の分化能を確認するため、脂肪細胞への分化誘導を行った。脂肪細胞への分化誘導には市販培地（Lonza Walkersville, Inc., MD）を使用し、両細胞を脂肪細胞誘導培地で４日間、続いて維持培地で３日間培養するサイクルを３回実施した後、両細胞を維持培地でさらに７日間培養した。また、脂肪滴の確認のため、 Oil Red O染色（和光純薬工業（株））を施した。
[Example 2]
Cell transplantation according to the present invention prepared by adding the prepared LH / P MPs (0.1 mg / ml), serum-free DMEM containing FGF-2 (5 ng / ml), and human plasma (volume ratio 4%) In order to confirm the differentiation potential of human bone marrow-derived mesenchymal stem cells and human adipose tissue-derived mesenchymal cells that have been amplified in vitro by three-dimensional culture in the composition for use, differentiation induction into adipocytes was performed. A commercial medium (Lonza Walkersville, Inc., MD) was used to induce differentiation into adipocytes, and after three cycles of culturing both cells in an adipocyte induction medium for 3 days followed by a maintenance medium for 3 days Both cells were further cultured in maintenance medium for 7 days. Moreover, Oil Red O dyeing | staining (Wako Pure Chemical Industries Ltd.) was performed for confirmation of a lipid droplet.
図５（ａ）は、３次元培養増幅したヒト骨髄由来間葉系幹細胞の脂肪細胞への分化誘導の試験結果であり、４８ウエルプレートの最初のＰ列は、購入細胞の分化誘導、２番目のＮ列は分化誘導培地を用いず、維持培地のみで培養したもの、３、４番目の−列は、体外２次元培養で増幅した細胞の分化能、５、６番目の＋列は体外３次元培養で増幅した細胞を示している。また、図５（ｂ）は、３次元培養増幅したヒト脂肪組織由来間葉系細胞の脂肪細胞への分化誘導の試験結果であり、４８ウエルプレートの最初のＰ列は、購入細胞の分化誘導、２番目のＮ列は分化誘導培地を用いず、維持培地のみで培養したもの、３、４番目の−列は、体外２次元培養で増幅した細胞の分化能、５、６番目の＋列は体外３次元培養で増幅した細胞を示している。 FIG. 5 (a) is a test result of differentiation induction of human bone marrow-derived mesenchymal stem cells amplified in three-dimensional culture into adipocytes. The first P row of the 48-well plate shows the differentiation induction of the purchased cells, the second The N column of the cells was cultured in the maintenance medium alone without using the differentiation-inducing medium, the third and fourth columns were the differentiation potential of the cells amplified in vitro two-dimensional culture, and the fifth and sixth columns were in vitro 3 Shows cells amplified in dimensional culture. FIG. 5 (b) is a test result of differentiation induction of human adipose tissue-derived mesenchymal cells amplified in three-dimensional culture into adipocytes. The first P row of the 48-well plate shows differentiation induction of purchased cells. The second row N was cultured in the maintenance medium alone without using the differentiation-inducing medium, the third and fourth rows were the differentiation potential of the cells amplified by in vitro two-dimensional culture, and the fifth and sixth rows. Indicates cells amplified by in vitro three-dimensional culture.
培養したヒト骨髄由来間葉系幹細胞及びヒト脂肪組織由来間葉系細胞は、何れも脂肪細胞に良好に分化誘導する分化能を保持していることが確認された。 It was confirmed that the cultured human bone marrow-derived mesenchymal stem cells and human adipose tissue-derived mesenchymal cells all retain the differentiation ability to induce differentiation into adipocytes.
［実施例３］
作製したＬＨ／Ｐ ＭＰｓ（０．１ｍｇ／ｍｌ）と、ＦＧＦ−２（５ｎｇ／ｍｌ）を含む無血清ＤＭＥＭと、ヒト血漿（容積比４％）とを加えて作製した本発明に係る細胞移植用組成物内で、体外３次元培養増幅させたヒト骨髄由来間葉系幹細胞及びヒト脂肪組織由来間葉系細胞の分化能を確認するため、骨芽細胞への分化誘導を行った。骨芽細胞への分化誘導には市販培地（Lonza Walkersville, Inc., MD）を使用し、両細胞を脂肪細胞誘導培地で４日間、続いて維持培地で３日間培養するサイクルを３回実施した後、両細胞を維持培地でさらに７日間培養した。また、骨芽細胞の誘導では、骨芽細胞のアルカリフォスファターゼ活性を調べるため、ＡＬＰ染色を実施した。
[Example 3]
Cell transplantation according to the present invention prepared by adding the prepared LH / P MPs (0.1 mg / ml), serum-free DMEM containing FGF-2 (5 ng / ml), and human plasma (volume ratio 4%) In order to confirm the differentiation potential of human bone marrow-derived mesenchymal stem cells and human adipose tissue-derived mesenchymal cells that were amplified in vitro in three-dimensional culture in the composition for use, differentiation induction into osteoblasts was performed. A commercially available medium (Lonza Walkersville, Inc., MD) was used for induction of differentiation into osteoblasts, and both cells were cultured for 3 days in an adipocyte induction medium, followed by 3 days in a maintenance medium for 3 cycles. Thereafter, both cells were further cultured in maintenance medium for 7 days. In the induction of osteoblasts, ALP staining was performed to examine the alkaline phosphatase activity of osteoblasts.
図６（ａ）は、３次元培養増幅したヒト骨髄由来間葉系幹細胞の骨芽細胞への分化誘導の試験結果であり、４８ウエルプレートの最初のＰ列は、購入細胞の分化誘導、２番目のＮ列は分化誘導培地を用いず、維持培地のみで培養したもの、３、４番目の−列は、体外２次元培養で増幅した細胞の分化能、５、６番目の＋列は体外３次元培養で増幅した細胞を示している。また、図５（ｂ）は、３次元培養増幅したヒト脂肪組織由来間葉系細胞の骨芽細胞への分化誘導の試験結果であり、４８ウエルプレートの最初のＰ列は、購入細胞の分化誘導、２番目のＮ列は分化誘導培地を用いず、維持培地のみで培養したもの、３、４番目の−列は、体外２次元培養で増幅した細胞の分化能、５、６番目の＋列は体外３次元培養で増幅した細胞を示している。 FIG. 6 (a) is a test result of differentiation induction of human bone marrow-derived mesenchymal stem cells amplified in three-dimensional culture into osteoblasts. The first P row of the 48-well plate shows differentiation induction of purchased cells, 2 The Nth row was cultured in the maintenance medium alone without using the differentiation induction medium, the 3rd and 4th rows were the differentiation potential of the cells amplified by in vitro two-dimensional culture, and the 5th and 6th rows were in vitro. Cells amplified in 3D culture are shown. FIG. 5 (b) is a test result of differentiation induction of human adipose tissue-derived mesenchymal cells amplified in three-dimensional culture into osteoblasts. The first P row of the 48-well plate shows the differentiation of purchased cells. Induction, the second row N was cultured in the maintenance medium alone without using the differentiation induction medium, and the fourth and fourth rows were the differentiation potential of the cells amplified by in vitro two-dimensional culture, and the fifth, sixth + Columns indicate cells amplified in in vitro 3D culture.
培養したヒト骨髄由来間葉系幹細胞及びヒト脂肪組織由来間葉系細胞は、何れも骨芽細胞に良好に分化誘導する分化能を保持していることが確認された。 It was confirmed that both the cultured human bone marrow-derived mesenchymal stem cells and human adipose tissue-derived mesenchymal cells retain the differentiation ability to induce differentiation into osteoblasts.
加えて、培地・血漿ゲル内でのヒト皮膚線維芽細胞３次元培養は、５日目以降顕著な血漿ゲル収縮とともに肉芽組織様形態を示した。また、ヒト皮膚毛細血管内皮細胞３次元培養は、２４時間以内に毛細血管様組織形成が観察された。 In addition, three-dimensional culture of human dermal fibroblasts in medium / plasma gel showed granulation tissue-like morphology with significant plasma gel contraction after day 5. In the three-dimensional culture of human skin capillary endothelial cells, capillary-like tissue formation was observed within 24 hours.
［実施例４］
作製したＬＨ／Ｐ ＭＰｓ（０．１ｍｇ／ｍｌ）と、サイトカインとしてＦＧＦ−２（５ｎｇ／ｍｌ）を含む無血清ＤＭＥＭ培地と、ヒト血漿（容積比４％）とを加えて作製したゲル状の細胞移植用組成物内を用いて、ヒト皮膚線維芽細胞及びヒト皮膚毛細血管内皮細胞に対する体外３次元培養増幅を実施した。
[Example 4]
A gel-like product prepared by adding the prepared LH / P MPs (0.1 mg / ml), serum-free DMEM medium containing FGF-2 (5 ng / ml) as a cytokine, and human plasma (volume ratio 4%). Using the inside of the composition for cell transplantation, in vitro three-dimensional culture amplification for human skin fibroblasts and human skin capillary endothelial cells was performed.
図７（ａ）に示すように、２４ウエル組織培養プレートに、約５０，０００のヒト皮膚線維芽細胞を作製した細胞移植用組成物内に加えて播種し、５日間体外３次元培養を行った（図中「■」）。比較対象として、比較対象として、ＦＧＦ−２（５ｎｇ／ｍｌ）のみを含むヒト血漿（容積比４％）ＤＭＥＭ（図中「□」）、ＬＨ／Ｐ ＭＰｓのみを含むヒト血漿（容積比４％）ＤＭＥＭ（図中「△」）、ＬＨ／Ｐ ＭＰｓ及びＦＧＦ−２の両者を含まないヒト血漿（容積比４％）ＤＭＥＭ（図中「●」）を３次元培養ネガティブコントロールとし、５日間の細胞増殖を比較した。
また、他の比較対象として、図７（ｂ）に示すように、ＬＨ／Ｐ ＭＰｓ及びＦＧＦ−２を含む無血清ＤＭＥＭ培地においてヒト血漿に代えてヒト血清（容積比４％）を加えたＤＭＥＭ（図中「■」）、ＦＧＦ−２（５ｎｇ／ｍｌ）のみを含むヒト血清（容積比４％）ＤＭＥＭ（図中「□」）、ＬＨ／Ｐ ＭＰｓのみを含むヒト血清（容積比４％）ＤＭＥＭ（図中「△」）、ＬＨ／Ｐ ＭＰｓ及びＦＧＦ−２の両者を含まないヒト血清（容積比１０％）ＤＭＥＭ（図中「○」）、ヒト血清（容積比４％）ＤＭＥＭ（図中「●」）を用いて、上記ヒト皮膚線維芽細胞を体外２次元培養し、５日間の細胞増殖を比較した。なお、各図中の縦軸は、細胞増幅（Cell growth ）を示す。
As shown in FIG. 7 (a), in a 24-well tissue culture plate, about 50,000 human dermal fibroblasts were seeded in addition to the prepared cell transplantation composition and subjected to in vitro three-dimensional culture for 5 days. ("■" in the figure). For comparison, human plasma containing only FGF-2 (5 ng / ml) (volume ratio 4%) DMEM (“□” in the figure), human plasma containing only LH / P MPs (volume ratio 4%) ) DMEM (“Δ” in the figure), human plasma not containing both LH / P MPs and FGF-2 (volume ratio 4%) DMEM (“●” in the figure) as a three-dimensional culture negative control for 5 days Cell growth was compared.
As another comparison object, as shown in FIG. 7 (b), DMEM in which human serum (volume ratio 4%) was added instead of human plasma in a serum-free DMEM medium containing LH / P MPs and FGF-2. (“■” in the figure), human serum containing only FGF-2 (5 ng / ml) (volume ratio 4%) DMEM (“□” in the figure), human serum containing only LH / P MPs (volume ratio 4%) ) DMEM (“△” in the figure), human serum not containing both LH / P MPs and FGF-2 (volume ratio 10%) DMEM (“◯” in the figure), human serum (volume ratio 4%) DMEM ( The human skin fibroblasts were cultured in vitro two-dimensionally using “●” in the figure, and the cell growth for 5 days was compared. In addition, the vertical axis | shaft in each figure shows cell amplification (Cell growth).
図８（ａ）は、ヒト皮膚線維芽細胞について上記作製した細胞移植用組成物内での体外３次元培養の培養３日目の顕微鏡写真であり、図８（ｂ）はＬＨ／Ｐ ＭＰｓ、ＦＧＦ−２及びヒト血清（容積比４％）を加えた２次元培養の培養３日目の顕微鏡写真である。 FIG. 8 (a) is a photomicrograph of the third day of culture of in vitro three-dimensional culture in the above-prepared cell transplant composition for human skin fibroblasts, and FIG. 8 (b) shows LH / P MPs, It is the microscope picture of the culture | cultivation 3rd day of the two-dimensional culture which added FGF-2 and human serum (volume ratio 4%).
図９（ａ）に示すように、２４ウエル組織培養プレートに、約５０，０００のヒト皮膚毛細血管内皮細胞を作製した細胞移植用組成物内に加えて播種し、５日間体外３次元培養を行った（図中「■」）。比較対象として、比較対象として、ＦＧＦ−２（５ｎｇ／ｍｌ）のみを含むヒト血漿（容積比４％）ＤＭＥＭ（図中「□」）、ＬＨ／Ｐ ＭＰｓのみを含むヒト血漿（容積比４％）ＤＭＥＭ（図中「△」）、ＬＨ／Ｐ ＭＰｓ及びＦＧＦ−２の両者を含まないヒト血漿（容積比４％）ＤＭＥＭ（図中「●」）を３次元培養ネガティブコントロールとし、５日間の細胞増殖を比較した。
また、他の比較対象として、図９（ｂ）に示すように、ＬＨ／Ｐ ＭＰｓ及びＦＧＦ−２を含む無血清ＤＭＥＭ培地においてヒト血漿に代えてヒト血清（容積比４％）を加えたＤＭＥＭ（図中「■」）、ＦＧＦ−２（５ｎｇ／ｍｌ）のみを含むヒト血清（容積比４％）ＤＭＥＭ（図中「□」）、ＬＨ／Ｐ ＭＰｓのみを含むヒト血清（容積比４％）ＤＭＥＭ（図中「△」）、ＬＨ／Ｐ ＭＰｓ及びＦＧＦ−２の両者を含まないヒト血清（容積比１０％）ＤＭＥＭ（図中「○」）、ヒト血清（容積比４％）ＤＭＥＭ（図中「●」）を用いて、上記ヒト皮膚毛細血管内皮細胞を体外２次元培養し、５日間の細胞増殖を比較した。なお、各図中の縦軸は、細胞増幅（Cell growth ）を示す。
As shown in FIG. 9 (a), in a 24-well tissue culture plate, about 50,000 human dermal capillary endothelial cells were seeded in addition to the prepared cell transplant composition, and in vitro three-dimensional culture was performed for 5 days. I went ("■" in the figure). For comparison, human plasma containing only FGF-2 (5 ng / ml) (volume ratio 4%) DMEM (“□” in the figure), human plasma containing only LH / P MPs (volume ratio 4%) ) DMEM (“Δ” in the figure), human plasma not containing both LH / P MPs and FGF-2 (volume ratio 4%) DMEM (“●” in the figure) as a three-dimensional culture negative control for 5 days Cell growth was compared.
As another comparison object, as shown in FIG. 9 (b), DMEM in which human serum (volume ratio 4%) was added instead of human plasma in a serum-free DMEM medium containing LH / P MPs and FGF-2. (“■” in the figure), human serum containing only FGF-2 (5 ng / ml) (volume ratio 4%) DMEM (“□” in the figure), human serum containing only LH / P MPs (volume ratio 4%) ) DMEM (“△” in the figure), human serum not containing both LH / P MPs and FGF-2 (volume ratio 10%) DMEM (“◯” in the figure), human serum (volume ratio 4%) DMEM ( Using the “●” in the figure, the human skin capillary endothelial cells were cultured in vitro two-dimensionally, and the cell growth for 5 days was compared. In addition, the vertical axis | shaft in each figure shows cell amplification (Cell growth).
図１０（ａ）は、ヒト皮膚毛細血管内皮細胞について上記作製した細胞移植用組成物内での体外３次元培養の培養３日目の顕微鏡写真であり、図４（ｂ）はＬＨ／Ｐ ＭＰｓ、ＦＧＦ−２及びヒト血清（容積比４％）を加えた２次元培養の培養３日目の顕微鏡写真である。 FIG. 10 (a) is a photomicrograph of the third day of culture of in vitro three-dimensional culture in the above-prepared cell transplant composition for human skin capillary endothelial cells, and FIG. 4 (b) shows LH / P MPs. FIG. 3 is a photomicrograph of the third day of culture of a two-dimensional culture in which FGF-2 and human serum (volume ratio 4%) are added.
なお、本実施例では、細胞培養後の細胞移植用組成物に対し、トリプシン・ＥＤＴＡ溶液で１０分以内に溶解してその中の増殖細胞を分雛し、血球計測器を用いて細胞数を計測した。 In this example, the cell transplanted composition after cell culture was dissolved in a trypsin / EDTA solution within 10 minutes, and the proliferating cells therein were classified, and the number of cells was determined using a hemocytometer. Measured.
図７〜１０に示すように、比較結果として、両細胞ともＬＨ／Ｐ ＭＰｓ及びＦＧＦ−２を含む無血清ＤＭＥＭ培地にヒト血漿（容積比４％）を加えた本発明の細胞移植用組成物内において最大の細胞増殖を示した。 As shown in FIGS. 7 to 10, as a comparison result, the composition for cell transplantation of the present invention in which human plasma (volume ratio 4%) is added to a serum-free DMEM medium containing LH / P MPs and FGF-2 for both cells. Showed maximum cell proliferation.
［実施例５］
作製したＬＨ／Ｐ ＭＰｓ（０．１ｍｇ／ｍｌ）と、１／４濃度のＳＣＦ（５ｎｇ／ｍｌ）・ＴＰＯ（１０ｎｇ／ｍｌ）・Ｆｌｔ−３ｌｉｇａｎｄ（１０ｎｇ／ｍｌ）の造血細胞増幅用サイトカインを含む無血清ＨＰＧＭと、ヒト血漿（容積比４％）とを加えて作製したゲル状の細胞移植用組成物内を用いて、ヒト骨髄由来ＣＤ３４陽性造血系前駆体細胞の３次元培養を実施した。
[Example 5]
Includes prepared LH / P MPs (0.1 mg / ml) and 1/4 concentration of SCF (5 ng / ml), TPO (10 ng / ml) and Flt-3 ligand (10 ng / ml) for hematopoietic cell amplification Three-dimensional culture of human bone marrow-derived CD34-positive hematopoietic progenitor cells was carried out using a gel-like cell transplant composition prepared by adding serum-free HPGM and human plasma (volume ratio 4%).
図１１に示すように、上記細胞移植用組成物を用いてヒト骨髄由来ＣＤ３４陽性造血系前駆体細胞について１０日間体外３次元培養を行った（図中「◆」）。比較対象として、ヒト血漿（ＨＰ：２％）を含む無血清ＨＰＧＭからなる培地・血漿ゲル（図中「■」) 、ＬＨ／Ｐ ＭＰｓ（０．１ｍｇ／ｍｌ）及びＨＰを含む無血清ＨＰＧＭからなる培地・血漿ゲル（図中「△」）、ＳＣＦ（１／４Ｘ；５ｎｇ／ｍｌ）・ＴＰＯ（１／４Ｘ；１０ｎｇ／ｍｌ）・Ｆｌｔ−３ ｌｉｇａｎｄ（１／４Ｘ；１０ｎｇ／ｍｌ）とヒト血漿を含む無血清ＨＰＧＭからなる培地・血漿ゲル内（図中「▲」）を３次元培養ネガティブコントロールとし、１０日間の細胞増殖を比較した。
また、他の比較対象として、ＳＣＦ（１Ｘ；２０ｎｇ／ｍｌ）・ＴＰＯ（１Ｘ；４０ｎｇ／ｍｌ・Ｆｌｔ−３ ｌｉｇａｎｄ（１Ｘ；４０ｎｇ／ｍｌ）のみを含んだＨＰＧＭ（図中「○」）、ＳＦＣ（１／４Ｘ）・ＴＰＯ（１／４Ｘ）・Ｆｌｔ−３ ｌｉｇａｎｄ（１／４Ｘ）のみを含んだＨＰＧＭ（図中「●」）、ＬＨ／Ｐ ＭＰｓと、ＳＦＣ（１／４Ｘ）・ＴＰＯ（１／４Ｘ）・Ｆｌｔ−３ ｌｉｇａｎｄ（１／４Ｘ）のみを含んだＨＰＧＭ（図中「□」）を体外２次元培養し、１０日間の細胞増殖を比較した。なお、各図中の縦軸は、細胞増幅（Cell growth ）を示す。
As shown in FIG. 11, human bone marrow-derived CD34-positive hematopoietic progenitor cells were subjected to in vitro three-dimensional culture for 10 days using the composition for cell transplantation (“♦” in the figure). As a comparison object, from a serum-free HPGM containing medium / plasma gel ("■" in the figure), serum-free HPGM containing human plasma (HP: 2%), LH / P MPs (0.1 mg / ml) and HP Medium, plasma gel (“Δ” in the figure), SCF (1 / 4X; 5 ng / ml), TPO (1 / 4X; 10 ng / ml), Flt-3 ligand (1 / 4X; 10 ng / ml) and human A medium / plasma gel consisting of serum-free HPGM containing plasma (“▲” in the figure) was used as a three-dimensional culture negative control, and cell growth for 10 days was compared.
Further, as other comparison targets, HPGM containing only SCF (1X; 20 ng / ml), TPO (1X; 40 ng / ml, Flt-3 ligand (1X; 40 ng / ml) (“◯” in the figure), SFC HPGM (“●” in the figure) including only (1 / 4X) · TPO (1 / 4X) · Flt-3 ligand (1 / 4X), LH / P MPs, and SFC (1 / 4X) · TPO ( HPGM (“□” in the figure) containing only ¼X) · Flt-3 ligand (1 / 4X) was cultured in vitro two-dimensionally and compared for 10 days of cell proliferation. Indicates cell growth.
また、図１２に示すように、上記細部移植用組成物内で増殖中のヒト骨髄由来ＣＤ３４陽性造血系前駆体細胞における０、４、１０日目にＣＤ３４陽性率をＰＥラベルヒート抗ＣＤ３４（BD Biosciences Pharmigen, USA ）を用いてEPICSRXL（Beckman Coulter, USA）によりフローサイトメトリーの計測結果である。図中「黒」は、ＬＨ／Ｐ ＭＰｓ（０．１ｍｇ／ｍｌ）とＳＦＣ（１／４Ｘ；５ｎｇ／ｍｌ）・ＴＰＯ（１／４Ｘ；１０ｎｇ／ｍｌ）・Ｆｌｔ−３ ｌｉｇａｎｄ（１／４Ｘ；１０ｎｇ／ｍｌ）を含む無血清ＨＰＧＭと、ヒト血漿（容積比３％）からなる細胞移植用組成物の３次元培養の結果であり、図中「灰色」は、ＬＨ／Ｐ ＭＰｓ（０．１ｍｇ／ｍｌ）とＳＦＣ（１／４Ｘ；５ｎｇ／ｍｌ）・ＴＰＯ（１／４Ｘ；１０ｎｇ／ｍｌ）・Ｆｌｔ−３ ｌｉｇａｎｄ（１／４Ｘ；１０ｎｇ／ｍｌ）を含む無血清ＨＰＧＭと、ヒト血清（容積比３％）からなる培地・血漿ゲルの２次元培養の結果であり、図中「白」は、コントロールとしてＳＦＣ（１／４Ｘ；５ｎｇ／ｍｌ）・ＴＰＯ（１／４Ｘ；１０ｎｇ／ｍｌ）・Ｆｌｔ−３ ｌｉｇａｎｄ（１／４Ｘ；１０ｎｇ／ｍｌ）のみを含む無血清ＨＰＧＭの体外２次元培養の結果である。 In addition, as shown in FIG. 12, the CD34 positive rate on the 0th, 4th, and 10th days of human bone marrow-derived CD34 positive hematopoietic progenitor cells proliferating in the above-described composition for detailed transplantation was expressed by PE label heat anti-CD34 (BD Biocytos Pharmigen, USA) and flow cytometry measurement results using EPICSRXL (Beckman Coulter, USA). In the figure, “black” indicates LH / P MPs (0.1 mg / ml), SFC (1 / 4X; 5 ng / ml), TPO (1 / 4X; 10 ng / ml), Flt-3 ligand (1 / 4X; 10 ng / ml) is a result of three-dimensional culture of a composition for cell transplantation consisting of serum-free HPGM containing 10 ng / ml) and human plasma (volume ratio 3%). In the figure, “grey” indicates LH / P MPs (0.1 mg Serum-free HPGM containing SFC (1 / 4X; 5 ng / ml), TPO (1 / 4X; 10 ng / ml), Flt-3 ligand (1 / 4X; 10 ng / ml), and human serum (volume) 3%) is a result of two-dimensional culture of a medium / plasma gel. In the figure, “white” indicates SFC (1 / 4X; 5 ng / ml), TPO (1 / 4X; 10 ng / ml) as a control, Flt-3 ligand (1 / 4X; 10 ng / ml) is a result of in vitro two-dimensional culture of serum-free HPGM.
図１３（ａ）はヒト骨髄由来ＣＤ３４陽性造血系前駆体細胞について上記細胞移植用組成物内での体外３次元培養の培養６日目の電子顕微鏡写真であり、図１３（ｂ）はヒト骨髄由来ＣＤ３４陽性造血系前駆体細胞についてヒト血清（容積比４％）を含む培地による２次元培養の培養６日目の電子顕微鏡写真である。 FIG. 13 (a) is an electron micrograph of 6th day culture of human bone marrow-derived CD34 positive hematopoietic progenitor cells cultured in vitro in the cell transplant composition, and FIG. 13 (b) is human bone marrow. It is the electron micrograph of the culture | cultivation 6th day of the two-dimensional culture | cultivation by the culture medium which contains human serum (volume ratio 4%) about origin CD34 positive hematopoietic progenitor cell.
図１１〜１３に示すように、本発明に係る細胞移植用組成物を用いた３次元細胞培養方法によるヒト骨髄由来ＣＤ３４陽性造血系前駆体細胞の培養では、ヒト骨髄由来ＣＤ３４陽性造血系前駆体細胞のコロニー形成を促進して最適な３次元増幅が行え、ヒト血漿に代えて等量の血清を添加した同じ培地を用いた２次元培養よりも有意に高い増殖率を得ることができた。また、３次元細胞培養方法で増幅させたヒト骨髄由来ＣＤ３４陽性造血系前駆体細胞は、同じ培地に等量の同種血清を添加した２次元培養よりも有意に高いＣＤ３４陽性率を維持した。 As shown in FIGS. 11 to 13, human bone marrow-derived CD34 positive hematopoietic progenitor cells are cultured by the three-dimensional cell culture method using the cell transplant composition according to the present invention. Cell colony formation was promoted to achieve optimal three-dimensional amplification, and a significantly higher growth rate was obtained than two-dimensional culture using the same medium supplemented with an equal amount of serum instead of human plasma. In addition, human bone marrow-derived CD34 positive hematopoietic progenitor cells amplified by the three-dimensional cell culture method maintained a significantly higher CD34 positive rate than two-dimensional culture in which an equal amount of allogeneic serum was added to the same medium.
［実施例６］
作製したＬＨ／Ｐ ＭＰｓ（０．１ｍｇ／ｍｌ）に、サイトカインとしてＩＬ−３（５ｎｇ／ｍｌ）・ＧＭ−ＣＳＦ（５ｎｇ／ｍｌ）を含む無血清ＤＭＥＭにヒト血漿（容積比４％）を加えて作製したゲル状の細胞移植用組成物内を用いて、ヒトＴＦ−１細胞の３次元培養を実施した。
[Example 6]
Human plasma (volume ratio 4%) was added to serum-free DMEM containing IL-3 (5 ng / ml) and GM-CSF (5 ng / ml) as cytokines to the prepared LH / P MPs (0.1 mg / ml). Using the gel-like composition for cell transplantation prepared as described above, three-dimensional culture of human TF-1 cells was performed.
図１４に示すように、上記細胞移植用組成物を用いてヒトＴＦ−１細胞について１０日間３次元培養を行った（図中「◆」）。比較対象として、ヒト血漿（ＨＰ：２％）を含む無血清ＤＭＥＭからなる培地・血漿ゲル（図中「■」) 、ＬＨ／Ｐ ＭＰｓ（０．１ｍｇ／ｍｌ）及びＨＰを含む無血清ＤＭＥＭからなる培地・血漿ゲル（図中「△」）、ＩＬ−３（５ｎｇ／ｍｌ）・ＧＭ−ＣＳＦ（５ｎｇ／ｍｌ）とヒト血漿を含む無血清ＤＭＥＭからなる培地・血漿ゲル内（図中「▲」）を３次元培養ネガティブコントロールとし、３日間の細胞増殖を比較した。
また、他の比較対象として、１０％ヒト血清（ＨＳ）のみを含んだＤＭＥＭ（図中「○」）、ＩＬ−３（５ｎｇ／ｍｌ）・ＧＭ−ＣＳＦ（５ｎｇ／ｍｌ）のみを含んだ無血清ＤＭＥＭ（図中「●」）、１０％ＨＳ及びＩＬ−３（５ｎｇ／ｍｌ）・ＧＭ−ＣＳＦ（５ｎｇ／ｍｌ）のみを含んだＤＭＥＭ（図中「□」）を体外２次元培養し、３日間の細胞増殖を比較した。なお、各図中の縦軸は、細胞増幅（Cell growth ）を示す。
As shown in FIG. 14, human TF-1 cells were three-dimensionally cultured for 10 days using the cell transplant composition (“♦” in the figure). As a comparison object, from a serum-free DMEM containing serum-free DMEM containing human plasma (HP: 2%), serum-free DMEM containing LH / P MPs (0.1 mg / ml) and HP Medium / plasma gel (“Δ” in the figure), IL-3 (5 ng / ml) / GM-CSF (5 ng / ml) and serum-free DMEM containing human plasma in the medium / plasma gel (“▲” in the figure) )) Was used as a three-dimensional culture negative control, and the cell growth for 3 days was compared.
In addition, as other comparison targets, DMEM containing only 10% human serum (HS) (“◯” in the figure), IL-3 (5 ng / ml), GM-CSF (5 ng / ml) alone were not included. Serum DMEM (“●” in the figure), DMEM (“□” in the figure) containing only 10% HS and IL-3 (5 ng / ml) and GM-CSF (5 ng / ml) were cultured in vitro two-dimensionally, Three day cell growth was compared. In addition, the vertical axis | shaft in each figure shows cell amplification (Cell growth).
図１５（ａ）はヒトＴＦ−１細胞について上記細胞移植用組成物内での体外３次元培養の培養２日目の電子顕微鏡写真であり、図１５（ｂ）はヒトＴＦ−１細胞についてヒト血清（容積比４％）を含む培地による２次元培養の培養２日目の電子顕微鏡写真である。 FIG. 15 (a) is an electron micrograph of the second day of culture of in vitro three-dimensional culture of the human TF-1 cells in the cell transplant composition, and FIG. 15 (b) is a human figure of human TF-1 cells. It is an electron micrograph of the culture | cultivation 2nd day of the two-dimensional culture | cultivation by the culture medium containing serum (volume ratio 4%).
図１４、１５に示すように、本発明に係る細胞移植用組成物を用いた３次元細胞培養方法によるヒトＴＦ−１細胞の培養では、ヒトＴＦ−１細胞のコロニー形成を促進して最適な３次元増幅が行え、ヒト血漿に代えて等量の血清を添加した同じ培地を用いた２次元培養よりも有意に高い増殖率を得ることができた。 As shown in FIGS. 14 and 15, human TF-1 cell culture by the three-dimensional cell culture method using the cell transplant composition according to the present invention promotes colony formation of human TF-1 cells and is optimal. Three-dimensional amplification could be performed, and a significantly higher growth rate could be obtained than two-dimensional culture using the same medium supplemented with an equal amount of serum instead of human plasma.
［実施例７］
ヒト肝癌細胞株（Ｈｕｈ７：５００，０００cells 及びＨｕｈ７：５, ０００, ０００cells ）を、ＬＨ／Ｐ ＭＰｓ（５ｍｇ／ｍｌ）及びＦＧＦ−２（１０μｇ／ｍｌ）を含む無血清ＤＭＥＭにヒト血漿（容積比５％）を加えて作製した細胞用移植用組成物に添加し、ヌードマウス皮下へ２００μｌ投与した異種腫瘍細胞移植実験を実施した。比較対象として、ＬＨ／Ｐ ＭＰｓ（５ｍｇ／ｍｌ）を腫瘍細胞浮遊液に加えた移植した（図中「＋ＬＨ／Ｐ ＭＰｓ」）。また、ＦＧＦ−２（１０μｇ／ｍｌ）を腫瘍細胞浮遊液に加えた移植した（図中「＋ＦＧＦ−２」）。さらに、ヒト血漿（容積比５％）を腫瘍細胞浮遊液に加えた移植した（図中「＋ＨＰ」）。また、ヒト血漿（容積比５％）及びＬＨ／Ｐ ＭＰｓ（５ｍｇ／ｍｌ）を腫瘍細胞浮遊液に加え移植した（図中「＋ＨＰ＋ＬＨ／Ｐ ＭＰｓ」）。さらに、ヒト血漿（容積比５％）とＬＨ／Ｐ ＭＰｓ（５ｍｇ／ｍｌ）とＦＧＦ−２（１０μｇ／ｍｌ）を腫瘍細胞浮遊液に加え移植した（図中「＋ＨＰ＋ＬＨ／Ｐ ＭＰｓ＋ＦＧＦ−２」）。コントロールとして、上記腫瘍細胞を生理食塩水に浮遊させ移植した（図中「Ｃｏｎｔｒｏｌ」）。
なお、腫傷サイズは、経時的に小型ノギスを用いて、縦・横・高さを測定し算出し、縦軸は成長した腫瘍容量（ｃｍ3 ）を示す。
[Example 7]
Human hepatoma cell lines (Huh7: 500,000 cells and Huh7: 5,000,000 cells) were transferred to human plasma (volume ratio) in serum-free DMEM containing LH / P MPs (5 mg / ml) and FGF-2 (10 μg / ml). 5%) was added to the cell transplantation composition prepared, and a heterogeneous tumor cell transplantation experiment was performed in which 200 μl was administered subcutaneously to nude mice. For comparison, LH / P MPs (5 mg / ml) was added to the tumor cell suspension and transplanted (“+ LH / P MPs” in the figure). In addition, FGF-2 (10 μg / ml) was transplanted in tumor cell suspension (“+ FGF-2” in the figure). Further, transplantation was performed by adding human plasma (volume ratio 5%) to the tumor cell suspension (“+ HP” in the figure). Further, human plasma (volume ratio 5%) and LH / P MPs (5 mg / ml) were added to the tumor cell suspension and transplanted (“+ HP + LH / P MPs” in the figure). Furthermore, human plasma (5% by volume), LH / P MPs (5 mg / ml) and FGF-2 (10 μg / ml) were added to the tumor cell suspension and transplanted (“+ HP + LH / P MPs + FGF-2” in the figure). . As a control, the tumor cells were suspended in physiological saline and transplanted (“Control” in the figure).
The tumor size was calculated by measuring the length, width, and height with a small caliper over time, and the vertical axis represents the tumor volume (cm 3 ) that has grown.
図１６（ａ）に示すように、Ｈｕｈ７細胞のみと、Ｈｕｈ７細胞＋ＦＧＦ−２投与群では、腫傷の生着及び成長は観察されなかったが、Ｈｕｈ７細胞を添加した上記細胞移植用組成物のヌードマウス皮下へ投与では、最適な生着及び腫傷成長を認められた。また、図１６（ｂ）に示すように、Ｈｕｈ７細胞（５, ０００, ０００cells ／２００μｌ）の移植実験では、コントロールでも細胞の生着は認められるものの、Ｈｕｈ７細胞を添加した上記細胞移植用組成物のヌードマウス皮下へ投与は、最適な腫癌成長を認められた。 As shown in FIG. 16 (a), tumor engraftment and growth were not observed in the Huh7 cells alone and in the Huh7 cells + FGF-2 administration group, but the above-mentioned cell transplantation composition to which Huh7 cells were added was used. When engrafted subcutaneously in nude mice, optimal engraftment and tumor growth were observed. In addition, as shown in FIG. 16 (b), in the transplantation experiment of Huh7 cells (5,000,000 cells / 200 μl), although the cell engraftment was observed even in the control, the above-mentioned composition for cell transplantation to which Huh7 cells were added Administration of nude mice subcutaneously showed optimal tumor growth.
［実施例８］
本発明による３次元細胞培養方法により増幅させたマウス骨髄由来ＣＤ３４陽性造血系前駆体細胞を含んだ細胞移植用組成物をＸ線照射により骨髄死を引き起こしたマウスの骨内へ投与した。その結果、末梢血での白血球や血小板の有意に高い回復が観察され、優れた骨内造血系前駆体細胞移植のための運搬システムとして適用の可能性が示された。
[Example 8]
The composition for cell transplantation containing mouse bone marrow-derived CD34 positive hematopoietic progenitor cells amplified by the three-dimensional cell culture method according to the present invention was administered into the bones of mice that caused bone marrow death by X-ray irradiation. As a result, a significantly high recovery of leukocytes and platelets in peripheral blood was observed, indicating the possibility of application as an excellent delivery system for intraosseous hematopoietic precursor cell transplantation.
本発明は、用途に応じて任意に選択される接着性細胞や造血系浮遊細胞等の各種細胞と、安全性が確認されている低分子へパリンとプロタミンとから構成される平均粒径が０．１μｍ〜５μｍ未満のＬＨ／Ｐ ＭＰｓと、適切なサイトカイン、培地及びヒト血漿を含んでなるゲル状の細胞移植用組成物を用いた３次元細胞培養方法により、特にＣＤ３４陽性造血系前駆体細胞の体外大量増幅システムとしての有用性が極めて高い。また、細胞移植用組成物は、注射器やカテーテル等を介して、生体局所に投与する医療用途へ安定的に供給する細胞運搬法として適用することができる。 The present invention has an average particle size of 0 consisting of various cells such as adhesive cells and hematopoietic suspension cells arbitrarily selected according to the use, and low molecular weight heparin and protamine that have been confirmed to be safe. In particular, a CD34 positive hematopoietic progenitor cell by a three-dimensional cell culture method using LH / P MPs of 1 μm to less than 5 μm and a gel-like cell transplantation composition comprising an appropriate cytokine, medium and human plasma Is extremely useful as an in vitro mass amplification system. Moreover, the composition for cell transplantation can be applied as a cell delivery method that can be stably supplied to a medical application administered locally to a living body via a syringe, a catheter, or the like.
さらに、血漿中に含まれる生理活性物質を安定に保持し、目的部位で担持した活性分子を徐放できるドラッグデリバリー用薬物包接担体としても使用できる。また、ヌードマウス等に局所注入した細胞移植用組成物内で癌細胞等の腫瘍細胞を担持した状態で効率よく増幅させることができるため、抗ガン剤等の新薬の開発に大きく貢献できる。そして、ヒト自己骨髄間葉系幹細胞或いは自己脂肪組織由来間葉系細胞に本細胞デリバリー担体を適用することで、美容形成外科領域の育毛剤及び雛取り等美容形成治療法への適用に加え、血管新生促進製剤、皮膚等の難治性創傷治癒促進剤として、さらには他の細胞導入を伴う再生医療への応用が可能となる。 Furthermore, it can also be used as a drug inclusion carrier for drug delivery that can stably hold a physiologically active substance contained in plasma and can slowly release an active molecule carried at a target site. In addition, since it can be efficiently amplified in a state in which tumor cells such as cancer cells are carried in a cell transplantation composition locally injected into nude mice or the like, it can greatly contribute to the development of new drugs such as anticancer agents. And by applying this cell delivery carrier to human autologous bone marrow mesenchymal stem cells or autologous adipose tissue-derived mesenchymal cells, in addition to application to beauty shaping treatment methods such as hair restorer and chick removal in the cosmetic plastic surgery area, As an angiogenesis-promoting preparation, an intractable wound healing promoter for skin, etc., it can be applied to regenerative medicine with other cell introduction.
Claims (4)
低分子ヘパリンとプロタミンとからなる平均粒径が０．１〜５μｍであるＬＨ／Ｐ ＭＰｓと、
前記接着性細胞の用途に応じて任意に選択されるサイトカインと、
容積比０．５〜２５％のヒト血漿と、
で構成されるゲル状組成物内で前記接着性細胞の培養を行うことを特徴とする３次元細胞培養方法。 Adherent cells including mesenchymal stem cells and tumor cells used in regenerative medicine,
LH / P MPs having an average particle size of 0.1 to 5 μm consisting of low molecular weight heparin and protamine;
A cytokine arbitrarily selected according to the use of the adhesive cells;
Human plasma with a volume ratio of 0.5-25%,
A three-dimensional cell culture method comprising culturing the adherent cells in a gel composition comprising:
低分子ヘパリンとプロタミンとからなる平均粒径が０．１〜５μｍであるＬＨ／Ｐ ＭＰｓと、
前記接着性細胞の用途に応じて任意に選択されるサイトカインと、
容積比０．５〜２５％のヒト血漿と、
で構成されるゲル状組成物であることを特徴とする細胞移植用組成物。 Adherent cells including mesenchymal stem cells and tumor cells used in regenerative medicine,
LH / P MPs having an average particle size of 0.1 to 5 μm consisting of low molecular weight heparin and protamine;
A cytokine arbitrarily selected according to the use of the adhesive cells;
Human plasma with a volume ratio of 0.5-25%,
A composition for cell transplantation, characterized in that it is a gel composition comprising:
低分子ヘパリンとプロタミンとからなる平均粒径が０．１〜５μｍであるＬＨ／Ｐ ＭＰｓと、
前記造血系浮遊細胞の用途に応じて任意に選択されるサイトカインと、
容積比０．５〜２５％のヒト血漿と、
で構成されるゲル状組成物内で前記造血系浮遊細胞の培養を行うことを特徴とする３次元細胞培養方法。 Hematopoietic suspension cells including CD34 positive hematopoietic progenitor cells used in regenerative medicine;
LH / P MPs having an average particle size of 0.1 to 5 μm consisting of low molecular weight heparin and protamine;
A cytokine arbitrarily selected according to the use of the hematopoietic suspension cells,
Human plasma with a volume ratio of 0.5-25%,
A three-dimensional cell culture method, comprising culturing the hematopoietic suspension cells in a gel composition comprising:
低分子ヘパリンとプロタミンとからなる平均粒径が０．１〜５μｍであるＬＨ／Ｐ ＭＰｓと、
前記造血系浮遊細胞の用途に応じて任意に選択されるサイトカインと、
容積比０．５〜２５％のヒト血漿と、
で構成されるゲル状組成物であることを特徴とする細胞移植用組成物。 Hematopoietic suspension cells including CD34 positive hematopoietic progenitor cells used in regenerative medicine;
LH / P MPs having an average particle size of 0.1 to 5 μm consisting of low molecular weight heparin and protamine;
A cytokine arbitrarily selected according to the use of the hematopoietic suspension cells,
Human plasma with a volume ratio of 0.5-25%,
A composition for cell transplantation, characterized in that it is a gel composition comprising:
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2012171027A JP2012205599A (en) | 2012-08-01 | 2012-08-01 | Three-dimensional cell culturing method, and composition for cell transplantation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2012171027A JP2012205599A (en) | 2012-08-01 | 2012-08-01 | Three-dimensional cell culturing method, and composition for cell transplantation |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JP2012205599A true JP2012205599A (en) | 2012-10-25 |
Family
ID=47185970
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2012171027A Pending JP2012205599A (en) | 2012-08-01 | 2012-08-01 | Three-dimensional cell culturing method, and composition for cell transplantation |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2012205599A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2018513208A (en) * | 2015-04-23 | 2018-05-24 | ボーン セラピューティクス エス．エー． | In vitro storage of therapeutic cells |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005160669A (en) * | 2003-12-02 | 2005-06-23 | Olympus Corp | Manufacturing method of biological tissue prosthesis |
| JP2005170816A (en) * | 2003-12-09 | 2005-06-30 | Naoki Ishiguro | Material for restoring cartilage, and method for producing the same |
| JP2011024423A (en) * | 2008-06-04 | 2011-02-10 | Masayuki Ishihara | Particulate cell carrier and cell-introducing system containing the same and cell culture system |
-
2012
- 2012-08-01 JP JP2012171027A patent/JP2012205599A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005160669A (en) * | 2003-12-02 | 2005-06-23 | Olympus Corp | Manufacturing method of biological tissue prosthesis |
| JP2005170816A (en) * | 2003-12-09 | 2005-06-30 | Naoki Ishiguro | Material for restoring cartilage, and method for producing the same |
| JP2011024423A (en) * | 2008-06-04 | 2011-02-10 | Masayuki Ishihara | Particulate cell carrier and cell-introducing system containing the same and cell culture system |
Non-Patent Citations (3)
| Title |
|---|
| JPN6014006582; 荒井桃子ら: 膝 ３２（１）, 2007, ｐｐ．５-８ * |
| JPN6014006583; 加藤秀昭ら: 第３５回社団法人日本口腔インプラント学会総会・学術大会 プログラム・抄録集 , 2005, ｐ．１１６ * |
| JPN6014006584; 川澄本明ら: 日整会誌 ８１（１）, 2007, ｐ．Ｓ１１４４ * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2018513208A (en) * | 2015-04-23 | 2018-05-24 | ボーン セラピューティクス エス．エー． | In vitro storage of therapeutic cells |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Qin et al. | Urine-derived stem cells for potential use in bladder repair | |
| Dominici et al. | Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement | |
| Sart et al. | Preconditioning stem cells for in vivo delivery | |
| Yin et al. | Hydrogels for large-scale expansion of stem cells | |
| EP2422622B1 (en) | Methods of using adipose tissue-derived cells in the treatment of cardiovascular conditions | |
| JP6548897B2 (en) | Methods and compositions for the induction of medical applications of allogeneic cells and therapeutic applications | |
| Wang et al. | The combination of stem cells and tissue engineering: an advanced strategy for blood vessels regeneration and vascular disease treatment | |
| Bicer et al. | Impact of 3D cell culture on bone regeneration potential of mesenchymal stromal cells | |
| TW200817019A (en) | De novo formation and regeneration of vascularized tissue from tissue progenitor cells and vascular progenitor cells | |
| US20100035327A1 (en) | Use of rice-derived products in a universal cell culture medium | |
| JP2013510582A (en) | Spherical aggregate of mesenchymal stem cells | |
| AU2013250089B2 (en) | Method for manufacturing stem cell having appropriate size for intravascular administration | |
| Luan et al. | Hydrogel based 3D carriers in the application of stem cell therapy by direct injection | |
| Ishihara et al. | Biomaterials as cell carriers for augmentation of adipose tissue-derived stromal cell transplantation | |
| Su et al. | Membrane-binding adhesive particulates enhance the viability and paracrine function of mesenchymal cells for cell-based therapy | |
| Song et al. | Exosomes derived from embryonic stem cells as potential treatment for cardiovascular diseases | |
| Nazeer et al. | Neovascularization of engineered tissues for clinical translation: Where we are, where we should be? | |
| Willenberg et al. | Gelatinized copper–capillary alginate gel functions as an injectable tissue scaffolding system for stem cell transplants | |
| Xu et al. | Strategies of prevascularization in tissue engineering and regeneration of craniofacial tissues | |
| Desai et al. | Scaffold-free spheroids derived from stem cells for tissue-engineering applications | |
| JP2011024423A (en) | Particulate cell carrier and cell-introducing system containing the same and cell culture system | |
| Sudulaguntla et al. | Stem cells: Cultivation and routes of administration | |
| JP2012205599A (en) | Three-dimensional cell culturing method, and composition for cell transplantation | |
| Mukherjee et al. | Chemokine SDF1 mediated bone regeneration using biodegradable Poly (D, L-lactide-co-glycolide) 3D scaffolds and bone marrow-derived mesenchymal stem cells: implication for the development of an “Off-the-Shelf” pharmacologically active construct | |
| Pooria et al. | Animal‐and human‐based evidence for the protective effects of stem cell therapy against cardiovascular disorders |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20120801 |
|
| A521 | Written amendment |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20120801 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20140218 |
|
| A02 | Decision of refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A02 Effective date: 20140715 |