JP4755460B2 - Method for culturing mesenchymal stem cells - Google Patents
Method for culturing mesenchymal stem cells Download PDFInfo
- Publication number
- JP4755460B2 JP4755460B2 JP2005217835A JP2005217835A JP4755460B2 JP 4755460 B2 JP4755460 B2 JP 4755460B2 JP 2005217835 A JP2005217835 A JP 2005217835A JP 2005217835 A JP2005217835 A JP 2005217835A JP 4755460 B2 JP4755460 B2 JP 4755460B2
- Authority
- JP
- Japan
- Prior art keywords
- stem cells
- mesenchymal stem
- ratio
- hematopoietic stem
- hematopoietic
- 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.)
- Expired - Fee Related
Links
Images
Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
本発明は、間葉系幹細胞の培養方法に関する。 The present invention relates to a method for culturing mesenchymal stem cells.
間葉系幹細胞（ＭＳＣ）は、骨細胞、軟骨細胞、脂肪細胞、筋肉細胞、ストローマ細胞、神経細胞、腱細胞等、様々な細胞への分化能を有しているため、これら細胞からなる組織を再生することができる細胞として知られている。このため、間葉系幹細胞を用いて組織再生や治療を行う試みが多くなされている。 Mesenchymal stem cells (MSCs) have the ability to differentiate into various cells such as bone cells, chondrocytes, adipocytes, muscle cells, stromal cells, nerve cells, tendon cells, etc. Known as cells that can regenerate. For this reason, many attempts have been made to perform tissue regeneration and treatment using mesenchymal stem cells.
間葉系幹細胞は骨髄液に多く含まれている。しかしながら、骨髄液から採取可能な間葉系幹細胞はごく微量であり、組織の再生に必要な量の間葉系幹細胞を得るためには、骨髄液中の間葉系幹細胞を濃縮してから分離し、間葉系幹細胞を培養することにより増殖させる必要がある。 Many mesenchymal stem cells are contained in bone marrow fluid. However, the amount of mesenchymal stem cells that can be collected from the bone marrow fluid is very small, and in order to obtain the amount of mesenchymal stem cells necessary for tissue regeneration, the mesenchymal stem cells in the bone marrow fluid are concentrated and separated, It is necessary to proliferate by culturing mesenchymal stem cells.
従来、骨髄液中の間葉系幹細胞を濃縮・分離してから培養する方法としては、採取した骨髄液を遠心した後に、その上澄み液を除去し、残った沈殿部分のみを培養容器に播種して底面に接着させた状態で増殖させる方法が知られている。この場合に、培養容器の底面に接着している間葉系幹細胞の播種密度が適正となるように調節する培養方法が提案されている（例えば、特許文献１参照。）。
しかしながら、間葉系幹細胞を培養容器の底面に接着させて培養する場合、間葉系幹細胞の増殖に伴って、細胞の接着する底面の面積が足りなくなるため、より広い底面積を有する培養容器への切替作業あるいは、複数の培養容器への切替作業（いわゆる継代作業）を行う必要がある。継代作業は、トリプシンのようなタンパク質分解酵素を用いて間葉系幹細胞を培養容器の底面から剥離させる作業を伴うものであり、間葉系幹細胞に損傷を与える可能性がある。また、複数の培養容器の切替を伴うため、間葉系幹細胞が何らかの細菌や塵埃と接触する可能性が高くなるという不都合も考えられる。さらに、継代作業は、培養容器の底面に接着している間葉系幹細胞を剥離させ、新たな培養容器に接着させて培養を継続させる作業であるため、継代作業自体に時間がかかり、培養期間が長期化する不都合もある。 However, when culturing with mesenchymal stem cells adhered to the bottom surface of the culture container, the area of the bottom surface to which the cells adhere becomes insufficient as the mesenchymal stem cells proliferate, so the culture container has a wider bottom area. Switching work or switching work to a plurality of culture vessels (so-called subculture work) is required. The subculture operation involves an operation of detaching mesenchymal stem cells from the bottom surface of the culture container using a proteolytic enzyme such as trypsin, which may damage the mesenchymal stem cells. In addition, since switching of a plurality of culture vessels is involved, there is a possibility that the mesenchymal stem cells are likely to come into contact with some bacteria or dust. Furthermore, since the subculture work is an operation of detaching the mesenchymal stem cells adhering to the bottom surface of the culture vessel and continuing the culture by adhering to a new culture vessel, the subculture operation itself takes time, There is also a disadvantage that the culture period is prolonged.
本発明は上述した事情に鑑みてなされたものであって、継代作業をなくして、間葉系幹細胞へのダメージを軽減し、コンタミネーションのリスクを低減することができるとともに、培養操作を簡略化して培養期間を短縮し、効率的な増殖を図ることにより採取骨髄量を低減して患者にかかる負担を低減することができる間葉系幹細胞の培養方法を提供することを目的としている。 The present invention has been made in view of the above-described circumstances, and can eliminate passage work, reduce damage to mesenchymal stem cells, reduce the risk of contamination, and simplify culture operations. It is an object of the present invention to provide a method for culturing mesenchymal stem cells that can reduce the burden on patients by reducing the amount of collected bone marrow by shortening the culture period and achieving efficient proliferation.
上記目的を達成するために、本発明は以下の手段を提供する。
培地内に間葉系幹細胞と造血幹細胞とを浮遊させ、間葉系幹細胞と造血幹細胞との比率を１：１０〜１：１００の範囲に維持しつつ培養する間葉系幹細胞の培養方法を提供する。
In order to achieve the above object, the present invention provides the following means.
Provided is a method for culturing mesenchymal stem cells in which mesenchymal stem cells and hematopoietic stem cells are suspended in a medium and cultured while maintaining the ratio of mesenchymal stem cells to hematopoietic stem cells in the range of 1:10 to 1: 100. To do.
本発明によれば、間葉系幹細胞と造血幹細胞との比率が１：１０〜１：１００の範囲に維持される。間葉系幹細胞は、骨髄中においては種々の細胞とともに共存しており、間葉系幹細胞も浮遊した状態に維持されている。このことから、間葉系幹細胞を生きた生体内に近い状態で培養すれば、浮遊状態においても増殖することが推測され、研究の結果、間葉系幹細胞と造血幹細胞との比率を上記範囲に維持することにより、生体外においても生体内と同様の条件が達成されて、間葉系幹細胞を効率的に培養することができることが判明した。このようにすることで、間葉系幹細胞を培養容器の底面に接着させる必要がなく、継代作業が不要となり、間葉系幹細胞へのダメージを軽減し、コンタミネーションのリスクを低減し、培養操作を簡略化して培養期間を短縮し、効率的な増殖を図ることにより採取骨髄量を低減して患者にかかる負担を低減することができる。 According to the present invention, the ratio of mesenchymal stem cells to hematopoietic stem cells is maintained in the range of 1:10 to 1: 100. Mesenchymal stem cells coexist with various cells in the bone marrow, and mesenchymal stem cells are also maintained in a floating state. From this, it is speculated that if mesenchymal stem cells are cultured in a state close to living organisms, they will proliferate even in a floating state, and as a result of research, the ratio of mesenchymal stem cells to hematopoietic stem cells falls within the above range By maintaining, it was found that the same conditions as in vivo were achieved in vitro, and mesenchymal stem cells could be efficiently cultured. This eliminates the need for adhering mesenchymal stem cells to the bottom of the culture vessel, eliminating the need for passage work, reducing damage to mesenchymal stem cells, reducing the risk of contamination, and culturing. By simplifying the operation, shortening the culture period, and promoting efficient growth, the amount of collected bone marrow can be reduced and the burden on the patient can be reduced.
上記発明においては、培地内の間葉系幹細胞と造血幹細胞との比率を監視し、間葉系幹細胞の比率が造血幹細胞の１／１０倍より多い場合に、造血幹細胞の比率を増加させる液体因子を添加することとしてもよい。
このようにすることで、間葉系幹細胞の比率が増加した場合に液体因子を添加して造血幹細胞の比率を増加させ、生きた生体内に近い状態で間葉系幹細胞を効率的に増殖させることが可能となる。間葉系幹細胞と造血幹細胞との比率の監視は、フローサイトメトリー（ＦＡＣＳ）により行われる。例えば、間葉系幹細胞数は、細胞表面マーカであるＣＤ２９，ＣＤ９０，ＳＨ３で、造血幹細胞数は、Ｓｔｅｍ−ｋｉｔ（ＢＤ）でＦＡＣＳにより測定を行う。これにより両者の比率をモニタすることができる。
In the above invention, the liquid factor that monitors the ratio of mesenchymal stem cells to hematopoietic stem cells in the medium and increases the ratio of hematopoietic stem cells when the ratio of mesenchymal stem cells is more than 1/10 times that of hematopoietic stem cells It is good also as adding.
By doing this, when the ratio of mesenchymal stem cells increases, liquid factor is added to increase the ratio of hematopoietic stem cells, and the mesenchymal stem cells are efficiently proliferated in a state close to living organisms. It becomes possible. The ratio of mesenchymal stem cells to hematopoietic stem cells is monitored by flow cytometry (FACS). For example, the number of mesenchymal stem cells is measured by cell surface markers CD29, CD90, and SH3, and the number of hematopoietic stem cells is measured by FACS using Stem-kit (BD). Thereby, the ratio of both can be monitored.
この場合に、造血幹細胞の比率を増加させる液体因子が、１〜１００ｎｇ／ｍＬのＳＣＦ（Stem Cell Factor）、１〜５０ｎｇ／ｍＬのＩＬ−３（Interleukin-3）、１〜５０ｎｇ／ｍＬのＩＬ−６、１〜５０ｎｇ／ｍＬのＩＬ−１０、１０〜３００ｎｇ／ｍＬのＦＬ（Flt-3L）および１〜５０ｎｇ／ｍＬのＴＰＯ（Thrombopoietin）の混合液からなることが好ましい。 In this case, liquid factors that increase the ratio of hematopoietic stem cells are 1-100 ng / mL SCF (Stem Cell Factor), 1-50 ng / mL IL-3 (Interleukin-3), 1-50 ng / mL IL. -6, preferably composed of a mixture of 1-10 ng / mL IL-10, 10-300 ng / mL FL (Flt-3L) and 1-50 ng / mL TPO (Thrombopoietin).
また、上記発明においては、培地内の間葉系幹細胞と造血幹細胞との比率を監視し、造血幹細胞の比率が間葉系幹細胞の１００倍より多い場合に、間葉系幹細胞の比率を増加させる液体因子を添加することとしてもよい。
このようにすることで、造血幹細胞の比率が増加した場合に液体因子を添加して間葉系幹細胞の比率を増加させ、生きた生体内に近い状態で間葉系幹細胞を効率的に増殖させることが可能となる。
In the above invention, the ratio of mesenchymal stem cells to hematopoietic stem cells in the medium is monitored, and when the ratio of hematopoietic stem cells is more than 100 times that of mesenchymal stem cells, the ratio of mesenchymal stem cells is increased. It is good also as adding a liquid factor.
By doing this, when the ratio of hematopoietic stem cells increases, liquid factor is added to increase the ratio of mesenchymal stem cells, and the mesenchymal stem cells are efficiently proliferated in a state close to living organisms. It becomes possible.
この場合に、間葉系幹細胞の比率を増加させる液体因子が、１〜１００ｎｇ／ｍＬのＰＤＧＦ（Platelet-Derived Growth Factor）、１〜１００ｎｇ／ｍＬのｂＦＧＦ（Basic Fibroblast Growth Factor）および５〜３０００μｇ／ｍＬのビタミンＣの混合液からなることが好ましい。 In this case, the liquid factors that increase the ratio of mesenchymal stem cells are 1 to 100 ng / mL of PDGF (Platelet-Derived Growth Factor), 1 to 100 ng / mL of bFGF (Basic Fibroblast Growth Factor) and 5 to 3000 μg / mL. It preferably consists of a mixed solution of mL of vitamin C.
本発明によれば、継代作業をなくして、間葉系幹細胞へのダメージを軽減し、コンタミネーションのリスクを低減することができるとともに、培養操作を簡略化して培養期間を短縮し、効率的な増殖を図ることにより採取骨髄量を低減して患者にかかる負担を低減することができるという効果を奏する。 According to the present invention, passage work can be eliminated, damage to mesenchymal stem cells can be reduced, the risk of contamination can be reduced, and the culture period can be simplified to shorten the culture period. By effecting proper growth, the amount of collected bone marrow can be reduced and the burden on the patient can be reduced.
本発明の一実施形態に係る間葉系幹細胞の培養方法について、以下に説明する。
本実施形態に係る間葉系幹細胞の培養方法は、まず、培養容器内に貯留した培地内に患者から採取した骨髄液を投入し、３７℃に保った状態で、攪拌する。培養容器の内壁には、接着性の間葉系幹細胞が付着しないようにコーティングが施されている。
A method for culturing mesenchymal stem cells according to an embodiment of the present invention will be described below.
In the method for culturing mesenchymal stem cells according to the present embodiment, first, bone marrow fluid collected from a patient is put into a medium stored in a culture vessel, and the mixture is agitated while maintaining at 37 ° C. The inner wall of the culture vessel is coated so that adhesive mesenchymal stem cells do not adhere.
患者から採取した骨髄液内には、間葉系幹細胞と造血幹細胞とが、１：１０〜１：１００の割合で存在している。したがって、骨髄液を投入して開始される培養開始時には、培養容器内における間葉系幹細胞と造血幹細胞との比率が生体内に近い状態になっている。また、培養容器内の培地を攪拌することにより、間葉系幹細胞および造血幹細胞は培地内において浮遊し、しかも、培養容器の内壁に間葉系幹細胞の付着を防止するコーティングが施されているので、間葉系幹細胞は培地内において浮遊状態に維持される。これも、生体内に近い状態になっている。 In the bone marrow fluid collected from the patient, mesenchymal stem cells and hematopoietic stem cells are present in a ratio of 1:10 to 1: 100. Therefore, at the start of culturing started by introducing bone marrow fluid, the ratio of mesenchymal stem cells to hematopoietic stem cells in the culture vessel is in a state close to that in the living body. In addition, by stirring the medium in the culture container, the mesenchymal stem cells and hematopoietic stem cells float in the medium, and the inner wall of the culture container is coated to prevent the mesenchymal stem cells from attaching. The mesenchymal stem cells are maintained in a floating state in the medium. This is also close to the living body.
本実施形態に係る間葉系幹細胞の培養方法においては、培地内における間葉系幹細胞と造血幹細胞の比率を監視する。例えば、間葉系幹細胞数は、細胞表面マーカであるＣＤ２９，ＣＤ９０，ＳＨ３で、造血幹細胞数はＳｔｅｍ−Ｋｉｔ（ＢＤ）でＦＡＣＳにより測定を行うことで、培養物中の間葉系幹細胞数と造血幹細胞数を監視することができる。そして、培地中の造血幹細胞数が多くなってきたときは、間葉系幹細胞を増加させる液体因子を添加し、培地中の造血幹細胞数が少なくなってきたときは、造血幹細胞を増加させる液体因子を添加する。 In the mesenchymal stem cell culturing method according to the present embodiment, the ratio of mesenchymal stem cells to hematopoietic stem cells in the medium is monitored. For example, the number of mesenchymal stem cells is CD29, CD90, SH3 which are cell surface markers, and the number of hematopoietic stem cells is measured by FACS with Stem-Kit (BD), so that the number of mesenchymal stem cells and hematopoietic stem cells in the culture The number can be monitored. When the number of hematopoietic stem cells in the medium increases, a liquid factor that increases mesenchymal stem cells is added, and when the number of hematopoietic stem cells in the medium decreases, the liquid factor increases the hematopoietic stem cells. Add.
間葉系幹細胞を増加させる液体因子としては、１〜１００ｎｇ／ｍＬのＰＤＧＦ（Platelet-Derived Growth Factor）、１〜１００ｎｇ／ｍＬのｂＦＧＦ（Basic Fibroblast
Growth Factor）および５〜３０００μｇ／ｍＬのビタミンＣの混合液を挙げることができる。また、造血幹細胞を増加させる液体因子としては、１〜１００ｎｇ／ｍＬのＳＣＦ（Stem Cell Factor）、１〜５０ｎｇ／ｍＬのＩＬ−３（Interleukin-3）、１〜５０ｎｇ／ｍＬのＩＬ−６、１〜５０ｎｇ／ｍＬのＩＬ−１０、１０〜３００ｎｇ／ｍＬのＦＬ（Flt-3L）および１〜５０ｎｇ／ｍＬのＴＰＯ（Thrombopoietin）の混合液を挙げることができる。
As liquid factors for increasing mesenchymal stem cells, 1 to 100 ng / mL of PDGF (Platelet-Derived Growth Factor), 1 to 100 ng / mL of bFGF (Basic Fibroblast)
Growth factor) and a mixed solution of vitamin C of 5 to 3000 μg / mL. In addition, as a liquid factor for increasing hematopoietic stem cells, 1 to 100 ng / mL SCF (Stem Cell Factor), 1 to 50 ng / mL IL-3 (Interleukin-3), 1 to 50 ng / mL IL-6, A mixed solution of 1-50 ng / mL IL-10, 10-300 ng / mL FL (Flt-3L) and 1-50 ng / mL TPO (Thrombopoietin) can be mentioned.
このように、本実施形態に係る間葉系幹細胞の培養方法によれば、培養中における培地内の間葉系幹細胞と造血幹細胞との比率を常に生体内に近い状態に維持するので、培養容器に接着させることなく、生体内と同様に間葉系幹細胞を浮遊させたままの状態で、生体内と同様に効率的に間葉系幹細胞を増殖させることができる。 Thus, according to the method for culturing mesenchymal stem cells according to the present embodiment, the ratio of mesenchymal stem cells and hematopoietic stem cells in the medium during culture is always maintained in a state close to that in the living body. The mesenchymal stem cells can be efficiently proliferated in the same manner as in the living body in a state in which the mesenchymal stem cells are suspended in the same manner as in the living body.
すなわち、本実施形態に係る培養方法によれば、間葉系幹細胞を培養容器に接着させることなく浮遊させたまま培養するので、培養容器を切り替える継代作業が不要となり、該継代作業に伴う不都合、つまり、間葉系幹細胞に与えるダメージやコンタミネーションリスクの低減を図ることができるという利点がある。
また、手間と時間を要する継代作業を不要とすることにより、培養操作を簡便化することができるとともに、培養期間を短縮して早期に効率的に間葉系幹細胞を必要細胞数まで増殖させることができる。
That is, according to the culture method according to the present embodiment, the mesenchymal stem cells are cultured while being suspended without adhering to the culture container, so that the subculture work for switching the culture container becomes unnecessary, and accompanying the subculture work There is an inconvenience, that is, an advantage that damage to the mesenchymal stem cells and risk of contamination can be reduced.
In addition, it eliminates the need for time-consuming and time-consuming passage work, and simplifies the culturing operation, and shortens the culturing period to quickly and efficiently proliferate mesenchymal stem cells to the required number of cells. be able to.
さらに、生体内に近い状態で間葉系幹細胞を効率的に増殖させるので、骨髄液の採取量を低減することができ、したがって、患者にかかる負担を低減することができるという利点もある。 Furthermore, since the mesenchymal stem cells are efficiently proliferated in a state close to that in the living body, the amount of bone marrow fluid collected can be reduced, and thus there is an advantage that the burden on the patient can be reduced.
表１および図１に、間葉系幹細胞と造血幹細胞との比率を生体内と同様の状態に維持して浮遊培養した結果を示す。具体的には、１２５Ｕ／ｍＬのヘパリンを含む骨髄液を３７℃インキュベータ内で培養した結果、６８時間後の間葉系幹細胞の数は初期の２．３倍も増殖した。その結果、骨髄中の各細胞の比率、特に間葉系幹細胞と造血幹細胞との比率を生体内と同様の状態に維持することによって、浮遊状態でも間葉系幹細胞を増殖させることが可能であることがわかった。 Table 1 and FIG. 1 show the results of suspension culture while maintaining the ratio of mesenchymal stem cells and hematopoietic stem cells in the same state as in vivo. Specifically, bone marrow fluid containing 125 U / mL heparin was cultured in a 37 ° C. incubator, and as a result, the number of mesenchymal stem cells proliferated 68 times after the initial period of 68 hours. As a result, it is possible to proliferate mesenchymal stem cells even in a floating state by maintaining the ratio of each cell in the bone marrow, particularly the ratio of mesenchymal stem cells and hematopoietic stem cells, in the same state as in vivo. I understood it.
Claims (5)
間葉系幹細胞と造血幹細胞との比率を１：１０〜１：１００の範囲に維持しつつ培養する間葉系幹細胞の培養方法。 Suspend mesenchymal stem cells and hematopoietic stem cells in the medium,
A method for culturing mesenchymal stem cells, which is cultured while maintaining the ratio of mesenchymal stem cells to hematopoietic stem cells in the range of 1:10 to 1: 100.
間葉系幹細胞の比率が造血幹細胞の１／１０倍より多い場合に、造血幹細胞の比率を増加させる液体因子を添加する請求項１に記載の間葉系幹細胞の培養方法。 Monitor the ratio of mesenchymal stem cells to hematopoietic stem cells in the medium,
The method for culturing mesenchymal stem cells according to claim 1, wherein a liquid factor that increases the ratio of hematopoietic stem cells is added when the ratio of mesenchymal stem cells is more than 1/10 times that of hematopoietic stem cells.
造血幹細胞の比率が間葉系幹細胞の１００倍より多い場合に、間葉系幹細胞の比率を増加させる液体因子を添加する請求項１から請求項３のいずれかに記載の間葉系幹細胞の培養方法。 Monitor the ratio of mesenchymal stem cells to hematopoietic stem cells in the medium,
The culture of mesenchymal stem cells according to any one of claims 1 to 3, wherein a liquid factor that increases the ratio of mesenchymal stem cells is added when the ratio of hematopoietic stem cells is more than 100 times that of mesenchymal stem cells. Method.
Priority Applications (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2005217835A JP4755460B2 (en) | 2005-07-27 | 2005-07-27 | Method for culturing mesenchymal stem cells |
| PCT/JP2006/309320 WO2006121043A1 (en) | 2005-05-09 | 2006-05-09 | Culture method for mesenchymal stem cell and process for production of supporting material for biological tissue |
| US11/919,303 US7972767B2 (en) | 2005-05-09 | 2006-05-09 | Method for culturing mesenchymal stem cell and method for producing biological tissue prosthesis |
| EP09014695A EP2210937A1 (en) | 2005-05-09 | 2006-05-09 | Culture method for mesenchymal stem cell and process for production of supporting material for biological tissue |
| EP06746151A EP1881062B1 (en) | 2005-05-09 | 2006-05-09 | Methode for culturing mesenchymal stem cell and method for producing biological tissue prosthesis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2005217835A JP4755460B2 (en) | 2005-07-27 | 2005-07-27 | Method for culturing mesenchymal stem cells |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2007029009A JP2007029009A (en) | 2007-02-08 |
| JP4755460B2 true JP4755460B2 (en) | 2011-08-24 |
Family
ID=37789005
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2005217835A Expired - Fee Related JP4755460B2 (en) | 2005-05-09 | 2005-07-27 | Method for culturing mesenchymal stem cells |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP4755460B2 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP4166652A1 (en) | 2009-11-12 | 2023-04-19 | Technion Research & Development Foundation Ltd. | Culture media, cell cultures and methods of culturing pluripotent stem cells in an undifferentiated state |
| JP7217921B2 (en) * | 2018-05-24 | 2023-02-06 | 国立研究開発法人国立国際医療研究センター | Medium for maintaining and culturing hematopoietic stem cells, and culture method using the same |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1108011A2 (en) * | 1998-06-08 | 2001-06-20 | Osiris Therapeutics, Inc. | In vitro maintenance of hematopoietic stem cells |
| JP4353510B2 (en) * | 2002-09-09 | 2009-10-28 | 株式会社カネカ | Tissue regeneration support and method for producing the same |
| JP2004222502A (en) * | 2003-01-17 | 2004-08-12 | Asahi Kasei Corp | Method for amplifying hematopoietic stem cell |
| KR100833612B1 (en) * | 2004-12-29 | 2008-05-30 | 에프씨비파미셀 주식회사 | Pharmaceutical composition containing the neural cell differentated from mesenchymal stem cell for neurodegenerative disease |
-
2005
- 2005-07-27 JP JP2005217835A patent/JP4755460B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JP2007029009A (en) | 2007-02-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US10246682B2 (en) | Methods of culturing retinal pigmented epithelium cells, including Xeno-free production, RPE enrichment, and cryopreservation | |
| US7972767B2 (en) | Method for culturing mesenchymal stem cell and method for producing biological tissue prosthesis | |
| KR101211913B1 (en) | Medium for Culturing Mesenchymal Stem Cells Derived from Amnion and Method for Culturing Mesenchymal Stem Cells Derived from Amnion Using thereof | |
| KR100900309B1 (en) | Process for the high-purity isolation of mesenchymal stem cells derived from placental chorionic plate membrane | |
| JP5138367B2 (en) | Method for forming mesenchymal stem cells from embryonic stem cells | |
| Kanematsu et al. | Isolation and cellular properties of mesenchymal cells derived from the decidua of human term placenta | |
| JP5678360B2 (en) | Method for culturing mesenchymal stem cells | |
| Tarle et al. | Development of a serum‐free system to expand dental‐derived stem cells: PDLSCs and SHEDs | |
| KR20120137404A (en) | Cell preparation containing mesenchymal stem cell, and method for producing same | |
| Zeiter et al. | Effect of TGF β 1, BMP-2 and hydraulic pressure on chondrogenic differentiation of bovine bone marrow mesenchymal stromal cells | |
| Hutson et al. | Rapid isolation, expansion, and differentiation of osteoprogenitors from full-term umbilical cord blood | |
| JP5252411B2 (en) | Cell culture carrier and cell culture method | |
| Jorgenson et al. | Production of adult human synovial fluid‐derived mesenchymal stem cells in stirred‐suspension culture | |
| Arpornmaeklong et al. | Allogenic human serum, a clinical grade serum supplement for promoting human periodontal ligament stem cell expansion | |
| JP2023052609A (en) | Method for manufacturing cell culture | |
| CN110872574B (en) | Efficient and reliable hESC-MSC preparation method | |
| JP2011529340A (en) | Method for separating highly active stem cells from human stem cells and highly active stem cells isolated by the method | |
| JP4755460B2 (en) | Method for culturing mesenchymal stem cells | |
| US20150329826A1 (en) | Materials and methods for cell culture | |
| Schop | Growth and metabolism of mesenchymal stem cells cultivated on microcarriers | |
| JP4804031B2 (en) | Method for culturing mesenchymal stem cells and method for producing biological tissue complement | |
| Lee et al. | Minimal cube explant provides optimal isolation condition of mesenchymal stem cells from umbilical cord | |
| JP4646600B2 (en) | Method for culturing mesenchymal stem cells | |
| EP4134429A1 (en) | Stem cell culturing method for promoting initial yield of stem cel.ls | |
| WO2021227573A1 (en) | Xeno-free culture medium and method for expansion of mesenchymal stem cells by means of using same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20080507 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20101214 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20110510 |
|
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20110527 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20140603 Year of fee payment: 3 |
|
| R151 | Written notification of patent or utility model registration |
Ref document number: 4755460 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R151 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20140603 Year of fee payment: 3 |
|
| S531 | Written request for registration of change of domicile |
Free format text: JAPANESE INTERMEDIATE CODE: R313531 |
|
| R350 | Written notification of registration of transfer |
Free format text: JAPANESE INTERMEDIATE CODE: R350 |
|
| LAPS | Cancellation because of no payment of annual fees |