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JP7731661B2 - Frozen stem cell modification method - Google Patents

Frozen stem cell modification method

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JP7731661B2
JP7731661B2 JP2020142717A JP2020142717A JP7731661B2 JP 7731661 B2 JP7731661 B2 JP 7731661B2 JP 2020142717 A JP2020142717 A JP 2020142717A JP 2020142717 A JP2020142717 A JP 2020142717A JP 7731661 B2 JP7731661 B2 JP 7731661B2
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グラドコフ・アレクセイ
グラドコワ・ニナ
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本発明は、所定温度に冷凍された間葉系幹細胞を活性化させて活性化間葉系幹細胞に改質させる冷凍幹細胞改質方法に関する。 The present invention relates to a method for modifying frozen stem cells, which activates mesenchymal stem cells that have been frozen to a predetermined temperature and modifies them into activated mesenchymal stem cells.

プロナーゼ溶液を用いて幹細胞を剥離する剥離工程と、剥離した幹細胞を幹細胞保存媒体中で緩慢凍結させる凍結工程とを備え、幹細胞保存媒体が、ヒドロキシエチルスターチ(ＨＥＳ)とジメチルスルホキシド(ＤＭＳＯ)とエチレングリコール(ＥＧ)を含有する媒体、或いは、ヒドロキシエチルスターチ(ＨＥＳ)とジメチルスルホキシド(ＤＭＳＯ)と、培地（culture medium）もしくはアルブミン溶液とを含む媒体である幹細胞保存方法が開示されている（特許文献１参照）。 A stem cell preservation method has been disclosed that includes a detachment step in which stem cells are detached using a pronase solution, and a freezing step in which the detached stem cells are slowly frozen in a stem cell preservation medium, where the stem cell preservation medium is a medium containing hydroxyethyl starch (HES), dimethyl sulfoxide (DMSO), and ethylene glycol (EG), or a medium containing hydroxyethyl starch (HES), dimethyl sulfoxide (DMSO), and a culture medium or an albumin solution (see Patent Document 1).

国際公開ＷＯ２０１３／１８７０７７International Publication WO2013/187077

各種の幹細胞は、各種の疾患（心血管疾患や中枢神経系疾患等）の治療や再生医療における再生、美容等の非治療的用途に利用される。培養した幹細胞を冷凍保存する場合、所定の収容容器に幹細胞を収容しつつ、その収容容器にジメチルスルホキシド(ＤＭＳＯ)やヒドロキシエチルスターチ(ＨＥＳ)、エチレングリコール(ＥＧ)等の冷凍時の細胞破壊を防ぐ凍結保護剤が注入される。冷凍保存された冷凍幹細胞を使用する場合、その冷凍幹細胞を解凍する必要があるが、解凍時に凍結保護剤が除去されることはなく、凍結保護剤とともに解凍された幹細胞がそのまま使用される。 Various stem cells are used for non-therapeutic purposes such as treating various diseases (such as cardiovascular disease and central nervous system diseases), regeneration in regenerative medicine, and cosmetic treatments. When cultured stem cells are cryopreserved, they are placed in a designated container and a cryoprotectant such as dimethyl sulfoxide (DMSO), hydroxyethyl starch (HES), or ethylene glycol (EG) is injected into the container to prevent cell destruction during freezing. When cryopreserved stem cells are used, they must be thawed, but the cryoprotectant is not removed during thawing; the thawed stem cells, along with the cryoprotectant, are used as is.

冷凍幹細胞を解凍する際に凍結保護剤を除去しない場合、冷凍幹細胞をのうちの約５０％以上が死滅するとともに、幹細胞を十分に活性化させることができない。生存する幹細胞が減少し、幹細胞を十分に活性化させることができない場合、幹細胞による各種の疾患に対する治療の効果や再生医療における再生の効果、美容等の非治療的用途における効果が低下し、所望の効果が得られない場合がある。又、凍結保護剤を含有した状態の幹細胞を人体に投与する場合、凍結保護剤による悪影響が懸念される。 If the cryoprotectant is not removed when thawing frozen stem cells, approximately 50% or more of the frozen stem cells will die and the stem cells will not be able to be fully activated. If the number of surviving stem cells decreases and the stem cells cannot be fully activated, the effectiveness of stem cell treatments for various diseases, the regenerative effects in regenerative medicine, and the effectiveness in non-therapeutic applications such as cosmetic treatments will be reduced, and the desired effects may not be achieved. Furthermore, if stem cells containing a cryoprotectant are administered to the human body, there are concerns about the adverse effects of the cryoprotectant.

本発明の目的は、冷凍保存された間葉系幹細胞を解凍する場合、凍結保護剤を除去しつつ、大部分の間葉系幹細胞を生存させることができ、冷凍保存された間葉系幹細胞を十分な活性を有する活性化間葉系幹細胞に改質することができる冷凍幹細胞改質方法を提供することにある。本発明の他の目的は、冷凍保存された間葉系幹細胞を解凍する際に、各種の疾患に対する治療や再生医療における再生、美容等の非治療的用途における十分かつ高い効果を備え、所望の効果を得ることが可能な間葉系幹細胞を作ることができる冷凍幹細胞改質方法を提供することにある。 The object of the present invention is to provide a frozen stem cell modification method that, when thawing cryopreserved mesenchymal stem cells, can remove the cryoprotectant while preserving the survival of most mesenchymal stem cells and can modify cryopreserved mesenchymal stem cells into activated mesenchymal stem cells with sufficient activity. Another object of the present invention is to provide a frozen stem cell modification method that, when thawing cryopreserved mesenchymal stem cells, can produce mesenchymal stem cells that are sufficiently and highly effective in the treatment of various diseases, regeneration in regenerative medicine, and non-therapeutic uses such as cosmetics, and can achieve the desired effects.

前記課題を解決するための本発明の前提は、所定温度に冷凍された間葉系幹細胞を活性化させて活性化間葉系幹細胞に改質させる冷凍幹細胞改質方法である。 The premise of the present invention, which aims to solve the above problems, is a method for modifying frozen stem cells, which activates mesenchymal stem cells frozen to a predetermined temperature and modifies them into activated mesenchymal stem cells.

前記前提における本発明の特徴は、冷凍幹細胞改質方法が、ドナーから採取した骨髄液を培養することから作られた間葉系幹細胞を所定容積の冷凍保存容器に収容するとともに、間葉系幹細胞を収容した冷凍保存容器に凍結保護剤を注入し、冷凍保存容器に収容した間葉系幹細胞を使用するまでの間、冷凍保存容器に収容した間葉系幹細胞を凍結保護剤とともに所定温度で冷凍保存する冷凍保存工程と、冷凍保存工程によって冷凍保存した間葉系幹細胞を使用する場合、冷凍保存された間葉系幹細胞及び凍結保護剤を収容した冷凍保存容器を３５～３７℃に保持された温水中に２～３分浸水させて間葉系幹細胞を２～３℃の温度に解凍する幹細胞解凍工程と、幹細胞解凍工程によって冷凍状態から解凍された間葉系幹細胞及び凍結保護剤を収容した冷凍保存容器に洗浄液を注入し、洗浄液を注入した冷凍保存容器を攪拌して洗浄液によって冷凍保存容器内の間葉系幹細胞から凍結保護剤を除去する撹拌除去工程と、撹拌除去工程によって間葉系幹細胞から凍結保護剤を除去した後の冷凍保存容器を遠心分離器に設置し、冷凍保存容器内の間葉系幹細胞、凍結保護剤、洗浄液を層状に遠心分離し、遠心分離した後の冷凍保存容器の最下層に位置する間葉系幹細胞を抽出する間葉系幹細胞抽出工程と、間葉系幹細胞抽出工程によって抽出した間葉系幹細胞を所定容量かつ所定面積の底面を有する第１培養容器に収容するとともに、間葉系幹細胞を収容した第１培養容器に所定の培養液を注入し、間葉系幹細胞を第１培養容器において３６～３７℃で２４時間培養し、第１培養容器内の間葉系幹細胞を活性化させて活性化間葉系幹細胞に改質させる幹細胞活性化工程とを有することにある。 The present invention, based on the above premise, is characterized in that the frozen stem cell modification method includes a cryopreservation step in which mesenchymal stem cells prepared by culturing bone marrow fluid collected from a donor are placed in a cryopreservation container of a predetermined volume, a cryoprotectant is injected into the cryopreservation container containing the mesenchymal stem cells, and the mesenchymal stem cells contained in the cryopreservation container are frozen and stored together with the cryoprotectant at a predetermined temperature until the mesenchymal stem cells are to be used; a stem cell thawing step in which, when the mesenchymal stem cells frozen and stored in the cryopreservation container are to be used, the cryopreservation container containing the frozen and stored mesenchymal stem cells and the cryoprotectant is immersed in warm water maintained at 35-37°C for 2-3 minutes to thaw the mesenchymal stem cells to a temperature of 2-3°C; and a cleaning solution is injected into the cryopreservation container containing the mesenchymal stem cells and the cryoprotectant that have been thawed from a frozen state in the stem cell thawing step. The method comprises: an agitation and removal step in which the cryopreservation container containing the mesenchymal stem cells is agitated and a cleaning solution is used to remove the cryoprotectant from the mesenchymal stem cells in the cryopreservation container; a mesenchymal stem cell extraction step in which the cryopreservation container after the agitation and removal step has been completed is placed in a centrifuge and the mesenchymal stem cells, cryoprotectant, and cleaning solution in the cryopreservation container are centrifuged in layers to extract the mesenchymal stem cells located in the bottom layer of the cryopreservation container after centrifugation; and a stem cell activation step in which the mesenchymal stem cells extracted in the mesenchymal stem cell extraction step are placed in a first culture vessel having a predetermined volume and a predetermined bottom area, a predetermined culture medium is poured into the first culture vessel containing the mesenchymal stem cells, and the mesenchymal stem cells are cultured in the first culture vessel at 36-37°C for 24 hours to activate the mesenchymal stem cells in the first culture vessel and convert them into activated mesenchymal stem cells.

本発明の一例としては、冷凍幹細胞改質方法が、活性化させた活性化間葉系幹細胞を第１培養容器から抽出した後、活性化間葉系幹細胞を抽出後１８～２０時間以内に使用する場合、抽出した活性化間葉系幹細胞を２０～２１℃に保持した状態で１８～２０時間以内に活性化間葉系幹細胞の使用箇所まで搬送する幹細胞搬送工程を含む。 As an example of the present invention, the frozen stem cell modification method includes a stem cell transport step in which, after extracting activated mesenchymal stem cells from the first culture vessel, the extracted activated mesenchymal stem cells are transported to the site where the activated mesenchymal stem cells will be used within 18 to 20 hours while maintained at 20 to 21°C.

本発明の他の一例としては、幹細胞活性化工程が、ドナーから採取した骨髄液を培養する培養過程において生成された培養生成液を第１培養容器に注入し、培養液及び培養生成液を利用して間葉系幹細胞を第１培養容器において３５～３７℃の温度で２４時間培養する。 In another example of the present invention, the stem cell activation step involves injecting a culture product solution produced during the culture process of culturing bone marrow fluid collected from a donor into a first culture vessel, and culturing mesenchymal stem cells in the first culture vessel using the culture solution and culture product solution at a temperature of 35-37°C for 24 hours.

本発明の他の一例としては、幹細胞活性化工程が、第１培養容器を２～５度の角度に傾斜させた状態で第１培養容器を３５～３７℃の温度で２４時間静的に放置する。 In another example of the present invention, the stem cell activation step involves leaving the first culture vessel statically tilted at an angle of 2 to 5 degrees at a temperature of 35 to 37°C for 24 hours.

本発明の他の一例としては、幹細胞活性化工程が、第１培養容器を一方向へ２～５度の角度に傾斜させた状態で第１培養容器を３５～３７℃の温度で１２時間静的に放置し、一方向へ傾斜させた第１培養容器を一方向と反対の他方向へ２～５度の角度に傾斜させた状態で第１培養容器を３５～３７℃の温度で１２時間静的に放置する。 In another example of the present invention, the stem cell activation process involves statically leaving the first culture vessel tilted in one direction at an angle of 2 to 5 degrees at a temperature of 35 to 37°C for 12 hours, and then tilting the first culture vessel tilted in one direction at an angle of 2 to 5 degrees in the opposite direction at a temperature of 35 to 37°C for 12 hours.

本発明の他の一例としては、凍結保護剤が、ヒドロキシエチルスターチ(ＨＥＳ)とジメチルスルホキシド(ＤＭＳＯ)とエチレングリコール(ＥＧ)とのうちの少なくとも該ジメチルスルホキシド(ＤＭＳＯ)であり、洗浄液が、ダルベッコりん酸緩衝生理食塩水（Ｄ－ＰＢＳ）又はりん酸緩衝生理食塩水（ＰＢＳ）である。 In another example of the present invention, the cryoprotectant is at least dimethyl sulfoxide (DMSO) out of hydroxyethyl starch (HES), dimethyl sulfoxide (DMSO), and ethylene glycol (EG), and the washing solution is Dulbecco's phosphate buffered saline (D-PBS) or phosphate buffered saline (PBS).

本発明の他の一例としては、間葉系幹細胞が、ドナーから採取した骨髄液を層状に分離し、層状に分離させた骨髄液のうちの中間層に位置する中間層骨髄液を抽出し、所定容量かつ所定面積の底面を有する第２培養容器に中間層骨髄液と所定の培養液とを注入して中間層骨髄液に含まれる第１間葉系幹細胞を第２培養容器の底面に定着させる幹細胞第１定着工程と、幹細胞第１定着工程によって第１間葉系幹細胞を第２培養容器の底面に定着させた後、第２培養容器内の培養液を排出しつつあらたな培養液を第２培養容器に注入して第１間葉系幹細胞を培養し、第２培養容器の底面面積に対する第１間葉系幹細胞の総平面面積が第１目標割合に達するまで第１間葉系幹細胞を増殖させる幹細胞第１培養工程と、幹細胞第１培養工程によって増殖させた第１間葉系幹細胞及び培養液を収容した第２培養容器を遠心分離器に設置し、第２培養容器内の第１間葉系幹細胞、培養液を層状に遠心分離し、遠心分離した後の第２培養容器の最下層に位置する第２間葉系幹細胞を抽出する第２間葉系幹細胞第１抽出工程と、所定容量かつ所定面積の底面を有して第２培養容器よりも大きい容量の第３培養容器に第２間葉系幹細胞第１抽出工程によって抽出した第２間葉系幹細胞を収容しつつ、第３培養容器にあらたな培養液を注入し、第２間葉系幹細胞を第３培養容器の底面に定着させる幹細胞第２定着工程と、幹細胞第２定着工程によって第２間葉系幹細胞を第３培養容器の底面に定着させた後、第３培養容器内の培養液を排出しつつあらたな培養液を第３培養容器に注入して第２間葉系幹細胞を培養し、第３培養容器の底面面積に対する第２間葉系幹細胞の総平面面積が第２目標割合に達するまで第２幹細胞を増殖させる幹細胞第２培養工程と、幹細胞第２培養工程によって培養された第２間葉系幹細胞を含む第３培養容器を遠心分離器に設置し、第３培養容器内の第２間葉系幹細胞、培養液を層状に遠心分離し、遠心分離した後の第３培養容器の最下層に位置する第２間葉系幹細胞を抽出する第２間葉系幹細胞第２抽出工程とを有する間葉系幹細胞培養方法によって作られている。 Another example of the present invention is a method for culturing mesenchymal stem cells, comprising: separating bone marrow fluid collected from a donor into layers; extracting intermediate layer bone marrow fluid from the intermediate layer of the separated layers; injecting the intermediate layer bone marrow fluid and a predetermined culture medium into a second culture vessel having a predetermined volume and a predetermined bottom area, thereby causing first mesenchymal stem cells contained in the intermediate layer bone marrow fluid to settle on the bottom of the second culture vessel; and, after the first mesenchymal stem cells have settled on the bottom of the second culture vessel by the first stem cell settlement step, draining the culture medium from the second culture vessel. a first stem cell culture step of injecting the new culture medium into a second culture vessel to culture the first mesenchymal stem cells and proliferating the first mesenchymal stem cells until a total planar area of the first mesenchymal stem cells relative to a bottom area of the second culture vessel reaches a first target ratio; and a second mesenchymal stem cell culture step of placing the second culture vessel containing the first mesenchymal stem cells proliferated in the first stem cell culture step and the culture medium in a centrifuge, centrifuging the first mesenchymal stem cells and the culture medium in the second culture vessel in layers, and extracting second mesenchymal stem cells located in the bottom layer of the second culture vessel after centrifugation. a first cell extraction step, a second stem cell fixation step of placing the second mesenchymal stem cells extracted in the first second mesenchymal stem cell extraction step in a third culture vessel having a predetermined volume and a bottom surface with a predetermined area and a volume larger than that of the second culture vessel, injecting a new culture medium into the third culture vessel, and fixing the second mesenchymal stem cells to the bottom surface of the third culture vessel, and a second stem cell fixation step of fixing the second mesenchymal stem cells to the bottom surface of the third culture vessel after fixing the second mesenchymal stem cells to the bottom surface of the third culture vessel in the second stem cell fixation step, injecting a new culture medium into the third culture vessel while discharging the culture medium in the third culture vessel, The mesenchymal stem cells are produced by a mesenchymal stem cell culture method including: a second stem cell culture step of culturing cells and proliferating the second stem cells until the total planar area of the second mesenchymal stem cells relative to the bottom area of the third culture vessel reaches a second target ratio; and a second mesenchymal stem cell extraction step of placing the third culture vessel containing the second mesenchymal stem cells cultured in the second stem cell culture step in a centrifuge, centrifuging the second mesenchymal stem cells and culture solution in the third culture vessel in layers, and extracting the second mesenchymal stem cells located in the bottom layer of the third culture vessel after centrifugation.

本発明の他の一例としては、冷凍保存工程が、第２間葉系幹細胞第２抽出工程によって抽出された直後の第２間葉系幹細胞を冷凍保存容器に収容し、冷凍保存容器に収容した間葉系幹細胞を凍結保護剤とともに直ちに冷凍保存する。 In another example of the present invention, the cryopreservation step involves placing the second mesenchymal stem cells in a cryopreservation container immediately after extraction in the second mesenchymal stem cell extraction step, and immediately freezing and preserving the mesenchymal stem cells placed in the cryopreservation container together with a cryoprotectant.

本発明の他の一例としては、培養生成液が、第２培養容器又は第３培養容器から培養後の間葉系幹細胞を抽出した後に残った培養液であり、間葉系幹細胞の培養過程において間葉系幹細胞から分泌された所定の代謝物質を含む。 In another example of the present invention, the culture solution is the culture solution remaining after extracting cultured mesenchymal stem cells from the second culture vessel or the third culture vessel, and contains specific metabolic substances secreted from the mesenchymal stem cells during the mesenchymal stem cell culture process.

本発明に係る冷凍幹細胞改質方法によれば、冷凍状態から解凍された間葉系幹細胞及び凍結保護剤を収容した冷凍保存容器に洗浄液を注入し、洗浄液を注入した冷凍保存容器を攪拌して洗浄液によって冷凍保存容器内の間葉系幹細胞から凍結保護剤を除去し、冷凍保存容器内の間葉系幹細胞、凍結保護剤、洗浄液を層状に遠心分離し、遠心分離した後の冷凍保存容器の最下層に位置する間葉系幹細胞を抽出するから、冷凍保存された間葉系幹細胞を解凍する際に洗浄液を利用して凍結保護剤を除去することができるとともに、冷凍状態から解凍された間葉系幹細胞の大部分（７０～９０％）を生存させることができる。冷凍幹細胞改質方法は、冷凍状態から解凍された間葉系幹細胞を所定容量かつ所定面積の底面を有する第１培養容器に収容するとともに、間葉系幹細胞を収容した第１培養容器に所定の培養液を注入し、間葉系幹細胞を第１培養容器において３５～３７℃で２４時間培養し、第１培養容器内の間葉系幹細胞を活性化させるから、冷凍保存された間葉系幹細胞を解凍する際に、解凍された間葉系幹細胞を十分な活性を有する活性化間葉系幹細胞に改質することができる。冷凍幹細胞改質方法は、各種疾患（心血管疾患や中枢神経系疾患等）の治療や再生医療における再生、美容等の非治療的用途における十分かつ高い効果を有する活性化間葉系幹細胞を作ることができ、所望の効果を得ることが可能な活性化間葉系幹細胞を作ることができる。冷凍幹細胞改質方法は、各種疾患の治療に好適かつタイムリーに使用することが可能な活性化間葉系幹細胞を作ることができ、各種組織や各種臓器を再生に好適かつタイムリーに使用することが可能な活性化間葉系幹細胞を作ることができるとともに、美容等の非治療的用途に好適かつタイムリーに使用することが可能な活性化間葉系幹細胞を作ることができる。冷凍幹細胞改質方法は、解凍された活性化間葉系幹細胞に凍結保護剤が含まれていないから、冷凍幹細胞改質方法によって作られた活性化間葉系幹細胞を人体に投与下としても、人体に悪影響を与えることはない。 The frozen stem cell modification method of the present invention involves injecting a cleaning solution into a cryopreservation container containing mesenchymal stem cells and a cryoprotectant that have been thawed from a frozen state, agitating the cryopreservation container with the cleaning solution to remove the cryoprotectant from the mesenchymal stem cells in the cryopreservation container, centrifuging the mesenchymal stem cells, cryoprotectant, and cleaning solution in layers within the cryopreservation container, and extracting the mesenchymal stem cells located in the bottom layer of the cryopreservation container after centrifugation. Therefore, when thawing frozen mesenchymal stem cells, the cleaning solution can be used to remove the cryoprotectant, and the majority (70-90%) of the mesenchymal stem cells thawed from a frozen state can be kept alive. The frozen stem cell modification method involves placing mesenchymal stem cells thawed from a frozen state in a first culture vessel having a predetermined volume and a predetermined bottom area, pouring a predetermined culture medium into the first culture vessel containing the mesenchymal stem cells, and culturing the mesenchymal stem cells in the first culture vessel at 35-37°C for 24 hours to activate the mesenchymal stem cells in the first culture vessel, thereby enabling the thawed mesenchymal stem cells to be modified into activated mesenchymal stem cells with sufficient activity when thawed. The frozen stem cell modification method can produce activated mesenchymal stem cells that are sufficient and highly effective in the treatment of various diseases (such as cardiovascular disease and central nervous system disease), regeneration in regenerative medicine, and non-therapeutic uses such as cosmetics, and can produce activated mesenchymal stem cells that can achieve desired effects. The frozen stem cell modification method can produce activated mesenchymal stem cells that can be used appropriately and in a timely manner to treat various diseases, can produce activated mesenchymal stem cells that can be used appropriately and in a timely manner to regenerate various tissues and organs, and can produce activated mesenchymal stem cells that can be used appropriately and in a timely manner for non-therapeutic purposes such as cosmetic treatments. Because the frozen stem cell modification method does not contain a cryoprotectant in the thawed activated mesenchymal stem cells, administering activated mesenchymal stem cells produced by the frozen stem cell modification method to the human body will not have any adverse effects.

活性化させた活性化間葉系幹細胞を第１培養容器から抽出した後、活性化間葉系幹細胞を抽出後１８～２０時間以内に使用する場合、抽出した活性化間葉系幹細胞を２０～２１℃に保持した状態で１８～２０時間以内に活性化間葉系幹細胞の使用箇所まで搬送する幹細胞搬送工程を含む冷凍幹細胞改質方法は、活性化間葉系幹細胞を２０～２１℃に保持した状態で１８～２０時間以内に活性化間葉系幹細胞の使用箇所まで搬送することで、活性化間葉系幹細胞の活性が失われず、その活性化間葉系幹細胞を各種疾患（心血管疾患や中枢神経系疾患等）の治療に利用することができ、その活性化間葉系幹細胞を再生医療における再生に利用することができるとともに、その活性化間葉系幹細胞を美容等の非治療的用途に利用することができる。 When the activated mesenchymal stem cells are to be used within 18 to 20 hours after extraction from the first culture vessel, the frozen stem cell modification method includes a stem cell transport step in which the extracted activated mesenchymal stem cells are transported to the site of use within 18 to 20 hours while maintained at 20 to 21°C. By transporting the activated mesenchymal stem cells to the site of use within 18 to 20 hours while maintained at 20 to 21°C, the activity of the activated mesenchymal stem cells is not lost, and the activated mesenchymal stem cells can be used to treat various diseases (such as cardiovascular disease and central nervous system disease). The activated mesenchymal stem cells can be used for regeneration in regenerative medicine, and can also be used for non-therapeutic purposes such as cosmetic treatment.

幹細胞活性化工程において、ドナーから採取した骨髄液を培養する培養過程において生成された培養生成液を第１培養容器に注入し、培養液及び培養生成液を利用して間葉系幹細胞を第１培養容器において３５～３７℃の温度で２４時間培養する冷凍幹細胞改質方法は、冷凍保存前の間葉系幹細胞の培養過程において生成された培養生成液を利用することによって第１培養容器において間葉系幹細胞の活性化が確実に促進されるから、培養生成液を利用して解凍後の間葉系幹細胞を２４時間で活性化させることができ、十分な活性を有する活性化間葉系幹細胞を効率よく製造することができるとともに、凍結保護剤を除去した間葉系幹細胞を速やかに利用することができる。 In this method of modifying frozen stem cells, the stem cell activation step involves injecting the culture product solution produced during the culturing process of bone marrow fluid collected from a donor into a first culture vessel, and culturing mesenchymal stem cells in the first culture vessel at a temperature of 35-37°C for 24 hours using the culture solution and culture product solution. By utilizing the culture product solution produced during the culturing process of mesenchymal stem cells before cryopreservation, the activation of mesenchymal stem cells in the first culture vessel is reliably promoted. Therefore, the culture product solution can be used to activate thawed mesenchymal stem cells within 24 hours, allowing for the efficient production of activated mesenchymal stem cells with sufficient activity and the rapid use of mesenchymal stem cells from which the cryoprotectant has been removed.

幹細胞活性化工程において、第１培養容器を２～５度の角度に傾斜させた状態で第１培養容器を３５～３７℃の温度で２４時間静的に放置する冷凍幹細胞改質方法は、第１培養容器を２～５度の角度に傾斜させることで、第１培養容器内において解凍後の間葉系幹細胞、培養液（及び培養生成液）が第１培養容器の一方に偏り、第１培養容器の一方において間葉系幹細胞、培養液（及び培養生成液）の圧力が大きくなるとともに間葉系幹細胞が第１培養容器の一方に集中し、それによって間葉系幹細胞どうしの活性を高めることができ、解凍された間葉系幹細胞を十分な活性を有する活性化間葉系幹細胞に改質することができる。 In this method of modifying frozen stem cells, the stem cell activation step involves leaving the first culture vessel tilted at an angle of 2 to 5 degrees and statically storing it at a temperature of 35 to 37°C for 24 hours. By tilting the first culture vessel at an angle of 2 to 5 degrees, the thawed mesenchymal stem cells and culture medium (and culture product solution) are concentrated on one side of the first culture vessel, increasing the pressure of the mesenchymal stem cells and culture medium (and culture product solution) on one side of the first culture vessel and concentrating the mesenchymal stem cells on that side, thereby increasing the activity of the mesenchymal stem cells and modifying the thawed mesenchymal stem cells into activated mesenchymal stem cells with sufficient activity.

幹細胞活性化工程において、第１培養容器を一方向へ２～５度の角度に傾斜させた状態で第１培養容器を３５～３７℃の温度で１２時間静的に放置し、一方向へ傾斜させた第１培養容器を一方向と反対の他方向へ２～５度の角度に傾斜させた状態で第１培養容器を３５～３７℃の温度で１２時間静的に放置する冷凍幹細胞改質方法は、第１培養容器を一方向へ２～５度の角度に傾斜させ、更に、一方向へ傾斜させた第１培養容器を一方向と反対の他方向へ２～５度の角度に傾斜させることで、第１培養容器内において解凍後の間葉系幹細胞、培養液（及び培養生成液）が第１培養容器の一方又は他方へ偏り、第１培養容器の一方又は他方において間葉系幹細胞、培養液（及び培養生成液）の圧力が大きくなるとともに間葉系幹細胞が第１培養容器の一方又は他方へ満遍なく集中し、それによって間葉系幹細胞どうしの活性を高めることができ、解凍された間葉系幹細胞を十分な活性を有する活性化間葉系幹細胞に改質することができる。 In the stem cell activation process, the first culture vessel is tilted at an angle of 2 to 5 degrees in one direction and left statically at a temperature of 35 to 37°C for 12 hours, and the first culture vessel tilted in one direction is tilted at an angle of 2 to 5 degrees in the opposite direction from the first direction and left statically at a temperature of 35 to 37°C for 12 hours. The frozen stem cell modification method involves tilting the first culture vessel at an angle of 2 to 5 degrees in one direction and then tilting the first culture vessel tilted in one direction at an angle of 2 to 5 degrees in the opposite direction from the first direction. By tilting the container at this angle, the thawed mesenchymal stem cells and culture medium (and culture product solution) in the first culture container are biased to one side or the other of the first culture container, increasing the pressure of the mesenchymal stem cells and culture medium (and culture product solution) on one side or the other of the first culture container and causing the mesenchymal stem cells to concentrate evenly on one side or the other of the first culture container, thereby increasing the activity of the mesenchymal stem cells and converting the thawed mesenchymal stem cells into activated mesenchymal stem cells with sufficient activity.

凍結保護剤がヒドロキシエチルスターチ(ＨＥＳ)とジメチルスルホキシド(ＤＭＳＯ)とエチレングリコール(ＥＧ)とのうちの少なくともジメチルスルホキシド(ＤＭＳＯ)であり、洗浄液がダルベッコりん酸緩衝生理食塩水（Ｄ－ＰＢＳ）又はりん酸緩衝生理食塩水（ＰＢＳ）である冷凍幹細胞改質方法は、冷凍状態から解凍された間葉系幹細胞及び凍結保護剤を収容した冷凍保存容器に洗浄液（ダルベッコりん酸緩衝生理食塩水（Ｄ－ＰＢＳ）又はりん酸緩衝生理食塩水（ＰＢＳ））を注入し、洗浄液を注入した冷凍保存容器を攪拌して洗浄液によって冷凍保存容器内の間葉系幹細胞から凍結保護剤（ヒドロキシエチルスターチ(ＨＥＳ)とジメチルスルホキシド(ＤＭＳＯ)とエチレングリコール(ＥＧ)とのうちの少なくともジメチルスルホキシド(ＤＭＳＯ)）を除去し、冷凍保存容器内の間葉系幹細胞、凍結保護剤、洗浄液を層状に遠心分離し、遠心分離した後の冷凍保存容器の最下層に位置する間葉系幹細胞を抽出するから、冷凍保存された間葉系幹細胞を解凍する際に洗浄液（ダルベッコりん酸緩衝生理食塩水（Ｄ－ＰＢＳ）又はりん酸緩衝生理食塩水（ＰＢＳ））を利用して凍結保護剤（ヒドロキシエチルスターチ(ＨＥＳ)とジメチルスルホキシド(ＤＭＳＯ)とエチレングリコール(ＥＧ)とのうちの少なくともジメチルスルホキシド(ＤＭＳＯ)）を確実に除去することができ、解凍において大部分（７０～９０％）の間葉系幹細胞を生存させることができるとともに、解凍された間葉系幹細胞を十分な活性を有する活性化間葉系幹細胞に改質することができる。 The frozen stem cell modification method, in which the cryoprotectant is at least dimethyl sulfoxide (DMSO) of hydroxyethyl starch (HES), dimethyl sulfoxide (DMSO), and ethylene glycol (EG), and the washing solution is Dulbecco's phosphate buffered saline (D-PBS) or phosphate buffered saline (PBS), involves injecting the washing solution (Dulbecco's phosphate buffered saline (D-PBS) or phosphate buffered saline (PBS)) into a cryopreservation container containing mesenchymal stem cells thawed from a frozen state and the cryoprotectant, stirring the cryopreservation container into which the washing solution has been injected, and removing the cryoprotectant (at least dimethyl sulfoxide (DMSO) of hydroxyethyl starch (HES), dimethyl sulfoxide (DMSO), and ethylene glycol (EG)) from the mesenchymal stem cells in the cryopreservation container by the washing solution. The cryoprotectant (hydroxyethyl starch (HES), dimethyl sulfoxide (DMSO) and ethylene glycol (EG), at least dimethyl sulfoxide (DMSO)) is removed, and the mesenchymal stem cells, cryoprotectant and washing solution in the cryopreservation container are centrifuged in layers, and the mesenchymal stem cells located in the bottom layer of the cryopreservation container after centrifugation are extracted. Therefore, when thawing the frozen mesenchymal stem cells, the washing solution (Dulbecco's phosphate buffered saline (D-PBS) or phosphate buffered saline (PBS)) is used to reliably remove the cryoprotectant (hydroxyethyl starch (HES), dimethyl sulfoxide (DMSO) and ethylene glycol (EG)), allowing the majority (70-90%) of the mesenchymal stem cells to survive during thawing, and enabling the thawed mesenchymal stem cells to be converted into activated mesenchymal stem cells with sufficient activity.

間葉系幹細胞が、ドナーから採取した骨髄液を層状に分離し、層状に分離させた骨髄液のうちの中間層に位置する中間層骨髄液を抽出し、所定容量かつ所定面積の底面を有する第２培養容器に中間層骨髄液と所定の培養液とを注入して中間層骨髄液に含まれる第１間葉系幹細胞を第２培養容器の底面に定着させる幹細胞第１定着工程と、幹細胞第１定着工程によって第１間葉系幹細胞を第２培養容器の底面に定着させた後、第２培養容器内の培養液を排出しつつあらたな培養液を第２培養容器に注入して第１間葉系幹細胞を培養し、第２培養容器の底面面積に対する第１間葉系幹細胞の総平面面積が第１目標割合に達するまで第１間葉系幹細胞を増殖させる幹細胞第１培養工程と、幹細胞第１培養工程によって増殖させた第１間葉系幹細胞及び培養液を収容した第２培養容器を遠心分離器に設置し、第２培養容器内の第１間葉系幹細胞、培養液を層状に遠心分離し、遠心分離した後の第２培養容器の最下層に位置する第２間葉系幹細胞を抽出する第２間葉系幹細胞第１抽出工程と、所定容量かつ所定面積の底面を有して第２培養容器よりも大きい容量の第３培養容器に第２間葉系幹細胞第１抽出工程によって抽出した第２間葉系幹細胞を収容しつつ、第３培養容器にあらたな培養液を注入し、第２間葉系幹細胞を第３培養容器の底面に定着させる幹細胞第２定着工程と、幹細胞第２定着工程によって第２間葉系幹細胞を第３培養容器の底面に定着させた後、第３培養容器内の培養液を排出しつつあらたな培養液を第３培養容器に注入して第２間葉系幹細胞を培養し、第３培養容器の底面面積に対する第２間葉系幹細胞の総平面面積が第２目標割合に達するまで第２幹細胞を増殖させる幹細胞第２培養工程と、幹細胞第２培養工程によって培養された第２間葉系幹細胞を含む第３培養容器を遠心分離器に設置し、第３培養容器内の第２間葉系幹細胞、培養液を層状に遠心分離し、遠心分離した後の第３培養容器の最下層に位置する第２間葉系幹細胞を抽出する第２間葉系幹細胞第２抽出工程とを有する間葉系幹細胞培養方法によって作られている冷凍幹細胞改質方法は、第１間葉系幹細胞を第２培養容器の底面に定着させた後、培養液を第２培養容器から排出しつつ、あらたな培養液を第２培養容器に注入し、第２培養容器の底面に定着させた第１間葉系幹細胞をあらたな培養液を利用して培養し、第２培養容器において増殖させた第２間葉系幹細胞を第３培養容器に収容し、第２間葉系幹細胞を第３培養容器の底面に定着させた後、培養液を第３培養容器から排出しつつ、あらたな培養液を第３培養容器に注入し、第３培養容器の底面に定着させた第２間葉系幹細胞をあらたな培養液を利用して培養し、第３培養容器において第２間葉系幹細胞を増殖させるから、間葉系幹細胞を効率よく確実に増殖させることができ、十分な活性を有する間葉系幹細胞を効率よく作ることができる。 A first stem cell settling step in which bone marrow fluid collected from a donor is separated into layers, intermediate layer bone marrow fluid from the separated layers is extracted, and the intermediate layer bone marrow fluid and a predetermined culture medium are poured into a second culture vessel having a predetermined volume and a predetermined bottom area, thereby causing first mesenchymal stem cells contained in the intermediate layer bone marrow fluid to settle on the bottom of the second culture vessel; and a first stem cell culture in which, after the first mesenchymal stem cells have settled on the bottom of the second culture vessel in the first stem cell settling step, the culture medium in the second culture vessel is discharged and a new culture medium is poured into the second culture vessel to culture the first mesenchymal stem cells, and the first mesenchymal stem cells are proliferated until the total planar area of the first mesenchymal stem cells relative to the bottom area of the second culture vessel reaches a first target ratio. a second mesenchymal stem cell first extraction step of placing a second culture vessel containing the first mesenchymal stem cells proliferated in the first stem cell culture step and a culture medium in a centrifuge, centrifuging the first mesenchymal stem cells and the culture medium in the second culture vessel in layers, and extracting the second mesenchymal stem cells located in the bottom layer of the second culture vessel after centrifugation; a stem cell second fixation step of placing the second mesenchymal stem cells extracted in the first second mesenchymal stem cell extraction step in a third culture vessel having a predetermined volume and a predetermined bottom area and a larger volume than the second culture vessel, and injecting a new culture medium into the third culture vessel to fix the second mesenchymal stem cells to the bottom surface of the third culture vessel; The method for modifying frozen stem cells is produced by a mesenchymal stem cell culture method including a second stem cell culture step in which, after the first mesenchymal stem cells have settled, the culture medium in the third culture vessel is discharged and a new culture medium is poured into the third culture vessel to culture the second mesenchymal stem cells, and the second stem cells are proliferated until the total planar area of the second mesenchymal stem cells relative to the bottom area of the third culture vessel reaches a second target ratio; and a second mesenchymal stem cell extraction step in which the third culture vessel containing the second mesenchymal stem cells cultured in the second stem cell culture step is placed in a centrifuge, the second mesenchymal stem cells and the culture medium in the third culture vessel are centrifuged in layers, and the second mesenchymal stem cells located in the bottom layer of the third culture vessel after centrifugation are extracted. After the first mesenchymal stem cells have settled to the bottom surface of the second culture vessel, the culture medium is discharged from the second culture vessel and new culture medium is poured into the second culture vessel. The first mesenchymal stem cells that have settled to the bottom surface of the second culture vessel are cultured in the new culture medium. The second mesenchymal stem cells that have proliferated in the second culture vessel are placed in a third culture vessel. After the second mesenchymal stem cells have settled to the bottom surface of the third culture vessel, the culture medium is discharged from the third culture vessel and new culture medium is poured into the third culture vessel. The second mesenchymal stem cells that have settled to the bottom surface of the third culture vessel are cultured in the new culture medium. The second mesenchymal stem cells are proliferated in the third culture vessel. This allows mesenchymal stem cells to be proliferated efficiently and reliably, and mesenchymal stem cells with sufficient activity can be efficiently produced.

冷凍保存工程において、第２間葉系幹細胞第２抽出工程によって抽出された直後の第２間葉系幹細胞を冷凍保存容器に収容し、冷凍保存容器に収容した間葉系幹細胞を凍結保護剤とともに直ちに冷凍保存する冷凍幹細胞改質方法は、間葉系幹細胞培養方法によって作られた十分な活性を有する間葉系幹細胞を直ちに冷凍保存するから、冷凍保存容器に収容された間葉系幹細胞の大部分を生きたまま冷凍保存することができ、間葉系幹細胞培養方法によって作られた間葉系幹細胞を長期間保存することができる。 In the cryopreservation step, the second mesenchymal stem cells immediately after extraction in the second mesenchymal stem cell extraction step are placed in a cryopreservation container, and the mesenchymal stem cells placed in the cryopreservation container are immediately frozen and preserved together with a cryoprotectant. This frozen stem cell modification method immediately freezes and preserves mesenchymal stem cells with sufficient activity produced by the mesenchymal stem cell culture method, allowing the majority of the mesenchymal stem cells placed in the cryopreservation container to be frozen and preserved alive, and mesenchymal stem cells produced by the mesenchymal stem cell culture method to be preserved for long periods of time.

培養生成液が第２培養容器又は第３培養容器から培養後の間葉系幹細胞を抽出した後に残った培養液であり、間葉系幹細胞の培養過程において間葉系幹細胞から分泌された所定の代謝物質を含む冷凍幹細胞改質方法は、間葉系幹細胞の培養過程において間葉系幹細胞から分泌された所定の代謝物質を含むから、間葉系幹細胞自体の代謝物質がトリガーとなり、間葉系幹細胞が速やかに活性を開始するとともに、その培養生成液を利用して間葉系幹細胞を速やかに活性化させることができ、活性化間葉系幹細胞を短時間に効率よく作ることができる。 In this frozen stem cell modification method, the culture product solution is the culture solution remaining after extracting cultured mesenchymal stem cells from the second or third culture vessel, and contains specific metabolic substances secreted by mesenchymal stem cells during the mesenchymal stem cell culture process. Because the culture product solution contains specific metabolic substances secreted by mesenchymal stem cells during the mesenchymal stem cell culture process, the metabolic substances in the mesenchymal stem cells themselves act as a trigger, causing the mesenchymal stem cells to quickly begin to become active. The culture product solution can then be used to quickly activate the mesenchymal stem cells, allowing activated mesenchymal stem cells to be produced efficiently in a short period of time.

一例として示す冷凍幹細胞改質システムの概略構成図。1 is a schematic diagram of an example frozen stem cell reforming system. 間葉系幹細胞培養方法における骨髄液分離工程の一例を示す説明図An explanatory diagram showing an example of a bone marrow fluid separation step in a mesenchymal stem cell culture method. 図２から続く骨髄液分離工程の説明図。An explanatory diagram of the bone marrow fluid separation process, continuing from Figure 2. 形状変形第１観察工程の一例を示す説明図。FIG. 10 is an explanatory diagram showing an example of a first shape deformation observation step. 第１扁平培養容器（第２培養容器）の側面図。FIG. 2 is a side view of the first flat culture vessel (second culture vessel). 間葉系幹細胞培養方法における第２間葉系幹細胞第１抽出工程の一例を示す説明図。FIG. 2 is an explanatory diagram showing an example of a first extraction step of second mesenchymal stem cells in the mesenchymal stem cell culture method. 間葉系幹細胞培養方法における形状変形第２観察工程の一例を示す説明図。FIG. 10 is an explanatory diagram showing an example of a second shape deformation observation step in the mesenchymal stem cell culture method. 第２扁平培養容器（第３培養容器）の側面図。FIG. 10 is a side view of the second flat culture vessel (third culture vessel). 冷凍幹細胞改質方法における冷凍保存工程の一例を示す説明図。FIG. 1 is an explanatory diagram showing an example of the cryopreservation process in the frozen stem cell modification method. 冷凍幹細胞改質方法における幹細胞解凍工程の一例を示す説明図。FIG. 1 is an explanatory diagram showing an example of a stem cell thawing process in a frozen stem cell modification method. 冷凍幹細胞改質方法における撹拌除去工程の一例を示す説明図。FIG. 1 is an explanatory diagram showing an example of the stirring and removal process in the frozen stem cell modification method. 冷凍幹細胞改質方法における間葉系幹細胞抽出工程の一例を示す説明図。FIG. 1 is an explanatory diagram showing an example of a mesenchymal stem cell extraction process in a frozen stem cell modification method. 冷凍幹細胞改質方法における幹細胞活性化工程の一例を示す説明図。FIG. 1 is an explanatory diagram showing an example of a stem cell activation step in a frozen stem cell modification method. 第３扁平培養容器（第１培養容器）の側面図。FIG. 10 is a side view of the third flat culture vessel (first culture vessel). 第２間葉系幹細胞の平面形状の一例を示す部分拡大図。FIG. 10 is a partially enlarged view showing an example of the planar shape of the second mesenchymal stem cells. 第２間葉系幹細胞の平面形状の他の一例を示す部分拡大図。FIG. 10 is a partially enlarged view showing another example of the planar shape of the second mesenchymal stem cells. 冷凍幹細胞改質方法における幹細胞搬送工程の一例を示す説明図。FIG. 1 is an explanatory diagram showing an example of a stem cell transport process in a frozen stem cell modification method.

一例として示す冷凍幹細胞改質システム１０の概略構成図である図１等の添付の図面を参照し、本発明に係る冷凍幹細胞改質方法の詳細を説明すると、以下のとおりである。尚、図２は、間葉系幹細胞培養方法における骨髄液分離工程の一例を示す説明図であり、図３は、図２から続く骨髄液分離工程の説明図である。図４は、形状変形第１観察工程の一例を示す説明図であり、図５は、第１扁平培養容器（第２培養容器）の側面図である。 The frozen stem cell modification method of the present invention will be described in detail below with reference to the accompanying drawings, such as Figure 1, which shows a schematic diagram of an example frozen stem cell modification system 10. Figure 2 is an explanatory diagram showing an example of the bone marrow fluid separation step in the mesenchymal stem cell culture method, and Figure 3 is an explanatory diagram of the bone marrow fluid separation step continuing from Figure 2. Figure 4 is an explanatory diagram showing an example of the first shape deformation observation step, and Figure 5 is a side view of the first flat culture vessel (second culture vessel).

冷凍幹細胞改質システム１０は、幹細胞培養方法によって特定種類の単一種間葉系幹細胞を培養（製造）し、冷凍保存された単一種の間葉系幹細胞を冷凍幹細胞改質方法によって活性化間葉系幹細胞に改質する。幹細胞培養方法は、複数のドナー（人）から採取した第１骨髄液を利用し、幹細胞第１定着工程、幹細胞第１培養工程、第２間葉系幹細胞第１抽出工程、幹細胞第２定着工程、幹細胞第２培養工程、第２間葉系幹細胞第２抽出工程を実施することにより、第１骨髄液に含まれる複数種類の間葉系幹細胞の中から特定種類の単一種間葉系幹細胞を培養（製造）する。 The frozen stem cell modification system 10 cultures (manufactures) a specific type of single-species mesenchymal stem cells using a stem cell culture method, and modifies cryopreserved single-species mesenchymal stem cells into activated mesenchymal stem cells using the frozen stem cell modification method. The stem cell culture method uses first bone marrow fluid collected from multiple donors (people) and performs a first stem cell colonization step, a first stem cell culture step, a first second mesenchymal stem cell extraction step, a second stem cell colonization step, a second stem cell culture step, and a second second mesenchymal stem cell extraction step to culture (manufacture) a specific type of single-species mesenchymal stem cells from the multiple types of mesenchymal stem cells contained in the first bone marrow fluid.

冷凍幹細胞改質システム１０は、管理サーバ１１、管理サーバ１１にインターフェイス（無線または有線）を介して接続されたＩＣタグリーダ／ライタ１２、管理サーバ１１にインターフェイス（無線または有線）を介して接続された電子顕微鏡１３、管理サーバ１１にインターフェイス（無線または有線）を介して接続された冷蔵庫１４又は冷凍庫１４から形成されている。電子顕微鏡１３は、撮像素子によって被写体の拡大画像を撮影する画像撮影機能を有するとともに、その拡大画像を管理サーバ１１に送信する画像送信機能を有する。 The frozen stem cell modification system 10 is made up of a management server 11, an IC tag reader/writer 12 connected to the management server 11 via an interface (wireless or wired), an electron microscope 13 connected to the management server 11 via an interface (wireless or wired), and a refrigerator 14 or freezer 14 connected to the management server 11 via an interface (wireless or wired). The electron microscope 13 has an image capture function that captures a magnified image of a subject using an image sensor, and an image transmission function that transmits the magnified image to the management server 11.

管理サーバ１１は、中央処理部（ＣＰＵ又はＭＰＵ、仮想ＣＰＵ又は仮想ＭＰＵ）とメモリ（メインメモリ及びキャッシュメモリ、仮想メインメモリ及び仮想キャッシュメモリ）とを備えた物理的なコンピュータ又はクラウドコンピューティングであり、大容量記憶領域又は大容量仮想記憶領域を内蔵している。クラウドとしては、Infrastructure as a Service（IaaS）、Platform as a Service（PaaS）、Software as a Service（SaaS）を利用することができる。管理サーバ１１には、キーボード１５やマウス１６等の入力装置、ディスプレイ１７やプリンタ（図示せず）等の出力装置がインターフェイス（無線または有線）を介して接続されている。 The management server 11 is a physical computer or cloud computing device equipped with a central processing unit (CPU or MPU, virtual CPU or virtual MPU) and memory (main memory and cache memory, virtual main memory and virtual cache memory), and has a large-capacity storage area or a large-capacity virtual storage area built in. Infrastructure as a Service (IaaS), Platform as a Service (PaaS), or Software as a Service (SaaS) can be used as the cloud. Input devices such as a keyboard 15 and mouse 16, and output devices such as a display 17 and printer (not shown) are connected to the management server 11 via interfaces (wireless or wired).

冷凍幹細胞改質システム１０では、各種のドナーデータ（ドナー特定情報）がＩＣタグ１８（ＩＣチップ）を利用して管理され、間葉系幹細胞に関する幹細胞データがＩＣタグ１８を利用して管理される。ドナーデータには、ドナーの氏名、住所、電話番号、生年月日、性別、血液型、身長、体重、メールアドレス等があり、幹細胞データには、幹細胞特定情報、幹細胞製造年月日、培養生成液製造年月日、培養生成液特定情報等がある。幹細胞第１定着工程では、担当者（医師や看護師、研究者、バイオ技術者等）がドナーから第１骨髄液を２～３ｃｃ（２～３ｍｌ）採取する。担当者は、第１骨髄液の採取と同時に、管理サーバ１１において冷凍幹細胞改質システム１０を起動し、キーボード１５やマウス１６等の入力装置を利用してドナーデータや幹細胞データを管理サーバ１１に適宜入力する。 In the frozen stem cell reforming system 10, various donor data (donor identification information) are managed using an IC tag 18 (IC chip), and stem cell data related to mesenchymal stem cells is also managed using the IC tag 18. Donor data includes the donor's name, address, telephone number, date of birth, gender, blood type, height, weight, email address, etc., while stem cell data includes stem cell identification information, stem cell production date, culture product fluid production date, culture product fluid identification information, etc. In the first stem cell settlement process, a person in charge (such as a doctor, nurse, researcher, or bioengineer) collects 2-3 cc (2-3 ml) of first bone marrow fluid from the donor. Simultaneously with the collection of the first bone marrow fluid, the person in charge starts the frozen stem cell reforming system 10 on the management server 11 and appropriately inputs donor data and stem cell data into the management server 11 using input devices such as the keyboard 15 and mouse 16.

管理サーバ１１は、ドナーデータが入力される度毎（ドナーから第１骨髄液を採取する度毎）に各ドナーを特定するユニークなドナー識別子を生成する。管理サーバ１１は、入力されたドナーデータや幹細胞データをＩＣタグリーダ／ライタ１２を利用してＩＣタグ１８に書き込む。管理サーバ１１は、ドナーデータ及び幹細胞データをドナー識別子及びＩＣタグ１８のＩＣタグ特定情報（ＩＣタグ識別子）に関連付けた状態で大容量記憶領域又は大容量仮想記憶領域に格納（記憶）する（ドナーデータ記憶工程）。 The management server 11 generates a unique donor identifier that identifies each donor each time donor data is input (each time first bone marrow fluid is collected from the donor). The management server 11 writes the input donor data and stem cell data to the IC tag 18 using the IC tag reader/writer 12. The management server 11 stores (memorizes) the donor data and stem cell data in a large-capacity storage area or large-capacity virtual storage area while associating them with the donor identifier and the IC tag identification information (IC tag identifier) of the IC tag 18 (donor data storage process).

ドナーから採取された第１骨髄液１９は、図２に示すように、上下方向へ長いガラス試験管２０（分離容器）内に注入（収容）される。尚、２～３ｃｃの第１骨髄液１９には、０．５～１ｍｌ（約５×１０７（ｃｅｌｌｓ／ｍｌ））の複数種類の間葉系幹細胞が含まれる。ガラス試験管２０の外周面には、ＩＣタグ１８が貼付されている。尚、管理サーバ１１は、ＩＣタグ１８に書き込まれたドナーデータや幹細胞データがＩＣタグリーダ／ライタ１２によって読み取られた場合、そのドナーデータや幹細胞データをディスプレイ１７に表示する。第１骨髄液１９を注入したガラス試験管２０は、試験管立て（図示せず）にセットされ、試験管立てとともに恒温槽（図示せず）の内部に収容される。 As shown in FIG. 2, first bone marrow fluid 19 collected from a donor is injected (contained) into a vertically long glass test tube 20 (separation container). Note that 2-3 cc of first bone marrow fluid 19 contains 0.5-1 ml (approximately 5 x 10 cells/ml) of multiple types of mesenchymal stem cells. An IC tag 18 is affixed to the outer surface of the glass test tube 20. Note that when the donor data and stem cell data written on the IC tag 18 are read by the IC tag reader/writer 12, the management server 11 displays the donor data and stem cell data on the display 17. The glass test tube 20 into which the first bone marrow fluid 19 has been injected is set in a test tube rack (not shown), and the test tube rack is then housed in a thermostatic bath (not shown).

第１骨髄液１９を注入したガラス試験管２０は、恒温槽において所定時間（約２時間）静的に放置（動かすことなく静かに放置）される。恒温槽内の温度は、体温と略同一の約３５～３７℃に保持されている。ガラス試験管２０を恒温槽に所定時間（約２時間）静的に放置することで、図３に示すように、ガラス試験管２０に注入された第１骨髄液１９がガラス試験管２０内において上下方向へ何層か（３層）の層状に分離する（骨髄液分離工程）。第１骨髄液１９を層状に分離させた後、恒温槽の内部から試験管立てを取り出し、試験管立てからガラス試験管２０を引き抜き、層状に分離した第１骨髄液１９の特定の層（３層（層状）に分離した中間層（第２層））に存在する第２骨髄液２１（中間層骨髄液）を注射器（図示せず）又はピペット（図示せず）を利用して抽出（吸引）する（骨髄液抽出工程）。 The glass test tube 20 into which the first bone marrow fluid 19 has been injected is left stationary (left undisturbed) in an incubator for a predetermined time (approximately two hours). The temperature inside the incubator is maintained at approximately 35-37°C, which is roughly the same as body temperature. By leaving the glass test tube 20 stationary in the incubator for a predetermined time (approximately two hours), the first bone marrow fluid 19 injected into the glass test tube 20 separates into several layers (three layers) vertically within the glass test tube 20, as shown in Figure 3 (bone marrow fluid separation process). After the first bone marrow fluid 19 has separated into layers, the test tube stand is removed from inside the incubator, the glass test tube 20 is pulled out of the test tube stand, and the second bone marrow fluid 21 (intermediate layer bone marrow fluid) present in a specific layer (the intermediate layer (second layer) of the three (layers) separated) of the separated first bone marrow fluid 19 is extracted (aspirated) using a syringe (not shown) or pipette (not shown) (bone marrow fluid extraction process).

第１骨髄液１９から中間層に位置する特定の第２骨髄液２１（中間層骨髄液）を抽出した後、第２骨髄液２１及び培養液２６を第１扁平培養容器２２（第２培養容器）（細胞培養容器）に注入（収容）し、培養容器２２を体温と略同一の温度（約３５～３７℃）に保持しつつ、１２～２４時間静的に放置（動かすことなく静かに放置）し、１２～２４時間の間において約１～２時間間隔で培養容器２２内の第２骨髄液２１に含まれる第１間葉系幹細胞２１の初期平面形状からの変形をディスプレイ１７（電子顕微鏡）で観察し、第１間葉系幹細胞２１が第１扁平培養容器２２の底面２３に定着したか否かを判断する（形状変形第１観察工程）。第１扁平培養容器２２の底面２３（底壁外面）には、ドナーデータや幹細胞データが書き込まれたＩＣタグ１８が取り付けられている。 After extracting a specific second bone marrow fluid 21 (intermediate layer bone marrow fluid) located in the intermediate layer from the first bone marrow fluid 19, the second bone marrow fluid 21 and culture medium 26 are poured (contained) into a first flat culture vessel 22 (second culture vessel) (cell culture vessel). The culture vessel 22 is maintained at a temperature approximately equal to body temperature (approximately 35-37°C) and left statically (left undisturbed) for 12-24 hours. The deformation of the first mesenchymal stem cells 21 contained in the second bone marrow fluid 21 in the culture vessel 22 from their initial planar shape is observed on the display 17 (electron microscope) at approximately 1-2 hour intervals over the 12-24 hour period, and it is determined whether the first mesenchymal stem cells 21 have settled on the bottom surface 23 of the first flat culture vessel 22 (first shape deformation observation process). An IC tag 18 containing donor data and stem cell data is attached to the bottom surface 23 (outer surface of the bottom wall) of the first flat culture vessel 22.

第１扁平培養容器２２（第２培養容器）は、透明なガラス又は透明なプラスチックから作られ、小容量かつ所定面積の底面２３を有する平面形状が略正四角形の扁平な容器である。第１扁平培養容器２２の注入口２４は、蓋２５によって水密に閉塞されている。第１扁平培養容器２２は、その容量が約２０～３０ｃｃ（好ましくは、２５ｃｃ）であり、その底面２３の面積が約２５～３６ｍｍ２であるとともに、その一辺の長さが５～６ｍｍである。尚、第１扁平培養容器２２として小容量かつ所定面積の底面を有する平面形状が円形や楕円形の扁平な容器を使用することもできる。 The first flat culture vessel 22 (second culture vessel) is made of transparent glass or transparent plastic, and is a flat vessel with a small capacity and a predetermined area on the bottom 23, with a roughly square planar shape. The inlet 24 of the first flat culture vessel 22 is watertightly closed by a lid 25. The first flat culture vessel 22 has a capacity of approximately 20 to 30 cc (preferably 25 cc), an area of the bottom 23 of approximately 25 to 36 mm2 , and a side length of 5 to 6 mm. Note that a flat vessel with a small capacity, a predetermined area on the bottom, and a circular or elliptical planar shape can also be used as the first flat culture vessel 22.

担当者は、注入口２４から蓋２５を取り外し、注射器又はピペットに吸引された第２骨髄液２１を第１扁平培養容器２２の注入口２４から培養容器２２の内部に注入（収容）するとともに、培養液２６を培養容器２２の内部に注入（収容）し、蓋２５によって注入口２４を閉塞する。培養液２６には、ペニシリン（約１００Ｕ／ｍｌ）、アムホテリシン（約１００ｎｇ／ｍｌ）、ストレプトマイシン（約１００ｍｋｇ／ｍｌ）、Ｌ－グルタミン（約２～４ｍｌ）、２０％ウシ胎児血清を添加したミネラル塩溶液およびアミノ酸が含まれる。 The technician removes the lid 25 from the inlet 24, and injects (contains) the second bone marrow fluid 21 drawn into the syringe or pipette into the first flat culture vessel 22 through the inlet 24, while also injecting (contains) the culture medium 26 into the culture vessel 22, and then closes the inlet 24 with the lid 25. The culture medium 26 contains penicillin (approximately 100 U/ml), amphotericin (approximately 100 ng/ml), streptomycin (approximately 100 mkg/ml), L-glutamine (approximately 2-4 ml), a mineral salt solution supplemented with 20% fetal bovine serum, and amino acids.

尚、培養液２６には、Ｄｕｌｂｅｃｃｏ’ｓ Ｍｏｄｉｆｉｅｄ Ｅａｇｌｅ’ｓ Ｍｅｄｉｕｍ（ＤＭＥＭ）、Ｇｒａｓｇｏｗ Ｍｉｎｉｍｕｍ Ｅｓｓｅｎｔｉａｌ Ｍｅｄｉｕｍ（ＧＭＥＭ）、ＲＰＭＩ６４０等を使用することもできる。培養液２６には、インスリン、トランスフェリン、エタノールアミン、セレニウム、２－メルカプトエタノール、Ｌ－アラニル－Ｌ－グルタミン、ピルビン酸ナトリウム、Ｌ－アラニン、Ｌ－アスパラギン、Ｌ－アスパラチン酸、グリシン、Ｌ－プロリン、Ｌ－セリン等を添加することもできる。第１扁平培養容器２２に注入された第２骨髄液２１に含まれる第１間葉系幹細胞２１は、時間の経過とともに第１扁平培養容器２２の底面２３に定着しつつ、培養液２６によって培養され、培養容器２２の底面２３において次第に増殖（分化）してコロニーを形成する。 In addition, Dulbecco's Modified Eagle's Medium (DMEM), Grasgow Minimum Essential Medium (GMEM), RPMI 640, etc. can also be used for the culture medium 26. Insulin, transferrin, ethanolamine, selenium, 2-mercaptoethanol, L-alanyl-L-glutamine, sodium pyruvate, L-alanine, L-asparagine, L-aspartic acid, glycine, L-proline, L-serine, etc. can also be added to the culture medium 26. The first mesenchymal stem cells 21 contained in the second bone marrow fluid 21 injected into the first flat culture vessel 22 settle on the bottom surface 23 of the first flat culture vessel 22 over time, are cultured in the culture medium 26, and gradually proliferate (differentiate) on the bottom surface 23 of the culture vessel 22 to form colonies.

第２骨髄液２１及び培養液２６を第１扁平培養容器２２に注入した後、培養容器２２を電子顕微鏡１３の試料ホルダ２７に設置（セット）する。電子顕微鏡１３の試料ホルダ２７の上面２８と第１扁平培養容器２２の底部２９との間にスペーサー３０を介在させ、培養容器２２の底部２９をスペーサー３０によって持ち上げた状態に保持し、培養容器２２の底部２９が上となり培養容器２２の頂部３１（注入口２４）が下となるように、培養容器２２を所定角度に傾斜させた状態に保持する。尚、第１扁平培養容器２２の頂部３１をスペーサー３０によって持ち上げた状態に保持し、培養容器２２の頂部３１が上となり培養容器２２の底部２９が下となるように、培養容器２２を所定角度に傾斜させた状態に保持してもよい。試料ホルダ２７の上面２８に対する第１扁平培養容器２２の傾斜角度α１は、２～５°の範囲にあり、好ましくは、２～３°の範囲にある。 After the second bone marrow fluid 21 and culture medium 26 are injected into the first flat culture vessel 22, the culture vessel 22 is placed (set) in the sample holder 27 of the electron microscope 13. A spacer 30 is interposed between the upper surface 28 of the sample holder 27 of the electron microscope 13 and the bottom 29 of the first flat culture vessel 22, and the bottom 29 of the culture vessel 22 is held elevated by the spacer 30. The culture vessel 22 is held tilted at a predetermined angle so that the bottom 29 of the culture vessel 22 is facing up and the top 31 (inlet 24) of the culture vessel 22 is facing down. Alternatively, the top 31 of the first flat culture vessel 22 may be held elevated by the spacer 30, and the culture vessel 22 may be held tilted at a predetermined angle so that the top 31 of the culture vessel 22 is facing up and the bottom 29 of the culture vessel 22 is facing down. The inclination angle α1 of the first flat culture vessel 22 relative to the upper surface 28 of the sample holder 27 is in the range of 2 to 5°, and preferably in the range of 2 to 3°.

試料ホルダ２７の上面２８に対して第１扁平培養容器２２を前記傾斜角度で傾斜させることで、培養容器２２内において第２骨髄液２１（又は第１間葉系幹細胞２１）及び培養液２６が培養容器２２の頂部３１の側（又は底部２９の側）に偏り、培養容器２２の頂部３１の側（又は底部２９の側）において第２骨髄液２１（又は第１間葉系幹細胞２１）と培養液２６との水圧が大きくなって第２骨髄液２１（又は第１間葉系幹細胞２１）が培養容器２２の頂部３１の側に集中し、それによって第１間葉系幹細胞２１どうしの活性が高まり、培養容器２２の底面２３において第１間葉系幹細胞２１を容易かつ迅速に定着（又は増殖（分化））させることができる。 By tilting the first flat culture vessel 22 at the above-mentioned tilt angle relative to the upper surface 28 of the sample holder 27, the second bone marrow fluid 21 (or first mesenchymal stem cells 21) and culture medium 26 are concentrated toward the top 31 side (or bottom 29 side) of the culture vessel 22 within the culture vessel 22. This increases the hydraulic pressure between the second bone marrow fluid 21 (or first mesenchymal stem cells 21) and culture medium 26 on the top 31 side (or bottom 29 side) of the culture vessel 22, causing the second bone marrow fluid 21 (or first mesenchymal stem cells 21) to concentrate on the top 31 side of the culture vessel 22. This increases the activity of the first mesenchymal stem cells 21, allowing the first mesenchymal stem cells 21 to easily and quickly settle (or proliferate (differentiate)) on the bottom surface 23 of the culture vessel 22.

電子顕微鏡１３は、第１扁平培養容器２２に注入された第２骨髄液２１に含まれる第１間葉系幹細胞２１の平面形状の拡大画像を約１～２時間間隔で撮影し、撮影した第１間葉系幹細胞２１の平面形状の拡大画像を約１～２時間間隔で管理サーバ１１に送信する。管理サーバ１１は、電子顕微鏡１３から送信された第１間葉系幹細胞２１の平面形状の拡大画像と撮影時間とをＩＣタグ特定情報（ＩＣタグ識別子）及びドナー識別子に関連付けた状態で記憶領域に格納（記憶）する。管理サーバ１１は、電子顕微鏡１３から送信された第１間葉系幹細胞２１の平面形状の拡大画像と撮影時間とをディスプレイ１７に出力（表示）する。担当者（医師や看護師、研究者、バイオ技術者等）は、ディスプレイ１７に表示された第１間葉系幹細胞２１の平面形状の拡大画像を１２～２４時間の間において約１～２時間間隔で確認（視認）し、第２骨髄液２１に含まれる第１間葉系幹細胞２１の平面形状の変化を観察する。 The electron microscope 13 captures enlarged images of the planar shape of the first mesenchymal stem cells 21 contained in the second bone marrow fluid 21 injected into the first flat culture vessel 22 at approximately 1-2 hour intervals, and transmits the captured enlarged images of the planar shape of the first mesenchymal stem cells 21 to the management server 11 at approximately 1-2 hour intervals. The management server 11 stores (memorizes) the enlarged images of the planar shape of the first mesenchymal stem cells 21 transmitted from the electron microscope 13 and the time of the image capture in a memory area, associated with the IC tag identification information (IC tag identifier) and donor identifier. The management server 11 outputs (displays) the enlarged images of the planar shape of the first mesenchymal stem cells 21 transmitted from the electron microscope 13 and the time of the image capture on the display 17. The person in charge (doctor, nurse, researcher, bioengineer, etc.) checks (visually confirms) the enlarged image of the planar shape of the first mesenchymal stem cells 21 displayed on the display 17 at approximately 1-2 hour intervals over a period of 12-24 hours, and observes any changes in the planar shape of the first mesenchymal stem cells 21 contained in the second bone marrow fluid 21.

図示はしていないが、第１間葉系幹細胞２１の初期平面形状は略円形であり、第１間葉系幹細胞２１の平面形状が略円形の場合、第１間葉系幹細胞２１が第１扁平培養容器２２の底面２３（底壁内面）に定着しておらず、第１間葉系幹細胞２１が増殖（分化）を開始していない。第１間葉系幹細胞２１の変形後の平面形状は定着前の略円形を核として第１間葉系幹細胞２１が一方向（所定方向）へ不定形に伸張（拡張）した扁平形状であり、第１間葉系幹細胞２１が第１扁平培養容器２２の底面２３（底壁内面）に定着し、第１間葉系幹細胞２１が増殖（分化）を開始している。 Although not shown in the figure, the initial planar shape of the first mesenchymal stem cells 21 is approximately circular. When the planar shape of the first mesenchymal stem cells 21 is approximately circular, the first mesenchymal stem cells 21 have not yet settled on the bottom surface 23 (inner surface of the bottom wall) of the first flat culture vessel 22, and the first mesenchymal stem cells 21 have not yet begun to proliferate (differentiate). The planar shape of the first mesenchymal stem cells 21 after deformation is a flattened shape in which the first mesenchymal stem cells 21 have irregularly stretched (expanded) in one direction (predetermined direction) using the approximately circular shape before settling as a nucleus. The first mesenchymal stem cells 21 have settled on the bottom surface 23 (inner surface of the bottom wall) of the first flat culture vessel 22, and the first mesenchymal stem cells 21 have begun to proliferate (differentiate).

形状変形第１観察工程における観察の結果、ディスプレイ１７に出力（表示）された第１間葉系幹細胞２１の平面形状が略円形から略円形を核として不定形の扁平形状に変形した場合、第１間葉系幹細胞２１が第１扁平培養容器２２の底面２３に定着したと判断する（幹細胞第１定着工程）。形状変形第１観察工程における観察の結果、第１間葉系幹細胞２１が略円形（初期平面形状）から略円形を核として不定形の扁平形状に変形し、第１間葉系幹細胞２１の第１扁平培養容器２２（第１培養容器）の底面２３への定着を確認した後、総平面面積第１観察工程が行われる。 If, as a result of the observation in the first shape deformation observation step, the planar shape of the first mesenchymal stem cells 21 output (displayed) on the display 17 changes from an approximately circular shape to an amorphous flat shape with an approximately circular nucleus, it is determined that the first mesenchymal stem cells 21 have settled on the bottom surface 23 of the first flat culture vessel 22 (first stem cell settlement step). After the observation in the first shape deformation observation step confirms that the first mesenchymal stem cells 21 have changed from an approximately circular shape (initial planar shape) to an amorphous flat shape with an approximately circular nucleus and that the first mesenchymal stem cells 21 have settled on the bottom surface 23 of the first flat culture vessel 22 (first culture vessel), the first total planar area observation step is carried out.

担当者は、第１扁平培養容器２２内に注入されている培養液２６を培養容器２２から排出し、培養容器２２内にあらたな培養液２６を注入（収容）する。次に、第１扁平培養容器２２を体温と略同一の温度（約３５～３７℃）で３６～４８時間静的に放置（動かすことなく静かに放置）して第１間葉系幹細胞２１を培養しつつ、３６～４８時間の間において約１～２時間間隔で培養容器２２の底面２３に定着した第１間葉系幹細胞２１の培養容器２２の底面面積に対する総平面面積をディスプレイ１７（電子顕微鏡１３）で観察し、第１間葉系幹細胞２１の総平面面積が培養容器２２の底面面積に対して第１目標割合に達したか否かを判断する（総平面面積第１観察工程）。第１扁平培養容器２２の底面面積に対する第１間葉系幹細胞２１の総平面面積の第１目標割合は、７０～８０％（７０～８０％コンフルエント）である。 The person in charge drains the culture medium 26 that has been poured into the first flat culture vessel 22 from the culture vessel 22 and pours (contains) new culture medium 26 into the culture vessel 22. Next, the person in charge leaves the first flat culture vessel 22 statically (left quietly without moving) at a temperature approximately equal to body temperature (approximately 35-37°C) for 36-48 hours to culture the first mesenchymal stem cells 21, while observing the total planar area of the first mesenchymal stem cells 21 that have settled on the bottom surface 23 of the culture vessel 22 relative to the bottom surface area of the culture vessel 22 on the display 17 (electron microscope 13) at approximately 1-2 hour intervals during the 36-48 hours, and determines whether the total planar area of the first mesenchymal stem cells 21 relative to the bottom surface area of the culture vessel 22 has reached a first target ratio (first total planar area observation step). The first target ratio of the total planar area of the first mesenchymal stem cells 21 to the bottom area of the first flat culture vessel 22 is 70-80% (70-80% confluent).

総平面面積第１観察工程では、第１扁平培養容器２２に注入した培養液２６を培養容器２２から排出し、あらたな培養液２６を培養容器２２に注入（収容）し、あらたな培養液２６を注入した第１扁平培養容器２２を電子顕微鏡１３の試料ホルダ２７に設置（セット）する。電子顕微鏡１３の試料ホルダ２７の上面２８と第１扁平培養容器２２の底部２９との間にスペーサー３０を介在させ、培養容器２２の底部２９をスペーサー３０によって持ち上げた状態に保持し、培養容器２２の底部２９が上となり培養容器２２の頂部３１（注入口２４）が下となるように、培養容器２２を所定角度に傾斜させた状態に保持する（図６参照）。試料ホルダ２７の上面２８に対する第１扁平培養容器２２の傾斜角度α１は、２～５°の範囲にあり、好ましくは、２～３°の範囲にある。 In the first observation step of the total planar area, the culture medium 26 poured into the first flat culture vessel 22 is discharged from the culture vessel 22, new culture medium 26 is poured (contained) into the culture vessel 22, and the first flat culture vessel 22 with the poured new culture medium 26 is placed (set) in the specimen holder 27 of the electron microscope 13. A spacer 30 is interposed between the upper surface 28 of the specimen holder 27 of the electron microscope 13 and the bottom 29 of the first flat culture vessel 22, and the bottom 29 of the culture vessel 22 is held elevated by the spacer 30. The culture vessel 22 is held tilted at a predetermined angle so that the bottom 29 of the culture vessel 22 is facing up and the top 31 (inlet 24) of the culture vessel 22 is facing down (see Figure 6). The tilt angle α1 of the first flat culture vessel 22 relative to the upper surface 28 of the specimen holder 27 is in the range of 2 to 5°, preferably 2 to 3°.

電子顕微鏡１３は、第１扁平培養容器２２内の第１間葉系幹細胞２１の平面形状の拡大画像を約１～２時間間隔で撮影し、撮影した第１間葉系幹細胞２１の平面形状の拡大画像を約１～２時間間隔で管理サーバ１１に送信する。管理サーバ１１は、電子顕微鏡１３から送信された第１間葉系幹細胞２１の平面形状の拡大画像と撮影時間とをＩＣタグ特定情報（ＩＣタグ識別子）及びドナー識別子に関連付けた状態で記憶領域に格納（記憶）する。管理サーバ１１は、電子顕微鏡１３から送信された第１間葉系幹細胞２１の平面形状の拡大画像と撮影時間とをディスプレイ１７に出力（表示）する。 The electron microscope 13 captures enlarged images of the planar shape of the first mesenchymal stem cells 21 in the first flat culture vessel 22 at approximately 1-2 hour intervals, and transmits the captured enlarged images of the planar shape of the first mesenchymal stem cells 21 to the management server 11 at approximately 1-2 hour intervals. The management server 11 stores (memorizes) the enlarged images of the planar shape of the first mesenchymal stem cells 21 transmitted from the electron microscope 13 and the time of the image capture in a memory area, associated with the IC tag identification information (IC tag identifier) and donor identifier. The management server 11 outputs (displays) the enlarged images of the planar shape of the first mesenchymal stem cells 21 transmitted from the electron microscope 13 and the time of the image capture on the display 17.

担当者（医師や看護師、研究者、バイオ技術者等）は、ディスプレイ１７に表示された第１間葉系幹細胞２１の平面形状の拡大画像を３６～４８時間の間において約１～２時間間隔で確認（視認）し、第１扁平培養容器２２の底面２３に定着した第１間葉系幹細胞２１の培養容器２２の底面面積に対する総平面面積を観察しつつ、第１間葉系幹細胞２１の総平面面積が第１扁平培養容器２２の底面面積に対して第１目標割合（７０～８０％コンフルエント）に達したか否かを判断する。 The person in charge (doctor, nurse, researcher, bioengineer, etc.) checks (visually confirms) the enlarged image of the planar shape of the first mesenchymal stem cells 21 displayed on the display 17 at approximately 1-2 hour intervals over a period of 36-48 hours, and while observing the total planar area of the first mesenchymal stem cells 21 that have settled on the bottom surface 23 of the first flat culture vessel 22 relative to the bottom surface area of the culture vessel 22, determines whether the total planar area of the first mesenchymal stem cells 21 relative to the bottom surface area of the first flat culture vessel 22 has reached a first target ratio (70-80% confluence).

第１間葉系幹細胞２１は、第１扁平培養容器２２の底面２３（底壁内面）において増殖してコロニーを形成する（幹細胞第１培養工程）。第１間葉系幹細胞２１がコロニーを形成し、総平面面積第１観察工程における観察の結果、第１間葉系幹細胞２１の平面形状が拡張し、ディスプレイ１７に表示された第１間葉系幹細胞２１の第１扁平培養容器２２の底面面積に対する総平面面積が第１目標割合（７０～８０％コンフルエント）に達した場合、培養容器２２から第１間葉系幹細胞２１を抽出する。 The first mesenchymal stem cells 21 proliferate and form colonies on the bottom surface 23 (inner surface of the bottom wall) of the first flat culture vessel 22 (first stem cell culture step). The first mesenchymal stem cells 21 form colonies, and as a result of observation in the first total planar area observation step, the planar shape of the first mesenchymal stem cells 21 expands. When the total planar area of the first mesenchymal stem cells 21 relative to the bottom surface area of the first flat culture vessel 22 displayed on the display 17 reaches a first target ratio (70-80% confluent), the first mesenchymal stem cells 21 are extracted from the culture vessel 22.

第１扁平培養容器２２に注入されている総平面面積第１観察工程時の培養液２６（第１培養生成液３２）を第１扁平培養容器２２から排出し、培養容器２２をりん酸緩衝生理食塩水（ＰＢＳ）で洗浄した後、トリプシン液を培養容器２２内に注入する。第１扁平培養容器２２にトリプシン液を注入すると、培養容器２２の底面２３に定着した第１間葉系幹細胞２１がトリプシン液によって底面２３から剥離し、トリプシン液の水面に浮上する。担当者は、浮上した第１間葉系幹細胞２１をピペットを利用して吸引する。 The culture medium 26 (first culture product liquid 32) during the first observation step (total planar area) injected into the first flat culture vessel 22 is discharged from the first flat culture vessel 22, the culture vessel 22 is washed with phosphate-buffered saline (PBS), and then trypsin solution is injected into the culture vessel 22. When the trypsin solution is injected into the first flat culture vessel 22, the first mesenchymal stem cells 21 that have settled on the bottom surface 23 of the culture vessel 22 are detached from the bottom surface 23 by the trypsin solution and rise to the surface of the trypsin solution. The person in charge uses a pipette to aspirate the floating first mesenchymal stem cells 21.

尚、第１扁平培養容器２２（第２培養容器）から排出された培養液２６は、培養容器２２から第１間葉系幹細胞２１を抽出した後に残った培養液２６であり、第１間葉系幹細胞２１の培養過程において第１間葉系幹細胞２１から分泌された所定の代謝物質を含む第１培養生成液３２（培養生成液）に変化している。代謝物質を含む第１培養生成液３２は、活性が低下した間葉系幹細胞を活性化させる。第１培養生成液３２は、ＩＣタグ１８が取り付けられた後記する冷凍保存容器４３に注入されて急速冷凍機（図示せず）によって急速冷凍された後、冷凍保存容器４３に収容された状態で冷蔵庫１４又は冷凍庫１４において所定期間、所定温度（０～５℃のチルド保存又は０℃以下の冷凍保存）で保存される。 The culture medium 26 discharged from the first flat culture vessel 22 (second culture vessel) is the culture medium 26 remaining after the first mesenchymal stem cells 21 are extracted from the culture vessel 22, and has changed into a first culture product solution 32 (culture product solution) containing specific metabolic substances secreted from the first mesenchymal stem cells 21 during the culture process of the first mesenchymal stem cells 21. The first culture product solution 32 containing metabolic substances activates mesenchymal stem cells with reduced activity. The first culture product solution 32 is poured into a cryopreservation container 43 (described below) to which an IC tag 18 is attached, and is quick-frozen in a quick-freezer (not shown). It is then stored in the cryopreservation container 43 at a predetermined temperature (chilled storage at 0-5°C or frozen storage at 0°C or below) for a predetermined period of time in the refrigerator 14 or freezer 14.

図６は、間葉系幹細胞培養方法における第２間葉系幹細胞第１抽出工程の一例を示す説明図であり、図７は、間葉系幹細胞培養方法における形状変形第２観察工程の一例を示す説明図であり、図８は、第２扁平培養容器３３（第３培養容器）の側面図である。担当者（医師や看護師、研究者、バイオ技術者等）は、第１扁平培養容器２２から第１間葉系幹細胞２１を抽出した後、第１間葉系幹細胞２１をガラス試験管３４に注入（収容）し、ガラス試験管３４を遠心分離器（図示せず）に設置（セット）する。第１間葉系幹細胞２１を遠心分離器によって所定時間遠心分離した後、ガラス試験管３４を遠心分離器から取り出す。ガラス試験管３４内の第１間葉系幹細胞２１は、遠心分離器によって上下方向へ２層の層状に遠心分離される。 Figure 6 is an explanatory diagram showing an example of the first extraction step of second mesenchymal stem cells in the mesenchymal stem cell culture method, Figure 7 is an explanatory diagram showing an example of the second shape deformation observation step in the mesenchymal stem cell culture method, and Figure 8 is a side view of the second flat culture vessel 33 (third culture vessel). After extracting the first mesenchymal stem cells 21 from the first flat culture vessel 22, a person in charge (doctor, nurse, researcher, bioengineer, etc.) injects (contains) the first mesenchymal stem cells 21 into a glass test tube 34 and places (sets) the glass test tube 34 in a centrifuge (not shown). After centrifuging the first mesenchymal stem cells 21 in the centrifuge for a predetermined time, the glass test tube 34 is removed from the centrifuge. The first mesenchymal stem cells 21 in the glass test tube 34 are centrifuged vertically in the centrifuge to form two layers.

第１間葉系幹細胞２１を層状に分離させた後、図６に示すように、層状に分離した第１間葉系幹細胞２１から下層（最下層）に位置する第２間葉系幹細胞３５を注射器（図示せず）又はピペット（図示せず）を利用して抽出（吸引）する抽出する（第２間葉系幹細胞第１抽出工程）。不要な幹細胞を含む第１間葉系幹細胞２１を遠心分離器で遠心分離して上下方向へ層状に分離させ、層状に遠心分離した第１間葉系幹細胞２１のうちの下層（最下層）に位置する第２間葉系幹細胞３５を抽出することで、第１間葉系幹細胞２１から特定の第２間葉系幹細胞３５を確実に抽出することができ、第１間葉系幹細胞２１から不要な間葉系幹細胞を除去することができる。 After separating the first mesenchymal stem cells 21 into layers, as shown in Figure 6, second mesenchymal stem cells 35 located in the lower layer (bottom layer) are extracted (aspirated) from the separated layer of first mesenchymal stem cells 21 using a syringe (not shown) or a pipette (not shown) (first second mesenchymal stem cell extraction step). The first mesenchymal stem cells 21 containing unwanted stem cells are centrifuged in a centrifuge to separate them into layers in the vertical direction, and the second mesenchymal stem cells 35 located in the lower layer (bottom layer) of the centrifuged layered first mesenchymal stem cells 21 are extracted, thereby ensuring the specific second mesenchymal stem cells 35 are extracted from the first mesenchymal stem cells 21 and removing unwanted mesenchymal stem cells from the first mesenchymal stem cells 21.

第１間葉系幹細胞２１から下層（最下層）に位置する特定の第２間葉系幹細胞３５を抽出した後、形状変形第２観察工程が行われる。形状変形第２観察工程では、第２間葉系幹細胞３５及び培養液３６を第２扁平培養容器３３（第３培養容器）（細胞培養容器）に注入（収容）し、培養容器３３を体温と略同一の温度（約３５～３７℃）で３６～４８時間静的に放置（動かすことなく静かに放置）しつつ、３６～４８時間の間において約１～２時間間隔で培養容器３３内の第２間葉系幹細胞３５（間葉系幹細胞）の初期平面形状からの変形をディスプレイ１７（電子顕微鏡）で観察し、第２間葉系幹細胞３５が培養容器３３の底面３７に定着したか否かを判断する（形状変形第２観察工程）。第２扁平培養容器３３の底面３７（底壁外面）には、ドナーデータや幹細胞データが書き込まれたＩＣタグ１８が取り付けられている。 After specific second mesenchymal stem cells 35 located in the lower layer (bottom layer) are extracted from the first mesenchymal stem cells 21, a second shape deformation observation process is performed. In the second shape deformation observation process, the second mesenchymal stem cells 35 and culture medium 36 are injected (contained) into a second flat culture vessel 33 (third culture vessel) (cell culture vessel), and the culture vessel 33 is left statically (left undisturbed) for 36 to 48 hours at a temperature approximately equal to body temperature (approximately 35 to 37°C). During this 36 to 48-hour period, the deformation of the second mesenchymal stem cells 35 (mesenchymal stem cells) from their initial planar shape within the culture vessel 33 is observed on the display 17 (electron microscope) at approximately 1-2 hour intervals, and it is determined whether the second mesenchymal stem cells 35 have settled on the bottom surface 37 of the culture vessel 33 (second shape deformation observation process). An IC tag 18 containing donor data and stem cell data is attached to the bottom surface 37 (outer surface of the bottom wall) of the second flat culture vessel 33.

第２扁平培養容器３３（第３培養容器）は、透明なガラス又は透明なプラスチックから作られ、小容量かつ所定面積の底面を有する平面形状が略正四角形の扁平な容器であり、その容積が第１扁平培養容器２２（第２培養容器）のそれよりも大きい。第２扁平培養容器３３の注入口３８は、蓋３９によって水密に閉塞されている。第２扁平培養容器３３は、その容量が約４０～６０ｃｃ（好ましくは、５０ｃｃ）であり、その底面面積が約５０～７２ｍｍ２であるともに、その一辺の長さが約７～８．５ｍｍである。尚、第２扁平培養容器３３として小容量かつ所定面積の底面を有する平面形状が円形や楕円形の扁平な容器を使用することもできる。第２扁平培養容器３３に注入された第２間葉系幹細胞３５は、時間の経過とともに培養容器３３の底面３７に定着しつつ、培養液３６によって培養され、培養容器３３の底面３７において次第に増殖（分化）してコロニーを形成する。 The second flat culture vessel 33 (third culture vessel) is made of transparent glass or transparent plastic, is a flat vessel with a small capacity and a predetermined bottom area, and has a planar shape of an approximately square. Its volume is larger than that of the first flat culture vessel 22 (second culture vessel). The injection port 38 of the second flat culture vessel 33 is watertightly closed by a lid 39. The second flat culture vessel 33 has a capacity of approximately 40 to 60 cc (preferably 50 cc), a bottom area of approximately 50 to 72 mm2 , and a side length of approximately 7 to 8.5 mm. Note that a flat vessel with a small capacity, a predetermined bottom area, and a planar shape of a circle or oval can also be used as the second flat culture vessel 33. The second mesenchymal stem cells 35 injected into the second flat culture vessel 33 settle on the bottom surface 37 of the culture vessel 33 over time, are cultured in the culture medium 36, and gradually proliferate (differentiate) on the bottom surface 37 of the culture vessel 33 to form colonies.

担当者は、注入口３８から蓋３９を取り外し、注射器又はピペットに吸引された第２間葉系幹細胞３５を第２扁平培養容器３３の注入口３８から培養容器３３の内部に注入（収容）するとともに、培養液３６を培養容器３３の内部に注入（収容）し、蓋３９によって注入口３８を閉塞する。培養液３６は、形状変形第１観察工程において注入されたそれと同一である。担当者は、第２間葉系幹細胞３５及び培養液３６を第２扁平培養容器３３に注入した後、培養容器３３を電子顕微鏡１３の試料ホルダ２７に設置（セット）する。 The technician removes the lid 39 from the injection port 38, and injects (contains) the second mesenchymal stem cells 35 aspirated into the syringe or pipette into the interior of the second flat culture vessel 33 through the injection port 38 of the culture vessel 33. At the same time, the technician injects (contains) the culture medium 36 into the interior of the culture vessel 33, and then closes the injection port 38 with the lid 39. The culture medium 36 is the same as that injected in the first shape deformation observation step. After injecting the second mesenchymal stem cells 35 and culture medium 36 into the second flat culture vessel 33, the technician places (sets) the culture vessel 33 in the specimen holder 27 of the electron microscope 13.

電子顕微鏡１３の試料ホルダ２７の上面２８と第２扁平培養容器３３の底部４０との間にスペーサー３０を介在させ、培養容器３３の底部４０をスペーサー３０によって持ち上げた状態に保持し、培養容器３３の底部４０が上となり培養容器３３の頂部４１（注入口３８）が下となるように、培養容器３３を所定角度に傾斜させた状態に保持する。又、電子顕微鏡１３の試料ホルダ２７の上面２８と第２扁平培養容器３３の頂部４１との間にスペーサー３０を介在させ、培養容器３３の頂部４１をスペーサー３０によって持ち上げた状態に保持し、培養容器３３の頂部４１が上となり培養容器３３の底部４０が下となるように、培養容器３３を所定角度に傾斜させた状態に保持してもよい。試料ホルダ２７の上面２８に対する第２扁平培養容器３３の傾斜角度α２は、２～５°の範囲にあり、好ましくは、２～３°の範囲にある。 A spacer 30 is interposed between the upper surface 28 of the sample holder 27 of the electron microscope 13 and the bottom 40 of the second flat culture vessel 33, and the bottom 40 of the culture vessel 33 is held elevated by the spacer 30, with the culture vessel 33 held tilted at a predetermined angle so that the bottom 40 of the culture vessel 33 is on top and the top 41 (inlet 38) of the culture vessel 33 is on the bottom. Alternatively, a spacer 30 may be interposed between the upper surface 28 of the sample holder 27 of the electron microscope 13 and the top 41 of the second flat culture vessel 33, and the top 41 of the culture vessel 33 is held elevated by the spacer 30, with the culture vessel 33 held tilted at a predetermined angle so that the top 41 of the culture vessel 33 is on top and the bottom 40 of the culture vessel 33 is on the bottom. The inclination angle α2 of the second flat culture vessel 33 relative to the upper surface 28 of the sample holder 27 is in the range of 2 to 5°, and preferably in the range of 2 to 3°.

試料ホルダ２７の上面２８に対して第２扁平培養容器３３を前記傾斜角度で傾斜させることで、培養容器３３内において第２間葉系幹細胞３５及び培養液３６が培養容器３３の頂部４１の側（又は底部４０の側）に偏り、培養容器３３の頂部４１の側（又は底部４０の側）において第２間葉系幹細胞３５と培養液３６との水圧が大きくなって第２間葉系幹細胞３５が培養容器３３の頂部４１の側（又は底部４０の側）に集中し、それによって第２間葉系幹細胞３５どうしの活性が高まり、培養容器３３の底面３７において第２間葉系幹細胞３５を容易かつ迅速に定着（又は増殖（分化））させることができる。 By tilting the second flat culture vessel 33 at the above-mentioned tilt angle relative to the upper surface 28 of the sample holder 27, the second mesenchymal stem cells 35 and culture medium 36 within the culture vessel 33 are biased toward the top 41 side (or bottom 40 side) of the culture vessel 33, and the water pressure between the second mesenchymal stem cells 35 and the culture medium 36 on the top 41 side (or bottom 40 side) of the culture vessel 33 increases, concentrating the second mesenchymal stem cells 35 on the top 41 side (or bottom 40 side) of the culture vessel 33. This increases the activity of the second mesenchymal stem cells 35, allowing the second mesenchymal stem cells 35 to easily and quickly settle (or proliferate (differentiate)) on the bottom surface 37 of the culture vessel 33.

電子顕微鏡１３は、第２扁平培養容器３３に注入された第２間葉系幹細胞３５の平面形状の拡大画像を約１～２時間間隔で撮影し、撮影した第２間葉系幹細胞３５の平面形状の拡大画像を約１～２時間間隔で管理サーバ１１に送信する。管理サーバ１１は、電子顕微鏡１３から送信された第２間葉系幹細胞３５の平面形状の拡大画像と撮影時間とをＩＣタグ特定情報（ＩＣタグ識別子）及びドナー識別子に関連付けた状態で記憶領域に格納（記憶）する。管理サーバ１１は、電子顕微鏡１３から送信された第２間葉系幹細胞３５の平面形状の拡大画像と撮影時間とをディスプレイ１７に表示する。担当者は、ディスプレイ１７に表示された第２間葉系幹細胞３５の平面形状の拡大画像を３６～４８時間の間において約１～２時間間隔で確認（視認）し、第２間葉系幹細胞３５の平面形状の変化を観察する。 The electron microscope 13 captures enlarged images of the planar shape of the second mesenchymal stem cells 35 injected into the second flat culture vessel 33 at approximately 1-2 hour intervals and transmits the captured enlarged images of the planar shape of the second mesenchymal stem cells 35 to the management server 11 at approximately 1-2 hour intervals. The management server 11 stores (memorizes) the enlarged images of the planar shape of the second mesenchymal stem cells 35 transmitted from the electron microscope 13 and the time of the image capture in a memory area, associated with the IC tag identification information (IC tag identifier) and donor identifier. The management server 11 displays the enlarged images of the planar shape of the second mesenchymal stem cells 35 transmitted from the electron microscope 13 and the time of the image capture on the display 17. The person in charge checks (visually confirms) the enlarged images of the planar shape of the second mesenchymal stem cells 35 displayed on the display 17 at approximately 1-2 hour intervals over a period of 36-48 hours to observe changes in the planar shape of the second mesenchymal stem cells 35.

第２間葉系幹細胞３５の初期平面形状は略円形であり、第２間葉系幹細胞３５の平面形状が略円形の場合、第２間葉系幹細胞３５が第２扁平培養容器３３の底面３７（底壁内面）に定着しておらず、第２間葉系幹細胞３５が増殖（分化）を開始していない。第２間葉系幹細胞３５の変形後の平面形状は定着前の略円形を核として第２間葉系幹細胞３５が一方向へ不定形に伸張した扁平形状であり、第２間葉系幹細胞３５が第２扁平培養容器３３の底面３７（底壁内面）に定着し、第２間葉系幹細胞３５が増殖（分化）を開始している。 The initial planar shape of the second mesenchymal stem cells 35 is approximately circular. When the planar shape of the second mesenchymal stem cells 35 is approximately circular, the second mesenchymal stem cells 35 have not yet settled on the bottom surface 37 (inner surface of the bottom wall) of the second flat culture vessel 33, and the second mesenchymal stem cells 35 have not yet begun proliferation (differentiation). The planar shape of the second mesenchymal stem cells 35 after deformation is a flattened shape in which the second mesenchymal stem cells 35 have stretched indefinitely in one direction, with the approximately circular shape before settling as a nucleus. The second mesenchymal stem cells 35 have settled on the bottom surface 37 (inner surface of the bottom wall) of the second flat culture vessel 33, and the second mesenchymal stem cells 35 have begun proliferation (differentiation).

形状変形第２観察工程における観察の結果、ディスプレイ１７に表示された第２間葉系幹細胞３５の平面形状が略円形から略円形を核として不定形の扁平形状に変形した場合、第２間葉系幹細胞３５が第２扁平培養容器３３の底面３７に定着したと判断する（幹細胞第２定着工程）。形状変形第２観察工程における観察の結果、第２間葉系幹細胞３５が略円形（初期平面形状）から略円形を核として不定形の扁平形状に変形し、第２間葉系幹細胞３５の第２扁平培養容器３３（第３培養容器）の底面３７への定着を確認した後、総平面面積第２観察工程が行われる。 If, as a result of the observation in the second shape deformation observation step, the planar shape of the second mesenchymal stem cells 35 displayed on the display 17 changes from an approximately circular shape to an amorphous flat shape with an approximately circular nucleus, it is determined that the second mesenchymal stem cells 35 have settled on the bottom surface 37 of the second flat culture vessel 33 (second stem cell settlement step). After the observation in the second shape deformation observation step confirms that the second mesenchymal stem cells 35 have changed from an approximately circular shape (initial planar shape) to an amorphous flat shape with an approximately circular nucleus and that the second mesenchymal stem cells 35 have settled on the bottom surface 37 of the second flat culture vessel 33 (third culture vessel), the second total planar area observation step is performed.

第２扁平培養容器３３内に注入されている培養液３６を培養容器３３から排出し、培養容器３３にあらたな培養液３６を注入（収容）する。次に、第２扁平培養容器３３を体温と略同一の温度（約３５～３７℃）で３６～４８時間静的に放置（動かすことなく静かに放置）して第２間葉系幹細胞３５を培養しつつ、３６～４８時間の間において約１～２時間間隔で培養容器３３の底面３７に定着した第２間葉系幹細胞３５の培養容器３３の底面面積に対する総平面面積をディスプレイ１７（電子顕微鏡）で観察し、第２間葉系幹細胞３５の総平面面積が第２扁平培養容器３３の底面面積に対して第２目標割合に達したか否かを判断する（総平面面積第２観察工程）。第２扁平培養容器３３の底面面積に対する第２間葉系幹細胞３５の総平面面積の第２目標割合は、８８～９２％（８８～９２％コンフルエント）である。 The culture medium 36 injected into the second flat culture vessel 33 is discharged from the culture vessel 33, and new culture medium 36 is injected (contained) into the culture vessel 33. Next, the second flat culture vessel 33 is left statically (left quietly without moving) for 36 to 48 hours at a temperature approximately equal to body temperature (approximately 35 to 37°C) to culture the second mesenchymal stem cells 35, while the total planar area of the second mesenchymal stem cells 35 that have settled on the bottom surface 37 of the culture vessel 33 relative to the bottom surface area of the culture vessel 33 is observed on the display 17 (electron microscope) at approximately 1 to 2 hour intervals during the 36 to 48 hours, and it is determined whether the total planar area of the second mesenchymal stem cells 35 relative to the bottom area of the second flat culture vessel 33 has reached a second target ratio (second total planar area observation step). The second target ratio of the total planar area of the second mesenchymal stem cells 35 to the bottom area of the second flat culture vessel 33 is 88-92% (88-92% confluent).

総平面面積第２観察工程では、第２扁平培養容器３３に注入した培養液３６を培養容器３３から排出し、あらたな培養液３６を培養容器３３に注入（収容）し、あらたな培養液３６を注入した培養容器３３を電子顕微鏡１３の試料ホルダ２７に設置（セット）する。電子顕微鏡１３の試料ホルダ２７の上面２８と第２扁平培養容器３３の底部４０との間にスペーサー３０を介在させ、培養容器３３の底部４０をスペーサー３０によって持ち上げた状態に保持し、培養容器３３の底部４０が上となり培養容器３３の頂部４１（注入口３８）が下となるように、第２扁平培養容器３３を所定角度に傾斜させた状態に保持する（図８参照）。試料ホルダ２７の上面２８に対する第２扁平培養容器３３の傾斜角度α１は、２～５°の範囲にあり、好ましくは、２～３°の範囲にある。 In the second observation step of the total planar area, the culture medium 36 poured into the second flat culture vessel 33 is discharged from the culture vessel 33, new culture medium 36 is poured (contained) into the culture vessel 33, and the culture vessel 33 with the poured new culture medium 36 is placed (set) on the specimen holder 27 of the electron microscope 13. A spacer 30 is interposed between the upper surface 28 of the specimen holder 27 of the electron microscope 13 and the bottom 40 of the second flat culture vessel 33, and the bottom 40 of the culture vessel 33 is held elevated by the spacer 30. The second flat culture vessel 33 is held tilted at a predetermined angle so that the bottom 40 of the culture vessel 33 is facing up and the top 41 (inlet 38) of the culture vessel 33 is facing down (see Figure 8). The tilt angle α1 of the second flat culture vessel 33 relative to the upper surface 28 of the specimen holder 27 is in the range of 2 to 5°, preferably 2 to 3°.

電子顕微鏡１３は、第２扁平培養容器３３内の第２間葉系幹細胞３５の平面形状の拡大画像を約１～２時間間隔で撮影し、撮影した第２間葉系幹細胞３５の平面形状の拡大画像を約１～２時間間隔で管理サーバ１１に送信する。管理サーバ１１は、電子顕微鏡１３から送信された第２間葉系幹細胞３５の平面形状の拡大画像と撮影時間とをＩＣタグ特定情報（ＩＣタグ識別子）及びドナー識別子に関連付けた状態で記憶領域に格納（記憶）する。管理サーバ１１は、電子顕微鏡１３から送信された第２間葉系幹細胞３５の平面形状の拡大画像と撮影時間とをディスプレイ１７に出力（表示）する。 The electron microscope 13 captures enlarged images of the planar shape of the second mesenchymal stem cells 35 in the second flat culture vessel 33 at approximately 1-2 hour intervals, and transmits the captured enlarged images of the planar shape of the second mesenchymal stem cells 35 to the management server 11 at approximately 1-2 hour intervals. The management server 11 stores (memorizes) the enlarged images of the planar shape of the second mesenchymal stem cells 35 transmitted from the electron microscope 13 and the time of imaging in a memory area, associated with the IC tag identification information (IC tag identifier) and donor identifier. The management server 11 outputs (displays) the enlarged images of the planar shape of the second mesenchymal stem cells 35 transmitted from the electron microscope 13 and the time of imaging on the display 17.

担当者（医師や看護師、研究者、バイオ技術者等）は、ディスプレイ１７に表示された第２間葉系幹細胞３５の平面形状の拡大画像を３６～４８時間の間において約１～２時間間隔で確認（視認）し、第２扁平培養容器３３の底面３７に定着した第２間葉系幹細胞３５の培養容器３３の底面面積に対する総平面面積を観察しつつ、第２間葉系幹細胞３５の総平面面積が培養容器３３の底面面積に対して第２目標割合（７０～８０％コンフルエント）に達したか否かを判断する。 The person in charge (doctor, nurse, researcher, bioengineer, etc.) checks (visually confirms) the enlarged image of the planar shape of the second mesenchymal stem cells 35 displayed on the display 17 at approximately 1-2 hour intervals over a period of 36-48 hours, and while observing the total planar area of the second mesenchymal stem cells 35 that have settled on the bottom surface 37 of the second flat culture vessel 33 relative to the bottom surface area of the culture vessel 33, determines whether the total planar area of the second mesenchymal stem cells 35 relative to the bottom surface area of the culture vessel 33 has reached a second target ratio (70-80% confluence).

第２間葉系幹細胞３５は、第２扁平培養容器３３の底面３７（底壁内面）において増殖してコロニーを形成する（幹細胞第２培養工程）。第２間葉系幹細胞３５がコロニーを形成し、第２間葉系幹細胞３５の平面形状が拡張し、総平面面積第２観察工程における観察の結果、ディスプレイ１７に表示された第２間葉系幹細胞３５の第２扁平培養容器３３の底面面積に対する総平面面積が第２目標割合（７０～８０％コンフルエント）に達した場合、培養容器３３から第２間葉系幹細胞を抽出する。 The second mesenchymal stem cells 35 proliferate and form colonies on the bottom surface 37 (inner surface of the bottom wall) of the second flat culture vessel 33 (second stem cell culture step). The second mesenchymal stem cells 35 form colonies, the planar shape of the second mesenchymal stem cells 35 expands, and, as a result of observation in the second total planar area observation step, the total planar area of the second mesenchymal stem cells 35 relative to the bottom surface area of the second flat culture vessel 33 displayed on the display 17 reaches a second target ratio (70-80% confluent), and the second mesenchymal stem cells are extracted from the culture vessel 33.

第２扁平培養容器３３に注入されている総平面面積第２観察工程時の培養液３６を培養容器３３から排出し、培養容器３３をりん酸緩衝生理食塩水（ＰＢＳ）で洗浄した後、トリプシン液を培養容器３３内に注入する。第２扁平培養容器３３にトリプシン液を注入すると、培養容器３３の底面３７に定着した第２間葉系幹細胞３５がトリプシン液によって底面３７から剥離し、トリプシン液の水面に浮上する。担当者は、浮上した第２間葉系幹細胞３５をピペットを利用して吸引する（第２間葉系幹細胞第２抽出工程）。 The culture medium 36 injected into the second flat culture vessel 33 during the second observation step is discharged from the culture vessel 33, the culture vessel 33 is washed with phosphate-buffered saline (PBS), and then trypsin solution is injected into the culture vessel 33. When the trypsin solution is injected into the second flat culture vessel 33, the second mesenchymal stem cells 35 that have settled on the bottom surface 37 of the culture vessel 33 are detached from the bottom surface 37 by the trypsin solution and rise to the surface of the trypsin solution. The person in charge then aspirates the floating second mesenchymal stem cells 35 using a pipette (second mesenchymal stem cell second extraction step).

尚、第２扁平培養容器３３（第３培養容器）から排出された培養液３６は、培養容器３３から第２間葉系幹細胞３５を抽出した後に残った培養液３６であり、第２間葉系幹細胞３５の培養過程において第２間葉系幹細胞３５から分泌された所定の代謝物質を含む第２培養生成液４２（培養生成液）に変化している。代謝物質を含む第２培養生成液４２は、活性が低下した間葉系幹細胞を活性化させる。第２培養生成液４２は、ＩＣタグ１８が取り付けられた冷凍保存容器４３に注入されて急速冷凍機によって急速冷凍された後、冷凍保存容器４３に収容された状態で冷蔵庫１４又は冷凍庫１４において所定期間、所定温度（０～５℃のチルド保存又は０℃以下の冷凍保存）で保存される。 The culture medium 36 discharged from the second flat culture vessel 33 (third culture vessel) is the culture medium 36 remaining after the second mesenchymal stem cells 35 are extracted from the culture vessel 33, and has changed into a second culture product liquid 42 (culture product liquid) containing specific metabolic substances secreted from the second mesenchymal stem cells 35 during the culture process of the second mesenchymal stem cells 35. The second culture product liquid 42 containing metabolic substances activates mesenchymal stem cells with reduced activity. The second culture product liquid 42 is poured into a freezing storage container 43 equipped with an IC tag 18 and flash-frozen in a flash freezer. The second culture product liquid 42 is then stored in the freezing storage container 43 at a predetermined temperature (chilled storage at 0-5°C or below 0°C) for a predetermined period of time in the refrigerator 14 or freezer 14.

冷凍幹細胞改質方法は、第１間葉系幹細胞２１を第１扁平培養容器２２（第２培養容器）の底面２３に定着させた後、培養液２６を培養容器２２から排出しつつ、あらたな培養液２６を培養容器２２に注入し、培養容器２２の底面２３に定着させた第１間葉系幹細胞２１をあらたな培養液２６を利用して培養し、培養容器２２において増殖させた第２間葉系幹細胞３５及び培養液３６を第２扁平培養容器３３（第３培養容器）に収容し、第２間葉系幹細胞３５を培養容器３３の底面３７に定着させた後、培養液３６を培養容器３３から排出しつつ、あらたな培養液３６を培養容器３３に注入し、培養容器３３の底面３７に定着させた第２間葉系幹細胞３５をあらたな培養液３６を利用して培養し、培養容器３３において第２間葉系幹細胞３５を増殖させるから、間葉系幹細胞３５を効率よく確実に増殖させることができ、十分な活性を有する間葉系幹細胞３５を効率よく作ることができる。 The frozen stem cell modification method involves allowing first mesenchymal stem cells 21 to settle on the bottom surface 23 of a first flat culture vessel 22 (second culture vessel), then discharging the culture medium 26 from the culture vessel 22 and injecting new culture medium 26 into the culture vessel 22, culturing the first mesenchymal stem cells 21 that have settled on the bottom surface 23 of the culture vessel 22 using the new culture medium 26, and placing the second mesenchymal stem cells 35 that have proliferated in the culture vessel 22 and the culture medium 36 in a second flat culture vessel 33 (third culture vessel) to produce the second mesenchymal stem cells 35. After the second mesenchymal stem cells 35 have settled on the bottom surface 37 of the culture vessel 33, the culture medium 36 is discharged from the culture vessel 33 and new culture medium 36 is poured into the culture vessel 33. The second mesenchymal stem cells 35 that have settled on the bottom surface 37 of the culture vessel 33 are cultured using the new culture medium 36, and the second mesenchymal stem cells 35 are proliferated in the culture vessel 33. This allows the mesenchymal stem cells 35 to proliferate efficiently and reliably, and mesenchymal stem cells 35 with sufficient activity to be efficiently produced.

図９は、冷凍幹細胞改質方法における冷凍保存工程の一例を示す説明図であり、図１０は、冷凍幹細胞改質方法における幹細胞解凍工程の一例を示す説明図である。図１１は、冷凍幹細胞改質方法における撹拌除去工程の一例を示す説明図であり、図１２は、冷凍幹細胞改質方法における間葉系幹細胞抽出工程の一例を示す説明図である。図１３は、冷凍幹細胞改質方法における幹細胞活性化工程の一例を示す説明図であり、図１４は、第３扁平培養容器（第１培養容器）の側面図である。図１５は、第２間葉系幹細胞３５の平面形状の一例を示す部分拡大図であり、図１６は、第２間葉系幹細胞３５の平面形状の他の一例を示す部分拡大図である。図１７は、冷凍幹細胞改質方法における幹細胞搬送工程の一例を示す説明図である。図１５，１６は、電子顕微鏡１３によって撮影された第２間葉系幹細胞３５の平面形状の拡大画像を示す。 Figure 9 is an explanatory diagram showing an example of the cryopreservation process in the frozen stem cell modification method, and Figure 10 is an explanatory diagram showing an example of the stem cell thawing process in the frozen stem cell modification method. Figure 11 is an explanatory diagram showing an example of the stirring and removal process in the frozen stem cell modification method, and Figure 12 is an explanatory diagram showing an example of the mesenchymal stem cell extraction process in the frozen stem cell modification method. Figure 13 is an explanatory diagram showing an example of the stem cell activation process in the frozen stem cell modification method, and Figure 14 is a side view of a third flat culture vessel (first culture vessel). Figure 15 is a partially enlarged view showing an example of the planar shape of second mesenchymal stem cells 35, and Figure 16 is a partially enlarged view showing another example of the planar shape of second mesenchymal stem cells 35. Figure 17 is an explanatory diagram showing an example of the stem cell transport process in the frozen stem cell modification method. Figures 15 and 16 show enlarged images of the planar shape of second mesenchymal stem cells 35 photographed by electron microscope 13.

冷凍幹細胞改質方法は、幹細胞培養方法によって培養した第２間葉系幹細胞３５（間葉系幹細胞）を冷凍保存工程を実施することによって冷凍保存し、冷凍保存した後の第２間葉系幹細胞３５（間葉系幹細胞）を幹細胞解凍工程、撹拌除去工程、間葉系幹細胞抽出工程、幹細胞活性化工程、幹細胞搬送工程を実施することによって活性化間葉系幹細胞５５に改質する。 The frozen stem cell modification method involves carrying out a cryopreservation process to freeze and preserve second mesenchymal stem cells 35 (mesenchymal stem cells) cultured by a stem cell culture method, and then modifying the frozen and preserved second mesenchymal stem cells 35 (mesenchymal stem cells) into activated mesenchymal stem cells 55 by carrying out a stem cell thawing process, a stirring and removal process, a mesenchymal stem cell extraction process, a stem cell activation process, and a stem cell transport process.

担当者（医師や看護師、研究者等）は、第２間葉系幹細胞３５（培養された間葉系幹細胞）をピペットから所定容積の冷凍保存容器４３に注入（収容）する。冷凍保存容器４３に注入された第２間葉系幹細胞３５は、不要な間葉系幹細胞が除去された活性を有する培養対象の特定種類（略単一種）のピュアな間葉系幹細胞である。冷凍保存容器４３の外周面には、ドナーデータや幹細胞データが格納されたＩＣタグ１８が取り付けられている。 A person in charge (doctor, nurse, researcher, etc.) injects (contains) second mesenchymal stem cells 35 (cultured mesenchymal stem cells) from a pipette into a cryopreservation container 43 of a predetermined volume. The second mesenchymal stem cells 35 injected into the cryopreservation container 43 are active pure mesenchymal stem cells of a specific type (substantially a single type) to be cultured, from which unnecessary mesenchymal stem cells have been removed. An IC tag 18 containing donor data and stem cell data is attached to the outer surface of the cryopreservation container 43.

冷凍保存容器４３には、凍結保護剤４４（ヒドロキシエチルスターチ(ＨＥＳ)とジメチルスルホキシド(ＤＭＳＯ)とエチレングリコール(ＥＧ)とのうちの少なくともジメチルスルホキシド(ＤＭＳＯ)）が注入される。第２間葉系幹細胞３５及び凍結保護剤４４を収容した冷凍保存容器４３は、急速冷凍機（図示せず）によって急速冷凍する。急速冷凍された第２間葉系幹細胞３５は、それが使用されるまでの間、図９に示すように、冷凍保存容器４３に収容された状態で冷蔵庫１４又は冷凍庫１４において所定期間、所定温度（０～５℃のチルド保存又は０℃以下の冷凍保存）で保存される（冷凍保存工程）。 A cryoprotectant 44 (at least dimethyl sulfoxide (DMSO) of hydroxyethyl starch (HES), dimethyl sulfoxide (DMSO), and ethylene glycol (EG)) is poured into the cryopreservation container 43. The cryopreservation container 43 containing the second mesenchymal stem cells 35 and the cryoprotectant 44 is then rapidly frozen using a rapid freezer (not shown). The rapidly frozen second mesenchymal stem cells 35 are stored in the cryopreservation container 43 at a predetermined temperature (chilled storage at 0-5°C or frozen storage at 0°C or below) in a refrigerator 14 or freezer 14 for a predetermined period of time until they are used, as shown in Figure 9 (freezing storage process).

冷蔵庫１４又は冷凍庫１４で冷凍保存された第２間葉系幹細胞３５を使用する場合（冷凍保存工程によって冷凍保存した間葉系幹細胞３５を使用する場合）、図１０に示すように、温度コントロールが可能な所定容積の保温容器４５（恒温槽）を用意し、その保温容器４５の温水中に冷凍保存容器４３を２～３分間漬け（浸水させ）、第２間葉系幹細胞３５を２～３℃の温度に解凍する（幹細胞解凍工程）。保温容器４５では、温水の温度が３５～３７℃に保持される。尚、第１培養生成液３２及び第２培養生成液４２を収容した冷凍保存容器４３を保温容器４５の温水中に２～３分間漬け（浸水させ）、第１及び第２培養生成液３２，４２を２～３℃の温度に解凍する。 When using second mesenchymal stem cells 35 that have been frozen and stored in a refrigerator 14 or freezer 14 (using mesenchymal stem cells 35 that have been frozen and stored through a cryopreservation process), as shown in FIG. 10, a temperature-controllable insulated container 45 (constant temperature bath) of a predetermined volume is prepared, and the cryopreservation container 43 is immersed (submerged) in the warm water of the insulated container 45 for 2-3 minutes to thaw the second mesenchymal stem cells 35 to a temperature of 2-3°C (stem cell thawing process). The temperature of the warm water in the insulated container 45 is maintained at 35-37°C. The cryopreservation container 43 containing the first culture product solution 32 and the second culture product solution 42 is immersed (submerged) in the warm water of the insulated container 45 for 2-3 minutes to thaw the first and second culture product solutions 32, 42 to a temperature of 2-3°C.

次に、保温容器４５から冷凍保存容器４３を取り出し、冷凍状態から解凍された第２間葉系幹細胞３５及び凍結保護剤４４を収容した冷凍保存容器４３に洗浄液４６（ダルベッコりん酸緩衝生理食塩水（Ｄ－ＰＢＳ）又はりん酸緩衝生理食塩水（ＰＢＳ））を注入する。冷凍保存容器４３に洗浄液４６を注入した後、図１１に示すように、洗浄液４６を注入した冷凍保存容器４３を左右に振って冷凍保存容器４３を攪拌し、洗浄液４６（ダルベッコりん酸緩衝生理食塩水（Ｄ－ＰＢＳ）又はりん酸緩衝生理食塩水（ＰＢＳ））によって冷凍保存容器４３内の第２間葉系幹細胞３５から凍結保護剤４４を除去（分離）する（撹拌除去工程）。 Next, the cryopreservation container 43 is removed from the insulated container 45, and a washing solution 46 (Dulbecco's phosphate-buffered saline (D-PBS) or phosphate-buffered saline (PBS)) is poured into the cryopreservation container 43 containing the thawed second mesenchymal stem cells 35 and the cryoprotectant 44. After pouring the washing solution 46 into the cryopreservation container 43, as shown in FIG. 11 , the cryopreservation container 43 containing the washing solution 46 is shaken side to side to agitate the cryopreservation container 43, and the cryoprotectant 44 is removed (separated) from the second mesenchymal stem cells 35 in the cryopreservation container 43 by the washing solution 46 (Dulbecco's phosphate-buffered saline (D-PBS) or phosphate-buffered saline (PBS)) (agitation and removal process).

担当者（医師や看護師、研究者等）は、撹拌除去工程によって第２間葉系幹細胞３５から凍結保護剤４４を除去（分離）した後、第２間葉系幹細胞３５と間葉系幹細胞３５から分離された凍結保護剤４４と洗浄液４６とを含む混合液を収容した冷凍保存容器４３を遠心分離器４７に設置（セット）する。混合液を遠心分離器４７によって所定時間遠心分離し、冷凍保存容器４３内の第２間葉系幹細胞３５、凍結保護剤４４、洗浄液４６を層状に遠心分離する。冷凍保存容器４３内の混合液は、上下方向へ２層の層状に遠心分離される。 After removing (separating) the cryoprotectant 44 from the second mesenchymal stem cells 35 through the stirring and removal process, the person in charge (doctor, nurse, researcher, etc.) places (sets) the cryopreservation container 43 containing a mixture containing the second mesenchymal stem cells 35, the cryoprotectant 44 separated from the mesenchymal stem cells 35, and the washing solution 46 in the centrifuge 47. The mixture is centrifuged for a predetermined time in the centrifuge 47, and the second mesenchymal stem cells 35, cryoprotectant 44, and washing solution 46 in the cryopreservation container 43 are centrifuged in layers. The mixture in the cryopreservation container 43 is centrifuged in two layers, one above the other.

担当者は、混合液を２層の層状に分離させた後、図１２に示すように、層状に分離した混合液から下層（最下層）に位置する第２間葉系幹細胞３５を注射器（図示せず）又はピペット（図示せず）を利用して抽出（吸引）する抽出する（間葉系幹細胞抽出工程）。間葉系幹細胞抽出工程によって抽出された第２間葉系幹細胞３５は、その冷凍時に注入された凍結保護剤４４（ヒドロキシエチルスターチ(ＨＥＳ)とジメチルスルホキシド(ＤＭＳＯ)とエチレングリコール(ＥＧ)とのうちの少なくともジメチルスルホキシド(ＤＭＳＯ)）が除去（分離）されている。 After separating the mixture into two layers, the person in charge extracts (sucks) the second mesenchymal stem cells 35 located in the lower (bottom) layer from the separated mixture using a syringe (not shown) or pipette (not shown) as shown in Figure 12 (mesenchymal stem cell extraction process). The second mesenchymal stem cells 35 extracted by the mesenchymal stem cell extraction process have had the cryoprotectant 44 (at least dimethyl sulfoxide (DMSO) of hydroxyethyl starch (HES), dimethyl sulfoxide (DMSO), and ethylene glycol (EG)) injected during freezing removed (separated) from them.

混合液から下層（最下層）に位置する特定の第２間葉系幹細胞３５を抽出した後、幹細胞活性化工程が行われる。幹細胞活性化工程では、洗浄後の第２間葉系幹細胞３５と培養液４８と第１培養生成液３２及び／又は第２培養生成液４２とを第３扁平培養容器４９（第１培養容器）に注入（収容）する。第３扁平培養容器４９に注入される第１培養生成液３２は、解凍された第２間葉系幹細胞３５（間葉系幹細胞）の元となる第１間葉系幹細胞２１の培養過程において第１間葉系幹細胞２１から分泌された所定の代謝物質を含む培養生成液であり、第３扁平培養容器４９に注入される第２培養生成液４２は、解凍された第２間葉系幹細胞３５（間葉系幹細胞）の培養過程において第２間葉系幹細胞３５から分泌された所定の代謝物質を含む培養生成液である。尚、第３扁平培養容器４９に注入する培養生成液３２，４２の注入割合は、培養容器４９に注入する培養液４８の総注入量を１００％としたときに５～１５％、好ましくは、８～１２％、より好ましくは、１０％である。 After specific second mesenchymal stem cells 35 located in the lower layer (bottom layer) are extracted from the mixed solution, a stem cell activation process is carried out. In the stem cell activation process, the washed second mesenchymal stem cells 35, culture solution 48, and first culture product solution 32 and/or second culture product solution 42 are injected (contained) into a third flat culture vessel 49 (first culture vessel). The first culture product solution 32 injected into the third flat culture vessel 49 is a culture product solution containing specific metabolic substances secreted from the first mesenchymal stem cells 21 during the culture process of the first mesenchymal stem cells 21 that are the source of the thawed second mesenchymal stem cells 35 (mesenchymal stem cells). The second culture product solution 42 injected into the third flat culture vessel 49 is a culture product solution containing specific metabolic substances secreted from the second mesenchymal stem cells 35 during the culture process of the thawed second mesenchymal stem cells 35 (mesenchymal stem cells). The ratio of the culture solution 32, 42 injected into the third flat culture vessel 49 is 5-15%, preferably 8-12%, and more preferably 10%, when the total amount of culture solution 48 injected into the culture vessel 49 is taken as 100%.

第３扁平培養容器４９（第１培養容器）は、透明なガラス又は透明なプラスチックから作られ、小容量かつ所定面積の底面を有する平面形状が略正四角形の扁平な容器である。第３扁平培養容器４９の注入口５０は、蓋５１によって水密に閉塞されている。第３扁平培養容器４９は、その容量が約２０～３０ｃｃ（好ましくは、２５ｃｃ）であり、その底面面積が約２５～３６ｍｍ２であるとともに、その一辺の長さが５～６ｍｍである。尚、第３扁平培養容器４９として小容量かつ所定面積の底面を有する平面形状が円形や楕円形の扁平な容器を使用することもできる。第３扁平培養容器４９の底面５２（底壁外面）には、ドナーデータや幹細胞データが書き込まれたＩＣタグ１８が取り付けられている。 The third flat culture vessel 49 (first culture vessel) is made of transparent glass or transparent plastic, is a flat vessel with a small capacity, a predetermined bottom area, and a roughly square planar shape. The inlet 50 of the third flat culture vessel 49 is watertightly closed by a lid 51. The third flat culture vessel 49 has a capacity of approximately 20-30 cc (preferably 25 cc), a bottom area of approximately 25-36 mm2 , and a side length of 5-6 mm. It should be noted that a flat vessel with a small capacity, a predetermined bottom area, and a circular or elliptical planar shape can also be used as the third flat culture vessel 49. An IC tag 18, on which donor data and stem cell data are written, is attached to the bottom surface 52 (outer surface of the bottom wall) of the third flat culture vessel 49.

洗浄後の第２間葉系幹細胞３５、培養液４８、第１培養生成液３２及び／又は第２培養生成液４２を収容した後、第３扁平培養容器４９を電子顕微鏡１３の試料ホルダ２７に設置（セット）する。電子顕微鏡１３の試料ホルダ２７の上面２８と第３扁平培養容器４９の底部５３との間にスペーサー３０を介在させ、培養容器４９の底部５３をスペーサー３０によって持ち上げた状態に保持し、培養容器４９の底部５３が上となり培養容器４９の頂部５４（注入口５０）が下となるように、培養容器４９を所定角度に傾斜させた（一方向へ傾斜させた）状態に保持する。尚、第３扁平培養容器４９の頂部５４をスペーサー３０によって持ち上げた状態に保持し、培養容器４９の頂部５４が上となり培養容器４９の底部５３が下となるように、培養容器４９を所定角度に傾斜させた（一方向へ傾斜させた）状態に保持してもよい。試料ホルダ２７の上面２８に対する第３扁平培養容器４９の一方向への傾斜角度α３は、２～５°の範囲にあり、好ましくは、２～３°の範囲にある。 After containing the washed second mesenchymal stem cells 35, culture medium 48, first culture production medium 32, and/or second culture production medium 42, the third flat culture vessel 49 is placed (set) in the specimen holder 27 of the electron microscope 13. A spacer 30 is interposed between the upper surface 28 of the specimen holder 27 of the electron microscope 13 and the bottom 53 of the third flat culture vessel 49, and the bottom 53 of the culture vessel 49 is held elevated by the spacer 30. The culture vessel 49 is held tilted at a predetermined angle (tilted in one direction) so that the bottom 53 of the culture vessel 49 is facing up and the top 54 (inlet 50) of the culture vessel 49 is facing down. The top 54 of the third flat culture vessel 49 may be held elevated by the spacer 30, and the culture vessel 49 may be held tilted at a predetermined angle (tilted in one direction) so that the top 54 of the culture vessel 49 is on top and the bottom 53 of the culture vessel 49 is on the bottom. The tilt angle α3 of the third flat culture vessel 49 in one direction relative to the upper surface 28 of the sample holder 27 is in the range of 2 to 5 degrees, and preferably in the range of 2 to 3 degrees.

第３扁平培養容器４９を電子顕微鏡１３の試料ホルダ２７に傾斜させた状態で設置（セット）した後、第２間葉系幹細胞３５、培養液４８、第１培養生成液３２及び／又は第２培養生成液４２を収容した第３扁平培養容器４９を体温と略同一の温度（約３５～３７℃）で２４時間静的に放置（動かすことなく静かに放置）し、第２間葉系幹細胞３５（間葉系幹細胞）を第３扁平培養容器４９（第１培養容器）において３６～３７℃で２４時間培養する。 After the third flat culture vessel 49 is placed (set) in an inclined position on the specimen holder 27 of the electron microscope 13, the third flat culture vessel 49 containing the second mesenchymal stem cells 35, culture medium 48, first culture product solution 32 and/or second culture product solution 42 is left statically (left unmoved) for 24 hours at a temperature approximately equal to body temperature (approximately 35-37°C), and the second mesenchymal stem cells 35 (mesenchymal stem cells) are cultured in the third flat culture vessel 49 (first culture vessel) at 36-37°C for 24 hours.

第３扁平培養容器４９を電子顕微鏡１３の試料ホルダ２７に設置（セット）した後、１２時間経過した場合、電子顕微鏡１３の試料ホルダ２７の上面２８と培養容器４９の底部５３との間に介在させたスペーサー３０を取り外し、そのスペーサー３０を電子顕微鏡１３の試料ホルダ２７の上面２８と培養容器４９の頂部５４との間に介在させ、培養容器４９の頂部５４をスペーサー３０によって持ち上げた状態に保持し、培養容器４９の頂部５４が上となり培養容器４９の底部５３が下となるように、培養容器４９を所定角度に傾斜させた（他方向へ傾斜させた）状態に保持する。尚、最初に第３扁平培養容器４９の頂部５４が上となり培養容器４９の底部５３が下となるように傾斜させた場合、スペーサー３０を電子顕微鏡１３の試料ホルダ２７の上面２８と培養容器４９の底部５３との間に介在させ、培養容器４９の底部５３をスペーサー３０によって持ち上げた状態に保持し、培養容器４９の底部５３が上となり培養容器４９の頂部５４が下となるように、培養容器４９を所定角度に傾斜させた（他方向へ傾斜させた）状態に保持する。 If 12 hours have passed since the third flat culture vessel 49 was installed (set) on the specimen holder 27 of the electron microscope 13, the spacer 30 interposed between the upper surface 28 of the specimen holder 27 of the electron microscope 13 and the bottom 53 of the culture vessel 49 is removed, and the spacer 30 is interposed between the upper surface 28 of the specimen holder 27 of the electron microscope 13 and the top 54 of the culture vessel 49, the top 54 of the culture vessel 49 is held elevated by the spacer 30, and the culture vessel 49 is held tilted at a predetermined angle (tilted in the other direction) so that the top 54 of the culture vessel 49 is on top and the bottom 53 of the culture vessel 49 is on the bottom. Furthermore, if the third flat culture vessel 49 is initially tilted so that the top 54 of the culture vessel 49 is on top and the bottom 53 of the culture vessel 49 is on the bottom, a spacer 30 is interposed between the top surface 28 of the specimen holder 27 of the electron microscope 13 and the bottom 53 of the culture vessel 49, and the bottom 53 of the culture vessel 49 is held elevated by the spacer 30, and the culture vessel 49 is held tilted at a predetermined angle (tilted in the other direction) so that the bottom 53 of the culture vessel 49 is on top and the top 54 of the culture vessel 49 is on the bottom.

試料ホルダ２７の上面２８に対する第３扁平培養容器４９の他方向への傾斜角度α３は、２～５°の範囲にあり、好ましくは、２～３°の範囲にある。尚、第３扁平培養容器４９を一方向（又は他方向）へ継続して傾斜させた状態で体温と略同一の温度（約３５～３７℃）で２４時間静的に放置（動かすことなく静かに放置）し、第２間葉系幹細胞３５（間葉系幹細胞）を第３扁平培養容器４９（第１培養容器）において３６～３７℃で２４時間培養する場合もある。 The tilt angle α3 of the third flat culture vessel 49 in the other direction relative to the upper surface 28 of the specimen holder 27 is in the range of 2 to 5°, and preferably in the range of 2 to 3°. In some cases, the third flat culture vessel 49 may be left statically (left unmoved) for 24 hours at a temperature approximately equal to body temperature (approximately 35 to 37°C) while continuously tilted in one direction (or the other direction), and the second mesenchymal stem cells 35 (mesenchymal stem cells) may be cultured in the third flat culture vessel 49 (first culture vessel) at 36 to 37°C for 24 hours.

試料ホルダ２７の上面２８に対して第３扁平培養容器４９を前記傾斜角度で一方向及び他方向へ傾斜させることで、培養容器４９内において第２間葉系幹細胞３５（間葉系幹細胞）、培養液４８、培養生成液３２，４２が培養容器４９の頂部５４の側（又は底部５３の側）に偏り、培養容器４９の頂部５４の側（又は底部５３の側）において第２間葉系幹細胞３５、培養液４８、培養生成液３２，４２の水圧が大きくなって第２間葉系幹細胞３５が培養容器４９の頂部５４の側（又は底部５３の側）に集中し、それによって第２間葉系幹細胞３５どうしの活性が高まり、培養容器４９の底面５２において第２間葉系幹細胞３５を容易かつ迅速に活性化（定着）させることができる。 By tilting the third flat culture vessel 49 in one direction and the other relative to the upper surface 28 of the sample holder 27 at the tilt angle, the second mesenchymal stem cells 35 (mesenchymal stem cells), culture medium 48, and culture products 32, 42 are biased toward the top 54 side (or bottom 53 side) of the culture vessel 49 within the culture vessel 49, and the water pressure of the second mesenchymal stem cells 35, culture medium 48, and culture products 32, 42 on the top 54 side (or bottom 53 side) of the culture vessel 49 increases, concentrating the second mesenchymal stem cells 35 on the top 54 side (or bottom 53 side) of the culture vessel 49, thereby increasing the activity of the second mesenchymal stem cells 35 among themselves and enabling the second mesenchymal stem cells 35 to be easily and quickly activated (established) on the bottom surface 52 of the culture vessel 49.

電子顕微鏡１３は、第３扁平培養容器４９に注入された第２間葉系幹細胞３５の平面形状の拡大画像を約１～２時間間隔で撮影し、撮影した第２間葉系幹細胞３５の平面形状の拡大画像を約１～２時間間隔で管理サーバ１１に送信する。管理サーバ１１は、電子顕微鏡１３から送信された第２間葉系幹細胞３５の平面形状の拡大画像と撮影時間とをＩＣタグ特定情報（ＩＣタグ識別子）及びドナー識別子に関連付けた状態で記憶領域に格納（記憶）する。管理サーバ１１は、電子顕微鏡１３から送信された第２間葉系幹細胞３５の平面形状の拡大画像と撮影時間とをディスプレイ１７に出力（表示）する。担当者（医師や看護師、研究者、バイオ技術者等）は、ディスプレイ１７に表示された第２間葉系幹細胞３５の平面形状の拡大画像を１２～２４時間の間において約１～２時間間隔で確認（視認）し、第２間葉系幹細胞３５の平面形状の変化を観察し、第２間葉系幹細胞３５が活性化したか（第３扁平培養容器４９の底面５２に定着したか）否かを判断する（形状変形第３観察工程）。 The electron microscope 13 captures enlarged images of the planar shape of the second mesenchymal stem cells 35 injected into the third flat culture vessel 49 at approximately 1-2 hour intervals, and transmits the captured enlarged images of the planar shape of the second mesenchymal stem cells 35 to the management server 11 at approximately 1-2 hour intervals. The management server 11 stores (memorizes) the enlarged images of the planar shape of the second mesenchymal stem cells 35 transmitted from the electron microscope 13 and the time of photographing in a memory area, associated with the IC tag identification information (IC tag identifier) and donor identifier. The management server 11 outputs (displays) the enlarged images of the planar shape of the second mesenchymal stem cells 35 transmitted from the electron microscope 13 and the time of photographing on the display 17. The person in charge (doctor, nurse, researcher, bioengineer, etc.) checks (visually confirms) the enlarged image of the planar shape of the second mesenchymal stem cells 35 displayed on the display 17 at approximately 1-2 hour intervals over a period of 12-24 hours, observes changes in the planar shape of the second mesenchymal stem cells 35, and determines whether the second mesenchymal stem cells 35 have been activated (whether they have settled on the bottom surface 52 of the third flat culture vessel 49) (third shape deformation observation process).

解凍後の第２間葉系幹細胞３５の平面形状は略円形であり、第２間葉系幹細胞３５の平面形状が略円形の場合、第２間葉系幹細胞３５が活性化しておらず（第３扁平培養容器４９の底面５２（底壁内面）に定着しておらず）、第２間葉系幹細胞３５が活性化を開始していない。第２間葉系幹細胞３５の変形後の平面形状は定着前の略円形を核として第２間葉系幹細胞３５が一方向（所定方向）へ不定形に伸張（拡張）した扁平形状であり、第２間葉系幹細胞３５が第３扁平培養容器４９の底面５２（底壁内面）に定着し、第２間葉系幹細胞３５が活性化し、培養容器内４９（第１培養容器内）の第２間葉系幹細胞３５（間葉系幹細胞）が活性化間葉系幹細胞に改質している。 After thawing, the second mesenchymal stem cells 35 have a roughly circular planar shape. When the second mesenchymal stem cells 35 have a roughly circular planar shape, the second mesenchymal stem cells 35 are not activated (they have not settled on the bottom surface 52 (inner bottom wall surface) of the third flat culture vessel 49) and have not yet begun activation. The planar shape of the second mesenchymal stem cells 35 after deformation is a flattened shape in which the second mesenchymal stem cells 35 have irregularly stretched (expanded) in one direction (a predetermined direction) using the roughly circular shape before settlement as a nucleus. The second mesenchymal stem cells 35 have settled on the bottom surface 52 (inner bottom wall surface) of the third flat culture vessel 49, the second mesenchymal stem cells 35 have been activated, and the second mesenchymal stem cells 35 (mesenchymal stem cells) in the culture vessel 49 (in the first culture vessel) have been converted into activated mesenchymal stem cells.

担当者は、形状変形第３観察工程における観察の結果、図１４に示すように、ディスプレイ１７に表示された第２間葉系幹細胞３５の平面形状が略円形のまま観察される場合、第２間葉系幹細胞３５が第３扁平培養容器４９の底面５２（底壁内面）に定着していないと判断し、第２間葉系幹細胞３５の平面形状の変化を約１～２時間間隔で継続して観察する。担当者は、形状変形第３観察工程における観察の結果、図１５に示すように、ディスプレイ１７に出力（表示）された第２間葉系幹細胞３５の平面形状が略円形から略円形を核として不定形の扁平形状に変形した場合、第２間葉系幹細胞３５が活性化し（第３扁平培養容器の底面に定着し）、第２間葉系幹細胞３５が活性化間葉系幹細胞５５に改質したと判断する（幹細胞活性化工程）。 If, as a result of the observation in the third shape transformation observation step, the second mesenchymal stem cells 35 displayed on the display 17 remain approximately circular in plan view as shown in Figure 14, the person in charge determines that the second mesenchymal stem cells 35 have not settled on the bottom surface 52 (inner surface of the bottom wall) of the third flat culture vessel 49, and continues to observe changes in the planar shape of the second mesenchymal stem cells 35 at intervals of approximately 1 to 2 hours. If, as a result of the observation in the third shape transformation observation step, the second mesenchymal stem cells 35 output (displayed) on the display 17 change from approximately circular to an amorphous flat shape with the approximately circular shape as the nucleus as shown in Figure 15, the person in charge determines that the second mesenchymal stem cells 35 have been activated (settled on the bottom surface of the third flat culture vessel) and have been transformed into activated mesenchymal stem cells 55 (stem cell activation step).

第２間葉系幹細胞３５が活性化間葉系幹細胞５５に改質したと判断した後、第３扁平培養容器４９に注入されている培養液４８及び培養生成液３２，４２を培養容器４９から排出し、培養容器４９をりん酸緩衝生理食塩水（ＰＢＳ）で洗浄した後、トリプシン液を培養容器４９内に注入する。第３扁平培養容器４９にトリプシン液を注入すると、培養容器４９の底面５２に定着した活性化間葉系幹細胞５５がトリプシン液によって底面５２から剥離し、トリプシン液の水面に浮上する。担当者は、浮上した活性化間葉系幹細胞５５をピペットを利用して吸引し、その活性化間葉系幹細胞５５を第３扁平培養容器４９（第１培養容器）から抽出し、その活性化間葉系幹細胞５５を保存容器５６に収容する。 After determining that the second mesenchymal stem cells 35 have been transformed into activated mesenchymal stem cells 55, the culture medium 48 and culture products 32 and 42 injected into the third flat culture vessel 49 are discharged from the culture vessel 49, the culture vessel 49 is washed with phosphate-buffered saline (PBS), and then trypsin solution is injected into the culture vessel 49. When the trypsin solution is injected into the third flat culture vessel 49, the activated mesenchymal stem cells 55 that have settled on the bottom surface 52 of the culture vessel 49 are detached from the bottom surface 52 by the trypsin solution and rise to the surface of the trypsin solution. The person in charge aspirates the floating activated mesenchymal stem cells 55 using a pipette, extracts the activated mesenchymal stem cells 55 from the third flat culture vessel 49 (first culture vessel), and stores the activated mesenchymal stem cells 55 in a storage container 56.

活性化間葉系幹細胞５５を抽出後１８～２０時間以内に使用する場合、担当者は、図１７に示すように、活性化間葉系幹細胞５５を収容した保存容器５６を持ち運び可能な恒温ケース５７に収容し、恒温ケース５７内の温度を２０～２１℃に保持し、抽出した活性化間葉系幹細胞５５を２０～２１℃に保持した状態で、１８～２０時間以内に活性化間葉系幹細胞５５の使用箇所まで搬送する（幹細胞搬送工程）。使用箇所まで搬送された活性化間葉系幹細胞５５は、恒温ケース５７から取り出され、各種の疾患（心血管疾患や中枢神経系疾患等）の治療や再生医療における再生、美容等の非治療的用途に使用される。 When the activated mesenchymal stem cells 55 are to be used within 18 to 20 hours after extraction, the person in charge places the storage container 56 containing the activated mesenchymal stem cells 55 in a portable thermostatic case 57, as shown in Figure 17, maintains the temperature inside the thermostatic case 57 at 20 to 21°C, and transports the extracted activated mesenchymal stem cells 55, while maintained at 20 to 21°C, to the site of use within 18 to 20 hours (stem cell transport process). The activated mesenchymal stem cells 55 transported to the site of use are removed from the thermostatic case 57 and used for the treatment of various diseases (such as cardiovascular disease and central nervous system disease), regeneration in regenerative medicine, and non-therapeutic purposes such as cosmetic treatment.

冷凍幹細胞改質方法（冷凍幹細胞改質システム１０）は、冷凍状態から解凍された第２間葉系幹細胞３５（間葉系幹細胞）及び凍結保護剤４４（ヒドロキシエチルスターチ(ＨＥＳ)とジメチルスルホキシド(ＤＭＳＯ)とエチレングリコール(ＥＧ)とのうちの少なくともジメチルスルホキシド(ＤＭＳＯ)）を収容した冷凍保存容器４３に洗浄液４６（ダルベッコりん酸緩衝生理食塩水（Ｄ－ＰＢＳ）又はりん酸緩衝生理食塩水（ＰＢＳ））を注入し、洗浄液４６を注入した冷凍保存容器４３を攪拌して洗浄液４６によって冷凍保存容器４３内の第２間葉系幹細胞３５から凍結保護剤４４を除去（分離）し、冷凍保存容器４３内の第２間葉系幹細胞３５、凍結保護剤４４、洗浄液４６を層状に遠心分離し、遠心分離した後の冷凍保存容器４３の最下層に位置する第２間葉系幹細胞３５を抽出するから、冷凍保存された第２間葉系幹細胞３５を解凍する際に洗浄液４６を利用して凍結保護剤４４を除去することができるとともに、冷凍状態から解凍された第２間葉系幹細胞３５の大部分（７０～９０％）を生存させることができる。 The frozen stem cell modification method (frozen stem cell modification system 10) involves injecting a cleaning solution 46 (Dulbecco's phosphate buffered saline (D-PBS) or phosphate buffered saline (PBS)) into a cryopreservation container 43 containing second mesenchymal stem cells 35 (mesenchymal stem cells) that have been thawed from a frozen state and a cryoprotectant 44 (hydroxyethyl starch (HES), dimethyl sulfoxide (DMSO), and ethylene glycol (EG), at least dimethyl sulfoxide (DMSO)), and agitating the cryopreservation container 43 containing the cleaning solution 46 to remove the cleaning solution 46. 6 removes (separates) the cryoprotectant 44 from the second mesenchymal stem cells 35 in the cryopreservation container 43, then the second mesenchymal stem cells 35, cryoprotectant 44, and washing solution 46 in the cryopreservation container 43 are centrifuged in layers, and the second mesenchymal stem cells 35 located in the bottom layer of the cryopreservation container 43 after centrifugation are extracted. Therefore, when thawing the frozen-preserved second mesenchymal stem cells 35, the cryoprotectant 44 can be removed using the washing solution 46, and the majority (70-90%) of the second mesenchymal stem cells 35 thawed from a frozen state can survive.

冷凍幹細胞改質方法（冷凍幹細胞改質システム１０）は、冷凍状態から解凍された第２間葉系幹細胞３５（間葉系幹細胞）を所定容量かつ所定面積の底面を有する第３扁平培養容器４９（第１培養容器）に収容するとともに、第２間葉系幹細胞３５を収容した培養容器４９に所定の培養液４８及び培養生成液３２，４２を注入し、第２間葉系幹細胞３５を培養容器４９において３５～３７℃で２４時間培養し、培養容器４９内の第２間葉系幹細胞３５を活性化させるから、冷凍保存された第２間葉系幹細胞３５を解凍する際に、解凍された第２間葉系幹細胞３５を十分な活性を有する活性化間葉系幹細胞５５に改質することができる。 The frozen stem cell modification method (frozen stem cell modification system 10) involves placing second mesenchymal stem cells 35 (mesenchymal stem cells) thawed from a frozen state in a third flat culture vessel 49 (first culture vessel) with a predetermined volume and a predetermined bottom area, injecting a predetermined culture medium 48 and culture generating solutions 32, 42 into the culture vessel 49 containing the second mesenchymal stem cells 35, and culturing the second mesenchymal stem cells 35 in the culture vessel 49 at 35-37°C for 24 hours to activate the second mesenchymal stem cells 35 in the culture vessel 49. Therefore, when the frozen stored second mesenchymal stem cells 35 are thawed, the thawed second mesenchymal stem cells 35 can be modified into activated mesenchymal stem cells 55 with sufficient activity.

冷凍幹細胞改質方法（冷凍幹細胞改質システム１０）は、各種疾患（心血管疾患や中枢神経系疾患等）の治療や再生医療における再生、美容等の非治療的用途における十分かつ高い効果を有する活性化間葉系幹細胞５５を作ることができ、所望の効果を得ることが可能な活性化間葉系幹細胞５５を作ることができる。冷凍幹細胞改質方法（冷凍幹細胞改質システム１０）は、各種疾患の治療に好適かつタイムリーに使用することが可能な活性化間葉系幹細胞５５を作ることができ、各種組織や各種臓器を再生に好適かつタイムリーに使用することが可能な活性化間葉系幹細胞５５を作ることができるとともに、美容等の非治療的用途に好適かつタイムリーに使用することが可能な活性化間葉系幹細胞５５を作ることができる。冷凍幹細胞改質方法（冷凍幹細胞改質システム１０）は、活性化された活性化間葉系幹細胞５５に凍結保護剤４４が含まれていないから、冷凍幹細胞改質方法によって作られた活性化間葉系幹細胞５５を人体に投与下としても、人体に悪影響を与えることはない。 The frozen stem cell modification method (frozen stem cell modification system 10) can produce activated mesenchymal stem cells 55 that are sufficiently effective for the treatment of various diseases (such as cardiovascular disease and central nervous system disease), regeneration in regenerative medicine, and non-therapeutic uses such as cosmetics, and can produce activated mesenchymal stem cells 55 that can achieve the desired effects. The frozen stem cell modification method (frozen stem cell modification system 10) can produce activated mesenchymal stem cells 55 that can be used suitably and in a timely manner for the treatment of various diseases, can produce activated mesenchymal stem cells 55 that can be used suitably and in a timely manner for the regeneration of various tissues and organs, and can produce activated mesenchymal stem cells 55 that can be used suitably and in a timely manner for non-therapeutic uses such as cosmetics. Because the frozen stem cell modification method (frozen stem cell modification system 10) does not contain a cryoprotectant 44 in the activated mesenchymal stem cells 55, administering the activated mesenchymal stem cells 55 produced by the frozen stem cell modification method to the human body will not have any adverse effects.

冷凍幹細胞改質方法（冷凍幹細胞改質システム１０）は、培養生成液３２，４２が第１扁平培養容器２２（第２培養容器）又は第２扁平培養容器３３（第３培養容器）から培養後の第１及び第２間葉系幹細胞２１，３５（間葉系幹細胞）を抽出した後に残った培養液であり、間葉系幹細胞２１，３５の培養過程において間葉系幹細胞２１，３５から分泌された所定の代謝物質を含むから、間葉系幹細胞２１，３５自体の代謝物質がトリガーとなり、第２間葉系幹細胞３５が速やかに活性を開始するとともに、その培養生成液３２，４２を利用して第２間葉系幹細胞３５を速やかに培養することができ、活性化間葉系幹細胞５５を短時間に効率よく作ることができる。 In the frozen stem cell modification method (frozen stem cell modification system 10), the culture product solution 32, 42 is the culture solution remaining after extracting cultured first and second mesenchymal stem cells 21, 35 (mesenchymal stem cells) from the first flat culture vessel 22 (second culture vessel) or the second flat culture vessel 33 (third culture vessel). The culture product solution 32, 42 contains specific metabolic substances secreted by the mesenchymal stem cells 21, 35 during the culture process. Therefore, the metabolic substances of the mesenchymal stem cells 21, 35 themselves act as a trigger, causing the second mesenchymal stem cells 35 to rapidly begin activation. The second mesenchymal stem cells 35 can then be rapidly cultured using the culture product solution 32, 42, enabling activated mesenchymal stem cells 55 to be produced efficiently in a short period of time.

冷凍幹細胞改質方法（冷凍幹細胞改質システム１０）は、活性化間葉系幹細胞５５を抽出後１８～２０時間以内に使用する場合、活性化間葉系幹細胞５５を２０～２１℃に保持した状態で１８～２０時間以内に活性化間葉系幹細胞５５の使用箇所まで搬送することで、活性化間葉系幹細胞５５の活性が失われず、その活性化間葉系幹細胞５５を各種疾患（心血管疾患や中枢神経系疾患等）の治療に利用することができ、その活性化間葉系幹細胞５５を再生医療における再生に利用することができるとともに、その活性化間葉系幹細胞５５を美容等の非治療的用途に利用することができる。 When the activated mesenchymal stem cells 55 are to be used within 18 to 20 hours after extraction, the frozen stem cell modification method (frozen stem cell modification system 10) allows the activated mesenchymal stem cells 55 to be maintained at 20 to 21°C and transported to the site of use within 18 to 20 hours, thereby preventing the activation of the activated mesenchymal stem cells 55 and allowing the activated mesenchymal stem cells 55 to be used in the treatment of various diseases (such as cardiovascular disease and central nervous system disease).The activated mesenchymal stem cells 55 can be used for regeneration in regenerative medicine, and the activated mesenchymal stem cells 55 can also be used for non-therapeutic purposes such as cosmetic treatment.

１０ 冷凍幹細胞改質システム
１１ 管理サーバ
１２ ＩＣタグリーダ／ライタ
１３ 電子顕微鏡
１４ 冷蔵庫又は冷凍庫
１５ キーボード
１６ マウス
１７ ディスプレイ
１８ ＩＣタグ
１９ 第１骨髄液
２０ ガラス試験管
２１ 第２骨髄液（第１間葉系幹細胞）
２２ 第１扁平培養容器（第２培養容器）
２３ 底面
２４ 注入口
２５ 蓋
２６ 培養液
２７ 試料ホルダ
２８ 上面
２９ 底部
３０ スぺーサー
３１ 頂部
３２ 第１培養生成液
３３ 第２扁平培養容器（第３培養容器）
３４ ガラス試験管
３５ 第２間葉系幹細胞
３６ 培養液
３７ 底面
３８ 注入口
３９ 蓋
４０ 底部
４１ 頂部
４２ 第２培養生成液
４３ 冷凍保存容器
４４ 凍結保護剤
４５ 保温容器
４６ 洗浄剤
４７ 遠心分離器
４８ 培養液
４９ 第３扁平培養容器（第１培養容器）
５０ 注入口
５１ 蓋
５２ 底面
５３ 底部
５４ 頂部
５５ 活性化間葉系幹細胞
５６ 保存容器
５７ 恒温ケース 10 Frozen stem cell modification system 11 Management server 12 IC tag reader/writer 13 Electron microscope 14 Refrigerator or freezer 15 Keyboard 16 Mouse 17 Display 18 IC tag 19 First bone marrow fluid 20 Glass test tube 21 Second bone marrow fluid (first mesenchymal stem cells)
22 First flat culture container (second culture container)
23 Bottom surface 24 Inlet 25 Lid 26 Culture medium 27 Sample holder 28 Top surface 29 Bottom 30 Spacer 31 Top 32 First culture product medium 33 Second flat culture vessel (third culture vessel)
34 Glass test tube 35 Second mesenchymal stem cells 36 Culture medium 37 Bottom surface 38 Inlet 39 Lid 40 Bottom 41 Top 42 Second culture product solution 43 Cryopreservation container 44 Cryoprotectant 45 Insulating container 46 Detergent 47 Centrifuge 48 Culture medium 49 Third flat culture vessel (first culture vessel)
50 Inlet 51 Lid 52 Bottom 53 Bottom 54 Top 55 Activated mesenchymal stem cells 56 Storage container 57 Constant temperature case

Claims (6)

所定温度に冷凍された間葉系幹細胞を活性化させて活性化間葉系幹細胞に改質させる冷凍幹細胞改質方法において、
前記冷凍幹細胞改質方法が、ドナーから採取した骨髄液を培養することから作られた間葉系幹細胞を所定容積の冷凍保存容器に収容するとともに、前記間葉系幹細胞を収容した前記冷凍保存容器に凍結保護剤を注入し、前記冷凍保存容器に収容した間葉系幹細胞を使用するまでの間、該冷凍保存容器に収容した該間葉系幹細胞を前記凍結保護剤とともに所定温度で冷凍保存する冷凍保存工程と、
前記冷凍保存工程によって冷凍保存した前記間葉系幹細胞を使用する場合、冷凍保存された前記間葉系幹細胞及び前記凍結保護剤を収容した前記冷凍保存容器を３５～３７℃に保持された温水中に２～３分浸水させて該間葉系幹細胞を２～３℃の温度に解凍する幹細胞解凍工程と、
前記幹細胞解凍工程によって冷凍状態から解凍された前記間葉系幹細胞及び前記凍結保護剤を収容した前記冷凍保存容器に洗浄液を注入し、前記洗浄液を注入した前記冷凍保存容器を攪拌して該洗浄液によって該冷凍保存容器内の間葉系幹細胞から凍結保護剤を除去する撹拌除去工程と、
前記撹拌除去工程によって間葉系幹細胞から凍結保護剤を除去した後の前記冷凍保存容器を遠心分離器に設置し、前記冷凍保存容器内の前記間葉系幹細胞、前記凍結保護剤、前記洗浄液を層状に遠心分離し、前記遠心分離した後の前記冷凍保存容器の最下層に位置する間葉系幹細胞を抽出する間葉系幹細胞抽出工程と、
前記間葉系幹細胞抽出工程によって抽出した間葉系幹細胞を所定容量かつ所定面積の底面を有する第１培養容器に収容するとともに、前記間葉系幹細胞を収容した前記第１培養容器に所定の培養液を注入し、前記間葉系幹細胞を収容した前記第１培養容器を一方向へ２～５度の角度に傾斜させた状態で該第１培養容器を３５～３７℃の温度で１２時間静的に放置し、前記一方向へ傾斜させた前記第１培養容器を該一方向と反対の他方向へ２～５度の角度に傾斜させた状態で該第１培養容器を３５～３７℃の温度で１２時間静的に放置して該間葉系幹細胞を培養し、該第１培養容器内の該間葉系幹細胞を活性化させて前記活性化間葉系幹細胞に改質させる幹細胞活性化工程と、
前記活性化させた活性化間葉系幹細胞を前記第１培養容器から抽出した後、該活性化間葉系幹細胞を抽出後１８～２０時間以内に使用する場合、前記活性化間葉系幹細胞を収容した保存容器を持ち運び可能な恒温ケースに収容し、前記恒温ケース内の温度を２０～２１℃に保持し、抽出した活性化間葉系幹細胞を２０～２１℃に保持した状態で１８～２０時間以内に該活性化間葉系幹細胞の使用箇所まで搬送する幹細胞搬送工程とを有することを特徴とする冷凍幹細胞改質方法。 A method for modifying frozen stem cells, which activates mesenchymal stem cells frozen to a predetermined temperature and modifies them into activated mesenchymal stem cells, comprising:
The frozen stem cell modification method includes a cryopreservation step of storing mesenchymal stem cells prepared by culturing bone marrow fluid collected from a donor in a cryopreservation container of a predetermined volume, injecting a cryoprotectant into the cryopreservation container containing the mesenchymal stem cells, and freezing and storing the mesenchymal stem cells stored in the cryopreservation container together with the cryoprotectant at a predetermined temperature until the mesenchymal stem cells stored in the cryopreservation container are used;
a stem cell thawing step in which, when the mesenchymal stem cells cryopreserved in the cryopreservation step are to be used, the cryopreservation container containing the cryopreserved mesenchymal stem cells and the cryoprotectant is immersed in warm water maintained at 35 to 37°C for 2 to 3 minutes to thaw the mesenchymal stem cells to a temperature of 2 to 3°C;
a stirring and removal step of injecting a washing solution into the cryopreservation container containing the mesenchymal stem cells thawed from a frozen state in the stem cell thawing step and the cryoprotectant, and stirring the cryopreservation container into which the washing solution has been injected to remove the cryoprotectant from the mesenchymal stem cells in the cryopreservation container by the washing solution;
a mesenchymal stem cell extraction step of placing the cryopreservation container after the cryoprotectant has been removed from the mesenchymal stem cells by the stirring and removal step in a centrifuge, centrifuging the mesenchymal stem cells, the cryoprotectant, and the washing solution in the cryopreservation container in layers, and extracting the mesenchymal stem cells located in the bottom layer of the cryopreservation container after the centrifugation;
a stem cell activation step of accommodating the mesenchymal stem cells extracted in the mesenchymal stem cell extraction step in a first culture vessel having a predetermined volume and a predetermined bottom area, pouring a predetermined culture medium into the first culture vessel containing the mesenchymal stem cells, tilting the first culture vessel containing the mesenchymal stem cells in one direction at an angle of 2 to 5 degrees, and leaving the first culture vessel statically at a temperature of 35 to 37°C for 12 hours, and then tilting the first culture vessel tilted in one direction at an angle of 2 to 5 degrees in the opposite direction, and leaving the first culture vessel statically at a temperature of 35 to 37°C for 12 hours to culture the mesenchymal stem cells, thereby activating the mesenchymal stem cells in the first culture vessel and converting them into the activated mesenchymal stem cells;
After extracting the activated mesenchymal stem cells from the first culture vessel, if the activated mesenchymal stem cells are to be used within 18 to 20 hours after extraction, the method for modifying frozen stem cells comprises a stem cell transport step of placing the storage container containing the activated mesenchymal stem cells in a portable thermostatic case, maintaining the temperature within the thermostatic case at 20 to 21°C, and transporting the extracted activated mesenchymal stem cells, while maintained at 20 to 21°C, to a site where the activated mesenchymal stem cells will be used within 18 to 20 hours. 前記幹細胞活性化工程が、前記ドナーから採取した骨髄液を培養する培養過程において生成された培養生成液を前記第１培養容器に注入し、前記培養液及び前記培養生成液を利用して前記間葉系幹細胞を前記第１培養容器において３５～３７℃の温度で２４時間培養する請求項１に記載の冷凍幹細胞改質方法。 The frozen stem cell modification method of claim 1, wherein the stem cell activation step comprises injecting a culture product solution produced during a culture process in which bone marrow fluid collected from the donor is cultured into the first culture vessel, and culturing the mesenchymal stem cells in the first culture vessel at a temperature of 35-37°C for 24 hours using the culture solution and the culture product solution. 前記凍結保護剤が、ヒドロキシエチルスターチ(ＨＥＳ)とジメチルスルホキシド(ＤＭＳＯ)とエチレングリコール(ＥＧ)とのうちの少なくとも該ジメチルスルホキシド(ＤＭＳＯ)であり、前記洗浄液が、ダルベッコりん酸緩衝生理食塩水（Ｄ－ＰＢＳ）又はりん酸緩衝生理食塩水（ＰＢＳ）である請求項２に記載の冷凍幹細胞改質方法。 The frozen stem cell modification method of claim 2, wherein the cryoprotectant is at least dimethyl sulfoxide (DMSO) of hydroxyethyl starch (HES), dimethyl sulfoxide (DMSO), and ethylene glycol (EG), and the washing solution is Dulbecco 's phosphate buffered saline (D-PBS) or phosphate buffered saline (PBS). 前記間葉系幹細胞が、前記ドナーから採取した骨髄液を層状に分離し、層状に分離させた前記骨髄液のうちの中間層に位置する中間層骨髄液を抽出し、所定容量かつ所定面積の底面を有する第２培養容器に前記中間層骨髄液と所定の培養液とを注入して該中間層骨髄液に含まれる第１間葉系幹細胞を前記第２培養容器の底面に定着させる幹細胞第１定着工程と、前記幹細胞第１定着工程によって前記第１間葉系幹細胞を前記第２培養容器の底面に定着させた後、前記第２培養容器内の培養液を排出しつつあらたな培養液を該第２培養容器に注入して前記第１間葉系幹細胞を培養し、前記第２培養容器の底面面積に対する前記第１間葉系幹細胞の総平面面積が第１目標割合に達するまで該第１間葉系幹細胞を増殖させる幹細胞第１培養工程と、前記幹細胞第１培養工程によって増殖させた前記第１間葉系幹細胞及び前記培養液を収容した第２培養容器を遠心分離器に設置し、前記第２培養容器内の前記第１間葉系幹細胞、前記培養液を層状に遠心分離し、前記遠心分離した後の前記第２培養容器の最下層に位置する第２間葉系幹細胞を抽出する第２間葉系幹細胞第１抽出工程と、所定容量かつ所定面積の底面を有して前記第２培養容器よりも大きい容量の第３培養容器に前記第２間葉系幹細胞第１抽出工程によって抽出した第２間葉系幹細胞を収容しつつ、前記第３培養容器にあらたな培養液を注入し、前記第２間葉系幹細胞を前記第３培養容器の底面に定着させる幹細胞第２定着工程と、前記幹細胞第２定着工程によって前記第２間葉系幹細胞を前記第３培養容器の底面に定着させた後、前記第３培養容器内の培養液を排出しつつあらたな培養液を該第３培養容器に注入して前記第２間葉系幹細胞を培養し、前記第３培養容器の底面面積に対する前記第２間葉系幹細胞の総平面面積が第２目標割合に達するまで該第２間葉系幹細胞を増殖させる幹細胞第２培養工程と、前記幹細胞第２培養工程によって培養された前記第２間葉系幹細胞を含む前記第３培養容器を遠心分離器に設置し、前記第３培養容器内の前記第２間葉系幹細胞、前記培養液を層状に遠心分離し、前記遠心分離した後の前記第３培養容器の最下層に位置する第２間葉系幹細胞を抽出する第２間葉系幹細胞第２抽出工程とを有する間葉系幹細胞培養方法によって作られている請求項３に記載の冷凍幹細胞改質方法。 The mesenchymal stem cells are obtained by a first stem cell settling step of separating bone marrow fluid collected from the donor into layers, extracting intermediate layer bone marrow fluid located in the intermediate layer of the separated layers of bone marrow fluid, and injecting the intermediate layer bone marrow fluid and a predetermined culture medium into a second culture vessel having a predetermined volume and a predetermined bottom area to settling first mesenchymal stem cells contained in the intermediate layer bone marrow fluid on the bottom surface of the second culture vessel; and after the first mesenchymal stem cells have settled on the bottom surface of the second culture vessel by the first stem cell settling step, discharging the culture medium in the second culture vessel and injecting a new culture medium into the second culture vessel. a first stem cell culture step of culturing the first mesenchymal stem cells in a culture medium containing the first mesenchymal stem cells and growing the first mesenchymal stem cells until a ratio of a total planar area of the first mesenchymal stem cells to a bottom area of the second culture vessel reaches a first target ratio; a first second mesenchymal stem cell extraction step of placing the second culture vessel containing the first mesenchymal stem cells grown in the first stem cell culture step and the culture medium in a centrifuge, centrifuging the first mesenchymal stem cells and the culture medium in the second culture vessel in layers, and extracting second mesenchymal stem cells located in the bottom layer of the second culture vessel after the centrifugation; a second stem cell settling step in which the second mesenchymal stem cells extracted in the first second mesenchymal stem cell extraction step are placed in a third culture vessel having a bottom surface of a predetermined area and a volume larger than the second culture vessel, a new culture medium is poured into the third culture vessel, and the second mesenchymal stem cells are allowed to settle on the bottom surface of the third culture vessel; and after the second mesenchymal stem cells have settled on the bottom surface of the third culture vessel in the second stem cell settling step, the culture medium in the third culture vessel is discharged and a new culture medium is poured into the third culture vessel to culture the second mesenchymal stem cells, and the bottom surface of the third culture vessel is settling. 4. The method for modifying frozen stem cells according to claim 3, wherein the frozen stem cells are produced by a mesenchymal stem cell culture method comprising: a second stem cell culture step of proliferating the second mesenchymal stem cells until the total planar area of the second mesenchymal stem cells relative to the volume of the third culture vessel reaches a second target ratio; and a second mesenchymal stem cell extraction step of placing the third culture vessel containing the second mesenchymal stem cells cultured by the second stem cell culture step in a centrifuge, centrifuging the second mesenchymal stem cells and the culture solution in the third culture vessel in layers, and extracting the second mesenchymal stem cells located in the bottom layer of the third culture vessel after the centrifugation. 前記冷凍保存工程が、前記第２間葉系幹細胞第２抽出工程によって抽出された直後の前記第２間葉系幹細胞を前記冷凍保存容器に収容し、該冷凍保存容器に収容した該間葉系幹細胞を前記凍結保護剤とともに直ちに冷凍保存する請求項４に記載の冷凍幹細胞改質方法。 The frozen stem cell modification method according to claim 4, wherein the cryopreservation step comprises placing the second mesenchymal stem cells in the cryopreservation container immediately after extraction in the second mesenchymal stem cell second extraction step, and immediately freezing and preserving the mesenchymal stem cells placed in the cryopreservation container together with the cryoprotectant. 前記培養生成液が、前記第２培養容器又は前記第３培養容器から培養後の間葉系幹細胞を抽出した後に残った培養液であり、前記間葉系幹細胞の培養過程において該間葉系幹細胞から分泌された所定の代謝物質を含む請求項５に記載の冷凍幹細胞改質方法。


 The frozen stem cell modification method described in claim 5, wherein the culture solution is the culture solution remaining after extracting cultured mesenchymal stem cells from the second culture vessel or the third culture vessel, and contains specific metabolic substances secreted from the mesenchymal stem cells during the culture process of the mesenchymal stem cells.
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