JP6741597B2 - 幹細胞の安定性増進用組成物 - Google Patents
幹細胞の安定性増進用組成物 Download PDFInfo
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- A01N1/0205—Chemical aspects
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
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- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/35—Fat tissue; Adipocytes; Stromal cells; Connective tissues
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/38—Albumins
- A61K38/385—Serum albumin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/46—Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
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Description
脂肪吸引術(Liposuction)により腹部脂肪から得られたヒト脂肪組織を分離し、PBSで洗浄した。組織を細切した後、I型コラゲナーゼ(1mg/ml)を添加したDMEM培地を用いて37℃で2時間組織を分解させた。コラゲナーゼ処理された組織をPBSで洗浄した後、1000rpmで5分間遠心分離して上清を除去し、ペレットをPBSで洗浄してから、1000rpmで5分間遠心分離した。100μmのメッシュで濾過して浮遊物を除去した後、PBSで洗浄し、10%のFBS、2mMのNAC(N‐acetyl‐Lcysteine)、0.2mMのアスコルビン酸が添加されたDMEM培地で培養した。一晩経過後に付着されていない細胞をPBSで洗浄し、5%のFBS、2mMのNAC、0.2mMのアスコルビン酸、0.09mMのカルシウム、5ng/mlのrEGF、5μg/mlのインスリン、10ng/mlのbFGF、及び74ng/mlのヒドロコルチゾンを含有するケラチノサイト‐SFM培地を2日毎に交換しながら継代培養して、脂肪組織由来間葉系幹細胞を分離した。
2‐1:実験群の構成
実施例1の方法により分離されたそれぞれ異なる4種の脂肪幹細胞を4℃で冷蔵保存し、冷蔵剤形の細胞治療剤注射製品に使用される幹細胞の生存率を分析した。
本実施例では、幹細胞保存安定性増進用組成物の賦形剤のpHを分析した。
実施例1の方法により分離された脂肪幹細胞をPBSで洗浄した後、0.3%のアルブミンが含まれた生理食塩水に浮遊させ、それぞれ10、20、30%の自家及び他家ヒト血清を添加して、実施例2の対照群及び実験群を構成した。
細胞1としては、0.5×107の幹細胞を、0.3%のアルブミンが含まれた生理食塩水に浮遊させた対照群、及びそれぞれ10、20、30%の自家血清/0.3%のアルブミンが含まれた生理食塩水に浮遊させた3つの幹細胞実験群を準備した。
細胞3としては、0.5×107の幹細胞を、0.3%のアルブミンが含まれた生理食塩水に浮遊させた対照群、及びそれぞれ10、20、30%の他家血清/0.3%のアルブミンが含まれた生理食塩水に浮遊させた3つの幹細胞実験群を準備した。
実施例3と同様の条件で、時間による幹細胞の生存率を分析した。
前記実施例において、自家血清と他家血清の間に効果の差がないことを確認した後、血清濃度及び血漿による細胞安定性の効果を調べるための実験群を構成した。
実施例1の方法により分離されたそれぞれ異なる2種の脂肪幹細胞を4℃で冷蔵保存し、冷蔵剤形の細胞治療剤注射製品に使用される幹細胞の生存率を分析した。
血清を分離するために、抗凝固剤が入っていない採血チューブを用いて血液10mlを採取した後、冷蔵で10分間立てて置いた。1000RCFで15分間遠心分離(ブレーキ0)してフィブリンを除去し、分離された上清である血清を回収した。回収された血清は1700rpmで5分間遠心分離(ブレーキ0)した後、上清を回収して0.2μm注射器フィルターを用いて滅菌して準備した。
実施例2‐2のように、それぞれの実験群毎に、幹細胞保存安定性増進用組成物の賦形剤のpHを分析した。
実施例1の方法により分離された脂肪幹細胞をPBSで洗浄した後、実施例5‐1の方法により細胞を準備した。2種の幹細胞に対して、表11のように対照群及び6つの実験群を構成し、9日まで細胞数、生存率、及び細胞サイズを調べた。
実施例6において、幹細胞#1(図5)及び幹細胞#2(図6)の生存率を分析した結果、細胞1と細胞2の間には差がほとんどないことが確認されたため、この実施例では、細胞1に及ぶ安定剤毎の効果を比較した。
Claims (6)
- i)脂肪由来間葉系幹細胞;
ii)10〜30%(v/v)含量の血清;
iii)0.1〜1%(v/v)含量のアルブミン;及び
iv)生理食塩水、ハルトマン‐D溶液及びPBSからなる群から選択される何れか1つ以上を含有し、
0.1〜10℃で72〜120時間、冷蔵保存安定性を有して、体内投与が可能な脂肪由来間葉系幹細胞を含む治療剤組成物。 - 前記冷蔵保存温度の範囲は、0.1〜10℃であることを特徴とする請求項1に記載の脂肪由来間葉系幹細胞を含む治療剤組成物。
- 前記アルブミンの含量は、0.2〜0.5%(v/v)であることを特徴とする請求項1又は2に記載の脂肪由来間葉系幹細胞を含む治療剤組成物。
- 前記幹細胞の濃度は、1.0×105〜1.0×109cell/mlであることを特徴とする請求項1〜3の何れか一項に記載の脂肪由来間葉系幹細胞を含む治療剤組成物。
- 請求項1〜4の何れか一項に記載の脂肪由来間葉系幹細胞を含む治療剤組成物を含有する細胞治療用注射製品。
- 懸濁剤、溶解補助剤、安定化剤、等張化剤、保存剤、吸着防止剤、界面活性剤、希釈剤、pH調整剤、無痛化剤、緩衝剤、含硫黄還元剤、及び酸化防止剤からなる群より選択された一つ以上をさらに含有することを特徴とする請求項5に記載の細胞治療用注射製品。
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KR20140085020 | 2014-07-08 | ||
KR10-2014-0085020 | 2014-07-08 | ||
PCT/KR2015/000595 WO2016006782A1 (ko) | 2014-07-08 | 2015-01-21 | 줄기세포의 보관 안정성 증진용 조성물 |
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JP2017521378A JP2017521378A (ja) | 2017-08-03 |
JP6741597B2 true JP6741597B2 (ja) | 2020-08-19 |
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US (1) | US10172347B2 (ja) |
EP (1) | EP3178318B1 (ja) |
JP (1) | JP6741597B2 (ja) |
KR (1) | KR101597604B1 (ja) |
CN (1) | CN107072189B (ja) |
WO (1) | WO2016006782A1 (ja) |
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EP3581644A4 (en) * | 2017-02-10 | 2020-01-29 | FUJIFILM Corporation | PRESERVATIVES FOR HUMAN STEM CELLS, SUSPENSION FOR HUMAN STEM CELLS AND METHOD FOR THE PRESERVATION OF HUMAN STEM CELLS |
US20210008120A1 (en) | 2018-03-13 | 2021-01-14 | Oxacell Ag | Cell products with improved stability and uses thereof |
JP6594578B1 (ja) * | 2018-04-25 | 2019-10-23 | セルトラスト・アニマル・セラピューティクス株式会社 | 細胞の保存方法および細胞懸濁液 |
CN108849857A (zh) * | 2018-07-20 | 2018-11-23 | 吉林济惠生物科技有限公司 | 一种脐带间充质干细胞的运输保护液 |
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DK0647267T3 (da) * | 1992-06-25 | 2000-12-18 | David Scheer | Opbevaring af stamceller |
US5827640A (en) * | 1996-06-14 | 1998-10-27 | Biostore New Zealand Limited | Methods for the preservation of cells and tissues using trimethylamine oxide or betaine with raffinose or trehalose |
JP3364680B2 (ja) | 1998-10-07 | 2003-01-08 | 山梨県 | 豚***保存用希釈液および豚***の希釈保存法 |
KR100394430B1 (ko) | 1999-12-14 | 2003-08-09 | (주)이노테크 메디칼 | 인간 혈청을 포함하는 인간 세포 배양용 배지 및 이를이용한 인간 세포의 배양 방법 |
CA2587885C (en) * | 2004-11-19 | 2013-05-21 | Jms Co., Ltd. | Human serum for cell culture |
US9034316B2 (en) * | 2006-10-24 | 2015-05-19 | Amorcyte, Llc | Infarct area perfusion-improving compositions and methods of vascular injury repair |
MY147516A (en) * | 2005-11-07 | 2012-12-31 | Amorcyte Inc | Compositions and method of vascular injury repair cross-reference to related applications |
RU2303631C1 (ru) * | 2006-05-19 | 2007-07-27 | Общество с ограниченной ответственностью "Транс-Технологии" | Среда для криоконсервирования мезенхимальных стволовых клеток и биотрансплантат с ее использованием |
KR20080103637A (ko) | 2007-05-25 | 2008-11-28 | 주식회사 알앤엘바이오 | 지방유래 줄기세포를 함유하는 사지말단부 허혈성 질환의세포치료용 조성물 |
EP2248887A4 (en) * | 2008-10-17 | 2013-04-17 | Affiliated Hospital Of Ningxia Medical University | METHOD FOR PRODUCING HUMAN MESENCHYMAL PLAZENTAZELL LIBRARIES FOR CLINICAL APPLICATIONS |
KR101358973B1 (ko) | 2010-11-24 | 2014-02-11 | 중앙대학교 산학협력단 | 트리할로오스를 이용한 정원줄기세포의 동결보존 방법 |
KR20120057784A (ko) * | 2010-11-29 | 2012-06-07 | 주식회사 알앤엘바이오 | 줄기세포의 안정성 증진용 조성물 |
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KR102024740B1 (ko) * | 2012-11-09 | 2019-09-24 | 주식회사 휴림바이오셀 | 간엽줄기세포의 냉동보존용 배지 및 이를 이용한 간엽줄기세포의 냉동보존방법 |
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- 2015-01-21 EP EP15818206.3A patent/EP3178318B1/en active Active
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EP3178318A4 (en) | 2017-12-13 |
EP3178318A1 (en) | 2017-06-14 |
EP3178318B1 (en) | 2020-08-26 |
CN107072189B (zh) | 2021-07-20 |
KR20160006127A (ko) | 2016-01-18 |
US10172347B2 (en) | 2019-01-08 |
CN107072189A (zh) | 2017-08-18 |
JP2017521378A (ja) | 2017-08-03 |
US20170105406A1 (en) | 2017-04-20 |
KR101597604B1 (ko) | 2016-02-26 |
WO2016006782A1 (ko) | 2016-01-14 |
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