JP5859532B2 - 滅菌されたヒト細胞懸濁液の製造法 - Google Patents
滅菌されたヒト細胞懸濁液の製造法 Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
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- 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
- C12N5/067—Hepatocytes
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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
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
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Description
本発明はまた、以下に関する。
[1]
以下のステップ:
a)液状抗生物質組成物による、哺乳動物に由来する組織の灌流、
b)単一細胞懸濁液を獲得するための、組織の酵素処理の実施、及び
c)滅菌された調製品の獲得
を含む、哺乳動物の組織に由来する滅菌された調製品の製造法。
[2]
組織が肝組織である、[1]に記載の方法。
[3]
ステップb)に基づく酵素処理がステップa)と同時に行われる、[1]又は[2]に記載の方法。
[4]
ステップb)に基づく酵素処理がステップa)に続いて行われる、[1]又は[2]に記載の方法。
[5]
酵素処理がコラゲナーゼ処理及び/又はプロテイナーゼ処理である、[1]〜[4]のいずれかに記載の方法。
[6]
ステップa)での灌流時間が30〜120分であり、灌流の間に温度が高められる、[1]〜[5]のいずれかに記載の方法。
[7]
液状抗生物質組成物が少なくとも一つのコラゲナーゼ及び/又は少なくとも一つのプロテイナーゼを含有する、[1]〜[6]のいずれかに記載の方法。
[8]
液状抗生物質組成物が、少なくとも一つの灌流用緩衝液中に存在し、少なくとも一つの抗生物質を含む、[1]〜[7]のいずれかに記載の方法。
[9]
液状抗生物質組成物が、少なくとも二つの灌流用緩衝液中、詳しくは少なくとも一つのEGTA又はEDTA含有リン酸-HEPES-緩衝液、及び少なくとも一つのリン酸-HEPES-緩衝液中に存在する、[1]〜[8]のいずれかに記載の方法。
[10]
ステップa)に先立ち、液状抗生物質組成物により組織の表面が洗浄される、[1]〜[9]のいずれかに記載の方法。
[11]
ステップb)に続いて少なくとも一回の、抗生物質を含まない洗浄用緩衝液による洗浄ステップが実施される、[1]〜[10]のいずれかに記載の方法。
[12]
[1]〜[11]のいずれかに記載の方法で製造された、滅菌された哺乳動物細胞調製品。
A.実施
肝臓に由来する滅菌された肝細胞懸濁液を製造するために、以下の抗生物質組成物を製造した:
抗生物質は、記載されるすべての灌流用緩衝液に以下の濃度で加えた:
1.EDTA含有リン酸-HEPES-緩衝液5L;灌流10分;20〜40℃;
2.リン酸-HEPES-緩衝液5L;灌流10分;25〜40℃;
3.コラゲナーゼ/プロテアーゼ含有リン酸-HEPES-緩衝液3L;灌流24分;25〜40℃
コラゲナーゼ/プロテアーゼ濃度:2000ヴュンシュユニットのコラゲナーゼI及び400〜2500ユニットのプロテアーゼ(クロストリジオペプチダーゼI)からなる組合せ。
輸送培地中には、以下の微生物が検出された:エンテロバクター・クロアカ(Enterobacter cloacae)、アシネトバクター・バウマニ(Acinetobacter baumannii)、ただし、微生物的な成長が認められるまでの、自動インキュベーションの時間、いわゆる「陽性となるまでの時間(Time to positivity)」は、1.67時間であった。
A.実施
全臓器移植には適さない複数ドナー肝臓をまず表面的に、抗生物質濃度が2倍のリン酸-HEPES-緩衝液5Lを用いて、室温におき8分洗浄し、続いて二ステップにおき抗生物質含有緩衝液を用いて、さらなる一ステップにおきコラゲナーゼを含み抗生物質を含まない緩衝液を用いて以下の体積、pH7.25〜7.45で灌流した:
1.EDTA含有リン酸-HEPES-緩衝液10L;灌流68分;20〜40℃;
2.リン酸-HEPES-緩衝液5L;灌流10分;25〜40℃;
3.コラゲナーゼ/プロテアーゼを含み抗生物質を含まないリン酸-HEPES-緩衝液3L;灌流24分;25〜40℃
コラゲナーゼ/プロテアーゼ濃度:2000ヴュンシュユニットのコラゲナーゼI及び400〜2500ユニットのプロテアーゼ(クロストリジオペプチダーゼI)からなる組合せ。
輸送培地中には、黄色ブドウ球菌(Staphylococcus Aureus)を検出することができた。微生物的な成長が認められるまでの、自動インキュベーションの時間、いわゆる「陽性となるまでの時間(Time to positivity)」は、3.85時間であった。
Claims (7)
- 以下のステップ:
a)少なくとも一つの灌流用緩衝液中に存在する、少なくとも一つの抗生物質を含む、酵素を含まない液状抗生物質組成物による、哺乳動物に由来する組織の灌流、
b)単一細胞懸濁液を獲得するための、組織の、抗生物質を含まない酵素処理の実施、及び
c)滅菌された調製品の獲得
を含み、ステップb)に基づく酵素処理がステップa)に続いて行われる、哺乳動物の組織に由来する滅菌された調製品の製造法。 - 組織が肝組織である、請求項1に記載の方法。
- 酵素処理がコラゲナーゼ処理及び/又はプロテイナーゼ処理である、請求項1又は2に記載の方法。
- ステップa)での灌流時間が30〜120分であり、灌流の間に温度が高められる、請求項1〜3のいずれか一項に記載の方法。
- 液状抗生物質組成物が、少なくとも二つの灌流用緩衝液中、詳しくは少なくとも一つのEGTA又はEDTA含有リン酸-HEPES-緩衝液、及び少なくとも一つのリン酸-HEPES-緩衝液中に存在する、請求項1〜4のいずれか一項に記載の方法。
- ステップa)に先立ち、液状抗生物質組成物により組織の表面が洗浄される、請求項1〜5のいずれか一項に記載の方法。
- ステップb)に続いて少なくとも一回の、抗生物質を含まない洗浄用緩衝液による洗浄ステップが実施される、請求項1〜6のいずれか一項に記載の方法。
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PCT/EP2011/003838 WO2012022429A1 (de) | 2010-08-14 | 2011-07-30 | Verfahren zur herstellung desinfizierter humaner zellsuspensionen |
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US4695536A (en) | 1984-01-10 | 1987-09-22 | Lindstrom Richard L | Corneal storage system |
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US5741782A (en) | 1996-03-29 | 1998-04-21 | Cryolife, Inc. | Antibiotic cocktail and method of use |
US6613278B1 (en) * | 1998-11-13 | 2003-09-02 | Regeneration Technologies, Inc. | Tissue pooling process |
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CN103124493A (zh) | 2013-05-29 |
ZA201300532B (en) | 2014-03-26 |
DK2603074T3 (en) | 2016-05-23 |
DE102010034330B4 (de) | 2013-10-31 |
WO2012022429A1 (de) | 2012-02-23 |
EP2603074B1 (de) | 2016-02-24 |
ES2571241T3 (es) | 2016-05-24 |
CA2807890C (en) | 2019-06-25 |
CA2807890A1 (en) | 2012-02-23 |
AU2011291058A1 (en) | 2013-02-07 |
BR112013003484B1 (pt) | 2019-04-24 |
PL2603074T3 (pl) | 2016-08-31 |
AU2011291058B2 (en) | 2015-10-15 |
IL224648A (en) | 2016-10-31 |
KR101822961B1 (ko) | 2018-01-29 |
US10017742B2 (en) | 2018-07-10 |
KR20130098990A (ko) | 2013-09-05 |
AR082668A1 (es) | 2012-12-26 |
EP2603074A1 (de) | 2013-06-19 |
RU2563806C2 (ru) | 2015-09-20 |
US20130143324A1 (en) | 2013-06-06 |
BR112013003484A2 (pt) | 2016-09-13 |
HK1181258A1 (zh) | 2013-11-08 |
US8999710B2 (en) | 2015-04-07 |
US20150072428A1 (en) | 2015-03-12 |
RU2013111300A (ru) | 2014-09-20 |
DE102010034330A1 (de) | 2012-02-16 |
JP2013535217A (ja) | 2013-09-12 |
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