WO2012133575A1 - Procédé d'inhibition de la mort cellulaire de cellules souches mésenchymateuses - Google Patents

Procédé d'inhibition de la mort cellulaire de cellules souches mésenchymateuses Download PDF

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Publication number
WO2012133575A1
WO2012133575A1 PCT/JP2012/058225 JP2012058225W WO2012133575A1 WO 2012133575 A1 WO2012133575 A1 WO 2012133575A1 JP 2012058225 W JP2012058225 W JP 2012058225W WO 2012133575 A1 WO2012133575 A1 WO 2012133575A1
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mesenchymal stem
stem cells
aqueous solution
trehalose
physiological aqueous
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PCT/JP2012/058225
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English (en)
Japanese (ja)
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英司 小林
工 寺谷
廣 野島
効志 岩田
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株式会社大塚製薬工場
学校法人自治医科大学
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Publication of WO2012133575A1 publication Critical patent/WO2012133575A1/fr

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    • C—CHEMISTRY; METALLURGY
    • 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
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/34—Sugars

Definitions

  • the present invention relates to a method for subculture while suppressing cell death of mesenchymal stem cells, and an agent used therefor.
  • Mesenchymal stem cells exist in mammalian bone marrow and are known as stem cells that differentiate into adipocytes, chondrocytes, bone cells, and the like. Mesenchymal stem cells are attracting attention as transplant materials for regenerative medicine in many tissues because of their multipotency. That is, “regenerative medicine by cell transplantation” that uses mesenchymal stem cells to regenerate a tissue that has not been regenerated by a conventional treatment method and restores a function lost by a disease or a disorder.
  • transplantation of bone marrow mesenchymal stem cells to patients with lower limb ischemia (Burger's disease)
  • transplantation of bone marrow mesenchymal stem cells to the affected area of periodontal disease transplantation of bone marrow mesenchymal stem cells to patients with osteoarthritis Treatment such as transplantation is started or planned.
  • mesenchymal stem cells are extremely small, and the amount that can be collected from one individual is small. Therefore, in order to provide a sufficient number of mesenchymal stem cells for practicing regenerative medicine, it is necessary to stably culture and proliferate them in a state with a high survival rate.
  • trehalose is a kind of disaccharide formed by combining glucose with 1,1-glycoside.
  • Trehalose is sweet and has a high water retention capacity, so it is used in various foods and cosmetics.
  • Trehalose stabilizes cell membranes and suppresses cell damage, and is therefore used as an active ingredient in organ protective solutions when organs are transplanted.
  • Excellent organ preservation solutions containing trehalose such as ET-Kyoto solution and New ET-Kyoto solution have been developed (Patent Documents 1 and 2, Non-Patent Document 1).
  • mesenchymal stem cells are adherent cells, when they are passaged, the cells are first detached from the plate with trypsin, and proteolysis with trypsin is stopped with a medium or solution supplemented with serum or trypsin inhibitor. Then, the detached cells are collected by centrifugation, and further washed with PBS ( ⁇ ) or physiological saline to remove residual inactivated trypsin and contaminants. Sowing on a plate.
  • PBS ⁇
  • physiological saline physiological saline
  • An object of the present invention is to provide a technique for subculturing mesenchymal stem cells while suppressing cell death and a technique for recovering a large amount of living cells necessary for transplantation.
  • the present invention relates to the following.
  • a method for in vitro passage of mesenchymal stem cells comprising washing mesenchymal stem cells detached from a cell culture vessel by proteolytic enzyme treatment with a physiological aqueous solution containing trehalose.
  • the trehalose concentration in the physiological aqueous solution is 3 to 6 (w / v)%.
  • An agent for washing mesenchymal stem cells detached from a cell culture vessel by proteolytic enzyme treatment in vitro passage of mesenchymal stem cells comprising a physiological aqueous solution containing trehalose.
  • a physiological aqueous solution containing trehalose comprising a physiological aqueous solution containing trehalose.
  • mesenchymal stem cells can be passaged while suppressing cell death. Therefore, the present invention contributes to the provision of high-quality mesenchymal stem cells in regenerative medicine.
  • the effect of trehalose on the survival rate of mesenchymal stem cells is shown.
  • a) shows the measurement result of direct survival rate.
  • b) shows a relative value when the survival rate immediately after peeling is 100%.
  • c) shows a relative value when the survival rate of the PBS ( ⁇ ) washing treatment group is 100%.
  • the effect of trehalose on the survival rate of mesenchymal stem cells is shown.
  • a) shows the results of comparing the direct survival rate between the trehalose-containing group and the trehalose-free group.
  • b) shows the results of comparing the direct survival rate between the group having a trehalose content of 3 to 6 (w / v)% and the group having a content of 6.5 to 10 (w / v)%.
  • the present invention relates to a method for in vitro passage of mesenchymal stem cells, comprising washing mesenchymal stem cells detached from a cell culture vessel by proteolytic enzyme treatment with a physiological aqueous solution containing trehalose. It is to provide.
  • Mesenchymal stem cell broadly means a population of stem cells or progenitor cells that proliferate in an undifferentiated state and can differentiate into all or some of bone cells, chondrocytes and adipocytes.
  • the mesenchymal stem cell used in the method of the present invention is not particularly limited as long as it is a vertebrate cell.
  • the vertebrates include mammals, birds, fish, amphibians and reptiles.
  • mammals include, for example, laboratory animals such as rodents and rabbits such as mice, rats, hamsters, and guinea pigs, domestic animals such as pigs, cows, goats, horses, sheep and minks, pets such as dogs and cats, humans, Primates such as monkeys, rhesus monkeys, marmosets, orangutans and chimpanzees.
  • the vertebrate is preferably a mammal, more preferably a rodent (such as a rat) or a primate (such as a human).
  • mesenchymal stem cells are present at a low frequency in bone marrow, peripheral blood, umbilical cord blood, and adipose tissue.
  • Mesenchymal stem cells can be isolated or purified from these tissues by a known method. “Isolated or purified” means that the artificially placed in a state different from the naturally occurring state, for example, an operation to remove components other than the desired component from the naturally occurring state.
  • Human mesenchymal stem cells can be isolated from bone marrow fluid by the Percoll gradient method (Hum. Cell, vol.10, p.45-50, 1997).
  • human mesenchymal stem cells can be isolated by culture and passage of hematopoietic stem cells after bone marrow puncture (Journalourof Autoimmunity, ity30 (2008) 163-171).
  • Mesenchymal stem cells are usually adherently cultured in an appropriate medium in a culture container such as a petri dish, a plate, or a bottle.
  • a basal medium of a medium used for culturing mesenchymal stem cells those commonly used for maintaining or culturing mammalian adherent cells can be used, and are not particularly limited.
  • DMEM, EMEM, RPMI -1640, ⁇ -MEM, F-12, F-10, M-199 and the like can be used, and are not particularly limited.
  • a medium modified for mesenchymal stem cell culture or the like may be used, or a mixture of the above basal medium may be used.
  • the medium used for culturing mesenchymal stem cells may contain serum.
  • the serum is not particularly limited as long as it is a serum derived from a mammal, but is preferably a serum derived from the mammal (for example, fetal bovine serum, human serum, etc.).
  • serum supplements for example, Knockout Serum Replacement (KSR) (Invitrogen)
  • KSR Knockout Serum Replacement
  • concentration of serum is not particularly limited, but is usually in the range of 0.1 to 30 (v / v)%.
  • the medium used for culturing mesenchymal stem cells can contain additives known per se.
  • the additive include, but are not particularly limited to, for example, growth factors (such as insulin), iron sources (such as transferrin), minerals (such as sodium selenate), saccharides (such as glucose), organic acids (such as pyruvic acid, Lactic acid etc.), serum proteins (eg albumin etc.), amino acids (eg L-glutamine etc.), reducing agents (eg 2-mercaptoethanol etc.), vitamins (eg ascorbic acid, d-biotin etc.), antibiotics (eg streptomycin, etc.) , Penicillin, gentamicin, etc.), buffering agents (eg, HEPES, etc.) and the like.
  • Each of the additives is preferably contained within a concentration range known per se.
  • the culture temperature is usually in the range of about 30 to 40 ° C, preferably about 37 ° C.
  • the CO 2 concentration is usually in the range of about 1-10%, preferably about 5%.
  • the humidity is usually in the range of about 70-100%, preferably about 95-100%.
  • the medium is removed with an aspirator or the like.
  • the mesenchymal stem cells are washed with a physiological aqueous solution not containing the protein, and the remaining medium is removed.
  • the physiological aqueous solution is not particularly limited as long as it does not inhibit the activity of the proteolytic enzyme and has no toxicity to mesenchymal stem cells.
  • physiological saline phosphate buffered saline, Tris Buffered saline, HEPES buffered saline, Ringer's solution, 5% glucose aqueous solution, liquid medium for mammalian culture, aqueous solution of isotonic agents (glucose, D-sorbitol, D-mannitol, lactose, sodium chloride, etc.) And an isotonic aqueous solution.
  • PBS is used.
  • the physiological aqueous solution preferably does not contain calcium ions.
  • the mesenchymal stem cells adhered to the surface of the culture container are treated with a proteolytic enzyme so as to be detached from the surface of the culture container.
  • Treatment of mesenchymal stem cells with proteolytic enzymes is carried out by bringing mesenchymal stem cells into contact with a physiological aqueous solution containing proteolytic enzymes.
  • proteolytic enzymes include trypsin, pronase, pepsin, elastase, collagenase and the like, but preferably trypsin is used.
  • the physiological aqueous solution include those described above, and preferably PBS is used.
  • the physiological aqueous solution preferably does not contain calcium ions.
  • the concentration of the proteolytic enzyme in the physiological aqueous solution can be appropriately set depending on the type of the proteolytic enzyme, and can be appropriately set to detach mesenchymal stem cells. / V) in the range of%.
  • the exfoliation of mesenchymal stem cells can be promoted by further containing a calcium ion chelator in a physiological aqueous solution containing a proteolytic enzyme and removing the calcium ions.
  • a calcium ion chelator examples include EDTA and EGTA, and EDTA is preferable.
  • the concentration of calcium ion chelator is usually in the range of 0.1 to 2 mM.
  • the temperature during the proteolytic enzyme treatment is usually within a range of 20 to 37 ° C.
  • the treatment time with the proteolytic enzyme is not particularly limited as long as it is sufficient to detach the cells from the culture vessel, but is usually 15 seconds to 15 minutes, preferably 30 seconds to 10 minutes. If mesenchymal stem cells are treated with proteolytic enzymes over a long period of time, the cells may be damaged and the survival rate may be reduced, so treatment with proteolytic enzymes while observing the state of the cells with the naked eye or under a microscope When the cell separation is confirmed, it is preferable to proceed to the next step in as short a time as possible.
  • the resulting mesenchymal stem cell suspension contains a sufficient amount of protein to suppress the activity of proteolytic enzymes
  • a physiological aqueous solution the culture medium for culture
  • the obtained mesenchymal stem cell suspension is subjected to centrifugation, and the supernatant is discarded to separate the detached mesenchymal stem cells.
  • Washing the exfoliated mesenchymal stem cells with a physiological aqueous solution containing trehalose is performed by suspending the mesenchymal stem cells separated with the physiological aqueous solution containing trehalose, and subjecting the obtained suspension to centrifugation. Discard the supernatant.
  • the physiological aqueous solution is not particularly limited as long as it is not toxic to mesenchymal stem cells.
  • physiological saline phosphate buffered saline, Tris buffered saline, HEPES buffered physiology Saline, Ringer's solution, 5% glucose aqueous solution, liquid culture medium for mammalian culture, isotonic aqueous solutions such as aqueous solutions of isotonic agents (glucose, D-sorbitol, D-mannitol, lactose, sodium chloride, etc.), the above-mentioned media, etc. Can be mentioned.
  • the trehalose content in the physiological aqueous solution is not limited as long as it increases the survival rate of mesenchymal stem cells, but is usually 3 to 10 (w / v)%, preferably 3 to 6 (w / v)%. . If the trehalose concentration is too high, it may affect the cell morphology, such as the size of the mesenchymal stem cells becoming smaller. If the trehalose concentration is too low, the effect of increasing the survival rate of the mesenchymal stem cells is sufficiently achieved. There is a possibility that it will not be.
  • ET-Kyoto solution Japanese Patent No. 3253131
  • the ET-Kyoto solution is commercially available from Otsuka Pharmaceutical Factory.
  • the trehalose content in the ET-Kyoto solution is 4.53 (w / v)%.
  • the number of washing operations of exfoliated mesenchymal stem cells with a physiological aqueous solution containing trehalose may be performed only once or a plurality of times (for example, 2 to 4 times). If the number of washings is too large, the viability of the mesenchymal stem cells may decrease, and the number of washings is usually 1 to 2 times, preferably 1 time.
  • the mesenchymal stem cells are suspended in an appropriate concentration in the above-mentioned fresh medium and seeded in a new culture vessel.
  • the present invention also provides an agent for washing mesenchymal stem cells detached from a cell culture vessel by proteolytic enzyme treatment in vitro passage of mesenchymal stem cells, comprising the physiological aqueous solution containing trehalose described above. It is to provide.
  • an agent for washing mesenchymal stem cells detached from a cell culture vessel by proteolytic enzyme treatment in vitro passage of mesenchymal stem cells comprising the physiological aqueous solution containing trehalose described above. It is to provide.
  • Example 1 Human adipose tissue-derived mesenchymal stem cells (passage number 6) (1.3 ⁇ 10 6 cells) were cultured in ⁇ MEM (GIBCO) containing 10 (w / v)% FBS (GIBCO) on a culture dish. After removing the medium and washing the mesenchymal stem cell surface with PBS, 0.25 (w / v)% trypsin (GIBCO) was added dropwise to the dish and allowed to stand at room temperature. After confirming that the cells were detached from the dish, ⁇ MEM containing 10 (w / v)% FBS was immediately added and pipetting to stop the trypsin degradation and collect the cells.
  • ⁇ MEM containing 10 (w / v)% FBS was immediately added and pipetting to stop the trypsin degradation and collect the cells.
  • trehalose suppresses a decrease in the survival rate of mesenchymal stem cells within a range of at least 3 to 10 (w / v)%, suggesting that 3 to 6 (w / v)% is a preferable concentration range. It was done.
  • mesenchymal stem cells can be collected and passaged while suppressing cell death. Therefore, the present invention contributes to the provision of high-quality mesenchymal stem cells in regenerative medicine.

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Abstract

La présente invention concerne un procédé de repiquage in vitro de cellules souches mésenchymateuses, ledit procédé comprenant une étape consistant à laver les cellules souches mésenchymateuses détachées d'un flacon de culture cellulaire à l'issue d'un traitement par une enzyme protéolytique, au moyen d'une solution physiologique aqueuse contenant du tréhalose. La présente invention concerne, en outre, un agent de lavage de cellules souches mésenchymateuses détachées d'un flacon de culture cellulaire à l'issue d'un traitement par une enzyme protéolytique dans le cadre du repiquage in vitro de cellules souches mésenchymateuses, ledit agent de lavage contenant ladite solution physiologique aqueuse comprenant du tréhalose.
PCT/JP2012/058225 2011-03-29 2012-03-28 Procédé d'inhibition de la mort cellulaire de cellules souches mésenchymateuses WO2012133575A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014208053A1 (fr) 2013-06-28 2014-12-31 株式会社大塚製薬工場 Solution contenant un tréhalose et un dextrane pour la transplantation de cellules de mammifères
US9040300B2 (en) 2012-09-28 2015-05-26 Otsuka Pharmaceutical Factory, Inc. Method of washing adherent cell using trehalose-containing cell-washing solution

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ATSUSHI OHATA ET AL.: "Usagi Mocho Fukuheki Sakka Model o Mochiita Trehalose Seizai no Yuchaku Keigen Koka", JOURNAL OF ABDOMINAL EMERGENCY MEDICINE, vol. 31, no. 2, 17 February 2011 (2011-02-17), pages 434 - 464 *
CHIKAKO SOMEI ET AL.: "Effect of the tooth cryopreservation for the cell activity of periodontal membrane - Hozon Eki to shite no Trehalose no Koka", JAPANESE SOCIETY OF CONSERVATIVE DENTISTRY 2009 NENDO SHUNKI GAKUJUTSU TAIKAI (DAI 130 KAI) PROGRAM, KOEN SHOROKUSHU, 11 June 2009 (2009-06-11), pages 204 *
DI, G. ET AL.: "Development and evaluation of a trehalose-contained solution formula to preserve hUC-MSCs at 4°C", JOURNAL OF CELLULAR PHYSIOLOGY, vol. 227, no. 3, 14 December 2011 (2011-12-14), pages 879 - 884, Retrieved from the Internet <URL:http://onlinelibrary.wiley.com/doi/10.1002/jcp.23066> *
LIU, Y. ET AL.: "Cryopreservation of human bone marrow-derived mesenchymal stem cells with reduced dimethylsulfoxide and well-defined freezing solutions", BIOTECHNOLOGY PROGRESS, vol. 26, no. 6, November 2010 (2010-11-01), pages 1635 - 1643, XP055102221, doi:10.1002/btpr.464 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9040300B2 (en) 2012-09-28 2015-05-26 Otsuka Pharmaceutical Factory, Inc. Method of washing adherent cell using trehalose-containing cell-washing solution
US20150224145A1 (en) * 2012-09-28 2015-08-13 Otsuka Pharmaceutical Factory, Inc. Method of washing adherent cell using trehalose-containing cell-washing solution
US9498500B2 (en) 2012-09-28 2016-11-22 Otsuka Pharmaceutical Factory, Inc. Method of washing adherent cell using trehalose-containing cell-washing solution
WO2014208053A1 (fr) 2013-06-28 2014-12-31 株式会社大塚製薬工場 Solution contenant un tréhalose et un dextrane pour la transplantation de cellules de mammifères
JP5820958B2 (ja) * 2013-06-28 2015-11-24 株式会社大塚製薬工場 トレハロース及びデキストラン含有哺乳動物細胞移植用溶液
KR20160026863A (ko) 2013-06-28 2016-03-09 가부시기가이샤오오쓰까세이야꾸고오죠오 트레할로스 및 덱스트란 함유 포유동물 세포 이식용 용액
JP2016034279A (ja) * 2013-06-28 2016-03-17 株式会社大塚製薬工場 トレハロース及びデキストラン含有哺乳動物細胞移植用溶液
JPWO2014208053A1 (ja) * 2013-06-28 2017-02-23 株式会社大塚製薬工場 トレハロース及びデキストラン含有哺乳動物細胞移植用溶液
US10945427B2 (en) 2013-06-28 2021-03-16 Otsuka Pharmaceutical Factory, Inc. Trehalose and dextran-containing solution for transplanting mammalian cells

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