JP6054223B2 - Stem cell culture apparatus and stem cell culture method - Google Patents
Stem cell culture apparatus and stem cell culture method Download PDFInfo
- Publication number
- JP6054223B2 JP6054223B2 JP2013060669A JP2013060669A JP6054223B2 JP 6054223 B2 JP6054223 B2 JP 6054223B2 JP 2013060669 A JP2013060669 A JP 2013060669A JP 2013060669 A JP2013060669 A JP 2013060669A JP 6054223 B2 JP6054223 B2 JP 6054223B2
- Authority
- JP
- Japan
- Prior art keywords
- carrier
- stem cell
- culture
- stem cells
- low
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 210000000130 stem cell Anatomy 0.000 title claims description 215
- 238000004113 cell culture Methods 0.000 title claims description 30
- 230000021164 cell adhesion Effects 0.000 claims description 45
- 239000000126 substance Substances 0.000 claims description 43
- 238000000034 method Methods 0.000 claims description 37
- 238000012258 culturing Methods 0.000 claims description 23
- 108010085895 Laminin Proteins 0.000 claims description 17
- 239000001963 growth medium Substances 0.000 claims description 15
- 102000000905 Cadherin Human genes 0.000 claims description 10
- 108050007957 Cadherin Proteins 0.000 claims description 10
- 239000011248 coating agent Substances 0.000 claims description 6
- 238000000576 coating method Methods 0.000 claims description 6
- 108010082117 matrigel Proteins 0.000 claims description 6
- 230000002062 proliferating effect Effects 0.000 claims description 2
- 230000004956 cell adhesive effect Effects 0.000 claims 1
- 210000004027 cell Anatomy 0.000 description 79
- 239000000243 solution Substances 0.000 description 40
- 239000000758 substrate Substances 0.000 description 27
- 239000011521 glass Substances 0.000 description 17
- 239000000463 material Substances 0.000 description 16
- 239000004793 Polystyrene Substances 0.000 description 15
- 239000002245 particle Substances 0.000 description 15
- 229920002223 polystyrene Polymers 0.000 description 15
- 238000003756 stirring Methods 0.000 description 15
- 238000004114 suspension culture Methods 0.000 description 15
- 230000003746 surface roughness Effects 0.000 description 13
- 238000012136 culture method Methods 0.000 description 12
- 241000699666 Mus <mouse, genus> Species 0.000 description 10
- 239000002609 medium Substances 0.000 description 9
- 238000010008 shearing Methods 0.000 description 9
- 239000000969 carrier Substances 0.000 description 8
- 230000000052 comparative effect Effects 0.000 description 8
- 238000004519 manufacturing process Methods 0.000 description 8
- 210000001778 pluripotent stem cell Anatomy 0.000 description 8
- 238000007796 conventional method Methods 0.000 description 7
- 230000004069 differentiation Effects 0.000 description 7
- 108090000623 proteins and genes Proteins 0.000 description 7
- 210000002469 basement membrane Anatomy 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 239000003112 inhibitor Substances 0.000 description 6
- 239000003550 marker Substances 0.000 description 6
- 239000008188 pellet Substances 0.000 description 5
- 238000011084 recovery Methods 0.000 description 5
- 238000003466 welding Methods 0.000 description 5
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 description 4
- 102000004058 Leukemia inhibitory factor Human genes 0.000 description 4
- 108090000581 Leukemia inhibitory factor Proteins 0.000 description 4
- 239000000427 antigen Substances 0.000 description 4
- 102000036639 antigens Human genes 0.000 description 4
- 108091007433 antigens Proteins 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 4
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 3
- 239000002585 base Substances 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 210000004748 cultured cell Anatomy 0.000 description 3
- 210000001671 embryonic stem cell Anatomy 0.000 description 3
- 238000013508 migration Methods 0.000 description 3
- 238000000465 moulding Methods 0.000 description 3
- 238000010899 nucleation Methods 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 230000003068 static effect Effects 0.000 description 3
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 2
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 102100021253 Antileukoproteinase Human genes 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 102000004877 Insulin Human genes 0.000 description 2
- 108090001061 Insulin Proteins 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 2
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 2
- 229930182816 L-glutamine Natural products 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 description 2
- 102000004338 Transferrin Human genes 0.000 description 2
- 108090000901 Transferrin Proteins 0.000 description 2
- 108010076089 accutase Proteins 0.000 description 2
- 230000001464 adherent effect Effects 0.000 description 2
- 238000013019 agitation Methods 0.000 description 2
- 230000000890 antigenic effect Effects 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 230000012292 cell migration Effects 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- 239000000835 fiber Substances 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 229940125396 insulin Drugs 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000005012 migration Effects 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 230000010412 perfusion Effects 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 229910052711 selenium Inorganic materials 0.000 description 2
- 239000011669 selenium Substances 0.000 description 2
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 239000012581 transferrin Substances 0.000 description 2
- LAQPKDLYOBZWBT-NYLDSJSYSA-N (2s,4s,5r,6r)-5-acetamido-2-{[(2s,3r,4s,5s,6r)-2-{[(2r,3r,4r,5r)-5-acetamido-1,2-dihydroxy-6-oxo-4-{[(2s,3s,4r,5s,6s)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxy}hexan-3-yl]oxy}-3,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy}-4-hydroxy-6-[(1r,2r)-1,2,3-trihydrox Chemical compound O[C@H]1[C@H](O)[C@H](O)[C@H](C)O[C@H]1O[C@H]([C@@H](NC(C)=O)C=O)[C@@H]([C@H](O)CO)O[C@H]1[C@H](O)[C@@H](O[C@]2(O[C@H]([C@H](NC(C)=O)[C@@H](O)C2)[C@H](O)[C@H](O)CO)C(O)=O)[C@@H](O)[C@@H](CO)O1 LAQPKDLYOBZWBT-NYLDSJSYSA-N 0.000 description 1
- HJCMDXDYPOUFDY-WHFBIAKZSA-N Ala-Gln Chemical compound C[C@H](N)C(=O)N[C@H](C(O)=O)CCC(N)=O HJCMDXDYPOUFDY-WHFBIAKZSA-N 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 108091016585 CD44 antigen Proteins 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010042086 Collagen Type IV Proteins 0.000 description 1
- 102000004266 Collagen Type IV Human genes 0.000 description 1
- CKLJMWTZIZZHCS-UHFFFAOYSA-N D-OH-Asp Natural products OC(=O)C(N)CC(O)=O CKLJMWTZIZZHCS-UHFFFAOYSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-UHFFFAOYSA-N D-alpha-Ala Natural products CC([NH3+])C([O-])=O QNAYBMKLOCPYGJ-UHFFFAOYSA-N 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 108010067306 Fibronectins Proteins 0.000 description 1
- 102000016359 Fibronectins Human genes 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 102000008055 Heparan Sulfate Proteoglycans Human genes 0.000 description 1
- 229920002971 Heparan sulfate Polymers 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000976622 Homo sapiens Zinc finger protein 42 homolog Proteins 0.000 description 1
- QNAYBMKLOCPYGJ-UWTATZPHSA-N L-Alanine Natural products C[C@@H](N)C(O)=O QNAYBMKLOCPYGJ-UWTATZPHSA-N 0.000 description 1
- CKLJMWTZIZZHCS-UWTATZPHSA-N L-Aspartic acid Natural products OC(=O)[C@H](N)CC(O)=O CKLJMWTZIZZHCS-UWTATZPHSA-N 0.000 description 1
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- 229930182821 L-proline Natural products 0.000 description 1
- 102000007547 Laminin Human genes 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 101000976618 Mus musculus Zinc finger protein 42 Proteins 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 101710150336 Protein Rex Proteins 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 239000006146 Roswell Park Memorial Institute medium Substances 0.000 description 1
- 206010039491 Sarcoma Diseases 0.000 description 1
- 108090000054 Syndecan-2 Proteins 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 102100023550 Zinc finger protein 42 homolog Human genes 0.000 description 1
- HUXIAXQSTATULQ-UHFFFAOYSA-N [6-bromo-3-[(2-methoxyphenyl)carbamoyl]naphthalen-2-yl] dihydrogen phosphate Chemical compound COC1=CC=CC=C1NC(=O)C1=CC2=CC(Br)=CC=C2C=C1OP(O)(O)=O HUXIAXQSTATULQ-UHFFFAOYSA-N 0.000 description 1
- 102000019997 adhesion receptor Human genes 0.000 description 1
- 108010013985 adhesion receptor Proteins 0.000 description 1
- 229960003767 alanine Drugs 0.000 description 1
- 229960002648 alanylglutamine Drugs 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 229960005261 aspartic acid Drugs 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 239000012888 bovine serum Substances 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 230000006727 cell loss Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000000919 ceramic Substances 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 230000005757 colony formation Effects 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000000151 deposition Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 229910003460 diamond Inorganic materials 0.000 description 1
- 239000010432 diamond Substances 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 210000002615 epidermis Anatomy 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 238000004299 exfoliation Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- -1 first Proteins 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 229960002449 glycine Drugs 0.000 description 1
- 210000004263 induced pluripotent stem cell Anatomy 0.000 description 1
- 238000001746 injection moulding Methods 0.000 description 1
- 239000011256 inorganic filler Substances 0.000 description 1
- 229910003475 inorganic filler Inorganic materials 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 150000002739 metals Chemical class 0.000 description 1
- 239000007758 minimum essential medium Substances 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000012766 organic filler Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 229920002635 polyurethane Polymers 0.000 description 1
- 239000004814 polyurethane Substances 0.000 description 1
- 229960002429 proline Drugs 0.000 description 1
- 238000004080 punching Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 229960001153 serine Drugs 0.000 description 1
- 229940054269 sodium pyruvate Drugs 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 239000013638 trimer Substances 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Description
本発明は、幹細胞を未分化状態で増殖させる培養装置および培養方法に関する。 The present invention relates to a culture apparatus and a culture method for growing stem cells in an undifferentiated state.
幹細胞は、血球、粘膜上皮、表皮等特定の機能を有する細胞に分化する能力を、細胞***後も保持し続ける細胞である。幹細胞は、個体組織の発生メカニズムの研究に重用される。また医療分野では、治療対象の組織や器官の幹細胞を培養して患者に移植することが期待される。 Stem cells are cells that retain the ability to differentiate into cells with specific functions, such as blood cells, mucosal epithelium, and epidermis, even after cell division. Stem cells are heavily used to study the mechanism of development of individual tissues. In the medical field, it is expected that stem cells of tissues or organs to be treated are cultured and transplanted to patients.
受精卵から発生する胚性幹細胞（Emblyonic Stem cell, ＥＳ細胞）は、あらゆる細胞に分化しうる多能性を有する。従って、ＥＳ細胞から分化させた任意の細胞を利用することにより、難病の原因解明や薬物の安全確認を分子レベルで行える可能性が高まっている。 Embryonic stem cells (ES cells) generated from fertilized eggs have pluripotency that can be differentiated into all types of cells. Therefore, the use of arbitrary cells differentiated from ES cells has increased the possibility of elucidating the cause of intractable diseases and confirming drug safety at the molecular level.
上記の基礎研究、臨床研究の進展や実用化には、所望の細胞に分化誘導させ得る未分化の幹細胞の量産が前提となる。かかる背景により、幹細胞の量産可能な培養方法の確立が、一層望まれる。 Progress and practical application of the basic research and clinical research described above are premised on the mass production of undifferentiated stem cells that can be induced to differentiate into desired cells. With this background, establishment of a culture method capable of mass-producing stem cells is further desired.
従来、幹細胞の培養方法の例として、静置培養法がある。静置培養法は、シャーレ、6〜24穴のプレート、或いはＴ−フラスコ等の底面に細胞を付着させて行われる。しかし、この方法では、シャーレ上で得られる幹細胞の細胞密度が小さいため、大量培養が難しい。 Conventionally, there is a stationary culture method as an example of a stem cell culture method. The static culture method is performed by attaching cells to the bottom of a petri dish, a 6-24 hole plate, a T-flask, or the like. However, in this method, since the cell density of the stem cells obtained on the petri dish is small, mass culture is difficult.
例えば、直径20μｍのマウス幹細胞を培養する場合、シャーレ上で静置培養される細胞の密度は、6×10４Cells/ｃｍ２である。上記の方法によると、10８Cells培養するために、約2000ｃｍ２の平面が必要になる。 For example, when culturing mouse stem cells having a diameter of 20 μm, the density of cells that are statically cultured on a petri dish is 6 × 10 4 cells / cm 2 . According to the above method, a plane of about 2000 cm 2 is required for culturing 10 8 cells.
細胞の培養方法の他の例として浮遊培養法がある。浮遊培養法は、培養容器内に満たした培地を撹拌、振とう等することにより細胞を浮遊させて培養する細胞培養方法である。浮遊培養法は、高い細胞密度で培養することができる。 Another example of the cell culture method is a suspension culture method. The suspension culture method is a cell culture method in which cells are suspended and cultured by stirring or shaking a medium filled in a culture vessel. The suspension culture method can be cultured at a high cell density.
上記の静置培養法の例で説明したものと同様のマウス幹細胞を浮遊培養する場合、培養される細胞の密度は１×10６ Cells/ｃｍ2である。この場合、100ｍLの培養漕で10８Cells培養することができる。すなわち、浮遊培養法は、比較的小さな装置で大量培養を行うことができる。 When mouse stem cells similar to those described in the example of the stationary culture method are subjected to suspension culture, the density of the cultured cells is 1 × 10 6 Cells / cm 2 . In this case, 10 8 Cells can be cultured in a 100 mL culture vessel. That is, the suspension culture method can perform mass culture with a relatively small apparatus.
特許文献１は、浮遊培養による胚性幹細胞培養方法の例である。特許文献１では、目的の細胞以外の細胞が培養液中に実質的に存在しない条件下で、胚性幹細胞の球状の細胞塊を形成させ、該球状体で細胞を増殖させることが開示される。 Patent document 1 is an example of the embryonic stem cell culture method by suspension culture. Patent Document 1 discloses that a spherical cell mass of embryonic stem cells is formed under the condition that cells other than the target cell are not substantially present in the culture solution, and the cells are proliferated in the spherical body. .
しかし幹細胞の浮遊培養法では、幹細胞を担体に付着させても、培養液が撹拌等されることにより幹細胞に対しせん断力が働き、幹細胞が担体から剥離しやすい。したがって、幹細胞の大量培養においては、幹細胞の担体からの剥離を抑制することが求められる。 However, in the stem cell suspension culture method, even if the stem cells are attached to the carrier, the shearing force acts on the stem cells when the culture solution is stirred, and the stem cells are easily detached from the carrier. Therefore, in large-scale culture of stem cells, it is required to suppress detachment of stem cells from the carrier.
また、未分化状態を維持させて幹細胞を培養することが困難な場合がある。すなわち、幹細胞の培養では、未分化状態の幹細胞を得るために、培地に分化抑制剤を含有させることがある。幹細胞は付着性が高いため、浮遊させた幹細胞同士が付着し、大きな細胞塊が形成されやすい。大きな細胞塊の中心部には、分化抑制剤が侵入しにくい。その結果、細胞塊の中心部から分化が進行しやすくなる。 In addition, it may be difficult to culture stem cells while maintaining an undifferentiated state. That is, in the culture of stem cells, in order to obtain undifferentiated stem cells, the culture medium may contain a differentiation inhibitor. Since stem cells are highly adherent, suspended stem cells adhere to each other and a large cell mass is likely to be formed. It is difficult for a differentiation inhibitor to enter the center of a large cell mass. As a result, differentiation is likely to proceed from the center of the cell mass.
加えて、大きな細胞塊が形成されると、細胞回収時の遠心分離工程前に大きな細胞塊をほぐさなければならず、工程数が多くなる。 In addition, if a large cell mass is formed, the large cell mass must be loosened before the centrifugation step at the time of cell recovery, which increases the number of steps.
本発明が解決しようとする課題は、幹細胞を、未分化状態を維持しながら効率的に大量培養しうる培養装置および培養方法を提供することである。 The problem to be solved by the present invention is to provide a culture apparatus and a culture method capable of efficiently mass-culturing stem cells while maintaining an undifferentiated state.
本発明者らは、鋭意研究の結果、その表面に所定の高低を有し、かつ幹細胞付着性物質をコートさせた担体を用い、幹細胞を未分化状態で維持して大量培養させうる培養装置および培養方法を発明するに至った。 As a result of diligent research, the present inventors have used a carrier having a predetermined height on its surface and coated with a stem cell adhesion substance, a culture device capable of maintaining a stem cell in an undifferentiated state and performing large-scale culture, and It came to invent the culture method.
本発明に用いられる担体は、その表面に高低を有し、かつ幹細胞付着性物質がコートされる。該担体は幹細胞非剥離性を有し、その表面で幹細胞を増殖させる。担体表面に高低を形成させる低部は、担体表面積1000000μｍ２あたり400〜3000000個存存在する。該低部は、1000000〜3000000個存在することが好ましく、400〜14000個存在することも好ましい。該担体表面にコートさせる幹細胞付着性物質としては、少なくともＥ−カドヘリン、マトリゲル、ラミニンのいずれか一つ以上が選択される。 The carrier used in the present invention has a height on its surface and is coated with a stem cell adhesion substance. The carrier has stem cell non-peeling property, and proliferates stem cells on its surface. There are 400 to 3000000 low parts per 1 million μm 2 of the surface area of the carrier that form the height on the carrier surface. The lower part is preferably 1000000-3000000, more preferably 400-14000. As the stem cell adhesion substance to be coated on the surface of the carrier, at least one of E-cadherin, Matrigel and laminin is selected.
本発明は、幹細胞を播種させた培養液中に、その表面に高低を有し、かつ幹細胞付着性物質をコートさせた担体を浮遊させる工程と、前記担体表面に幹細胞付着性物質を介して分散状態で付着させた幹細胞を、該担体表面から剥離させず、かつ未分化状態を維持させて増殖させる工程とを含む幹細胞培養方法を包含する。 The present invention includes a step of suspending a carrier having a height on its surface and coated with a stem cell adhesion substance in a culture medium seeded with stem cells, and dispersing the stem cell adhesion substance on the carrier surface. A stem cell culturing method comprising a step of allowing stem cells attached in a state to be detached from the surface of the carrier and proliferating while maintaining an undifferentiated state.
上記培養方法においては、担体表面に高低を形成させる低部が担体表面積1000000μｍ２あたり400〜3000000個存在する担体が好ましく用いられる。担体表面に高低を形成させる低部が担体表面積1000000μｍ２あたり1000000〜3000000個存在する担体や、400〜14000個存在する担体を用いることも好ましい。 In the above culture method, a carrier in which 400 to 3000000 low portions for forming a height on the surface of the carrier are present per 1 million μm 2 of the carrier surface area is preferably used. It is also preferable to use a carrier having 1000000 to 3000000 low portions for forming a height on the surface of the carrier, or 400 to 14000 carriers per 1000000 μm 2 of the carrier surface area.
本発明に係る幹細胞培養装置は、幹細胞の担体表面への付着性が良好である。本発明で発揮される担体の幹細胞非剥離性は、特に浮遊培養法に適用する場合に有用である。該装置は培養終了後の幹細胞の回収を簡便に行うことができる。従って、本発明に係る幹細胞培養方法は、未分化状態が維持された幹細胞を小さな装置で効率的に大量培養させることができる。 The stem cell culture apparatus according to the present invention has good adhesion of stem cells to the carrier surface. The stem cell non-detachability of the carrier exhibited in the present invention is particularly useful when applied to the suspension culture method. The apparatus can easily collect stem cells after culturing. Therefore, the stem cell culturing method according to the present invention can efficiently cultivate a large amount of stem cells maintained in an undifferentiated state with a small apparatus.
本発明は、特にＥＳ細胞の大量培養や、未分化状態で培養されたＥＳ幹細胞の効率的な回収に寄与する。マウス等の哺乳動物の幹細胞をはじめ、ヒトＥＳ細胞にも適用しうる。 The present invention contributes particularly to mass culture of ES cells and efficient recovery of ES stem cells cultured in an undifferentiated state. It can be applied to human ES cells as well as mammalian stem cells such as mice.
本発明に係る幹細胞培養装置は、培養漕と、培養槽を満たす培養液と、幹細胞付着性物質をコートさせ、かつ所定の表面形状を形成させた担体とを備える。 The stem cell culture apparatus according to the present invention includes a culture vessel, a culture solution that fills the culture tank, and a carrier that is coated with a stem cell adhesion substance and has a predetermined surface shape.
本発明に用いられる担体は、その表面に高低を有し、かつ幹細胞付着性物質がコートされる。担体表面に高低を形成させる低部は、担体表面積1000000μｍ２あたり400〜3000000個存在する。本発明にかかる担体表面上の低部存在数は、培養対象となる幹細胞の種類により適宜決定される。 The carrier used in the present invention has a height on its surface and is coated with a stem cell adhesion substance. There are 400 to 3000000 low parts per 1 million μm 2 of the surface area of the carrier that form the height on the carrier surface. The number of low parts present on the surface of the carrier according to the present invention is appropriately determined depending on the type of stem cells to be cultured.
図１〜３は、本発明の幹細胞培養装置の例を示す模式図である。図１は培養液５を撹拌させて担体４を浮遊させる幹細胞培養装置１の例である。図２は、培養液５を振とうさせて担体４を浮遊させる幹細胞培養装置２である。図３は、培養液５を灌流させて担体４を浮遊させる幹細胞培養装置３の例である。図３に示される矢印は灌流方向を示す。図１〜３において４は担体、５は培養液、６は培養槽である。 1 to 3 are schematic views showing examples of the stem cell culture apparatus of the present invention. FIG. 1 shows an example of a stem cell culture apparatus 1 that stirs a culture solution 5 to float a carrier 4. FIG. 2 shows a stem cell culture apparatus 2 that floats the carrier 4 by shaking the culture solution 5. FIG. 3 shows an example of the stem cell culture device 3 in which the culture medium 5 is perfused to float the carrier 4. The arrows shown in FIG. 3 indicate the perfusion direction. 1-3, 4 is a support | carrier, 5 is a culture solution, 6 is a culture tank.
図４は本発明に係る担体の一例の断面を示す模式図である。図４において、４は本発明に用いられる担体である。該担体の表面は後に説明する幹細胞付着性物質（不図示）でコートされる。担体表面は、複数の高部７と低部８とにより形成される高低を有する。Ｃは、高部７を足場にして担体４に付着した幹細胞である。 FIG. 4 is a schematic view showing a cross section of an example of a carrier according to the present invention. In FIG. 4, 4 is a carrier used in the present invention. The surface of the carrier is coated with a stem cell adhesion substance (not shown) described later. The carrier surface has a height formed by a plurality of high portions 7 and low portions 8. C is a stem cell attached to the carrier 4 using the high part 7 as a scaffold.
上記担体は、担体表面に高低を形成させる低部により担体表面と幹細胞との平坦な接着面積を確保することができる。該低部は、少なくとも従来公知の平坦な表面からなる担体と同様の幹細胞付着性を有する。 The carrier can secure a flat adhesion area between the carrier surface and the stem cells by the low part that forms the height on the carrier surface. The lower part has at least the same stem cell adhesion as a carrier having a conventionally known flat surface.
従来公知の平坦な表面からなる担体は、浮遊培養法に適用される場合、担体表面に付着させた幹細胞が、培養液中に生じるせん断力を直接受けるため、剥離しやすい。 When a carrier having a conventionally known flat surface is applied to a suspension culture method, stem cells attached to the surface of the carrier are directly subjected to a shearing force generated in the culture solution, and thus are easily detached.
これに対し本発明に用いられる担体は、低部が周囲を高部により囲まれている。そのため、培養液の撹拌等により生じるせん断力は該高部によって受け止められ、低部内では低減される。これにより本発明は、担体低部に付着する幹細胞がせん断力により担体表面から剥離することが抑制される。 In contrast, in the carrier used in the present invention, the lower part is surrounded by the high part. For this reason, the shearing force generated by stirring the culture medium is received by the high part and reduced in the low part. Thereby, the present invention suppresses the stem cells adhering to the lower part of the carrier from being detached from the surface of the carrier due to the shearing force.
すなわち本発明は、静置培養法に加え、浮遊培養法においても優れた幹細胞非剥離性を発揮する。本発明に用いられる担体において「非剥離性」とは幹細胞が全く剥離しない場合に加え、「担体から幹細胞が剥離しにくい」ことを包含する。 That is, the present invention exhibits excellent non-detachability of stem cells in the suspension culture method in addition to the stationary culture method. In the carrier used in the present invention, “non-peeling” includes not only the case where the stem cells do not peel at all, but also “the stem cells are difficult to peel from the carrier”.
上記「担体から幹細胞が剥離しにくい」とは、担体に付着したと推測される細胞数に対し、担体に付着後剥離したと推測される細胞数が10％程度であることを意味する。上記細胞数の推測値は、担体の表面形状、培養対象となる細胞の種類、播種密度等を考慮し、培養を終了させ担体を培養槽から取り出した直後の培養槽内に残った細胞数等から導き出される。 The above “stem cells are difficult to detach from the carrier” means that the number of cells presumed to be detached after attaching to the carrier is about 10% of the number of cells presumed to be attached to the carrier. Estimate of the number of cells, the surface shape of the support, the type of cells to be cultured target seeding density such consideration, the number of cells such that the carrier end the culture was left in the culture tank immediately after taken out from the culture tank Derived from.
低部と共に担体表面に高低を形成させる高部は、培地に播種された幹細胞を担体に付着させる足場として機能する。すなわち、培地に播種された幹細胞は、担体表面に付着すると、近傍にある高部をとらえて伸展する。
本発明は、担体表面に足場として機能する高部が形成されることにより、幹細胞が伸展しやすくなる。該幹細胞は、該高部にフィットして担体表面に良好に付着する。従って本発明に用いる担体に付着させた幹細胞は、せん断力が生じる培養液中においても剥離しにくい。
The high part that forms the height on the surface of the carrier together with the low part functions as a scaffold for attaching the stem cells seeded in the medium to the carrier. That is, when the stem cells seeded in the medium adhere to the surface of the carrier, the stem cells catch the high part in the vicinity and expand.
In the present invention, stem cells are easily extended by forming a high part that functions as a scaffold on the surface of a carrier. The stem cell fits to the high part and adheres well to the surface of the carrier. Therefore, the stem cells attached to the carrier used in the present invention are not easily detached even in a culture solution in which shearing force is generated.
本発明で用いられる担体の表面には、幹細胞の表面に発現する所定の基底膜成分と親和性を有する幹細胞付着性物質をコートさせる。特に担体に形成される高部は、幹細胞付着性物質のコート量が比較的多量になる傾向がある。これにより本発明は、幹細胞の担体への付着性を向上させる。 The surface of the carrier used in the present invention is coated with a stem cell adhesion substance having an affinity for a predetermined basement membrane component expressed on the surface of the stem cells. In particular, the high part formed on the carrier tends to have a relatively large amount of coat of the stem cell adhesion substance. Thereby, this invention improves the adhesiveness to the support | carrier of a stem cell.
上記の担体を培養液中にいれると、担体表面をコートする幹細胞付着性物質の親水性により、担体は培養槽底部へ沈降する。しかし培養液を撹拌等させることにより、沈降した担体を培養液内で浮遊させることができる。 When the above carrier is placed in the culture solution, the carrier settles to the bottom of the culture tank due to the hydrophilicity of the stem cell adhesion substance that coats the surface of the carrier. However, by stirring the culture solution, the precipitated carrier can be suspended in the culture solution.
浮遊させた該担体は、培養液中に播種された幹細胞と自然に接触する。担体表面の高部の頂点間の距離が幹細胞の直径より小さい領域では、幹細胞が低部に入り込まず、各頂点上に支持されるように接触させることができる。
担体表面の高部の頂点間の距離が幹細胞の直径より大きい領域では、幹細胞が低部に分散状態で付着する。低部では、幹細胞と担体表面との接着面積を広く確保できる。
The suspended carrier naturally comes into contact with stem cells seeded in the culture medium. In a region where the distance between the high vertices on the surface of the carrier is smaller than the diameter of the stem cells, the stem cells do not enter the low part and can be contacted so as to be supported on each vertex.
In a region where the distance between the apexes of the high part on the surface of the carrier is larger than the diameter of the stem cell, the stem cells adhere to the low part in a dispersed state. In the low part, a wide adhesion area between the stem cells and the carrier surface can be secured.
本発明においては、担体表面に形成される高部と低部とのいずれにおいても幹細胞が付着しやすく、かつ剥離しにくい。
担体表面の高部においては該頂点が幹細胞付着の足場として機能することと比較的多量にコートされる幹細胞付着性物質とにより、良好な幹細胞付着性を発揮する。担体表面の低部においては、広い接着面積を確保することにより良好な幹細胞付着性を発揮する。
In the present invention, stem cells are likely to adhere to both the high part and the low part formed on the surface of the carrier and are difficult to peel off.
In the high part of the carrier surface, the apex functions as a scaffold for stem cell adhesion and a stem cell adhesion substance coated in a relatively large amount exhibits good stem cell adhesion. In the lower part of the surface of the carrier, good stem cell adhesion is exhibited by securing a wide adhesion area.
本発明において発揮される付着性は、少なくとも培養液の撹拌等により生じるせん断力に対抗しうる。すなわち本発明は、担体に付着する幹細胞が上記せん断力により担体から剥離することを抑制することができる。 The adhesion exhibited in the present invention can counter at least the shearing force generated by stirring the culture solution. That is, the present invention can suppress the stem cells adhering to the carrier from being detached from the carrier by the shearing force.
本発明に用いられる担体は、その表面に幹細胞付着性物質がコートされているため、幹細胞が、該幹細胞付着性物質のコート領域に分散状態で生育させることができる。 Since the surface of the carrier used in the present invention is coated with a stem cell adhesion substance, the stem cells can be grown in a dispersed state in the coat region of the stem cell adhesion substance.
本発明の培養装置を説明する。
［担体］
本発明に用いられる担体４は、その表面に複数の高部７と低部８とが存在する。該複数の高部と低部とは、本発明の担体表面に高低を形成させる。本発明において「高低を有する」とは、担体の表面が平坦でないことを意味する。
The culture apparatus of the present invention will be described.
[Carrier]
The carrier 4 used in the present invention has a plurality of high portions 7 and low portions 8 on its surface. The plurality of high portions and low portions form a height on the carrier surface of the present invention. In the present invention, “having high and low” means that the surface of the carrier is not flat.
本発明に用いられる担体の高低を有する表面を、図４を用いて説明する。図４において、Ｐ１、Ｐ２およびＰ３は、各高部の頂点である。Ｂ１、Ｂ２、Ｂ３は各低部の最低点である。ｇ１、ｇ２、ｇ３は、高部と該高部と隣接する低部との高低差を表す。「高低を有する」とは、高部の頂点と該高部と隣接する低部の最低点との高低差の平均値が0.5μｍより大きいことを包含する。 The surface having the height of the carrier used in the present invention will be described with reference to FIG. In FIG. 4, P1, P2 and P3 are the vertices of each high part. B1, B2, and B3 are the lowest points of each low part. g1, g2, and g3 represent height differences between a high portion and a low portion adjacent to the high portion. “Having a height” includes that the average value of the height difference between the apex of the high part and the lowest point of the low part adjacent to the high part is greater than 0.5 μm.
なお、図４において、ｄ１はＰ１とＰ２との間の隣り合う頂点間の距離であり、ｄ２はＰ２とＰ３との間の頂点間距離である。 In FIG. 4, d1 is the distance between adjacent vertices between P1 and P2, and d2 is the distance between vertices between P2 and P3.
上記担体は、目的となる幹細胞の直径や柔軟性に対応させた所定の高低からなる表面粗さを有するため、培養液中に懸濁される幹細胞を担体表面にフィットさせやすい。担体表面に形成される高部は、幹細胞を支持する足場として機能する。また幹細胞剥離抑制作用を奏する。これにより、本発明は、良好な幹細胞付着性を有する。 Since the carrier has a predetermined surface roughness corresponding to the diameter and flexibility of the target stem cell, it is easy to fit the stem cell suspended in the culture medium onto the carrier surface. The high part formed on the surface of the carrier functions as a scaffold for supporting stem cells. In addition, it exhibits an effect of suppressing stem cell detachment. Thereby, this invention has favorable stem cell adhesiveness.
培養終了後は、該担体を培地から取り出して、常法に従い幹細胞を回収できる。本発明は、幹細胞を担体上に分散状態で付着させて培養するため、大きな細胞塊は形成されない。したがって、回収工程を簡便に行うことができる。 After completion of the culture, the carrier can be removed from the medium, and stem cells can be collected according to a conventional method. In the present invention, stem cells are cultured in a dispersed state on a carrier, so that a large cell mass is not formed. Therefore, the recovery process can be easily performed.
本発明に用いられる担体表面の高低は、担体表面積1000000μｍ２あたりの低部の存在数で表現されうる。本発明において該低部の存在数は、担体表面積1000000μｍ２あたりおよそ400〜3000000個である。
好ましくは、担体表面積1000000μｍ２あたり1000000〜3000000個であり、より好ましくは1300000〜1700000個であり、さらに好ましくは1500000個である。他の好ましい低部存在数は、担体表面積1000000μｍ２あたり400〜14000個であり、より好ましくは4000〜10000個である。
該低部の存在数を上記の好ましい範囲内で形成させる方法は特に限定されない。該担体の製造方法は、後にその例を挙げて説明する。
The height of the support surface used in the present invention can be expressed by the number of low portions per 1000000 μm 2 of the support surface area. In the present invention, the number of the low portions is about 400 to 3000000 per 1 million μm 2 of the carrier surface area.
The number is preferably 1000000 to 3000000 per carrier surface area of 1000000 μm 2 , more preferably 1300,000 to 1700,000, and even more preferably 150,000. The other preferable low part existence number is 400 to 14000 per carrier surface area of 1000000 μm 2 , more preferably 4000 to 10,000.
There is no particular limitation on the method for forming the number of the low portions within the above-mentioned preferable range. The method for producing the carrier will be described later with examples.
該担体の表面粗さは、図４においてｄ１又はｄ２で例示される高部の頂点間距離によっても表現されうる。上記隣り合う高部の頂点間の好ましい距離は、培養される幹細胞の直径や柔軟性等により適宜決定される。頂点間の距離は、幹細胞の直径より1〜5％、或いは50〜200％程度の大きさであることが好ましい。 The surface roughness of the carrier can also be expressed by the distance between the apexes at the high portion exemplified by d1 or d2 in FIG. A preferable distance between the apexes of the adjacent high portions is appropriately determined depending on the diameter and flexibility of the cultured stem cells. The distance between the apexes is preferably about 1 to 5%, or about 50 to 200%, from the diameter of the stem cells.
図４においてｄ１、ｄ２により例示される頂点間距離が小さすぎる担体や、ｇ１、ｇ２およびｇ３により例示される表面高低差の平均値が、0.5μｍより小さな担体は、各高部の頂点と幹細胞とのそれぞれの接触面積が小さくなりやすい。そのため、担体表面の高部の足場として機能が低くなり、幹細胞が担体表面から剥離しやすくなる。上記のような滑らかな担体表面を有する担体の多くは、低部の存在数が3000000個より多い場合に認められる。 In FIG. 4, a carrier whose vertex-to-vertex distance exemplified by d1 and d2 is too small, and a carrier whose average surface height difference exemplified by g1, g2 and g3 is smaller than 0.5 μm The contact area with each other tends to be small. Therefore, the function as a scaffold on the high part of the carrier surface is reduced, and the stem cells are easily detached from the carrier surface. Many of the carriers having the smooth carrier surface as described above are recognized when the number of low portions is more than 3000000.
マウス多能性幹細胞の培養に用いられる担体の場合、好ましい頂点間距離は、0.1〜2.7μｍであり、より好ましくは、0.5〜2.0μｍである。他の好ましい頂点間距離は、5μm〜40μmである。担体表面の平均高低差は、0.5〜2.7μｍが好ましく、0.5〜2.0μｍが好ましい。 In the case of a carrier used for culturing mouse pluripotent stem cells, a preferable distance between vertices is 0.1 to 2.7 μm, and more preferably 0.5 to 2.0 μm. Another preferable distance between the vertices is 5 μm to 40 μm. The average height difference on the carrier surface is preferably 0.5 to 2.7 μm, more preferably 0.5 to 2.0 μm.
担体表面の低部の存在数すなわち表面粗さの程度は、培養対象となる幹細胞の種類に対応して適宜決定される。具体的な考慮すべき要素としては、幹細胞のサイズや、幹細胞の移動速度等が挙げられる。 The number of low portions on the surface of the carrier, that is, the degree of surface roughness, is appropriately determined according to the type of stem cells to be cultured. Specific factors to be considered include stem cell size, stem cell migration speed, and the like.
低部の存在数と高部の存在数は比例する。すなわち低部の存在数が少ない場合、高部の存在数も少なくなる。高部の頂点間距離と幹細胞直径との差がほとんどない担体は、幹細胞の足場が少ない。そのため、幹細胞を複数の高部で支持しがたくなる。その場合、浮遊培養法による所望の幹細胞培養が困難になる。 The number of low parts and the number of high parts are proportional. That is, when the number of low portions is small, the number of high portions is also small. A carrier having little difference between the distance between the apexes of the high part and the stem cell diameter has few stem cell scaffolds. Therefore, it becomes difficult to support stem cells at a plurality of high portions. In that case, desired stem cell culture by the suspension culture method becomes difficult.
幹細胞は培養基材表面上を移動して増殖するが、その速度はその培養基材表面性状により異なる。細胞の移動速度が速い場合、細胞は急速に伸張、収縮する。急速に収縮して球形に近い形状になった細胞は、培養基材との接触面積が減少する。したがって好ましい付着性を得難くなる。 Stem cells migrate on the surface of the culture substrate and proliferate, but the speed varies depending on the surface properties of the culture substrate. When the moving speed of the cell is high, the cell rapidly expands and contracts. Cells that rapidly shrink and have a nearly spherical shape have a reduced contact area with the culture substrate. Therefore, it becomes difficult to obtain preferable adhesion.
移動速度が速い細胞を培養する場合は、上記に推察される低付着性を補完する観点から、幹細胞の伸張時に足場となる高部を提供しうる担体を用いることが好ましいと考えられる。すなわち移動速度が速い細胞を培養する場合、本発明所定の範囲内で比較的担体表面の低部の存在数が多い担体を用いることは、好ましい選択である。 In the case of culturing cells having a high migration speed, it is considered preferable to use a carrier that can provide a high part that serves as a scaffold when stem cells are stretched, from the viewpoint of complementing the low-adhesiveness estimated above. That is, when culturing cells having a high migration speed, it is a preferable choice to use a carrier having a relatively large number of low portions on the surface of the carrier within the predetermined range of the present invention.
コロニー形成を防止するためには、培養対象となる幹細胞のサイズに対し適切な表面粗さを備える担体を用いるが好ましい。播種密度が過度に高い培養液で頂点間距離が大きな担体を用いると、低部に多数の幹細胞が入り込みコロニーを形成する可能性が高まる。コロニーになった幹細胞は、コロニー中心部から分化が進む傾向がある。したがって幹細胞のサイズを考慮して担体の表面粗さを選択することが、未分化細胞培養の観点から好ましい。 In order to prevent colony formation, it is preferable to use a carrier having a surface roughness appropriate for the size of the stem cells to be cultured. When a culture medium with an excessively high seeding density and a carrier with a large distance between vertices is used, the possibility that a large number of stem cells enter the lower part to form colonies increases. Stem cells that have become colonies tend to differentiate from the center of the colony. Therefore, it is preferable from the viewpoint of undifferentiated cell culture to select the surface roughness of the carrier in consideration of the size of the stem cells.
担体基材としては、幹細胞、特にマウスＥＳ細胞やヒトＥＳ細胞の培養に使用する公知の材料を用いることができる。本発明の担体はその表面に起伏を形成させるため、基材には成型加工が容易な材料を用いることが好ましい。好ましくはポリスチレン、ニトロセルロース、ナイロン、ポリウレタン等を用いることができる。 As the carrier substrate, known materials used for culturing stem cells, particularly mouse ES cells and human ES cells can be used. In order to form undulations on the surface of the carrier of the present invention, it is preferable to use a material that can be easily molded for the substrate. Preferably, polystyrene, nitrocellulose, nylon, polyurethane and the like can be used.
担体の形状は、培養液中に浮遊させた担体同士が接触しにくい形状が選択される。担体同士の接触を抑制することにより、担体に付着させた幹細胞の剥落を抑制できる。 As the shape of the carrier, a shape in which the carriers suspended in the culture solution do not easily come into contact with each other is selected. By suppressing contact between the carriers, exfoliation of the stem cells attached to the carriers can be suppressed.
好ましい担体の形状としては、球状、ディスク型、リング型、ファイバー型等が例示される。特にディスク型担体は、担体同士が接触しにくく、接触した場合も接触面積が少ないため、幹細胞の剥離を抑制することができる。ディスク型担体は、製造コスト面からも好ましい形状の担体である。 Examples of preferable carrier shapes include a spherical shape, a disk shape, a ring shape, and a fiber shape. In particular, the disc-type carrier is difficult to contact with each other and has a small contact area even when contacted with each other. The disk-type carrier is a carrier having a preferable shape from the viewpoint of manufacturing cost.
担体のサイズは、培地中で接触しにくいサイズが適宜選択される。3〜20ｍｌ／ｗｅｌｌの培養漕内で用いられる担体のサイズとしては、ディスク型担体の場合、外径1〜6ｍｍが好ましく、4〜6ｍｍがより好ましい。また厚さは0.01〜0.03ｍｍが好ましい。ファイバー型担体の場合、断面直径0.5〜1ｍｍ、長さ1〜6ｍｍ程度が好ましい。 As the size of the carrier, a size that is difficult to contact in the medium is appropriately selected. In the case of a disk-type carrier, the size of the carrier used in a 3 to 20 ml / well culture basket is preferably 1 to 6 mm in outer diameter, and more preferably 4 to 6 mm. The thickness is preferably 0.01 to 0.03 mm. In the case of a fiber type carrier, a cross-sectional diameter of 0.5 to 1 mm and a length of about 1 to 6 mm are preferable.
本発明に用いられる担体の製造方法は、本発明の作用効果を阻害しない限り特に限定されない。通常は所定の高低を有する担体基材を製造し、得られた担体基材に幹細胞付着性物質をコートさせることにより製造することができる。 The method for producing the carrier used in the present invention is not particularly limited as long as the effects of the present invention are not inhibited. Usually, it can be produced by producing a carrier substrate having a predetermined height and coating the obtained carrier substrate with a stem cell adhesion substance.
表面に本発明所定の高低を有する担体基材の成型方法としては、担体基材にガラス板を圧接させる方法や、射出成型法が挙げられる。上記の圧接による製造方法を行う場合、圧接材は、選択される担体基材の硬度に対応して適宜選択される。好ましい圧接材としては、ガラス、ステンレス、鉄等の金属、セラミックを挙げることができる。 Examples of the method for molding the carrier substrate having the predetermined height according to the present invention on the surface include a method in which a glass plate is pressed against the carrier substrate, and an injection molding method. When performing the manufacturing method by said pressure welding, a pressure welding material is suitably selected according to the hardness of the support base material selected. Preferred examples of the pressure contact material include metals such as glass, stainless steel, and iron, and ceramics.
圧接材には、平均粒子径0.1〜2μｍの粒子が含有される。該粒子は、圧接材の担体基材との接触面に適宜露出する。該粒子の露出部分が担体基材に圧接されることにより、担体基材表面に高低が形成される。 The pressure contact material contains particles having an average particle size of 0.1 to 2 μm. The particles are appropriately exposed on the contact surface of the pressure contact material with the carrier substrate. When the exposed portion of the particles is pressed against the carrier substrate, the height is formed on the surface of the carrier substrate.
担体基材にポリスチレンを選択する場合、圧接材にはガラスが好ましく用いられる。以下にポリスチレンからなる担体基材の製造方法の一例を説明する。まず、一組のガラス板の間にポリスチレンペレットを挟み、各ガラス板の一面をポリスチレンペレットに圧接させる。 When polystyrene is selected as the carrier substrate, glass is preferably used as the pressure contact material. An example of a method for producing a carrier substrate made of polystyrene will be described below. First, polystyrene pellets are sandwiched between a pair of glass plates, and one surface of each glass plate is pressed against the polystyrene pellets.
ガラス板は、上記の所定の平均粒子径の範囲内で所望の表面粗さを形成させるのに適した粒子径を含有させたものが選択される。したがって、ガラス板の粒子露出面をポリスチレンペレットに加熱下で圧接させることにより、ポリスチレンペレットはフィルム状に変形され、該ポリスチレンフィルムに所望の高低を有する表面を形成させることができる。 The glass plate is selected to have a particle size suitable for forming a desired surface roughness within the range of the predetermined average particle size. Therefore, when the particle exposed surface of the glass plate is pressed into contact with the polystyrene pellet under heating, the polystyrene pellet is deformed into a film shape, and a surface having a desired height can be formed on the polystyrene film.
ポリスチレンフィルム表面において、ガラス板の粒子露出部を圧接させた部分が低部となり、他の部分が高部となる。したがって、担体表面積あたりの低部の存在数は、圧接材に含有させる粒子数により調節することができる。 On the surface of the polystyrene film, the portion where the exposed portion of the glass plate is pressed is the low portion, and the other portion is the high portion. Therefore, the number of low portions per surface area of the carrier can be adjusted by the number of particles contained in the pressure contact material.
粒子露出面を有するガラス板は、一組のガラス板のうち一方でもよく双方でもよい。双方に粒子露出面を有するガラス板を用いれば、担体基材の両面に表面粗さを形成することができる。 The glass plate having the particle exposed surface may be one or both of a pair of glass plates. If glass plates having particle-exposed surfaces on both sides are used, surface roughness can be formed on both sides of the carrier substrate.
本発明に用いられる担体は、本発明の作用効果を十分に引き出すため、基材の全表面に均質に上記所定の表面粗さを備えることが好ましい。かかる観点から、本発明にかかる担体の成型に用いられるガラス板は、含有される粒子のガラス板表面への露出部分が均等に分散していることが好ましい。しかし本発明は、その作用効果を阻害しない限り、担体の表面粗さが不均質に形成されるものも包含する。従って、上記ガラス板は、該粒子の露出部分が不均等に分散されるものを用い得る。 The carrier used in the present invention preferably has the above-mentioned predetermined surface roughness uniformly over the entire surface of the substrate in order to sufficiently bring out the effects of the present invention. From this point of view, it is preferable that the glass plate used for molding the carrier according to the present invention has evenly dispersed portions of the contained particles exposed to the glass plate surface. However, the present invention includes those in which the surface roughness of the carrier is formed inhomogeneously as long as the function and effect are not inhibited. Therefore, the glass plate may be one in which the exposed portions of the particles are dispersed unevenly.
圧接材に含有させる上記粒子は、加熱圧接時にその硬度が担体基材表面の硬度より硬い材料であって、担体表面に高部と低部とを形成させることができるものであれば、公知の有機フィラーや無機フィラー等を限定することなく用いることができる。例としては、ダイヤモンド、アルミナを挙げられる。 The particles to be contained in the pressure welding material are known as long as the hardness is higher than the hardness of the surface of the carrier base material during heating and pressure welding and can form a high part and a low part on the surface of the carrier. An organic filler, an inorganic filler, etc. can be used without limiting. Examples include diamond and alumina.
圧接時の処理温度は、選択される担体基材と圧接材とにより適宜調整される。ガラス板によりポリスチレンからなる担体基材を成型する場合の処理温度は、130〜170℃が好ましく、150〜170℃がより好ましい。 The treatment temperature at the time of pressure contact is appropriately adjusted depending on the selected carrier substrate and pressure contact material. The processing temperature when molding a carrier substrate made of polystyrene with a glass plate is preferably from 130 to 170 ° C, more preferably from 150 to 170 ° C.
所望の高部と低部とを表面に形成させたポリスチレンフィルムは、室温で5〜10分間静置する。その後、得られたポリスチレンフィルムをパンチしてディスク状にする等、上記に説明した好ましい形状に適宜成型する。成型された所定の高低を有する基材担体に、以下に説明する幹細胞付着性物質をコートさせることにより、本発明に用いる担体を得ることができる。 The polystyrene film having the desired high and low portions formed on the surface is allowed to stand at room temperature for 5 to 10 minutes. Thereafter, the obtained polystyrene film is appropriately formed into the preferred shape described above, such as punching into a disk shape. The carrier used in the present invention can be obtained by coating the base material carrier having a predetermined height with a stem cell adhesion substance described below.
本発明に用いられる担体の表面にコートさせる当該幹細胞付着性物質の例としては、フィブロネクチン、コラーゲン等の細胞結合物質が好ましく、特にＥ−カドヘリン、マトリゲル、ラミニン等がより好ましく用いられる。本発明において、上記の幹細胞付着性物質は一種を用いてもよく、二種以上を混合させて用いてもよい。該幹細胞付着性物質は、常法にしたがって製造してもよく、市販品を用いてもよい。 As examples of the stem cell adhesion substance to be coated on the surface of the carrier used in the present invention, cell binding substances such as fibronectin and collagen are preferable, and in particular, E-cadherin, matrigel, laminin and the like are more preferably used. In the present invention, the above stem cell adhesion substance may be used alone or in combination of two or more. The stem cell adhesion substance may be produced according to a conventional method, or a commercially available product may be used.
担体表面に接触した幹細胞は、幹細胞表面に発現する所定の基底膜成分と、担体表面にコートさせた幹細胞付着性物質とが互いの親和性により強固に結合するため、担体表面に十分に接着させることができる。幹細胞付着性物質として例示したＥ−カドヘリン、マトリゲル、ラミニンについて説明する。 Stem cells that are in contact with the surface of the carrier are sufficiently adhered to the surface of the carrier because a predetermined basement membrane component expressed on the surface of the stem cell and the stem cell adhesion substance coated on the surface of the carrier are strongly bonded to each other due to their affinity. be able to. E-cadherin, matrigel, and laminin exemplified as stem cell adhesion substances will be described.
Ｅ−カドヘリンは、未分化のマウスＥＳ細胞で発現するタンパク質の一種で、多能性幹細胞の付着性にかかわる物質として知られている。 E-cadherin is a kind of protein expressed in undifferentiated mouse ES cells, and is known as a substance involved in the adhesion of pluripotent stem cells.
マトリゲルは、マウスＥＨＳ肉腫から抽出、生成される基底膜成分である。マトリゲルにはＩＶ型コラーゲン、ラミニン、ヘパラン硫酸プロテオグリカン、エンタクテン等が含有される。 Matrigel is a basement membrane component extracted and generated from mouse EHS sarcoma. Matrigel contains type IV collagen, laminin, heparan sulfate proteoglycan, entactene and the like.
ラミニンは、基底膜を構成する主要タンパク質の一つであり、細胞の接着、遊走、増殖に関与すると考えられている。ラミニンは、α、β、γ鎖の３本のサブユニット鎖からなるヘテロ三量体である。ヒトラミニンの数種は、その組み換えタンパク質が、ヒトＥＳ細胞が発現する接着受容体と高い親和性を有することが解明されている。そのようなラミニンとして、本発明においては、ラミニン、ヒトラミニン、ヒトラミニンα５β１γ１、ヒトラミニンα３β３γ２が、好ましく用いられる。 Laminin is one of the major proteins that make up the basement membrane, and is thought to be involved in cell adhesion, migration, and proliferation. Laminin is a heterotrimer composed of three subunit chains of α, β, and γ chains. Several human laminins have been elucidated that their recombinant proteins have a high affinity for adhesion receptors expressed by human ES cells. As such laminin, laminin, human laminin, human laminin α5β1γ1, and human laminin α3β3γ2 are preferably used in the present invention.
上記に例示したラミニンを組み換えタンパク質として製造する場合、まず、目的ラミニンのα鎖、β鎖、γ鎖の各タンパク質をコードするＤＮＡを取得し、それぞれ発現ベクターに挿入する。得られた各発現ベクターを宿主細胞に共導入して発現させ、三量体を形成しているタンパク質を精製する。これにより目的のラミニンを得ることができる。本発明に用いられるラミニンについて、上記の製造方法は一つの例である。したがって、目的のラミニンを得られる方法であれば、製造方法は上記に限定されない。 When the above-exemplified laminin is produced as a recombinant protein, first, DNAs encoding the target laminin α chain, β chain, and γ chain proteins are obtained and inserted into expression vectors, respectively. Each of the obtained expression vectors is co-introduced into a host cell and expressed, and a protein forming a trimer is purified. Thereby, the target laminin can be obtained. The above manufacturing method is an example of laminin used in the present invention. Accordingly, the production method is not limited to the above as long as the target laminin can be obtained.
幹細胞付着性物質のコート量は、付着性向上の観点から、担体に対しコートさせ得る最
大量とすることが好ましい。本発明に用いる担体は表面に所定の高低を有するため、表面積が大きい。したがって幹細胞付着性物質を多量にコートさせることができる。担体基材表面に対する幹細胞付着性物質のコート量は、0.6〜1.1μｇ/ｃｍ２が好ましく、0.8〜1.1μｇ／ｃｍ２がより好ましい。
The coating amount of the stem cell adhesion substance is preferably the maximum amount that can be coated on the carrier from the viewpoint of improving adhesion. Since the carrier used in the present invention has a predetermined height on the surface, the surface area is large. Therefore, a large amount of the stem cell adhesion substance can be coated. Coating amount of stem cells adherent material to the support substrate surface, preferably 0.6~1.1μg / cm 2, 0.8~1.1μg / cm 2 is more preferable.
幹細胞付着性物質を担体表面にコートさせる方法は特に限定されない。一例としては、幹細胞付着性物質を蒸留水またはＰｈｏｓｐｈａｔｅ Ｂｕｆｆｅｒｅｄ Ｓａｌｉｎ（ＰＢＳ）で10μｇ／ｍＬに調製後、ろ過、滅菌した溶液に担体基材を接触させ、室温35℃〜37℃で30〜60分間静置させる方法を挙げることができる。 The method for coating the surface of the carrier with the stem cell adhesion substance is not particularly limited. As an example, after preparing the stem cell adhesion substance to 10 μg / mL with distilled water or Phosphate Buffered Salin (PBS), the carrier substrate is brought into contact with the filtered and sterilized solution, and the room temperature is 35 to 37 ° C. for 30 to 60 minutes. The method of leaving still can be mentioned.
担体基材と所定の幹細胞付着性物質とを接触させることにより、担体基材に対し、幹細胞付着性物質を吸着させたり、共有結合させたりすることにより、幹細胞付着性物質が担体基材に定着し、本発明に用いられる担体を得ることができる。 By contacting the carrier substrate with a predetermined stem cell adhesion substance, the stem cell adhesion substance is fixed to the carrier substrate by adsorbing or covalently bonding the stem cell adhesion substance to the carrier substrate. Thus, the carrier used in the present invention can be obtained.
幹細胞付着性物質を担体基材に接触させる前に、幹細胞付着性物質に予め抗原性分子を結合させておくと共に、担体基材表面に該抗原性分子と特異的に結合する抗体分子を付加させておくことも好ましい。これにより幹細胞付着性物質中の抗原と担体基材表面の抗体とが特異的に結合し、担体表面に幹細胞付着物質を良好に定着させることができる。 Before bringing the stem cell adhesion substance into contact with the carrier substrate, an antigenic molecule is previously bound to the stem cell adhesion substance, and an antibody molecule that specifically binds to the antigenic molecule is added to the surface of the carrier substrate. It is also preferable to keep it. As a result, the antigen in the stem cell adhesion substance specifically binds to the antibody on the surface of the carrier substrate, and the stem cell adhesion substance can be well fixed on the carrier surface.
［培養液］
本発明の培養漕を満たす培養液は、従来公知の幹細胞培養液を用いることができる。従来公知の培養液の例としては、Ｄｕｌｂｅｃｃｏ’ｓ Ｍｏｄｉｆｉｅｄ Ｅａｇｌｅ’ｓ Ｍｅｄｉｕｍ（ＤＭＥＭ）、Ｇｒａｓｇｏｗ Ｍｉｎｉｍｕｍ Ｅｓｓｅｎｔｉａｌ Ｍｅｄｉｕｍ（ＧＭＥＭ）、ＲＰＭＩ６４０等を挙げることができる。
[Culture medium]
A conventionally known stem cell culture solution can be used as the culture solution satisfying the culture cake of the present invention. Examples of conventionally known culture solutions include Dulbecco's Modified Eagle's Medium (DMEM), Grasgo Minimum Essential Medium (GMEM), RPMI 640, and the like.
上記に例示した培地にＬ−グルタミン、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−セリン等公知の成分を添加することができる。培地には、ウシ胎児血清（ＦＢＳ）等添加できるが、無血清培養してもよい。 In the medium exemplified above, L-glutamine, insulin, transferrin, ethanolamine, selenium, 2-mercaptoethanol, L-alanyl-L-glutamine, sodium pyruvate, L-alanine, L-asparagine, L-aspartic acid, Known components such as glycine, L-proline and L-serine can be added. Although fetal bovine serum (FBS) can be added to the medium, serum-free culture may be performed.
上記の培地に、分化抑制剤として白血病抑制因子（ＬＩＦ）を添加してもよい。
ヒト多能性幹細胞にはＬＩＦ反応性がない。従ってヒト多能性幹細胞を培養する場合、未分化状態を維持する目的で、マウスやヒト由来の線維芽細胞をフィーダー細胞として用いる共培養法を適用しうる。
A leukemia inhibitory factor (LIF) may be added to the above medium as a differentiation inhibitor.
Human pluripotent stem cells are not LIF responsive. Therefore, when culturing human pluripotent stem cells, a co-culture method using mouse or human-derived fibroblasts as feeder cells can be applied for the purpose of maintaining an undifferentiated state.
しかしヒト多能性幹細胞を再生医療に応用する観点からは、共培養法は、目的となる多能性幹細胞に対する影響が懸念される。そのため、近年、フィーダー細胞を用いないヒト多能性幹細胞の培養方法に関する知見が得られている。従って、上記の知見に基づく手法を適用することにより、本発明においても培養液に分化抑制剤を添加することなく未分化状態を維持してヒト多能性幹細胞を培養しうる。 However, from the viewpoint of applying human pluripotent stem cells to regenerative medicine, there is a concern that the coculture method has an influence on the target pluripotent stem cells. Therefore, in recent years, knowledge about a method for culturing human pluripotent stem cells without using feeder cells has been obtained. Therefore, by applying a technique based on the above findings, human pluripotent stem cells can be cultured in the present invention while maintaining an undifferentiated state without adding a differentiation inhibitor to the culture medium.
[培養漕]
本発明の培養装置に用いる培養漕は、撹拌培養、灌流培養、振とう・ゆらし培養等に適した公知の培養漕を用いることができる。浮遊培養法に用いる培養漕の内壁には、幹細胞の付着を抑制する成分が塗布されうる。
[Culture culture]
As the culture rod used in the culture apparatus of the present invention, a known culture rod suitable for stirring culture, perfusion culture, shaking / shaking culture, or the like can be used. A component that suppresses adherence of stem cells can be applied to the inner wall of the culture tub used in the suspension culture method.
［培養方法］
続いて、本発明に係る幹細胞培養方法について説明する。
上記に説明した幹細胞培養に適する培養液を培養漕に満たし、本発明所定の担体を培養液中に入れる。該担体は、親水性の幹細胞付着性物質でコートされているため、培養槽の底部に沈降しやすい。培養液量3〜20ｍｌに対する担体の個数は、3〜100個が好ましく、6〜50個がより好ましい。
[Culture method]
Subsequently, the stem cell culture method according to the present invention will be described.
The culture solution suitable for the stem cell culture described above is filled in the culture vessel, and the predetermined carrier of the present invention is put into the culture solution. Since the carrier is coated with a hydrophilic stem cell adhesion substance, it tends to settle at the bottom of the culture tank. The number of carriers with respect to 3 to 20 ml of the culture solution is preferably 3 to 100, more preferably 6 to 50.
該培養液に、単一細胞に分散させた幹細胞を播種する。播種される幹細胞は特に限定されないが、本発明は、特にヒト、マウス、ウシ等種々の哺乳動物由来のＥＳ細胞、人工多能性幹細胞（ｉＰＳ細胞）、多能性生殖幹細胞（ｍＧＳ細胞）等に好適である。
上記の幹細胞の本発明の担体に対する播種密度は、5〜100ｃｅｌｌｓ／ｃｍ２が好ましく、20〜50ｃｅｌｌｓ／ｃｍ２がより好ましい。
Stem cells dispersed in single cells are seeded in the culture solution. The stem cells to be seeded are not particularly limited. In the present invention, ES cells derived from various mammals such as humans, mice and cows, induced pluripotent stem cells (iPS cells), pluripotent germ stem cells (mGS cells), etc. It is suitable for.
Seeding density to the carrier of the present invention the stem cells are preferably 5~100cells / cm 2, 20~50cells / cm 2 is more preferable.
培養漕内の培養液を、撹拌、振とう、或いは灌流させることにより、幹細胞を培養液に懸濁させ、かつ培養漕の底部に沈降した担体を浮遊させる。 By stirring, shaking, or perfusing the culture solution in the culture vessel, the stem cells are suspended in the culture solution, and the carrier that has settled at the bottom of the culture vessel is suspended.
本発明に用いられる担体の表面には、播種される幹細胞のサイズに対応して適切な接触面積を提供する高部と低部とが形成される。また、該担体表面には、播種される幹細胞の表面に発現する基底膜成分と親和性を有する幹細胞付着性物質がコートされる。 On the surface of the carrier used in the present invention, a high part and a low part that provide an appropriate contact area corresponding to the size of the stem cells to be seeded are formed. The surface of the carrier is coated with a stem cell adhesion substance having an affinity for a basement membrane component expressed on the surface of the seeded stem cells.
本発明においては担体表面に形成される高部を足場にできるため、担体に幹細胞を付着させやすい。撹拌等させた培養液中で該担体表面に接触した幹細胞は、担体の高部を足場として担体表面に付着する。さらに幹細胞付着性物質と幹細胞の基底膜成分とが結合する。 In the present invention, since the high part formed on the surface of the carrier can be used as a scaffold, stem cells can be easily attached to the carrier. Stem cells that have contacted the surface of the carrier in the agitated culture solution adhere to the surface of the carrier using the high part of the carrier as a scaffold. Further, the stem cell adhesion substance and the basement membrane component of the stem cell are combined.
その結果、本発明は幹細胞が担体表面に良好に付着させることができ、撹拌等により生じるせん断力によって担体からの幹細胞の剥離を抑制することができる。すなわち本発明は幹細胞のロスを低減し、効率的に幹細胞培養を行うことができる。本発明の培養方法における幹細胞の上記担体に対する接着効率は、50〜100％である。 As a result, according to the present invention, the stem cells can adhere well to the surface of the carrier, and the peeling of the stem cells from the carrier can be suppressed by the shearing force generated by stirring or the like. That is, the present invention can reduce stem cell loss and efficiently perform stem cell culture. The adhesion efficiency of stem cells to the carrier in the culture method of the present invention is 50 to 100%.
本発明においては、幹細胞を担体表面の幹細胞付着性物質の存在領域に分散状態で付着させる。本発明において分散状態とは、担体表面の幹細胞付着性物質コート領域に、幹細胞を単一細胞として個々に付着させた状態を意味し、さらに各幹細胞が明確なコロニーを形成しない程度に互いの接触領域が少ない状態で付着させることを包含する。したがって本発明により培養される幹細胞は細胞塊になりにくく未分化状態を維持しやすい。 In the present invention, the stem cells are attached in a dispersed state to the existing region of the stem cell adhesion substance on the surface of the carrier. In the present invention, the dispersed state means a state in which the stem cells are individually attached as single cells to the stem cell adhesion substance coat region on the surface of the carrier, and further contact with each other to the extent that each stem cell does not form a clear colony. It includes depositing with less area. Therefore, the stem cells cultured according to the present invention are unlikely to become a cell mass and easily maintain an undifferentiated state.
上記の方法により、コンフルエントになるまで担体上で幹細胞の培養を続ける。培養条件は、従来公知の浮遊培養条件を適用することができる。例えば、本発明の撹拌培養装置でマウスＥＳ細胞を培養する場合、温度条件35〜37℃、2〜5％ＣＯ２の環境下で行うことができる。 Stem cell culture is continued on the carrier until confluent by the above method. Conventionally known suspension culture conditions can be applied to the culture conditions. For example, when culturing mouse ES cells with the stirring culture apparatus of the present invention, it can be carried out in an environment of temperature conditions of 35 to 37 ° C. and 2 to 5% CO 2 .
培養液の撹拌条件は、培養液中に本発明所定の担体を浮遊させ得ることが求められる。撹拌が弱すぎると担体を浮遊させることが困難になる他、培養液への酸素導入が不十分になる問題がある。しかし撹拌が強すぎると、培養液のせん断力が大きくなるため、幹細胞が担体から剥離しやすくなる。 The stirring conditions for the culture solution are required to allow the carrier of the present invention to float in the culture solution. If the agitation is too weak, it is difficult to float the carrier, and oxygen introduction into the culture solution is insufficient. However, if the agitation is too strong, the shear force of the culture medium increases, and the stem cells are easily detached from the carrier.
従って本発明においては、担体を浮遊させ得る撹拌条件であって、かつ酸素導入およびせん断力抑制の観点から好ましい撹拌条件が選択される。撹拌培養や振とう培養を行う場合、好ましい振とう数は60〜80ｒｐｍであり、65〜75ｒｐｍがより好ましい。 Therefore, in the present invention, stirring conditions that can float the carrier and preferable stirring conditions are selected from the viewpoint of introducing oxygen and suppressing shear force. When stirring culture or shaking culture is performed, the preferable number of shaking is 60 to 80 rpm, and 65 to 75 rpm is more preferable.
本発明を用いて培養された幹細胞の未分化率は、85〜90％程度であると推察される。かかる未分化率は、カドヘリンをコートさせた担体を用いて分散状態で幹細胞を培養させた場合の、公知の未分化率である。幹細胞を分散状態で培養させる場合、幹細胞同士の接触領域が少ない。そのため幹細胞がコロニーを形成しにくい。従って、担体に付着させた全ての幹細胞に十分に分化抑制剤を作用させることができる。その結果、幹細胞の未分化状態を維持させることができる。 It is estimated that the undifferentiated rate of stem cells cultured using the present invention is about 85 to 90%. Such an undifferentiation rate is a known undifferentiation rate when stem cells are cultured in a dispersed state using a carrier coated with cadherin. When culturing stem cells in a dispersed state, the contact area between the stem cells is small. Therefore, it is difficult for stem cells to form colonies. Therefore, the differentiation inhibitor can sufficiently act on all the stem cells attached to the carrier. As a result, the undifferentiated state of the stem cell can be maintained.
例えば特許第4688793号には、幹細胞の未分化状態が維持される例として、分化抑制剤（LIF）の濃度が100Ｕ/ｍＬの場合に、細胞未分化率約90％で培養しうることが開示されている。ただし、特許第4688793号で用いる担体は、表面に所定の高低が形成されていない点で本発明と異なる。 For example, Japanese Patent No. 4687993 discloses that, as an example of maintaining the undifferentiated state of stem cells, when the concentration of the differentiation inhibitor (LIF) is 100 U / mL, it can be cultured at a cell undifferentiation rate of about 90%. Has been. However, the carrier used in Japanese Patent No. 4687793 differs from the present invention in that a predetermined height is not formed on the surface.
本発明は、その表面に所定の高低を有する担体を用いることにより、幹細胞を良好に付着させることができる。従って、静置培養法だけでなく、浮遊培養法でせん断力により幹細胞が担体から剥離しやすい条件下においても、未分化状態を維持した幹細胞を培養することができる。 In the present invention, a stem cell can be favorably adhered by using a carrier having a predetermined height on the surface thereof. Therefore, stem cells that have maintained an undifferentiated state can be cultured not only in stationary culture methods but also in conditions in which stem cells are easily detached from the carrier due to shearing force in suspension culture methods.
培養細胞の未分化状態の確認は、後に詳説するＥＳ細胞の種類に対応するＡＬＰ活性や特異的マーカー遺伝子の発現量を測定することにより行うことができる。上記特異的マーカーとしては、Ｏｃｔ−３／４やＲｅｘ１／Ｚｆｐ４２等の遺伝子産物の発現を利用することができる。 Confirmation of the undifferentiated state of the cultured cells can be performed by measuring the ALP activity corresponding to the type of ES cell, which will be described in detail later, and the expression level of a specific marker gene. As the specific marker, expression of gene products such as Oct-3 / 4 and Rex1 / Zfp42 can be used.
ＡＬＰ活性の検出の一例は、培養したＥＳ細胞をＰＢＳで洗浄後、66％アセトン／3％ホルマリンを含むクエン酸溶液で固定し、ＰＢＳで洗浄後、ｎａｐｈｔｈｏｌ ＡＳ−ＢＩ ｐｈｏｓｐｈａｔｅ ａｌｋａｌｉｎｅ染色液で15分間処理し、発色反応させることにより行われる。 An example of detection of ALP activity is that cultured ES cells are washed with PBS, fixed with a citric acid solution containing 66% acetone / 3% formalin, washed with PBS, and washed with naphthol AS-BI phosphate alkali for 15 minutes. It is carried out by processing and color reaction.
また、抗原分子を未分化マーカーとして利用してもよい。各種抗原分子の低減、消失によってもＥＳ細胞の分化を確認することができるからである。そのような抗原分子の例として、マウスＥＳ細胞の場合は、ＳＳＥＡ−１、ヒトＥＳ細胞の場合は、ＳＳＥＡ−３、ＳＳＥＡ−４、ＴＲＡ−１−６０、ＴＲＡ−１−８１、ＧＣＴＭ−２等がある。 Moreover, you may utilize an antigen molecule as an undifferentiation marker. This is because ES cell differentiation can also be confirmed by the reduction or disappearance of various antigen molecules. Examples of such antigen molecules include SSEA-1 for mouse ES cells, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, GCTM-2 for human ES cells. Etc.
未分化マーカーの利用例として以下の方法がある。まず、培養されたＥＳ細胞からＲＮＡを調整し、常法に従ってｃＤＮＡを合成する。該ｃＤＮＡを鋳型としてＰＣＲにより、所定の未分化マーカーの遺伝子の断片を増幅させる。得られたＰＣＲ産物を染色し検出する。未分化マーカーが有意に検出されることにより、培養されたＥＳ細胞が未分化であることを確認できる。 The following method is an example of using an undifferentiated marker. First, RNA is prepared from cultured ES cells, and cDNA is synthesized according to a conventional method. A predetermined undifferentiated marker gene fragment is amplified by PCR using the cDNA as a template. The resulting PCR product is stained and detected. When the undifferentiation marker is detected significantly, it can be confirmed that the cultured ES cells are undifferentiated.
培養液がコンフルエントになった場合、本発明は常法により幹細胞を継代させることができる。継代は少なくとも3〜10継代まで行い得る。 When the culture solution becomes confluent, the present invention allows passage of stem cells by a conventional method. Passaging can be performed up to at least 3 to 10 passages.
培養した幹細胞を回収する場合、培養液中から幹細胞を分散状態で付着させた担体を回収し、常法により担体から単一細胞を得る。培養液の撹拌等を停止させると担体は３〜４時間で自然に培養槽の底部に沈降する。したがって、本発明において担体の回収は、該沈降した担体を培養液から取り出すことにより、極めて簡単に行うことができる。 When recovering the cultured stem cells, a carrier on which the stem cells are attached in a dispersed state is recovered from the culture solution, and single cells are obtained from the carrier by a conventional method. When the stirring of the culture solution is stopped, the carrier naturally settles to the bottom of the culture tank in 3 to 4 hours. Therefore, in the present invention, recovery of the carrier can be performed very easily by taking out the precipitated carrier from the culture solution.
単一細胞は、常法に従って得ることができる。一例として、タンパク質分解酵素溶液を用いる方法がある。この方法においては、まず培養幹細胞が分散状態で付着する担体を洗浄し、洗浄後の担体に該トリプシン等のタンパク質分解酵素溶液を加え、35〜37℃の温度条件で3〜10分間培養する。続いて該幹細胞を本発明で用いる上記培養液に懸濁させて、単一細胞を得る。 Single cells can be obtained according to conventional methods. As an example, there is a method using a proteolytic enzyme solution. In this method, first, a carrier to which cultured stem cells adhere in a dispersed state is washed, and a protease solution such as trypsin is added to the washed carrier, followed by culturing at a temperature of 35 to 37 ° C. for 3 to 10 minutes. Subsequently, the stem cells are suspended in the culture medium used in the present invention to obtain single cells.
他の回収方法の例としては、エチレンジアミン四酢酸（ＥＤＴＡ）溶液を用いる方法がある。この方法においては、上記方法と同様にして担体を洗浄後、ＥＤＴＡ溶液を濃度が0.01〜100ｍＭ程度になるように添加し、35〜37℃の温度条件で１〜60分間処理して担体から幹細胞を剥離させ、上記本発明で用いる培養液に懸濁させて単一細胞を得る。 An example of another recovery method is a method using an ethylenediaminetetraacetic acid (EDTA) solution. In this method, after washing the carrier in the same manner as in the above method, an EDTA solution is added to a concentration of about 0.01 to 100 mM and treated at a temperature of 35 to 37 ° C. for 1 to 60 minutes. Is detached and suspended in the culture medium used in the present invention to obtain single cells.
以下に実施例を挙げて本発明をさらに説明する。ただし本発明は下記の実施例に限定されるものではない。 The following examples further illustrate the present invention. However, the present invention is not limited to the following examples.
［実施例１］
平均粒子径0.5〜2μｍのガラス粒子を含有させたガラス製の圧接材（8ｃｍ２）２枚を150℃に加熱した。直径0.3ｃｍのポリスチレンペレットを、上記圧接材２枚の粒子露出面に接触するようにして挟み、最大375ｋＰａで徐々に加圧して薄く延ばした。直径2.5ｃｍの表面粗さ＃14000のポリスチレンフィルムを作製した。
得られた表面粗さ＃14000のポリスチレンフィルムをパンチングして、外径6ｍｍのディスク状担体を30個作製した。表面粗さ＃14000の担体表面１ｍｍ２に存在する低部数を目視で数え、該低部数を担体表面積1000000ｍ２あたりの低部数に換算したところ、該低部数は、2000000個であった。
[Example 1]
Two glass pressure welding materials (8 cm 2 ) containing glass particles having an average particle diameter of 0.5 to 2 μm were heated to 150 ° C. A polystyrene pellet having a diameter of 0.3 cm was sandwiched so as to be in contact with the two exposed surfaces of the pressure contact material, and was gradually pressed at a maximum of 375 kPa to be thinly extended. A polystyrene film having a surface roughness # 14000 of 2.5 cm in diameter was produced.
The obtained polystyrene film having a surface roughness of # 14000 was punched to produce 30 disk-shaped carriers having an outer diameter of 6 mm. When the number of low parts present on 1 mm 2 of the carrier surface with surface roughness # 14000 was visually counted and the low parts were converted into low parts per 1 million m 2 of the surface area of the carrier, the number of low parts was 2000000.
Ｅ−カドヘリンをＰＢＳで10μｇ／ｍＬに希釈し、ろ過、滅菌した溶液を調製した。該Ｅ−カドヘリン溶液に得られたディスク状担体を浸漬させ、室温35℃〜37℃で約30〜60分間静置させ、担体基材にＥ−カドヘリンをコートさせ、本発明の実施例１に用いる担体を得た。 E-cadherin was diluted to 10 μg / mL with PBS, and a filtered and sterilized solution was prepared. The obtained disk-shaped carrier was immersed in the E-cadherin solution and allowed to stand at room temperature of 35 ° C. to 37 ° C. for about 30 to 60 minutes to coat the carrier substrate with E-cadherin. The carrier used was obtained.
0.1％ウシ血清由来アルブミン（ＢＳＡ）溶液に30分以上浸漬させて該ＢＳＡコート処理した24Ｗｅｌｌのプレートに、Ｌ−グルタミン、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からなる培地１ｍＬを添加し、上記担体を2個、該24Ｗｅｌｌプレートに入れた。さらに該プレートに、マウスＥＳ細胞を3000細胞播種した。 Add 1 mL of a medium consisting of L-glutamine, insulin, transferrin, ethanolamine, selenium, 2-mercaptoethanol to a 24-well plate immersed in 0.1% bovine serum-derived albumin (BSA) for 30 minutes or longer and treated with BSA. Two of the above carriers were placed in the 24 Well plate. Further, 3000 cells of mouse ES cells were seeded on the plate.
上記プレートを振とう機（アズワン株式会社製 ミニウェーブＷＥＶ-03）のインキュベータ内に載置し、37℃、2％ＣＯ２条件下で振とう回転数70ｒｐｍで振とうさせ、幹細胞の培養を開始した。 Place the above plate in the incubator of a shaker (Mini Wave WEV-03, manufactured by AS ONE Corporation), shake at 37 ° C and 2% CO 2 at a rotation speed of 70 rpm, and start stem cell culture. did.
常法に従って継代操作を行いながら培養を続け、３日後、振とうを停止させて培養を終了した。振とうの終了により、プレート内の担体は自然に沈降した。該担体をプレート内から取り出して洗浄し、担体に付着する幹細胞を回収した。幹細胞の回収は、プレートから取り出した担体にＡｃｃｕｔａｓｅを添加し、37℃で10分間程度インキュベートした後、上記培養液に懸濁させて単一細胞とすることにより行った。 Cultivation was continued while performing subculture operations according to a conventional method, and after 3 days, shaking was stopped and the cultivation was terminated. Upon completion of the shaking, the carrier in the plate naturally settled. The carrier was removed from the plate and washed, and stem cells adhering to the carrier were collected. Stem cells were collected by adding Accutase to the carrier removed from the plate, incubating at 37 ° C. for about 10 minutes, and then suspending in the culture medium to form single cells.
［実施例２、３および比較例１〜５］
用いる担体を表１に示す表面粗さのものに変えた他は、上記実施例１と同条件にして幹細胞培養を行い、実施例２、３および比較例１〜４とした。比較例５は、Ｅ−カドヘリンをコートさせた平坦な付着面からなる培養漕を用いて静置培養した比較例である。下記に説明する方法により実施例１〜３および比較例１〜５により得られた幹細胞数を測定した。測定結果を表１および図５に示す。
[Examples 2 and 3 and Comparative Examples 1 to 5]
Except that the carrier used was changed to one having the surface roughness shown in Table 1, stem cell culture was carried out under the same conditions as in Example 1 above to give Examples 2, 3 and Comparative Examples 1-4. Comparative Example 5 is a comparative example in which static culture was performed using a culture rod having a flat adhesion surface coated with E-cadherin. The number of stem cells obtained in Examples 1 to 3 and Comparative Examples 1 to 5 was measured by the method described below. The measurement results are shown in Table 1 and FIG.
表１および図５によれば、実施例１は、静置培養法を行った比較例５より多くの細胞を培養することができた。比較例１〜４は、培養液の撹拌中に細胞が剥離し、培養細胞を効率的に回収することができなかった。 According to Table 1 and FIG. 5, Example 1 was able to culture more cells than Comparative Example 5 in which static culture was performed. In Comparative Examples 1 to 4, the cells were detached during the stirring of the culture solution, and the cultured cells could not be efficiently collected.
すなわち、本発明は、所定の高低を有する担体を用いることにより、幹細胞と担体との付着性が良好で、浮遊培養法で培養液に生じるせん断力に対抗しうる非剥離性を発揮する。上記担体表面に形成される高部の頂点間の距離は幹細胞直径よりやや小さいため、低部で細胞塊が形成されることがない。また幹細胞を担体表面に分散状態で生育しやすい。 That is, according to the present invention, by using a carrier having a predetermined height, the adherence between the stem cell and the carrier is good, and the non-peeling property that can counter the shear force generated in the culture medium by the suspension culture method is exhibited. Since the distance between the apexes of the high part formed on the surface of the carrier is slightly smaller than the stem cell diameter, no cell mass is formed in the low part. In addition, stem cells are likely to grow in a dispersed state on the carrier surface.
［細胞数の測定方法］
細胞数の測定は、Ａｃｃｕｔａｓｅで分散した細胞を回収し、２４Ｗｅｌｌプレートにいれ、１５〜３０分静置し、細胞の沈降を待った。細胞が底部に沈降したら、顕微鏡で写真撮影を3枚異なる視野で行い、細胞数の平均値を計数した。総細胞数は
計数平均値×２４Ｗｅｌｌプレート１Ｗｅｌｌの面積／視野面積
で算出した。
[Method for measuring cell number]
For the measurement of the number of cells, the cells dispersed with Accutase were collected, placed in a 24 well plate, allowed to stand for 15 to 30 minutes, and the cells were allowed to settle. When the cells settled to the bottom, three photographs were taken with a microscope and the average number of cells was counted. The total number of cells was calculated by (count average value × 24 well plate 1 well area / field of view area).
１、２、３ 幹細胞培養装置
４ 担体
５ 培養液
６ 培養漕
７ 高部
８ 低部
Ｐ１、Ｐ２、Ｐ３ 担体表面に形成される高部の頂点
Ｂ１、Ｂ２、Ｂ３ 担体表面に形成される低部の最低点
ｄ１ Ｐ１とＰ２との頂点間距離
ｄ２ Ｐ２とＰ３との頂点間距離
ｇ１ Ｐ１とＢ１との高低差
ｇ２ Ｐ２とＢ２との高低差
ｇ３ Ｐ３とＢ３との高低差
Ｃ 幹細胞
1, 2, 3 Stem cell culturing apparatus 4 Carrier 5 Culture solution 6 Culture basket 7 High part 8 Low part P1, P2, P3 High apex B1, B2, B3 formed on the carrier surface Low part formed on the carrier surface The lowest point d1 between the vertices of P1 and P2, the distance between the vertices d1 between P2 and P3, the difference in height between P1 and B1, the difference in height between P2 and B2, and the difference in height between P2 and B3.
Claims (14)
前記担体表面の前記高部と前記低部との両方に幹細胞付着性物質を介して分散状態で付着させた幹細胞を、該担体表面から剥離させず、かつ未分化状態を維持させて増殖させる工程とを含む幹細胞培養方法。 The culture solution was seeded with stem cells, on the surface thereof, a plurality of high and low which is formed by a plurality of higher part and lower part, and Ri Na by coating the stem cell adhesive substances, the average height of the surface Floating a carrier whose difference is smaller than the diameter of the stem cell ;
A step of proliferating stem cells attached in a dispersed state to both the high part and the low part of the carrier surface via a stem cell adhesion substance while maintaining the undifferentiated state without detaching from the carrier surface. A stem cell culture method comprising:
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2013060669A JP6054223B2 (en) | 2013-03-22 | 2013-03-22 | Stem cell culture apparatus and stem cell culture method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2013060669A JP6054223B2 (en) | 2013-03-22 | 2013-03-22 | Stem cell culture apparatus and stem cell culture method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2014183770A JP2014183770A (en) | 2014-10-02 |
| JP6054223B2 true JP6054223B2 (en) | 2016-12-27 |
Family
ID=51831986
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2013060669A Active JP6054223B2 (en) | 2013-03-22 | 2013-03-22 | Stem cell culture apparatus and stem cell culture method |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP6054223B2 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP6345826B2 (en) * | 2016-03-10 | 2018-06-20 | 株式会社金太郎ＣｅｌｌｓＰｏｗｅｒ | Production method of culture product |
| EP3766958B1 (en) * | 2018-03-12 | 2022-11-09 | Kyushu University, National University Corporation | Culture substrate, method for manufacturing culture substrate, and culturing method and culturing device for stem cell |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SG150567A1 (en) * | 2004-03-23 | 2009-03-30 | Akaike Toshihiro | Pluripotent stem cell growing method |
| US20120156777A1 (en) * | 2010-12-16 | 2012-06-21 | General Electric Company | Cell carrier, associated methods for making cell carrier and culturing cells using the same |
-
2013
- 2013-03-22 JP JP2013060669A patent/JP6054223B2/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| JP2014183770A (en) | 2014-10-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102307991B (en) | Multipotential stem cell culture on microcarrier | |
| US8802431B2 (en) | Population of multipotent cardiac precursor cells derived from human blastocysts derived stem cells | |
| Marinho et al. | Xeno-free production of human embryonic stem cells in stirred microcarrier systems using a novel animal/human-component-free medium | |
| US9534206B2 (en) | Cell carrier, associated methods for making cell carrier and culturing cells using the same | |
| JP5252411B2 (en) | Cell culture carrier and cell culture method | |
| US9957478B2 (en) | Cell carrier, associated methods for making cell carrier and culturing cells using the same | |
| JP2009538617A (en) | Method for culturing human embryonic stem cells | |
| JP6247932B2 (en) | Cell culture equipment | |
| JP2017526378A (en) | Three-dimensional cell culture system and cell culture method using the same | |
| JP6054223B2 (en) | Stem cell culture apparatus and stem cell culture method | |
| WO2011016485A1 (en) | METHOD FOR INDUCING DIFFERENTIATION OF iPS CELLS INTO HEPATIC PARENCHYMAL CELLS | |
| US9428728B2 (en) | Carrier for undifferentiated cell culture and subculture method thereof | |
| JP6616559B2 (en) | Method for producing sheet cell culture | |
| WO2023037544A1 (en) | Method for producing pluripotent stem cells | |
| JP2019080561A (en) | Porous substrate for culturing pluripotent stem cells and method for culturing pluripotent stem cells while maintaining undifferentiation | |
| WO2023208112A1 (en) | Animal origin-free cell sheet, preparation method therefor, and use thereof | |
| JP2023135601A (en) | Cell collection method and cell collection device | |
| JP2014143970A (en) | Method of producing sheet-like cell culture | |
| Sarwar et al. | Adhesion strength of various mammalian cell lines, Part II: Effect of polystyrene, glass and ageing on various mammalian cell lines | |
| JP2015126720A (en) | Cell culture method and cell culture apparatus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20151030 |
|
| A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20160629 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20160726 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20160923 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20161108 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20161130 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 6054223 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |