CN115466717A - 一种非人灵长类胚胎体外培养试剂盒和体外培养方法 - Google Patents
一种非人灵长类胚胎体外培养试剂盒和体外培养方法 Download PDFInfo
- Publication number
- CN115466717A CN115466717A CN202211148926.6A CN202211148926A CN115466717A CN 115466717 A CN115466717 A CN 115466717A CN 202211148926 A CN202211148926 A CN 202211148926A CN 115466717 A CN115466717 A CN 115466717A
- Authority
- CN
- China
- Prior art keywords
- vitro culture
- human primate
- embryo
- culture
- vitro
- 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.)
- Pending
Links
- 238000000338 in vitro Methods 0.000 title claims abstract description 76
- 238000012136 culture method Methods 0.000 title claims abstract description 17
- 210000002257 embryonic structure Anatomy 0.000 title claims description 45
- 210000001161 mammalian embryo Anatomy 0.000 claims abstract description 59
- VOXZDWNPVJITMN-ZBRFXRBCSA-N 17β-estradiol Chemical compound OC1=CC=C2[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 VOXZDWNPVJITMN-ZBRFXRBCSA-N 0.000 claims abstract description 38
- RJKFOVLPORLFTN-LEKSSAKUSA-N Progesterone Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H](C(=O)C)[C@@]1(C)CC2 RJKFOVLPORLFTN-LEKSSAKUSA-N 0.000 claims abstract description 38
- 241000282567 Macaca fascicularis Species 0.000 claims abstract description 31
- 108010082117 matrigel Proteins 0.000 claims abstract description 31
- 239000001963 growth medium Substances 0.000 claims abstract description 26
- 229960005309 estradiol Drugs 0.000 claims abstract description 21
- PWKSKIMOESPYIA-BYPYZUCNSA-N L-N-acetyl-Cysteine Chemical compound CC(=O)N[C@@H](CS)C(O)=O PWKSKIMOESPYIA-BYPYZUCNSA-N 0.000 claims abstract description 19
- 229960003387 progesterone Drugs 0.000 claims abstract description 19
- 239000000186 progesterone Substances 0.000 claims abstract description 19
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 claims abstract description 18
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims abstract description 16
- 239000000203 mixture Substances 0.000 claims abstract description 16
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 claims abstract description 15
- 229930182816 L-glutamine Natural products 0.000 claims abstract description 15
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 13
- 238000000034 method Methods 0.000 claims abstract description 12
- 229960002743 glutamine Drugs 0.000 claims abstract description 9
- 229960005322 streptomycin Drugs 0.000 claims abstract description 9
- 229930182555 Penicillin Natural products 0.000 claims abstract description 8
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 claims abstract description 8
- 229940049954 penicillin Drugs 0.000 claims abstract description 8
- 210000002459 blastocyst Anatomy 0.000 claims description 20
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 16
- 239000008103 glucose Substances 0.000 claims description 16
- 239000002609 medium Substances 0.000 claims description 12
- 239000011159 matrix material Substances 0.000 claims description 9
- 230000008859 change Effects 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 6
- 230000004720 fertilization Effects 0.000 claims description 6
- -1 koSR Substances 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
- 239000012530 fluid Substances 0.000 claims description 4
- 238000011161 development Methods 0.000 abstract description 19
- 230000008569 process Effects 0.000 abstract description 5
- 210000004291 uterus Anatomy 0.000 abstract description 4
- 230000009286 beneficial effect Effects 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 21
- 230000018109 developmental process Effects 0.000 description 19
- 239000012091 fetal bovine serum Substances 0.000 description 14
- 210000004027 cell Anatomy 0.000 description 12
- 238000002513 implantation Methods 0.000 description 12
- 241000699666 Mus <mouse, genus> Species 0.000 description 9
- 239000007788 liquid Substances 0.000 description 8
- 101000687905 Homo sapiens Transcription factor SOX-2 Proteins 0.000 description 7
- 241000699670 Mus sp. Species 0.000 description 7
- 102100024270 Transcription factor SOX-2 Human genes 0.000 description 7
- 230000013020 embryo development Effects 0.000 description 7
- 238000007789 sealing Methods 0.000 description 7
- 241000288906 Primates Species 0.000 description 6
- 238000010586 diagram Methods 0.000 description 6
- 230000002357 endometrial effect Effects 0.000 description 6
- 238000001727 in vivo Methods 0.000 description 6
- 210000002966 serum Anatomy 0.000 description 6
- 230000012010 growth Effects 0.000 description 5
- 229940088597 hormone Drugs 0.000 description 5
- 239000005556 hormone Substances 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 241000283690 Bos taurus Species 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 238000007865 diluting Methods 0.000 description 4
- 229930182833 estradiol Natural products 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 210000000056 organ Anatomy 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 description 4
- 238000010257 thawing Methods 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 210000004340 zona pellucida Anatomy 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 108010003272 Hyaluronate lyase Proteins 0.000 description 3
- 102000001974 Hyaluronidases Human genes 0.000 description 3
- 102000008730 Nestin Human genes 0.000 description 3
- 108010088225 Nestin Proteins 0.000 description 3
- 102100035423 POU domain, class 5, transcription factor 1 Human genes 0.000 description 3
- 101710126211 POU domain, class 5, transcription factor 1 Proteins 0.000 description 3
- 229930040373 Paraformaldehyde Natural products 0.000 description 3
- 229920001213 Polysorbate 20 Polymers 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 238000004140 cleaning Methods 0.000 description 3
- 230000008143 early embryonic development Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 229960002773 hyaluronidase Drugs 0.000 description 3
- 238000010166 immunofluorescence Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 210000005055 nestin Anatomy 0.000 description 3
- 229920002866 paraformaldehyde Polymers 0.000 description 3
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 3
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 3
- 238000011282 treatment Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 210000001325 yolk sac Anatomy 0.000 description 3
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 description 2
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 2
- CYDQOEWLBCCFJZ-UHFFFAOYSA-N 4-(4-fluorophenyl)oxane-4-carboxylic acid Chemical compound C=1C=C(F)C=CC=1C1(C(=O)O)CCOCC1 CYDQOEWLBCCFJZ-UHFFFAOYSA-N 0.000 description 2
- IDDDVXIUIXWAGJ-DDSAHXNVSA-N 4-[(1r)-1-aminoethyl]-n-pyridin-4-ylcyclohexane-1-carboxamide;dihydrochloride Chemical compound Cl.Cl.C1CC([C@H](N)C)CCC1C(=O)NC1=CC=NC=C1 IDDDVXIUIXWAGJ-DDSAHXNVSA-N 0.000 description 2
- 102100030942 Apolipoprotein A-II Human genes 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 101100298998 Caenorhabditis elegans pbs-3 gene Proteins 0.000 description 2
- 108010062540 Chorionic Gonadotropin Proteins 0.000 description 2
- 102000011022 Chorionic Gonadotropin Human genes 0.000 description 2
- 208000032170 Congenital Abnormalities Diseases 0.000 description 2
- 206010010356 Congenital anomaly Diseases 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 101000793406 Homo sapiens Apolipoprotein A-II Proteins 0.000 description 2
- 241000218204 Nigella Species 0.000 description 2
- 235000007413 Nigella arvensis Nutrition 0.000 description 2
- 235000016698 Nigella sativa Nutrition 0.000 description 2
- 102100024616 Platelet endothelial cell adhesion molecule Human genes 0.000 description 2
- 206010042573 Superovulation Diseases 0.000 description 2
- 239000013504 Triton X-100 Substances 0.000 description 2
- 229920004890 Triton X-100 Polymers 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000007698 birth defect Effects 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000011010 flushing procedure Methods 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 2
- 239000000017 hydrogel Substances 0.000 description 2
- 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 2
- 239000003550 marker Substances 0.000 description 2
- 230000008774 maternal effect Effects 0.000 description 2
- 210000004379 membrane Anatomy 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 210000000754 myometrium Anatomy 0.000 description 2
- 210000005036 nerve Anatomy 0.000 description 2
- 210000003061 neural cell Anatomy 0.000 description 2
- 210000001020 neural plate Anatomy 0.000 description 2
- 210000002569 neuron Anatomy 0.000 description 2
- 239000011664 nicotinic acid Substances 0.000 description 2
- 230000035935 pregnancy Effects 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 230000008844 regulatory mechanism Effects 0.000 description 2
- 239000001540 sodium lactate Substances 0.000 description 2
- 229940005581 sodium lactate Drugs 0.000 description 2
- 235000011088 sodium lactate Nutrition 0.000 description 2
- 229940054269 sodium pyruvate Drugs 0.000 description 2
- 230000003068 static effect Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000002381 testicular Effects 0.000 description 2
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 101100468640 Danio rerio rhcgl2 gene Proteins 0.000 description 1
- 208000012239 Developmental disease Diseases 0.000 description 1
- 241000283073 Equus caballus Species 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 208000012902 Nervous system disease Diseases 0.000 description 1
- 208000025966 Neurological disease Diseases 0.000 description 1
- 108010072194 Ovidrel Proteins 0.000 description 1
- 239000005662 Paraffin oil Substances 0.000 description 1
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 description 1
- 102000004338 Transferrin Human genes 0.000 description 1
- 108090000901 Transferrin Proteins 0.000 description 1
- 206010000210 abortion Diseases 0.000 description 1
- 231100000176 abortion Toxicity 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000019552 anatomical structure morphogenesis Effects 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 210000000625 blastula Anatomy 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 229940015047 chorionic gonadotropin Drugs 0.000 description 1
- 230000001143 conditioned effect Effects 0.000 description 1
- 239000006059 cover glass Substances 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 210000003981 ectoderm Anatomy 0.000 description 1
- 230000032692 embryo implantation Effects 0.000 description 1
- 210000001900 endoderm Anatomy 0.000 description 1
- 230000037149 energy metabolism Effects 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- 235000020774 essential nutrients Nutrition 0.000 description 1
- 230000012173 estrus Effects 0.000 description 1
- 239000003172 expectorant agent Substances 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 108010006578 follitropin alfa Proteins 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000007045 gastrulation Effects 0.000 description 1
- 229940057854 gonal f Drugs 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 210000004524 haematopoietic cell Anatomy 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000011132 hemopoiesis Effects 0.000 description 1
- 229940084986 human chorionic gonadotropin Drugs 0.000 description 1
- 238000003125 immunofluorescent labeling Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000036244 malformation Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 210000003716 mesoderm Anatomy 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 229940066491 mucolytics Drugs 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000005305 organ development Effects 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- 210000002220 organoid Anatomy 0.000 description 1
- 239000002357 osmotic agent Substances 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 229940112876 ovidrel Drugs 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 230000000270 postfertilization Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 101150053759 rhcg gene Proteins 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 229910052711 selenium Inorganic materials 0.000 description 1
- 239000011669 selenium Substances 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 230000026799 smooth muscle cell apoptotic process Effects 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000001550 testis Anatomy 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 239000012581 transferrin Substances 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000011277 treatment modality Methods 0.000 description 1
- 239000006216 vaginal suppository Substances 0.000 description 1
- 229940120293 vaginal suppository Drugs 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
Images
Classifications
-
- 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
- C12N5/0603—Embryonic cells ; Embryoid bodies
-
- 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
-
- 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
- C12N2500/32—Amino acids
-
- 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
-
- 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
- C12N2500/35—Polyols, e.g. glycerin, inositol
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/999—Small molecules not provided for elsewhere
-
- 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
- C12N2513/00—3D culture
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Developmental Biology & Embryology (AREA)
- Reproductive Health (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Gynecology & Obstetrics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明涉及胚胎培养技术领域,尤其涉及一种非人灵长类胚胎体外培养试剂盒和体外培养方法。本发明提供一种非人灵长类胚胎体外培养试剂盒,包括第一培养基;所述第一培养基包括独立包装的Matrigel和混合试剂;所述混合试剂为IVC1与Geltrex的混合物,且Geltrex在混合试剂中的含量为9‑11%(V/V);所述IVC1与Geltrex的混合物中IVC1的成分为:DMEM/F12,热灭活FBS,L‑谷氨酰胺,青霉素,链霉素,ITS‑X,β‑雌二醇,孕酮,N‑乙酰‑L‑半胱氨酸。本发明的试剂盒有助于模拟食蟹猴胚胎植入子宫过程,以促进胚胎持续发育。
Description
技术领域
本发明涉及胚胎培养技术领域,尤其涉及一种非人灵长类胚胎体外培养试剂盒和体外培养方法。
背景技术
早期胚胎发育关乎生命健康本源,但人类早期胚胎发育调控机制受限于技术和伦理尚未得到全面解析。高等灵长类(人与非人灵长类)胚胎在发育的第3周和第4周完成了外胚层、中胚层和内胚层三胚层的形成,并形成各种器官的雏形。该发育过程失败和畸形往往会导致早期妊娠流产和出生缺陷。灵长类与人共享众多发育相关保守机制,体外培养研究灵长类早期胚胎能为洞悉人类胚胎发育调控机制和防治出生缺陷及多种发育源性疾病提供重要理论支撑和可行路径。
2014年,英国剑桥大学Magdalena Zernicka-Goetz教授团队开发出一个2D小鼠胚胎体外培养体系,在两种按顺序使用的培养基IVC1(高级DMEM/F12,热灭活FBS,L-谷氨酰胺,青霉素,链霉素,ITS-X,β-雌二醇,孕酮,N-乙酰-L-半胱氨酸)和IVC2(高级DMEM/F12,KoSR,L-谷氨酰胺,青霉素,链霉素,ITS-X,β-雌二醇,孕酮,N-乙酰-L-半胱氨酸)中,允许小鼠胚胎从E3.5发育到卵圆筒阶段(Nature Protocols,2014,9(12):2732–2739)。2016年,Magdalena Zernicka-Goetz和Ali H.Brivanlou等人在此系统基础上将人类胚胎在体外培养到受精后第12天,揭示了人类胚胎早期形态发生关键事件是胚胎自主调控的,母体组织不是必须条件(Nature Cell Biology,2016,18(6):700–708;Nature,2016,533(7602):251–254)。2019年,昆明理工大学李天晴课题组基于Magdalena Zernicka-Goetz等人的研究改进了人类胚胎培养系统:mIVC1(高级DMEM/F12,DFBS,L-谷氨酰胺,ITS-X,β-雌二醇,孕酮,N-乙酰-L-半胱氨酸,乳酸钠,丙酮酸钠,Y27632)和mIVC2(高级DMEM/F12,KOSR,L-谷氨酰胺,ITS-X,β-雌二醇,孕酮,N-乙酰-L-半胱氨酸,乳酸钠,丙酮酸钠,Y27632),通过在mIVC2中加添10%Matrigel为胚胎提供了一个3D生长环境,首次阐述了人胚胎三维条件原肠发育(Nature,2020,577(7791):537–542)。
Magdalena Zernicka-Goetz等人在2014年开发的培养体系只允许小鼠胚胎在体外发育到卵圆筒阶段,2D的体外培养环境和刚性附着面与真实的体内3D柔软子宫环境差异很大,限制了胚胎3D生长潜力。尽管Magdalena Zernicka-Goetz和李天晴等人在2016、2019年分别通过2D和3D体系将人类胚胎在体外培养到原条发生前,但总的来说,这些培养体系下的胚胎发育率都不是很高,仍有巨大提升空间。
在2019年发明人课题组和中科院动物所王红梅课题组同时开发了食蟹猴胚胎体外培养体系之前,学界尚没有合适的非人灵长类胚胎体外培养体系。但在该体系中所有食蟹猴胚胎最长只能在体外生长约20天,反映了该体系的技术局限性。因此,需要进一步优化培养体系,以支持食蟹猴胚胎在原肠发育后继续生长,并达到早期器官阶段。
发明内容
本发明提供一种非人灵长类胚胎体外培养试剂盒和体外培养方法,用以解决现有技术中食蟹猴胚胎体外培养不成熟的缺陷。
本发明提供一种非人灵长类胚胎体外体外培养试剂盒,包括第一培养基;
所述第一培养基包括独立包装的Matrigel和混合试剂;
所述混合试剂为IVC1与Geltrex的混合物,且Geltrex在混合试剂中的含量为9-11%(V/V)。该体外培养基下层为孵育成胶后的Matrigel,类似子宫肌层,上层中的10%Geltrex(V/V)类似柔软的子宫内膜组织,两者有助于模拟食蟹猴胚胎植入子宫过程,以促进胚胎持续发育,大幅改善食蟹猴胚胎在19-20d.p.f后的生长状况。
所述IVC1与Geltrex的混合物中IVC1的成分为:DMEM/F12,热灭活FBS,L-谷氨酰胺,青霉素,链霉素,ITS-X,β-雌二醇,孕酮,N-乙酰-L-半胱氨酸。
优选的,每10mL IVC1含有7.06-7.51mL DMEM/F12、1.8-2.2mL热灭活FBS、90-110uL ITS-X、1.8-2.2mM L-谷氨酰胺、7.2-8.8nMβ-雌二醇、180-220ng/mL孕酮、22.5-27.5uM N-乙酰-L-半胱氨酸。
优选的,所述IVC1中包括PS。
进一步优选的,每10mL IVC1含有40-60IU PS。
根据本发明提供的非人灵长类胚胎体外培养试剂盒,还包括独立包装的MIVC1、MIVC2中的一种或两种;
所述MIVC1包括3.8-4.2%Matrigel(V/V);
所述MIVC2包括3.8-4.2%Matrigel(V/V)。本发明在培养基中添加Matrigel使其稠度增加,更类似于子宫内液体。
根据本发明提供的非人灵长类胚胎体外培养试剂盒,所述MIVC1还包括DMEM/F12、热灭活FBS、青霉素,链霉素、ITS-X、L-glutamine、β-estradiol、Progesterone、N-acetyl-L-cysteine。
和/或,所述MIVC2还包括DMEM/F12、KoSR、青霉素、链霉素、ITS-X、L-glutamine、β-estradiol、Progesterone、N-acetyl-L-cysteine。
优选的,每10mL MIVC1含有7.06-7.51mL DMEM/F12、1.8-2.2mL热灭活FBS、40-60IU PS、90-110uL ITS-X、1.8-2.2mML-glutamine、7.2-8.8nMβ-estradiol、180-220ng/mLProgesterone、22.5-27.5uM N-acetyl-L-cysteine、3.8-4.2%Matrigel(V/V)。
和/或,每10mLMIVC2含有5.06-7.51mLDMEM/F12、2-4mLKoSR、40-60IU PS、90-110uLITS-X、1.8-2.2mM L-glutamine、7.2-8.8nMβ-estradiol、180-220ng/mLProgesterone、22.5-27.5uMN-acetyl-L-cysteine、3.8-4.2%Matrigel(V/V)。
根据本发明提供的非人灵长类胚胎体外试剂盒,还包括独立包装的第二培养基和/或第三培养基;
所述第二培养基含有MIVC2和葡萄糖,所述葡萄糖与MIVC2的比例为2.9-3.1mg/mL(优选3mg/mL);
所述第三培养基为含有MIVC2和葡萄糖,所述葡萄糖与MIVC2的比例为3.4-3.6mg/mL(优选3.5mg/mL)。
本发明还提供一种非人灵长类胚胎体外培养方法,利用上述体外培养试剂盒。
根据本发明提供的非人灵长类胚胎体外培养方法,包括以下步骤:
1)将Matrigel孵育;
2)将IVC1与Geltrex混合均匀得到混合物,将上述混合物铺在步骤1)孵育后的Matrigel上;
3)将囊胚转移至步骤2)中IVC1与Geltrex的混合物中,孵育至包裹囊胚的Geltrex成为果冻样。
根据本发明提供的非人灵长类胚胎的体外培养方法,还包括以下步骤:
4)向步骤3)得到的产物中加入MIVC1;
5)体外培养第2天开始用MIVC2换液。
根据本发明提供的非人灵长类胚胎的体外培养方法,步骤5)中,体外培养第2天到第8天用MIVC2进行换液,第9天到第13天用第二培养基进行换液,第14-19天用第三培养基进行换液。本发明中胚胎培养方式是仿生状态的嵌入模式:利用高浓度的Matrigel原溶液铺板类似子宫肌层,9-11%(尤其是10%)Geltrex类似柔软的子宫内膜组织,两者有助于模拟食蟹猴胚胎植入子宫过程,以促进胚胎持续发育。本发明中胚胎培养基组成成分具有以下优点:培养基中添加4%Matrigel使其稠度增加,更类似于子宫内液体;在胚胎培养过程中,随着发育进程推进,逐步添加葡萄糖可以支持胚胎器官发生过程中的能量需求。
根据本发明提供的非人灵长类胚胎体外培养方法,步骤3)中,所述囊胚为受精后第6-7天的囊胚。
根据本发明提供的非人灵长类胚胎体外培养方法,所述非人灵长类为食蟹猴。
根据本发明提供的非人灵长类胚胎体外培养方法,步骤1)中,将Matrigel在36.5-37.5℃下孵育28-32min,优选37℃下孵育30min。
根据本发明提供的非人灵长类胚胎体外培养方法,步骤4)中,所述MIVC1为预平衡后的MIVC1。
优选的,所述预平衡时间为1h以上,优选1-2h。本发明的预平衡能够中和培养基中的pH值,稳定培养基组分,促进胚胎的发育。
本发明提供了一种非人灵长类植入后胚胎嵌入式-3D体外培养方法,建立了一种较成熟的体外培养体系,使在体外培养的食蟹猴胚胎发育进程能够更接近于体内时期。为研究非人灵长类胚胎的早期发育搭建相关的科研平台。
与已建立的灵长类体外培养体系相比,本发明开发的嵌入式嵌入式-3D体外培养方法,更类似于体内胚胎着床方式。此外本体系可以支持非人灵长类胚胎体外发育至早期器官阶段,与2019年发表的食蟹猴2D培养方式(所有食蟹猴胚胎在19-20d.p.f后均塌陷)比较,延长了胚胎体外存活时间。此外本发明具有如下优点:
1.利用该培养体系可研究早期器官发生的空间信息和分子机制,对类器官研究具有理论指导作用。
2.该体系能够支持灵长类胚胎进行早期神经细胞特化,可为早期胚胎发育源性神经性疾病提供研究平台。
附图说明
为了更清楚地说明本发明或现有技术中的技术方案,下面将对实例或现有技术描述中所需要使用的附图作简单介绍。显而易见地,下面描述中的附图是本发明的一些实例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1是本发明小鼠胚胎体外培养发育图;
图2是本发明嵌入式-3D体外培养方式中食蟹猴胚胎发育图;
图3是本发明神经细胞在EPI位置特化图;
图4是本发明神经板特化图
图5是本发明25d.p.f.卵黄囊造血细胞图;
图6是本发明食蟹猴胚胎嵌入式-3D体外培养体系建立示意图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚,下面将结合本发明中的附图,对本发明中的技术方案进行清楚、完整地描述,显然,所描述的实施例是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
设备:
CO2培养箱,Thermo fisher Scientific,3111
生物安全柜,Thermo fisher Scientific,1389
倒置显微镜,Leica,2010
4℃医用冷藏冰箱,海尔,HYC-360
-20℃冰柜,海尔,HYC-40l262
ibiTreatμ-slide 8-well(80826,Ibidi)
试剂:
rhFSH,Merck Serono,Gonal F:食蟹猴超排。
rhCG,Merck Serono,OVIDREL:食蟹猴超排。
H9+G-2TMPLUS,VitroLife,10132:植入前食蟹猴胚胎培养。
牛睾丸透明质酸酶,Sigma-Aldrich,H3506:去除胚胎透明带。
Matrigel,康宁,354277:基底胶。
Geltrex,Gibco,A1413202:水凝胶。
β-Estradiol,Sigma-Aldrich,E8875:β-雌二醇,子宫内膜容受性调节关键激素,用于模拟围植入期及植入后阶段胚胎所处子宫内膜微环境,促进胚胎发育。
Progesterone,Sigma-Aldrich,P0130:孕酮,子宫内膜容受性调节关键激素,用于模拟围植入期及植入后阶段胚胎所处子宫内膜微环境,促进胚胎发育。
N-acetyl-L-cysteine,Sigma-Aldrich,A7250:N-乙酰-L-半胱氨酸,一种含巯基的抗氧化剂和粘液溶解剂,能增加细胞库的自由基捕获。据报道,有防止神经细胞凋亡,诱导平滑肌细胞凋亡,抑制HIV复制作用。
Fetal bovine serum(FBS),即胎牛血清,Corning,35-076-CV:富含血浆蛋白、多肽、生长因子、激素等细胞生长必须营养成份和生物活性物质。
KoSR,即Knock Out血清替代物,Thermo Fisher,10828028:血清替代物,化学成分明确,性能相比FBS更加稳定可靠,用于代替血清添加在DMEM/F12中用于植入后胚胎培养。
Penicillin/streptomycin(PS),青霉素/链霉素,Thermo Fisher Scientific,15070-063:抗生素,抑制细菌增殖。
ITS-X,胰岛素-转铁蛋白-硒-乙醇胺添加剂,Thermo fisher Scientific,51500-056:胰岛素、转铁蛋白、硒、乙醇胺混合添物,广泛用于哺乳动物无血清细胞培养,对细胞能量代谢、抗氧化、铁离子运输、氧分压调节、细胞器组成具有十分重要的作用,能进一步降低细胞常规维持和低密度生长所需胎牛血清浓度。
L-glutamine,L-谷氨酰胺,Thermo fisher Scientific,25030:为细胞提供能量和氮源,作为有机渗透剂调节细胞对高渗透压环境的适应性。
DMEM/F12,Thermo fisher Scientific,11320-033:一种广泛应用的细胞培养基础培养液,含有无机盐、氨基酸、维生素、高浓度葡萄糖等多种营养成分。
葡萄糖,Sigma-Aldrich,G7021:供能。
小分子稀释
80μM雌二醇配置。称取0.0218g雌二醇溶于10mL无水乙醇中,制备8mM雌二醇溶液,用20um滤器过滤去除细菌。吸取10uL 8mM雌二醇溶液与990uL无水乙醇混匀,10uL/管分装,-20℃下保存,避免反复冻融。
200ug/mL孕酮配置。称取20mg孕酮溶于1mL无水乙醇中,制备20mg/mL溶液,用20um滤器过滤去除细菌。吸取10uL 20mg/mL孕酮溶液与990uL无水乙醇混匀,20uL/管分装,-20℃下保存,避免反复冻融。
250mM N-乙酰-L-半胱氨酸配置。称取0.266gN-乙酰-L-半胱氨酸溶于10mL细胞级双蒸水中,制备250mM N-乙酰-L-半胱氨酸溶液,用20um滤器过滤去除细菌。将溶液按10uL/管分成等份放入离心管中,可在-20℃保存2个月以上,避免反复冻融。
胎牛血清热灭活处理
按照逐步解冻法(-2O℃至4℃至室温)解冻FBS,解冻后将其摇匀,使温度及成分均一,减少沉淀发生。解冻前选用一个同规格FBS瓶对照瓶,并在其中加入与血清等体积的水。在对照瓶内插入2支温度计,将血清瓶与带温度计的对照瓶放入水浴锅中,设定水浴锅温度为56℃。待温度计所示温度上升至56℃时,计时30分钟,灭活结束。灭活后的FBS分装并保存在-20~-70℃冰柜中。
表1培养液组成
食蟹猴胚胎体外长时程培养:嵌入式-3D
在接种食蟹猴囊胚之前,将μ-slide 8-well和200ul枪头在-20℃预冷10min,后将未稀释的Matrigel融溶后均匀铺于8孔板底部,每孔50μL,然后将8孔板放入37℃恒温培养箱孵育30分钟。
在配置好的IVC1(不含有4%Matrigel)中加入10%(v/v)融溶的Geltrex,混匀后加到铺好Matrigel的8孔板内,每孔50μL。
将受精后第6-7天(d.p.f.6-7)食蟹猴囊胚转移到牛睾丸透明质酸酶内,使用口吸管吹吸30S后移入H9+G-2TMPLUS培养基内中和,然后再移入含有10%Geltrex的IVC1滴内,放入37℃恒温培养箱凝胶40分钟(待囊胚基本固定不晃动)后加入提前平衡好的含有4%Matrigel的IVC1(300μL每孔)。在体外长时程培养过程中,体外培养第1天静置培养不换液,第2天到第8天用含有4%Matrigel的IVC2进行半量换液,第9天到第13天在4%Matrigel的IVC2中加入3mg/mL葡萄糖,第14到19天将4%Matrigel的IVC2中的葡萄糖浓度提高到3.5mg/mL。
实例1:利用小鼠胚胎筛选3D体外培养体系的支架材料
1.材料
ICR小鼠,由昆明医科大学动物部提供:SCXK(滇)K2015-0002;
M2,Sigma,M7167
Acidic Tyrode’s(AT)solution,Sigma-Aldrich,T1788
KSOM,millipore,MR-106-D
石蜡油,Ovoil,510140
其余试剂与方案中一致。
2.实验方法
2.1小鼠超数排卵
选择处于发情前期的健康适龄雌性ICR小鼠进行超数排卵。每只小鼠通过腹腔注射10U的马属动物绒毛膜促性腺激素(PMSG,宁波第二激素厂),46-48h后注射同等剂量的人绒毛膜促性腺激素(HCG,宁波第二激素厂),然后将其与雄性ICR小鼠合笼。第二天早上检查阴道栓判断雌性ICR鼠受精情况,见栓记为受精后0.5天。妊娠第3.5天用M2培养基冲洗子宫角收集囊胚。
2.2培养基mIVC配制
表2小鼠体外培养液配制
所配置培养液可在4℃储存2周。
2.3小鼠胚胎培养
组1:μ-slide 8孔板未作处理(二维培养,2D)
在ICR小鼠受精后3.5天,使用M2培养基冲洗子宫角获得囊胚后,放入酸性台式液中去除囊胚透明带,将提前配置完成的IVC1(与mIVC1的区别为:不含4%Matrigel)培养基放入恒温培养箱(37℃,21%O2,5%CO2)内平衡至少1小时以上,将平衡好的IVC1培养基直接加入8孔板内(每孔300μL IVC1培养基),接入无透明带的囊胚,放入恒温培养箱中静置培养。待胚胎完全贴壁后进行IVC2(与mIVC2的区别为:不含4%Matrigel)换液,在体外培养3天,回收胚胎进行免疫荧光鉴定,并与体内正常发育胚胎进行对照。
组2:使用50uL未稀释的Matrigel原胶铺板,培养箱37℃孵育30min。(三维培养,3D)
将提前配置完成的IVC1培养基放入恒温培养箱内平衡至少1小时以上,然后在孵育好的含有Matrigel的8孔板内每孔加入300μL含有4%Matrigel的IVC1培养基,再将去完透明带的小鼠囊胚接入孔内,每孔接入6-8枚胚胎,待胚胎完全贴壁后进行IVC2(含4%Matrigel)换液,在体外培养3天,回收胚胎进行免疫荧光鉴定,并与体内正常发育胚胎进行对照。
组3:使用50uL未稀释的Matrigel原胶铺板,培养箱37℃孵育30min。将IVC1(不包含Matrigel)与未稀释的水凝胶Geltrex混合成10%IVC1含有Geltrex,吸取50μL铺在孵育后的八孔板中。(嵌入式-3D)
获得囊胚并且去除透明带后,将囊胚接入10%IVC1含有Geltrex的胶滴内,再次放入恒温培养箱内孵育40分钟以后,待胚胎在胶滴内固定不晃动后,加入提前平衡至少1小时以上的4%Matrigel的IVC1(每孔300μL),第2天开始即可正常更换IVC2(含4%Matrigel)培养基,在体外培养3天,回收胚胎进行免疫荧光鉴定,并与体内正常发育胚胎进行对照。
3实验结果
3.1培养板不同处理方式对植入后小鼠胚胎发育影响如图1所示:
3.2培养板不同处理方式对植入后小鼠胚胎发育影响如表3所示:
表3培养板不同处理方式的植入后小鼠胚胎发育率
4结论
嵌入式-3D培养是一种仿生状态的胚胎体外培养方式,模拟了胚胎着床的子宫环境,胚胎形态和发育率明显优于2D和3D方式。
实例2:
1.材料与试剂
6-7d.p.f.食蟹猴囊胚(由昆明理工大学灵长类转化医学研究院提供);
玻片,CITOTEST,1A5105;
OCT包埋剂,Sakura Finetek;
4%多聚甲醛,biosharp,BL539A;
PBS,Meilunbio,MA0008;
Triton X-100,Amresco,0694;
Tween 20,Sigma-Aldrich,P1379;
BSA,Sigma-Aldrich,B2064
OCT4,R&D systems,AF1579,1:400稀释于封闭液(PBS+10%FBS+3%BSA);
SOX2,Abcam,ab137385,1:200稀释于封闭液;
NESTIN,BD,644658,1:500稀释于封闭液;
CD31,R&D systems,AF3628,1:200稀释于封闭液;
RUNX1-3,Abcam,ab220117,1:50稀释于封闭液;
APOA2,Abcam,ab92478,1:50稀释于封闭液;
DAPI,Sigma,D5943,1:500稀释于封闭液;
其余试剂与方案中一致。
2.实验方法
2.1培养基MIVC配制
表4食蟹猴体外培养液配制
所配置培养液可在4℃储存2周。
2.2利用嵌入式-3D方式体外培养食蟹猴胚胎
在8孔板底部铺50uL未稀释的Matrigel,培养箱37℃孵育30min。将IVC1(与MIVC1的区别为:不包含matrigel)与10%Geltrex混合均匀,50μL铺在孵育后的八孔板中。
将胚胎置于来源于牛睾丸的透明质酸酶中处理30s,去除透明带。将去除透明带的囊胚转移至含10%Geltrex的IVC1微滴中。37℃恒温培养箱孵育中40min,直至包裹囊胚的Geltrex成为果冻样,加入至少预平衡1h以上的MIVC1,每孔300μL。体外培养第一天不进行换液,之后每天进行MIVC2半量换液。
2.3葡萄糖添加
体外培养第9-13天向MIVC2加入3mg/mL葡萄糖,体外培养第14-19天将葡萄糖浓度提高至3.5mg/mL。
2.4食蟹猴胚胎冰冻切片制备
胚胎固定:在含有4%多聚甲醛的PBS中,4℃过夜固定。
清洗:固定的胚胎用冷PBS冲洗3次,并浸泡在PBS中数小时,以尽可能干净地去除残留多聚甲醛。
包埋:将胚胎包埋在OCT包埋剂中,冷冻保存于-20℃。
切片制备:样品在预处理的载玻片制备厚度为8μm的冷冻切片,并存储在-80℃冰箱。
2.5食蟹猴胚胎冰冻切片免疫荧光染色
复温:从-80℃冰箱中取出冷冻的胚胎切片,室温复温30min,用PBS浸泡和冲洗去除包埋剂。
透膜:样品在室温下用PBST(0.3%Triton X-100PBS)细胞透膜30分钟。
封闭:用含有3%(w/v)BSA和10%(w/vol)FBS的PBS覆盖样本,4℃过夜。
一抗孵育:样本与一抗4℃孵育过夜。
清洗:用TBS(PBS+0.1%吐温20)清洗2次,PBS 3次去除未结合的一抗。
二抗孵育:带有荧光结合的二抗和DAPI与载玻片在室温黑暗中孵育2h。
清洗:用TBS(PBS+0.1%吐温20)清洗2次,PBS 3次去除未结合的二抗。
封片:在样本上滴加10uL抗荧光淬灭剂,上层覆盖盖玻片,透膜指甲油封闭四周。
3实验结果
3.1本发明中嵌入式-3D体外培养方式中食蟹猴胚胎发育明场图如图2所示。
3.2本发明中嵌入式-3D体外培养方式的食蟹猴胚胎发育率统计如表5所示。
表5食蟹猴胚胎发育率
3.3本发明中所得食蟹猴胚胎能够进行神经特化
如图3、图4所示,在18d.p.f,发现OCT4和SOX2在EPI中共表达且水平相似。在19d.p.f时,EPI细胞亚群中OCT4表达下降,SOX2水平保持不变。随着发育时程推进,在23d.p.f时,则观察到在EPI中间位置出现OCT4-/SOX2+细胞,标志着神经细胞逐步特化。
为了进一步研究体外培养的猴子胚胎中神经的分化,检测了NESTIN(神经板标记物)在SOX2阳性细胞中的表达情况,结果显示在19d.p.f.,少数SOX2+细胞中初步检测到微弱NESTIN表达,但在23d.p.f,则与SOX2重叠表达。
3.4本发明中所得食蟹猴胚胎能够分化形成造血细胞
如图5所示,在25d.p.f.,在胚胎样本中检测到RUNX1-3与CD31双阳性细胞,且与卵黄囊标记物APOA2共标,证明已出现造血祖细胞。
本培养体系能够在没有母体组织的基础下,正常维持食蟹猴胚胎发育至早期器官形成,包括神经诱导、两波卵黄囊造血等胚胎事件。
最后应说明的是:以上实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。
Claims (10)
1.一种非人灵长类胚胎体外培养试剂盒,其特征在于,包括第一培养基;
所述第一培养基包括独立包装的Matrigel和混合试剂;
所述混合试剂为IVC1与Geltrex的混合物,且Geltrex在混合试剂中的含量为9-11%(V/V);
所述IVC1与Geltrex的混合物中IVC1的成分为:DMEM/F12,热灭活FBS,L-谷氨酰胺,青霉素,链霉素,ITS-X,β-雌二醇,孕酮,N-乙酰-L-半胱氨酸。
2.根据权利要求1所述的非人灵长类胚胎体外培养试剂盒,其特征在于,还包括独立包装的MIVC1、MIVC2中的一种或两种;
所述MIVC1包括3.8-4.2%Matrigel(V/V);
所述MIVC2包括3.8-4.2%Matrigel(V/V)。
3.根据权利要求2所述的非人灵长类胚胎体外培养试剂盒,其特征在于,所述MIVC1还包括DMEM、热灭活FBS、PS、ITS-X、L-glutamine、β-estradiol、Progesterone、N-acetyl-L-cysteine;
和/或,所述MIVC2还包括DMEM、KoSR、PS、ITS-X、L-glutamine、β-estradiol、Progesterone、N-acetyl-L-cysteine;
优选的,每10mL MIVC1含有7.06-7.51mL DMEM/F12、1.8-2.2mL热灭活FBS、40-60IUPS、90-110uL ITS-X、1.8-2.2mML-glutamine、7.2-8.8nMβ-estradiol、180-220ng/mLProgesterone、22.5-27.5uM N-acetyl-L-cysteine、3.8-4.2%Matrigel(V/V);
和/或,每10mLMIVC2含有5.06-7.51mLDMEM/F12、2-4mLKoSR、40-60IU PS、90-110uLITS-X、1.8-2.2mM L-glutamine、7.2-8.8nMβ-estradiol、180-220ng/mLProgesterone、22.5-27.5uMN-acetyl-L-cysteine、3.8-4.2%Matrigel(V/V)。
4.根据权利要求1-3任一项所述的非人灵长类胚胎体外培养试剂盒,其特征在于,还包括独立包装的第二培养基和/或第三培养基;
所述第二培养基含有MIVC2和葡萄糖,所述葡萄糖与MIVC2的比例为2.9-3.1mg/mL;
所述第三培养基为含有MIVC2和葡萄糖,所述葡萄糖与MIVC2的比例为3.4-3.6mg/mL。
5.一种非人灵长类胚胎体外培养方法,其特征在于,利用权利要求1-4任一项所述的体外培养试剂盒。
6.根据权利要求5所述的非人灵长类胚胎体外培养方法,其特征在于,包括以下步骤:
1)将Matrigel孵育;
2)将IVC1与Geltrex混合均匀得到混合物,将上述混合物铺在步骤1)孵育后的Matrigel上;
3)将囊胚转移至步骤2)中IVC1与Geltrex的混合物中,孵育至包裹囊胚的Geltrex成为果冻样。
7.根据权利要求6所述的非人灵长类胚胎体外培养方法,其特征在于,还包括以下步骤:
4)向步骤3)得到的产物中加入MIVC1;
5)体外培养第2天开始用MIVC2换液。
8.根据权利要求7所述的非人灵长类胚胎体外培养方法,其特征在于,步骤5)中,体外培养第2天到第8天用MIVC2进行换液,第9天到第13天用第二培养基进行换液,第14-19天用第三培养基进行换液。
9.根据权利要求6-8任一项所述的非人灵长类胚胎体外培养方法,其特征在于,步骤3)中,所述囊胚为受精后第6-7天的囊胚。
10.根据权利要求5-9任一项所述的非人灵长类胚胎体外培养方法,其特征在于,所述非人灵长类为食蟹猴。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202211148926.6A CN115466717A (zh) | 2022-09-20 | 2022-09-20 | 一种非人灵长类胚胎体外培养试剂盒和体外培养方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202211148926.6A CN115466717A (zh) | 2022-09-20 | 2022-09-20 | 一种非人灵长类胚胎体外培养试剂盒和体外培养方法 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN115466717A true CN115466717A (zh) | 2022-12-13 |
Family
ID=84334852
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202211148926.6A Pending CN115466717A (zh) | 2022-09-20 | 2022-09-20 | 一种非人灵长类胚胎体外培养试剂盒和体外培养方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN115466717A (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115161267A (zh) * | 2022-08-18 | 2022-10-11 | 中国医学科学院医学生物学研究所 | 一种食蟹猴的未成熟卵母细胞和胚胎的体外培养液及其应用 |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20130136721A1 (en) * | 2010-02-09 | 2013-05-30 | The Johns Hopkins University | Compositions and Methods of Generating a Differentiated Mesodermal Cell |
CN112574944A (zh) * | 2020-12-14 | 2021-03-30 | 广州医科大学附属第三医院(广州重症孕产妇救治中心、广州柔济医院) | 基于体外诱导eps发育形成类囊胚结构的方法 |
CN113999404A (zh) * | 2021-10-09 | 2022-02-01 | 昆明理工大学 | 一种用于骨关节炎的双交联干细胞球水凝胶的制备方法 |
WO2022099498A1 (en) * | 2020-11-11 | 2022-05-19 | Guangzhou Institutes Of Biomedicine And Health, Chinese Academy Of Sciences | Media and methods for establishing and maintaining early embryo-like cells |
WO2023173370A1 (en) * | 2022-03-17 | 2023-09-21 | Guangzhou Institutes Of Biomedicine And Health, Chinese Academy Of Sciences | Media and methods for producing human cells and tissues from teratoma, organoids and embryoid bodies |
-
2022
- 2022-09-20 CN CN202211148926.6A patent/CN115466717A/zh active Pending
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20130136721A1 (en) * | 2010-02-09 | 2013-05-30 | The Johns Hopkins University | Compositions and Methods of Generating a Differentiated Mesodermal Cell |
WO2022099498A1 (en) * | 2020-11-11 | 2022-05-19 | Guangzhou Institutes Of Biomedicine And Health, Chinese Academy Of Sciences | Media and methods for establishing and maintaining early embryo-like cells |
CN117120064A (zh) * | 2020-11-11 | 2023-11-24 | 深圳华大科技控股集团有限公司 | 用于建立和维持早期胚胎样细胞的培养基和方法 |
CN112574944A (zh) * | 2020-12-14 | 2021-03-30 | 广州医科大学附属第三医院(广州重症孕产妇救治中心、广州柔济医院) | 基于体外诱导eps发育形成类囊胚结构的方法 |
CN113999404A (zh) * | 2021-10-09 | 2022-02-01 | 昆明理工大学 | 一种用于骨关节炎的双交联干细胞球水凝胶的制备方法 |
WO2023173370A1 (en) * | 2022-03-17 | 2023-09-21 | Guangzhou Institutes Of Biomedicine And Health, Chinese Academy Of Sciences | Media and methods for producing human cells and tissues from teratoma, organoids and embryoid bodies |
Non-Patent Citations (3)
Title |
---|
LIFENG XIANG等: "A developmental landscape of 3D-cultured human pre-gastrulation embryos", NATURE, 12 November 2019 (2019-11-12), pages 1 - 2 * |
MARTINA ZAMPONI等: "Combined 3D bioprinting and tissue-specific ECM system reveals the influence of brain matrix on stem cell differentiation", FRONT CELL DEV BIOL, 25 October 2023 (2023-10-25) * |
邵红莲等: "小鼠植入后胚胎体外延时培养体系的应用进展", 中国比较医学杂志, vol. 30, no. 8, 24 July 2020 (2020-07-24) * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115161267A (zh) * | 2022-08-18 | 2022-10-11 | 中国医学科学院医学生物学研究所 | 一种食蟹猴的未成熟卵母细胞和胚胎的体外培养液及其应用 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Bukovsky et al. | Oogenesis in cultures derived from adult human ovaries | |
Lee et al. | In vitro differentiation of germ cells from nonobstructive azoospermic patients using three-dimensional culture in a collagen gel matrix | |
US20200190479A1 (en) | 3d microphysiologic system | |
AU2013221839B2 (en) | Feeder-free method for culture of bovine and porcine spermatogonial stem cells | |
Kossowska-Tomaszczuk et al. | Cells with stem cell characteristics in somatic compartments of the ovary | |
US20140249364A1 (en) | Oocytes derived from ovarian culture initially containing no oocytes | |
Cadenas et al. | Relationship between follicular dynamics and oocyte maturation during in vitro culture as a non-invasive sign of caprine oocyte meiotic competence | |
Głabowski et al. | Growth factors effects on preimplantation development of mouse embryos exposed to tumor necrosis factor alpha | |
Balaban et al. | Progression to the blastocyst stage of embryos derived from testicular round spermatids | |
CN115466717A (zh) | 一种非人灵长类胚胎体外培养试剂盒和体外培养方法 | |
Martins et al. | Development of goat primordial follicles after in vitro culture of ovarian tissue in Minimal Essential Medium supplemented with coconut water | |
Pessoa et al. | Effect of morphological integrity, period, and type of culture system on the in vitro development of isolated caprine preantral follicles | |
Souza-Fabjan et al. | In vitro production of small ruminant embryos: Latest improvements and further research | |
Desai et al. | New hyaluronan-based biomatrix for 3-D follicle culture yields functionally competent oocytes | |
Rahmani et al. | Transplantation of mouse iPSCs into testis of azoospermic mouse model: in vivo and in vitro study | |
Coleman et al. | In vitro maturation and early developmental capacity of bovine oocytes cultured in pure follicular fluid and supplementation with follicular wall | |
Zhou et al. | Development of properly-polarized trophoblast stem cell-derived organoids to model early human pregnancy | |
Davachi et al. | Effects of co-incubation with conspecific ampulla oviductal epithelial cells and media composition on cryotolerance and developmental competence of in vitro matured sheep oocytes | |
Watanabe et al. | Variation of cholesterol contents in porcine cumulus-oocyte complexes is a key factor in regulation of fertilizing capacity | |
WO2009138855A2 (en) | Methods and systems for the production of granulosa cells | |
Le Saint et al. | Autologous endometrial cell co-culture improves human embryo development to high-quality blastocysts: a randomized controlled trial | |
Wang et al. | Effects of changing culture medium on preimplantation embryo development in rabbit | |
Rezaei-Tazangi et al. | Effects of kisspeptin on the maturation of human ovarian primordial follicles in vitro | |
KR100715188B1 (ko) | 소 수정란의 체외배양용 배지조성물 및 이를 이용한체외배양 방법 | |
Dolanbay et al. | Expression of trophinin and dipeptidyl peptidase IV in endometrial co-culture in the presence of an embryo: A comparative immunocytochemical study |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination |