CN114292804B - Vascularized fat organoid culture method - Google Patents
Vascularized fat organoid culture method Download PDFInfo
- Publication number
- CN114292804B CN114292804B CN202111511138.4A CN202111511138A CN114292804B CN 114292804 B CN114292804 B CN 114292804B CN 202111511138 A CN202111511138 A CN 202111511138A CN 114292804 B CN114292804 B CN 114292804B
- Authority
- CN
- China
- Prior art keywords
- fat
- adipose
- culture
- vascularized
- cell
- 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
- 210000002220 organoid Anatomy 0.000 title claims abstract description 89
- 238000012136 culture method Methods 0.000 title claims abstract description 7
- 210000000130 stem cell Anatomy 0.000 claims abstract description 75
- 241000699670 Mus sp. Species 0.000 claims abstract description 45
- 210000001596 intra-abdominal fat Anatomy 0.000 claims abstract description 39
- 239000001963 growth medium Substances 0.000 claims abstract description 29
- 238000012258 culturing Methods 0.000 claims abstract description 12
- 238000000034 method Methods 0.000 claims abstract description 12
- 210000004027 cell Anatomy 0.000 claims description 51
- 239000002609 medium Substances 0.000 claims description 42
- 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 description 42
- 230000006698 induction Effects 0.000 claims description 40
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 24
- 210000000577 adipose tissue Anatomy 0.000 claims description 24
- 239000000243 solution Substances 0.000 claims description 23
- 210000001789 adipocyte Anatomy 0.000 claims description 22
- 229930182555 Penicillin Natural products 0.000 claims description 21
- 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 description 21
- 230000029087 digestion Effects 0.000 claims description 21
- 229940049954 penicillin Drugs 0.000 claims description 21
- 229960005322 streptomycin Drugs 0.000 claims description 21
- 230000009278 visceral effect Effects 0.000 claims description 21
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 20
- 239000012091 fetal bovine serum Substances 0.000 claims description 19
- 239000008188 pellet Substances 0.000 claims description 18
- 238000004113 cell culture Methods 0.000 claims description 17
- 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 claims description 16
- 239000007788 liquid Substances 0.000 claims description 15
- 238000012423 maintenance Methods 0.000 claims description 14
- 241000699666 Mus <mouse, genus> Species 0.000 claims description 13
- 239000006285 cell suspension Substances 0.000 claims description 12
- 238000001179 sorption measurement Methods 0.000 claims description 12
- 230000002792 vascular Effects 0.000 claims description 11
- 210000003556 vascular endothelial cell Anatomy 0.000 claims description 11
- 239000000872 buffer Substances 0.000 claims description 10
- 238000011010 flushing procedure Methods 0.000 claims description 10
- 108010082117 matrigel Proteins 0.000 claims description 9
- 229930183010 Amphotericin Natural products 0.000 claims description 8
- QGGFZZLFKABGNL-UHFFFAOYSA-N Amphotericin A Natural products OC1C(N)C(O)C(C)OC1OC1C=CC=CC=CC=CCCC=CC=CC(C)C(O)C(C)C(C)OC(=O)CC(O)CC(O)CCC(O)C(O)CC(O)CC(O)(CC(O)C2C(O)=O)OC2C1 QGGFZZLFKABGNL-UHFFFAOYSA-N 0.000 claims description 8
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 8
- 229930182566 Gentamicin Natural products 0.000 claims description 8
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 claims description 8
- 102000004877 Insulin Human genes 0.000 claims description 8
- 108090001061 Insulin Proteins 0.000 claims description 8
- 229940009444 amphotericin Drugs 0.000 claims description 8
- APKFDSVGJQXUKY-INPOYWNPSA-N amphotericin B Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C=C/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-INPOYWNPSA-N 0.000 claims description 8
- 229960002518 gentamicin Drugs 0.000 claims description 8
- JYGXADMDTFJGBT-VWUMJDOOSA-N hydrocortisone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 JYGXADMDTFJGBT-VWUMJDOOSA-N 0.000 claims description 8
- 229940125396 insulin Drugs 0.000 claims description 8
- 239000011159 matrix material Substances 0.000 claims description 8
- 230000005305 organ development Effects 0.000 claims description 7
- 230000023895 stem cell maintenance Effects 0.000 claims description 7
- YASAKCUCGLMORW-UHFFFAOYSA-N Rosiglitazone Chemical compound C=1C=CC=NC=1N(C)CCOC(C=C1)=CC=C1CC1SC(=O)NC1=O YASAKCUCGLMORW-UHFFFAOYSA-N 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- 210000001519 tissue Anatomy 0.000 claims description 6
- 239000012981 Hank's balanced salt solution Substances 0.000 claims description 5
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 claims description 5
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 claims description 5
- 230000002293 adipogenic effect Effects 0.000 claims description 5
- 239000012894 fetal calf serum Substances 0.000 claims description 5
- 230000001939 inductive effect Effects 0.000 claims description 5
- 238000007789 sealing Methods 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- APIXJSLKIYYUKG-UHFFFAOYSA-N 3 Isobutyl 1 methylxanthine Chemical compound O=C1N(C)C(=O)N(CC(C)C)C2=C1N=CN2 APIXJSLKIYYUKG-UHFFFAOYSA-N 0.000 claims description 4
- 102000029816 Collagenase Human genes 0.000 claims description 4
- 108060005980 Collagenase Proteins 0.000 claims description 4
- 102000009024 Epidermal Growth Factor Human genes 0.000 claims description 4
- 101800003838 Epidermal growth factor Proteins 0.000 claims description 4
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 claims description 4
- 229960005070 ascorbic acid Drugs 0.000 claims description 4
- 235000010323 ascorbic acid Nutrition 0.000 claims description 4
- 239000011668 ascorbic acid Substances 0.000 claims description 4
- 229960002424 collagenase Drugs 0.000 claims description 4
- 229960003957 dexamethasone Drugs 0.000 claims description 4
- UREBDLICKHMUKA-CXSFZGCWSA-N dexamethasone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)C[C@@H]2O UREBDLICKHMUKA-CXSFZGCWSA-N 0.000 claims description 4
- 229940116977 epidermal growth factor Drugs 0.000 claims description 4
- 210000003743 erythrocyte Anatomy 0.000 claims description 4
- 229960000890 hydrocortisone Drugs 0.000 claims description 4
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 claims description 4
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 claims description 3
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 claims description 3
- 210000000683 abdominal cavity Anatomy 0.000 claims description 3
- 239000007640 basal medium Substances 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 238000012835 hanging drop method Methods 0.000 claims description 3
- 229960004586 rosiglitazone Drugs 0.000 claims description 3
- 239000008223 sterile water Substances 0.000 claims description 3
- 150000002632 lipids Chemical class 0.000 claims description 2
- 230000001502 supplementing effect Effects 0.000 claims description 2
- 230000015572 biosynthetic process Effects 0.000 claims 5
- 108010019160 Pancreatin Proteins 0.000 claims 2
- 230000003321 amplification Effects 0.000 claims 2
- 238000003199 nucleic acid amplification method Methods 0.000 claims 2
- 229940055695 pancreatin Drugs 0.000 claims 2
- 241000894006 Bacteria Species 0.000 claims 1
- 101000599951 Homo sapiens Insulin-like growth factor I Proteins 0.000 claims 1
- 102100037852 Insulin-like growth factor I Human genes 0.000 claims 1
- 230000001580 bacterial effect Effects 0.000 claims 1
- 239000012888 bovine serum Substances 0.000 claims 1
- 239000013592 cell lysate Substances 0.000 claims 1
- 230000003833 cell viability Effects 0.000 claims 1
- 238000001514 detection method Methods 0.000 claims 1
- 238000000926 separation method Methods 0.000 claims 1
- 239000000725 suspension Substances 0.000 claims 1
- 241001465754 Metazoa Species 0.000 abstract description 10
- 238000011161 development Methods 0.000 abstract description 6
- 238000011160 research Methods 0.000 abstract description 6
- 238000001727 in vivo Methods 0.000 abstract description 5
- 208000008589 Obesity Diseases 0.000 abstract description 4
- 235000020824 obesity Nutrition 0.000 abstract description 4
- 239000003102 growth factor Substances 0.000 abstract description 2
- 238000010874 in vitro model Methods 0.000 abstract 1
- 230000006444 vascular growth Effects 0.000 abstract 1
- 235000019197 fats Nutrition 0.000 description 53
- 230000011759 adipose tissue development Effects 0.000 description 11
- 210000000593 adipose tissue white Anatomy 0.000 description 6
- 102000004142 Trypsin Human genes 0.000 description 5
- 108090000631 Trypsin Proteins 0.000 description 5
- 230000000903 blocking effect Effects 0.000 description 5
- 230000018109 developmental process Effects 0.000 description 5
- 239000012588 trypsin Substances 0.000 description 5
- 229930040373 Paraformaldehyde Natural products 0.000 description 4
- 102100024616 Platelet endothelial cell adhesion molecule Human genes 0.000 description 4
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 229920002866 paraformaldehyde Polymers 0.000 description 4
- 239000002243 precursor Substances 0.000 description 4
- 210000002536 stromal cell Anatomy 0.000 description 4
- 208000009415 Spinocerebellar Ataxias Diseases 0.000 description 3
- 238000007664 blowing Methods 0.000 description 3
- 239000006143 cell culture medium Substances 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 230000008021 deposition Effects 0.000 description 3
- 201000010063 epididymitis Diseases 0.000 description 3
- 230000035755 proliferation Effects 0.000 description 3
- 201000003624 spinocerebellar ataxia type 1 Diseases 0.000 description 3
- 210000004003 subcutaneous fat Anatomy 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 102000007372 Ataxin-1 Human genes 0.000 description 2
- 108010032963 Ataxin-1 Proteins 0.000 description 2
- 108050007372 Fibroblast Growth Factor Proteins 0.000 description 2
- 102000018233 Fibroblast Growth Factor Human genes 0.000 description 2
- 206010021143 Hypoxia Diseases 0.000 description 2
- 210000004204 blood vessel Anatomy 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 238000007334 copolymerization reaction Methods 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 206010012601 diabetes mellitus Diseases 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 229940126864 fibroblast growth factor Drugs 0.000 description 2
- 238000005206 flow analysis Methods 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 239000000017 hydrogel Substances 0.000 description 2
- 230000007954 hypoxia Effects 0.000 description 2
- 210000002865 immune cell Anatomy 0.000 description 2
- 238000010166 immunofluorescence Methods 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- RCEBHKDQZCVBTC-UHFFFAOYSA-N 2-acetamidothiophene-3-carboxylic acid Chemical compound CC(=O)NC=1SC=CC=1C(O)=O RCEBHKDQZCVBTC-UHFFFAOYSA-N 0.000 description 1
- 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 1
- 235000019737 Animal fat Nutrition 0.000 description 1
- 201000001320 Atherosclerosis Diseases 0.000 description 1
- 210000003771 C cell Anatomy 0.000 description 1
- 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 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 208000004930 Fatty Liver Diseases 0.000 description 1
- 108010087819 Fc receptors Proteins 0.000 description 1
- 102000009109 Fc receptors Human genes 0.000 description 1
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 1
- 206010019708 Hepatic steatosis Diseases 0.000 description 1
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 1
- 101000935043 Homo sapiens Integrin beta-1 Proteins 0.000 description 1
- 101000917858 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-A Proteins 0.000 description 1
- 101000917839 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-B Proteins 0.000 description 1
- 101000692455 Homo sapiens Platelet-derived growth factor receptor beta Proteins 0.000 description 1
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 description 1
- 208000031226 Hyperlipidaemia Diseases 0.000 description 1
- 206010020880 Hypertrophy Diseases 0.000 description 1
- 206010022489 Insulin Resistance Diseases 0.000 description 1
- 102100025304 Integrin beta-1 Human genes 0.000 description 1
- 102100029185 Low affinity immunoglobulin gamma Fc region receptor III-B Human genes 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 102100026547 Platelet-derived growth factor receptor beta Human genes 0.000 description 1
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 230000009815 adipogenic differentiation Effects 0.000 description 1
- 210000003486 adipose tissue brown Anatomy 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000003592 biomimetic effect Effects 0.000 description 1
- 210000001593 brown adipocyte Anatomy 0.000 description 1
- 239000002771 cell marker Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- 230000003511 endothelial effect Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000000918 epididymis Anatomy 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 208000010706 fatty liver disease Diseases 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000002710 gonadal effect Effects 0.000 description 1
- 238000003125 immunofluorescent labeling Methods 0.000 description 1
- 238000012606 in vitro cell culture Methods 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 230000004132 lipogenesis Effects 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 208000030159 metabolic disease Diseases 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000005087 mononuclear cell Anatomy 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 210000002747 omentum Anatomy 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000003087 receptor blocking agent Substances 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 231100000240 steatosis hepatitis Toxicity 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 150000003626 triacylglycerols Chemical class 0.000 description 1
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 1
- 210000000636 white adipocyte Anatomy 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a method for culturing vascularized fat organoids, which comprises the steps of separating and amplifying visceral fat stem cells of mice and culturing vascularized fat organoids. The invention uses the pro-vascular growth factor and the vascularized fat organoid culture medium to induce the visceral fat stem cells of the mice to spontaneously form the vascularized fat organoid under the three-dimensional culture condition, thereby improving the traditional fat three-dimensional culture method. The method has strong operability, high experimental repeatability and lower cost, provides a new in vitro model which is simple and convenient to operate, easy to quantitatively analyze and can reflect the in vivo fat development mode of animals for fat biology and obesity research.
Description
Technical Field
The invention belongs to the technical field of cell culture, and particularly relates to a method for culturing a fat organoid.
Background
Obesity and its induced diabetes, fatty liver, hyperlipidemia and other diseases have become worldwide problems threatening human health. The research on fat development and the metabolic mechanism thereof has important significance for developing lipid-lowering medicaments and inhibiting obesity. The fat tissue of mammals is mainly classified into subcutaneous fat and visceral fat according to the deposition location, the visceral fat is classified into mesenteric fat, omentum fat, and gonadal fat, etc., and the subcutaneous fat is classified into shoulder nail visceral fat, inguinal visceral fat, etc., and furthermore, fat cells can be ectopically deposited in other organs such as muscle to form intramuscular fat. Adipose tissue is classified into white adipose tissue and brown adipose tissue according to functions. White adipocytes store excess energy of the body's metabolism mainly by synthesizing triglycerides, while brown adipocytes are rich in mitochondria and can generate heat by consuming energy in fatty acids. Prior studies have shown that animal adipocytes develop primarily from mesodermal cells, which develop to form blood vessels, and that perivascular stromal cells subsequently develop to form adipocytes under the induction of adipogenic signals. Perivascular stromal cells capable of developing into adipocytes typically express markers such as CD29, CD34, SCA1, PDGFR alpha, and the like. However, there is heterogeneity between these stromal cells, and the current research on this heterogeneity and the changes that occur during its adipogenesis are not deep enough. Animal fat deposition mainly comprises proliferation and hypertrophy of fat cells, and compared with subcutaneous fat, animal visceral fat tissue is more easily deposited with age, and metabolic diseases such as insulin resistance, diabetes, vascular atherosclerosis and the like are caused. Therefore, research on the development and fat deposition and metabolism of the fat stem cells in visceral fat has more important significance for researching the fat-reducing medicaments and promoting human health.
Currently, in vitro studies of fat biology have been mainly performed on the 3T3-L1,3T3-F442A precursor adipocyte line isolated from mouse embryos, on the Ob17 precursor adipocyte line isolated from mouse epididymal adipose tissue, and on primary adipose vascular matrix fractions isolated from mouse and human adipose tissue (stromal vascular fractions, SVF). The precursor fat cell lines which are successfully constructed are fewer, the precursor fat cell lines have single composition, the microenvironment formed by various cells in the adipose tissue in vivo is difficult to simulate, and the primary fat SVF cells are difficult to proliferate for a high time in vitro and maintain strong adipogenic differentiation capacity. It is also difficult to reproduce single-chamber mature adipocytes and their secretory properties in adipose tissue in animals by conventional two-dimensional cell culture.
Although the mice are used as model animals, the individuals are small, the growth speed is high, the genetic background is clear, and the mice are good animal models for researching human obesity. However, due to the characteristics of fat-rich adipose tissue, overlarge mature adipose cell volume and the like, the in-vivo adipose cells are difficult to observe and quantitatively detect in real time, and meanwhile, compared with in-vitro cell culture, the in-vivo adipose development process of animals is slower, and the experimental period required for researching the biological functions of the animals is relatively longer. These have hampered research into fat development and function.
In recent years, as organoid technology has evolved and matured, it has become an important tool （KIM J, KOO B K, KNOBLICH J A. Human organoids: model systems for human biology and medicine[J]. Nat Rev Mol Cell Bio, 2020, 21(10): 571-84.SHAMIR E R, EWALD A J.Three-dimensional organotypic culture: experimental models of mammalian biology and disease [J]. Nat Rev Mol Cell Bio, 2014, 15(10): 647-64.）. for studying organ development, tissue microenvironment, drug effectiveness and safety, but in the field of fat research, there is a lack of mature organoid models. Although three-dimensional culture of human and mouse adipocyte lines and adipose-derived vascular matrix components （LOUIS F, PANNETIER P, SOUGUIR Z, et al. A biomimetic hydrogel functionalized with adipose ECM components as a microenvironment for the 3Dculture of human and murine adipocytes [J]. Biotechnology and Bioengineering, 2017, 114(8): 1813-24.）, and lipogenesis induction have been performed by using magnetic beads, hydrogels, and other materials, these models have not been able to effectively simulate the development timing of cells in adipose tissue in vivo, multiple cell types and microenvironments in which they are located, and are complex to operate, and have obvious culture batch properties, and lack of standardized culture schemes.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a low-cost and standardized method for culturing the fat organoids.
In order to achieve the above experimental purposes, the technical scheme provided by the invention is as follows:
Fetal Bovine Serum (FBS), DMEM/F12, trypLE and type I collagenase in the culture method are purchased from ThermoFisher; the vascular endothelial growth factor, alkaline fibroblast growth factor, epidermal growth factor, R3-insulin-like growth factor-1, ascorbic acid, hydrocortisone, gentamicin/amphotericin, and vascular endothelial cell basal medium EBM-2 are purchased from Lonza corporation; the ultra-low adsorption plate and the matrigel are purchased from Corning company.
The vascularized fat organoid culture method comprises two parts of i and ii:
i. Separating and obtaining visceral fat stem cells of mice:
Before separating visceral adipose stem cells of mice, firstly preparing adipose tissue digestion liquid and flushing liquid, wherein the adipose tissue digestion liquid contains 2% diabody, 97% HBSS buffer solution, 1% BSA by mass fraction and 1mg/ml type I collagenase by mass concentration, and the flushing liquid contains 2% fetal bovine serum, 2% penicillin/streptomycin diabody and 96% HBSS buffer solution by volume ratio.
Preferably, the fat tissue digestion solution and the rinse solution are prepared and then filtered through a 0.22 μm filter and kept in a refrigerator at 4 ℃.
More preferably, penicillin/streptomycin diabodies in adipose tissue digestion solution and flushing solution can be replaced by gentamicin/amphotericin, and the two are in corresponding proportion: 10000U penicillin/10 mg streptomycin: 3mg gentamicin/1.5 μg amphotericin.
Opening the abdominal cavity of a mouse under the aseptic condition, separating the white adipose tissue on the epididymis of the mouse, placing the white adipose tissue on a 10cm cell culture dish filled with flushing liquid, transferring the white adipose tissue into an ultra-clean bench, flushing the white adipose tissue with the flushing liquid for 2-3 times, and removing the dirt of the visceral adipose tissue. Then transferring the visceral adipose tissue into a 5ml centrifuge tube, fully cutting by using surgical scissors, adding an equal volume of visceral adipose tissue digestion solution, and placing the mixture at 37 ℃ for digestion for 30min by a 150 rpm shaker. Taking out digestion liquid containing adipose tissues, stopping digestion by using an equal volume of DMEM/F12 culture medium containing 10% fetal calf serum, filtering twice by using a filter screen with 100 mu m and 70 mu m, centrifuging for 300g for 5min, washing precipitated cells twice by using the DMEM/F12 culture medium in a resuspension mode, finally, transferring the cells into a cell culture plate after the cells are resuspended by using the DMEM/F12 complete culture medium containing 10% fetal calf serum, culturing for 4-6h under the condition of 37 ℃ and 5% CO 2, and changing the liquid after 4-6h according to the characteristic of strong adherence of the adipose-derived stem cells, thus obtaining the visceral adipose-derived stem cells of the mice.
Vascularized adipose organoid culture:
the vascularized adipose organoid culture medium comprises a adipose stem cell maintenance medium, a adipose stem cell vascularization induction medium, a vascularized adipose organoid adipogenesis directional induction medium, a vascularized adipose organoid adipogenesis induction maintenance medium and a vascularized adipose organoid maintenance medium.
Adipose stem cell maintenance media include DMEM/F12 medium, fetal bovine serum, basic fibroblast growth factor, penicillin and streptomycin. Penicillin concentration 10U/ml, streptomycin 10 μg/ml, fetal bovine serum volume fraction 12%, alkaline fibroblast growth factor mass concentration in the culture medium 10ng/ml.
Adipose-derived stem cell vascularization induction medium: including fetal bovine serum, vascular endothelial growth factor, basic fibroblast growth factor, epidermal growth factor, R3-insulin-like growth factor-1, ascorbic acid, hydrocortisone, gentamicin/amphotericin and vascular endothelial cell basal medium EBM-2. The volume ratio of fetal bovine serum in the culture medium is 5%, and the mass concentrations of vascular endothelial growth factor, epidermal growth factor, R3-insulin-like growth factor, ascorbic acid, hydrocortisone, gentamicin and amphotericin are respectively 0.5ng/ml, 5ng/ml, 20ng/ml, 1 mug/ml, 0.2 mug/ml, 30 mug/ml and 15ng/ml.
The vascularized fat organoid adipogenesis directional induction culture medium consists of DMEM/F12, insulin, dexamethasone, rosiglitazone, 3-isobutyl-1-methylxanthine, fetal bovine serum and penicillin/streptomycin double antibodies, wherein the volume ratio of the fetal bovine serum in the culture medium is 15%, the concentration of insulin, dexamethasone, rosiglitazone, 3-isobutyl-1-methylxanthine, penicillin and streptomycin is respectively 10 mu g/ml, 1 mu M, 0.5mM, 10U/ml and 10 mu g/ml, and the rest is DMEM/F12;
The vascularized fat organoid adipogenesis induction maintenance culture medium consists of DMEM/F12, insulin, fetal bovine serum and penicillin/streptomycin double antibodies, wherein the volume ratio of the fetal bovine serum in the culture medium is 12%, the concentrations of the insulin, the penicillin and the streptomycin are 5 mug/ml, 10U/ml and 10 mug/ml respectively, and the rest of the DMEM/F12 is the component;
the vascularized fat organoid maintenance medium comprises 10% fetal bovine serum by volume and 1% penicillin/streptomycin diabody by volume, and the balance of DMEM/F12 medium.
The isolated visceral adipose-derived stem cells of mice were cultured and expanded at 37℃in a 5% CO 2 using an adipose-derived stem cell maintenance medium.
Preferably, the visceral adipose stem cells of the mice are cultured under a 5% o 2 hypoxia environment.
And (3) culturing and expanding the visceral fat stem cells of the mice to 2 to 3 generations, and preparing the three-dimensional visceral fat stem cell pellets when the cell number reaches more than 10 6 by adopting a hanging drop method.
The cultured and expanded visceral adipose stem cells of mice were digested with 0.25% trypsin for 1min, then the digestion was stopped with twice the volume of complete medium, and the single cell suspension obtained after mixing was blown off, and 20. Mu.L was taken for cell counting. 300g,5min centrifugation to remove supernatant, adding appropriate amount of complete medium to cell pellet to resuspend according to cell count result, adjusting cell suspension concentration to 4×10 5~5×105/ml, using a pipette, preparing hanging drop at the inner side of 10cm cell culture plate top cover with 20 μl each hanging drop, and adding sterile water at the bottom of cell culture plate to maintain humidity.
Preferably, the TrypLE is used for replacing trypsin to digest and passge the visceral fat stem cells of the mice, so that the influence on the proliferation activity of the visceral fat stem cells can be reduced.
The fat stem cells with formed cell pellets are transferred to a 24-hole ultra-low adsorption plate containing 500 mu L of fat stem cell vascularization induction medium by using a 1mL gun head, 3-4 cell pellets are arranged in each hole, and the ultra-low adsorption plate is placed at 37 ℃ for 2 days under the condition of 5% CO 2, so that the visceral fat stem cells of the mice are induced to be oriented to vascular endothelial cells.
And placing Paraflim sealing films on 384-hole PCR plates to press and construct a porous mold, transferring three-dimensional fat stem cell spheres into the mold, embedding the cell spheres by adopting matrigel with the concentration of 8-12mg/ml, then transferring the mold into a cell incubator with the temperature of 37 ℃ for 30min, transferring the mold into an ultralow adsorption plate after solidification, and continuously inducing for 4-5 days by using a vascular endothelial promoting culture medium, and changing liquid once during the period to induce the cell spheres to form a branched vascular structure.
The adipose organoids with the vascular structures are cultured by adopting a vascularized adipose organoid adipogenic induction directional culture medium for 3 days, and the perivascular stromal cells are induced to orient to adipocytes.
The vascularized fat organoid is used to form fat induction maintenance medium to culture fat organoid for 10-12 days, and the liquid is changed every 2-3 days to induce the fat organoid to form mature fat cells with the diameter of more than 40 mu m.
The vascularized adipose organoids that have formed mature adipocytes are continued to be cultured using the adipose organoid maintenance medium.
The beneficial effects of the invention are as follows:
(1) The method for culturing vascularized fat organoids has low cost and less matrigel consumption for each culture.
(2) The vascularized adipose organoids obtained by the culture method of the invention have a vascular structure similar to that of adipose tissues in animals, and contain cell types in various animal adipose tissues such as vascular endothelial cells, mature adipocytes and the like.
(3) The vascularized fat organoid model constructed by the present invention contains mature adipocytes that are greater than 40 microns in diameter.
Drawings
The invention will be further described with reference to the drawings and examples.
FIG. 1 shows the procedure for isolation of visceral adipose stem cells and culture of vascularized adipose organoids in mice in example 1 and example 2.
FIG. 2 is a diagram of visceral adipose stem cells of mice used for culturing vascularized adipose organoids in example 1.
Fig. 3.1 and 3.2 are respectively a front-back flow analysis and identification chart of the difference adherence of the visceral adipose stem cells of the mice obtained in example 1 (CD 45 and CD31 are respectively immune cell and vascular endothelial cell markers, and SCA1 is an adipose stem cell marker).
FIG. 4 is an immunofluorescence of example 2 after 4 days of vascularization induction of vascularized visceral fat organoids.
FIG. 5 is a diagram showing the morphology of blood vessels after 7 days of induction of vascularization of a vascularized visceral fat organovessel in the mice of example 2.
FIG. 6 is a morphology of example 2 following induction of vascularized fat organogenesis for 11 days.
FIG. 7 is an immunofluorescence of example 2 after 24 days of vascularized adipose organoids culture.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention.
Example 1: separating and obtaining the visceral fat stem cells of the mice
As shown in FIG. 1, before separating visceral fat stem cells of mice, an adipose tissue-digested solution containing 1mg/ml type I collagenase, 1% BSA by mass, 2% penicillin/streptomycin diabody by volume and 97% HBSS buffer by volume and a wash solution containing 2% fetal bovine serum, 2% penicillin/streptomycin diabody and 96% HBSS buffer by volume were prepared.
Preferably, the fat tissue digestion solution and the rinse solution are prepared and then filtered through a 0.22 μm filter and kept in a refrigerator at 4 ℃.
More preferably, penicillin/streptomycin diabodies in adipose tissue digestion solution and flushing solution can be replaced by gentamicin/amphotericin, and the two are in corresponding proportion: 10000U penicillin/10 mg streptomycin: 3mg gentamicin/1.5 μg amphotericin.
4-6C 57BL6/J mice were sacrificed by cervical dislocation approved by the university of Zhejiang animal ethics committee and then sterilized by immersing in 75% alcohol for 10min. The abdominal cavity of the mice was opened under aseptic conditions, and the epididymal white adipose tissues of the mice were isolated. Then transferring the visceral adipose tissue of the mice into a 5ml centrifuge tube, fully cutting the visceral adipose tissue by using surgical scissors, adding the equal volume of visceral adipose tissue digestion solution, and placing the mixture at 37 ℃ for digestion for 30min by using a 150 rpm shaker. Digestion with adipose tissue was stopped by taking out the digests containing adipose tissue, stopping digestion with an equal volume of DMEM/F12 medium containing 10% fetal bovine serum, re-suspending after twice filtration through 100 μm and 70 μm sieves, centrifuging for 300g, removing supernatant in 5min, and re-suspending and washing the cell pellet twice with DMEM/F12 medium. Adding 1ml of erythrocyte lysate to the washed cell pellet, resuspending the cells, lysing for 1-2min, removing the erythrocytes, centrifuging for 5min to remove the supernatant, finally, resuspending the cells with 1ml of DMEM/F12 complete medium containing 12% fetal calf serum, transferring the cells into a cell culture plate, culturing under the condition of 37 ℃ and 5% CO 2, and changing the adipose-derived stem cell maintenance medium after 4-6h according to the characteristic of strong adherence of the adipose-derived stem cells to obtain adherent mouse visceral adipose-derived stem cells, as shown in figure 2.
Preferably, the visceral adipose stem cells of mice are cultured under the condition of 5% O 2 hypoxia, so that the proliferation capacity of the cells is stronger.
The visceral adipose stem cells of the mice obtained by the method are identified, because the matrix vascular components of the adipose tissue of the mice contain immune cells such as mononuclear cells/macrophages, vascular endothelial cells, adipose stem cells and other cells, in order to obtain the visceral adipose stem cells with higher purity at lower cost, the visceral adipose stem cells are obtained by a differential adherence method in the example, and are analyzed and identified by flow.
The identification step of the visceral adipose stem cells of the mice: (1) The visceral fat stem cells of the mice are digested for 1min by using 0.25% trypsin, then the digestion is stopped by using a complete culture medium with twice volume, a single cell suspension is prepared by blowing by a liquid-transfering gun, and 10 mu L of the single cell suspension is taken for cell counting; (2) 300g, centrifuging for 5min to remove supernatant, and re-suspending cell sediment with flow buffer (PBS containing 2% fetal calf serum and 1mM EDTA) according to cell counting result, and adjusting cell concentration to about 0.5-1×10 6/ml; (3) Blocking cells with Fc receptor blocker (anti-mouse CD16/32, biolegend) for 10-15min; (4) Adding specific fluorescent conjugated flow antibody (CD 31/PECAM-1-PE-Cy7, invitrogen, CD-PE-Cy 7, invitrogen, SCA/Ly 6a-APC, biolegend) to the blocked single cell suspension, and incubating for 20-30 min in the absence of light; (5) After washing the cells twice with flow buffer centrifugation (300 g,5 min) (6) flow cytometry was performed.
As shown in fig. 3.1 and 3.2, the results of the flow analysis showed that the proportion of cd45+cd31+ in the fat SVF was 44.2% and the proportion of adipose stem cells of CD45-CD31-sca1+ was 48.7% before differential adherence was performed; the fat stem cells obtained after differential adherence have the proportion of CD45+CD31+ of 11.4 percent and the proportion of CD45-CD31-SCA1+ of 78.7 percent, and the mouse visceral fat stem cells with higher purity are obtained by a differential adherence method.
Example 2: vascularized adipose organoid culture
As shown in FIG. 1, the visceral adipose-derived stem cells of mice are obtained by culturing, amplifying and separating the visceral adipose-derived stem cells at 37 ℃ under the condition of 5% CO 2 by using an adipose-derived stem cell maintenance medium.
Adipose-derived stem cell maintenance medium with specific composition shown in table 1:
TABLE 1
The visceral fat stem cells of the mice are cultured and expanded to 2 to 3 generations, and when the cell number reaches more than 10 6, the three-dimensional visceral fat stem cell pellets are prepared by adopting a hanging drop method.
The cultured and expanded visceral fat stem cells of mice were digested with 0.25% trypsin for 1min, then digested with twice the volume of complete medium, cell counted by taking 20. Mu.L of single cell suspension after being blown and mixed uniformly, and simultaneously supernatant was removed by centrifugation for 5min, an appropriate amount of complete medium was added to cell pellet according to the result of cell counting to resuspension, the cell suspension concentration was adjusted to 4X 10 5~5×105/ml, then the hanging drop was placed inside the top cover of a 10cm cell culture plate in an amount of 20. Mu.L per hanging drop using a pipette, and sterile water was added to the bottom of the cell culture dish to maintain humidity, and the hanging drop culture of visceral fat stem cells of mice was carried out on the cell culture dish containing the hanging drop of visceral fat stem cells of mice under the condition of 5% CO 2 at 37 ℃.
Preferably, tryPLE is used instead of trypsin to digest visceral adipose stem cells in mice.
The visceral fat stem cells of the mice were cultured in hanging drops for 2-3 days to form cell pellets with more regular morphology.
Preparation of a mice visceral adipose stem cell vascularization induction medium (table 2): wherein the VEGF concentration is within 1-2 ng/ml:
TABLE 2
The fat stem cells with formed cell pellets are transferred to a 24-hole ultra-low adsorption plate containing 500 mu L of fat stem cell vascularization induction medium by using a 1mL gun head, 3-4 cell pellets are arranged in each hole, and the ultra-low adsorption plate is placed at 37 ℃ for 2 days under the condition of 5% CO 2, so that the visceral fat stem cells of the mice are induced to be oriented to vascular endothelial cells.
Placing Paraflim sealing films on 384-hole PCR plates, superposing 384-hole ultralow adsorption plates on the sealing films, pressing with force to construct a porous mold, removing paper sheets on the sealing films, transferring three-dimensional adipose-derived stem cell spheres into the mold, embedding the cell spheres by using 6-8 mu L of matrix gel with the concentration of 8-12mg/ml in each hole, placing the mold at the bottom of a10 cm cell culture dish, transferring the mold into a 37 ℃ cell culture box for 30min, absorbing the adipose-derived stem cell vascularization induction culture medium by using a 200 mu L pipetting gun after the adipose-derived stem cell culture medium is solidified, carefully blowing matrix gel agglomerates solidified in the mold to fall into the 24-hole ultralow adsorption plates, supplementing the vascular endothelial cell culture medium for continuous culture for 4-5 days, changing liquid once in the period, and inducing the adipose-derived stem cell culture medium to form a branched vascular structure. The morphology after 7 days of vascularization induction of vascularized adipose organoids is shown in figure 5.
The vascularized adipose organogenesis directional induction medium (table 3) and the vascularized adipose organogenesis induction maintenance medium (table 4) were prepared, and the vascularized adipose organogenesis directional induction medium may also perform adipogenesis induction on the vascularized adipose organogenesis using only DMEM/F12 complete medium with insulin or insulin + dexamethasone + 3-isobutyl-1-methylxanthine as adipogenesis inducer.
TABLE 3 Table 3
TABLE 4 Table 4
The vascularized fat organoid adipogenesis directional induction culture medium and adipogenesis induction maintenance culture medium are used for adipogenesis induction for 13 to 15 days, liquid exchange is carried out every two to three days, and the adipogenesis organoids containing mature fat cells are induced. The morphology of the vascularized adipose organoids after 11 days of adipogenic induction is shown in figure 6.
Preparing a vascularized fat organoid maintenance medium, which comprises the following components: 89% DMEM/F12 medium, 10% fetal bovine serum, 1% penicillin/streptomycin diabody.
Mice were cultured for 6 days using vascularized adipose organoid maintenance medium with fluid changes every two to three days. Finally, dead and alive dyes are adopted to perform dead and alive dyeing on the organoid cells, and the proportion of the dead and alive cells is detected, so that the result shows that the proportion of the living cells in the fat organoid is more than 90%.
Identification of vascularized adipose organoids by immunofluorescence staining:
(1) 4 days after vascularization induction of vascularized adipose organoids. The medium in the ultra-low adsorption plate was carefully removed using a 200. Mu.L gun head, and then the fat organoids were aspirated using a 1ml gun head and transferred to the center of a 35mm confocal dish (Cellvis D-10-0-N). The copolymerization Jiao Min containing the vascularized fat organoids is placed in a refrigerator at 4 ℃ to dissolve the matrigel embedding the vascularized fat organoids.
The vascularized fat organoids were blown two to three times with a pipette gun, excess matrigel on the vascularized fat organoids was removed, excess medium around the vascularized fat organoids in a confocal dish was aspirated, the vascularized fat organoids were fixed with 200ul of 4% paraformaldehyde at room temperature for 30min, then the paraformaldehyde was removed, and the vascularized fat organoids were rinsed two to three times with PBS. The vascularized fat organoids were permeabilized at room temperature for 10-15min with 0.2% TritonX-100 solution and rinsed twice to three times with PBS. The vascularized adipose organoids were then blocked for 30min-45 mm using PBS blocking solution with 2.5% bsa, and the blocking solution was removed by two to three washes with pbst buffer. After immersing vascularized adipose organoids with 100 μl PBS, 1% anti-mouse CD31 antibody was added to PBS and incubated overnight at 4 ℃. The vascularized adipose organoids were washed two to three times with PBST buffer. Vascularized adipose organoids were incubated with 1% Alexa flow 594 or dysight 549 in PBS for 2 hours, and after two to three PBST washes, vascularized adipose organoids were immersed in 100 μl PBS.
The immunofluorescent-stained vascularized adipose organoids were imaged using a LSM880 (Zeiss, germany) 20-fold air mirror and a 40-fold water mirror. As shown in fig. 4.
(2) After 15 days of induction of vascularized adipogenic organoids. The medium in the ultra-low adsorption plate was carefully removed using a 200. Mu.L gun head, and then the fat organoids were aspirated using a 1ml gun head and transferred to the center of a 35mm confocal dish (Cellvis D-10-0-N). The copolymerization Jiao Min containing the vascularized fat organoids is placed in a refrigerator at 4 ℃ to dissolve the matrigel embedding the vascularized fat organoids.
Blowing the vascularized fat organoid twice to three times by a pipette gun, removing redundant matrigel on the vascularized fat organoid, sucking redundant culture medium around the vascularized fat organoid in a confocal dish, fixing the vascularized fat organoid for 30min at room temperature by 200ul of 4% paraformaldehyde, removing the paraformaldehyde, and flushing the vascularized fat organoid twice to three times by PBS. The vascularized fat organoids were permeabilized at room temperature for 10-15min with 0.2% TritonX-100 solution and rinsed twice to three times with PBS. The vascularized adipose organoids were then blocked for 30min-45 mm using PBS blocking solution containing 2.5% bsa, and the vascularized adipose organoids were washed two to three times with pbst buffer to remove the blocking solution. After immersing vascularized adipose organoids with 100 μl PBS, 1% anti-mouse CD31 antibody was added to PBS and incubated overnight at 4 ℃. The vascularized adipose organoids were washed two to three times with PBST buffer. Vascularized adipose organoids were incubated with a PBS solution containing 1% Alexa Flour594 or Dyight 549 for 2 hours, after two to three washes with PBST, the lipid droplets of vascularized adipose organoids were stained with a PBS solution containing 1. Mu.g/ml Bodipy for 30min, after two to three washes with PBS, the vascularized adipose organoid nuclei were stained with a PBS solution containing 1. Mu.g/ml DAPI for 10 min, and finally washed twice with PBS, and vascularized adipose organoids were immersed with 100. Mu.L PBS.
The immunofluorescent-stained vascularized adipose organoids were imaged using a LSM880 (Zeiss, germany) 20-fold air mirror and a 40-fold water mirror. As shown in fig. 7.
The embodiments in the foregoing description may be further combined or replaced, and the embodiments are merely illustrative of the preferred embodiments of the present invention and are not intended to limit the spirit and scope of the present invention, and various changes and modifications made by those skilled in the art to which the present invention pertains without departing from the spirit of the present invention. The scope of the invention is given by the appended claims and any equivalents thereof.
Claims (3)
1. The vascularized fat organoid culture method is characterized by comprising two parts of separation and amplification of visceral fat stem cells of mice and vascularized fat organoid culture, and comprises the following specific steps:
mice were sacrificed in humane ways;
Obtaining adipose tissue of a mouse: opening the abdominal cavity of the mouse under the aseptic condition to obtain visceral adipose tissue, flushing the visceral adipose of the mouse for 2-3 times by using adipose tissue flushing liquid at the temperature of 2-8 ℃ to remove dirt;
Digesting adipose tissue: the ophthalmic scissors cut the adipose tissue, the adipose tissue is digested by using a constant temperature shaking table comprising 1mg/ml type I collagenase, 2% penicillin/streptomycin double antibody by volume, 1% BSA by volume and 97% HBSS buffer adipose tissue digestion solution by volume at 37 ℃,30min and 150 rpm;
single cell suspension was obtained: stopping digestion of the complete medium of the same volume DMEM/F12, filtering the fat tissue digestion solution twice by using a 100 mu m and 70 mu m cell filter screen, centrifuging for 300g for 5min to remove the supernatant, and using a red blood cell lysate to lyse red blood cells, and re-suspending the cells by using the medium to obtain single cell suspension;
2D culture expansion of adipose-derived stem cells: inoculating the obtained single cell suspension to a tissue treatment cell culture dish, supplementing a culture medium, putting the culture dish into a 37 ℃ CO 2 or 37 ℃ CO 2,5%O2 incubator with 5% CO 2, changing the liquid after 4-6 hours to obtain high-purity adipose-derived stem cells, and continuing to culture and amplify the adipose-derived stem cells after 2-3 days;
Hanging drop culture: after the pancreatin or TrypLE of the adipose-derived stem cells after culture and amplification are digested, the complete culture medium is used for stopping digestion, and a hanging drop method is adopted to enable the visceral adipose-derived stem cells of the mice to form three-dimensional adipose-derived stem cell spheres which are cultured for 2 to 3 days in hanging drops, wherein the number of cells contained in each cell sphere is 8000 to 10000;
Vascularization induction: culturing and inducing the visceral fat stem cells of the mice in a suspension manner in an ultralow adsorption plate for 2 days by using a fat stem cell vascularization induction culture medium, inducing the visceral fat stem cells of the mice to orient towards vascular endothelial cells, constructing a porous mold by using a Paraflim sealing film and a 384-hole PCR plate, transferring the visceral fat stem cell pellets of the mice cultured by using the fat stem cell vascularization induction culture medium to the mold, placing one fat stem cell pellet in each hole of the mold, embedding the cell pellet by using matrix gel with the concentration of 8-12 mg/ml, placing the mold at the bottom of a bacterial or cell culture dish, inversely placing the mold in a cell culture dish for 30 minutes at the temperature of 37 ℃, transferring matrix gel containing the visceral fat stem cell pellets of the mice to the ultralow adsorption plate by using the fat stem cell vascularization induction culture medium after the matrix gel is solidified, and continuously suspending and inducing for 4-5 days, and changing liquid for 2-3 days to form a branched vascular structure;
Lipid formation induction: performing fat formation induction orientation on the mouse fat organoid for 3 days by using a vascularized fat organoid fat formation orientation induction culture medium, and then continuing to perform fat formation induction on the vascularized fat organoid by using a vascularized fat organoid fat formation induction maintenance culture medium for 10-12 days to form a vascularized fat organoid with mature fat cells with the diameter of more than 40 mu m;
Maintenance culture: culturing the vascularized adipose organoids for 6 days by using a vascularized adipose organoid maintenance medium for subsequent cell viability and phenotype detection, wherein the vascularized adipose organoids obtained by culture contain vascular endothelial cells and mature adipocytes;
the 2D culture expansion adipose-derived stem cells use an adipose-derived stem cell maintenance medium, which comprises 12% fetal calf serum, 1% penicillin/streptomycin double antibody, 10ng/ml basic fibroblast growth factor and 87% DMEM/F12 medium;
The complete culture medium used for hanging drop culture comprises 15% fetal bovine serum by volume, 1% penicillin/streptomycin double antibody by volume and 84% DMEM/F12 by volume;
The adipose-derived stem cell vascularization induction culture medium used for vascularization induction comprises vascular endothelial growth factor 1ng/ml, epidermal growth factor 5ng/ml, insulin-like growth factor-1 of 20ng/mlR, ascorbic acid 1 mug/ml, hydrocortisone 0.2 mug/ml, gentamicin 30 mug/ml, amphotericin 15ng/ml and vascular endothelial cell basal medium EBM-2 with volume ratio of 5% bovine serum;
embedding the mouse visceral adipose-derived stem cell pellets by using matrigel 2 days after the three-dimensional adipose-derived stem cell pellets are induced by vascularization, wherein the amount of matrigel used for embedding each cell pellet is 6-8 mu L;
The vascularized fat tube adipogenic directional induction culture medium comprises fetal bovine serum with the volume ratio of 15%, penicillin/streptomycin double antibody with the volume ratio of 1%, insulin with the volume ratio of 10 mug/ml, dexamethasone with the volume ratio of 1 mug, rosiglitazone with the volume ratio of 1 mug, 3-isobutyl-1-methylxanthine with the volume ratio of 0.5mM and DMEM/F12 culture medium with the volume ratio of 84%; the vascularized fat organogenesis-induced maintenance medium comprises fetal bovine serum at a volume ratio of 12%, 1% penicillin/streptomycin diabody, 5 μg/ml insulin, and DMEM/F12 medium at a volume ratio of 87%.
2. The method of claim 1, wherein the adipose tissue wash comprises 2% fetal bovine serum by volume, 2% penicillin/streptomycin diabody by volume and 96% hbss buffer by volume.
3. The method according to claim 1, wherein the 2D culture expanded adipose stem cells are prepared by pancreatin digestion before hanging drop culture, single cell suspension is prepared, the cell concentration is 4 x 10 5~5×105/ml, the inside of the top cover of the cell culture dish is inoculated with 20 μl single cell suspension, hanging drops are prepared, and sterile water is added to the bottom of the bacteria or cell culture plate to maintain the humidity.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111511138.4A CN114292804B (en) | 2021-12-11 | 2021-12-11 | Vascularized fat organoid culture method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111511138.4A CN114292804B (en) | 2021-12-11 | 2021-12-11 | Vascularized fat organoid culture method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114292804A CN114292804A (en) | 2022-04-08 |
| CN114292804B true CN114292804B (en) | 2024-05-10 |
Family
ID=80968389
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111511138.4A Active CN114292804B (en) | 2021-12-11 | 2021-12-11 | Vascularized fat organoid culture method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114292804B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118272309A (en) * | 2023-07-14 | 2024-07-02 | 北京嘉士腾医学检验实验室有限公司 | Efficient digestive tract tumor organoid culture method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108350423A (en) * | 2015-07-10 | 2018-07-31 | 法国血液机构 | Method for obtaining mankind's brown/cream-coloured adipocyte |
| CN113474002A (en) * | 2018-10-12 | 2021-10-01 | 索尔克生物学研究所 | Cells, islets and organoids evading immunodetection and autoimmunity, method for producing same and use thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA3025344A1 (en) * | 2016-05-25 | 2017-11-30 | Salk Institute For Biological Studies | Compositions and methods for organoid generation and disease modeling |
-
2021
- 2021-12-11 CN CN202111511138.4A patent/CN114292804B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108350423A (en) * | 2015-07-10 | 2018-07-31 | 法国血液机构 | Method for obtaining mankind's brown/cream-coloured adipocyte |
| CN113474002A (en) * | 2018-10-12 | 2021-10-01 | 索尔克生物学研究所 | Cells, islets and organoids evading immunodetection and autoimmunity, method for producing same and use thereof |
Non-Patent Citations (2)
| Title |
|---|
| A Straightforward Approach to Engineer Vascularized Adipose Tissue Using Microvascular Fragments;Francisca M Acosta 等;TISSUE ENGINEERING: Part A;第26卷;第906页 右栏 第2段，第906页 右栏 第4段-第907页 左栏 第2段，图1 * |
| Human adipose stromal-vascular fraction self-organizes to form vascularized adipose tissue in 3D cultures;Sandra Muller 等;Scientific Reports;第9卷;摘要，第7页最后一段-第8页第4段 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114292804A (en) | 2022-04-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Li et al. | Human cord cell hematopoiesis in three-dimensional nonwoven fibrous matrices: in vitro simulation of the marrow microenvironment | |
| JP2009515558A (en) | Extracellular matrix components for proliferation or differentiation of hepatic progenitor cells | |
| CN112048470B (en) | Method for preparing clinical grade mesenchymal stem cell preparation by using human induced pluripotent stem cells | |
| WO2006001473A1 (en) | Selective culture method and separation method for small hepatocytes with the use of hyaluronic acid | |
| CN112226409B (en) | Method for differentiating embryonic stem cells into CD34+ hematopoietic progenitor cells | |
| CN100478441C (en) | Stem cell separating liquid and its separating method | |
| US20220395537A1 (en) | Methods of stem cell culture for obtaining products, and implementations thereof | |
| CN103013917A (en) | Method for inducing human amniotic mesenchymal stem cells to differentiate into neuron-like cells | |
| CN114292804B (en) | Vascularized fat organoid culture method | |
| CN111534477B (en) | Method for culturing primary epithelial stem cell balls of lung tissue of mouse | |
| WO2023016029A1 (en) | Method for separating fibroblasts derived from human induced pluripotent stem cells, and use thereof | |
| CN110904026A (en) | Preparation method and application of hepatic precursor-like cells from different sources | |
| CN108070560A (en) | A kind of isolation and culture method of the primary stomach cancer cell of people | |
| CN115927164B (en) | Culture method and application of vascularized tumor organoids | |
| Medvinsky et al. | Analysis and manipulation of hematopoietic progenitor and stem cells from murine embryonic tissues | |
| CN110951686A (en) | Hematopoietic stem cell in-vitro amplification culture system and method | |
| CN110499279A (en) | A method of induce human urine derived stem cells to hepatocyte differentiation | |
| RU2628092C1 (en) | Method for obtaining of msc-associated non-differentiated hemopoietic precursor cells with cd34+/cd133+ phenotype | |
| Joplin et al. | Human intrahepatic biliary epithelial cell lineages: studies in vitro | |
| CN110592007B (en) | Mesenchymal stem cell and preparation method and application thereof | |
| CN109897817B (en) | Method for separating immune cells and application thereof | |
| CN112375732A (en) | 3D suspension culture method for in-vitro amplification of spermatogonial stem cells | |
| Wencel et al. | Dried human skin fibroblasts as a new substratum for functional culture of hepatic cells | |
| CN104818245A (en) | Liver stem cell culture medium and culture method | |
| CN111979176B (en) | Preparation method of human corneal epithelial cells, conditioned medium thereof and preparation method thereof |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |