CN115340977B - 一种血管化胰岛及其制备方法 - Google Patents
一种血管化胰岛及其制备方法 Download PDFInfo
- Publication number
- CN115340977B CN115340977B CN202211277768.4A CN202211277768A CN115340977B CN 115340977 B CN115340977 B CN 115340977B CN 202211277768 A CN202211277768 A CN 202211277768A CN 115340977 B CN115340977 B CN 115340977B
- Authority
- CN
- China
- Prior art keywords
- islet
- cells
- vascular
- cell
- vascularized
- 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
- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- 210000004153 islets of langerhan Anatomy 0.000 claims abstract description 69
- 230000002792 vascular Effects 0.000 claims abstract description 29
- 230000004069 differentiation Effects 0.000 claims abstract description 27
- 210000000056 organ Anatomy 0.000 claims abstract description 6
- 210000004027 cell Anatomy 0.000 claims description 49
- 210000005167 vascular cell Anatomy 0.000 claims description 30
- 210000000130 stem cell Anatomy 0.000 claims description 19
- 238000000034 method Methods 0.000 claims description 18
- 230000001939 inductive effect Effects 0.000 claims description 11
- 238000012258 culturing Methods 0.000 claims description 5
- 238000004114 suspension culture Methods 0.000 claims description 5
- 230000004931 aggregating effect Effects 0.000 claims description 4
- 230000004927 fusion Effects 0.000 claims description 4
- 239000000017 hydrogel Substances 0.000 claims description 4
- 230000008569 process Effects 0.000 claims description 4
- 238000007499 fusion processing Methods 0.000 claims description 2
- 239000000463 material Substances 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims 1
- 239000002243 precursor Substances 0.000 abstract description 20
- 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 abstract description 18
- 210000004204 blood vessel Anatomy 0.000 abstract description 15
- 238000000338 in vitro Methods 0.000 abstract description 12
- 230000004083 survival effect Effects 0.000 abstract description 11
- 102000004877 Insulin Human genes 0.000 abstract description 9
- 108090001061 Insulin Proteins 0.000 abstract description 9
- 229940125396 insulin Drugs 0.000 abstract description 9
- 230000015572 biosynthetic process Effects 0.000 abstract description 7
- 230000000694 effects Effects 0.000 abstract description 7
- VOUAQYXWVJDEQY-QENPJCQMSA-N 33017-11-7 Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)NCC(=O)NCC(=O)N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N1[C@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O)CCC1 VOUAQYXWVJDEQY-QENPJCQMSA-N 0.000 abstract description 4
- 108010075254 C-Peptide Proteins 0.000 abstract description 4
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 238000003786 synthesis reaction Methods 0.000 abstract description 2
- 210000000227 basophil cell of anterior lobe of hypophysis Anatomy 0.000 description 22
- 210000002220 organoid Anatomy 0.000 description 22
- 238000010166 immunofluorescence Methods 0.000 description 19
- 230000006698 induction Effects 0.000 description 19
- 210000002889 endothelial cell Anatomy 0.000 description 11
- 230000024245 cell differentiation Effects 0.000 description 10
- 239000002609 medium Substances 0.000 description 7
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 6
- 210000002242 embryoid body Anatomy 0.000 description 6
- 239000012091 fetal bovine serum Substances 0.000 description 6
- 238000011081 inoculation Methods 0.000 description 6
- 238000002054 transplantation Methods 0.000 description 6
- 239000003550 marker Substances 0.000 description 5
- 210000000496 pancreas Anatomy 0.000 description 5
- 239000000654 additive Substances 0.000 description 4
- 239000011324 bead Substances 0.000 description 4
- 239000006285 cell suspension Substances 0.000 description 4
- 239000008188 pellet Substances 0.000 description 4
- 210000003556 vascular endothelial cell Anatomy 0.000 description 4
- 101000583553 Homo sapiens Phosphoglucomutase-1 Proteins 0.000 description 3
- 241000124008 Mammalia Species 0.000 description 3
- 102100030999 Phosphoglucomutase-1 Human genes 0.000 description 3
- 239000002870 angiogenesis inducing agent Substances 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 210000001671 embryonic stem cell Anatomy 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 238000010899 nucleation Methods 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 102100028098 Homeobox protein Nkx-6.1 Human genes 0.000 description 2
- 101000578254 Homo sapiens Homeobox protein Nkx-6.1 Proteins 0.000 description 2
- 102100024616 Platelet endothelial cell adhesion molecule Human genes 0.000 description 2
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 description 2
- 238000004220 aggregation Methods 0.000 description 2
- 210000000013 bile duct Anatomy 0.000 description 2
- 239000006143 cell culture medium Substances 0.000 description 2
- 239000002771 cell marker Substances 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000003511 endothelial effect Effects 0.000 description 2
- 108010082117 matrigel Proteins 0.000 description 2
- 230000001338 necrotic effect Effects 0.000 description 2
- 230000000250 revascularization Effects 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 102000029816 Collagenase Human genes 0.000 description 1
- 108060005980 Collagenase Proteins 0.000 description 1
- 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 description 1
- 241000282412 Homo Species 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 230000019552 anatomical structure morphogenesis Effects 0.000 description 1
- 230000001772 anti-angiogenic effect Effects 0.000 description 1
- 230000001363 autoimmune Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 230000008614 cellular interaction Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 229960002424 collagenase Drugs 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 230000001079 digestive effect Effects 0.000 description 1
- 208000016097 disease of metabolism Diseases 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 201000001421 hyperglycemia Diseases 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 208000030159 metabolic disease Diseases 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 210000004923 pancreatic tissue Anatomy 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 210000003240 portal vein Anatomy 0.000 description 1
- 230000001023 pro-angiogenic effect Effects 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 210000005166 vasculature Anatomy 0.000 description 1
- 230000035899 viability Effects 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
- C12N5/0676—Pancreatic cells
- C12N5/0677—Three-dimensional culture, tissue culture or organ culture; Encapsulated cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0697—Artificial constructs associating cells of different lineages, e.g. tissue equivalents
-
- 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
- C12N5/0676—Pancreatic cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/069—Vascular Endothelial cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/117—Keratinocyte growth factors (KGF-1, i.e. FGF-7; KGF-2, i.e. FGF-12)
-
- 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
- C12N2501/10—Growth factors
- C12N2501/15—Transforming growth factor beta (TGF-β)
-
- 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
- C12N2501/10—Growth factors
- C12N2501/16—Activin; Inhibin; Mullerian inhibiting substance
-
- 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
- C12N2501/30—Hormones
- C12N2501/38—Hormones with nuclear receptors
- C12N2501/385—Hormones with nuclear receptors of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
-
- 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
- C12N2501/30—Hormones
- C12N2501/38—Hormones with nuclear receptors
- C12N2501/395—Thyroid hormones
-
- 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
- C12N2501/40—Regulators of development
- C12N2501/41—Hedgehog proteins; Cyclopamine (inhibitor)
-
- 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
- C12N2501/70—Enzymes
- C12N2501/72—Transferases (EC 2.)
- C12N2501/727—Kinases (EC 2.7.)
-
- 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
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/22—Coculture with; Conditioned medium produced by pancreatic cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/28—Vascular endothelial cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2513/00—3D culture
-
- 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
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Vascular Medicine (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
本发明属于血管化器官制备技术领域,具体涉及一种血管化胰岛及其制备方法,包括细胞球诱导步骤与细胞球融合培养步骤。其有益效果在于,制备得到的胰岛中形成明显的网络状血管结构,包覆于胰岛组织中;血管网络的形成促进了胰岛球在体外培养时的存活率;血管化促进胰腺前体细胞的分化效率;血管可以明显的促进胰岛素前体C‑peptide的合成。操作简单,具有较高的可重复性,从而可以批量生成所需数量的血管化胰岛;获得的血管化胰岛相比于无血管的胰岛有更强的细胞活性,可以持续长时间培养。
Description
技术领域
本发明属于血管化器官制备技术领域,具体涉及一种血管化胰岛及其制备方法。
背景技术
糖尿病是由胰岛内产生胰岛素的β细胞功能丧失所导致的慢性代谢疾病,全球至少有4.6亿人受此病痛的折磨。而1型糖尿病 ( Type 1 diabetes, T1D ) 是由自身免疫系统破坏β细胞导致病人高血糖。现在虽然可以通过门静脉移植胰岛可以缓解病人对胰岛素的依赖,但由于供体的短缺,移植物移植后即刻失去功能等问题,迫使研究者们探究有效的治疗手段。
胰岛具有高度的血管化,这使其具有快速分泌胰岛素以应对血糖变化的重要能力。虽然胰岛只占胰腺组织的1-2%,但它却接收了5-10%的胰腺血流。胰岛内的血管密度比周围的外分泌组织的血管密度更大大。
在胰岛发育的发育过程中,内皮细胞-内分泌细胞的相互作用指导胰腺分化和形态发生。但是之前关于胰岛细胞的体外分化缺少血管的介入,导致分化效率不理想,因此合适的血管网络对于胰岛的分化值得深入探究。
对于体外培养的胰岛或诱导形成的胰岛细胞团,随着胰岛球体积的增大,胰岛中心形成缺氧核心或坏死中心,这会造成胰岛碎片化,影响其功能和活性,不利于后续的研究及移植。在胰岛移植手术中,移植后的胰岛数天内无血管,这是导致大量移植的胰岛死亡的原因。因此,对胰岛进行体外血管重建是非常必要的,这对提升胰岛存活率和胰岛移植的预后均有帮助。
为解决上述问题,现有技术中提出了如下方法:
方法一:增加促血管生成因子的作用或抑制抗血管生成因子,从而刺激内皮细胞的增殖、迁移和成熟。
这种方法虽获得了一定的成效,但要形成成熟的、功能完整的胰岛血管系统,可能需要精确控制移植后血管生成因子作用的时间、剂量和持续时间,重建过程比较困难。
方法二:直接聚焦于内皮细胞,使其形成成熟、有功能的血管,这可能涉及到添加预先活化的内皮细胞或某些类型的内皮祖细胞群。
但是这些都无法形成良好的血管网络,也不能完全模拟真实的血管结构。因此,建立一种血管化的胰岛,对实现移植后快速血运重建、维持胰岛活力具有重要意义。
发明内容
本发明的目的在于构建一种血管化的胰岛,其血管网络的特点为功能化可灌注,可以促进胰岛的活性。
为实现上述目的,本发明采用的技术方案为:
一种血管化胰岛的制备方法,包括如下步骤:
步骤1、分别制备血管细胞球及胰岛细胞球;其中,血管细胞球是由能够分化为血管谱系细胞的细胞诱导分化形成,胰岛细胞球是由哺乳类器官分离获取或由具有分化为胰岛细胞的细胞诱导分化形成;
步骤2、将步骤1制备的一个或多个血管细胞球和1个或多个胰岛细胞球融合、并培养后制备得到血管化胰岛类器官;
进一步的、步骤2制备血管细胞球的制备过程为将能够分化为血管谱系细胞的细胞培养形成拟球体后分化为血管细胞球;或先在2D条件下将干细胞诱导分化为血管细胞后再聚集成球;
进一步的、步骤2制备胰岛细胞球的制备过程为将能够分化为胰岛细胞的细胞培养形成拟球体后分化为胰岛细胞球;或先在2D条件下将干细胞诱导分化为胰岛细胞后再聚集成球;或在哺乳类胰腺器官分离提取获得胰岛;
优选的,当细胞同时能够分化为血管谱系细胞和胰岛细胞时,步骤2制备血管细胞球的制备过程为先将细胞培养成拟球体(EBs)后分为两份,并分别诱导分化为血管细胞球和胰岛细胞球;
进一步的,步骤2中所述的融合过程,采用血管细胞球和胰岛细胞球在悬浮培养条件或包埋入水凝胶等材料中融合;
优选的,所述融合步骤采用悬浮培养条件下融合。
进一步的,步骤2中,所述的胰岛细胞球和血管细胞球的数量比例为1:1~10:1。
进一步的,步骤2中,所述胰岛细胞球的数量为1~10个。
进一步的,步骤2中,所述血管细胞球的数量为1~5个。
优选的,步骤2中,所述胰岛细胞球的数量为5个;所述血管细胞球的数量为5个。
进一步的,步骤2中,所述培养的时间为3~5天,所述的血管化胰岛直径尺寸为100-500μm。
本发明还公开了一种血管化胰岛,采用上述方法制备而成。
本发明的有益效果在于:
1、制备得到的胰岛器官中形成明显的网络状血管结构,包覆于胰岛组织中;
2、血管网络的形成促进了胰岛球在体外培养时的存活率;
3、血管化促进胰腺前体细胞的分化效率;
4、血管可以明显的促进胰岛素前体C-peptide的合成;
5、操作简单,具有较高的可重复性,从而可以批量生成所需数量的血管化胰岛类器官;
6、获得的血管化胰岛类器官相比于无血管的胰岛有更强的细胞活性,可以持续长时间培养;
7、血管可以促进胰岛祖细胞和胰岛β细胞的分化。
附图说明
图1为实施例1、实施例2使用的hiPSC诱导胰岛β细胞的诱导体系图;
图2为实施例1、实施例2实施例所使用的血管内皮细胞的诱导方法图;
图3为实施例1的hiPSC形成的EBs和诱导成的胰腺前体细胞球的明场图;
图4为实施例1的血管化胰岛类器官PI染色免疫荧光照片及统计图;
图5为实施例1的血管促进胰岛祖细胞分化的免疫荧光照片及统计图;
图6为实施例1的血管促进胰岛β细胞分化的免疫荧光照片及统计图;
图7为实施例1的血管化的胰岛类器官免疫荧光照片;
图8为实施例2的hiPSC诱导形成的胰腺前体细胞和内皮细胞的明场图;
图9为实施例2的hiPSC诱导形成的胰腺前体细胞和内皮细胞鉴定图;
图10为实施例2的血管化胰岛类器官聚集成团的明场图;
图11为实施例2的血管促进胰岛祖细胞分化的免疫荧光照片及其统计图;
图12为实施例2的血管促进胰岛β细胞分化的免疫荧光照片及其统计图;
图13为实施例2的血管化的胰岛类器官免疫荧光照片;
图14为实施例3的分离得到的小鼠胰岛DTZ染色图;
图15为实施例3的体外培养血管化/无血管化胰岛球存活率及无血管化的胰岛坏死中心图;
图16为实施例3血管化胰岛的免疫荧光图;
图17为实施例3的体外培养血管化/无血管化胰岛球后insulin的免疫荧光表达图;
图18为实施例4胰腺来源的胰岛单细胞与干细胞诱导的血管系细胞重聚成团的形态图;
图19为实施例4重聚的血管化胰岛团的免疫荧光图。
具体实施方式
下面结合实施例对本发明的具体实施方式作进一步描述,以下实施例仅用于更加清楚地说明本发明的技术实施例,而不能以此来限制本发明的保护范围。
实施例1
一种血管化胰岛类器官的制备方法,包括如下步骤:
(1)将hiPSC消化成单细胞悬液,按照1:3的比例传代至超低吸附孔板中,使用干细胞培养基PGM1培养1天,使其形成拟胚体(EBs);
(2)EBs形成2天后开始使用成分明确的胰岛祖细胞诱导体系开始为期8天(前3阶段)的胰岛祖细胞的诱导,此时记为诱导前第8天(Day-8);
本实施例所使用的胰岛细胞分化方法中分化阶段、分化天数、分化培养基、分化所使用的添加剂如附图1和图2所示,添加剂所使用的浓度为表1所示;
(3)EBs形成2天后开始使用成分明确的血管细胞球诱导体系开始为期8天的血管细胞球的诱导,此时记为第0天(Day0);
本实施例所使用的血管细胞球的诱导方法如图2所示;
(4)步骤1获得EBs细胞球、步骤2所获得的胰岛细胞球、步骤3所获得的血管细胞球如图3所示;
(5)将步骤2和3获得的1个胰岛祖细胞球和1个血管细胞球接种到超低吸附的96孔板的一个孔中(如图3);
(5)在接种后添加胰岛类器官的培养基并添加10%的胎牛血清(FBS)放入培养箱中过夜,促进细胞的存活和细胞球的融合;
(6)在接种的第2天开始,撤去FBS,使用PI染色发现血管的加入可以促进细胞的存活(如图3);
(7)换成成分明确的第4阶段诱导培养基继续胰岛前体细胞的诱导,在接种的第5天(Day5),利用免疫荧光技术检测胰岛前体细胞的标志物Nkx6.1,发现血管可以促进胰岛前体细胞的分化,血管化胰岛类器官中胰岛前体细胞的比例显著高于无血管化组(23% vs16%)(如图4);
(8)更换第7阶段胰岛β细胞诱导培养基继续诱导,接种第20天(Day20),利用免疫荧光技术检测胰岛β细胞的标志物C-peptide,发现血管可以进一步促进胰岛β细胞的分化,血管化胰岛类器官中胰岛前体细胞的比例显著高于无血管化组(30% vs 20%)(如图5);
(9)在血管化胰岛类器官形成的第20天,利用免疫荧光技术对血管内皮细胞(CD31阳性)和胰岛β细胞(c-peptide阳性)染色,发现本实施例构建了一种血管网络包覆的血管化胰岛类器官(如图6)。
(10)通过上述的实验结果可知,血管的加入可以促进胰岛细胞的存活,在胰岛细胞分化过程中,血管可以不仅可以促进胰岛前体细胞(胰岛祖细胞)的分化,而且会进一步促进胰岛β细胞的分化,证明了血管化对于胰岛类器官构架的重要作用。
本实施例所述的,hiPSC可以为任一多能诱导干细胞系或具有与hiPSC相似性质的人胚胎干细胞系(hESC)。
实施例2
一种血管化胰岛类器官的制备方法,包括如下步骤:
(1)将人胚胎干细胞(hESC)H1细胞系(H1)消化成单细胞悬液,按照1×105的接种密度传代至Matrigel包被的孔板中,使用干细胞培养基PGM1培养1-2天,培养至细胞80-90%的融合度,开始诱导β细胞,此时记为诱导前第8天(Day-8);
(2)将H1消化单细胞悬液,按照1x105的接种密度传代至Matrigel包被的孔板中,使用干细胞培养基PGM1培养1-2天,培养至细胞70-80%的融合度,开始诱导内皮细胞,此时记为诱导前第8天(Day-8);
本实施例所使用的胰岛细胞分化方法中分化阶段、分化天数、分化培养基、分化所使用的添加剂如附图1所示,添加剂所使用的浓度为表1所示;
本实施例所使用的血管内皮细胞的诱导方法如图2所示;
(3)分化至胰腺祖细胞阶段(Day0),通过形态学与免疫荧光鉴定其分化程度(如图7);
(4)将步骤2中分化得到的Day-8的细胞使用CD31磁珠纯化后得到的内皮细胞通过形态学和免疫荧光鉴定内皮细胞标志物(CD31),证明本实施例获得了高纯度的内皮细胞(如图8);
(4)将胰岛祖细胞球与内皮细胞球按照3:1的比例混合,将其包埋于活性水凝胶中使其聚集成团。在接种后添加胰岛类器官的培养基并添加10%的胎牛血清(FBS)放入培养箱中过夜,促进细胞的存活和细胞球的融合(如图9);
(5)在接种的第2天开始,撤去FBS,换成成分明确的第4阶段诱导培养基继续胰岛前体细胞的诱导;
(6)换成成分明确的第4阶段诱导培养基继续胰岛前体细胞的诱导,在接种的第5天(Day5),利用免疫荧光技术检测胰岛前体细胞的标志物Nkx6.1,发现血管可以促进胰岛前体细胞的分化,血管化胰岛类器官中胰岛前体细胞的比例显著高于无血管化组(25% vs16%)(如图10);
(8)更换第7阶段胰岛β细胞诱导培养基继续诱导,接种第20天(Day20),利用免疫荧光技术检测胰岛β细胞的标志物C-peptide,发现血管可以进一步促进胰岛β细胞的分化,血管化胰岛类器官中胰岛前体细胞的比例显著高于无血管化组(27% vs 18%)(如图11);
(9)在血管化胰岛类器官形成的第20天,利用免疫荧光技术对血管内皮细胞(CD31阳性)和胰岛β细胞(c-peptide阳性)染色,发现本实施例构建了一种血管网络包覆的血管化胰岛类器官(如图12)。
(10)通过上述的实验结果可知,血管的加入可以促进胰岛细胞的存活,在胰岛细胞分化过程中,血管可以不仅可以促进胰岛前体细胞(胰岛祖细胞)的分化,而且会进一步促进胰岛β细胞的分化,证明了血管化对于胰岛类器官构架的重要作用。
本实施例所述的,hiPSC可以为任一多能诱导干细胞系或具有与hiPSC相似性质的人胚胎干细胞系(hESC)。
实施例3
一种血管化胰岛的制备方法,包括如下步骤:
(1)按照实施例1或实施例2制备获得血管细胞球;
(2)按照已有的胰岛分离纯化的步骤提取小鼠的胰岛;简述的,脊椎处死小鼠后,消毒后打开腹腔暴露出胆管,经胆管缓慢向胰腺注射胶原酶P使其充盈,取下充分灌注的胰腺进行酶消化,消化后摇匀并终止消化,离心过细胞筛,将筛选到的组织经过梯度离心,获得胰岛(如图13);
(3)将已获得的血管细胞球及小鼠胰岛按照4:1比例在体外悬浮培养;
(4)在体外培养1周后,通过检测胰岛球的存活率可知,在血管细胞球的存在下,大部分胰岛以球的形式至少可以培养一周,而不加血管细胞球的胰岛则至少有40%会碎片化,还会形成较大的坏死中心(如图14);
(5)在体外培养2周后,通过检测胰岛球内部胰岛β细胞的标志物insulin的表达可知,在血管细胞球的存在下,存活胰岛球insulin的表达会显著高于不加血管细胞球的胰岛的表达(如图15);
(6)通过上述的实验结果可知,血管的加入不仅可以促进胰岛细胞的存活,还可以维持胰岛β细胞的活性。
本实施例所述的,分离纯化的胰岛不仅仅限于小鼠胰岛,也可以是从任何哺乳类动物中原代分离的胰岛,包括从人体中取出的胰岛。
实施例4
一种血管化胰岛的制备方法,包括如下步骤:
(1)按照实施例1或实施例2制备获得血管细胞球;
(2)按照实施例3分离纯化获得胰岛;
(3)将纯化得到的胰岛至于0.25%的胰酶消化液中,37℃培养箱中消化3分钟,消化终止后,将胰岛细胞重悬为单细胞悬液并计数;
(4)将已获得的血管细胞球及胰岛细胞按照细胞数比例1:5比例在体外悬浮培养;
(5)体外培养2周后,从形态学观察,并不是所有的胰岛细胞都能与血管细胞球结合,只有少量的细胞与血管细胞球聚集形成团(如图16);
(6)通过免疫荧光检测内皮细胞的标记物CD31和胰岛β细胞的标志物insulin可知,重新聚团的胰岛球能与血管球聚集生长且不丢失其标记物的表达,但是胰岛团的血管化程度较低,可能需要优化培养方法(如图17);
(7)通过上述的实验结果可知,当将分离的胰岛消化为单细胞加入到血管细胞球的培养体系中,按照上述细胞比例,胰岛细胞只有少部分会生长到血管球内部,这可能需要进一步优化培养聚集的体系。
本实施例所述的,分离纯化的胰岛不仅仅限于小鼠胰岛,也可以是从任何哺乳类动物中原代分离的胰岛,包括从人体中取出的胰岛。
最后应说明的是:以上各实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述各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,或者对其中部分或者全部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范围。
Claims (6)
1.一种血管化胰岛的制备方法,其特征在于,包括如下步骤:
步骤1、分别制备血管细胞球及胰岛细胞球;其中,血管细胞球是由能分化为血管谱系的细胞诱导分化形成,胰岛细胞球是由哺乳类器官分离获取或由能分化为胰岛细胞的细胞诱导分化形成;
步骤2、将步骤1制备的1个或多个血管细胞球和1个或多个胰岛细胞球融合、并培养后制备得到血管化胰岛;
步骤1制备血管细胞球的制备过程为将能分化为血管谱系的细胞培养形成拟球体后分化为血管细胞球;或先在2D条件下将干细胞诱导分化为血管细胞后再聚集成球;
步骤1制备胰岛细胞球的制备过程为将能分化为胰岛细胞的细胞培养形成拟球体后分化为胰岛细胞球;或先在2D条件下将干细胞诱导分化为胰岛细胞后再聚集成球;
当细胞同时具有分化为血管谱系细胞和胰岛细胞的能力时,步骤1制备血管细胞球和胰岛细胞球的制备过程为先将细胞培养成拟球体后分为两份,并分别诱导分化为血管细胞球和胰岛细胞球。
2.根据权利要求1所述的血管化胰岛的制备方法,其特征在于,步骤2中所述的融合过程,采用血管细胞球和胰岛细胞球在悬浮培养条件或包埋于水凝胶等材料中融合。
3.根据权利要求1所述的血管化胰岛的制备方法,其特征在于,步骤2中,所述胰岛细胞球和血管细胞球的数量比为1:1~10:1。
4.根据权利要求1所述的血管化胰岛的制备方法,其特征在于,步骤2中,所述胰岛细胞球的数量为1~10个。
5.根据权利要求1所述的血管化胰岛的制备方法,其特征在于,步骤2中,所述胰岛细胞球的数量为5个;所述血管细胞球的数量为5个。
6.根据权利要求1所述的血管化胰岛的制备方法,其特征在于,步骤2中,融合得到的血管化胰岛球大小为100-500μm。
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202211277768.4A CN115340977B (zh) | 2022-10-19 | 2022-10-19 | 一种血管化胰岛及其制备方法 |
US18/381,660 US20240228974A9 (en) | 2022-10-19 | 2023-10-19 | Vascularized islet and preparation method thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202211277768.4A CN115340977B (zh) | 2022-10-19 | 2022-10-19 | 一种血管化胰岛及其制备方法 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN115340977A CN115340977A (zh) | 2022-11-15 |
CN115340977B true CN115340977B (zh) | 2023-03-31 |
Family
ID=83957177
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202211277768.4A Active CN115340977B (zh) | 2022-10-19 | 2022-10-19 | 一种血管化胰岛及其制备方法 |
Country Status (2)
Country | Link |
---|---|
US (1) | US20240228974A9 (zh) |
CN (1) | CN115340977B (zh) |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2008111064A1 (en) * | 2007-03-12 | 2008-09-18 | Technion Research & Development Foundation Ltd. | Vascularized islets and methods of producing same |
WO2014141528A1 (ja) * | 2013-03-15 | 2014-09-18 | 国立大学法人佐賀大学 | 心臓又は血管組織型スフェロイド |
CN114107173A (zh) * | 2021-11-15 | 2022-03-01 | 北京赛拉达生物科技有限公司 | 一种血管化胰岛微器官及其构建方法 |
-
2022
- 2022-10-19 CN CN202211277768.4A patent/CN115340977B/zh active Active
-
2023
- 2023-10-19 US US18/381,660 patent/US20240228974A9/en active Pending
Also Published As
Publication number | Publication date |
---|---|
CN115340977A (zh) | 2022-11-15 |
US20240132850A1 (en) | 2024-04-25 |
US20240228974A9 (en) | 2024-07-11 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
KR100374976B1 (ko) | 기능성랑게르한스섬의시험관내성장및그생체내사용방법 | |
Ricordi et al. | Automated islet isolation from human pancreas | |
US20020187551A1 (en) | Method for culturing langerhans islets and islet autotransplantation islet regeneration | |
WO2022183740A1 (zh) | 一种制备胰腺beta细胞的方法及其应用 | |
KR101293364B1 (ko) | 조직공학적 인간 각막 내피의 재건 방법 | |
CN110283777B (zh) | 一种对虾细胞连续培养方法 | |
US11453863B2 (en) | Pancreatic islet-like cell structures and a method of preparing thereof | |
CN113215094A (zh) | 逆转2型糖尿病胰岛β细胞去分化的间充质干细胞外泌体及其制备方法和应用 | |
CN114107173A (zh) | 一种血管化胰岛微器官及其构建方法 | |
CN103845362A (zh) | 一种用于治疗糖尿病的干细胞制剂及其制备方法 | |
WO2023217132A1 (zh) | 胆囊前体样细胞的制备方法和应用 | |
US20230323263A1 (en) | Special culture apparatus for 3d biological tissue, and method for preparing block-shaped cultured meat | |
CN115340977B (zh) | 一种血管化胰岛及其制备方法 | |
CN116121173B (zh) | 一种眼组织类器官及其衍生细胞系、其制备方法和应用 | |
CN112760289A (zh) | 一种乳腺癌类器官专用培养基及3d培养方法 | |
CN112592883B (zh) | 一种小鼠胰腺类器官培养基及其应用 | |
CN110923192B (zh) | 一种成熟肝细胞的长期体外培养方法 | |
CN113101302A (zh) | 一种自体MSCs体内胰岛归巢分化治疗糖尿病的研究方法 | |
CN113106066A (zh) | 一种肉瘤细胞培养基以及采用该培养基体外生产Matrigel原液的方法 | |
KR20150115304A (ko) | 중간엽 줄기세포 스페로이드로부터 분화된 인슐린 분비세포의 제조방법 및 그의 용도 | |
CN117187174B (zh) | 一种Muse细胞培养基以及一种脂肪Muse细胞的提取方法 | |
CN109486770A (zh) | 一种microRNA-181c-5p促进人源iPS定向分化为胰岛β细胞的方法 | |
CN117286108B (zh) | 一种乳腺癌类器官专用培养基及培养方法 | |
WO2024088255A1 (zh) | 一种自发永生化的猪肌源性干细胞系及其应用 | |
CN109055305B (zh) | 一种奶牛肝干细胞的分离提取方法 |
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 |