CN102105581A - 人工肾前体及其制备方法 - Google Patents
人工肾前体及其制备方法 Download PDFInfo
- Publication number
- CN102105581A CN102105581A CN2009801261256A CN200980126125A CN102105581A CN 102105581 A CN102105581 A CN 102105581A CN 2009801261256 A CN2009801261256 A CN 2009801261256A CN 200980126125 A CN200980126125 A CN 200980126125A CN 102105581 A CN102105581 A CN 102105581A
- Authority
- CN
- China
- Prior art keywords
- metanephros
- precursor
- erythropoietin
- kidney
- mammals
- 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.)
- Granted
Links
- 210000003734 kidney Anatomy 0.000 title claims abstract description 66
- 239000002243 precursor Substances 0.000 title claims abstract description 61
- 238000000034 method Methods 0.000 title claims abstract description 37
- 238000007710 freezing Methods 0.000 claims abstract description 27
- 230000008014 freezing Effects 0.000 claims abstract description 27
- 238000010257 thawing Methods 0.000 claims abstract description 6
- 102000003951 Erythropoietin Human genes 0.000 claims description 40
- 108090000394 Erythropoietin Proteins 0.000 claims description 40
- 229940105423 erythropoietin Drugs 0.000 claims description 40
- OXCMYAYHXIHQOA-UHFFFAOYSA-N potassium;[2-butyl-5-chloro-3-[[4-[2-(1,2,4-triaza-3-azanidacyclopenta-1,4-dien-5-yl)phenyl]phenyl]methyl]imidazol-4-yl]methanol Chemical compound [K+].CCCCC1=NC(Cl)=C(CO)N1CC1=CC=C(C=2C(=CC=CC=2)C2=N[N-]N=N2)C=C1 OXCMYAYHXIHQOA-UHFFFAOYSA-N 0.000 claims description 40
- 241000124008 Mammalia Species 0.000 claims description 32
- 210000000130 stem cell Anatomy 0.000 claims description 27
- 210000001161 mammalian embryo Anatomy 0.000 claims description 22
- 238000012545 processing Methods 0.000 claims description 4
- 210000002901 mesenchymal stem cell Anatomy 0.000 abstract description 5
- 238000000338 in vitro Methods 0.000 abstract 2
- 241001465754 Metazoa Species 0.000 description 14
- 210000004027 cell Anatomy 0.000 description 11
- 238000004321 preservation Methods 0.000 description 11
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 9
- 239000007788 liquid Substances 0.000 description 7
- 241000282693 Cercopithecidae Species 0.000 description 6
- 208000007502 anemia Diseases 0.000 description 6
- 210000000056 organ Anatomy 0.000 description 6
- 201000010099 disease Diseases 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 108700018351 Major Histocompatibility Complex Proteins 0.000 description 4
- 206010058116 Nephrogenic anaemia Diseases 0.000 description 4
- 230000035479 physiological effects, processes and functions Effects 0.000 description 4
- 230000020382 suppression by virus of host antigen processing and presentation of peptide antigen via MHC class I Effects 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 241000283707 Capra Species 0.000 description 3
- 241000699800 Cricetinae Species 0.000 description 3
- 241000283073 Equus caballus Species 0.000 description 3
- 241000282326 Felis catus Species 0.000 description 3
- 241000282560 Macaca mulatta Species 0.000 description 3
- 241000772415 Neovison vison Species 0.000 description 3
- 241000283973 Oryctolagus cuniculus Species 0.000 description 3
- 241000282577 Pan troglodytes Species 0.000 description 3
- 241001494479 Pecora Species 0.000 description 3
- 241000282405 Pongo abelii Species 0.000 description 3
- 241000288906 Primates Species 0.000 description 3
- 241000283984 Rodentia Species 0.000 description 3
- 238000010171 animal model Methods 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 241001515942 marmosets Species 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 238000002054 transplantation Methods 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 208000001647 Renal Insufficiency Diseases 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 210000004204 blood vessel Anatomy 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 239000002577 cryoprotective agent Substances 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000007236 host immunity Effects 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 201000006370 kidney failure Diseases 0.000 description 2
- 108010082117 matrigel Proteins 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 210000003360 nephrocyte Anatomy 0.000 description 2
- 230000035764 nutrition Effects 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 238000011476 stem cell transplantation Methods 0.000 description 2
- 238000009631 Broth culture Methods 0.000 description 1
- 108091016585 CD44 antigen Proteins 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 239000006145 Eagle's minimal essential medium Substances 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 102000008055 Heparan Sulfate Proteoglycans Human genes 0.000 description 1
- 229920002971 Heparan sulfate Polymers 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 101000718529 Saccharolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2) Alpha-galactosidase Proteins 0.000 description 1
- 108090000054 Syndecan-2 Proteins 0.000 description 1
- 208000025865 Ulcer Diseases 0.000 description 1
- 210000000577 adipose tissue Anatomy 0.000 description 1
- NNISLDGFPWIBDF-MPRBLYSKSA-N alpha-D-Gal-(1->3)-beta-D-Gal-(1->4)-D-GlcNAc Chemical compound O[C@@H]1[C@@H](NC(=O)C)C(O)O[C@H](CO)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)O)[C@@H](O)[C@@H](CO)O1 NNISLDGFPWIBDF-MPRBLYSKSA-N 0.000 description 1
- 210000004381 amniotic fluid Anatomy 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000005784 autoimmunity Effects 0.000 description 1
- 230000035578 autophosphorylation Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000003995 blood forming stem cell Anatomy 0.000 description 1
- 238000009583 bone marrow aspiration Methods 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 210000001612 chondrocyte Anatomy 0.000 description 1
- 208000020832 chronic kidney disease Diseases 0.000 description 1
- 208000022831 chronic renal failure syndrome Diseases 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000002338 cryopreservative effect Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 210000004700 fetal blood Anatomy 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000002682 general surgery Methods 0.000 description 1
- 238000012252 genetic analysis Methods 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000006058 immune tolerance Effects 0.000 description 1
- 238000012744 immunostaining Methods 0.000 description 1
- 238000002513 implantation Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 210000002977 intracellular fluid Anatomy 0.000 description 1
- 230000003908 liver function Effects 0.000 description 1
- 210000003716 mesoderm Anatomy 0.000 description 1
- 239000007758 minimum essential medium Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000002988 nephrogenic effect Effects 0.000 description 1
- 108010008217 nidogen Proteins 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 210000002220 organoid Anatomy 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 231100000397 ulcer Toxicity 0.000 description 1
- 230000002620 ureteric effect Effects 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/12—Drugs for disorders of the urinary system of the kidneys
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
- A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
- A61F2/02—Prostheses implantable into the body
- A61F2/04—Hollow or tubular parts of organs, e.g. bladders, tracheae, bronchi or bile ducts
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/22—Urine; Urinary tract, e.g. kidney or bladder; Intraglomerular mesangial cells; Renal mesenchymal cells; Adrenal gland
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/54—Biologically active materials, e.g. therapeutic substances
-
- 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
- C12N5/0684—Cells of the urinary tract or kidneys
- C12N5/0686—Kidney cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified 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
- C12N2502/02—Coculture with; Conditioned medium produced by 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
- C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
- C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal 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
- C12N2523/00—Culture process characterised by temperature
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Cell Biology (AREA)
- Urology & Nephrology (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Epidemiology (AREA)
- Transplantation (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Pharmacology & Pharmacy (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Dermatology (AREA)
- Virology (AREA)
- Immunology (AREA)
- Developmental Biology & Embryology (AREA)
- Heart & Thoracic Surgery (AREA)
- Pulmonology (AREA)
- Cardiology (AREA)
- Gastroenterology & Hepatology (AREA)
- Vascular Medicine (AREA)
- Molecular Biology (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Botany (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了包含从生物体中取出的非人类哺乳动物后肾的人工肾前体,其中所述后肾在生物体外已受到冷冻和解冻处理,并且包含在生物体外转移入其中的哺乳动物间充质干细胞。
Description
技术领域
本发明涉及人工肾前体及其制备方法。
背景技术
肾是产生红细胞生成素的主要器官;伴随肾衰竭的红细胞生成素产生的减少引起作为并发症的肾性贫血。
近年来,已提出作为用于肾性贫血的下一代治疗方法的方法,其中通过再生医学技术制备具有产生红细胞生成素潜力的人工肾或其前体,并将其移植至肾性贫血患者。例如,专利文件1公开了一种用于制备产生红细胞生成素的类器官(人工肾)前体的方法,其包括以下步骤:将分离的得自哺乳动物的间充质干细胞移植入怀孕的哺乳动物宿主体内的胚胎中或分离自怀孕的哺乳动物宿主的胚胎中,从而诱导间充质干细胞分化,其中将间充质干细胞移植入的部位为胚胎的肾发生部位,并且移植的时机对应于其中宿主的免疫***仍为免疫耐受的时期。
然而该方法中存在的问题为程序复杂,因为其需要正好在将人工肾前体移植至患者之前自怀孕的哺乳动物中采集羊水,并确认前体的生物安全性。此外,间充质干细胞必须注入胚胎中的可在将来形成肾的合适部位,这种操作需要高技巧。另外,因为间充质干细胞必须在宿主的免疫***处于免疫耐受状态时注入胚胎中,所以整个治疗时间表的灵活性低。在该方法中,除非进行全胚胎培养,否则难以得到有功能的人工肾前体,这就产生由于培养而引起污染的风险,并且在培养后需要进行复杂的纯化程序。
因此,存在对开发以下制备人工肾前体的方法的需要,所述方法中1)在移植至患者之前可容易地测试人工肾前体的生物安全性,2)操作简单,3)整个治疗时间表的灵活性可保证,以及4)不需要培养操作。
现有技术文件
专利文件
专利文件1:WO 2008/004598
发明概述
技术问题
本发明的目标是提供制备人工肾前体的方法,其中1)在移植至患者之前可容易地测试人工肾前体的生物安全性,2)操作简单,3)整个治疗时间表的灵活性可保证,以及4)不需要培养操作。
解决问题的方案
为实现上述目标,本发明人进行了广泛的研究;结果,本发明人发现上述问题可通过使用以下人工肾前体解决,所述人工肾前体通过冷冻和解冻分离自胚胎的后肾,并将得自患者的间充质干细胞转移入解冻的后肾得到,从而开发出本发明。
因此,本发明涉及以下内容。
[1]一种包含从生物体中分离出的非人类哺乳动物后肾的人工肾前体,其中所述后肾在生物体外已受到冷冻和解冻处理,并且包含在生物体外转移入的哺乳动物间充质干细胞。
[2][1]中描述的人工肾前体,其中所述后肾为处于MHC表达仍未成熟时期的哺乳动物胚胎中的后肾。
[3][1]中描述的人工肾前体,其中所述前体当移植入哺乳动物的网膜时能获得产生红细胞生成素的潜力。
[4]一种制备人工肾前体的方法,其包括以下步骤:
(I)冷冻从生物体中分离出的哺乳动物后肾;
(II)解冻所述冷冻的哺乳动物后肾;以及
(III)在冷冻(I)之前或解冻(II)之后,在生物体外将哺乳动物间充质干细胞转移入后肾中。
[5][4]中描述的方法,其中用于(I)中的后肾为从处于MHC表达仍未成熟时期的哺乳动物胚胎中分离出的后肾。
[6][4]中描述的方法,其中所述人工肾前体在移植入哺乳动物的网膜时能获得产生红细胞生成素的潜力。
发明效果
利用本发明的方法,可在移植前容易地检测人工肾前体的生物安全性。利用本发明的方法,通过简单操作就有可能制备人工肾前体。利用本发明的方法,可灵活性地设计整个治疗时间表,因为通过使用冷冻保存的后肾可在所需时机制备人工肾前体。此外,因为本发明的方法基本避免了对培养操作的需要,所以其并不包含污染的风险,也无需在培养后进行复杂的纯化程序。
实施方案的描述
本发明提供包含从生物体中分离出的哺乳动物后肾的人工肾前体,其中所述后肾在生物体外已受到冷冻和解冻处理,并且包含在生物体外转移入的哺乳动物间充质干细胞。
在本发明中,“人工肾”指的是非单个细胞的细胞组织,其具有产生红细胞生成素的潜力,这是肾天然具有的功能。本发明中的“人工肾”可能不具有过滤血液中的废料和水并排出尿的功能。除在其表面上的细胞之外,人工肾的各个细胞在三维上相互接触,相邻细胞通过各种胞间连接交换信息,并且可具有调节红细胞生成素产生的潜力。
“前体”指的是当置于生物体内时变成人工肾的组织。尽管“前体”具有产生红细胞生成素和调节红细胞生成素产生的潜力必需的因子,但是其处于由于环境因素而未显示产生红细胞生成素和调节红细胞生成素产生的潜力的状态。人工肾前体在移植至哺乳动物的网膜时能获得产生红细胞生成素的潜力。
“调节红细胞生成素产生的潜力”意指以下能力:当受体(患者)的身体需要比正常状态更大量的红细胞生成素时(贫血时等),产生大量的红细胞生成素(对改善贫血所需的量),以及在当受体(患者)处于正常状态时(贫血已改善时等),产生对维持身体中的正常状态所需量的红细胞生成素。也就是说,本发明的人工肾比常规产生红细胞生成素的细胞更具优势,这是因为其能产生受体(患者)所需量的红细胞生成素,并且能通过从网膜的血管中获取必需营养而长时间连续产生红细胞生成素。
在本发明中,“后肾”指的是在哺乳动物胚胎中对应于肾发生的部位。本发明中所用的后肾优选在开始发育前肾母细胞处于萌芽状态的后肾。后肾位于哺乳动物胚胎中的输尿管芽萌芽部位附近,更具体的是在体节和侧板之间。后肾优选为生后肾的中胚层。
作为从其中获得后肾的哺乳动物的实例,可指实验动物,例如啮齿动物例如小鼠、大鼠、仓鼠和豚鼠,以及兔;家畜,例如猪、牛、山羊、马、绵羊和水貂;伴侣动物(companion animal),例如狗和猫;灵长类,例如人、猴子、恒河猴、狨猴、猩猩和黑猩猩等。哺乳动物优选大鼠或猪。
后肾在生物体外从哺乳动物胚胎中分离出。就胚胎而言,其中MHC(主要组织相容性复合体)的表达仍未成熟的胚胎为适合使用的。通过使用处于该时期的后肾,可避免将人工肾前体移植至受体所伴随的免疫反应。当胚胎为非灵长类哺乳动物的胚胎时,优选在外源性抗原(糖链抗原例如α-Gal)出现前的时期内分离出后肾。例如,在使用大鼠的实验中,通常使用E(胚胎期天数)9-16、优选E10-15、更优选E11-14的胚胎。对于其它哺乳动物也一样,处于同等时期的胚胎可为适宜使用的。然而,只要条件为选定的,也可利用之前或之后的时期。可使用立体显微镜等进行从胚胎中分离后肾。
后肾的冷冻处理在常规用于冷冻哺乳动物组织或细胞的冷冻保存液中进行。冷冻保存液通常包含冷冻保护剂(cryopreservative)例如DMSO、甘油等。冷冻保护剂的浓度范围通常为5-30%重量,优选10-20%重量。保存液可包含血清。血清的浓度通常为5-50%重量,优选10-30%重量。作为优选的冷冻保存液,可指含DMSO和FCS的ET-KYOTO溶液、CELLBANKER(注册商标)等。
通过冷冻后肾,可抑制伴随着将人工肾前体移植至受体(患者)的免疫反应。
冷冻的后肾可以冷冻状态几乎永久地保存,并可根据需要在解冻和复苏后使用。冷冻保存期间的温度通常为-80℃至-200℃,优选-196℃(液氮中)。因为由于后肾的冷冻保存而可在所需时机制备人工肾前体,所以可灵活性地设计整个治疗时间表。
根据常规方法,在生理培养基中使冷冻的后肾受到解冻处理。解冻的方法没有具体的限制;例如,随着冷冻管在37℃调温水浴中漂浮时,通过向其中以滴状加入生理培养基解冻冷冻的后肾。为了除去冷冻保存液污染物,优选将解冻的后肾用生理肉汤培养基洗涤。
本发明的人工肾前体中所用的后肾包含在生物体外转移入的哺乳动物间充质干细胞。
“间充质干细胞”广泛地意指以未分化状态增殖并能分化成成骨细胞、成软骨细胞、成脂肪细胞等中的全部或一些的干细胞或其祖细胞群体。用于本发明的间充质干细胞具有分化成产生红细胞生成素的肾细胞的潜力。
作为从其中获得间充质干细胞的哺乳动物的实例,可指实验动物,例如啮齿动物例如小鼠、大鼠、仓鼠和豚鼠,以及兔;家畜,例如猪、牛、山羊、马、绵羊和水貂;伴侣动物,例如狗和猫;灵长类,例如人、猴子、恒河猴、狨猴、猩猩和黑猩猩等。哺乳动物优选大鼠或人。
从其中获得间充质干细胞的哺乳动物优选与意图将本发明人工肾前体移植入的受体在动物种上相同的动物。更优选的是,使用受体自身的间充质干细胞。
间充质干细胞可通过公知的普通方法收集自哺乳动物骨髓液、外周血、脐带血等。例如,可通过培养和传代通过骨髓穿刺获得的造血干细胞等分离人间充质干细胞(Journal of Autoimmunity,30(2008)163-171)。
分离自生物体的间充质干细胞还可通过在合适的培养基中进行粘附培养,在体外扩繁,同时保存其分化潜力。因此,间充质干细胞在体外的扩繁也落入本发明的范围内。就培养基而言,例如使用DMEM、EMEM、RPMI-1640、F-12、α-MEM、MSC生长培养基(BioWhittaker)等。培养温度通常为约30-40℃的范围内,优选约37℃。CO2浓度通常为约1-10%的范围内,优选约5%。湿度范围通常为约70-100%,优选约95-100%。为了维持高分化潜力,优选培养不延续超过2-5代以上。
将间充质干细胞转移入后肾中在冷冻后肾前或解冻后肾后进行。例如,将间充质干细胞在立体显微镜下使用微量吸液管等注入后肾中。转移入的细胞的数量根据后肾的大小等合适地确定;通常每个大鼠后肾注入约104-106个(例如,5×104个)间充质干细胞。
尽管悬浮于生理培养基中的间充质干细胞可用于转移,但是从防止在冷冻保存后的穿刺时漏出胞内液的观点看,适宜使用包封在含细胞外基质成分(层粘连蛋白、胶原IV型、巢蛋白、硫酸乙酰肝素蛋白聚糖、Matrigel(注册商标)等)的凝胶中的间充质干细胞。
本发明人工肾前体的大小无需接近于肾(产生红细胞生成素的器官)的大小;整个肾大小的1/50至1/10为足够的。
本发明的人工肾前体可通过进行以下方法制备:
(I)冷冻从生物体中分离出的哺乳动物后肾;
(II)解冻所述冷冻的哺乳动物后肾;以及
(III)在冷冻(I)之前或解冻(II)之后,将哺乳动物间充质干细胞在生物体外转移入后肾中。
各个术语的定义和操作的细节如上所述。
尽管通过上述方法得到的本发明人工肾前体可经过器官培养,但优选不进行器官培养,因为这样没有污染的风险,并且在培养后无需进行复杂的纯化程序。本发明的人工肾前体的优势在于其在移植入哺乳动物时无需经过所述器官培养程序,就能获得足够的产生红细胞生成素和调节红细胞生成素产生的潜力。可通过将人工肾前体放置在过滤器上,向其下的皿中加入合适的培养基,并将所述皿静置于培养箱中,来进行器官培养。
当将本发明的人工肾前体移植至受体哺乳动物的体内时,该前体移入(engraft)受体体内,而且该前体中所含的间充质干细胞增殖并分化成产生红细胞生成素的肾细胞。因此,人工肾前体分化成具有产生红细胞生成素和调节红细胞生成素产生的潜力的人工肾,其立即能通过从血管中获取必要的营养而长时间连续地在生物体内产生红细胞生成素。因此,本发明的人工肾前体可用于治疗红细胞生成素相关疾病。通过将本发明的人工肾前体移植至哺乳动物,可治疗哺乳动物中的红细胞生成素相关疾病。
在本发明中,“红细胞生成素相关疾病”为与红细胞生成素产生的量减少有关的疾病,包括与肾衰竭、糖尿病、溃疡、癌症、感染、透析、外科手术或化学治疗有关的贫血。具体而言,在由于慢性肾衰竭产生的贫血的情况下,由于肾实质和肝功能的进行性破坏导致不能产生红细胞生成素,因而在循环中的红细胞生成素浓度不会上升;这是主要的问题。
作为受体哺乳动物的实例,可指实验动物,例如啮齿动物例如小鼠、大鼠、仓鼠和豚鼠,以及兔;家畜,例如猪、牛、山羊、马、绵羊和水貂;伴侣动物,例如狗和猫;灵长类,例如人、猴子、恒河猴、狨猴、猩猩和黑猩猩等。受体哺乳动物优选大鼠或人。
尽管将本发明的人工肾前体移植至哺乳动物中的方法没有具体的限制,只要该前体可移入受体体内并获得产生红细胞生成素和调节红细胞生成素产生的潜力即可,但是优选将人工肾前体移植至受体哺乳动物的网膜中。可通过普通手术方法移植至网膜中;例如,可指以下方法,其包括用锋利的钳状骨针夹起要被移植的组织,用钳状骨针尖端在网膜中的脂肪组织表面上切个小切口,并将组织植入切口内。本发明的人工肾前体还可借助于内窥镜移植入网膜内。
本发明人工肾前体的剂量根据所给予的受试者、目标疾病、症状等而变化;一般而言,将制备自猪后肾的人工肾前体移植至成人肾性贫血患者(假定60kg体重)中时,在一次手术中移植约10个后肾并在监测患者贫血的严重程度时根据需要逐步增加后肾的数量,这都是合宜的。在其它动物的情况下,可移植根据60kg体重计算的量。
在下文中借助于以下实施例更详细地描述本发明,然而,不应以任何方式将本发明限制于以下实施例。
实施例
在怀孕14天时从大鼠胚胎中分离出后肾,然后在ET-Kyoto+10%DMSO+20%FCS中冷冻保存。直径为约2mm。
在冷冻保存2天后,将冷冻的后肾解冻。将大鼠间充质干细胞包封于Matrigel中,然后以1×104个细胞/后肾注入后肾中。此后,将后肾移植至同系大鼠的网膜中。
移植7天后,将已成长的后肾从接受后肾移植的大鼠中分离出。接受后肾移植的动物数量为2,其中各自从7个后肾当中分别成长出3个和6个。后肾长成直径6-8mm。红细胞生成素在已生长的人工肾前体中的产生通过与Transplantation VOL85,No11,June 15 2008中所述方法一样的方法证实,即对产生红细胞生成素的细胞进行免疫染色和遗传分析。
工业实用性
利用本发明的方法,在移植前可容易地测试人工肾前体的生物安全性。利用本发明的方法,通过简单操作就有可能制备人工肾前体。利用本发明的方法,可灵活性地设计整个治疗时间表,因为通过使用冷冻保存的后肾可在所需时机制备人工肾前体。此外,因为本发明的方法基本避免了对培养操作的需求,其不牵涉污染的风险,也无需在培养后进行复杂的纯化程序。
本申请基于在日本申请的专利申请号2008-173935(申请日:2008年7月2日),其内容通过本引用全文结合于本文中。
Claims (6)
1.一种包含从生物体中分离出的非人类哺乳动物后肾的人工肾前体,其中所述后肾在生物体外已受到冷冻和解冻处理,并且包含在生物体外转移入的哺乳动物间充质干细胞。
2.权利要求1的人工肾前体,其中所述后肾为处于MHC表达仍未成熟时期的哺乳动物胚胎中的后肾。
3.权利要求1的人工肾前体,其中所述前体在移植入哺乳动物的网膜时能获得产生红细胞生成素的潜力。
4.一种制备人工肾前体的方法,所述方法包括以下步骤:
(I)冷冻从生物体中分离出的哺乳动物后肾;
(II)解冻所述冷冻的哺乳动物后肾;以及
(III)在冷冻(I)之前或解冻(II)之后,在生物体外将哺乳动物间充质干细胞转移入后肾中。
5.权利要求4的方法,其中(I)中所用的后肾为从处于MHC表达仍未成熟时期的哺乳动物胚胎中分离出的后肾。
6.权利要求4的方法,其中所述人工肾前体在移植入哺乳动物的网膜时能获得产生红细胞生成素的潜力。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2008173935 | 2008-07-02 | ||
JP2008-173935 | 2008-07-02 | ||
PCT/JP2009/062098 WO2010001951A1 (ja) | 2008-07-02 | 2009-07-02 | 人工腎臓前駆体およびその製造方法 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN102105581A true CN102105581A (zh) | 2011-06-22 |
CN102105581B CN102105581B (zh) | 2013-09-04 |
Family
ID=41466042
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN2009801261256A Expired - Fee Related CN102105581B (zh) | 2008-07-02 | 2009-07-02 | 人工肾前体及其制备方法 |
Country Status (13)
Country | Link |
---|---|
US (2) | US20110104656A1 (zh) |
EP (1) | EP2298866A4 (zh) |
JP (1) | JP5388138B2 (zh) |
KR (1) | KR20110043580A (zh) |
CN (1) | CN102105581B (zh) |
AU (1) | AU2009266761A1 (zh) |
CA (1) | CA2729765A1 (zh) |
HK (1) | HK1159171A1 (zh) |
IL (1) | IL210227A0 (zh) |
NZ (1) | NZ590122A (zh) |
RU (1) | RU2511395C2 (zh) |
TW (1) | TWI389696B (zh) |
WO (1) | WO2010001951A1 (zh) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109642210A (zh) * | 2016-06-29 | 2019-04-16 | 横尾隆 | 一种肾脏的制造方法 |
US11607425B2 (en) | 2016-06-29 | 2023-03-21 | Takashi Yokoo | Kidney production method |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP6185902B2 (ja) * | 2014-12-18 | 2017-08-23 | オートリブ ディベロップメント エービー | カーテンエアバッグ装置 |
US10335263B2 (en) | 2014-12-19 | 2019-07-02 | Bios Co., Ltd. | Organ for transplantation and organ structure |
Family Cites Families (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5217860A (en) * | 1991-07-08 | 1993-06-08 | The American National Red Cross | Method for preserving organs for transplantation by vitrification |
US6087164A (en) | 1997-10-03 | 2000-07-11 | Yissum Research Development Company Of The Hebrew University Of Jerusalem | Methods and compositions for inducing tumor-specific cytotoxicity |
WO2002061053A1 (en) * | 2001-01-31 | 2002-08-08 | The General Hospital Corporation | Renal stem cells and uses thereof |
CN100335015C (zh) * | 2001-05-17 | 2007-09-05 | 钟渊化学工业株式会社 | 用于构建具有蛋白代谢功能的体内型人工肾的组合 |
CN1879901A (zh) * | 2005-03-24 | 2006-12-20 | 彭罗民 | 多功能便移式人工肾 |
WO2006117889A1 (ja) * | 2005-04-28 | 2006-11-09 | Stemcell Institute Inc. | 移植用臓器の調製方法 |
WO2007025233A1 (en) * | 2005-08-26 | 2007-03-01 | Regents Of The University Of Minnesota | Decellularization and recellularization of organs and tissues |
CA2676546A1 (en) * | 2006-04-24 | 2007-11-08 | Stemcell Institute Inc. | Method for preparing an organ for transplantation |
US20070292401A1 (en) | 2006-06-20 | 2007-12-20 | Harmon Alexander M | Soft tissue repair and regeneration using stem cell products |
CA2658060A1 (en) * | 2006-07-04 | 2008-01-10 | Stemcell Institute Inc. | Erythropoietin-producing organoid precursor, production method thereof, and method for treating erythropoietin-related disorder |
JP2008173935A (ja) | 2007-01-22 | 2008-07-31 | Funai Electric Co Ltd | 画像形成装置 |
-
2009
- 2009-07-02 TW TW98122386A patent/TWI389696B/zh not_active IP Right Cessation
- 2009-07-02 CN CN2009801261256A patent/CN102105581B/zh not_active Expired - Fee Related
- 2009-07-02 JP JP2010519101A patent/JP5388138B2/ja active Active
- 2009-07-02 NZ NZ590122A patent/NZ590122A/en not_active IP Right Cessation
- 2009-07-02 EP EP09773529.4A patent/EP2298866A4/en not_active Withdrawn
- 2009-07-02 AU AU2009266761A patent/AU2009266761A1/en not_active Abandoned
- 2009-07-02 KR KR1020117000152A patent/KR20110043580A/ko not_active Application Discontinuation
- 2009-07-02 RU RU2011103549/10A patent/RU2511395C2/ru not_active IP Right Cessation
- 2009-07-02 WO PCT/JP2009/062098 patent/WO2010001951A1/ja active Application Filing
- 2009-07-02 CA CA2729765A patent/CA2729765A1/en not_active Abandoned
- 2009-07-02 US US13/001,764 patent/US20110104656A1/en not_active Abandoned
-
2010
- 2010-12-23 IL IL210227A patent/IL210227A0/en unknown
-
2011
- 2011-12-15 HK HK11113578.0A patent/HK1159171A1/xx not_active IP Right Cessation
-
2014
- 2014-12-09 US US14/565,104 patent/US9758766B2/en active Active
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109642210A (zh) * | 2016-06-29 | 2019-04-16 | 横尾隆 | 一种肾脏的制造方法 |
US11559605B2 (en) | 2016-06-29 | 2023-01-24 | Takashi Yokoo | Kidney production method |
US11607425B2 (en) | 2016-06-29 | 2023-03-21 | Takashi Yokoo | Kidney production method |
Also Published As
Publication number | Publication date |
---|---|
JPWO2010001951A1 (ja) | 2011-12-22 |
CN102105581B (zh) | 2013-09-04 |
TWI389696B (zh) | 2013-03-21 |
RU2511395C2 (ru) | 2014-04-10 |
NZ590122A (en) | 2012-02-24 |
EP2298866A4 (en) | 2013-11-20 |
JP5388138B2 (ja) | 2014-01-15 |
RU2011103549A (ru) | 2012-08-10 |
IL210227A0 (en) | 2011-03-31 |
AU2009266761A1 (en) | 2010-01-07 |
TW201010709A (en) | 2010-03-16 |
EP2298866A1 (en) | 2011-03-23 |
US9758766B2 (en) | 2017-09-12 |
CA2729765A1 (en) | 2010-01-07 |
WO2010001951A1 (ja) | 2010-01-07 |
US20110104656A1 (en) | 2011-05-05 |
US20150104434A1 (en) | 2015-04-16 |
HK1159171A1 (en) | 2012-07-27 |
KR20110043580A (ko) | 2011-04-27 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
KR101840078B1 (ko) | 가이드를 갖는 이식용 재생 기관 원기의 제조방법, 해당 방법에 의해 제조되는, 가이드를 갖는 이식용 재생 기관 원기를 포함하는 조성물 및 가이드를 갖는 이식용 재생 기관 원기의 이식방법 | |
CN101330935A (zh) | 自脐带羊膜分离和培养干/祖细胞及其分化的细胞的应用 | |
EP3124600B1 (en) | Method for generating a cell condensate for self-organisation | |
KR20000052709A (ko) | 와튼제대교질에서 분리된 세포를 이용하여 연골 조직을 생산 | |
US20040052768A1 (en) | Vascularised tissue graft | |
US10633634B2 (en) | Method for preparing bone marrow cell aggregate | |
CN102105581B (zh) | 人工肾前体及其制备方法 | |
EP2014316A1 (en) | Method of preparing organ for transplantation | |
CA2419923C (en) | Vascularised tissue graft | |
Ajmal et al. | Organ regeneration through stem cells and tissue engineering | |
KR102286779B1 (ko) | 자궁내막 손상의 치료용 자궁내막복합체, 상기 자궁내막복합체의 제조방법 및 상기 방법으로 제조된 자궁내막복합체 | |
JP2009011588A (ja) | 人工皮膚およびその製造方法 | |
CN105963795A (zh) | 一种基于胶原制备组织工程表皮的方法 | |
JP4876275B2 (ja) | 生物の組織を薄切した切片からなる動物細胞の培養担体と、この担体を用いる動物細胞の培養方法および移植方法 | |
CN105039239A (zh) | 细胞转化诱导液及其应用 | |
Li et al. | The histocompatibility research of hair follicle stem cells with bladder acellular matrix | |
CN109355257A (zh) | 不同组织来源的间充质干细胞混合培养方法 | |
WO2016088373A1 (ja) | 移植用培養細胞シート、移植用培養細胞シートの製造方法、及び、移植用の骨組織作製方法 | |
Isaac et al. | Construction of a skin substitute composed of porcine collagen matrix populated with human dermal fibroblasts and keratinocytes: histological evaluation | |
Malik et al. | Organ, Histotypic and Organotypic Culture, and Tissue Engineering | |
CN1432645A (zh) | 人细胞库及其在自体细胞移植和组织修复中的应用 | |
AU2001283687B2 (en) | Vascularised tissue graft | |
CN116200334A (zh) | 一种三维毛***细胞培养体系及其构建方法 | |
CN101912636A (zh) | 迷你皮肤结构及其制备方法和用途 | |
Sreekanth et al. | PERIODONTAL REGENERATION THROUGH CELL SHEET ENGINEERING-A NOVEL APPROACH |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
REG | Reference to a national code |
Ref country code: HK Ref legal event code: DE Ref document number: 1159171 Country of ref document: HK |
|
C14 | Grant of patent or utility model | ||
GR01 | Patent grant | ||
REG | Reference to a national code |
Ref country code: HK Ref legal event code: GR Ref document number: 1159171 Country of ref document: HK |
|
CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130904 Termination date: 20150702 |
|
EXPY | Termination of patent right or utility model |