CN106581763A - Method for preparing stem cell human miniature organs - Google Patents

Method for preparing stem cell human miniature organs Download PDF

Info

Publication number
CN106581763A
CN106581763A CN201611231369.9A CN201611231369A CN106581763A CN 106581763 A CN106581763 A CN 106581763A CN 201611231369 A CN201611231369 A CN 201611231369A CN 106581763 A CN106581763 A CN 106581763A
Authority
CN
China
Prior art keywords
cell
stem cell
cells
collagen
miniature
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201611231369.9A
Other languages
Chinese (zh)
Inventor
张孝兵
袁卫平
万谦
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Traceability Life Polytron Technologies Inc
Original Assignee
Traceability Life Polytron Technologies Inc
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Traceability Life Polytron Technologies Inc filed Critical Traceability Life Polytron Technologies Inc
Priority to CN201611231369.9A priority Critical patent/CN106581763A/en
Publication of CN106581763A publication Critical patent/CN106581763A/en
Pending legal-status Critical Current

Links

Classifications

    • 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
    • A61L27/3886—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 comprising two or more cell types
    • 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
    • A61L27/3804—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 characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney 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
    • A61L27/36—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
    • A61L27/3804—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 characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem 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
    • A61L27/36—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
    • A61L27/3839—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 characterised by the site of application in the body
    • 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
    • C12N5/067—Hepatocytes
    • C12N5/0671—Three-dimensional culture, tissue culture or organ culture; Encapsulated 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
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
    • 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
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/426—Immunomodulating agents, i.e. cytokines, interleukins, interferons
    • 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
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/28—Materials or treatment for tissue regeneration for liver reconstruction
    • 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
    • C12N2501/12—Hepatocyte growth factor [HGF]
    • 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
    • C12N2501/415—Wnt; Frizzeled
    • 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
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
    • C12N2502/1352—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
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/28—Vascular endothelial cells

Abstract

The invention discloses a method for preparing stem cell human miniature organs, and relates to the technical field of miniature organ transplantation. The method mainly includes the steps that mesenchymal stem cells, endothelial progenitor cells and cytokines, like collagen or fibrous protein, in hydrogel promote formation of new vessels, and cross-linked collagen microspheres secrete Wnt2, HGF and other factors 2-4 weeks after blood vascular system reconstruction so that proliferation of liver cells can be promoted. The method has the advantages that a replacement therapy method or even a cure method is provided for patients who cannot find organ donors. One to two years after transplantation, abdomen miniature organs possibly grow to remarkable sizes and can replace livers with functions damaged and failed. Lives of people who wait for the organ donors are sustained, and the miniature organs can provide support for survival. Besides, the human miniature organs are used for toxicologic tests and other functional tests, tests can be conducted on rats or mice with immunodeficiency through the same method, and tools are provided for clinical drug research.

Description

A kind of preparation method of the miniature organ of stem cell people
Technical field：
The present invention relates to a kind of preparation method of the miniature organ of stem cell people, more particularly to miniature organ transplantation technique neck Domain.
Background technology：
Organ transplantation is the unique method of the diseases such as the various organ failuries for the treatment of.However, donor is limited；Using the thin of itself The stem cell in born of the same parents source produces the new method of miniature organ needed for patient itself and is provided new hope for survival.In recent years, profit An extensive research topic is become with the de- organelle transplanting that isolated organ and patients own cells originate.However, this Plant not through the organ evolved and remodeling process is produced, its permanent function is a controversial topic.From patient certainly The miniature organ of body generation, undoubtedly a kind of better method, it can improve the symptoms such as organ failure.In addition, by base Because of modification and some regeneration factors of damaged organ itself secretion, miniature organ be likely to grow into one it is relatively significantly bigger It is little.
The content of the invention：
For the problems referred to above, the technical problem to be solved in the present invention is to provide a kind of preparation side of the miniature organ of stem cell people Method.
To solve above-mentioned technical problem, a kind of preparation method of the miniature organ of stem cell people of the present invention is：Fill using Cytokine in matter stem cell, endothelial progenitor cells, hydrogel such as collagen or fibrin promote the formation of new vesselses, in blood 2-4 after guard system is rebuild is all, and the factor such as crosslinked with collagen microsphere secretion Wnt2 and HGF can promote hepatocellular propagation；Specifically Method be:Step one：The propagation of mescenchymal stem cell：Induced multi-potent stem cell under standard conditions, E are formed latter 5-9 days, are used Collagenase or dispersed protein enzyme isolate EBs cells, use MSC culture medium culturings, contain PDGF in culture medium, EGF and FGF, the concentration of every kind of factor are 10ng/ml, and after 2 weeks, higher than 1000 times, the cell of wherein most all can table for MSC propagation Up to the mark similar to CD73, CD105, CD29, CD44, MSC etc.；
Step 2：The propagation of endothelial progenitor cells：After induced multi-potent stem cell EB at the standard conditions forms latter 5-9 days, EBs cell lines are isolated with Collagenase or dispersed protein enzyme, with the culture medium culturing of endotheliocyte, containing dense eventually in culture Spend for 10ng/ml VEGF and FGF, in 1-100ng/ml, after 2 weeks, the quantity of EPC increases above 1000 times to concentration range, big portion The cell for dividing is expressed as CD31lowKDR+VE-Cadherin+This kind of EPC surface markers；In the previous day of cell transplantation, will With SDF-1, VEGF, HGF, minicircle dna etc. are contaminated by consideration convey carries out transient transfection to EPC cells, the table of these factors after transfection Danone continues one week；
Step 3：The culture of MSC and EPC, survival rate is relatively low after the transfer with other cells to separate single MSC, so And, these cells carry out transplanting the survival rate and its function in organism that can significantly improve cell with microspheres form, therefore, In stem cell transplantation the previous day, transplanted after sessile drop method or pressure centrifuging make 100-10000 cell microsphere；
Step 4:It is prepared by hydrogel:Endothelial cell growth medium glue is hydrogel, using concentration in 1-2mg/mL people Collagen protein, under the conditions of blood heat, a hour can just form gel；
Step 5：Transplanting, 1mL hydrogels are made the microsphere for mixing with MSC, EPC and hepatocyte, and 37 DEG C are incubated 1h to reach To optimum combination pattern, then, this gel is transplanted to by the specific position of surgical procedure postabdomen, or in ultrasound Under mediation, used in cell aqueous gel mixture is injected directly into abdominal part or liver；
Step 6：Two weeks after is transplanted, once main vascular system is set up, sustained release factor WNT2 and HGF can promote Hepatocellular propagation, for the somatomedin of some sustained releases, adds 1-10 μ in collagen protein of the concentration for 1-2mg/mL The WNT2 and HGF of g, is subsequently adding fluid, and with the collagen microsphere of 50-100 μm optimum of speed concussion generation, adds penta 2 Aldehyde crosslinked with collagen microsphere, endothelial cells secrete MMP degraded crosslinked with collagen discharge WNT2 and HGF and induce the amplification of stem cell, The sustained release of siRNA, knocks out WW45 genes, can significantly increase the dirty size of Hepar Mus, and sustained release WW45, siRNA can To promote stem cell, the amplification of ovum and increase miniature organ size, cell transfecting one week is interior, and nutrient substance and oxygen can only Obtained by simple diffusion, therefore, the cell density of high concentration can cause most cells dead, and optimum cell density is 1- 5x106Individual/mL, 0.1-0.5x 106In/mL hepatocyte, the ratio of MSCs and EPCs is with 1:1-1:5 are mixed.
Beneficial effects of the present invention：(1) provide a kind for the treatment of for those patients that can not find organ donor even to control Curing method.After transplanting 1-2, the miniature organ of abdominal part probably grows into a significant size, it is possible to which alternative functions are received Damage the liver with exhaustion.(2) those are maintained to wait the life of organ donor, miniature organ provide support for survival.(3) The function tests such as toxicology are carried out using human body micro-organs, can also on the rat or mice of immunodeficiency with same method Tested.When organ donation can obtain, organ transplantation provides a kind of Therapeutic Method for organ failure patient.However, same After kind of allosome organ transplantation, long-term use immunosuppressant can increase considerably accordingly suffer from Other diseases such as severe infections with The chance of cancer.Another method of organizational project is in de- organelle to inject cell.Organ Reconstruction is using in vitro Together with patients own cells' In vitro culture, this de- cytoskeletal method has obvious limitation to organ：Only normal organ The function of 1-2%, after transplanting, they can only at most survive several hours.The miniature organ produced using the present invention and vitro tissue Engineering is compared with more preferable function, because they start anew to produce, additionally, for autologous organ there is no need with exempting from Epidemic disease inhibitor.
Specific embodiment：
This specific embodiment is employed the following technical solutions：Using in mescenchymal stem cell, endothelial progenitor cells, hydrogel Cytokine such as collagen or fibrin promote the formation of new vesselses, and the 2-4 after vascular system is rebuild is all, and crosslinked with collagen is micro- The factors such as ball secretion Wnt2 and HGF can promote hepatocellular propagation；Specifically method is:Step one：Mescenchymal stem cell Propagation：Induced multi-potent stem cell under standard conditions, E are formed latter 5-9 days, are isolated with Collagenase or dispersed protein enzyme EBs cells, use MSC culture medium culturings, contain PDGF, EGF and FGF in culture medium, and the concentration of every kind of factor is 10ng/ml, and 2 Zhou Hou, MSC propagation higher than 1000 times, can all express similar to CD73, CD105, CD29, CD44, MSC by the cell of wherein most Deng mark；
Step 2：The propagation of endothelial progenitor cells：After induced multi-potent stem cell EB at the standard conditions forms latter 5-9 days, EBs cell lines are isolated with Collagenase or dispersed protein enzyme, with the culture medium culturing of endotheliocyte, containing dense eventually in culture Spend for 10ng/ml VEGF and FGF, in 1-100ng/ml, after 2 weeks, the quantity of EPC increases above 1000 times to concentration range, big portion The cell for dividing is expressed as CD31lowKDR+VE-Cadherin+This kind of EPC surface markers；In the previous day of cell transplantation, will With SDF-1, VEGF, HGF, minicircle dna etc. are contaminated by consideration convey carries out transient transfection to EPC cells, the table of these factors after transfection Danone continues one week；
Step 3：The culture of MSC and EPC, survival rate is relatively low after the transfer with other cells to separate single MSC, so And, these cells carry out transplanting the survival rate and its function in organism that can significantly improve cell with microspheres form, therefore, In stem cell transplantation the previous day, transplanted after sessile drop method or pressure centrifuging make 100-10000 cell microsphere；
Step 4:It is prepared by hydrogel:Endothelial cell growth medium glue is hydrogel, using concentration in 1-2mg/mL people Collagen protein, under the conditions of blood heat, a hour can just form gel；
Step 5：Transplanting, 1mL hydrogels are made the microsphere for mixing with MSC, EPC and hepatocyte, and 37 DEG C are incubated 1h to reach To optimum combination pattern, then, this gel is transplanted to by the specific position of surgical procedure postabdomen, or in ultrasound Under mediation, used in cell aqueous gel mixture is injected directly into abdominal part or liver；
Step 6：Two weeks after is transplanted, once main vascular system is set up, sustained release factor WNT2 and HGF can promote Hepatocellular propagation, for the somatomedin of some sustained releases, adds 1-10 μ in collagen protein of the concentration for 1-2mg/mL The WNT2 and HGF of g, is subsequently adding fluid, and with the collagen microsphere of 50-100 μm optimum of speed concussion generation, adds penta 2 Aldehyde crosslinked with collagen microsphere, endothelial cells secrete MMP degraded crosslinked with collagen discharge WNT2 and HGF and induce the amplification of stem cell, The sustained release of siRNA, knocks out WW45 genes, can significantly increase the dirty size of Hepar Mus, and sustained release WW45, siRNA can To promote stem cell, the amplification of ovum and increase miniature organ size, cell transfecting one week is interior, and nutrient substance and oxygen can only Obtained by simple diffusion, therefore, the cell density of high concentration can cause most cells dead, and optimum cell density is 1- 5x106Individual/mL, 0.1-0.5x 106In/mL hepatocyte, the ratio of MSCs and EPCs is with 1:1-1:5 are mixed.
The ultimate principle and principal character and advantages of the present invention of the present invention has been shown and described above.The technology of the industry Personnel it should be appreciated that the present invention is not restricted to the described embodiments, the simply explanation described in above-described embodiment and description this The principle of invention, without departing from the spirit and scope of the present invention, the present invention also has various changes and modifications, these changes Change and improvement is both fallen within scope of the claimed invention.The claimed scope of the invention by appending claims and its Equivalent thereof.

Claims (1)

1. the preparation method of the miniature organ of a kind of stem cell people, it is characterised in that：Its method is：Using mescenchymal stem cell, Cytokine in endothelial progenitor cells, hydrogel such as collagen or fibrin promote the formation of new vesselses, in vascular system weight 2-4 after building is all, and the factor such as crosslinked with collagen microsphere secretion Wnt2 and HGF can promote hepatocellular propagation；Specific method step Suddenly it is:
Step one：The propagation of mescenchymal stem cell：Induced multi-potent stem cell under standard conditions, E are formed latter 5-9 days, use collagen Protease or dispersed protein enzyme isolate EBs cells, use MSC culture medium culturings, contain PDGF, EGF and FGF, often in culture medium The concentration for planting the factor is 10ng/ml, and after 2 weeks, higher than 1000 times, the cell of wherein most can all express similar to MSC propagation In the mark of CD73, CD105, CD29, CD44, MSC etc.；
Step 2：The propagation of endothelial progenitor cells：After induced multi-potent stem cell EB at the standard conditions forms latter 5-9 days, glue is used Former protease or dispersed protein enzyme isolate EBs cell lines, with the culture medium culturing of endotheliocyte, containing final concentration of in culture 10ng/ml VEGF and FGF, in 1-100ng/ml, after 2 weeks, the quantity of EPC increases above 1000 times to concentration range, most Cell is expressed as CD31lowKDR+VE-Cadherin+This kind of EPC surface markers；In the previous day of cell transplantation, will use SDF-1, VEGF, HGF, minicircle dna etc. carry out transient transfection to EPC cells by consideration convey dye, the expression of these factors after transfection One week can be continued；
Step 3：The culture of MSC and EPC, survival rate is relatively low after the transfer with other cells to separate single MSC, however, this A little cells carry out transplanting the survival rate and its function in organism that can significantly improve cell with microspheres form, therefore, dry Cell transplantation the previous day, transplanted after sessile drop method or pressure centrifuging make 100-10000 cell microsphere；
Step 4:It is prepared by hydrogel:Endothelial cell growth medium glue is hydrogel, using concentration 1-2mg/mL people glue Former albumen, under the conditions of blood heat, a hour can just form gel；
Step 5：Transplanting, 1mL hydrogels are made the microsphere for mixing with MSC, EPC and hepatocyte, and 37 DEG C are incubated 1h to reach most Excellent integrated mode, then, this gel is transplanted to by the specific position of surgical procedure postabdomen, or in ultrasonic mediation Under, used in cell aqueous gel mixture is injected directly into abdominal part or liver；
Step 6：Two weeks after is transplanted, once main vascular system is set up, sustained release factor WNT2 and HGF can promote liver thin The propagation of born of the same parents, for the somatomedin of some sustained releases, adds 1-10 μ g's in collagen protein of the concentration for 1-2mg/mL WNT2 and HGF, is subsequently adding fluid, and with the collagen microsphere of 50-100 μm optimum of speed concussion generation, adds glutaraldehyde Crosslinked with collagen microsphere, endothelial cells secrete MMP degraded crosslinked with collagen discharge WNT2 and HGF and induce the amplification of stem cell, The sustained release of siRNA, knocks out WW45 genes, can significantly increase the dirty size of Hepar Mus, and sustained release WW45, siRNA can To promote stem cell, the amplification of ovum and increase miniature organ size, cell transfecting one week is interior, and nutrient substance and oxygen can only Obtained by simple diffusion, therefore, the cell density of high concentration can cause most cells dead, and optimum cell density is 1- 5x106Individual/mL, 0.1-0.5x106In/mL hepatocyte, the ratio of MSCs and EPCs is with 1:1-1:5 are mixed.
CN201611231369.9A 2016-12-28 2016-12-28 Method for preparing stem cell human miniature organs Pending CN106581763A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201611231369.9A CN106581763A (en) 2016-12-28 2016-12-28 Method for preparing stem cell human miniature organs

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201611231369.9A CN106581763A (en) 2016-12-28 2016-12-28 Method for preparing stem cell human miniature organs

Publications (1)

Publication Number Publication Date
CN106581763A true CN106581763A (en) 2017-04-26

Family

ID=58604475

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201611231369.9A Pending CN106581763A (en) 2016-12-28 2016-12-28 Method for preparing stem cell human miniature organs

Country Status (1)

Country Link
CN (1) CN106581763A (en)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101437528A (en) * 2004-10-28 2009-05-20 梅迪沃什有限公司 Bioactive wound dressings and implantable devices and methods of use
CN101534747A (en) * 2006-07-10 2009-09-16 哥伦比亚大学 De novo formation and regeneration of vascularized tissue from tissue progenitor cells and vascular progenitor cells
US20100215715A1 (en) * 2009-02-19 2010-08-26 University Of Southern California Gel delivery system for tissue repair
US20110064810A1 (en) * 2010-11-18 2011-03-17 Jalaledin Ghanavi Tissue engineering method for organ reconstruction using injectable matrix
CN104651300A (en) * 2015-02-11 2015-05-27 南开大学 Three-dimensional complex cell aggregate model as well as preparation method and application thereof

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101437528A (en) * 2004-10-28 2009-05-20 梅迪沃什有限公司 Bioactive wound dressings and implantable devices and methods of use
CN101534747A (en) * 2006-07-10 2009-09-16 哥伦比亚大学 De novo formation and regeneration of vascularized tissue from tissue progenitor cells and vascular progenitor cells
US20100215715A1 (en) * 2009-02-19 2010-08-26 University Of Southern California Gel delivery system for tissue repair
US20110064810A1 (en) * 2010-11-18 2011-03-17 Jalaledin Ghanavi Tissue engineering method for organ reconstruction using injectable matrix
CN104651300A (en) * 2015-02-11 2015-05-27 南开大学 Three-dimensional complex cell aggregate model as well as preparation method and application thereof

Similar Documents

Publication Publication Date Title
Lombaert et al. Concise review: salivary gland regeneration: therapeutic approaches from stem cells to tissue organoids
Ji et al. Promising therapeutic strategies for mesenchymal stem cell-based cardiovascular regeneration: from cell priming to tissue engineering
Caron et al. A new three dimensional biomimetic hydrogel to deliver factors secreted by human mesenchymal stem cells in spinal cord injury
Stoltz et al. Stem cells and regenerative medicine: myth or reality of the 21th century
Ansari et al. Muscle tissue engineering using gingival mesenchymal stem cells encapsulated in alginate hydrogels containing multiple growth factors
Liu et al. Autologous stem cell transplantation for myocardial repair
Baiguera et al. Endothelialization approaches for viable engineered tissues
Hsu et al. Self‐assembled adult adipose‐derived stem cell spheroids combined with biomaterials promote wound healing in a rat skin repair model
KR101613478B1 (en) Composition comprising mesenchymal stem cell-hydrogel and preparation method thereof
US20220090008A1 (en) Colony forming medium and use thereof
WO2016168950A9 (en) Method for in vitro constructing salivary gland organoids and acinus-like
Ghoneim et al. From mesenchymal stromal/stem cells to insulin-producing cells: progress and challenges
Takaku et al. In vivo cell tracking by bioluminescence imaging after transplantation of bioengineered cell sheets to the knee joint
Huang et al. A translational approach in using cell sheet fragments of autologous bone marrow-derived mesenchymal stem cells for cellular cardiomyoplasty in a porcine model
Sierra Parraga et al. Effects of normothermic machine perfusion conditions on mesenchymal stromal cells
Aframian et al. Current status of the development of an artificial salivary gland
Wang et al. A new scaffold containing small intestinal submucosa and mesenchymal stem cells improves pancreatic islet function and survival in vitro and in vivo
KR101585032B1 (en) Composition comprising mesenchymal stem cell-hydrogel and preparation method thereof
CN105705633A (en) Culture medium composition for improving regenerative capacity of stem cells, and stem cell culturing method using same
Deng et al. Narrative review of the choices of stem cell sources and hydrogels for cartilage tissue engineering
Pan et al. In-vivo organ engineering: Perfusion of hepatocytes in a single liver lobe scaffold of living rats
Ishihara et al. Biomaterials as cell carriers for augmentation of adipose tissue-derived stromal cell transplantation
Rufaihah et al. The effect of scaffold modulus on the morphology and remodeling of fetal mesenchymal stem cells
Ermis Photo-crosslinked gelatin methacrylate hydrogels with mesenchymal stem cell and endothelial cell spheroids as soft tissue substitutes
Sudulaguntla et al. Stem cells: Cultivation and routes of administration

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
RJ01 Rejection of invention patent application after publication
RJ01 Rejection of invention patent application after publication

Application publication date: 20170426