CN108685948A - A kind of preparation method of new medical cell repair agent - Google Patents
A kind of preparation method of new medical cell repair agent Download PDFInfo
- Publication number
- CN108685948A CN108685948A CN201810540720.5A CN201810540720A CN108685948A CN 108685948 A CN108685948 A CN 108685948A CN 201810540720 A CN201810540720 A CN 201810540720A CN 108685948 A CN108685948 A CN 108685948A
- Authority
- CN
- China
- Prior art keywords
- cell
- amnion
- stem cell
- mesenchymal stem
- preparation
- 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
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/1808—Epidermal growth factor [EGF] urogastrone
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
- A61K31/425—Thiazoles
- A61K31/429—Thiazoles condensed with heterocyclic ring systems
- A61K31/43—Compounds containing 4-thia-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula, e.g. penicillins, penems
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/7036—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin having at least one amino group directly attached to the carbocyclic ring, e.g. streptomycin, gentamycin, amikacin, validamycin, fortimicins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
- A61K31/728—Hyaluronic acid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- A61K31/734—Alginic acid
-
- 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
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/1825—Fibroblast growth factor [FGF]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/1858—Platelet-derived growth factor [PDGF]
- A61K38/1866—Vascular endothelial growth factor [VEGF]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0014—Skin, i.e. galenical aspects of topical compositions
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
-
- 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
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0668—Mesenchymal stem cells from other natural sources
-
- 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
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Immunology (AREA)
- Developmental Biology & Embryology (AREA)
- Biomedical Technology (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biotechnology (AREA)
- Dermatology (AREA)
- Cell Biology (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Rheumatology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Hematology (AREA)
- Virology (AREA)
- Vascular Medicine (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The present invention relates to the medicaments for repairing wound, more particularly, to a kind of preparation method of new medical cell repair agent.Specific preparation process is that human amnion mesenchymal stem cell isolating and purifying and cultivating, the preparation of sodium alginate complexing agent, the preparation of cell repair agent.The present invention establishes human amnion mesenchymal stem cell preparation method and corresponding stem cell bank, prepares human amnion mesenchymal stem cell renovation agent for fairly large production and its application is got ready.
Description
Technical field:
The present invention relates to the medicaments for repairing wound, more particularly, to a kind of preparation method of new medical cell repair agent.
Background technology:
Skin ultrastructure is a complicated biological process, it starts the biological event of a series of complex, including
Inflammation, tissue new life and reconstructed tissue.Although many researchs treat skin ultrastructure, healing quality using different methods
It is low, accessory organ is non-renewable etc. is still critical issue during skin wound healing.Currently, dermatoplasty is to repair greatly
The most effectual way of areas of skin damage.In recent years, artificial synthesized skin is already used to treatment skin injury, but is not one
The perfect Graftskin of kind, there is also a series of problems:Artificial synthesized skin seed cells used at present are self
Or the epidermal cell of allosome.Autologous epidermis cell quantity is insufficient, and then there is rejections for allosome epidermal cell;It is used at present
There is living cells in artificial synthesized skin, but it is a problem to preserve, if extemporaneous preparation needs the time longer, and skin injury
It needs to use artificial synthetic skin within the time as soon as possible;Artificial synthesized skin used at present is formed without the new rawhide of the surface of a wound
The ability of blood vessel is easy necrosis.And mescenchymal stem cell has proven to be the germinative seed cell of skin ultrastructure.
Having many experimental studies results confirms that BMSCs plays a positive role in terms of wound healing.
Bone Marrow Mesenchymal Stem Cells Transplantation to the deep ii degree burn surface of a wound, is it is found that it is obviously promoted the healing of the surface of a wound by Badiavas.It pays small
BMSCs is migrated to pig deep burn wound by soldier etc., it is found that healing speed is obviously accelerated, and epidermis thickens, and corium nerve is fine
Dimension increases, and substantially improves wound healing quality.Document report BMSCs also participates in the reconstruction of epidermis, sweat gland and surface of a wound blood vessel.This
A little results of study show that BMSCs is extraordinary skin tissue engineering seed cells.There is growth in each stage of wound healing
The participation and regulation and control of the factor, growth factor have wide answer to treating chronic refractory conjunction skin wounds and large-area burn wound
Use foreground.But as clinical application, formality is more complicated when obtaining mesenchymal stem cell, and supplier is more painful.This
Outside, the mescenchymal stem cell of derived from bone marrow is with the increase at supplier's age, and quantity can be reduced, corresponding differentiation capability
It can die down.In recent years, the stem cell in placenta affiliated group source, especially amnion mesenchymal stem cell are found, is filled between marrow
The good substitute of matter stem cell.2004, the human amnion mesenchymal stem cell that In t Anker etc. are turned out from amnion
(hAMSCs), immunophenotype is similar to mesenchymal stem cell (BMSCs), and it has stronger amplification energy than BMSCs
Power.HAMSCs not only has cells and characteristic of stem, and shows low immunogenicity and immunosuppressive action, is ideal exempt from
Epidemic disease absolves cell, is the germinative seed cell of skin ultrastructure.It is not only more original, and growth ability is strong, and rejection
Also smaller.Amnion tissue is originally Biohazard Waste, derives from a wealth of sources and is easy to get.
It discloses application No. is 200810136415.6 patent of invention and a kind of uses methylcellulose as cellular matrix
Human umbilical cord mesenchymal stem cells wound surface smearing agent preparation method, but the invention contains antibiotics gentamycin, is not suitable for
In antibiotic allergic human population;The use that stem cell is filled between people's umbilical cord does not have distribution type, might have rejection, and liniment is fast
Speed contains animal derived components fetal calf serum in preparing, it is possible to cause allergy and have animal derived security risk.
Invention content:
The present invention is exactly in view of the above-mentioned problems, provide a kind of preparation method of new medical cell repair agent.
To achieve the goals above, the present invention adopts the following technical scheme that, specific preparation process is,
1, the preparation of sodium alginate complexing agent
The anhydrous sodium alginates of 5g are weighed, are added in 100ml vials, are subsequently added into 40ml deionized waters uniform stirring extremely
Dissolving, adds 20ml glycerine mixings, and the serum-free mescenchymal stem cell culture medium that 40ml is then added mixes well;Again toward glass
Epidermal growth factor EGF 1mg, basic fibroblast growth factor bFGF 2mg, angiogenesis factor are added in glass bottle
VEGF2mg obtains sodium alginate complexing agent after stirring evenly;
2, the preparation of cell repair agent
1.0 × 10 are added into trehalose complexing agent made from 1ml steps 16Cell/ml human amnion mesenchymal stem cells are outstanding
Liquid 1ml adds the Sodium Hyaluronate liquid that 1ml mass concentrations are 1%, up to cell repair agent after stirring evenly.
The preparation process of human amnion mesenchymal stem cell is,
(1) it isolates and purifies
Aseptic collection amnion tissue, Biohazard Safety Equipment is taken to be built in the PBS of precooling and rinse, if the related chorion of placenta,
Chorion and amnion need to be first detached, amnion is taken out, abandons chorion;The small item of amnion that amnion tissue is 2cm × 2cm is cut, bodies are waited
2U/ml neutral protein enzymic digestion 30min are added in product, then add the 5.5mg/ml collagen type vs enzyme and 2U/ml of two sesquialter amnion volumes
Hyaluronidase 1:1 mixed liquor digests 3.5h;The cell suspension that digestion finishes is collected, it is multiple that the equilibrium liquid without calcium ions and magnesium ions is added
Cleaning counts mononuclearcell after glacial acetic acid dilution, obtains cell total amount;
Cell concentration is adjusted as 1.0 × 105A/ml is added serum-free mescenchymal stem cell culture medium, is placed in 37 DEG C,
A concentration of 5%CO2 is cultivated in the incubator of saturated humidity, and liquid is changed 1 time per 2d, and it is dry thin to obtain primary amnion mesenchymal after a week
Born of the same parents;When the 80-90% of cell growth to culture vessel floor space, when a large amount of cell masses are linked to be piece, with concentration 0.05%g/L pancreases
It is passed on after protease digestion, cell is placed in the culture bottle for being covered with L- poly-D-lysines and carries out secondary culture, passed on
Amnion mesenchymal stem cell;
2, quality testing is carried out to cell before freezing
(1) culture solution before being collected to cell carries out infectious disease pathogens detection, including hepatitis B, hepatitis, syphilis, AIDS
Substance;
(2) while cell cryopreservation, cell is taken to carry out microculture, carries out microorganism detection;
3, commercialization serum-free cell frozen stock solution is chosen, by 5 × 106The human amnion mesenchymal stem cell of a/ml is added to 1ml
It in frozen stock solution, is put into cryopreservation tube, is first placed in refrigerator, 8min is preserved at 4 DEG C, then be adjusted to preserve 12-18h at -80 DEG C, then
It is put into the human amniotic mesenchymal stem cell bank equipped with liquid nitrogen container;
4, it is preserved according to neonatal sex and ABO/Rh partings and HLA partings, establishes cellular informatics archives, construct people sheep
Film mesenchyma stem cell.
When specifically used, the cell rapid configuration method of culture period can be used as needed or freeze the cell rapid configuration of phase
Method.
1, the cell rapid configuration method of culture period
Cultivate cell in advance according to vitro growth rates, collect cell, after being washed using 20ml phosphate buffers (PBS) from
The heart, centrifugal force 400g centrifuge 5min, remove impurity and cell fragment ingredient;Cell is counted, it is qualified to be detected by Cell viability
Afterwards, the Sodium Hyaluronate liquid for being immediately 1% with sodium alginate complexing agent and mass concentration mixes in equal volume, is loaded on sterile glass
Bottle, you can use;
2, the cell rapid configuration of phase is frozen
The cell for freezing the phase uses rapid fluid resuscitation method, cryopreservation tube is taken out from liquid nitrogen in 37 DEG C of water-bath recovery cells,
It is added into mixing in the centrifuge tube of the culture medium of mescenchymal stem cell containing 10ml to centrifuge, centrifugal force 400g centrifuges 5min, repeats
2 times, count cell;After Cell viability detection is qualified, be with sodium alginate complexing agent and mass concentration immediately 1% it is transparent
Matter acid sodium liquid mixes in equal volume, is loaded on sterile glass vials, you can uses.
Beneficial effects of the present invention:
1, human amnion mesenchymal stem cell preparation method is improved, cell repair agent uses human amnion mesenchymal stem cell.
2, it is preserved according to neonatal sex and ABO/Rh partings and HLA partings, establishes cellular informatics archives, construct people sheep
Film mesenchyma stem cell.Simultaneously as mescenchymal stem cell antigenicity itself is weaker and has immunosuppressive action, organizing
It can inhibit in the process with organ transplant or prevent graft versus host disease(GVH disease) (GVHD), therefore rejection is smaller.
3, it uses sodium alginate soln to make mother liquor and mesenchymal cell culture medium fully to mix as matrix, then adds epidermis
Growth factor, basic fibroblast growth factor, angiogenesis factor, penicillin element 100U/ml, streptomysin 0.1mg/ml,
Obtain sodium alginate complexing agent.Finally again with human amnion mesenchymal stem cell suspension and hyaluronic acid solution shape according to a certain percentage
At a kind of paste.Can skin wound uniformly be applied to and form a kind of covering.Using the agent of this cell repair, it can induce and formed
Newborn epidermis and corium, and itself skin at surface of a wound edge is promoted to be grown into the surface of a wound.
Description of the drawings:
Fig. 1 is the cellular morphology figure in amnion mesenchymal stem cell culture.A is that the part Epithelial that primary cell obtains is thin
Born of the same parents' form, B are the fibroblast form obtained when second generation culture.
Fig. 2 is flow cytometer showed cell surface molecule CD34, CD44, CD45, CD73, CD90, CD105, CD326, HLA-DR.
Fig. 3 is that dyeing immunofluorescence cell analyzes Vimentin ingredients in cytoplasm.
Fig. 4 is cell to osteoblast Induction of committed differentiation.
Fig. 5 is cell to lipoblast Induction of committed differentiation.
Specific implementation mode:
Embodiment 1
1, the preparation of human amnion mesenchymal stem cell
Aseptic collection amnion tissue, Biohazard Safety Equipment is taken to be built in the PBS of precooling and rinse, if the related chorion of placenta,
Chorion and amnion need to be first detached, amnion is taken out, abandons chorion;The small item of amnion that amnion tissue is 2cm × 2cm is cut, bodies are waited
2U/ml neutral protein enzymic digestion 30min are added in product, then add the 5.5mg/ml collagen type vs enzyme and 2U/ml of two sesquialter amnion volumes
Hyaluronidase 1:1 mixed liquor digests 3.5h;The cell suspension that digestion finishes is collected, it is multiple that the equilibrium liquid without calcium ions and magnesium ions is added
Cleaning counts mononuclearcell after glacial acetic acid dilution, obtains cell total amount;Cell concentration is adjusted as 1.0 × 105A/ml, adds
Enter serum-free mescenchymal stem cell culture medium, be placed in 37 DEG C, a concentration of 5%CO2, cultivated in the incubator of saturated humidity, per 2d
It changes liquid 1 time, obtains primary amnion mesenchymal stem cell after a week;When the 80-90% of cell growth to culture vessel floor space,
When a large amount of cell masses are linked to be piece, to be passed on after concentration 0.05%g/L trypsin digestions, cell are placed in and is covered with the bad ammonia of L- polies
Secondary culture is carried out in the culture bottle of acid, the amnion mesenchymal stem cell passed on.Quality testing is carried out to cell before freezing:
Culture solution before being collected to cell carries out infectious disease pathogens detection:Such as hepatitis B, hepatitis, syphilis, AIDS;In the same of cell cryopreservation
When, it takes a small amount of cell to carry out microculture, carries out microorganism detection.Commercialization serum-free cell frozen stock solution is chosen, by 5 ×
106The human amnion mesenchymal stem cell of a/ml is added in 1ml frozen stock solutions, is put into cryopreservation tube, is first placed in refrigerator, is protected at 4 DEG C
8min is deposited, then is adjusted to preserve 12-18h at -80 DEG C, is then placed in the human amniotic mesenchymal stem cell bank equipped with liquid nitrogen container;Root
It is preserved according to neonatal sex and ABO/Rh partings and HLA partings, establishes cellular informatics archives, it is dry thin to construct human amnion mesenchymal
Born of the same parents library.
2, the preparation of sodium alginate complexing agent
The anhydrous sodium alginates of 3g are weighed, are added in 100ml vials, are subsequently added into 40ml deionized waters uniform stirring extremely
Dissolving, adds 20ml glycerine mixings, and the serum-free mescenchymal stem cell culture medium that 40ml is then added mixes well;Again toward glass
Epidermal growth factor EGF 1mg, basic fibroblast growth factor bFGF 2mg, angiogenesis factor are added in glass bottle
VEGF2mg obtains sodium alginate complexing agent after stirring evenly;
3, the preparation of cell repair agent
1.0 × 10 are added into trehalose complexing agent made from 1ml steps 16Cell/ml human amnion mesenchymal stem cells are outstanding
Liquid 1ml adds the Sodium Hyaluronate liquid that 1ml mass concentrations are 1%, up to cell repair agent after stirring evenly.
For the client of acute skin injury or large area skin wounds, using the cell rapid configuration for the phase that freezes, according to
The gender and ABO/Rh partings and HLA partings of user selects suitable cell seed from amniotic mesenchymal cell library.From liquid nitrogen
Middle taking-up cryopreservation tube is added into the centrifuge tube of the culture medium of mescenchymal stem cell containing 10ml and mixes in 37 DEG C of water-bath recovery cells
Even centrifugation, centrifugal force 400g centrifuge 5min, are repeated 2 times, and count cell;After Cell viability detection is qualified, immediately with sea
The Sodium Hyaluronate liquid that mosanom complexing agent and mass concentration are 1% mixes in equal volume, is loaded on sterile glass vials, you can uses.
Embodiment 2
1, the preparation of human amnion mesenchymal stem cell
Aseptic collection amnion tissue, Biohazard Safety Equipment is taken to be built in the PBS of precooling and rinse, if the related chorion of placenta,
Chorion and amnion need to be first detached, amnion is taken out, abandons chorion;The small item of amnion that amnion tissue is 2cm × 2cm is cut, bodies are waited
2U/ml neutral protein enzymic digestion 30min are added in product, then add the 5.5mg/ml collagen type vs enzyme and 2U/ml of two sesquialter amnion volumes
Hyaluronidase 1:1 mixed liquor digests 3.5h;The cell suspension that digestion finishes is collected, it is multiple that the equilibrium liquid without calcium ions and magnesium ions is added
Cleaning counts mononuclearcell after glacial acetic acid dilution, obtains cell total amount;Cell concentration is adjusted as 1.0 × 105A/ml, adds
Enter serum-free mescenchymal stem cell culture medium, be placed in 37 DEG C, a concentration of 5%CO2, cultivated in the incubator of saturated humidity, per 2d
It changes liquid 1 time, obtains primary amnion mesenchymal stem cell after a week;When the 80-90% of cell growth to culture vessel floor space,
When a large amount of cell masses are linked to be piece, to be passed on after concentration 0.05%g/L trypsin digestions, cell are placed in and is covered with the bad ammonia of L- polies
Secondary culture is carried out in the culture bottle of acid, the amnion mesenchymal stem cell passed on.Quality testing is carried out to cell before freezing:
Culture solution before being collected to cell carries out infectious disease pathogens detection:Such as hepatitis B, hepatitis, syphilis, AIDS;In the same of cell cryopreservation
When, it takes a small amount of cell to carry out microculture, carries out microorganism detection.Commercialization serum-free cell frozen stock solution is chosen, by 5 ×
106The human amnion mesenchymal stem cell of a/ml is added in 1ml frozen stock solutions, is put into cryopreservation tube, is first placed in refrigerator, is protected at 4 DEG C
8min is deposited, then is adjusted to preserve 12-18h at -80 DEG C, is then placed in the human amniotic mesenchymal stem cell bank equipped with liquid nitrogen container;Root
It is preserved according to neonatal sex and ABO/Rh partings and HLA partings, establishes cellular informatics archives, it is dry thin to construct human amnion mesenchymal
Born of the same parents library.
2, the preparation of sodium alginate complexing agent
The anhydrous sodium alginates of 3g are weighed, are added in 100ml vials, are subsequently added into 40ml deionized waters uniform stirring extremely
Dissolving, adds 20ml glycerine mixings, and the serum-free mescenchymal stem cell culture medium that 40ml is then added mixes well;Again toward glass
Epidermal growth factor EGF 1mg, basic fibroblast growth factor bFGF 2mg, angiogenesis factor are added in glass bottle
VEGF2mg obtains sodium alginate complexing agent after stirring evenly;
3, the preparation of cell repair agent
1.0 × 10 are added into trehalose complexing agent made from 1ml steps 16Cell/ml human amnion mesenchymal stem cells are outstanding
Liquid 1ml adds the Sodium Hyaluronate liquid that 1ml mass concentrations are 1%, up to cell repair agent after stirring evenly.
For chronic skin injury or the client of small area skin injury, using culture period cell rapid configuration, according to
The gender and ABO/Rh partings and HLA partings at family select suitable cell seed from amniotic mesenchymal cell library.According to cell
The speed of growth cultivates cell in advance, and cell is collected when density 80%.First 20ml phosphate buffers (PBS) is used to wash, 400g,
5min is centrifuged, and removes impurity and cell fragment ingredient.Count cell, after Cell viability detection is qualified, immediately with alginic acid
Sodium complexing agent mixes, and is loaded on sterile glass vials, gives client's use.
3 application effect of embodiment detects
1, human amnion mesenchymal stem cell biological property made from embodiment 1:
A. flow cytometer cellular elements surface markers are identified:
Take the good 3rd generation amnion mesenchymal stem cell of growth conditions to do flow cytometry, see its whether express CD34,
The surface moleculars such as CD44, CD45, CD73, CD90, CD105, CD326, HLA-DR.
B. immunofluorescent staining is tested:
The good 3rd generation amnion mesenchymal stem cell of growth conditions is taken, cell concentration is adjusted, does cell climbing sheet, cell is long
It is fixed with 4% paraformaldehyde after full, does immunofluorescent staining, whether detection contains fluorescence microscopy into the cell under the microscope
Vimentin。
C. external Multidirectional Differentiation experiment:
Human amnion mesenchymal stem cell is lured in dexamethasone, the skeletonization such as β-phosphatide glycerine, ascorbate after secondary culture
It is cultivated in conductor system to osteoblast differentiation, is identified with alizarin red and alkaline phosphatase staining;It is filled between secondary culture descendant's amnion
Matter stem cell is cultivated in the adipogenic induction system containing 3-isobutyl-1-methylxanthine, indoles magnesium zinc to lipoblast
Differentiation, is identified with oil red O stain.
2, the detection method of wound surface smearing agent application effect:
Transplantation experiments in a bodies
The 1.5cmX1.5cm full thickness skin surface of a wound is cut off in 18 nude mice back surgeries, nude mice is randomly divided into 3 groups, every group 6
Only:One group of smear cell renovation agent;One group is only smeared sodium alginate complexing agent;One group is only smeared physiological saline.0,7,14,
21,28 days, one was put to death respectively.Wound tissue, 4% paraformaldehyde is taken to fix, specimens paraffin embedding slices carry out Yihong bush uniformly dyeing
Color, immunohistochemistry and identified by immunofluorescence carry out wound tissue histology inspection:
Wound healing time, cicatrization and skin suppleness;
The detection of donor's cells in receptor wound tissue:
The identification of green fluorescent protein:In order to study whether human amnion mesenchymal stem cell participates in wound repair, with green
Fluorescin plasmid transfected with human amnion mesenchymal stem cell, recipient mice wound cambium is solid with 4% paraformaldehyde after 2 weeks
Fixed, with frozen section embedding medium, -20 DEG C of cryostat microtome slices detect human amnion mesenchymal stem cell with fluorescence microscope
The expression of green fluorescent protein.
The detection of cytokeratin and collagen:In order to study whether human amnion mesenchymal stem cell breaks up in vivo
Chrotoplast forms collagen, and the new raw slicers of Recipient mice do rabbit-anti human keratin (pan-CK) and the anti-human 1 Collagen Type VI egg of rabbit respectively
White polyclonal antibody is incubated jointly, and the goat anti-rabbit immunoglobulin G (IgG) that fluorescence rhodophyll marks then is used to be incubated, 0,
It is detected with fluorescence microscope within 7,14,21,28 days.
As a result, it has been found that:
1, the 3rd generation amnion mesenchymal stem cell is taken to be detected with stream type cell analyzer.As a result it shows:Express interstitial cell table
Face indicates CD44, CD73, CD90, CD105, does not express hematopoietic cell mark CD34, CD45, does not express epithelial cell mark
CD326 is not expressed and is repelled the surface marker HLA-DR for reacting closely related with trnasplantion immunity.
2, the 3rd generation amnion mesenchymal stem cell is taken to do immunofluorescence dyeing.The results show that cells express cell skelemin
Vimentin。
3, human amnion mesenchymal stem cell can be divided into osteoblast and lipoblast.
4, cell repair agent can be excellently attached to wound tissue, and participate in the reparation of epidermis and corium:
(1) using 14 days after cell repair agent, wound healing, flexibility is preferable;And the wound of control group then needs 21
It could heal, and have extensive cicatricial contraction.
(2) 7 days after using cell repair agent, visible 4~6 layers of new skin of wound tissue Yihong haematoxylin dyeing, after 21 days
Reach 8~10 layers;Collagen queueing discipline in corium, and control group wound needs 21 days ability epithelializations.
(3) after using cell repair agent after 14 days, find have containing green fluorescent protein in recipient mice wound tissue
Donor's cells exist, and control group is then feminine gender.Prove that donor's cells transplant successfully in recipient's body.
(4) after using cell repair agent after 14 days, it is found that 1 Collagen Type VI obviously increases in intradermal expression, and keratin
It expresses in epidermis and obviously increases, it was demonstrated that donor's cells can be divided into epithelium and secrete collagen.
As shown in Figure 2, amnion mesenchymal stem cell height expresses interstitial cell surface marker CD44, CD73, CD90, CD105,
Hematopoietic cell mark CD34, CD45 are not expressed, does not express epithelial cell mark CD326, are not expressed and are repelled generation with trnasplantion immunity
React closely related surface marker HLA-DR.
From the figure 3, it may be seen that containing a large amount of cytoskeletal protein Vimentin in amnion mesenchymal stem cell cytoplasm.
As shown in Figure 4, it takes on a red color tubercle through Alizarin red staining.This illustrates that amnion mesenchymal stem cell has Osteoblast Differentiation
Potential.
As shown in Figure 5, amnion mesenchymal stem cell has a large amount of red fat drips, this explanation to have to adipocyte point into the cell
The potential of change.
Claims (2)
1. a kind of preparation method of new medical cell repair agent, which is characterized in that specific preparation process is,
(1) preparation of sodium alginate complexing agent
The anhydrous sodium alginates of 1~10g are weighed, are added in 100ml vials, are subsequently added into 40ml deionized waters uniform stirring extremely
Dissolving, adds 20ml glycerine mixings, and the serum-free mescenchymal stem cell culture medium that 40ml is then added mixes well;Again toward glass
Epidermal growth factor EGF 1mg, basic fibroblast growth factor bFGF 2mg, angiogenesis factor VEGF are added in glass bottle
2mg obtains sodium alginate complexing agent after stirring evenly;
(2) preparation of cell repair agent
1.0 × 10 are added into trehalose complexing agent made from 1ml steps 16Cell/ml human amnion mesenchymal stem cell suspensions
1ml adds the Sodium Hyaluronate liquid that 1ml mass concentrations are 1%, up to cell repair agent after stirring evenly.
2. the preparation method of cell repair agent according to claim 1, which is characterized in that human amnion mesenchymal stem cell
Preparation process is,
(1) it isolates and purifies
Aseptic collection amnion tissue, Biohazard Safety Equipment is taken to be built in the PBS of precooling and rinse, if the related chorion of placenta, needs elder generation
Chorion and amnion are detached, amnion is taken out, abandons chorion;The small item of amnion that amnion tissue is 2cm × 2cm is cut, it is isometric to add
Enter 2U/ml neutral protein enzymic digestion 30min, then adds the 5.5mg/mlV Collagenase Types and 2U/ml hyalomitomes of two sesquialter amnion volumes
Sour enzyme 1:1 mixed liquor digests 3.5h;The cell suspension that digestion finishes is collected, the equilibrium liquid without calcium ions and magnesium ions is added and is cleaned multiple times,
Mononuclearcell is counted after glacial acetic acid dilution, obtains cell total amount;
Cell concentration is adjusted as 1.0 × 105A/ml is added serum-free mescenchymal stem cell culture medium, is placed in 37 DEG C, a concentration of
5%CO2 is cultivated in the incubator of saturated humidity, is changed liquid 1 time per 2d, is obtained primary amnion mesenchymal stem cell after a week;It waits for thin
Intracellular growth to culture vessel floor space 80-90% when, when a large amount of cell masses are linked to be piece, with concentration 0.05%g/L trypsase
It is passed on after digestion, cell is placed in the culture bottle for being covered with L- poly-D-lysines and carries out secondary culture, between the amnion passed on
Mesenchymal stem cells;
(2) quality testing is carried out to cell before freezing
A. the culture solution before being collected to cell carries out infectious disease pathogens detection, including hepatitis B, hepatitis, syphilis, AIDS pathogen;
B. while cell cryopreservation, cell is taken to carry out microculture, carries out microorganism detection;
(3) commercialization serum-free cell frozen stock solution is chosen, by 5 × 106The human amnion mesenchymal stem cell of a/ml is added to 1ml and freezes
It in liquid, is put into cryopreservation tube, is first placed in refrigerator, 8min is preserved at 4 DEG C, then be adjusted to preserve 12-18h at -80 DEG C, be then placed in
In human amniotic mesenchymal stem cell bank equipped with liquid nitrogen container;
(4) it is preserved according to neonatal sex and ABO/Rh partings and HLA partings, establishes cellular informatics archives, construct people's amnion
Mesenchyma stem cell.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810540720.5A CN108685948B (en) | 2018-05-30 | 2018-05-30 | Preparation method of medical cell repairing agent |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810540720.5A CN108685948B (en) | 2018-05-30 | 2018-05-30 | Preparation method of medical cell repairing agent |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108685948A true CN108685948A (en) | 2018-10-23 |
| CN108685948B CN108685948B (en) | 2020-10-30 |
Family
ID=63849176
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810540720.5A Active CN108685948B (en) | 2018-05-30 | 2018-05-30 | Preparation method of medical cell repairing agent |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108685948B (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110179807A (en) * | 2019-05-17 | 2019-08-30 | 温州医科大学附属第二医院、温州医科大学附属育英儿童医院 | Application of the trehalose in the drug that preparation promotes ultra long random flap survival |
| CN111139221A (en) * | 2020-01-09 | 2020-05-12 | 赛瑞诺(北京)生物科技有限公司 | Culture and cryopreservation method of amniotic mesenchymal stem cells |
| CN111378616A (en) * | 2020-01-14 | 2020-07-07 | 河南省银丰生物工程技术有限公司 | Research method for repairing skin by adopting prepared stem cell freeze-dried powder |
| CN111939176A (en) * | 2020-08-27 | 2020-11-17 | 北京瀚梅生物科技有限公司 | Skin injury repairing agent containing adipose-derived stem cells and preparation method thereof |
| CN111956668A (en) * | 2020-08-27 | 2020-11-20 | 北京瀚梅生物科技有限公司 | Skin regeneration and repair cell composition and preparation method thereof |
| CN112048469A (en) * | 2020-09-11 | 2020-12-08 | 山东大学 | Method for enhancing secretion function of mesenchymal stem cells and application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102670654A (en) * | 2012-04-28 | 2012-09-19 | 北京汉氏联合干细胞研究院有限公司 | Stem cells preparation for repairing wound surface and preparation method thereof |
-
2018
- 2018-05-30 CN CN201810540720.5A patent/CN108685948B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102670654A (en) * | 2012-04-28 | 2012-09-19 | 北京汉氏联合干细胞研究院有限公司 | Stem cells preparation for repairing wound surface and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| KAIRINOS N等: "Negative pressure wound therapy I:the paradox of negativ-pressure wound therapy", 《PLAST RECONSTR SURG.》 * |
| 周武: "封装VEGF及bFGF的海藻酸钠凝胶负荷内皮祖细胞修复大鼠皮肤创面的实验研究", 《中国博士学位论文全文数据库》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110179807A (en) * | 2019-05-17 | 2019-08-30 | 温州医科大学附属第二医院、温州医科大学附属育英儿童医院 | Application of the trehalose in the drug that preparation promotes ultra long random flap survival |
| CN111139221A (en) * | 2020-01-09 | 2020-05-12 | 赛瑞诺(北京)生物科技有限公司 | Culture and cryopreservation method of amniotic mesenchymal stem cells |
| CN111139221B (en) * | 2020-01-09 | 2022-05-10 | 赛瑞诺(北京)生物科技有限公司 | Culture and cryopreservation method of amniotic mesenchymal stem cells |
| CN111378616A (en) * | 2020-01-14 | 2020-07-07 | 河南省银丰生物工程技术有限公司 | Research method for repairing skin by adopting prepared stem cell freeze-dried powder |
| CN111939176A (en) * | 2020-08-27 | 2020-11-17 | 北京瀚梅生物科技有限公司 | Skin injury repairing agent containing adipose-derived stem cells and preparation method thereof |
| CN111956668A (en) * | 2020-08-27 | 2020-11-20 | 北京瀚梅生物科技有限公司 | Skin regeneration and repair cell composition and preparation method thereof |
| CN111939176B (en) * | 2020-08-27 | 2021-05-04 | 上海咏俪玺辰护肤品有限公司 | Skin injury repairing agent containing adipose-derived stem cells and preparation method thereof |
| CN112048469A (en) * | 2020-09-11 | 2020-12-08 | 山东大学 | Method for enhancing secretion function of mesenchymal stem cells and application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108685948B (en) | 2020-10-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108685948A (en) | A kind of preparation method of new medical cell repair agent | |
| CN105713871A (en) | Human chorion mesenchymal stem cell isolated culture method | |
| CN103352026A (en) | Method for cultivating autologous umbilical cord mesenchymal stem cells by adopting human umbilical cord blood rich platelet lysate | |
| CN107494517A (en) | Serum-free frozen stock solution and its application in mescenchymal stem cell is frozen | |
| CN104342402B (en) | A kind of bone marrow dedifferentes the cultural method of mescenchymal stem cell | |
| US20150231244A1 (en) | Regenerative cell and adipose-derived stem cell processing system and method | |
| CN110079498B (en) | Human placenta mesenchymal stem cell and preparation method and application thereof | |
| US20200078411A1 (en) | Biological Scaffolds, Products Containing Biological Scaffolds and Methods of Using the Same | |
| CN108192862A (en) | A kind of preparation method of pilose antler stem cell, pilose antler stem cell and its application | |
| Akhavan-Tavakoli et al. | In vitro differentiation of menstrual blood stem cells into keratinocytes: a potential approach for management of wound healing | |
| CN107385517A (en) | The construction method of mesenchyma stem cell | |
| CN105331579A (en) | Separation and culture method and application of human testis mesenchymal stem cells | |
| CN105238749A (en) | Method for resuscitating mesenchymal stem cells | |
| Bolte et al. | Two‐step stem cell therapy improves bone regeneration compared to concentrated bone marrow therapy | |
| CN108938669B (en) | Stem cell ointment for treating skin injury and preparation method thereof | |
| CN106614524B (en) | Preservation solution and preservation method for mesenchymal stem cells | |
| Linon et al. | Engraftment of autologous bone marrow cells into the injured cranial cruciate ligament in dogs | |
| Trombi et al. | Human autologous plasma‐derived clot as a biological scaffold for mesenchymal stem cells in treatment of orthopedic healing | |
| CN110693911A (en) | Menstrual blood-derived endometrial stem cell preparation and preparation method and application thereof | |
| CN107349220A (en) | A kind of preparation comprising fibroblast excretion body and application thereof | |
| CN110172445A (en) | Expand method, culture medium and the application of mesenchymal stem cell | |
| Bodhak et al. | Combinatorial cassettes to systematically evaluate tissue-engineered constructs in recipient mice | |
| US9434923B2 (en) | Preparation of parental cell bank from foetal tissue | |
| TWI640625B (en) | Method for rapid mincing of adipose tissues to isolate alive cells in vitro | |
| CN103173407B (en) | Method for induced differentiation of liver cells by using endometrium stem cells |
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 |