CN105779384A - Seed cell screening and culturing cryopreservation technical method of human amniotic mesenchymal stem cells for tissue engineering - Google Patents
Seed cell screening and culturing cryopreservation technical method of human amniotic mesenchymal stem cells for tissue engineering Download PDFInfo
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- CN105779384A CN105779384A CN201610194898.XA CN201610194898A CN105779384A CN 105779384 A CN105779384 A CN 105779384A CN 201610194898 A CN201610194898 A CN 201610194898A CN 105779384 A CN105779384 A CN 105779384A
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- 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
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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- 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
- C12N5/0603—Embryonic cells ; Embryoid bodies
- C12N5/0605—Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly
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- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/02—Coculture with; Conditioned medium produced by embryonic cells
- C12N2502/025—Coculture with; Conditioned medium produced by embryonic cells extra-embryonic cells, e.g. amniotic epithelium, placental cells, Wharton's jelly
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- 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
- C12N2502/1388—Mesenchymal stem cells from other natural sources
Abstract
The invention discloses a seed cell screening and culturing cryopreservation technical method of human amniotic mesenchymal stem cells for tissue engineering, and belongs to the field of biological stem cell medical medicines. The amniotic mesenchymal stem cells are derived from multipotential stem cells of mesoblast. The invention describes and particularly discloses an innovative stem cell acquisition, separation, extraction and cryopreservation technique and a placenta amniotic layer high-simulation in-vivo extracellular matrix acquisition and preparation technique, the techniques are significant points of innovative techniques of hAMSCs adhesion extraction and preparation, multi-combined culture liquid gradient serum-free habituated culture system design is also a key technique needed to particularly acquire hAMSCs, and the key technique combined extraction culture is efficient bionic biotechnology invention. The acquired stem cells are accordant with the international society for cellular therapy (ISCT) standard and has high purity, stem cell proliferation amplification and good cell activity, meanwhile design and development of future PDB-MSCs stem cell clinical medicines can be met, stem cell recycling requirements are provided in medical rescue, and multipurpose requirements of seed stem cells needed to make tissue engineering skin are provided.
Description
Technical field
The invention belongs to biological stem cells technology, field of tissue engineering technology.Relate in a kind of external structure height analogue body
Attaching environment separation is screened, the cultivation of human amnion mesenchymal stem cell, amplification, Techniques of preserving.
The amnion-derived mescenchymal stem cell of people (human amniotic mesenchymal stem cells, hAMSCs)
Derive from mesoblastic pluripotent stem cell, it is recognized that there is height self-renewal capacity and multi-lineage potential.This
Invention illustrates and the acquisition innovation separation and Extraction Cryopreservation Technology of this stem cell of specifically disclosed hAMSCs, Placenta Hominis sheep
Film floor height intend cells in vivo epimatrix obtain and technology of preparing, be hAMSCs attachment extract preparation be innovative technology
Key takeaway, many combination culture fluid gradient serum-frees domestication cultivating systems designs are also exclusive acquisition hAMSCs
Required core technology, it is efficient bionical biotechnology inventive technique that cultivation is extracted in key technology combination.
Background technology
In recent years, international and domestic stem cell regenerating medical research field is increasingly paid attention to, cell therapy regeneration doctor
Learnt unprecedented propelling, scientific research field be washed in be able under the Chaoyang in epoch flourish.Between especially
Mesenchymal stem cells has become as a kind of new treatment means and extensively applies the impaired or histoorgan of pathological changes of reparation, controls
Treat Cardial or cerebral vascular diseases, nervous system disease, clinical Bone Defect Repari, repair of cartilage, bone marrow regeneration, muscle again
Life, lipogenesis, tendon repair, gene therapy, blood vessel support, corneal injury, empyrosis wound repair
Etc. multiple disease.Stem cell after cultured and amplified in vitro is opened up in organizational project regeneration and Various Tissues are repaired
Illustrating and make researcher impressive treatment popularity potential, this potential is that scientific research field is mathematical
Developing direction.
Mescenchymal stem cell (Mesenchymal Stem Cells MSCs) has self renewal and Multidirectional Differentiation
Ability.Derive from a fetal development mesoblastic class pluripotent stem cell in early days.It is a kind of non-hematogenic multi-functional
Stem cell, is widely present in human body different tissues, such as bone marrow, fat, periodontal membrane and umbilical cord, placenta tissue
Deng.Scholar was had to find its main source mesenchymal stem cells MSCs (bone marrow in recent years
Mesenchymal stemcells, BMSc) can with autotransplantation, it is to avoid the immunologic rejection after transplanting, bone marrow
The amplification in vitro of mescenchymal stem cell is easier, and have interdepartmental or across differentiation of germinal layers be different tissue sources
The potential of cell, so being applied to the multisystems such as central nervous system, cardiovascular and immune system in clinic
The treatment of disease, also relates in cancer and hereditary research.BMSc can Differentiation Induction in vitro for becoming
Osteocyte, chondrocyte, adipose cell, myoblast induction under certain conditions can be divided into nerve
Cell, and autotransplantation can be carried out, it is to avoid immunologic rejection is it is considered to be treat the good cell of multiple disease
Source.Also having researcher to think in placenta tissue and also contain abundant mescenchymal stem cell, prompting can be by it
Extract the alternative cell as cellular replacement therapy.Placenta mesenchyma stem cell has collection convenience, is prone to body
Outer cultivation, expand and the characteristic such as induction is it is considered to be a kind of preferable seed cell of stem-cell research.
Mesenchymal stem cells MSCs, enters a phase at U.S. FDA approved clinical in terms of Cardiac Stem Cells transplanting
Test, this fully illustrates the huge applications prospect of MSCs.But mesenchymal stem cells MSCs containing in bone marrow
Measure less, and along with the increase at age, the content in bone marrow gradually decrease, its proliferation and differentiation ability also by
The most weak；Drawing materials of mesenchymal stem cells MSCs is invasive store period simultaneously, adds the misery of donor
With infect chance, so the clinical practice of mesenchymal stem cells MSCs receives certain restriction, and sufferer
Severe infections, hematological malignancy or with advancing age, bone marrow cell and propagation, differentiation capability are the brightest
When aobvious decline, patient just cannot obtain enough BMSCs that can be used for treatment smoothly, necessary for this
Find new MSCs source.
Placental origin, in embryonic development period extraembryonic mesoderm, is made up of interstitial, blood vessel, trophocyte, contains
Substantial amounts of mesenchyme composition, also containing substantial amounts of stem cell.Umbilical blood and umbilical cord China Tong Shi glue all may contain MSCs,
And closely coupled with umbilical cord tissue, after fetal birth, many Placenta Hominiss being dropped together with umbilical cord as refuse also contain
Have and the cell of mesenchymal stem cells MSCs similar characteristic, placenta tissue but contains these precursors, greatly
Solve stem cell in degree carrys out source problem.
Mature Placenta Hominis is the garbage of childbirth, draws materials easily, donor does not cause damage, without ethical issues, and
And Placenta Hominis volume is big, it is thus achieved that stem cell is the most, so placenta mesenchyma stem cell is more convenient is applicable to clinic.
There is scholar to be successfully separated at fetus decidua side placenta tissue and turn out placenta mesenchyma stem cell, and it is biological to observe it
Learn characteristic and anti-apoptotic cytokine vascular endothelial cell growth factor, insulin like growth factor, hepatocyte life
The secretion level of the long factor, result confirms that the biological characteristics of placenta mesenchyma stem cell is dry with medulla mesenchyma thin
Born of the same parents are similar, and can secrete vascular endothelial cell growth factor, insulin like growth factor and hepatocyte growth factor.
Study confirmation placenta mesenchyma stem cell and there is the function similar with mesenchymal stem cells MSCs and characteristic, two
The multi-lineage potential of person is also similar to, and the multiplication capacity of the mescenchymal stem cell in Placenta Hominis source is higher.Both at home and abroad
Many researchers finds, Placenta Hominis derived mesenchymal stem cell can be induced to differentiate into mesoblastic in testing in vitro
Myocardial cell, smooth muscle cell, osteoblast, adipose cell, endoblastic islet cells, hepatocyte and
Ectodermic neuron, astrocyte, chondrocyte, and these results are equally at myocardial infarction and handkerchief
In the In vivo model such as the gloomy rat model of gold, diabetic mouse model and spinal cord injury primate model, treatment is aobvious
Effect is confirmed.Also have been reported that Placenta Hominis derived mesenchymal stem cell further applies reparation human knee joint in the future
Degenerative change or injury of meniscus of knee joint scientific experiment.
The present invention tastes directly separating mesenchymal stem cell from placenta tissue, and including Human plactnta source, Cord blood comes
Source, the patent of invention of the mescenchymal stem cell of amniotic fluid-derived, and the biological characteristics of gained cell is reflected
Fixed, for research Derived from Mesenchymal Stem Cells ability, carrying out clinical application research as seed cell provides solely
Special thinking is to obtain system foundation.Scientific research basis is established for mescenchymal stem cell clinical treatment and basic research.
Mescenchymal stem cell is the essentialspecies daughter cell of current organizational project, after the research of multidigit scholar for many years,
Although organizational project has been obtained for remarkable progress, but, but owing to lacking at present preferable screening technique, fill
Matter stem cell is difficult to problems such as obtaining, amplification in vitro is difficult so that it is the application fed back in clinical rank is restricted.
Therefore, find a kind of be easily obtained, immunogenicity is low, the seed of the clinical treatment rank that is prone to amplification in vitro is thin
Born of the same parents are one of current organizational project problems in the urgent need to address.
Regenerating tissues engineering cell is set up work and is started from this century (Harrison 1907, Carrel 1912),
Guidance leads each big linking fields such as biology, clinical medicine material, is referred to as one of important basic science,
Tissue construction and cell injuring model are it is critical that problem.Tissue, analogue body is taken from donor donor's live body
The serial specific micro-environment in vitro system such as interior physiological environment, carries out hatching and cultivates, makes histiocyte healthy growth.
Mescenchymal stem cell extracellular matrix stick strong rapid charater so that in-vitro screening cultivate people source between fill
Matter stem cell is able to method specificity development at home and abroad.And stem cell biology performance, can accomplish to promote merit
Can, it is to avoid potentially harmful gene pairs stem cell controls divergaence time and controls the impact of different differentiation directions etc.,
Basic scientific research, clinical treatment, stem-cell therapy research direction, regeneration injury repairing etc. all have the weight of period of history
Otherwise commutable directive significance.
Extracellular matrix (extracellular matrix, ECM) is widely present in being substantially filled with of intercellular substance
Composition is cell metabolism, grow, breed, break up, express, transmit, reciprocal relied on the heaviest
Want place.Researcher thinks in early days, and epimatrix is a kind of to organize simple support structure interior, extracellular.
Hauschka with Konigsberg finds the collagen filling interstice and to promote into flesh thin research in 1966
The phenomenon of dysuria with lower abdominal colic myotube, and proved after the two years, scholars are just to this stable outer material microenvironment
There are new understanding and thinking.
Hay reported ECM after 11 years and has important inducing action to embryo development procedure.ECM affects
The phenomenon of cell behavior is paid close attention to and is studied widely, but the theoretical basis of not molding is proved
ECM and the substantial connection of cell.Until nineteen eighty-two have scholar to bring forward proof and set up ECM and cytoskeleton and
The model of " the most reciprocal " between nuclear matrix.In this mould, ECM molecule is made mutually with cell surface receptor
With, then signal is entered in cytoplasm by cell membrane conduction, cause and from cytoskeleton to nuclear one be
Row change, thus cause the expression of some specific gene.
Study and prove that ECM can be had an effect again by the expression product of these genes in turn, being permitted of today
Many researchs confirm the reasonability of this model, and prove that cell and ECM interact, and directly take part in thin
The sticking of born of the same parents, grow, migration, differentiation and the process of programmed death (apoptosis).It is thin that outer medium participates in regulation
Intracellular cytokine, the activity of somatomedin, can also directly activate, signal transduction mechanism in active cell.Due to this
Planting the regulation to cell and the conduction to signal, therefore, we must be fully understood by the change of extracellular matrix
, physics, biological property and function and the effect in the formation and reparation of tissue thereof, could preferably answer
It is configured to tissue with these biomaterials accept, or even simulated tissue improves " the biological framework " of regeneration.I
Set up high intend that cell epimatrix material utilizes is just reverse theory, instruct us to carry out in a deep going way ECM
Understanding, simultaneously the most also retroaction obtains our available stem cell, this research mode promote subject to move ahead and
Development.
The culture medium of the most conventional a kind of hAMSCs of cultivation stem cell is exactly the culture medium being added with animal serum.
But, use the hAMSCs stem cell of this culture medium culturing, not only can occur growing because of addition problem
Fast or relatively slow phenomenon, and after exceeding several generations, its potential being divided into functioning cell is substantially reduced, animal
The comprehensive multiplication capacity of serum free culture system technology, immunoregulation capability, secrete cytokines this three aspect of ability equal
Can be changed by uncontrollable direction.The animal sources serum batch of different geographical vendor source exists potential both at home and abroad
The most repeated, also it is the technical bottleneck that have impact on cell amplification large-scale production；On the other hand, relative to people it is
Heterologous, foreign protein is mixed into potential cell therapy and has a uncertainty, thus limit its clinically should
With.Therefore, explore rapid amplifying hAMSCs stem cell and don't affect condition of culture and the cultivation thereof of its potential
The optimum organization of basis set one-tenth, is very important research contents.
Human amnion mesenchymal stem cell (human amniotic mesenchymal stem cells, hAMSCs) is
Derive from mesoblastic pluripotent stem cell, there is height self-renewal capacity and multi-lineage potential, be that cell moves
Plant the preferable seed cell with organizational project.The nothing of hAMSCs cellular environment in many combination cultivating system analogue bodies
Serum free culture system scheme is with traditional containing compared with animal serum culture medium, and offer of the present invention obtains hAMSCs stem cell
Not only purity performance high, adherent is strong, obtain cell can keep stem cell pedomorphism, have more preferable cell
Multiplication rate and cell dryness, and the cell performance that the cells show cultivating acquisition goes out excellence reduces training simultaneously
Form this.Therefore the inventive method obtains the hAMCs keeping stablizing cell characteristics, and this is for the most clinical real
Test research to have great importance.Present invention especially discloses hAMCs separation and Culture store method, biology
The research of characteristic, stem cell properties and its Differentiation Induction in vitro ability carries out detailed technology path, acquisition
HAMSCs meets (ISCT) standard and has the design meeting following exploitation PDB-MSCs stem cell clinical medicine
Exploitation；Medical treatment is carried out stem cell and is fed back requirement；And set up the seed stem cell needed for organization engineering skin
Multipurpose demand.
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Summary of the invention
One, placenta amnion floor height plan cells in vivo epimatrix kind:
It is characterized in that: for the current situation of the technology of the present invention, it is an object of the invention to provide a kind of high plan body
Interior Human plactnta rete layer attaches for screening as extracellular matrix, mescenchymal stem cell screening and separating and In vitro culture
Method, and make the high-purity mescenchymal stem cell of acquisition.
Invention belongs to biological stem cells technical field, and method feature includes: consubstantiality Human plactnta amniotic membrane pull-up is thin
Born of the same parents' substrate preparation；The separation of mescenchymal stem cell；Height is utilized to intend the screening of internal attaching substratum mescenchymal stem cell
And cultivate.
At present the most stablize subculture more than 16 (generations), before finding after testing to expand for 16 generations by this method
Mescenchymal stem cell still there is normal caryotype, change without exception.Confirm that this is thin through experiment in vivo and vitro
Born of the same parents are acellular poison, has stronger multiplication capacity, it is possible to form differentiation.Mescenchymal stem cell is in scientific research, religion
Learn and immeasurable effect is played in the application of clinic, breed huge academic direction and social benefit and warp simultaneously
Ji benefit, advances the academic Foundation of China's mescenchymal stem cell by the theoretical basis of this kind of method.
Two, placenta amnion floor height plan cells in vivo epimatrix is constituted:
Native three dimensional pore structure is remained containing multiple collagen through analyzing.
Three, placenta amnion floor height is intended cells in vivo epimatrix substrate pores rate and sticks surface character:
High plan substrate electron microscopic observation, the basement membrane pore diameter measuring the substrate tissue regeneration that this kind sticks is
30～211 μm.Three dimensions constructed by this porosity stick surface all be conducive to mescenchymal stem cell stick,
Apposition growth, breed, break up.
By method made above, the microcosmic framework of extracellular matrix and complete basement membrane are effectively kept it
Structure, its main component includes the insoluble matrixs such as each Collagen Type VI, elastin laminin, proteoglycan and glycosaminoglycan
Composition, can be that mescenchymal stem cell quickly adheres to, breeds extension offer strong support.Therefore, amniotic membrane floor height
Intend the solution that substrate is current optimal cell screening.
Four, placenta amnion confluent monolayer cells height is intended the preparation of cells in vivo epimatrix and is embodied as step and method:
One, following two step kind method is included:
Step one: placenta tissue disinfects process:
Placenta amnion floor height intends cells in vivo epimatrix processing procedure: takes donor operating-table aseptic condition and obtains strong
Health term birth baby's placenta tissue, 4 DEG C of preservations, normal saline dilution containing 0.05～0.1% benzalkonium bromide molten
In liquid 10～15min, fully cleaning the blood of residual, rinsing is disinfected to colourless, in superclean bench
Take out placenta tissue, divest amniotic membrane and cut into piece of tissue immersion, with physiological saline solution cleaning and removing residual benzene repeatedly
Prick bromine ammonium.
Step 2: low temperature-30～-100 DEG C of lyophilizations, vacuum reaches 10～130pa, sloughs amniotic membrane
Piece of tissue 90～more than 95% moisture, take out and seal and vacuum packaging, irradiated 15～80KGY doses of sterilization.
Whole product can preserve for a long time.Start and only need to soak 37 DEG C, PBS liquid or following described nutritional solution recovery 15～30min
Can apply, it is better that immersion extends to 10～12h.Use afterwards and cell rinsing liquid should be spent and (see below
Mutually it should be mentioned that the de-Cell sap being suitable in method) de-cell processes, again through said process immortality Reusability
Feature.
1: step 2 detailed description of the invention:
Above-mentioned mentioned de-Cell sap is prepared as described below:
Two, method:
Case method 1: amnion tissue block add 0.1～0.5% protease ice bath cold disappear 48～12h, with containing 0.25%
Glutaraldehyde phosphate buffer solution regulation PH rinses piece of tissue 3～6 times with crosslinked fluid between 7.20～7.40,
It is finally putting in crosslinked fluid crosslinking 4～6h, after terminating, repeatedly rinses 8～10 with ultra-pure water (or water for injection)
Secondary removal remains glutaraldehyde, can carry out freeze-drying and dehydrating encapsulation.
1.1: case method 1 detailed description of the invention:
Method one feature: cross-linking agent can effectively make the crosslinking framework of connective tissue protein's insolubility, strengthens
The anti-degradability of substrate, keeps substrate complete, and Protease Treatment substrate can make its soft wilful enhancing.
The basal cell layer obtained be cell enliven the most attachment, divide, break up place, organized layer space
Framework timbering material in vitro is difficult to simulation to be imitated.Therefore this type of placenta amnion floor height plan cells in vivo epimatrix is
Be suitable for mescenchymal stem cell adhere to, breed, grow, break up needed for ideal microenvironment place.
Case method 2: enzyme-nonionic surfactant-chelating agent combination method
0.1～0.25%DisPase neutral protease and 0.01～0.02%EDTA simultaneous digestion, neutral protein
Enzyme acts on basement membrane, optionally decomposes fibronectin and IV Collagen Type VI (acting on 48h at 4 DEG C), then
Application nonionic surfactant makes cell dissolved destruction TritonX-100 (Triton X-100)
The lipid bindings such as (0.1～0.8% solution act on 48～24h under room temperature) and the phospholipid in biomembrane are formed compound
Thing is dissolved in solution, and the oleophylic cardinal extremity in TritonX-100 also can combine broken with cell outer membrane protein simultaneously
Bad biomembrane, thus reach de-and abandon cytosis.
Carry out glutaraldehyde cross-linking step through method 1 after de-cell, freeze-drying and dehydrating encapsulation can be carried out.
2.1: case method 2 detailed description of the invention:
Case method 3: chelating agent salt adding-anion surfactant method
Utilize high permeating sodium chloride solution ((0.01～0.02%EDTA contain 0.1～1mol/L sodium chloride solution, 37 DEG C
Lower effect 24～12h)) make the hemi desmosome of anchor-shaped filament and Tissue Base cell separate, thus completely remove
Cell, then with rupture of membranes agent dodecyl sodium sulfate (0.1～0.8%SDS solution, under room temperature act on 60～30min)
Cell component is removed from amniotic membrane.
Carry out glutaraldehyde cross-linking step through method 1 after de-cell, freeze-drying and dehydrating encapsulation can be carried out.
3.1: case method 3 detailed description of the invention:
Case method 4: with 0.1～0.6% trypsin and 0.01～0.1%EDTA volume ratio (1:2)
Mixed liquor simultaneous digestion piece of tissue, 37 DEG C, rapid digestion 60～10min, the de-cell of rinsing, finally puts repeatedly
Enter and crosslinked fluid cross-links 4～6h, after terminating, repeatedly rinse 8～10 removals with ultra-pure water (or water for injection)
Residual glutaraldehyde, can carry out freeze-drying and dehydrating encapsulation.
4.1: case method 4 detailed description of the invention:
Above method all can be combined to be prepared outside placenta amnion floor height plan cells in vivo epimatrix high plan cells in vivo
Substrate carried out bolting house mesenchymal stem cells.
Said method prepares slightly difference, the time has different on cost but all can effectively make cell component quilt
Remove, the structural intergrity of substrate be retained, containing elastin, keratan sulfate, laminin, each Collagen Type VI,
Fibronectin, desmin, basement membrane structure composition all can completely retain.
Seed mescenchymal stem cell for tack provides favourable microenvironment so that it is expresses, grow, increase
Grow, differentiation etc. plays effect of crucial importance, the irreplaceable high simulation attachment state three dimensions of conventional screening assays
Framework, we term it " the black soil environment of cell seeding ".
Amniotic membrane height intends the mescenchymal stem cell culture method that cells in vivo epimatrix screening Human plactnta is amnion-derived
Five, the mescenchymal stem cell that Human plactnta is amnion-derived is drawn materials screening:
From operating-table aseptic condition, take healthy foot monthly output baby's placenta tissue be placed in culture fluid A, 4 DEG C of preservations.
In superclean bench, take out Placenta Hominis, divest amniotic membrane, fully clean the blood of residual with PBS, repeatedly
Rinse rinsing the most colourless, rinse well without being cut into 1mmx1mmx1mm with shears clip placenta amnion tissue after bloodstain
Fragment.
The amnion-derived mescenchymal stem cell of Human plactnta is drawn materials the placenta amnion layer tissue block in screening step.
Six, the mescenchymal stem cell screening and culturing step that Human plactnta is amnion-derived
Because the amnion-derived mescenchymal stem cell of Human plactnta has stronger adhesiveness, to extracellular to basement membrane
The adhesion speed of substrate is more faster than other cell type, and adherent growth habit is so utilizing this kind of characteristic to carry out
Cultivate screening.
Seven, the mescenchymal stem cell screening and culturing claimed method that Human plactnta is amnion-derived:
Preparation culture fluid A: placental peptide injection 0.06ml/L, EGF5～25ng/ml, stem cell growth because of
Son (SCF) 2～11ng/ml, transferrins 5～15 μ g/ml, adenine phosphate 0.5～1mg/L, vitamin
C1～4 μ g/ml, insulin 5～8 μ g/ml, penicillin 50～100u/ml, streptomycin 100u/m L, hydrogenation
Cortisone 0.4 μ g/ml, commercial culture medium-low sugar DMEM/F12 (3:1) constant volume to 500ml.PH controls
7.2～7.4.
A.1: preparation culture fluid A detailed description of the invention
Preparation culture fluid B: placental peptide injection 0.2ml/L, human serum albumin (HSA) 1～3g/L, people
Blood FN (FN) 1～5 μ g/L, human laminin (LN) 5～10 μ g/L, platelet derived growth factor
(PDGF) 1～10mg/L, placental growth factor (rhPIGF) 1～6mg/L, vascular endothelial cell growth factor
(VEGF) 1～6 μ g/L, EGF5～10ng/ml, vitamin B1～5mg/L, folic acid 1～10ng/ml, dimension
Raw element E 0.05～1mg/L, adenine phosphate 0.5～1mg/L, Progesterone 0.01～0.04 μ g/L, coenzyme
Q10 0.1～2mM/L, cholesterol 1～10mg/L, taurine 0.1～0.4g/L, heparin sodium 0.1～0.3g/L,
Insulin 5～6 μ g/ml, penicillin 100u/ml, streptomycin 100u/mL, commercial culture medium low sugar DMEM with
Commercial culture medium F12 (1:1) constant volume is to 500ml.PH controls 7.2～7.4.
B.1: preparation culture fluid B detailed description of the invention
Preparation culture fluid C: placental peptide injection 0.02～0.8ml/L, human serum albumin (HSA) 0.1～
6g/L, human blood FN (FN) 3～25 μ g/L, human laminin (LN) 10～60 μ g/L, restructuring
IGF-1 (IGF-1) 1～4mg/L, platelet derived growth factor (PDGF) 10～20mg/L,
Placental growth factor (rhPIGF) 6～10mg/L, vascular endothelial cell growth factor (VEGF) 6～10 μ g/L,
Recombinant human hepatocyte growth factor (HGF) 5～20mg/L, EGF, bFGF10～25mg/L, restructuring human brain source property
Neurotrophic factor (BDNF) 15～40ng/ml, vitamin B1～4mg/L, folic acid 1～15ng/ml, corpus luteum
Ketone 0.01～0.06 μ g/L, vitamin E 0.05～1.2mg/L, vitamin C 0.1～10mg/L, dimension are raw
Element B4 0.1～0.5mg/L, coenzyme Q10 0.1～2mM/L, cholesterol 1～10mg/L, taurine 0.1～
0.4g/L, heparin sodium 0.1～0.3g/L, reductive glutathione 3～6 μ g/L, oleic acid 0.5～7.5mg/L,
Transferrins 6～20mg/L, insulin 5～7mg/L, poly-D-lysine 4～6mg/L, penicillin 100u/ml,
Streptomycin 100u/mL, commercial culture medium low sugar DMEM and commercial culture medium F12 (1:1) constant volume to 500ml.
PH controls 7.2～7.4.
C.1: preparation culture fluid C detailed description of the invention
Illustrate: clinical cytology treatment feeds back phase cell amplification.Carry out descending domestication accordingly and cultivate corresponding reduction
Or reject the substance having pharmacological effect effect in the above formula.
2, Digestive system preparation method:
Digestive system preparation A: containing solute 0.1% neutral protease and 0.1% II Collagenase Type (volume ratio 1:1),
Aseptic is solvent containing dual anti-PBS, prepares solution.
Digestive system preparation B: containing solute 0.8%IV Collagenase Type and 0.1% II Collagenase Type (volume ratio 1:1),
The A of culture fluid without placental peptide injection is solvent, prepares solution.
Digestive system preparation C: containing solute 0.1% neutral protease and 0.5%IV Collagenase Type and 0.1% II type glue
Protoenzyme (volume ratio 1:1:1), is solvent without placental peptide injection culture fluid A, prepares solution.
3, prepared by the mescenchymal stem cell freeze thawing preservation liquid that screening Human plactnta is amnion-derived:
Freeze proof solute component includes: adenine phosphate 1～10 μ g/ml, vitamin C 1～4 μ g/ml, poly-
Ethylene glycol 4ml, (preferably autoserum) people's AB serum 10～15%, glycerol 20%, mannitol 3ml, thin
Born of the same parents' relaxin 2.7 μ g/ml, dimethyl sulfoxide (DMSO) 10%, chondroitin sulfate 3～6%, preserve liquid with
Preparation culture fluid ABC is that antifreeze solution prepared by solvent.PH controls to filter at 7.3～7.4,0.22 μm micropores
Degerming.
3.1:3 screening cell freeze thawing preserves liquid and prepares detailed description of the invention:
Explanation, it is characterised in that: this freezing liquid can be by filling between freezing long-term depositary's placenta amnion source
Matter Stem Cell Activity
5, the mescenchymal stem cell screening step method that Human plactnta is amnion-derived:
Step 1, take the amnion-derived mescenchymal stem cell of Human plactnta draw materials screening step in placenta amnion layer
Piece of tissue, with aseptic (penicillin 80～100u/ml and 80～100 μ g/ml streptomycin) PBS fully soak 3～
5min, rinses 3 times with PBS, digests 10～15min with Digestive system A room temperature, and tissue mass suspension is centrifuged
(900r/min is centrifuged 5～10min), discards Digestive system A, collects piece of tissue and precipitates with cell, with digestion
Liquid B or C (37 DEG C, digest 30min～60min under the conditions of vibration 120r/min), autologous containing 10% by several times
Serum or people AB serum free culture system liquid A washing terminate digestion, cross the steel of diameter 100 μm after full and uniform piping and druming
Screen filtration processed, then that the cell suspension being filtrated to get is centrifugal (900r/min is centrifuged 5～10min), abandon
Clear liquid, collects cell precipitation, moves into and be equipped with GE Ficoll separation of lymphocytes in advance after diluting by equivalent PBS
In the centrifuge tube of liquid, 1200～2500r/min are centrifuged 10～30min, draw interface cloud tunica albuginea layer temperature control
At 18～20 DEG C, PBS washs 2～3 times, collects the centrifugal stem cell obtained, heavier with appropriate culture fluid A
Outstanding cell, adjusts cell concentration, with 3x10 after counting chamber counting4/ml。
Step 2, take placenta amnion floor height intend cells in vivo epimatrix, with containing antibiotic PBS rinse 3 times,
Rinse 3 times with preparation culture fluid A again, in 37 DEG C, be immersed in culture fluid A recovery 10～12h standby.
The screening of the mescenchymal stem cell that step 3, Human plactnta are amnion-derived and cultivation: take ready Placenta Hominis
Amniotic membrane floor height is intended cells in vivo epimatrix and is put into 100mm plate addition preparation culture fluid B, adds cell concentration 3
×104/ ml fat stem cell suspension, 37 DEG C, gas phase 5%CO2Incubator cultivates 6～72h taking-ups, takes out
Transfer placenta amnion floor height is intended cells in vivo epimatrix, PBS or culture fluid B and is cleaned 3～6 times, target person tire
The adherent placenta amnion floor height that adheres to of the membrane derived mescenchymal stem cell of argali is intended in cells in vivo epimatrix.Change ware
Adding preparation culture fluid C to cultivate, change whole culture medium C behind 6～72h, the most every 2～3d change liquid one
Secondary continuation amplification cultivation.
Step 4, digestion enrichment Secondary Culture: the amnion-derived mescenchymal stem cell of above-mentioned Human plactnta is through amplification
After, when growth of mesenchymal stem cells to 80%～90% merges, add Digestive system preparation B or C and digest Placenta Hominis sheep
Film floor height intends cells in vivo epimatrix, and 37 DEG C of digestion 15～30min, by several times containing 10% autoserum or people AB
Serum free culture system liquid B terminate digestion, blow and beat a little, cell suspension be collected by filtration and transfer to centrifuge tube, 600～
1200r/min is centrifuged 4～6min, abandons supernatant, adds the culture fluid C of preparation, and re-suspended cell, with 1～3
×l06The density transfer of/ml is passed on and is cultivated according to above-mentioned steps, puts 37 DEG C, gas phase 5%CO2Incubator is cultivated,
Every 3～4d change culture fluid C processed once continues amplification cultivation.
6, Human plactnta is amnion-derived mesenchymal stem cell biological characteristic and immunohistochemical method are examined:
Cellular morphology: the cellular morphology that inverted microscope is observed, in cultivating 3d, attached cell starts division growth,
Cell merges monolayer and reaches 80% and be paved with and at this moment carry out had digestive transfer culture.Observing 3 generation hAMSCs cells is spindle shape
Fibroblast sample grows, and rapidly, purity is high in passage cell growth, hAMSCs visible cell row after fusion
Row closely arrangement or swirling colony growth in flat shape, similar to classical derived from bone marrow source MSCs.
Immunohistochemicalphenotypic: choose the cell application Flow cytometry research of 3 generations and find that hAMSCs is equal
Positive expression CD29, CD44, CD49d, CD71, CD73, CD90, CD105 and positive rate are equal
Up to 96.6～more than 98.6%, this is identical with the result of study of scholar De Ugarte etc..CD29 is that identification is whole
Close the albumen of element subunit, be widely present in except in exo-erythrocytic various tissues and cell；CD44 is mesh
Front found adult stem cell wide expression be distributed in after birth and endochylema mark, this positive expression enters one
Step proves the identity of stem cell；CD90 and CD105 is the specific phenotypes of mescenchymal stem cell.And all can
HAMSCs stem cell acquired in the confirmation of the high expressed positive has stronger adherent ability, and cytoactive is good.
Negative expression CD34, CD45, CD19, CD11b and HLA-DR.And vimentin positive and epithelium
Cell sign CK19 is negative.CD34 and CD45 is hematopoietic cell system molecular marker, illustrates that obtain does carefully
Born of the same parents have into fat and Osteoblast Differentiation potential.
Cytoactive is equal > 98%；
Can induce to becoming fat, skeletonization, nerve and islet-like cells respectively under hAMSCs specified conditions in vitro,
The Derived from Mesenchymal Stem Cells potential that Human plactnta is amnion-derived, hAMSCs adipogenic induction cultivates 4d continuously, under mirror
Visible cell third dimension has small fat to ooze in strengthening existing, and fat is gradually increased fusion after dripping 3 weeks, form is ellipse
Circle or polygon, oil red O staining method shows the lipid precipitation incarnadined.8d is cultivated after hAMSCs osteogenic induction,
Kytoplasm inner cell grain amount increases, and form is polygon, and after 2 weeks, cellular matrix mineralizer engenders, is formed
The little junction structure of multilamellar, cultivates more than 4 weeks, the obvious calcium scoring of observable, by ALP stained positive and thin
Born of the same parents' coloring degree is different, and the coloring of cell intensive place is deeper.Deposit in peony tuberosity after selecting Alizarin red staining.
After hAMSCs neurad cell induction neuroinduction culture fluid, 6h cellular morphology starts to change,
Substantially expanding in the middle of 24～48h cells, cell process spindle, under mirror, there is part point in visible lug tips
, flanking cell projection is linked to be net, and two ends grow similar aixs cylinder or dendron, neuron specific enolase
Changing enzyme immunohistochemical staining, kytoplasm is dyed to brown color, is positive.
Illustrate: placenta amnion exists the stem cell that can stably breed, and does not express hematopoietic cell relevant to transplant rejection
Cell surface marker, prompting is MSCs from amnion-derived stromal cell, and the characteristic with stem cell belongs to
Immunodeficiency cell, the clinical reinfusion transplantation treatment being suitable between Different Individual.Identified and the present invention
Cultivating system result shows the mescenchymal stem cell that the cell behaviour placenta amnion that this separation and Culture is obtained is originated,
Its ability of growing with extremely strong increasing and multi-lineage potential, be cell clinical reinfusion transplantation treatment and organizational project
Having promising seed stem cell, cell purity is higher.Promote target hAMSCs purity, repeatable enter
Row specific implementation method 3 step obtains the hAMSCs of higher purity.
Eight, the having the advantage that of the present invention
The invention discloses the cultivation of the amnion-derived mescenchymal stem cell of Human plactnta, screening, amplification technique,
These bottlenecks are always focus and the difficult point of stem cell field research biology, significantly stick currently with basement membrane
Characteristic carries out isolated and purified, uses three dimensions to cultivate amplification means and obtains.
This invention is suitable for relying on the adherent type cell of amplification and providing three-dimensional high cells in vivo epimatrix system of intending to carry out
The screening of target cell level with separate.
Attaching environment in the invention belongs to high analogue body, the biological cell of In vitro culture hAMSCs stem cell is new
Type culture technique.It is characterized in that: cellular matrix substrate be donor take off thin placenta amnion be matrix substrates screening
The good preparation method sticking place screening hAMSCs stem cell, corresponding placenta amnion height simulation substrate place ring
Screening and culturing target placenta amnion stem cell is sticked in border.The placenta amnion stem cell obtained by the method, has height
Passage capacity, high proliferation ability, and can environmental induction differentiation potential in vitro, the method ratio is traditional
The more scientific research of hAMSCs stem cell screening and teaching clinical practice meaning are further investigation stem cell growth propagation
Differentiation provides immeasurable scientific research learning value, has started the external high new century intending internal adherent attachment cultivation
Unit, the method proposes as to initiate both at home and abroad.
By the screening of this high target pattern, in conjunction with hAMSCs reduced immunogenicity, extend transplant time,
Increase organizational project and clinical practice, it is provided that the cell therapy solution of absolute advantages.
1, organization engineering skin: placenta amnion height intends cells in vivo epimatrix, and substrate attachment in-vitro screening method is to seed
HAMSCs carries out screening and is that current any method is not replaced in internal microenvironment concordance, and
Preparation simply can Reusability, be build containing hAMSCs stem cell tissue engineering skin basis；
2, high specificity, by SABC detection, transmission electron microscope observing and flow cytomery, result shows
The hAMSCs purity of the present invention is high；
3, the application in hAMSCs repair in trauma, in terms of gene studies, beauty and shaping reparation, oral disease, skin
Skin disease field；
4, hAMSCs stem cell scientific research value: the research of hAMSCs stem cells pedigree, further investigated is studied
The Scientific basis researchs such as hAMSCs sticks, grows, breeds, breaks up, Immune expression；
5, the method can be that the regeneration of other organ In vitro culture provides good scientific research theoretical basis；
6, regenerative medicine field of tissue engineering technology application seed hAMSCs stem cell bank.
Accompanying drawing explanation
Fig. 1 is the preparation flow that amniotic membrane floor height of the present invention intends cells in vivo epimatrix.
Fig. 2 is that the hAMSCs application amniotic membrane floor height of the present invention intends cells in vivo epimatrix screening and culturing method stream
Journey.
Claims (8)
1. clinical treatment level cell therapy employment placenta amnion source a mescenchymal stem cell preparation method, by with
Lower step forms；
(1) the Mesenchymal stem cell nutrient solution combination that Human plactnta is amnion-derived；(2) screening amniotic membrane floor height intends body
Prepared by extracellular matrix；(3) application amniotic membrane floor height plan cells in vivo epimatrix is amnion-derived with Human plactnta
Mesenchymal stem cell nutrient solution combined sorting cultivate the amnion-derived mescenchymal stem cell of Human plactnta, and
The amnion-derived long-term freeze thawing of mescenchymal stem cell of Human plactnta preserves liquid and prepares.
2. the Mesenchymal stem cell nutrient solution combination utilizing the Human plactnta described in claim 1 amnion-derived includes:
Preparation culture fluid A: placental peptide injection 0.06ml/L, EGF5～25ng/ml, stem cell growth
The factor (SCF) 2～11ng/ml, transferrins 5～15 μ g/ml, adenine phosphate 0.5～1mg/L, vitamin
C1～4 μ g/ml, insulin 5～8 μ g/ml, penicillin 50～100u/ml, streptomycin 100u/m L, hydrogen
Change cortisone 0.4 μ g/ml, commercial culture medium-low sugar DMEM/F12 (3:1) constant volume controls to 500ml, PH
7.2～7.4；
Preparation culture fluid B: placental peptide injection 0.2ml/L, human serum albumin (HSA) 1～3g/L, people
Blood FN (FN) 1～5 μ g/L, human laminin (LN) 5～10 μ g/L, platelet derived growth factor
(PDGF) 1～10mg/L, placental growth factor (rhPIGF) 1～6mg/L, vascular endothelial cell growth factor
(VEGF) 1～6 μ g/L, EGF5～10ng/ml, vitamin B1～5mg/L, folic acid 1～10ng/ml, dimension life
Element E 0.05～1mg/L, adenine phosphate 0.5～1mg/L, Progesterone 0.01～0.04 μ g/L, ubiquinone
10 0.1～2mM/L, cholesterol 1～10mg/L, taurine 0.1～0.4g/L, heparin sodium 0.1～0.3g/L,
Insulin 5～6 μ g/ml, penicillin 100u/ml, streptomycin 100u/mL, commercial culture medium low sugar DMEM with
Commercial culture medium F12 (1:1) constant volume is to 500ml.PH controls 7.2～7.4；
Preparation culture fluid C: placental peptide injection 0.02～0.8ml/L, human serum albumin (HSA) 0.1～
6g/L, human blood FN (FN) 3～25 μ g/L, human laminin (LN) 10～60 μ g/L, restructuring
IGF-1 (IGF-1) 1～4mg/L, platelet derived growth factor (PDGF) 10～20mg/L,
Placental growth factor (rhPIGF) 6～10mg/L, vascular endothelial cell growth factor (VEGF) 6～10 μ g/L,
Recombinant human hepatocyte growth factor (HGF) 5～20mg/L, EGF, bFGF10～25mg/L, restructuring human brain source property
Neurotrophic factor (BDNF) 15～40ng/ml, vitamin B1～4mg/L, folic acid 1～15ng/ml, corpus luteum
Ketone 0.01～0.06 μ g/L, vitamin E 0.05～1.2mg/L, vitamin C 0.1～10mg/L, dimension are raw
Element B4 0.1～0.5mg/L, coenzyme Q10 0.1～2mM/L, cholesterol 1～10mg/L, taurine 0.1～
0.4g/L, heparin sodium 0.1～0.3g/L, reductive glutathione 3～6 μ g/L, oleic acid 0.5～7.5mg/L,
Transferrins 6～20mg/L, insulin 5～7mg/L, poly-D-lysine 4～6mg/L, penicillin 100u/ml,
Streptomycin 100u/mL, commercial culture medium low sugar DMEM and commercial culture medium F12 (1:1) constant volume to 500ml.
PH controls 7.2～7.4.
3. utilize sieve amniotic membrane floor height described in claim 1 to intend a cells in vivo epimatrix preparation method, including
Wherein said amniotic membrane floor height intends cells in vivo epimatrix；Preparation comprises the following steps: A) lobate floss
Trichilemma tissue disinfection processes；B) amniotic membrane floor height plan cells in vivo epimatrix takes off cell process；C) will sterilization or
The amniotic membrane floor height processed through de-cell intends that cells in vivo epimatrix carries out low temperature further, dehydration, irradiation go out
Bacterium processes；Wherein said step B) method for removing cells selected from cold enzyme Crosslink bond type, enzyme-nonionic table
Face activating agent-chelating agent combination method, chelating agent salt adding-anion surfactant method or pancreatin associating
EDTA crosslinked fluid method.
4. B as claimed in claim 3) amniotic membrane floor height intends cells in vivo epimatrix and takes off cell and process step, and it is special
Levying and be, the cold enzyme Crosslink bond type that described amniotic membrane floor height is intended in the preparation method of cells in vivo epimatrix is:
Add 0.1～0.5% protease ice bath cold disappear 48～12h, with containing 0.25% glutaraldehyde phosphate buffer solution,
PH, between 7.20～7.40, cross-links 4～6h, and ultra-pure water or water for injection rinse 8～10 times；
Enzyme-nonionic surfactant-chelating agent combination method in described preparation method is: 0.1～0.25%
Neutral protease and 0.01～0.02%EDTA, 0.1～0.8%TritonX-100 solution, make under room temperature
With 48～24h；
Described chelating agent salt adding-anion surfactant method is: 0.01～0.02%EDTA containing 0.1～
1mol/L sodium chloride solution, acts on 24～12h, afterwards by 0.1～0.8%SDS solution, room temperature at 37 DEG C
Lower effect 60～30min；
Described pancreatin associating EDTA crosslinked fluid is: 0.1～0.6% trypsin and 0.01～0.1%EDTA,
Trypsin and EDTA solution are 1:2 proportioning according to volume ratio, 37 DEG C of rapid digestion 60～10min,
Crosslinked fluid cross-links 4～6h, rinses 8～10 times with ultra-pure water or water for injection after crosslinking.
5. C as claimed in claim 3) step low temperature, dehydration, irradiation sterilization, it is characterised in that low temperature-30～
-100 DEG C of lyophilizations, vacuum reaches 10～130pa, sloughs umbilical cord tissue height and intends the outer base of cells in vivo
Matter 90～more than 95% moisture, take out and seal and vacuum packaging, irradiated 15～80KGY doses of sterilization.
6. the application amniotic membrane floor height that a kind utilizes described in claim 1 is intended cells in vivo epimatrix and is come with Human plactnta amniotic membrane
The Mesenchymal stem cell nutrient solution combined sorting in source cultivates the side of the amnion-derived mescenchymal stem cell of Human plactnta
Method, including:
A) mesenchyma stem cell suspension that Human plactnta is amnion-derived is prepared: draw materials, Digestive system A, B or C bis-
Step digestion, with described digesting containing 10% human serum culture fluid A termination described in claim 2, filters
GE Ficoll separates liquid, collects cellular layer, and re-suspended cell counts, rear addition culture fluid A；
B) screening of the mescenchymal stem cell that Human plactnta is amnion-derived and cultivation: amniotic membrane floor height is intended internal carefully
Extracellular matrix adds the culture fluid B described in claim 2, adds cell suspension prepared by step a),
Cultivating, clean, the adherent amniotic membrane floor height that adheres to of the mescenchymal stem cell in target person placenta amnion source intends body
Extracellular matrix, adds the culture fluid C described in claim 2 and carries out amplification cultivation；
C) digestion enrichment Secondary Culture: Digestive system B or C digests, and terminates containing 10% human serum culture fluid B
Digestion, adds the culture fluid C described in claim 2 and carries out amplification cultivation after being centrifuged；
D) the long-term freeze thawing of mescenchymal stem cell that Human plactnta is amnion-derived preserves liquid and prepares.
7. Digestive system A, B, C that a kind utilizes described in claim 6, it is characterised in that component includes:
Digestive system preparation A: containing solute 0.1% neutral protease and 0.1% II Collagenase Type (volume ratio 1:1), nothing
Bacterium is solvent containing dual anti-PBS, prepares solution.
Digestive system preparation B: containing solute 0.8%IV Collagenase Type and 0.1% II Collagenase Type (volume ratio 1:1), nothing
Placental peptide injection culture fluid A is solvent, prepares solution.
Digestive system preparation C: containing solute 0.1% neutral protease and 0.5%IV Collagenase Type and 0.1% II Collagen Type VI
Enzyme (volume ratio 1:1:1), is solvent without placental peptide injection culture fluid A, prepares solution.
8. prepared by the mescenchymal stem cell freeze thawing preservation liquid utilizing the Human plactnta described in claim 1 amnion-derived,
It is characterized in that, freeze proof solute component includes: adenine phosphate 1～10 μ g/ml, vitamin C 1～4 μ g/ml,
Polyethylene Glycol 4ml, (preferably autoserum) people's AB serum 10～15%, glycerol 20%, mannitol 3ml,
Cytochalasin 2.7 μ g/ml, dimethyl sulfoxide (DMSO) 10%, chondroitin sulfate 3～6%, preservation is joined
Liquid processed prepares antifreeze solution with the culture fluid ABC of the told nonreactive of claim 2 for solvent.PH controls
7.3～7.4,0.22 μm micropore Entkeimung.
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| CN112574948A (en) * | 2020-08-18 | 2021-03-30 | 北京昱龙盛世生物科技有限公司 | Separation culture method of human amniotic mesenchymal stem cells |
| CN113549524A (en) * | 2021-07-21 | 2021-10-26 | 山东省泉溪生物技术有限公司 | Self-refrigerating low-temperature placenta collection bag convenient and timely to wash |
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| CN109182260A (en) * | 2018-09-11 | 2019-01-11 | 邵勇 | A kind of method of in vitro culture fetal membrane mescenchymal stem cell |
| CN110352951A (en) * | 2018-11-15 | 2019-10-22 | 崔磊 | A kind of serum-free is without DMSO tissue engineered bone frozen stock solution and its preparation and cryopreservation methods |
| CN111084905A (en) * | 2019-12-10 | 2020-05-01 | 肖雁冰 | Method for preparing artificial amnion by using amnion mesenchyme stem cell |
| CN110846229A (en) * | 2019-12-19 | 2020-02-28 | 广州傲农生物科技有限公司 | Preservation method and preservative for improving survival rate of bacillus coagulans |
| CN111248193A (en) * | 2020-04-18 | 2020-06-09 | 李刚 | Human amniotic mesenchymal stem cell cryopreservation solution and cryopreservation method thereof |
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| CN112574948A (en) * | 2020-08-18 | 2021-03-30 | 北京昱龙盛世生物科技有限公司 | Separation culture method of human amniotic mesenchymal stem cells |
| CN113549524A (en) * | 2021-07-21 | 2021-10-26 | 山东省泉溪生物技术有限公司 | Self-refrigerating low-temperature placenta collection bag convenient and timely to wash |
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