CN106701673A - Special culturing medium for human amniotic membrane mesenchymal stem cell - Google Patents

Special culturing medium for human amniotic membrane mesenchymal stem cell Download PDF

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CN106701673A
CN106701673A CN201710048550.4A CN201710048550A CN106701673A CN 106701673 A CN106701673 A CN 106701673A CN 201710048550 A CN201710048550 A CN 201710048550A CN 106701673 A CN106701673 A CN 106701673A
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stem cell
mesenchymal stem
human
amnion mesenchymal
culture media
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谷涌泉
刘�英
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Beijing Tian Sheng Yu Biological Technology Co Ltd
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Beijing Tian Sheng Yu Biological Technology Co Ltd
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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
    • C12N5/0652—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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/998—Proteins not provided for elsewhere

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Abstract

The invention discloses a special culturing medium for a human amniotic membrane mesenchymal stem cell, and belongs to the technical field of biology. The special culturing medium for the human amniotic membrane mesenchymal stem cell comprises an alpha-MEM culturing medium, a human plasma, and a human serum albumin, wherein the concentration of volume percent of the human plasma is 10%, and the concentration of the human serum albumin is 1%. The special culturing medium for the human amniotic membrane mesenchymal stem cell has the advantages that the special culturing medium is simple in ingredients and convenient in preparation, does not contain animal serums, can prompt the proliferation of the human amniotic membrane mesenchymal stem cell, is high in cell purity, meets the clinical condition of cell therapy, is safe and reliable, and can be used for amplification of the stem cell in vitro. The culturing medium is strong in selectivity, and can conduct specificity enrichment on the human amniotic membrane mesenchymal stem cell from a human amniotic membrane mononuclear cell.

Description

A kind of special culture media of human amnion mesenchymal stem cell
Technical field
The present invention relates to a kind of special culture media of human amnion mesenchymal stem cell, belong to biological technical field.
Background technology
People amnion stem cell has height self-renewing, propagation, implantable and polyphyly differentiation capability, and allogene is individual to be moved Without immunological rejection and oncogenicity risk after plant.And, people's amnion source of human stem cell is applied in the placenta discarded after parturient childbirth Clinic will not bring Medical Ethics to dispute on.Amnion stem cell have obvious Colony forming ability, multiplication capacity, embryo's dryness, Immunoregulation capability and without excellent biological characteristicses such as allotransplantation rejection and oncogenicity, can be utilized for many diseases Cell therapy, the reparation of injuries of tissues and organs with rebuild, be the preferable seed cell resource of regenerative medicine field, with wide Potential applicability in clinical practice.
In the last few years, it is substantial amounts of research show, amnion stem cell, both with to same germinal layer different type cell differentiation Multi-lineage potential, there is the interdepartmental ability across differentiation of germinal layers again, shows good plasticity.Such as, originate from mesoblastic cell Can break up to mesoblastemas such as skeletonization, cartilage, fat, also can be thin to the endoderm cells such as pancreatic cell, liver cell, nerve The ectoderm cells such as born of the same parents break up.
People's amnion stem cell not only has significant self-renewal capacity and interdepartmental across germinal layer polyphyly differentiation potential, and relatively Embryonic stem cell and other adult stem cells, its aboundresources are easily obtained, and without dispute of ethic, immunogenicity is low, without tumorigenesis wind Danger, also has a powerful paracrine or autocrine work(of the multiple biological activities factor such as secretory immune inhibiting factor, neurotrophic factor Energy.These characteristics impart it and face the correlative regeneration medical domain such as organizational project, cell therapy, gene therapy is immeasurable Bed application value.
In the prior art, the cultivating system of stem cell mainly application containing animal blood serum (such as FBS) culture medium, BMSCs and The culture of mesenchymal stem cells in umbilical cord blood also more uses FBS.The cultivating system of human amnion mesenchymal stem cell is usually LG-DMEM 10% FBS, 2mmol/L Glu, 1% nonessential amino acid is added in culture medium2 mercapto ethanol and 1mmol/L Sodium Pyruvates.But the application of the animal blood serums such as FBS, after stem cell transplantation, input foreign protei exists latent to patient Threat, anti-FBS antibody and the adverse reaction of other not known dawns can be produced, and be likely to result in disease between people and other species The propagation of poison infection.
The content of the invention
The invention solves the problems that technical problem is to provide a kind of the special of human amnion mesenchymal stem cell without animal blood serum Culture medium.
To achieve the above object, the present invention uses following technical scheme：
Human amnion mesenchymal stem cell special culture media, is made up of α-MEM culture mediums, human plasma and human serum albumin, its The concentration of volume percent of middle human plasma is 10%, and the concentration of human serum albumin is 1% (g/100ml).
α-MEM culture mediums are the basal medium of stem cell culture, and its composition and compound method are known in the art general knowledge. Voluntarily prepare or buy commercialized α-MEM culture mediums and be used equally to the present invention.
The human plasma behaviour AB type blood plasma.
The pH value of the human amnion mesenchymal stem cell special culture media is 7.2-7.4.
Human amnion mesenchymal stem cell special culture media of the present invention can be used for culture people's amnion mononuclearcell and/ Or human amnion mesenchymal stem cell.
It is present invention also offers a kind of method for cultivating human amnion mesenchymal stem cell, the in vitro single core of people's amnion is thin Born of the same parents pressIndividual cell/cm2It is inoculated in blake bottle, is incubated at 37 DEG C,Incubator, nutrient solution is people of the invention Amnion mesenchymal stem cell special culture media；Liquid is changed after 24 hours, non-adherent cell is abandoned, liquid is changed within every 2~3 days later；Treat people sheep Intermembranous mesenchymal stem cells grow into 70~80% fusions, usePancreatin digests (1~2 minute), by 0.8 × 104~1.0 × 104Individual cell/cm2Passage；Passed on once with the human amnion mesenchymal stem cell special culture media within every 2~3 days, make cell dense Degree maintains 5 × 105~10 × 105Individual cell/mL；The condition of each Secondary Culture is 37 DEG C,PassIn generation, obtains people Amnion mesenchymal stem cell.
Present invention also offers the people that a kind of use the present inventor amnion mesenchymal stem cell special culture media culture is obtained Amnion mesenchymal stem cell, is expressed as follows three kinds of mescenchymal stem cell membrane molecules：HL's differentiation antigen CD73, people are white Cell differentiation antigen CD90 and HL's differentiation antigenTwo kinds of membrane molecules are not expressed as follows：HL breaks up AntigenWith human leucocyte antigen (HLA) HLA-DR.
It is an advantage of the invention that：Special culture media composition of the invention is simple, and it is convenient to prepare, without animal blood serum, can Promote human amnion mesenchymal stem cell propagation, cell purity is high, can meet the clinical condition of cell therapy, safe and reliable, can use In ex vivo expansion of stem cell.Present invention culture based selective is strong, can from people's amnion mononuclearcell specific enrichment people's amnion Mescenchymal stem cell.
The present invention is elaborated with reference to the accompanying drawings and detailed description, not limitation of the invention, it is all according to The equivalent of any this area carried out according to disclosure file, belongs to protection scope of the present invention.
Brief description of the drawings
Situation when Figure 1A is just adherent human amnion mesenchymal stem cell
Figure 1B starts situation during growth for human amnion mesenchymal stem cell
Fig. 1 C are the situation after human amnion mesenchymal stem cell propagation
Fig. 2A is expression of the human amnion mesenchymal stem cell by flow cytomery HL's differentiation antigen CD73 Situation
Fig. 2 B are expression of the human amnion mesenchymal stem cell by flow cytomery HL's differentiation antigen CD90 Situation
Fig. 2 C are that human amnion mesenchymal stem cell passes through flow cytomery HL's differentiation antigenTable Up to situation
Fig. 2 D are that human amnion mesenchymal stem cell passes through flow cytomery HL's differentiation antigenExpression Situation
Fig. 2 E are expression feelings of the human amnion mesenchymal stem cell by flow cytomery human leucocyte antigen (HLA) HLA-DR Condition
Specific embodiment
Experimental technique used in following embodiments is conventional method unless otherwise specified.
Material used, reagent etc. in following embodiments, unless otherwise specified, commercially obtain.
α-MEM culture mediums are the product of Gibco companies, and its production code member is：11900024.
People AB blood plasma is purchased from BJ Red Cross Blood Center.
Injection human serum albumin (Chinese medicines quasi-word S20030043).
Embodiment 1, human amnion mesenchymal stem cell special culture media
Human amnion mesenchymal stem cell special culture media, by α-MEM culture mediums, people's AB types blood plasma and human serum albumin group Into the concentration of volume percent of wherein people AB types blood plasma is 10%, and the concentration of human serum albumin is 1% (g/100ml).
α-MEM culture mediums are the product of Gibco companies of the U.S., and goods number is 11900024.
The pH value of culture medium is 7.2-7.4
Embodiment 2, human amnion mesenchymal stem cell is separately cultured
1st, cell separation
(1) amnion takes from mature Cesarean esction health puerpera, after collectionTreatment.
(2) will be a diameter ofAmnion cleaned with 0.9% sodium chloride solution, shred to about 1mm × 1mm × 1mm, through 0.1% clostridiopetidase A and37 DEG C of digestion of pancreatin of %It is diluted to α-MEM (Gibco, USA) 40ml。
(3) aforesaid liquid is successively filtered with 100 mesh and 200 mesh filter screens, removes indigested tissue.
(4) the celliferous liquid after filtering is placed inIn centrifuge tube, be placed in refrigerated centrifuge after trim, with from Mental and physical efforts 300Xg, 4 ± 2 DEG C of temperature is centrifuged 8 minutes.
Gently take out centrifuge tube after shutdown to be placed on rack for test tube, centrifuge tube content is divided into two parts, upper strata is collagen Enzyme, pancreatin and α-MEM culture mediums, lower floor are tissue pieces and cell mixture.
Upper strata clostridiopetidase A, pancreatin and α-MEM culture mediums are suctioned out.
(7) 2000r/min is centrifuged 10min and washes after separated people's amnion mononuclearcell is diluted with α-MEM culture mediums It is secondary.
2nd, cell culture
People's amnion mononuclearcell of separation is pressedIndividual cell/cm2It is inoculated inPlastic culture bottle, culture In 37 DEG C,Incubator, nutrient solution is human amnion mesenchymal stem cell special culture media (being prepared by embodiment 1).24 hours After change liquid, abandon non-adherent cell, change liquid within every 2~3 days later；Treat that human amnion mesenchymal stem cell grows into 70~80% fusions, WithPancreatin (Sigma, USA) digests (1~2 minute), by 0.8 × 104~1.0 × 104Individual cell/cm2Passage.Every 2~ Passed on once with the human amnion mesenchymal stem cell special culture media within 3 days, cell concentration is maintained 5~10 × 105It is individual thin Born of the same parents/mL.The condition of each Secondary Culture is 37 DEG C,PassIn generation, obtains human amnion mesenchymal stem cell.
Situation when Figure 1A is just adherent human amnion mesenchymal stem cell, is that form is in tiny circle.
Figure 1B starts situation during growth for human amnion mesenchymal stem cell, and after 48 hours, cell starts propagation, Xiang Changsuo Shape changes.
Fig. 1 C are the situation after human amnion mesenchymal stem cell propagation, and cell starts propagation and forms what is differed in size after one week Cell colony.
Embodiment 3：FCM analysis
First, method
The human amnion mesenchymal stem cell that embodiment 2 is obtained is logical with anti-human CD73 monoclonal antibodies (BioLegend, USA) The expression of overflow-type cell instrument detection HL's differentiation antigen CD73, with anti-human CD90 monoclonal antibodies (BioLegend, USA) by the expression of flow cytomery HL's differentiation antigen CD90, with anti-human Monoclonal antibody (BioLegend, USA) passes through flow cytomery HL's differentiation antigenExpression, With anti-humanMonoclonal antibody (BioLegend, USA) passes through flow cytomery HL's differentiation antigen's Expression, flow cytometer (model is passed through with anti-human HLA HLA-DR monoclonal antibodies (BioLegend, USA) FACSCaliburBD companies) detection human leucocyte antigen (HLA) HLA-DR expression.
2nd, result
Result shows that human amnion mesenchymal stem cell expression people is white as shown in Fig. 2A, Fig. 2 B, Fig. 2 C, Fig. 2 D and Fig. 2 E Cell differentiation antigen CD73, HL's differentiation antigen CD90 and HL's differentiation antigenDo not express HL Differentiation antigenWith human leucocyte antigen (HLA) HLA-DR.Flow cytomery result shows the human amnion mesenchymal stem cell Purity reaches

Claims (10)

1. human amnion mesenchymal stem cell special culture media, it is characterised in that：By α-MEM culture mediums, human plasma and the white egg of human blood White composition.
2. the human amnion mesenchymal stem cell special culture media according to claim l, it is characterised in that：The human plasma Concentration of volume percent is 10%, and the concentration of human serum albumin is 1%.
3. human amnion mesenchymal stem cell special culture media according to claim 1 and 2, it is characterised in that：The human blood It is people's AB type blood plasma to starch.
4. human amnion mesenchymal stem cell special culture media according to claim 3, it is characterised in that：Between people's amnion The pH value of mesenchymal stem cells special culture media is 7.2-7.4.
5. human amnion mesenchymal stem cell special culture media according to claim 4, it is characterised in that：Described people's amnion Mescenchymal stem cell special culture media is used to cultivate people's amnion mononuclearcell.
6. human amnion mesenchymal stem cell special culture media according to claim 4, it is characterised in that：Described people's amnion Mescenchymal stem cell special culture media is used to cultivate human amnion mesenchymal stem cell.
7. it is a kind of cultivate human amnion mesenchymal stem cell method, it is characterised in that：In vitro people's amnion mononuclearcell is pressed 1 ×106Individual cell/cm2It is inoculated in blake bottle, is incubated at 37 DEG C, 5%CO2Incubator, nutrient solution is in Claims 1-4 Human amnion mesenchymal stem cell special culture media described in what one；Liquid is changed after 24 hours, non-adherent cell is abandoned, later every 2~3 It changes liquid；Treat that human amnion mesenchymal stem cell grows into 70~80% fusions, digested with 0.25% pancreatin, by 0.8 × 104~ 1.0×104Individual cell/cm2Passage；Passed on once with the human amnion mesenchymal stem cell special culture media within every 2~3 days, made thin Born of the same parents' concentration maintains 5 × 105~10 × 105Individual cell/mL；The condition of each Secondary Culture is 37 DEG C, 5%CO2。
8. it is according to claim 7 culture human amnion mesenchymal stem cell method, it is characterised in that：The Secondary Culture Human amnion mesenchymal stem cell is obtained to pass for 5 generations.
9. the human amnion mesenchymal stem cell for being obtained with the method for the culture human amnion mesenchymal stem cell of claim 7 or 8.
10. human amnion mesenchymal stem cell according to claim 9, it is characterised in that：Described human amnion mesenchymal is done Cell, is expressed as follows three kinds of mescenchymal stem cell membrane molecules：HL's differentiation antigen CD73, HL's differentiation antigen CD90 and HL's differentiation antigen CD105；Two kinds of membrane molecules are not expressed as follows：HL's differentiation antigen CD45 and people HLA HLA-DR.
CN201710048550.4A 2017-01-20 2017-01-20 Special culturing medium for human amniotic membrane mesenchymal stem cell Pending CN106701673A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
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CN107653223A (en) * 2017-11-06 2018-02-02 江西瑞济生物工程技术股份有限公司 A kind of amnion stem cell media and its cultural method

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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