CN106676063A - Separate culture method for human amniotic mesenchymal stem cells - Google Patents
Separate culture method for human amniotic mesenchymal stem cells Download PDFInfo
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- 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
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Abstract
The invention discloses a separate culture method for human amniotic mesenchymal stem cells, and belongs to the technical field of biology. The separate culture method comprises the following steps: (I) cell separation; (II) cell culture: inoculating a single separated human amniotic karyocyte into a plastic culture bottle of 75cm<2>, culturing in a 5 percent CO2 incubator at the temperature 37 DEG C, wherein a culture solution is a culture medium special for the human amniotic mesenchymal stem cell; replacing the culture solution 24 hours later, discarding non-adherent cells, and replacing the culture solution every 2 to 3 days; fusing the cells after the human amniotic mesenchymal stem cells grow to 70 to 80 percent, digesting with pancreatin, and performing passage; performing passage once in the culture medium special for the human amniotic mesenchymal stem cells every 2 to 3 days at the temperature of 37 DEG C and in 5 percent CO2. The obtained human amniotic mesenchymal stem cells express by the following three kinds of cell membrane molecules, namely, a human leukocyte differentiation antigen CD73, a human leukocyte differentiation antigen CD90 and a human leukocyte differentiation antigen CD105, and do not express a human leukocyte differentiation antigen CD45 and a human leucocyte antigen HLA-DR.
Description
Technical field
The present invention relates to a kind of isolated culture method of amnion mesenchymal stem cell, belongs to biological technical field.
Background technology
People amniotic membrane stem cell has height self renewal, propagation, implantable and polyphyly differentiation capability, and allogene is individual to be moved
Without immunologic rejection and oncogenicity risk after plant.And, the Placenta Hominiss that people's amniotic membrane source of human stem cell is discarded after parturient childbirth are applied to
Clinic will not bring Medical Ethics to dispute on.Amniotic membrane 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, amniotic membrane 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, hepatocyte, nerve
The ectoderm cells such as born of the same parents break up.
People's amniotic membrane stem cell not only has significant self-renewal capacity and interdepartmental across a germinal layer polyphyly differentiation potential, and relatively
Embryonic stem cell and other adult stem cells, its aboundresources is 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 inhibitive 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 using value.
In prior art, main culture medium of the application containing animal serum (such as FBS) of the cultivating system of stem cell, BMSCs and
The culture of mesenchymal stem cells in umbilical cord blood also more adopts FBS.The cultivating system of human amnion mesenchymal stem cell is usually LG-DMEM
10% FBS, 2mmol/L L-glutaminate, 1% non essential amino acid are added in culture medium/ L 2 mercapto ethanols and
1mmol/L Sodium Pyruvates.But the application of the animal serums such as FBS, after stem cell transplantation, input foreign protein exists latent to patient
Threat, can produce anti-FBS antibody and the untoward reaction of other not known dawns, and be likely to result in disease between people and other species
The propagation of poison infection.
The content of the invention
The technical problem to be solved in the present invention is to provide a kind of isolated culture method of new human amnion mesenchymal stem cell.
For achieving the above object, the present invention is employed the following technical solutions：
The isolated culture method of human amnion mesenchymal stem cell, step is as follows：
(1) cell separation
(1) amniotic membrane takes from mature Cesarean esction health puerpera, after collectionProcess；
(2) will be a diameter ofAmniotic membrane cleaned with 0.9% sodium chloride solution, shred to about 1mm × 1mm ×
The collagenase of 1mm, Jing 0.1% and37 DEG C of digestion of pancreatin of %40ml is diluted to α-MEM culture medium；
(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 on after trim in refrigerated centrifuge, 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 test tube rack, centrifuge tube content is divided into two parts, upper strata is collagen
Enzyme, pancreatin and α-MEM culture medium, lower floor is tissue pieces and cell mixture；
Upper strata collagenase, pancreatin and α-MEM culture medium are suctioned out；
(7) separated people's amniotic membrane mononuclearcell is washed with 2000r/min centrifugation 10min after the dilution of α-MEM culture medium
It is secondary；
(2) cell culture
Detached people's amniotic membrane mononuclearcell is pressedIndividual cell/cm2It is inoculated inPlastic culture bottle, culture
In 37 DEG C,Incubator, culture fluid is human amnion mesenchymal stem cell special culture media；Liquid is changed after 24 hours, non-adherent is abandoned
Cell, changed liquid per 2~3 days later；Treat that human amnion mesenchymal stem cell grows into 70~80% fusions, use% pancreatin disappears
Change 1~2 minute, by 0.8 × 104~1.0 × 104Individual cell/cm2Pass on；It is special with human amnion mesenchymal stem cell per 2~3 days
Pass in culture medium once, make cell concentration maintain 5 × 105~10 × 105Individual cell/M1；Every time the condition of Secondary Culture is equal
For 37 DEG C,PassIn generation, obtains human amnion mesenchymal stem cell.
The human amnion mesenchymal stem cell special culture media is made up of α-MEM culture medium, human plasma and Human Albumin,
Concentration of the human plasma in the human amnion mesenchymal stem cell special culture media is 10% (concentration expressed in percentage by volume), the white egg of human blood
The white concentration in the human amnion mesenchymal stem cell special culture media is 1% (concentration expressed in percentage by volume).
The human plasma is people's AB blood plasma.
Human amnion mesenchymal stem cell provided by the present invention, using the separation and Culture of above-mentioned human amnion mesenchymal stem cell
Method culture is obtained.
Human amnion mesenchymal stem cell of the present invention is expressed as follows three kinds of membrane molecules：Human leukocyte differentiation antigen
CD73, human leukocyte differentiation antigen CD90 and human leukocyte differentiation antigenHuman leukocyte differentiation antigen is not expressed
With human leucocyte antigen (HLA) HLA-DR.
Human amnion mesenchymal stem cell special culture media, is made up of α-MEM culture medium, human plasma and Human Albumin.People
Concentration of the blood plasma in the human amnion mesenchymal stem cell special culture media is 10% (concentration expressed in percentage by volume), Human Albumin
Concentration in the human amnion mesenchymal stem cell special culture media is 1% (concentration expressed in percentage by volume).
The invention provides the preparation method of human amnion mesenchymal stem cell：In vitro people's amniotic membrane mononuclearcell is as follows
Cultivate in human amnion mesenchymal stem cell special culture media and pass on and obtain human amnion mesenchymal stem cell：In α-MEM culture medium
The culture medium that middle addition human plasma and Human Albumin are obtained, the human plasma is in the special training of the human amnion mesenchymal stem cell
Concentration in foster base is 10% (concentration expressed in percentage by volume), and the Human Albumin is in the special training of the human amnion mesenchymal stem cell
Concentration in foster base is 1% (concentration expressed in percentage by volume).
It is an advantage of the invention that：The cultural method and culture medium of the present invention does not contain animal serum, can promote between people's amniotic membrane
Mesenchymal stem cell proliferation, cell purity is high, can meet the clinical condition of cell therapy, safe and reliable, can be used for stem cell in vitro
Amplification.This product additive uses people's product-derived, and reaches clinical practice rank；Prepare and buy convenient, fast.
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.
Description of the drawings
Figure 1A is situation when human amnion mesenchymal stem cell is just adherent
Figure 1B starts situation when growing 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 human leukocyte differentiation antigen CD73
Situation
Fig. 2 B are expression of the human amnion mesenchymal stem cell by flow cytomery human leukocyte differentiation antigen CD90
Situation
Fig. 2 C are that human amnion mesenchymal stem cell passes through flow cytomery human leukocyte differentiation antigenTable
Up to situation
Fig. 2 D are that human amnion mesenchymal stem cell passes through flow cytomery human leukocyte 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 if no special instructions, is conventional method.
Material used, reagent etc. in following embodiments, if no special instructions, commercially obtain.
α-MEM culture medium is the product of Gibco companies, and its production code member is：11900024；People AB blood plasma is red purchased from Beijing
Cross Blood Center；Injection Human Albumin (Chinese medicines quasi-word S20030043).
Human amnion mesenchymal stem cell special culture media：Add people AB blood plasma and people in α-MEM (Gibco, USA) culture medium
The culture medium of blood albumin, concentration of the people AB blood plasma in the human amnion mesenchymal stem cell special culture media is 10% (volume
Percentage concentration), concentration of the Human Albumin in the human amnion mesenchymal stem cell special culture media is that 1% (volume basis are dense
Degree), pH value is 7.2-7.4.
The separation and Culture of embodiment 1, human amnion mesenchymal stem cell
1st, cell separation
(1) amniotic membrane takes from mature Cesarean esction health puerpera, after collectionProcess.
(2) will be a diameter ofAmniotic membrane cleaned with 0.9% sodium chloride solution, shred to about 1mm × 1mm ×
The collagenase of 1mm, Jing 0.1% 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 on after trim in refrigerated centrifuge, 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 test tube rack, centrifuge tube content is divided into two parts, upper strata is collagen
Enzyme, pancreatin and α-MEM culture medium, lower floor is tissue pieces and cell mixture.
Upper strata collagenase, pancreatin and α-MEM culture medium are suctioned out.
(7) separated people's amniotic membrane mononuclearcell is washed with 2000r/min centrifugation 10min after the dilution of α-MEM culture medium
It is secondary.
2nd, cell culture
Detached people's amniotic membrane mononuclearcell is pressedIndividual cell/cm2It is inoculated inPlastic culture bottle, culture
In 37 DEG C,Incubator, culture fluid is human amnion mesenchymal stem cell special culture media.Liquid is changed after 24 hours, non-adherent is abandoned
Cell, changed liquid per 2~3 days later；Treat that human amnion mesenchymal stem cell grows into 70~80% fusions, use% pancreatin
(Sigma, USA) digests (1~2 minute), by 0.8 × 104~1.0 × 104Individual cell/cm2Pass on.Per 2~3 days with the people sheep
Pass on once in intermembranous mesenchymal stem cells special culture media, make cell concentration maintain 5 × 105~10 × 105Individual cell/mL.Often
The condition of secondary Secondary Culture is 37 DEG C,PassIn generation, obtains human amnion mesenchymal stem cell.
Situations of the Figure 1A for human amnion mesenchymal stem cell when just adherent is form in tiny circle.
Figure 1B starts situation when growing 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 to breed what formation was differed in size after one week
Cell colony.
Embodiment 2：FCM analysis
First, method
The human amnion mesenchymal stem cell that embodiment 1 is obtained is logical with anti-human CD73 monoclonal antibodies (BioLegend, USA)
Overflow-type cell instrument detects the expression of human leukocyte differentiation antigen CD73, with anti-human CD90 monoclonal antibodies
(BioLegend, USA) by the expression of flow cytomery human leukocyte differentiation antigen CD90, with anti-human
Monoclonal antibody (BioLegend, USA) passes through flow cytomery human leukocyte differentiation antigenExpression,
With anti-humanMonoclonal antibody (BioLegend, USA) passes through flow cytomery human leukocyte differentiation antigen's
Expression, with anti-human human leucocyte antigen HLA-DR monoclonal antibody (BioLegend, USA) flow cytometer (model is passed through
FACSCaliburBD companies) detection human leucocyte antigen (HLA) HLA-DR expression.
2nd, result
As a result as shown in Fig. 2A, Fig. 2 B, Fig. 2 C, Fig. 2 D and Fig. 2 E, show that human amnion mesenchymal stem cell expression people is white
Cell differentiation antigen CD73, human leukocyte differentiation antigen CD90 and human leukocyte differentiation antigenHuman leukocyte is not expressed
Differentiation antigenWith human leucocyte antigen (HLA) HLA-DR.Flow cytomery result shows the human amnion mesenchymal stem cell
Purity reach
Claims (10)
1. the isolated culture method of human amnion mesenchymal stem cell, it is characterised in that step is as follows：
(1) cell separation
(1) amniotic membrane takes from mature Cesarean esction health puerpera, 6~12h process after collection；
(2) amniotic membrane of a diameter of 6cm × 6cm is cleaned with 0.9% sodium chloride solution, is shredded to about 1mm × 1mm × 1mm, Jing
0.1% collagenase and 0.125% 37 DEG C of 30~60min of digestion of pancreatin, with α-MEM culture medium 40ml is diluted to；
(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 in 50ml centrifuge tubes, is placed on after trim in refrigerated centrifuge, with centrifugal force
300Xg, 4 ± 2 DEG C of temperature is centrifuged 8 minutes；
(5) gently take out centrifuge tube and be placed on test tube rack after shutting down, centrifuge tube content is divided into two parts, upper strata be collagenase,
Pancreatin and α-MEM culture medium, lower floor is tissue pieces and cell mixture；
(6) upper strata collagenase, pancreatin and α-MEM culture medium are suctioned out；
(7) separated people's amniotic membrane mononuclearcell is washed into secondary with 2000r/min centrifugation 10min after the dilution of α-MEM culture medium；
(2) cell culture
Detached people's amniotic membrane mononuclearcell is pressed into 1 × 106Individual cell/cm2It is inoculated in 75cm2Plastic culture bottle, is incubated at 37
DEG C, 5%CO2Incubator, culture fluid is human amnion mesenchymal stem cell special culture media；Liquid is changed after 24 hours, non-adherent is abandoned thin
Born of the same parents, changed liquid per 2~3 days later；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/cm2Pass on；Passed with human amnion mesenchymal stem cell special culture media per 2~3 days
In generation, once, makes cell concentration maintain 5 × 105~10 × 105Individual cell/Ml；Every time the condition of Secondary Culture is 37 DEG C, and 5%
CO2；Passing for 5 generations obtains human amnion mesenchymal stem cell.
2. the isolated culture method of human amnion mesenchymal stem cell according to claim 1, it is characterised in that：The people sheep
Intermembranous mesenchymal stem cells special culture media is made up of α-MEM culture medium, human plasma and Human Albumin.
3. the isolated culture method of human amnion mesenchymal stem cell according to claim 2, it is characterised in that：The human blood
Concentration of the slurry in human amnion mesenchymal stem cell special culture media is 10%, and Human Albumin is dry thin in the human amnion mesenchymal
Concentration in born of the same parents' special culture media is 1%.
4. the isolated culture method of the human amnion mesenchymal stem cell according to Claims 2 or 3, it is characterised in that：It is described
Human plasma is people's AB blood plasma.
5. the isolated culture method culture of the human amnion mesenchymal stem cell according to any one of claims 1 to 3 is obtained
The human amnion mesenchymal stem cell for arriving.
6. between the people's amniotic membrane for being obtained according to the isolated culture method culture of the human amnion mesenchymal stem cell described in claim 4
Mesenchymal stem cells.
7. human amnion mesenchymal stem cell according to claim 5, it is characterised in that：The human amnion mesenchymal stem cell
It is expressed as follows three kinds of membrane molecules：Human leukocyte differentiation antigen CD73, human leukocyte differentiation antigen CD90 and human leukocyte point
Change antigens c D105；Human leukocyte differentiation antigen CD45 and human leucocyte antigen (HLA) HLA-DR is not expressed.
8. human amnion mesenchymal stem cell according to claim 6, it is characterised in that：The human amnion mesenchymal stem cell
It is expressed as follows three kinds of membrane molecules：Human leukocyte differentiation antigen CD73, human leukocyte differentiation antigen CD90 and human leukocyte point
Change antigens c D105；Human leukocyte differentiation antigen CD45 and human leucocyte antigen (HLA) HLA-DR is not expressed.
9. human amnion mesenchymal stem cell special culture media, it is characterised in that：By α-MEM culture medium, human plasma and the white egg of human blood
White composition.
10. human amnion mesenchymal stem cell special culture media according to claim 9, it is characterised in that：The human plasma
Concentration in the human amnion mesenchymal stem cell special culture media is 10%, and Human Albumin is dry in the human amnion mesenchymal
Concentration in cell special culture media is 1%.
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