CN103966159A - Sub-totipotent stem cell of human placenta and stem cell bank construction method thereof - Google Patents
Sub-totipotent stem cell of human placenta and stem cell bank construction method thereof Download PDFInfo
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Abstract
The invention provides a sub-totipotent stem cell of a human placenta. The sub-totipotent stem cell of the human placenta is derived from stripped human placenta amnion, aiming at the characteristics of cells contained by an epithelial layer and a mesenchyme layer of the amnion, a step-by-step separation method is adopted, pollution of the sub-totipotent stem cell of the placenta by the mesenchyme layer of the amnion is reduced to the greatest extent, and the stemness of the sub-totipotent stem cell of the human placenta is improved effectively. The method has the characteristics of low cost, broad application prospect and capacity of supplying a large quantity of stem cell resources to clinic and research. The invention further provides a method for preparing the sub-totipotent stem cell of the human placenta from the human placenta amnion and constructing a stem cell bank.
Description
Technical field
The present invention relates to stem cell field, relate to particularly a kind of its and derive from the sub-myeloid-lymphoid stem cell of human placenta's amnion.Meanwhile, the present invention also relates to preparation method and the method for inducing differentiation of this Asia myeloid-lymphoid stem cell.
Background technology
Stem-cell research is the study hotspot emerging in recent years, and the progress that it is at full speed and wide application prospect are more and more attracted attention stem-cell research.Stem cell is the cell that a class has self and differentiation potential, according to source, can be divided into embryonic stem cell and adult stem cell.Wherein adult stem cell is not because have dispute of ethic, and easily obtains and be considered to the seed cell of potential cell therapy.
Recent study shows, in adult placenta tissue, is being rich in a kind of sub-myeloid-lymphoid stem cell with Multidirectional Differentiation ability.Placenta is between Mammals pregnancy duration mothers and sons, to exchange the transitional organ of material, and its physiological function is widely studied, but placenta as a kind of cell resource, its research is also in the starting stage.The sub-myeloid-lymphoid stem cell of placenta involved in the present invention is a kind of epithelial stem cell that derives from placenta amnion.Research shows, after the epithelial lining of amnion originates from zygote and forms 8 days, and the epiblast before Blastocyst formation.That is to say, the formation of epithelial lining early than interior, in, the generation of outer three germinal layers, therefore, the differentiation capability of the sub-totipotent cell of placenta is better than the multipotential stem cell that other originate from three germinal layers.According to existing, studies show that, the sub-totipotent cell of placenta can be inwardly, in, the cell of outer three germinal layers breaks up, for example, the sub-totipotent cell of placenta can be induced to break up and be become myocardial cell, neurocyte, liver cell, islet cells etc.In addition, multinomial experimentation on animals shows, the sub-totipotent cell of placenta can effectively be treated the illnesss such as pulmonary fibrosis, liver cirrhosis, Parkinson and multiple sclerosis.
Therefore, the sub-myeloid-lymphoid stem cell of placenta is a kind of important cell resource.Because it is positioned on amnion, because the sub-myeloid-lymphoid stem cell of this person's placenta belongs to adult stem cell category, avoided the dispute of ethic of embryonic stem cell, and also the obtaining easily of placenta, can not cause secondary injury to puerpera, there are boundless market outlook.
Because placenta amnion is except containing epithelial lining, also contain mesenchyme layer, it is closely connected under epithelial lining.Conventional cell dissociation method is difficult to two layers of tissue to distinguish and treat, and therefore can cause mesenchymal cell to pollute epithelial situation, and mesenchymal cell dryness is lower, thereby greatly reduces the dryness of the resulting cell of digestion.The mesenchymal cell growth that the digestion of mesenchyme layer obtains is in addition very fast, meeting and epithelial cell competitive growth in culturing process, thus epithelial cell (being the sub-totipotent cell of placenta) growth is suppressed.Therefore the conventional resulting cell of digestion method is difficult to reach the requirement that stores cell resource.
Summary of the invention
An object of the present invention is to provide the sub-myeloid-lymphoid stem cell of a kind of people's placenta, it derives from human placenta's amnion of peeling off, epithelial lining and the contained cell feature of mesenchyme layer for amnion, adopted the progressively method of separation, reduce to greatest extent the pollution of amnion mesenchymal confluent monolayer cells to the sub-totipotent cell of placenta, effectively improved the dryness of the sub-totipotent cell of people's placenta.The method have cost low, have a extensive future, can be clinical and research the feature of a large amount of stem cell resources is provided.
The sub-myeloid-lymphoid stem cell of people's placenta of the present invention derives from human placenta's amnion of peeling off, express marker molecule SSEA4, Sox2, Oct4, Nanog, TRA1-81, TRA1-60 and EpCAM, do not express CD45, can be in culture vessel adherent growth, can be in human body, in, ectoderm tissue differentiation.Wherein SSEA4, Sox2, Oct4, Nanog, TRA1-81, TRA1-60 are the special signs of reflection stem cell dryness, and EpCAM is a kind of epithelial sign, and CD45 is present in hemopoietic stem cell.
In order to preserve and use this outstanding stem cell resource, we have built the sub-myeloid-lymphoid stem cell storage vault of people's placenta.So-called stem cell bank is the place that (profound hypothermia) stores stem cell and collect storage stem cell resource dependency data in the liquid nitrogen of be about-196oC.A perfect stem cell bank should possess the ability that the healthy stem cell storing is provided to clinical use whenever and wherever possible.At present, there is no the placenta stem-cell storage vault that can preserve high dryness, therefore, build the sub-myeloid-lymphoid stem cell of people's placenta storehouse, the sub-myeloid-lymphoid stem cell of a large amount of high dryness that just have while being kept at baby due, is existing resource to be carried out to the important method of rational and efficient use.The foundation in the sub-myeloid-lymphoid stem cell of people's placenta storehouse can need to adopt the sub-myeloid-lymphoid stem cell of placenta to transplant and during the various diseases of correlation technique treatment, provide clinical adaptive type placenta stem-cell for storage I, family members and other people, and its application prospect is very wide.
A second aspect of the present invention provides a kind of to be prepared the sub-myeloid-lymphoid stem cell of people's placenta and sets up the method for stem cell bank from placenta amnion, and the method comprises the following steps:
(1) the sub-myeloid-lymphoid stem cell of separated people's placenta;
(2) the sub-myeloid-lymphoid stem cell of cultivator placenta;
(3) detect the sub-myeloid-lymphoid stem cell of people's placenta;
(4) the sub-myeloid-lymphoid stem cell of cryopreserved human placenta;
Wherein, step (3) and (4) are parallel carries out, result to be detected out after, to detect underproof cell according to its numbering, corresponding freeze-stored cell is together discarded together with cryopreservation tube, make a record, put into refuse bag, concentrate and be positioned over specified location, by technician, transport processing.
The sub-myeloid-lymphoid stem cell separation method of people's placenta of the present invention is used discarded placenta as raw material, and wide material sources are with low cost, and obtain easily, can not cause secondary injury to puerpera.
In the method for the sub-myeloid-lymphoid stem cell of separated people's placenta, with first with slide glass, the gelatinoid of amnion mesenchymal layer being scraped before protein enzyme solution digestion placenta amnion, and remove residual gelatinoid by ACETYLCYSTEINE.Described protein enzyme solution is that the Collagenase II-EDTA solution of 0.15% (w/v) and trypsinase-EDTA solution of 0.25% (w/v) mix with the volume ratio of 1:9.
In a specific embodiment, the method for the sub-myeloid-lymphoid stem cell of separated people's placenta comprises the following steps:
A) by amnion from ablatio placentae, D-Hank ' the s balanced salt solution that is 7.2-7.4 by pH value rinses repeatedly, removes residual blood, it is 5 * 5 cm that amnion is shredded
2fritter;
B) in the culture dish of 6-cm, splash into 1mL D-Hank ' s balanced salt solution, wetting culture dish bottom, is positioned over the epithelial lining of amnion downwards in culture dish, uses slide glass that the gelatinoid of mesenchyme layer is scraped;
C) amnion of having scraped glue is put into 10mL containing D-Hank ' the s balanced salt solution of 10% (w/v) ACETYLCYSTEINE, incubated at room 15 minutes; Remove mucus remaining on amnion, so that digestion completely;
D) to amnion, add about 20mL protein enzyme solution, under 37oC temperature condition, gas bath digestion is 30 minutes, collects Digestive system and adds 5mL foetal calf serum; Wherein said protein enzyme solution is that the Collagenase II-EDTA solution of 0.15% (w/v) and trypsinase-EDTA solution of 0.25% (w/v) mix with the volume ratio of 1:9;
E), after digestion finishes, use D-Hank ' s balanced salt solution repeatedly to rinse amnion;
F) Digestive system and the liquid washing down are merged, through 100 eye mesh screens, filter, repeat twice, obtain cell suspension;
G) cell suspension is placed in whizzer with the rotating speed of 2000 revs/min centrifugal 15 minutes, bottom cell D-Hank ' s balanced salt solution is blown and beaten repeatedly, then with the rotating speed of 1500 revs/min centrifugal 10 minutes, abandoning supernatant;
H) use 10mL D-Hank ' s balanced salt solution re-suspended cell, to cell suspension, slowly add 10mL Ficoll lymphocyte separation medium, with the speed of 250g centrifugal 8 minutes, do not use braking deceleration;
I) after centrifugal end, by the cell sucking-off between two-layer liquid level; Use D-Hank ' s balanced salt solution to clean the cell of sucking-off, with the rotating speeds of 1500 revs/min centrifugal 5 minutes, remove supernatant, obtain stem cell.
In a specific embodiment, the method for the sub-myeloid-lymphoid stem cell of cultivator placenta comprises the following steps:
A) stem cell is pressed to 2 * 10
5/ cm
2be inoculated in culturing bottle, add 10mL stem cell nutrient solution, be placed in 37oC, CO
2content is to cultivate in 5% incubator; Wherein, described stem cell nutrient solution is that DMEM/F12 nutrient solution adds 10% (v/v) foetal calf serum, 1% (w/v) pancreas islet plain sheet selenium transferrin (ITS), 10ng/mL Urogastron (EGF);
B) the stem cell nutrient solution more renewing after 4-5 days, discards non-adherent cell, within later every 3-4 days, changes stem cell nutrient solution once;
C) treat that Growth of Cells reaches 80% fusion, in culturing bottle, adding proteinase II solution digests, culturing bottle is put into 37 ° of C incubators 5 minutes, observation of cell under inverted microscope, with palm, pat gently culturing bottle and guarantee that cell and tissue block complete digestion get off, in each culturing bottle, add stem cell nutrient solution 10mL, piping and druming, cleans up the cell in culturing bottle repeatedly;
D) cell suspension washing down is moved in 50mL centrifuge tube, under the speed conditions of 1500 revs/min centrifugal 5 minutes, after centrifugal end, supernatant is abandoned in suction, in centrifuge tube, add the piping and druming of 30mL stem cell nutrient solution totally to mix cell, divide and cultivate in three culturing bottles, be placed in 37oC, CO
2content is to cultivate in 5% incubator, obtains the sub-myeloid-lymphoid stem cell of the people's placenta going down to posterity after cultivating.
In a specific embodiment, the method that detects the sub-myeloid-lymphoid stem cell of people's placenta comprises the following steps:
A) stem cell of cultivation is digested with proteinase II solution, with the resuspended cell suspension that is prepared into of stem cell nutrient solution,
B) draw and contain about 5 * 10
6the cell suspension of individual cell is sent into medicine non-clinical study quality control procedure standard laboratory and is carried out quality examination, comprise that aseptic detection, detection of mycoplasma, virus detection, immunophenotype detection, cell counting and vitality test, biological efficacy detect, cytodifferentiation ability detects, to determine whether it reaches criterion of acceptability;
Wherein, it is to detect hepatitis B virus, hepatitis C virus, human immunodeficiency virus by real-time fluorescent polyase chain reaction that described virus detects, and criterion is that indices is all negative; Described cell counting and vitality test refer to Trypan Blue and carry out cell counting, and criterion is that Cell viability is more than or equal to 90%; It is to detect by direct immunofluorescence that described immunophenotype detects, antibody used is monoclonal antibody SSEA4, Sox2, Oct4, Nanog, TRA1-81, TRA1-60, EpCAM, CD45, the HLA-DR of mouse anti human, criterion is that CD45, HLA-DR are negative, and SSEA4, Sox2, Oct4, Nanog, TRA1-81, TRA1-60, EpCAM are positive.
In a specific embodiment, the method for the sub-myeloid-lymphoid stem cell of cryopreserved human placenta comprises the following steps:
A) stem cell of cultivation is digested, the centrifugal supernatant that goes, adds foetal calf serum wherein, makes to meet 1 * 10
6-2 * 10
6cell/0.9mL foetal calf serum, fully mixes and obtains cell suspension;
B) on freeze pipe and freeze box, paste bar code, on freeze pipe, write same bar code number simultaneously;
C) above-mentioned cell suspension is joined in cryopreservation tube and mixed according to 1:1 ratio with frozen storing liquid, rapidly in immigration-80oC refrigerator;
D), after 24 hours, according to numbering, be put into and on corresponding freezing frame, move into the medium-term and long-term preservation of liquid nitrogen, storage condition periodic monitoring.
The present invention compared with prior art, has the following advantages:
The sub-myeloid-lymphoid stem cell of placenta is positioned at the epithelial lining of amnion, and the mesenchyme layer of amnion contains a large amount of gelatinoids, affects the digestion of trypsinase to epithelial lining.Use slide glass to scrape off the gelatinoid of mesenchyme layer, and remove residual gelatinoid by ACETYLCYSTEINE, coordinate fully digestive epithelium layer of protein enzyme solution that the present invention is special, obtain how highly purified placenta Asia myeloid-lymphoid stem cell.
Amnion, after digestion, can be isolated SSEA4
-mescenchymal stem cell, its density is different from sub-myeloid-lymphoid stem cell, by the centrifugal method of Ficoll, obtains highly purified sub-myeloid-lymphoid stem cell.
The sub-myeloid-lymphoid stem cell of the placenta that obtains, is preserved by building stem cell bank, and its source is abundant, and cell obtains easy, can obtain the sub-myeloid-lymphoid stem cell that a large amount of dryness are strong, and can preserve for a long time and do not lose its activity, is rich in application prospect.This storehouse is that healthy babies stores the sub-myeloid-lymphoid stem cell of placenta, for storage person is when suffering from the various diseases that need to adopt stem cell transplantation and correlation technique treatment, provide clinical adaptive type placenta sub-myeloid-lymphoid stem cell, and can be after obtaining license, the production by stored stem cell for other various stem cell medicines.
The invention has the beneficial effects as follows:
1. according to preparation method of the present invention, prepare SSEA4
+/ Sox2
+/ Oct4
+/ Nanog
+/ TRA1-81
+/ TRA1-60
+/ EpCAM
+/ CD45
-/ HLA-DR
-stem cell can be separated from autologous or other people placenta, can store as cell resource.
2. according to preparation method of the present invention, prepare SSEA4
+/ Sox2
+/ Oct4
+/ Nanog
+/ TRA1-81
+/ TRA1-60
+/ EpCAM
+/ CD45
-/ HLA-DR
-stem cell has Multidirectional Differentiation ability, can use for clinical treatment.
Accompanying drawing explanation
Fig. 1 amnion cell SSEA-4 expresses.
Fig. 2 A. amnion is HE dyeing picture after scraping off mesenchyme layer and ACETYLCYSTEINE processing; B. undressed amnion HE dyeing picture.
The HE dyeing photo of Fig. 3 amnion after protease digestion.
The SSEA-4 of the cell that Fig. 4 amnion epithelial lining and amnion mesenchymal layer obtain after digestion and EpCAM express.
Fig. 5 Ficoll is centrifugal, and rear SSEA-4 expresses.
The growthhabit of the sub-myeloid-lymphoid stem cell of Fig. 6 placenta.
The sub-myeloid-lymphoid stem cell growth curve of Fig. 7 placenta.
The sub-myeloid-lymphoid stem cell Phenotypic examination of Fig. 8 placenta.
Growthhabit after the sub-myeloid-lymphoid stem cell recovery of Fig. 9 placenta.
The sub-myeloid-lymphoid stem cell cells into cardiomyocytes induction of Figure 10 placenta.
The sub-myeloid-lymphoid stem cell of Figure 11 placenta is induced to liver cell.
The sub-myeloid-lymphoid stem cell neuralward of Figure 12 placenta cell induction.
Embodiment
Embodiment 1 gathers placenta
Puerpera is carried out to ABO/Rh Blood grouping, HLA(Human Leukocyte Antigen, human leucocyte antigen) detection of somatotype, microorganism immunodetection, health check-up HIV(Human Immunodeficiency Virus, human immunodeficiency virus), the abbreviation of HBV(Hepatitis B virus, hepatitis B virus), HCV(Hepatitis C virus, hepatitis C virus); Then in Operation theatre, gather puerpera's placenta.
Before placenta gathers, puerpera fills in Informed Consent Form, individual basic document, then puerpera is identified, gatherer process should strictly prevent the pollution of pathogenic micro-organism, requirement is carried out in aseptic place, then the placenta collecting is transported to appointed place, transportation environment temperature should be within the scope of 4-8oC; When placenta receives, should fill in and receive list, mark placenta bottle; Placenta carries out aseptic detection to placenta used after receiving, and guarantees the security of placenta.
Embodiment 2 obtains and the sub-myeloid-lymphoid stem cell of separated people's placenta
By amnion, from ablatio placentae, D-Hank ' the s balanced salt solution that is 7.2-7.4 by pH value rinses repeatedly, removes residual blood, and it is 5 * 5 cm that amnion is shredded
2fritter.Get a wherein amnion and make paraffin section, and carry out immunofluorescence dyeing (SSEA-4 is shown in Fig. 1).The demonstration of Fig. 1 result, SSEA-4 positive cell is mainly distributed in the outermost layer of amnion, i.e. amnion epithelial lining.
Amnion fragment is equally divided into two groups of A, B, and the method that wherein A group adopts the present invention to describe is carried out separation, and B group adopts ordinary method digestion separated (that epithelial lining is not separated with mesenchyme layer) as a comparison.
In the culture dish of 6-cm, splash into 1mL D-Hank ' s balanced salt solution, wetting culture dish bottom, the epithelial lining of A being organized to amnion is positioned over downwards in culture dish, uses slide glass that the gelatinoid of mesenchyme layer is scraped.
The amnion of having scraped glue is put into 10mL containing D-Hank ' the s balanced salt solution of 10% (w/v) ACETYLCYSTEINE, incubated at room 15 minutes.Remove mucus remaining on amnion, so that digestion completely.
Get part B and organize the amnion after untreated amnion and the processing of part A group, make frozen section, again through hematoxylin-eosin (HE) dyeing (see figure 2), in Fig. 2, result shows that B organizes undressed amnion and also has a large amount of gluey mesenchyme layers, and the amnion mesenchymal layer that A organizes after processing reduces in a large number, epithelial lining is intact.
To A group amnion, add about 20mL protein enzyme solution (the Collagenase II-EDTA solution of 0.15% (w/v) mixes with the volume ratio of 1:9 with trypsinase-EDTA solution of 0.25% (w/v)), under 37oC temperature condition, gas bath digestion is 30 minutes, after digestion finishes, use D-Hank ' s balanced salt solution repeatedly to rinse amnion.Part A group amnion is made to frozen section, and carry out HE dyeing (see figure 3), in figure, result shows after protein enzyme solution digestion, the complete digestion of the cell of epithelial lining, and mesenchyme layer is digested.After digestion finishes, collect the Digestive system of amnion and add 5mL foetal calf serum.Use D-Hank ' s balanced salt solution repeatedly to rinse amnion, Digestive system and the liquid washing down are merged, through 100 eye mesh screens, filter, obtain cell suspension.
To B group, add trypsinase-EDTA solution of 0.25% (w/v) of about 20mL to digest 30 minutes.Collect the Digestive system of amnion and add 5mL foetal calf serum.Use D-Hank ' s balanced salt solution repeatedly to rinse amnion, Digestive system and the liquid washing down are merged, through 100 eye mesh screens, filter.Repeat twice, obtain cell suspension.
The digestion mixed solution of two groups of amnions is placed in respectively to whizzer with the rotating speed of 2000 revs/min centrifugal 15 minutes, bottom cell D-Hank ' s balanced salt solution is blown and beaten repeatedly, then with the rotating speed of 1500 revs/min centrifugal 10 minutes, abandoning supernatant.
The cell that A, two groups of amnions of B are obtained is used respectively 10mL D-Hank ' s balanced salt solution re-suspended cell, to cell suspension, slowly adds 10mL lymphocyte separation medium, with the speed of 250g centrifugal 8 minutes, does not use braking deceleration.
After centrifugal end, by the cell sucking-off between two-layer liquid level.Use D-Hank ' s balanced salt solution to clean the cell of sucking-off, with the rotating speeds of 1500 revs/min centrifugal 5 minutes, obtain stem cell.
The stem cell that A, two kinds of methods of B are obtained is with 10
5/ hole is inoculated into stem cell nutrient solution (DMEM/F12 nutrient solution interpolation 10% (v/v) foetal calf serum in 24 orifice plates, 1% (w/v) pancreas islet plain sheet selenium transferrin (ITS), 10ng/mL Urogastron (EGF)) in, after cell attachment, carry out immunofluorescence dyeing, detect stem cell and epithelial cell sign (see figure 4), the result of Fig. 4 shows, the stem cell sign SSEA-4 of the cell that method A obtains and epithelial cell sign EpCAM expression rate all approach 100%, and the amnion of method B is because contain the cell that a large amount of mesenchyme layer digestion gets, the speed of growth is very fast, affect the sub-myeloid-lymphoid stem cell growth of epithelial lining, therefore substantially do not express SSEA-4 and EpCAM.
The sub-totipotent cell that method A is obtained is in the centrifugal front and back of separation of lymphocytes, and by its SSEA-4 expression rate (see figure 5) of Flow cytometry, after the result of Fig. 5 shows that Ficoll is centrifugal, the SSEA-4 expression rate of cell significantly promotes, and illustrates that cell purity improves.
Embodiment 3 cultivates the sub-myeloid-lymphoid stem cell of placenta
The stem cell obtaining is pressed to 2 * 10
5/ cm
2be inoculated in culturing bottle, add 10mL stem cell nutrient solution, be placed in 37oC, CO
2content is to cultivate in 5% incubator, and the stem cell nutrient solution more renewing after 4-5 days, discards non-adherent cell, within later every 3-4 days, changes stem cell nutrient solution once, treats that Growth of Cells reaches 80% fusion, adds proteinase II solution and digest in culturing bottle, culturing bottle is put into 37 ° of C incubators 5 minutes, observation of cell under inverted microscope, with palm, pat gently culturing bottle and guarantee that cell and tissue block complete digestion get off, in each culturing bottle, add stem cell nutrient solution 10mL, piping and druming repeatedly, cell in culturing bottle is cleaned up, the cell suspension washing down is moved in 50mL centrifuge tube, under the speed conditions of 1500 revs/min centrifugal 5 minutes, after centrifugal end, supernatant is abandoned in suction, in centrifuge tube, add the piping and druming of 30mL stem cell nutrient solution totally to mix cell, obtain the sub-myeloid-lymphoid stem cell of people's placenta after cultivating, divide and cultivate in three culturing bottles, be placed in 37oC, CO
2content is to cultivate in 5% incubator, by 1:3, goes down to posterity, and obtains the sub-myeloid-lymphoid stem cell of the people's placenta going down to posterity after cultivating, and cell cultures the results are shown in Figure 6.
The mensuration of cell growth curve is with after protein enzyme solution II digestion, by 1 * 10 by attached cell
4cells/well is seeded in 24 orifice plates, every 3 holes of digestion in 24 hours, and collecting cell, and with 0.4% Trypan Blue living cell counting, drafting growth curve, as shown in Figure 7, in figure, X-coordinate is incubation time, ordinate zou is number of cells.
Embodiment 4 detects, the sub-myeloid-lymphoid stem cell of cryopreserved human placenta
Twice of the sub-myeloid-lymphoid stem cell of people's placenta of using the washing of 10mL D-Hank ' s balanced salt solution to go down to posterity after cultivating, washing lotion is abandoned in suction, in each culturing bottle, add 1mL proteinase II solution, put into 37 ° of C incubators 5 minutes, observation of cell under inverted microscope, and with palm, pat gently culturing bottle and guarantee that cell complete digestion gets off; In each culturing bottle, add again 10mL stem cell nutrient solution, repeatedly piping and druming; With the sucking-off of micro sample-adding rifle, a little is placed in centrifuge tube and is used as cell counting, according to count results, draws and contains about 5 * 10
6the cell suspension of individual cell divide in another centrifuge tube for detection of, all the other are for frozen.Two tube cells are centrifugal 5 minutes with 1500 revs/min, supernatant is abandoned in suction, for detection of cell with nutrient solution, repeatedly blow and beat cell, mix, send into medicine non-clinical study quality control procedure standard laboratory and carry out quality examination, comprise that aseptic detection, detection of mycoplasma, virus detection, immunophenotype detection, cell counting and vitality test, biological efficacy detect, cytodifferentiation ability detects, to determine whether it reaches criterion of acceptability; Wherein, it is to detect hepatitis B virus, hepatitis C virus, human immunodeficiency virus by real-time fluorescent polyase chain reaction that virus detects, and criterion is that indices is all negative, is considered as qualified cell.
Cell counting and vitality test refer to Trypan Blue and carry out cell counting, and criterion is that Cell viability is more than or equal to 90%, is considered as qualified cell.It is to detect by direct immunofluorescence that immunophenotype detects, the monoclonal antibody SSEA4 that antibody used is mouse anti human, Sox2, Oct4, Nanog, TRA1-81, TRA1-60, EpCAM, CD45, HLA-DR.Cell adds antibody after washing with PBS, hatches 30 minutes, then wash twice with PBS under 4oC, with 500 μ L PBS, blows and beats, mixes cell, detects afterwards with flow cytometer.The flow cytometer detected result of the sub-myeloid-lymphoid stem cell immunophenotype of people's placenta that the method according to this invention obtains is as shown in table 1.Detected result shows, CD45, HLA-DR in the sub-myeloid-lymphoid stem cell of placenta are negative, and are less than 2%; SSEA4, Sox2, Oct4, Nanog, TRA1-81, TRA1-60, EpCAM are positive, and the sub-myeloid-lymphoid stem cell of this placenta is qualified cell.Immunophenotyping detects sees Fig. 8.
The immunophenotype of the sub-myeloid-lymphoid stem cell of table 1 people placenta
| Mark | Expression amount |
| SSEA4 | + |
| Sox2 | + |
| Oct4 | + |
| Nanog | + |
| TRA1-81 | + |
| TRA1-60 | + |
| EpCAM | + |
| CD45 | - |
| HLA-DR | - |
Frozen step is: in the sub-myeloid-lymphoid stem cell of the people's placenta obtaining, add foetal calf serum, make to meet 1 * 10
6-2 * 10
6cell/0.9mL foetal calf serum, fully mixes, stand-by; On freeze pipe and freeze box, paste bar code, on freeze pipe, write same bar code number simultaneously; The sub-myeloid-lymphoid stem cell suspension of above-mentioned people's placenta is joined in cryopreservation tube and mixed according to 1:1 ratio with frozen storing liquid, after first cell suspension being joined respectively in freeze pipe, then unify to add frozen storing liquid, capping, concussion, fully mixes, in be moved into rapidly-80oC refrigerator; After 24 hours, according to numbering, be put on corresponding freezing frame and move into the medium-term and long-term preservation of liquid nitrogen, during cell cryopreservation, have detailed frozen record, meet freezing preservation standard, storage condition periodic monitoring, the detection of the sub-myeloid-lymphoid stem cell of people's placenta and frozen parallel carrying out, result to be detected out after, will detect underproof cell and according to its numbering, corresponding freeze-stored cell together be discarded together with cryopreservation tube, make a record, put into refuse bag, concentrate and be positioned over specified location, by technician, transport processing.
The frozen impact on cytoactive and surface marker of embodiment 5
Frozen cell is placed in after 37oC water-bath recovery, uses 10mL stem cell nutrient solution to be inoculated in culturing bottle, at 37 ℃, 5%CO
2in incubator, cultivate.After cell attachment, the stem cell nutrient solution more renewing, discards non-adherent cell.To cell count and vitality test count results in Table 2, Fig. 9 is shown in by cell photo.
The frozen front and back of table 2 stem cell counting and vitality test result
| Before frozen | After frozen | |
| Cell count | 2×10 6 | 2×10 6 |
| Motility rate | 96% | 94% |
| Immunophenotype | SSEA4 +/Sox2 +/Oct4 +/Nanog +/ TRA1-81 +/TRA1-60 +/EpCAM +/CD45 - | SSEA4 +/Sox2 +/Oct4 +/Nanog +/ TRA1-81 +/TRA1-60 +/EpCAM +/CD45 - |
The sub-myeloid-lymphoid stem cell cells into cardiomyocytes induction of embodiment 6 placentas
Separated stem cell is inoculated in 24 orifice plates that added in advance stem cell nutrient solution with 5000 cells/well, after cell attachment, by stem cell broth out, add myocardial cell's nutrient solution (the DMEM/F12 nutrient solution that contains 1 mM Ascorbic acid), cultivate 28 days, within every 3 days, change liquid.After induction finishes, by immunofluorescence, detect Troponin T(TNNT), experimental result is shown in Figure 10.
The sub-myeloid-lymphoid stem cell of embodiment 7 placentas is induced to liver cell
Separated cell is inoculated in 5000 cells/well in 24 orifice plates that added in advance stem cell nutrient solution, after cell attachment, by stem cell broth out, adds myocardial cell's nutrient solution (to contain 0.1 μ M Insulin and 1 * 10
-7the DMEM/F12 nutrient solution of M Dexamethasone), cultivate 28 days, within every 3 days, change liquid.After induction finishes, (Cytokeratin 19, and CK19), experimental result is shown in Figure 11 by immunofluorescence, to detect human serum albumin (albumin, ALB) and cytokeratin.
The induction of the sub-myeloid-lymphoid stem cell neuralward of embodiment 8 placentas like cell
Separated cell is inoculated in 5000 cells/well in 24 orifice plates that added in advance stem cell nutrient solution, after cell attachment, by stem cell broth out, adds myocardial cell's nutrient solution (to contain 5 * 10
-5m all-trans-retinoic acid (All-trans retinoic acid), 10ng/mL fibroblast growth factor (Fibroblast growth factor 4, FGF-4), the Neurobasal Medium A nutrient solution of 1% (w/v) B27), cultivate 28 days, within every 3 days, change liquid.After induction finishes, by immunofluorescence, detect MAP2, GFAP, Tublin and Nestin, experimental result is shown in Figure 12.
Claims (11)
1. the sub-myeloid-lymphoid stem cell of people's placenta, it derives from human placenta's amnion of peeling off, is characterised in that and expresses marker molecule SSEA4, Sox2, Oct4, Nanog, TRA1-81, TRA1-60 and EpCAM, does not express CD45.
2. the sub-myeloid-lymphoid stem cell of people's placenta according to claim 1, its can be in culture vessel adherent growth, can be in human body, in, ectoderm tissue differentiation.
3. the sub-myeloid-lymphoid stem cell of people's placenta according to claim 2, it can be induced to differentiate into myocardial cell, liver cell, neural like cell.
4. the method for the sub-myeloid-lymphoid stem cell of the people's placenta described in a separated claim 1-3 any one; it is characterized in that with first with slide glass, the gelatinoid of amnion mesenchymal layer being scraped before protein enzyme solution digestion placenta amnion, and remove residual gelatinoid by ACETYLCYSTEINE.
5. method according to claim 4, wherein said protein enzyme solution is that the Collagenase II-EDTA solution of 0.15% (w/v) and trypsinase-EDTA solution of 0.25% (w/v) mix with the volume ratio of 1:9.
6. method according to claim 5, it comprises the following steps:
(1) by amnion from ablatio placentae, D-Hank ' the s balanced salt solution that is 7.2-7.4 by pH value rinses repeatedly, removes residual blood, it is 5 * 5cm that amnion is shredded
2fritter;
(2) in the culture dish of 6-cm, splash into 1mL D-Hank ' s balanced salt solution, wetting culture dish bottom, is positioned over the epithelial lining of amnion downwards in culture dish, uses slide glass that the gelatinoid of mesenchyme layer is scraped;
(3) amnion of having scraped glue is put into 10mL containing D-Hank ' the s balanced salt solution of 10% (w/v) ACETYLCYSTEINE, incubated at room 15 minutes; Remove mucus remaining on amnion, so that digestion completely;
(4) to amnion, add about 20mL protein enzyme solution, under 37 ℃ of temperature condition, gas bath digestion is 30 minutes, collects Digestive system and adds 5mL foetal calf serum; Wherein said protein enzyme solution is that the Collagenase II-EDTA solution of 0.15% (w/v) and trypsinase-EDTA solution of 0.25% (w/v) mix with the volume ratio of 1:9;
(5), after digestion finishes, use D-Hank ' s balanced salt solution repeatedly to rinse amnion;
(6) Digestive system and the liquid washing down are merged, through 100 eye mesh screens, filter, repeat twice, obtain cell suspension;
(7) cell suspension is placed in whizzer with the rotating speed of 2000 revs/min centrifugal 15 minutes, bottom cell D-Hank ' s balanced salt solution is blown and beaten repeatedly, then with the rotating speed of 1500 revs/min centrifugal 10 minutes, abandoning supernatant;
(8) use 10mL D-Hank ' s balanced salt solution re-suspended cell, to cell suspension, slowly add 10mL Ficoll lymphocyte separation medium, with the speed of 250g centrifugal 8 minutes, do not use braking deceleration;
(9) after centrifugal end, by the cell sucking-off between two-layer liquid level; Use D-Hank ' s balanced salt solution to clean the cell of sucking-off, with the rotating speeds of 1500 revs/min centrifugal 5 minutes, remove supernatant, obtain stem cell.
7. cultivate a method for the sub-myeloid-lymphoid stem cell of people's placenta described in claim 1-3 any one, it is characterized in that the stem cell nutrient solution using is that DMEM/F12 nutrient solution adds 10% (v/v) foetal calf serum, 1% (w/v) pancreas islet plain sheet selenium transferrin (ITS), 10ng/mL Urogastron (EGF).
8. according to the method for claim 7, it comprises the following steps:
(1) stem cell is pressed to 2 * 10
5/ cm
2be inoculated in culturing bottle, add 10mL stem cell nutrient solution, be placed in 37 ℃, CO
2content is to cultivate in 5% incubator;
(2) the stem cell nutrient solution more renewing after 4-5 days, discards non-adherent cell, within later every 3-4 days, changes stem cell nutrient solution once;
(3) treat that Growth of Cells reaches 80% fusion, in culturing bottle, adding proteinase II solution digests, culturing bottle is put into 37 ℃ of incubators 5 minutes, observation of cell under inverted microscope, with palm, pat gently culturing bottle and guarantee that cell and tissue block complete digestion get off, in each culturing bottle, add stem cell nutrient solution 10mL, piping and druming, cleans up the cell in culturing bottle repeatedly;
(4) cell suspension that washes down is moved in 50mL centrifuge tube, under the speed conditions of 1500 revs/min centrifugal 5 minutes, after centrifugal end, supernatant is abandoned in suction, in centrifuge tube, adds the piping and druming of 30mL stem cell nutrient solution totally to mix cell, divides and cultivates in three culturing bottles, be placed in 37 ℃, CO
2content is to cultivate in 5% incubator, obtains the sub-myeloid-lymphoid stem cell of the people's placenta going down to posterity after cultivating.
9. a method for the sub-myeloid-lymphoid stem cell of people's placenta described in test right requirement 1-3 any one, comprises the following steps:
(1) stem cell of cultivation is digested with proteinase II solution, with the resuspended cell suspension that is prepared into of stem cell nutrient solution,
(2) draw and contain about 5 * 10
6the cell suspension of individual cell is sent into medicine non-clinical study quality control procedure standard laboratory and is carried out quality examination, comprise that aseptic detection, detection of mycoplasma, virus detection, immunophenotype detection, cell counting and vitality test, biological efficacy detect, cytodifferentiation ability detects, to determine whether it reaches criterion of acceptability;
Wherein, described stem cell nutrient solution is that DMEM/F12 nutrient solution adds 10% (v/v) foetal calf serum, 1% (w/v) pancreas islet plain sheet selenium transferrin (ITS), 10ng/mL Urogastron (EGF); It is to detect hepatitis B virus, hepatitis C virus, human immunodeficiency virus by real-time fluorescent polyase chain reaction that described virus detects, and criterion is that indices is all negative; Described cell counting and vitality test refer to Trypan Blue and carry out cell counting, and criterion is that Cell viability is more than or equal to 90%; It is to detect by direct immunofluorescence that described immunophenotype detects, antibody used is monoclonal antibody SSEA4, Sox2, Oct4, Nanog, TRA1-81, TRA1-60, EpCAM, CD45, the HLA-DR of mouse anti human, criterion is that CD45, HLA-DR are negative, and SSEA4, Sox2, Oct4, Nanog, TRA1-81, TRA1-60, EpCAM are positive.
10. a method for the sub-myeloid-lymphoid stem cell of the people's placenta described in frozen claim 1-3 any one, comprises the following steps:
(1) stem cell of cultivation is digested, the centrifugal supernatant that goes, adds foetal calf serum wherein, makes to meet 1 * 10
6-2 * 10
6cell/0.9mL foetal calf serum, fully mixes and obtains cell suspension;
(2) on freeze pipe and freeze box, paste bar code, on freeze pipe, write same bar code number simultaneously;
(3) above-mentioned cell suspension is joined in cryopreservation tube and mixed according to 1:1 ratio with frozen storing liquid, move into rapidly in-80 ℃ of refrigerators;
After (4) 24 hours, according to numbering, be put into and on corresponding freezing frame, move into the medium-term and long-term preservation of liquid nitrogen, storage condition periodic monitoring.
The construction process in the 11. 1 kinds of sub-myeloid-lymphoid stem cell of people's placenta storehouses, comprises the following steps:
(1) according to the sub-myeloid-lymphoid stem cell of the separated people's placenta of the method described in claim 4-6 any one;
(2) according to the sub-myeloid-lymphoid stem cell of the method cultivator placenta described in claim 7 or 8;
(3) method according to claim 9 detects the sub-myeloid-lymphoid stem cell of people's placenta;
(4) the sub-myeloid-lymphoid stem cell of method cryopreserved human placenta according to claim 10;
Wherein, step (3) and (4) are parallel carries out, result to be detected out after, to detect underproof cell according to its numbering, corresponding freeze-stored cell is together discarded together with cryopreservation tube, make a record, put into refuse bag, concentrate and be positioned over specified location, by technician, transport processing.
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| CN107446881A (en) * | 2017-08-10 | 2017-12-08 | 河南省银丰生物工程技术有限公司 | A kind of isolation and purification method of placenta myeloid-lymphoid stem cell |
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| CN108239621B (en) * | 2018-01-24 | 2020-08-18 | 北京臻溪谷医学研究中心（有限合伙） | Method for separating, amplifying, cryopreserving and reviving placenta sub-totipotent stem cells |
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