CN103966159B - Human plactnta Subaerial blue green algae and stem cell bank construction process thereof - Google Patents
Human plactnta Subaerial blue green algae and stem cell bank construction process thereof Download PDFInfo
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Abstract
The invention provides a kind of Human plactnta Subaerial blue green algae, it derives from human placenta's amnion of stripping, for the cellular features contained by the epithelial lining of amnion and mesenchyme layer, have employed the method be progressively separated, decrease the pollution of amnion mesenchymal confluent monolayer cells to the sub-totipotent cell of placenta to greatest extent, effectively improve the dryness of the sub-totipotent cell of Human plactnta.The method have cost low, have a extensive future, can be clinical and research the feature of a large amount of stem cell resource is provided.Present invention also offers and a kind ofly prepare Human plactnta Subaerial blue green algae from placenta amnion and set up the method for stem cell bank.
Description
Technical field
The present invention relates to stem cell field, relate to a kind of its particularly and derive from the Subaerial blue green algae of human placenta's amnion.Meanwhile, the present invention also relates to preparation method and the method for inducing differentiation of this Subaerial blue green algae.
Background technology
Stem-cell research is the study hotspot emerged in recent years, and its progress at full speed and wide application prospect make stem-cell research more and more be attracted attention.Stem cell is the cell that a class has self and differentiation potential, can be divided into embryonic stem cell and adult stem cell according to source.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 rich in a kind of Subaerial blue green algae with Multidirectional Differentiation ability.Placenta is the transitional organ exchanging material between Mammals pregnancy duration mothers and sons, and its physiological function is widely studied, but placenta is as a kind of cellular resources, and its research is also in the starting stage.Placenta Subaerial blue green algae involved in the present invention is a kind of epithelial stem cell deriving from placenta amnion.Research shows, the epithelial lining of amnion originates from after zygote forms 8 days, the epiblast before Blastocyst formation.That is, 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 other multipotential stem cells originating from three germinal layers.According to existing research display, the sub-totipotent cell of placenta can inwardly, in, the cell of outer three germinal layers breaks up, such as, placenta Asia totipotent cell differentiation-inducingly can become myocardial cell, neurocyte, liver cell, islet cells etc.In addition, multinomial experimentation on animals shows, the sub-totipotent cell of placenta can effectively treat the illnesss such as pulmonary fibrosis, liver cirrhosis, Parkinson and multiple sclerosis.
Therefore, placenta Subaerial blue green algae is a kind of important cellular resources.Because it is positioned on amnion, therefore Human plactnta Subaerial blue green algae belongs to adult stem cell category, avoids the dispute of ethic of embryonic stem cell, and the acquisition of placenta is easy, can not cause secondary injury, have boundless market outlook to puerpera.
Because placenta amnion is except containing except epithelial lining, also contain mesenchyme layer, under it is close-coupled at epithelial lining.Conventional cell dissociation method is difficult to two layers of tissue to be distinguished treat, and mesenchymal cell therefore can be caused to pollute epithelial situation, and mesenchymal cell dryness is lower, thus greatly reduces the dryness of the cell that digestion obtains.To digest the mesenchymal cell growth obtained very fast for mesenchyme layer in addition, in culturing process can and epithelial cell competitive growth, thus epithelial cell (i.e. the sub-totipotent cell of placenta) is grown be suppressed.Therefore the cell that conventional digestion method obtains is difficult to reach the requirement storing cellular resources.
Summary of the invention
An object of the present invention is to provide a kind of Human plactnta Subaerial blue green algae, it derives from human placenta's amnion of stripping, for the cellular features contained by the epithelial lining of amnion and mesenchyme layer, have employed the method be progressively separated, decrease the pollution of amnion mesenchymal confluent monolayer cells to the sub-totipotent cell of placenta to greatest extent, effectively improve the dryness of the sub-totipotent cell of Human plactnta.The method have cost low, have a extensive future, can be clinical and research the feature of a large amount of stem cell resource is provided.
Human plactnta Subaerial blue green algae of the present invention derives from human placenta's amnion of stripping, express marker molecule SSEA4, Sox2, Oct4, Nanog, TRA1-81, TRA1-60 and EpCAM, do not express CD45, can in culture vessel adherent growth, can in human body, in, ectodermal histological differentiation.Wherein SSEA4, Sox2, Oct4, Nanog, TRA1-81, TRA1-60 are the specific marker of reflection stem cell dryness, and EpCAM is a kind of epithelial mark, and CD45 is present in hemopoietic stem cell.
In order to the stem cell resource of preserving and use this outstanding, we construct Human plactnta Subaerial blue green algae storage vault.So-called stem cell bank is that (profound hypothermia) stores stem cell and collect the place storing stem cell resource related data in the liquid nitrogen being about-196oC.A perfect stem cell bank should possess the ability healthy stem cell stored being provided Clinical practice whenever and wherever possible.At present, there is no the placenta stem-cell storage vault can preserving high dryness, therefore, build Human plactnta Subaerial blue green algae storehouse, the Subaerial blue green algae of a large amount of high dryness just had when being kept at baby due is the important method of existing resource being carried out rational and efficient use.The foundation in Human plactnta Subaerial blue green algae storehouse can for store I, family members and other people need to adopt placenta Subaerial blue green algae to transplant and the various disease of correlation technique treatment time, provide clinical adaptive type placenta stem-cell, its application prospect is very wide.
A second aspect of the present invention provides a kind ofly to be prepared Human plactnta Subaerial blue green algae from placenta amnion and sets up the method for stem cell bank, and the method comprises the following steps:
(1) Human plactnta Subaerial blue green algae is separated;
(2) cultivator placenta Subaerial blue green algae;
(3) Human plactnta Subaerial blue green algae is detected;
(4) cryopreserved human placenta Subaerial blue green algae;
Wherein, step (3) and (4) walk abreast and carry out, result to be detected out after, underproof cell will be detected 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, transport process by technician.
Human plactnta Subaerial blue green algae separation method of the present invention uses 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 being separated Human plactnta Subaerial blue green algae, first scrape with the gelatinoid of slide glass by amnion mesenchymal layer with 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) mixes with the volume ratio of 1:9 with the trypsin-EDTA solutions of 0.25% (w/v).
In a specific embodiment, the method being separated Human plactnta Subaerial blue green algae comprises the following steps:
A) by amnion from ablatio placentae, repeatedly rinse with D-Hank ' the s balanced salt solution that pH value is 7.2-7.4, remove residual blood, being shredded by amnion is 5 × 5 cm
2fritter;
B) in the culture dish of 6-cm, instill 1mL D-Hank ' s balanced salt solution, bottom wetting culture dish, the epithelial lining of amnion is positioned over downwards in culture dish, use slide glass to be scraped by the gelatinoid of mesenchyme layer;
C) amnion having scraped glue is put into D-Hank ' the s balanced salt solution of 10mL containing 10% (w/v) ACETYLCYSTEINE, incubated at room 15 minutes; Remove mucus remaining on amnion, so that digestion completely;
D) add about 20mL protein enzyme solution to amnion, under 37oC temperature condition, gas bath digests 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) mixes with the volume ratio of 1:9 with the trypsin-EDTA solutions of 0.25% (w/v);
E), after digestion terminates, D-Hank ' s balanced salt solution is used repeatedly to rinse amnion;
F) Digestive system and the liquid washed down are merged, filter through 100 eye mesh screens, repeat twice, obtain cell suspension;
G) cell suspension is placed in whizzer with the rotating speed of 2000 revs/min centrifugal 15 minutes, is is repeatedly blown and beaten by bottom cell D-Hank ' s balanced salt solution, 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, slowly add 10mL Ficoll lymphocyte separation medium to cell suspension, with the centrifugation 8 minutes of 250g, do not use braking deceleration;
I) after centrifugal end, by the cell sucking-off between two-layer liquid level; Use the cell of D-Hank ' s balanced salt solution cleaning sucking-off, with the rotating speed of 1500 revs/min centrifugal 5 minutes, remove supernatant, obtain stem cell.
In a specific embodiment, the method for cultivator placenta Subaerial blue green algae comprises the following steps:
A) stem cell is pressed 2 × 10
5/ cm
2be inoculated in culturing bottle, add 10mL stem cell medium, be placed in 37oC, CO
2content is cultivate in the incubator of 5%; Wherein, described stem cell medium 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) stem cell medium more renewed after 4-5 days, discards non-adherent cell, within every 3-4 days later, changes stem cell medium once;
C) treat that Growth of Cells reaches 80% fusion, in culturing bottle, add proteinase II solution digest, culturing bottle is put into 37 ° of C incubators 5 minutes, observation of cell under inverted microscope, pat culturing bottle gently with palm and guarantee that cell and tissue block complete digestion are got off, add stem cell medium 10mL in each culturing bottle, repeatedly blow and beat, the cell in culturing bottle is cleaned up;
D) cell suspension washed 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 medium totally mix cell, divide and cultivate in three culturing bottles, be placed in 37oC, CO
2content is cultivate in the incubator of 5%, obtains the Human plactnta Subaerial blue green algae after Secondary Culture.
In a specific embodiment, the method detecting Human plactnta Subaerial blue green algae comprises the following steps:
A) stem cell of cultivation proteinase II solution digestion is got off, is prepared into cell suspension with stem cell medium is resuspended,
B) draw containing 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 Sterility testing, detection of mycoplasma, Viral diagnosis, immunophenotype detect, cell counting and vitality test, biological efficacy detect, cell differentiation detects, to determine whether it reaches criterion of acceptability;
Wherein, described Viral diagnosis detects hepatitis B virus, hepatitis C virus, human immunodeficiency virus by real-time fluorescent polyase chain reaction, and criterion is that indices is feminine gender; Described cell counting and vitality test refer to and carry out cell counting with Trypan Blue, and criterion is that Cell viability is more than or equal to 90%; It is detect by direct immunofluorescence that described immunophenotype detects, antibody used is monoclonal antibody SSEA4, Sox2, Oct4, Nanog, TRA1-81, TRA1-60, EpCAM, CD45, 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 cryopreserved human placenta Subaerial blue green algae comprises the following steps:
A) stem cell of cultivation is digested, centrifugally remove supernatant, add foetal calf serum wherein, make to meet 1 × 10
6-2 × 10
6cell/0.9mL foetal calf serum, fully mixing 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 and frozen storing liquid are joined in cryopreservation tube according to 1:1 ratio mix, move into rapidly in-80oC refrigerator;
D), after 24 hours, be put on corresponding freezing frame according to numbering and 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:
Placenta Subaerial blue green algae 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 protein enzyme solution that the present invention is special can abundant digestive epithelium layer, obtain more how highly purified placenta Subaerial blue green algae.
Amnion, after digestion, can isolate SSEA4
-mescenchymal stem cell, its density is different from Subaerial blue green algae, obtains highly purified Subaerial blue green algae by the method that Ficoll is centrifugal.
The placenta Subaerial blue green algae obtained, is preserved, its abundance by building stem cell bank, and cell obtains easy, can obtain the Subaerial blue green algae that a large amount of dryness is strong, and can preserve for a long time and not lose its activity, is rich in application prospect.This storehouse is that healthy babies stores placenta Subaerial blue green algae, for storage person is when suffering from the various disease needing to adopt stem cell transplantation and correlation technique treatment, clinical adaptive type placenta Subaerial blue green algae is provided, and after obtaining license, stored stem cell can be used for the production of other various stem cell medicines.
The invention has the beneficial effects as follows:
1. prepare SSEA4 according to preparation method of the present invention
+/ 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 cellular resources.
2. prepare SSEA4 according to preparation method of the present invention
+/ Sox2
+/ Oct4
+/ Nanog
+/ TRA1-81
+/ TRA1-60
+/ EpCAM
+/ CD45
-/ HLA-DR
-stem cell has Multidirectional Differentiation ability, can be used for clinical treatment to use.
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 process; B. undressed amnion HE dyeing picture.
The HE stained photographs of Fig. 3 amnion after protease digestion.
SSEA-4 and EpCAM of the cell that Fig. 4 amnioic epithelium layer and amnion mesenchymal layer obtain after digestion expresses.
Fig. 5 Ficoll is centrifugal, and rear SSEA-4 expresses.
The growthhabit of Fig. 6 placenta Subaerial blue green algae.
Fig. 7 placenta Subaerial blue green algae growth curve.
Fig. 8 placenta Subaerial blue green algae Phenotypic examination.
Growthhabit after the recovery of Fig. 9 placenta Subaerial blue green algae.
Figure 10 placenta Subaerial blue green algae cells into cardiomyocytes is induced.
Figure 11 placenta Subaerial blue green algae is induced to liver cell.
Figure 12 placenta Subaerial blue green algae neuralward cell induction.
Embodiment
Embodiment 1 gathers placenta
ABO/Rh Blood grouping, HLA(Human Leukocyte Antigen are carried out to puerpera, 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 the placenta of puerpera.
Before placenta gathers, puerpera fills in Informed Consent Form, individual basic document, then puerpera is identified, gatherer process strictly should prevent the pollution of pathogenic micro-organism, require to carry out in aseptic place, then the placenta collected is transported to appointed place, transportation environment temperature should within the scope of 4-8oC; Should fill in when placenta receives and receive list, mark placenta bottle; Placenta carries out Sterility testing to placenta used, guarantees the security of placenta after receiving.
Embodiment 2 obtains and is separated Human plactnta Subaerial blue green algae
By amnion from ablatio placentae, repeatedly rinse with D-Hank ' the s balanced salt solution that pH value is 7.2-7.4, remove residual blood, being shredded by amnion is 5 × 5 cm
2fritter.Get wherein one piece of amnion and make paraffin section, and carry out immunofluorescence dyeing (SSEA-4 is shown in Fig. 1).Fig. 1 result shows, and SSEA-4 positive cell is mainly distributed in the outermost layer of amnion, i.e. amnioic epithelium layer.
Amnion fragment is equally divided into A, B two groups, the method that wherein A group adopts the present invention to describe is separated, and B group adopts ordinary method digestion to be separated (not being separated with mesenchyme layer by epithelial lining) as a comparison.
In the culture dish of 6-cm, instill 1mL D-Hank ' s balanced salt solution, bottom wetting culture dish, the epithelial lining of A group amnion is positioned over downwards in culture dish, use slide glass to be scraped by the gelatinoid of mesenchyme layer.
The amnion having scraped glue is put into D-Hank ' the s balanced salt solution of 10mL containing 10% (w/v) ACETYLCYSTEINE, incubated at room 15 minutes.Remove mucus remaining on amnion, so that digestion completely.
Get the untreated amnion of part B group and part A group process after amnion, make frozen section, to dye (see figure 2) through hematoxylin-eosin (HE) again, in Fig. 2, the undressed amnion of result display B group also has a large amount of gluey mesenchyme layer, and the amnion mesenchymal layer after the process of A group reduces in a large number, epithelial lining is intact.
About 20mL protein enzyme solution (the Collagenase II-EDTA solution of 0.15% (w/v) mixes with the volume ratio of 1:9 with the trypsin-EDTA solutions of 0.25% (w/v)) is added to A group amnion, under 37oC temperature condition, gas bath digests 30 minutes, after digestion terminates, D-Hank ' s balanced salt solution is used repeatedly to rinse amnion.Part A group amnion is made frozen section, and carries out HE dyeing (see figure 3), in figure, result display is after protein enzyme solution digestion, the cell complete digestion of epithelial lining, and mesenchyme layer is not digested.After digestion terminates, 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 washed down are merged, filters through 100 eye mesh screens, obtain cell suspension.
The trypsin-EDTA solutions adding 0.25% (w/v) of about 20mL to B group digests 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 washed down are merged, filters through 100 eye mesh screens.Repeat twice, obtain cell suspension.
The digestion mixed solution of two groups of amnions is placed in respectively 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, B two groups of amnions obtain is used respectively 10mL D-Hank ' s balanced salt solution re-suspended cell, slowly add 10mL lymphocyte separation medium to cell suspension, with the centrifugation 8 minutes of 250g, do not use braking deceleration.
After centrifugal end, by the cell sucking-off between two-layer liquid level.Use the cell of D-Hank ' s balanced salt solution cleaning sucking-off, with the rotating speed of 1500 revs/min centrifugal 5 minutes, obtain stem cell.
Stem cell A, B two kinds of methods obtained is with 10
5/ hole is inoculated into stem cell medium (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 mark (see figure 4), the result display of Fig. 4, the stem-cell marker SSEA-4 of the cell that method A obtains and epithelial cell mark EpCAM expression rate are all close to 100%, and the amnion of method B is because digest containing a large amount of mesenchyme layers the cell got, the speed of growth is very fast, affect the Subaerial blue green algae growth of epithelial lining, therefore substantially SSEA-4 and EpCAM is not expressed.
The sub-totipotent cell obtained by method A 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 display Ficoll is centrifugal, the SSEA-4 expression rate of cell significantly promotes, and illustrates that cell purity improves.
Embodiment 3 cultivates placenta Subaerial blue green algae
The stem cell obtained is pressed 2 × 10
5/ cm
2be inoculated in culturing bottle, add 10mL stem cell medium, be placed in 37oC, CO
2content is cultivate in the incubator of 5%, and the stem cell medium more renewed after 4-5 days, discards non-adherent cell, within every 3-4 days later, changes stem cell medium once, treats that Growth of Cells reaches 80% fusion, add 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, pat culturing bottle gently with palm and guarantee that cell and tissue block complete digestion are got off, stem cell medium 10mL is added in each culturing bottle, repeatedly blow and beat, cell in culturing bottle is cleaned up, the cell suspension washed 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 medium totally mix cell, obtain the Human plactnta Subaerial blue green algae after cultivating, divide and cultivate in three culturing bottles, be placed in 37oC, CO
2content is cultivate in the incubator of 5%, namely goes down to posterity by 1:3, and obtain the Human plactnta Subaerial blue green algae after Secondary Culture, cell cultures the results are shown in Figure 6.
The mensuration of cell growth curve is after being digested by attached cell protein enzyme solution II, by 1 × 10
4cells/well is seeded in 24 orifice plates, every 24 hours digestion 3 holes, 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, and ordinate zou is number of cells.
Embodiment 4 detects, cryopreserved human placenta Subaerial blue green algae
10mL D-Hank ' s balanced salt solution is used to wash Human plactnta Subaerial blue green algae after Secondary Culture twice, washing lotion is abandoned in suction, 1mL proteinase II solution is added in each culturing bottle, put into 37 ° of C incubators 5 minutes, observation of cell under inverted microscope, and pat culturing bottle gently with palm and guarantee that cell complete digestion is got off; In each culturing bottle, add 10mL stem cell medium again, repeatedly blow and beat; Be placed in centrifuge tube a little with the sucking-off of micro sample-adding rifle and be used as cell counting, according to count results, draw containing about 5 × 10
6the cell suspension of individual cell divides for detecting in another centrifuge tube, and all the other are for frozen.Two tube cells are centrifugal 5 minutes with 1500 revs/min, supernatant is abandoned in suction, cell nutrient solution for detecting blows and beats cell, mixing repeatedly, send into medicine non-clinical study quality control procedure standard laboratory and carry out quality examination, comprise Sterility testing, detection of mycoplasma, Viral diagnosis, immunophenotype detect, cell counting and vitality test, biological efficacy detect, cell differentiation detects, to determine whether it reaches criterion of acceptability; Wherein, Viral diagnosis detects hepatitis B virus, hepatitis C virus, human immunodeficiency virus by real-time fluorescent polyase chain reaction, and criterion is that indices is feminine gender, is considered as competent cells.
Cell counting and vitality test refer to and carry out cell counting with Trypan Blue, and criterion is that Cell viability is more than or equal to 90%, is considered as competent cells.It is detect by direct immunofluorescence that immunophenotype detects, and antibody used is monoclonal antibody SSEA4, Sox2, Oct4, Nanog, TRA1-81, TRA1-60, EpCAM, CD45, HLA-DR of mouse anti human.Add antibody after cell PBS washes, under 4oC, hatch 30 minutes, then wash twice with PBS, blow and beat with 500 μ L PBS, mix cell, use flow cytomery afterwards.The flow cytomery result of the Human plactnta Subaerial blue green algae immunophenotype that method according to the present invention obtains is as shown in table 1.Detected result shows, CD45, HLA-DR in placenta Subaerial blue green algae are negative, and are less than 2%; SSEA4, Sox2, Oct4, Nanog, TRA1-81, TRA1-60, EpCAM are positive, and this placenta Subaerial blue green algae is competent cells.Immunophenotyping detects sees Fig. 8.
The immunophenotype of table 1 Human plactnta Subaerial blue green algae
| Mark | Expression amount |
| SSEA4 | + |
| Sox2 | + |
| Oct4 | + |
| Nanog | + |
| TRA1-81 | + |
| TRA1-60 | + |
| EpCAM | + |
| CD45 | - |
| HLA-DR | - |
Cryopreservation step is: in the Human plactnta Subaerial blue green algae obtained, add foetal calf serum, make to meet 1 × 10
6-2 × 10
6cell/0.9mL foetal calf serum, fully mixes, stand-by; Freeze pipe and freeze box paste bar code, on freeze pipe, writes same bar code number simultaneously; Above-mentioned Human plactnta Subaerial blue green algae suspension and frozen storing liquid joined in cryopreservation tube according to 1:1 ratio and mix, first joined respectively by cell suspension after in freeze pipe, then unification adds frozen storing liquid, capping, concussion, fully mixes, is moved into rapidly in-80oC refrigerator; After 24 hours, be put on corresponding freezing frame according to numbering and move into the medium-term and long-term preservation of liquid nitrogen, have detailed frozen record during cell cryopreservation, meet freezen protective standard, storage condition periodic monitoring, the detection of Human plactnta Subaerial blue green algae and frozenly parallelly to carry out, result to be detected out after, underproof cell will be detected and according to its numbering, corresponding freeze-stored cell will together be discarded together with cryopreservation tube, make a record, put into refuse bag, concentrate and be positioned over specified location, transport process by technician.
The frozen impact on cytoactive and surface marker of embodiment 5
After frozen cell being placed in 37oC water-bath recovery, 10mL stem cell medium is used to be inoculated in culturing bottle, at 37 DEG C, 5%CO
2cultivate in incubator.After cell attachment, the stem cell medium more renewed, discards non-adherent cell.Cell is counted and vitality test count results in table 2, Fig. 9 is shown in by cell photo.
Table 2 frozen front and back stem cell count 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 - |
Embodiment 6 placenta Subaerial blue green algae cells into cardiomyocytes is induced
The stem cell of separation is inoculated in 24 orifice plates of pre-add stem cell medium with 5000 cells/well, after cell attachment, stem cell medium is removed, adds myocardial cells culture liquid (the DMEM/F12 nutrient solution containing 1 mM Ascorbic acid), cultivate 28 days, within every 3 days, change liquid.After induction terminates, by Immunofluorescence test Troponin T(TNNT), experimental result is shown in Figure 10.
Embodiment 7 placenta Subaerial blue green algae is induced to liver cell
The cell of separation is inoculated in 24 orifice plates of pre-add stem cell medium with 5000 cells/well, after cell attachment, stem cell medium is removed, adds myocardial cells culture liquid (containing 0.1 μM of Insulin and 1 × 10
-7the DMEM/F12 nutrient solution of M Dexamethasone), cultivate 28 days, within every 3 days, change liquid.After induction terminates, by Immunofluorescence test human serum albumin (albumin, ALB) and cytokeratin (Cytokeratin 19, CK19), experimental result is shown in Figure 11.
Embodiment 8 placenta Subaerial blue green algae neuralward like cell is induced
The cell of separation is inoculated in 24 orifice plates of pre-add stem cell medium with 5000 cells/well, after cell attachment, stem cell medium is removed, adds myocardial cells culture liquid (containing 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 terminates, by Immunofluorescence test MAP2, GFAP, Tublin and Nestin, experimental result is shown in Figure 12.
Claims (3)
1. be separated a method for Human plactnta Subaerial blue green algae, described Human plactnta Subaerial blue green algae derives from human placenta's amnion of stripping, expresses marker molecule SSEA4, Sox2, Oct4, Nanog, TRA1-81, TRA1-60 and EpCAM, does not express CD45; It can in culture vessel adherent growth, can in human body, in, ectodermal histological differentiation; And it can be induced to differentiate into myocardial cell, liver cell, neural-like cells;
It is characterized in that:
First scrape with the gelatinoid of slide glass by amnion mesenchymal layer with before protein enzyme solution digestion placenta amnion, and remove residual gelatinoid by ACETYLCYSTEINE.
2. method according to claim 1, it is characterized in that, wherein said protein enzyme solution is that the Collagenase II-EDTA solution of 0.15% (w/v) mixes with the volume ratio of 1:9 with the trypsin-EDTA solutions of 0.25% (w/v).
3. method according to claim 1 and 2, is characterized in that, comprises the following steps:
(1) by amnion from ablatio placentae, repeatedly rinse with D-Hank ' the s balanced salt solution that pH value is 7.2-7.4, remove residual blood, being shredded by amnion is 5 × 5cm
2fritter;
(2) in the culture dish of 6-cm, instill 1mL D-Hank ' s balanced salt solution, bottom wetting culture dish, the epithelial lining of amnion is positioned over downwards in culture dish, use slide glass to be scraped by the gelatinoid of mesenchyme layer;
(3) amnion having scraped glue is put into D-Hank ' the s balanced salt solution of 10mL containing 10% (w/v) ACETYLCYSTEINE, incubated at room 15 minutes; Remove mucus remaining on amnion, so that digestion completely;
(4) add about 20mL protein enzyme solution to amnion, under 37 DEG C of temperature condition, gas bath digests 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) mixes with the volume ratio of 1:9 with the trypsin-EDTA solutions of 0.25% (w/v);
(5), after digestion terminates, D-Hank ' s balanced salt solution is used repeatedly to rinse amnion;
(6) Digestive system and the liquid washed down are merged, filter through 100 eye mesh screens, repeat twice, obtain cell suspension;
(7) cell suspension is placed in whizzer with the rotating speed of 2000 revs/min centrifugal 15 minutes, is is repeatedly blown and beaten by bottom cell D-Hank ' s balanced salt solution, 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, slowly add 10mL Ficoll lymphocyte separation medium to cell suspension, with the centrifugation 8 minutes of 250g, do not use braking deceleration;
(9) after centrifugal end, by the cell sucking-off between two-layer liquid level; Use the cell of D-Hank ' s balanced salt solution cleaning sucking-off, with the rotating speed of 1500 revs/min centrifugal 5 minutes, remove supernatant, obtain stem cell.
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| CN105441387B (en) * | 2014-09-03 | 2019-01-25 | 黄福来 | Subaerial blue green algae special culture media and its dedicated cultural method |
| CN104974980B (en) * | 2015-05-21 | 2018-06-22 | 重庆市细胞生物工程技术有限公司 | A kind of separation method of human amniotic membrane epithelial cell |
| CN105200007B (en) * | 2015-10-20 | 2018-06-26 | 博雅干细胞科技有限公司 | The method that Subaerial blue green algae is extracted from placental fetal surface chorion |
| CN106701665A (en) * | 2016-12-30 | 2017-05-24 | 东莞惠恩生物工程有限公司 | Method for induced differentiation of human placenta sub-totipotent stem cells to obtain cardiac muscle cells and application thereof |
| 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 |
| 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 |
| CN110172441A (en) * | 2019-05-29 | 2019-08-27 | 内蒙古银宏干细胞生命科技投资有限公司 | A kind of separation method of Human plactnta Subaerial blue green algae |
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