CN104560870B - A kind of method for preparing decidua mescenchymal stem cell - Google Patents
A kind of method for preparing decidua mescenchymal stem cell Download PDFInfo
- Publication number
- CN104560870B CN104560870B CN201410795200.0A CN201410795200A CN104560870B CN 104560870 B CN104560870 B CN 104560870B CN 201410795200 A CN201410795200 A CN 201410795200A CN 104560870 B CN104560870 B CN 104560870B
- Authority
- CN
- China
- Prior art keywords
- decidua
- stem cell
- mescenchymal stem
- cell
- placenta
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Abstract
The invention provides a kind of method for preparing decidua mescenchymal stem cell, including the use of male's term fetus placenta as raw material, aseptic process placenta；Separate decidua tissue；Identify decidua tissue and obtain decidua mescenchymal stem cell whether the identification from maternal tissue；Cultivate decidua mescenchymal stem cell；Decidua mescenchymal stem cell freezes；The recovery of decidua mescenchymal stem cell.The method of the present invention has following technical characterstic：Gather fetus be male placenta, obtain decidua tissue after through sex identification whether the pollution from parent without daughter tissue；Germ contamination method is taken precautions against, pollution probability is reduced from collection source, repeatedly rinses surface, effectively reduce pollution probability；Using single enzymic digestion, flow can be simplified；Animal derived components are reduced using serum free medium culture to use, and cell stable performance, can be maintained decidua mescenchymal stem cell long-term cultivation process in vitro, be maintained cellular morphology, multiplication capacity, the expression of MSC surface markers, differentiation capability etc..
Description
Technical field
The present invention relates to a kind of method for preparing decidua mescenchymal stem cell, belong to stem cells technology field.
Background technology
Mescenchymal stem cell (mesenchymal stem cell, MSC) is derived from developing mesoblastic a kind of ability more
Cell, there is self-renewing and multi-lineage potential, it can be divided into polytype histocyte under specific inductive condition,
The Various Tissues such as bone, cartilage, fat, cardiac muscle can be formed.Mescenchymal stem cell is mainly derived from marrow earliest, but in people's marrow
Mescenchymal stem cell (BMMSC) content is extremely low.In recent years, possessed the mesenchymal cell of ancestral cells feature from peripheral blood,
Separated in the Various Tissues such as dental pulp, cartilage, muscle, umbilical cord and placenta.It can be separated from placenta tissue and come from son
Umbilical cord mesenchymal stem cells, amnion mesenchymal stem cell and the chorion mescenchymal stem cell of body, and sloughing off from parent
Intermembranous mesenchymal stem cells.The mesenchymal cell of adult can be divided into a variety of thin with specific function under different conditions
Born of the same parents, such as Gegenbaur's cell, cartilage cell, adipocyte, cardiac muscle cell, myocyte, there is huge potential medical treatment and storage
Value.Umbilical cord, amnion and the chorion of placenta can extract mescenchymal stem cell, as child's own cells transplanting or
Freeze.And the mescenchymal stem cell that the decidua of placenta extracts, from parent, it can be used for the transplanting of mother's own cells
Or freeze, there is specific aim.
Decidua mescenchymal stem cell obtains frequently with enzymic digestion decidua tissue, uses the medium culture containing 10%FBS, more
Frozen with the frozen stock solution containing 10%DMSO.
In the prior art, the method for preparing decidua mescenchymal stem cell usually there will be following weak point：
1) prior art of decidua tissue separation, can not peel off the simple decidua tissue for coming from parent, always be mixed with tire
The fine hair membrane tissue or amnion tissue from daughter of disk.
2) prior art of placenta collection and decidua separation, which lacks, effectively reduces pollution measure, and anti-pollution measure effect has
Limit, original cuiture pollution rate is high, lacks the method that pollution is reduced since being gathered source, at present in culture link addition antibiotic
Contaminated bacteria breeding can only be suppressed in incubation, it is impossible to effectively reduce pollution；
2) existing enzymic digestion method is complicated, needs multiple enzymic digestion；
3) operation such as enzymic digestion, cryopreservation resuscitation is likely to cause cellular damage to cell；
4) the easy aging of cell in stem cell incubation, cell is flat, and increment is slowly or stopping is bred, and loses differentiation capability
Deng, illustrated there has been no method or technique experience effectively maintain decidua mescenchymal stem cell form at present, prevent cell senescence, maintenance
Cell retains differentiation capability in breeding；
5) Telomerase inactivation may occur after mescenchymal stem cell Long Term Passages, oncogene expression increases expression of tumor suppressor gene
Decline, karyotypic alteration equivalent risk, lack the research data of decidua mescenchymal stem cell this respect at present.
The content of the invention
The purpose of the present invention is overcome the deficiencies in the prior art part, there is provided one kind prepares simple decidua mescenchymal stem cell
Method.
The method of the preparation decidua mescenchymal stem cell of the present invention, comprises the following steps：
1) male's term fetus placenta in 0~30min of childbirth discharge is used as raw material；Tire is rinsed with placenta cleaning fluid
Disk appearance 1-3 times, placenta is soaked in placenta cleaning fluid；
2) placenta being immersed in placenta cleaning fluid is transported to laboratory in 48h, takes out placenta, cleaned again with cleaning fluid
1-3 times, then from the careful separation decidua vera of placenta bottom, after cleaning up, be put into cleaning fluid and soak 0~30min, then by decidua
Tissue shear is broken into 1~3mm3The tissue block of size；
3) decidua tissue is kept sample and carries out sex identification, it is determined that the decidua tissue of collection comes from merely parent；
4) decidua mescenchymal stem cell is obtained from decidua tissue block by collagenase digestion；
5) use serum free medium culture decidua mescenchymal stem cell, wherein P0 for decidua mescenchymal stem cell cover with to
After 80% density, using 0.25% pancreatin digest, digestion time 3-5min, P0 instead of after, cell passage operate after 48-
Passed in 72h, it is desirable to which cell density 80%-90% when passing on, cell presses (3-8) × 10 after passage3Individual/cm2Density connects
Kind, the pH value change of observation culture medium is paid attention in incubation, once the addition fresh culture and to remove culture vessel medium of turning yellow
Old culture medium is measured, cell passes on from P0 generations, reaches any generation among P1~P30；
Wherein,
Cleaning fluid in described step 1) and step 2) is 0.9% physiological saline；
Clostridiopetidase A used is final concentration of 1-5mg/ml clostridiopetidase A in collagenase digestion in described step 4)
II；
Serum free medium in described step 5) consists of the following composition：DMEM/F12、PDGF-BB、bFGF、
(TGF)-β 1, EGF and transferrins Transferrin；Wherein, PDGF-BB contents are 50~100ng/mL；BFGF contents are 0
~50ng/mL；(TGF) contents of-β 1 are 0~20ng/mL；EGF contents are 0~30ng/mL；Transferrins Transferrin contains
Measure as 2.0~4.5mg/mL.
Preferably,
Cleaning fluid in described step 1) and step 2) also contains antibiotic, and described antibiotic is penicillin, strepto-
Element or amphotericin B.
The step of collagenase digestion in described step 4), is as follows：Decidua tissue block is put into 37 DEG C of temperature, shaken
15~60min of digestion is swung, the physiological saline that volume is 1~5 times of tissue block volume is added, is centrifuged with 2500 turns/min rotating speed
5min, supernatant is removed, product after bottom is digested, be transferred to culture vessel, 10ml serum free mediums are added by 1ml decidua tissues block
Ratio addition serum free medium, the serum free medium in described step 4) consists of the following composition：DMEM/F12、
PDGF-BB, bFGF, (TGF)-β 1, EGF and transferrins Transferrin；Wherein, PDGF-BB contents are 50~100ng/
mL；BFGF contents are 0~50ng/mL；(TGF) contents of-β 1 are 0~20ng/mL；EGF contents are 0~30ng/mL；Transferrins
Transferrin contents are 2.0~4.5mg/mL.
Serum free medium in described step 4) consists of the following composition：DMEM/F12+80ng/mL PDGF-BB+
20ng/mL bFGF+10ng/mL (TGF)-β 1+10ng/mL EGF+3.0mg/mL transferrins.
Antibiotic is also added with serum free medium in described step 4), described antibiotic is penicillin, chain
Mycin or amphotericin B.
Serum free medium in described step 5) consists of the following composition：DMEM/F12+80ng/mL PDGF-BB+
20ng/mL bFGF+10ng/mL (TGF)-β 1+10ng/mL EGF+3.0mg/mL transferrins.
For P0 in described step 5) for antibiotic is also added with the serum free medium of cell culture, described is anti-
Raw element is penicillin, streptomysin or amphotericin B.
The cryopreservation methods of described decidua mescenchymal stem cell are as follows：Every 106-107Cell adds 1ml frozen stock solutions, is put into jelly
Box is deposited, is transferred to -80 DEG C of refrigerators, is transferred in -196 DEG C of liquid nitrogen or gas nitrogen and saves backup after 12~24h；The frozen stock solution is by basal liquid
Formed with permeability cryoprotector, described permeability cryoprotection agent concentration is 1-1.4mol/L, and described basal liquid is
Nutrient solution DMEM/F12；Described permeability cryoprotector is dimethyl sulfoxide (DMSO).
The method of the preparation decidua mescenchymal stem cell of the present invention, has the following technical effect that：
1) selection fetus is raw material for the mature placenta of male, can take precautions against pollution method, and pollution probability is reduced from source,
Surface is repeatedly rinsed, effectively reduces pollution probability；
2) decidua materials region is limited:Most surface decidua tissue is softly stripped from placenta bottom, reduction mixes cell contamination；
Sex identification is carried out to the decidua tissue stripped down simultaneously, it is ensured that the decidua tissue stripped down does not have its hetero-organization of placenta
Pollution.
3), using single enzymic digestion, flow can be simplified when separating decidua mescenchymal stem cell in this patent method；
4) reduce animal derived components using serum free medium culture to use, cell according to said method can be passaged to from P0 generations
P30 maintains stable performance, can maintain decidua mescenchymal stem cell long-term cultivation process in vitro, maintains cellular morphology, propagation energy
Power, the expression of MSC surface markers, differentiation capability, can also maintain decidua mescenchymal stem cell long-term cultivation process in vitro, maintain end
Granzyme expression, the stable expression of oncogene, caryogram are stable；
5) in this method enzymic digestion and cryopreservation resuscitation to cell fanout free region.
Brief description of the drawings
Fig. 1 is the aspect graph of obtained decidua mescenchymal stem cell；
Fig. 2-1 is sex identification result of the decidua tissue with other placenta tissues of separation；
Fig. 2-2 is the surface markers testing result figure of obtained decidua mescenchymal stem cell；
Fig. 3 is the aspect graph through the postdigestive decidua mescenchymal stem cell of digestive ferment；
Fig. 4 is the growth curve testing result figure through the postdigestive decidua mescenchymal stem cell of digestive ferment；
Fig. 5 is P0~P10 using serum free medium culture for decidua mescenchymal stem cell aspect graph；
Fig. 6 be P2, P5 and P10 using serum free medium culture for decidua mescenchymal stem cell, with aged cells and
The compares figure of neoblast form；
Fig. 7 is growth curve measure testing result figure；
Fig. 8 is skeletonization, breaks up testing result figure into fat, into chondrocyte induction；
Fig. 9 is flow cytometer detection result figure；
Figure 10 is Telomerase activity result figure；
Figure 11 is oncogene testing result figure；
Figure 12 is G Banded karyotype analysis result figures.
Embodiment
Reagent used is purchased from the manufacturer in bracket behind reagent name in embodiment.
Embodiment 1 prepares decidua mescenchymal stem cell
The method of preparation decidua mescenchymal stem cell in the present embodiment, comprises the following steps：
1) male's term fetus placenta in 0~30min of childbirth discharge is used as raw material；Operating personnel sterile gloves
Taking placenta, prevent placenta contact from may bring the object (such as ground) of pollution in operating process, rinsed with placenta cleaning fluid
Appearance 3 times, cleaning fluid are 0.9% physiological saline (the Guizhou world), and antibiotic can be added or be added without in cleaning fluid, addition
Antibiotic is penicillin (gibco), streptomysin (gibco), amphotericin B (sigma).Surface is wiped with sterile gauze, is removed
Surface bloodstain, it is soaked in after being dripped to placenta surface no liquid in the placenta collecting cassette containing cleaning fluid, cleaning will be immersed in 48h
Placenta in liquid is transported to laboratory.
2) after placenta is transported to laboratory, it is positioned among the plate of 40cm × 40cm × 20cm (length × width × height), with nothing
Bacterium cleaning fluid rinses appearance 3 times, and cleaning fluid is 0.9% physiological saline (the Guizhou world), can add or be added without in cleaning fluid
Antibiotic, the antibiotic of addition is penicillin (gibco), streptomysin (gibco), amphotericin B (sigma).Placenta is inverted, from
Decidua is softly stripped in the outermost layer tissue of placenta bottom, the decidua tissue of clip is cleaned with cleaning fluid, and cleaning fluid is 0.9% physiology
Salt solution (the Guizhou world), add cleaning fluid immersion 30min.Decidua tissue is shredded into 1~3mm3The tissue block of size.
After the start to process of above-mentioned placenta source, in follow-up incubation, pollution rate effectively reduces.Placenta source does not disappear
Poison, subsequent contamination incidence are (25.3 ± 7.2%), and carrying out pollution incidence by this method is reduced to (2.4 ± 1.5%), shows
Work reduces pollution.
3) decidua tissue is kept sample and carries out sex identification, it is determined that the decidua tissue of collection comes from merely parent
Sex appraisal method：Tissue that needs are identified or cell is taken to be carried according to the method for kit (solarbio, D1800)
Total genome is taken, the conserved sequence SYB added on the reagent extension Y chromosome of PCR kit, agarose is carried out to result
(takara) gel electrophoresis.From the results, it was seen that decidua tissue SYB is feminine gender, come from maternal tissue i.e. decidua, and other
Positive is all autologous tissue.
For qualification result as shown in Fig. 2-1, it is the moon that the conserved sequence on Y chromosome is shown as from the decidua tissue of parent
Property, and the tissue characterization result from chorion, amnion, umbilical cord is the positive.As can be seen that decidua tissue is derived from parent.
4) decidua mescenchymal stem cell is separated from decidua tissue block
The volume ratio that 1-5ml digestive ferments are added by every 1ml tissue blocks adds digestive ferment (gibco), and described digestive ferment is
Final concentration of 1-5mg/ml clostridiopetidase A II (gibco).
Digestion step is as follows：The decidua tissue block for having added digestive ferment is placed in 37 DEG C of temperature, vibration digestion 15-60min,
Vibration is provided by oscillator.Add volume be 1-5 times of tissue block volume 0.9% physiological saline (the Guizhou world), 2500 turns
5min is centrifuged, removes supernatant.Product after bottom is digested, is transferred to culture vessel, and the training of 10ml serum-frees is added by 1ml decidua tissues block
Support the ratio addition serum free medium of base (gibco).Continue culture after being climbed out of to decidua mescenchymal stem cell 5~20 days.
The composition of serum free medium is：DMEM/F12(invitrogen)+80ng/mL PDGF-BB(gibco)+
20ng/mLbFGF(invitrogen)+10ng/mL(TGF)-β1(Peprotech)+10ng/mL EGF(gibco)+3.0mg/
ML transferrins (sigma).
Can be added in serum free medium containing antibiotic, antibiotic be penicillin (gibco), streptomysin (gibco),
Amphotericin B (sigma).
Using the above method, the operating time of every part of tissue is saved, reducing the consumable reagents such as centrifuge tube, physiological saline makes
With；Digestion time shortens, and 60min is shorten to by the 90min of two-step method.
5) decidua mescenchymal stem cell is cultivated
The serum free medium culture of decidua mescenchymal stem cell.
The composition of serum free medium is：DMEM/F12(invitrogen)+80ng/mL PDGF-BB(gibco)+
20ng/mLbFGF(invitrogen)+10ng/mL(TGF)-β1(Peprotech)+10ng/mL EGF(gibco)+3.0mg/
ML transferrins (sigma).
Culture P0 can add antibiotic for the serum free medium that decidua mescenchymal stem cell uses, and antibiotic is mould
Plain (gibco), streptomysin (gibco), amphotericin B (sigma).
Using serum free medium culture decidua mescenchymal stem cell, P0 is covered with to 80% close for decidua mescenchymal stem cell
After degree, digested using 0.25% pancreatin (gibco), digestion time 3-5min.Generation after P0 generations is used without antibiotic
Medium culture.
P0 instead of after, cell is passed in 48~72h after passage operates, it is desirable to which cell density reaches when passing on
80%-90%, cell presses 3 × 10 after passage3-8×103Individual/cm2 density inoculation.Observation culture medium pH value is paid attention in incubation
Change, the addition fresh culture once PH turns yellow.Taken pictures per 24h and observe and record cellular morphology.
As shown in Figure 1, it can be seen that it is good to obtain passage form；
As shown in Fig. 2-2, surface markers meet ISCT to mescenchymal stem cell examination criteria；
As shown in figure 3, after the direct function cells of digestive ferment, cell maintains good form；
As shown in figure 4, Cell growth ability is uninfluenced after digestion；
As shown in table 1, surface markers are uninfluenced.Illustrate this digestion formula of liquid to mescenchymal stem cell in decidua and decidua
Mescenchymal stem cell does not have an impact.
Table 1
It is as shown in figure 5, good for decidua mescenchymal stem cell form using P0~P10 of serum free medium culture.
P0~P30 generations it is middle it is any once can all carry out morphologic observation, propagation test, induction differentiation, streaming, Telomerase,
Oncogene expression, caryogram detection.
1. morphologic observation records cellular morphology, contrasted with aged cells and neoblast form, testing result is such as
Shown in Fig. 6, it can be seen that cellular morphology remains good after passage, unaged；
2. propagation test includes：Growth curve determines, and testing result is as shown in Figure 7, it can be seen that repeatedly cell after passage
Maintain stronger proliferation activity；
3. induction differentiation detection includes：Skeletonization, detect into fat, into cartilage, testing result is as shown in Figure 8, it can be seen that more
Cell maintains differentiation capability after secondary passage；
4. flow cytometer detection includes：CD73, CD90, CD105, CD14, CD34, CD45, CD79- α, HLA-DR, testing result
As shown in Figure 9, it can be seen that repeatedly cell surface marker expression remains stable after passage, meets MSC examination criterias；
5. Telomerase activity method is：UsingTelomerase Detection Kit (millipore) are tried
Agent box detects, and testing result is as shown in Figure 10, it can be seen that repeatedly cell does not lose telomerase activation after passage；
6. oncogene detection includes：Real-time PCR detection oncogenes (c-Myc, c-fos, k-ras) and tumor suppressor gene
(P53, P21, RB, P16) is expressed, and testing result is as shown in figure 11, it can be seen that oncogene and expression of tumor suppressor gene expression are more steady
It is fixed；
7. caryogram detects：Analyzed using G Banded karyotypes, testing result is as shown in figure 12, it can be seen that cell is through repeatedly passing
It is normal for rear caryogram, it is both the caryogram that cell caryogram should be women, sex chromosome XX.
Therefore, the decidua mescenchymal stem cell cell that preparation method of the invention obtains in succeeding generations, can be tieed up in vitro
Cellular morphology, multiplication capacity, the expression of MSC surface markers, differentiation capability are held, also decidua mescenchymal stem cell can be maintained to grow in vitro
Phase incubation, maintain the stable expression of Telomerase Expression, oncogene, caryogram stable.
6) decidua mescenchymal stem cell is frozen：
Every 106~107Cell adds 1ml frozen stock solutions, is put into freezing storing box, is transferred to -80 DEG C of refrigerators, -196 are transferred to after 12-24h
Preserved in DEG C liquid nitrogen or gas nitrogen.
Decidua mesenchymal stem cell cryopreserving liquid is made up of basal liquid and permeability cryoprotector, the freezing of described permeability
Protection agent concentration is 1mol/L, and described basal liquid is nutrient solution DMEM/F12 (invitrogen)；Described permeability freezing
Protective agent is dimethyl sulfoxide (DMSO) (gibco).
7) recovery decidua mescenchymal stem cell：
The decidua mescenchymal stem cell or decidua tissue block that will be recovered take out from liquid nitrogen container, are put into 37 DEG C of water-baths and incubate
2-3min is educated, after being washed with 0.9% physiological saline (the Guizhou world) of 4 DEG C of precoolings, supernatant is removed in centrifugation, adds free serum culture
Base.
Free serum culture based component used is identical with step 5) in this step.
Cryopreservation resuscitation success rate：Cryopreservation resuscitation 111 times, success rate 100%.Cellular morphology is good, flow cytometer detection is qualified, point
The detection of change ability is qualified.
The specific method step difference for the various detections being related in the present invention is as follows.Involved parameter is by following instruments
Measure：OLYMPUS inverted microscopes, Lycra just put (laica) microscope, FACSAira flow cytometers, UV, visible light point
Light photometer, Biorad fluorescent PCRs instrument, BioradPCR instrument, incubator, low temperature refrigerator, liquid nitrogen container etc..
(1) OLYMPUS microscopes are taken pictures
The P2 turned out is fused to more than 80-90% for decidua mescenchymal stem cell, is placed under inverted microscope and claps
According to cell shape is short fusiformis, and size is homogeneous, marshalling, as shown in Figure 6.
(2) drafting of growth curve
The cell prepared is taken, is digested to single cell suspension；Serum free medium adjusts concentration to 1 × 104/ ml, point add
Enter in 96 orifice plates；If 13 groups every group 8 multiple holes, per the μ l of hole 200, be placed in 37 DEG C, containing 5% CO2, saturated humidity incubator in
Culture；Change liquid within every 2 days, respectively after cultivating to 1-13 days, 20 μ l concentration is added per hole for 5mg/mL tetrazolium bromide (MTT)
(sigma, M5655), continue to cultivate；Culture supernatant is carefully removed after 4 hours；100 μ l dimethyl sulfoxide (DMSO) is added per hole
(takara, 67-68-5), micro oscillator shake 5 minutes；The light surveyed under 570nm or 490nm on ELIASA (Thermo) is put to inhale
Receipts value, growth curve is drawn out after statistical analysis.Digestion after cell growth curve as shown in figure 4, repeatedly passage after cell
Growth curve is as shown in Figure 7.
(3) Multidirectional Differentiation ability is identified
By 1 × 104/ ml P2 for cell suspension inoculation in 24 well culture plates, 0.5ml/ holes；Cell fusion is to 60-
80%, change osteogenic induction complete medium (gibco), 0.5ml/ holes into；Induction broth is changed per 3-4 days full doses；Induction 24
After it, taken pictures after alizarin red (Chinese medicines group) dyeing, it is seen that there are a large amount of calcium tubercles to produce, as shown in Figure 8.
By 1 × 104/ m P2 for cell suspension inoculation in 24 well culture plates, 0.5ml/ holes；Cell fusion is to 80-
90%, change adipogenic induction complete medium (gibco), 0.5ml/ holes into；Induction broth is changed per 3-4 days full doses；Induction 14
After it, taken pictures after oil red O (gibco) dyeing, it is seen that it is all to have fat drips generation into the cell, as shown in Figure 8.
By 5 × 105P2 for cell suspension inoculation in 15ml centrifuge tube, cultivate, change into within second day soft after centrifugation
Self-bone grafting complete medium (gibco), 0.5ml/ holes；Induction broth is changed per 3-4 days full doses；After induction 21 days, cut into slices, Ah
You take pictures in Xinlan's (Chinese medicines group) dyeing, it is seen that and it is all to have cartilage generation into the cell, as shown in Figure 8.
(4) FCM analysis
By 5 × 106Cell dissociation is into single cell suspension；Concentration is adjusted after being washed twice with PBS to 1 × 105/ml, streaming
Cell instrument (BD, FACSAira) detection cell surface marker positive indication CD90, CD73 and CD105, negative indication CD34,
CD14, CD45, CD79a, HLA-DR, more than 95%, negative indication meets less than 2% positive cell rate, meets mesenchyma and does
The feature of cell, as shown in Figure 9.
(5) Telomerase activity
Take 1*106Cell according toThe explanation of Telomerase Detection Kit (milipore) kit
Book extraction sample is identified that testing result is as shown in Figure 10, it can be seen that repeatedly cell does not lose telomerase activation after passage.
(6) oncogene and tumor repressive gene detects
Take decidua mescenchymal stem cell 1x106Cell extraction RNA (with reference to QIAGEN RNA extracts kits specification), it is inverse
CDNA (TOYOBO First Strand cDNA Synthesis Kit) is transcribed into, oncogene is added and tumor suppressor gene is carried out
Quantitative fluorescent PCR (biorad) detects.Use 2-△△CtMethod analyzes experimental data, detects related proto-oncogene and tumor suppressor gene
Expression whether significant changes occur.Real-time PCR detection oncogenes (c-Myc, c-fos, k-ras) and tumor suppressor gene
(P53, P21, RB, P16) is expressed, and testing result is as shown in figure 11, it can be seen that oncogene and expression of tumor suppressor gene expression are more steady
It is fixed.
(7) karyotyping
Take the decidua mescenchymal stem cell in culture to be handled with colchicine, digest, be collected by centrifugation, PBS washings.It is hypotonic
In 20-40 minutes, a small amount of fixer is added dropwise in KCL (0.075M) processing, processing time, and centrifugation 1500rpm/5min removes supernatant, protected
1ml supernatants are stayed, gently piping and druming is hanged, and is slowly added to fixer (methanol：Glacial acetic acid is according to 3：1 ratio mixes), the vibration of side edged,
Until full packages.Supernatant is removed in centrifugation, gently pats and hangs, and is slowly added to fixer, side edged vibration, until full packages.4 spend night,
Supernatant is removed in centrifugation.Piece is dripped, slide is placed on 0 degree of ice-water bath in advance, takes out and drips piece from eminence after slide.Quickly overdo or air is done
Dry, after Giemsa staining, (laica) is observed under mirror.
Claims (8)
- A kind of 1. method for preparing decidua mescenchymal stem cell, it is characterised in that comprise the following steps：1）Raw material is used as using male's term fetus placenta in 0~30min of childbirth discharge；Placenta is rinsed with placenta cleaning fluid Appearance 1-3 times, placenta is soaked in placenta cleaning fluid；2）The placenta being immersed in placenta cleaning fluid is transported to laboratory in 48h, placenta is taken out, cleans 1-3 with cleaning fluid again It is secondary, then from the careful separation decidua vera of placenta bottom, after cleaning up, be put into cleaning fluid and soak 0~30min, then by decidua group Knit and shred into 1~3mm3The tissue block of size；3）Decidua tissue is kept sample and carries out sex identification, it is determined that the decidua tissue of collection comes from merely parent；4）Decidua mescenchymal stem cell is obtained from decidua tissue block by collagenase digestion；5）Using serum free medium culture decidua mescenchymal stem cell, wherein P0 is covered with to 80% for decidua mescenchymal stem cell After density, digested using 0.25% pancreatin, digestion time 3-5min, P0 instead of after, cell enters in 48-72h after passage operates Row passage, it is desirable to which cell density 80%-90% when passing on, cell is pressed after passage（3-8）×103Individual/cm2Density is inoculated with, incubation It is middle to pay attention to observation medium pH value changes, once flavescence adds fresh culture and removes the old culture medium of culture vessel moderate, Cell passes on from P0 generations, reaches any generation among P1~P30；Wherein,Described step 1）With step 2）In cleaning fluid be 0.9% physiological saline；Described step 4）In collagenase digestion in used clostridiopetidase A be final concentration of 1-5mg/ml clostridiopetidase A II；Described step 5）In serum free medium consist of the following composition：DMEM/F12、PDGF-BB、 bFGF、(TGF)-β 1st, EGF and transferrins Transferrin；Wherein, PDGF-BB contents are 50~100 ng/mL；BFGF contents are 0~50 ng/mL；(TGF) contents of-β 1 are 0~20ng/mL；EGF contents are 0~30 ng/mL；Transferrins Transferrin contents are 2.0~4.5 mg/mL.
- 2. the method according to claim 1 for preparing decidua mescenchymal stem cell, it is characterised in that described step 1）With Step 2）In cleaning fluid also contain antibiotic, described antibiotic is penicillin, streptomysin or amphotericin B.
- 3. the method according to claim 1 for preparing decidua mescenchymal stem cell, it is characterised in that described step 4）In Collagenase digestion the step of it is as follows：Decidua tissue block is put into 37 DEG C of temperature, vibration 15~60min of digestion, adds body Product is the physiological saline of 1~5 times of tissue block volume, centrifuges 5min with 2500 turns/min rotating speed, supernatant is removed, after bottom is digested Product, culture vessel being transferred to, the ratio that 10ml serum free mediums are added in 1ml decidua tissues block adds serum free medium, Described serum free medium consists of the following composition：DMEM/F12, PDGF-BB, bFGF, (TGF)-β 1, EGF and turn iron egg White Transferrin；Wherein, PDGF-BB contents are 50~100 ng/mL；BFGF contents are 0~50 ng/mL；(TGF)-β1 Content is 0~20ng/mL；EGF contents are 0~30 ng/mL；Transferrins Transferrin contents are 2.0~4.5 mg/ mL。
- 4. the method according to claim 3 for preparing decidua mescenchymal stem cell, it is characterised in that described serum-free training Foster base consists of the following composition：DMEM/F12+80ng/mL PDGF-BB+ 20ng/mL bFGF+ 10ng/mL (TGF)-β1+ 10ng/mL EGF+3.0mg/mL transferrins.
- 5. the method for the preparation decidua mescenchymal stem cell according to claim 3 or 4, it is characterised in that the no blood Antibiotic is also added with clear culture medium, described antibiotic is penicillin, streptomysin or amphotericin B.
- 6. the method according to claim 1 for preparing decidua mescenchymal stem cell, it is characterised in that described step 5）In Serum free medium consist of the following composition：DMEM/F12+80ng/mL PDGF-BB+ 20ng/mL bFGF+ 10ng/mL (TGF)-β 1+10ng/mL EGF+3.0mg/mL transferrins.
- 7. the method for the preparation decidua mescenchymal stem cell according to claim 1 or 6, it is characterised in that described step 5）In P0 for antibiotic is also added with the serum free medium of cell culture, described antibiotic is penicillin, strepto- Element or amphotericin B.
- 8. the method according to claim 1 for preparing decidua mescenchymal stem cell, it is characterised in that filled between described decidua The cryopreservation methods of matter stem cell are as follows：Every 106-107Cell adds 1ml frozen stock solutions, is put into freezing storing box, is transferred to -80 DEG C of refrigerators, and 12 It is transferred in -196 DEG C of liquid nitrogen or gas nitrogen and saves backup after~24h, the frozen stock solution is by basal liquid and permeability cryoprotector group Into described permeability cryoprotection agent concentration is 1-1.4 mol/L, and described basal liquid is nutrient solution DMEM/F12；It is described Permeability cryoprotector be dimethyl sulfoxide (DMSO).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410795200.0A CN104560870B (en) | 2014-12-18 | 2014-12-18 | A kind of method for preparing decidua mescenchymal stem cell |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410795200.0A CN104560870B (en) | 2014-12-18 | 2014-12-18 | A kind of method for preparing decidua mescenchymal stem cell |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104560870A CN104560870A (en) | 2015-04-29 |
| CN104560870B true CN104560870B (en) | 2017-12-01 |
Family
ID=53077950
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410795200.0A Active CN104560870B (en) | 2014-12-18 | 2014-12-18 | A kind of method for preparing decidua mescenchymal stem cell |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104560870B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109097391A (en) * | 2018-07-11 | 2018-12-28 | 个体化细胞治疗技术国家地方联合工程实验室（深圳） | A kind of method and its application that decidua mescenchymal stem cell active factors are mass produced |
Families Citing this family (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105018421A (en) * | 2015-05-27 | 2015-11-04 | 贵州北科泛特尔生物科技有限公司 | Culturing method for human placental mesenchymal stem cells |
| CN105238748A (en) * | 2015-10-27 | 2016-01-13 | 上海百众源生物科技有限公司 | Preparation method of placenta-source decidua parietalis mesenchymal stem cells by separation and refrigeration |
| CN106434544A (en) * | 2016-11-08 | 2017-02-22 | 华南生物医药研究院 | Method for acquiring chorion mesenchymal stem cells and kit |
| CN107022521A (en) * | 2017-02-13 | 2017-08-08 | 广东唯泰生物科技有限公司 | Decidua vera tissue freezing, the method recovered and be separately cultured mescenchymal stem cell |
| CN106957814B (en) * | 2017-05-19 | 2020-09-04 | 广州赛莱拉干细胞科技股份有限公司 | Culture medium for amniotic mesenchymal stem cells and method for culturing amniotic mesenchymal stem cells |
| CN108251362A (en) * | 2017-12-27 | 2018-07-06 | 重庆斯德姆生物技术有限公司 | A kind of preparation method of decidua mescenchymal stem cell |
| CN108192861A (en) * | 2017-12-27 | 2018-06-22 | 重庆斯德姆生物技术有限公司 | A kind of preparation method of mescenchymal stem cell applied to climacteric syndrome |
| CN108103009A (en) * | 2018-01-31 | 2018-06-01 | 溯源生命科技股份有限公司 | A kind of preparation method of placenta mesenchyma stem cell |
| CN109182260A (en) * | 2018-09-11 | 2019-01-11 | 邵勇 | A kind of method of in vitro culture fetal membrane mescenchymal stem cell |
| CN109652363A (en) * | 2018-11-13 | 2019-04-19 | 广东唯泰生物科技有限公司 | Placenta decidua vera Derived from Mesenchymal Stem Cells is the method for endometrial epithelial cell |
| CN112011504A (en) * | 2020-09-09 | 2020-12-01 | 江苏育瑞康生物科技有限公司 | Preparation method of placenta decidua mesenchymal stem cells |
| CN113136364B (en) * | 2021-04-13 | 2022-05-03 | 广东唯泰生物科技有限公司 | Preparation method and recovery method of decidua-muralis mesenchymal stem cells |
| CN113322228B (en) * | 2021-04-13 | 2022-08-19 | 广东唯泰生物科技有限公司 | Culture medium and co-culture method of decidua-mesenchymal stem cells for treating tumors |
| CN114717185A (en) * | 2022-03-31 | 2022-07-08 | 秦岭大熊猫研究中心(陕西省珍稀野生动物救护基地) | Separation and culture method of giant panda placenta mesenchymal stem cells |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101974484A (en) * | 2010-11-03 | 2011-02-16 | 江苏省北科生物科技有限公司 | Method for preparing human umbilical cord mesenchymal stem cells |
-
2014
- 2014-12-18 CN CN201410795200.0A patent/CN104560870B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101974484A (en) * | 2010-11-03 | 2011-02-16 | 江苏省北科生物科技有限公司 | Method for preparing human umbilical cord mesenchymal stem cells |
Non-Patent Citations (2)
| Title |
|---|
| 人胎盘底蜕膜间充质干细胞的分离及其生物学特性研究;韩之波 等;《中国实验血液学杂志》;20131231;第21卷(第3期);754-759 * |
| 人胎盘底蜕膜间充质干细胞的分离培养及其多向分化潜能的实验研究;卢国辉 等;《南方医科大学学报》;20111231;第31卷(第2期);262-265 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109097391A (en) * | 2018-07-11 | 2018-12-28 | 个体化细胞治疗技术国家地方联合工程实验室（深圳） | A kind of method and its application that decidua mescenchymal stem cell active factors are mass produced |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104560870A (en) | 2015-04-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104560870B (en) | A kind of method for preparing decidua mescenchymal stem cell | |
| CN104560869B (en) | A kind of method for preparing chorion mescenchymal stem cell | |
| EP1594957B1 (en) | Progenitor cells from wharton's jelly of human umbilical cord | |
| TWI773693B (en) | A method of isolating mesenchymal stem cells from the amniotic membrane of the umbilical cord, a mesenchymal stem cell population isolated from the amniotic membrane of the umbilical cord and a cell culture medium for isolating mesenchymal stem cells from the amniotic membrane of the umbilical cord | |
| TWI535377B (en) | Storage, culture and application of umbilical cord tissue and its derived cells | |
| CN102851254A (en) | Procurement, isolation, and cryopreservation of endometrial/menstrual cells | |
| CN105713871A (en) | Human chorion mesenchymal stem cell isolated culture method | |
| CN107022521A (en) | Decidua vera tissue freezing, the method recovered and be separately cultured mescenchymal stem cell | |
| CN105950550A (en) | Mesenchymal stem cell serum-free medium and cell isolation and cultivation methods | |
| CN101629165A (en) | Preparation method of umbilical cord Huatong glue mesenchymal stem cells and product thereof | |
| CN104560871A (en) | Culturing method of mesenchymal stem cells of menstrual blood | |
| CN110079498B (en) | Human placenta mesenchymal stem cell and preparation method and application thereof | |
| CN104762259A (en) | Culture medium for mesenchymal stem cells and large-scale culture method thereof | |
| CN104630142B (en) | A kind of isolation and culture method of ox umbilical cord mesenchymal stem cells | |
| CN108184818A (en) | A kind of Human plactnta mesenchyma stem cell suspension protective agent | |
| KR20100084620A (en) | Cell composition for tissue regeneration | |
| Romanov et al. | Optimized protocol for isolation of multipotent mesenchymal stromal cells from human umbilical cord | |
| CN108938669B (en) | Stem cell ointment for treating skin injury and preparation method thereof | |
| CN104472474A (en) | Human adipose tissue-derived stromal cell frozen stock solution | |
| CN107349220A (en) | A kind of preparation comprising fibroblast excretion body and application thereof | |
| CN104726401A (en) | Method for improving success rate of umbilical cord blood mesenchymal stem cell culture by using placental mesenchymal stem cells | |
| CN101353644A (en) | Vascular endothelial cells, and preparation and use thereof | |
| CN109628388A (en) | With digestive enzyme compositions from placenta blood vessel separating mesenchymal stem cell | |
| CN109652362A (en) | A kind of method that umbilical cord film saves and prepares stem cell | |
| CN107058225A (en) | A kind of co-induction culture medium and using the culture medium inducing umbilical cord mesenchymal stem into neuron cell method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |